Polynucleotide sequence (options), polypeptide (options), fused protein, a pharmaceutical composition, a monoclonal antibody, a hybrid strain of cultured cells

 

(57) Abstract:

The invention relates to propertytaxsession systems that require cleavage product a predecessor to the new polypeptide capable of restoring dichloroindophenol and oxidized glutathione to DNA that encodes this polypetide, farmkompanijam comprising the polypeptide, monoclonal antibodies against the specified polypeptide. Polynucleotide sequence encoding a protein characterized by the structural formula a-b-C. And represents the nucleotide sequence 10-1311 in SEQ ID NO. In represents Asn-Cys-Ser-Phe-Gln. With is a sequence encoding a polypeptide KM31-7. Polypeptide KM31-7 has the amino acid sequence 1-526 in SEQ ID 12. The pharmaceutical composition contains polypetide KM3 1-7 in combination with a pharmaceutically acceptable carrier for him. Monoclonal antibody specifically interacts with the polypeptide KM31-7. It is produced by a strain of hybrid cultivated cells of Mus musculus ΜM 150-2 FERM BP-5086. Refers to the Ig isotype G1. The invention allows to obtain a new polypeptide with healing activity in vivo and can be used for the treatment of diseases caused by the relevant systems, requiring cleavage product's predecessor, as well as proteases used in these systems. In addition, the present invention relates to a new polypeptide capable of restoring dichloroindophenol and oxidized glutathione to DNA that encodes a specified new polypeptide, vectors containing the indicated DNA to the cell host transformed by these vectors, and to pharmaceutical compositions containing the specified polypeptide. The present invention also relates to monoclonal antibodies against the indicated polypeptide and to a method for isolation and purification of the polypeptide using the indicated antibodies.

Prior art

Potyvirus are a group of viruses that have a genome consisting of about 10,000 bases of single-stranded RNA, and that infect plants, such as plants of the family Solanaceae (Solancede). Genome potyvirus differs in that it has an exceptionally long open-reading frames (ORF) (Dougherty, W. G. & Hiebert, E. (1980) Virology 101, 466-474, Allison, R. et. al. (1986), Virology 154: 9-20). For the expression of individual proteins encoded in ORF, translated polyprotein digested two types of proteases, which also code which is a member of the family potyviruses and produces nuclear inclusions, which in infected cells can be painted Trifanova blue. These nuclear inclusions consist apparently of two kinds of protein, one of which, as shown, is a viral protease, designated "nuclear inclusion a" or Nla (J. Virol., 61 2540-2548 (1987)).

The protease potyviruses called nuclear inclusion a, recognize and break down the peptide sequence that includes one of the Gln-Cly, Gln-Ser, and Cln-Ala, and which is, obviously, a review of the sequence located at the C-end of the Nla inside polyprotein. This cleavage occurs between the two residues forming the above dimers.

Were determined the complete genomic sequence TEV virus and stem spots tobacco (TYMV), another member of the family potyviruses, and the homological studies of these sequences, it was found that Nla these viruses are localized in their respective genomes. (Virology 154, 9-20 (1986), Nucleic Acids Res., 14: 5417-5430 (1986)).

Virus yellow proalcool mosaic clover, or CYVV, is also potyviruses. Up to the present time was sequenced only gene located at the 3'-end of the genome CYVV, together with the protein shell, which atmlane, and, in addition, the inclusion of the Nla was not selected.

The production of exogenous proteins by expressing systems can be implemented directly using well known techniques. However, there are many polypeptides that cannot be easily expressed in exogenous systems. The problem is that these polypeptides can not be synthesized in large quantities, and simple introduction of the regulatory element of the above-mentioned gene does not lead to the desired result. Alternatively, it may be that posttranscriptional processing required to generate the Mature form of the protein, either not happening or going wrong.

For example, the translation of many eukaryotic polypeptides begins with N-terminal methionine, which is then deleterule with the formation of a Mature form. In prokaryotes such processing does not occur, and therefore it was necessary to find an alternative way to achieve the expression. One such method involves stitching the desired exogenous protein, for example, maltose-binding protein or glutathione-S-transferases, and then purification of the synthesized hybrid protein with poltronova method is it requires two stages of cleaning, which leads to a significant loss of the final product.

In U.S. patent N 5162601 disclosed the use of TEV protease in the synthesis of polyprotein containing between proteins of the linker sequence, which is desirable for the synthesis of the polypeptide, such as people tPA. However, this patent describes only the cloning multigene encoding specified polyprotein, in the host cell, but does not describe neither the expression nor the cleaning proteoliticeski split the final product.

Oxygen for metabolic energy, usually supplied oxidizing agents present in the environment of the cell environment. Activated form, in the form which is usually consumed oxygen, are free radicals such as superoxide (O2-), peroxide (H2O2), or hydroxyl radical (OH-), all these radicals, after use, are restored with the formation of water (H2O). Gaseous oxygen, in itself, is a very good oxidizing agent, however, used in the present description, the term "activated oxygen" refers to the oxygen or oxygen-containing molecules with more Vista is a free radical, which is a molecule or atom that has one or more unpaired electrons.

Free radicals usually are unstable, and without proper control, they can denaturing lipids, proteins and nucleic acids. Therefore, although the activated oxygen is necessary for life support, it can represent a significant danger to human health, resulting in its amount in the body should be strictly regulated. Due to its high reactivity of activated oxygen, even in very small quantities, can cause certain health problems. Therefore, a highly active form of oxygen even able to kill the living cell only if the cell does not possess the mechanisms of protection against specified invalid oxygen.

Environmental cell environment, localization, number and time of generation of activated oxygen must be carefully balanced against the ability of the cell to counteract the damaging effect. This ability is usually provided by the protection mechanisms of the cells, using for this purpose its own antioxidants or antioxidant enzymes. In ineni, having the ability to prevent or inhibit the auto-oxidation, for example, lipids. The term "antioxidant enzyme" means primarily an enzyme that catalyzes the reaction of elimination of activated oxygen, and the term "antitissue action" means an action for the elimination of the specified activated oxygen.

An excessive amount of activated oxygen in the body can induce a number of pathological phenomena in the body, similar to the phenomena caused by stress, taking drugs, Smoking, surgery, transplantation of any organ or phenomena caused by ischemia, or even a heart attack, brain or myocardium. Under large quantities of oxygen refers to the amount exceeding the level of oxygen that the regulatory system of the body is able to eliminate, and so the excess oxygen have a toxic effect on the body, causing serious disruption in the life of its cells. This toxic effect, otherwise called oxidative stress, is responsible for the emergence of many Patologicheskaya the production of low-density lipoprotein, which were oxidized with activated oxygen (Steinberg, D. (1983), Arterioslerosis 3, 283-301). It is also believed that oxidative stress is directly related to the mechanisms of occurrence of metabolic disorders and vascular complications of diabetes (Kondo, M. ed., "Approaches from Modern Medicine (4) Free Radicals", Medical View Pub., pp. 138-146).

Activated oxygen is also the cause of other pathological conditions and disorders, such as ischemic disorders (reperfusion injury, ischemic heart disease, cerebral ischemia, ischemic enteritis, etc.,), edema, vascular sorprendentemente, inflammatory diseases, gastric mucosal injury, acute pancreatitis, a disease KROK, ulcerative colitis, liver disease, disease Paraquat, emphysema, chemical carcinogenes, metastasis of cancer, respiratory distress syndrome in adults, disseminated intravascular coagulation (DIC), cataract, petrolatina fibroplasia, autoimmune diseases, porfiria, hemolysis, erythroblastopenia (Mediterranean) anemia, Parkinson's disease, Alzheimer's disease, epilepsy, disorders caused by ultraviolet radiation, disorders of radioactive protection mechanisms, focused exclusively on the elimination of activated oxygen generated inside the body.

It is known that intracellular remedies, namely antioxidants and antioxidant enzymes, some of which are listed below, disposed of and helps eliminate active oxygen. For example, in peroxisome cells is catalase, which restores and removes hydrogen peroxide. Glutathione peroxidase is present in the cytoplasm and mitochondria, catalyzes the rehabilitation and detoxification of hydrogen peroxide and lipid peroxides in the presence of reduced glutathione. Transferrin, ferritin, and lactoferrin, for example, inhibit the generation of activated oxygen species by stabilizing the iron ions, whereas ceruloplasmin carries out a similar function in respect of copper ions. In addition, in the cytoplasm and mitochondria include superoxide dismutase, which catalyzes the recovery of superoxide with the formation of hydrogen peroxide, which is then eliminated by catalase. In addition to the above enzymes, the ability to recover and elimination of activated oxygen have also vitamins C and E, restored glutathione and other disqualifica the glutathione peroxidase, and restored glutathione are present in the extracellular space, where they have the same functions as their intracellular counterparts described above. However, compared with the situation taking place inside the cells, in the extracellular environment is a much smaller number of different types of antioxidants and antioxidant enzymes, and, in addition, only a few of them have antioxidant effect.

Restored glutathione, the formula of which is presented below, plays a major role in maintaining recovery condition both outside and inside the cell. For the first time glutathione was detected in yeast in 1888, de-Rey-Pailhade, and its name glutathione received after his selection as connections in 1921 Hopkins.

< / BR>
Glutathione is composed of three amino acids: glutamic acid, cysteine and glycine. Thiol groups of two molecules of glutathione can be oxidized with the formation of disulfide bonds in the presence of activated oxygen, which leads to the recovery of the specified activated oxygen.

Glutathione is produced mainly in the liver, after which it enters the bloodstream and circulates in the body. In a normal body glutathione, the CIO regeneration restored form through the action of glutathione reductase in the presence of nicotinamide-dinucleotide (NADP). Thus, the restored glutathione protects cell membranes from the damaging action of activated oxygen by restoring the specified active oxygen and free radicals. Due to its antioxidant properties restored glutathione also has anti-radiation effect and can be used as a therapeutic tool against cataracts. In addition, recently it was found that patients suffering from AIDS, systemic levels of reduced glutathione reduced, indicating the critical role of glutathione in the body. However, in abnormal conditions, the number of activated oxygen may be so great that virtually all of glutathione present in the oxidized state, and the activated oxygen is removed much more slowly than it usually occurs in normal conditions.

A second example of connection with, due to its oxidizing properties, various physiological functions, is thioredoxin present both in the intracellular and extracellular space. Thioredoxin man (also known as factor T-cell leukemia is the second T-cell leukemia adult. This factor represents a thiol-dependent reductase, having in its active centre two cysteine residue and has the ability to restore active oxygen and free radicals.

In addition to induction 1L-2 receptor, thioredoxin person also has the effect of stimulating the growth of cells of B-cell strain 3B6 infected by the virus of Epstein - Barr (EBV), protective effect against tumor necrosis factor (TNF), derived from the monocytic cell line U937, and a protective effect against damage to vascular endothelial cells by neutrophils. In addition, due to its restorative activity within the cell, thioredoxin person affects the transcription factors NFkB, JUN and FOS, which helps to stimulate DNA-binding activity and thereby increase the transcriptional activity. Currently developing a way to use thioredoxin person as a protective agent against radiation injuries, and also as a therapeutic agent for the treatment of reperfusion injury, rheumatoid arthritis and inflammatory diseases, in a word, such injuries and diseases, which can be the mouth of the>As mentioned above, for normal physiological activity of the organism is extremely important that the intracellular and extracellular space were maintained in the reducing conditions that can be achieved by elimination of activated oxygen and free radicals. It is obvious that in the intracellular and extracellular space is much still unknown antioxidants and antioxidant enzymes that perform the function of removing active oxygen and free radicals. Therefore, it would be extremely important to detect these recovery agents, able to regenerate, for example, restored glutathione. Such substances could use for the treatment of various pathological conditions, such as diseases and disorders described above.

Brief description of the invention.

The first aim of the present invention to provide a new protease; a nucleotide sequence that encodes the protease; vector containing a DNA sequence encoding the indicated protease, and a host cell transformed by the specified vector.

The second objective of the present invention to provide a DNA that encodes DNA sequence, located between the two DNA sequences, also encodes a peptide cleaved specified by the protease, and all of these sequences are in the same open reading frame. In addition, the aim of the present invention to provide a protein encoded by a specified DNA and the vector containing this DNA and expressing the system includes the specified vector and the specified vector is capable of Autonomous replication in the appropriate cell host, for example, the cell containing the nucleotide sequence necessary for the implementation of Autonomous replication.

Alternatively, the first aim of the present invention to provide a nucleotide sequence that encodes a new polypeptide with healing activity in vivo. In addition, the present invention is also such a DNA sequence that encodes a peptide capable of restoring dichloroindophenol and oxidized glutathione.

Another objective of the present invention to provide a recombinant vector containing the above DNA and having ability to AB is providing the specified Autonomous replication.

Another objective of the present invention is the production of a microorganism host, transformed with the above recombinant vector. In addition, the aim of the present invention to provide the above-mentioned peptide as an expression product of a transformed host cell, and obtaining monoclonal antibodies against this peptide.

In this work, we identified and cloned a new CYVV-protease (Nla), and at the same time, suddenly discovered that CYVV-Nla can be used as part of a hybrid protein is able to be expressed in such a host like E. coli, which allows to produce large quantities of the selected hybrid protein, which, in addition, has the ability to samaradarley with the formation of the desired exogenous product. This CYVV-Nla gene may be stably maintained and expressed in Esherichia coli, and expressed Nla-product retains its activity as a specific protease activity, even if this protein is only part of this hybrid protein.

In addition, we also found a DNA sequence, encoding a new polypeptide with the ability of chloro-4-(4-hydroxyphenyl)imino-2,5-cyclohexadiene-1-one), and oxidized glutathione, and the specified polypeptide can be produced in large quantities by using techniques of genetic engineering. This polypeptide can be particularly effectively used for the treatment of diseases caused or mediated oxidative stress, or any other diseases caused by active oxygen, such as atherosclerosis, diabetes, and coronary heart condition (including reperfusion disorders, ischemic heart disease, cerebroside and ischemic enteritis).

For example, in the first aspect of the first version of its implementation, the present invention relates to a polynucleotide sequence which, in the direction 5_ 3 and in the same open reading frame, includes:

a) a sequence encoding a protein "nuclear inclusions and" virus yellow prozhilkovo mosaic clover, or a mutant or variant with the same proteolytic specificity, and protein nuclear inclusions and" virus yellow prozhilkovo mosaic clover, (b) a sequence encoding a peptide that is recognized and cleaved by the specified protein nuclear inclusions and" virus yellow prozhilkovo mosaic clover, or egoscue the invention also relates to the sequence, encodes a protein with nuclear inclusions and" virus yellow prozhilkovo mosaic clover, or a mutant or variant with the same proteolytic specificity of the indicated protein nuclear inclusions and" virus yellow prozhilkovo mosaic clover.

In addition, the present invention relates to a vector, in particular by expressing the vector containing the sequence defined above.

The present invention also relates to host transformed by the vector, as defined above; by expressing the system includes the specified host and the specified expressing vector, and the polypeptide produced specified expressing system.

In the first alternative aspect, the present invention relates to a polynucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acids 1-526 of sequence ID No. 12, or encodes a mutant or variant of a specified polypeptide, provided that the polypeptide encoded a specified polynucleotide sequence, has the ability to restore dichloroindophenol and oxidized glutathione.terashima sequence, defined above.

The present invention also relates to host transformed by the vector, as defined above, by expressing the system includes the specified host and the specified expressing vector, and the polypeptide produced specified expressing system.

In addition, the present invention relates to the aforementioned polypeptide, which can be used for therapeutic purposes, the use of such polypeptide for the treatment and prevention of diseases or conditions caused or mediated oxidative stress, or any other diseases caused by active oxygen, as well as to pharmaceutical compositions containing the specified polypeptide.

In addition to the above, the present invention relates to a monoclonal antibody and its equivalents, directed against the specified polypeptide, the method of production of this antibody, as well as to the method of purification of the polypeptide using the indicated antibodies.

Brief description of Fig. 1 to 7, 8A, 8b, 9 - 13

The present invention is illustrated in Fig. 1 to 7, 8A, 8b, 9 - 13, described below.

In Fig. 1 shows Rennie plasmids pKN15', containing the 5'-region of the nuclear inclusion a (Nla).

In Fig. 3 illustrates the construction of plasmids pKN151L containing part 1L-11-gene and 5'-region Nla.

In Fig. 4 shows the primers that were used to obtain 5' 1L-DNA fragment and C1N3-DNA fragment and the 3'-end of the Nla gene was cross-linked to the 5'-end of 1L-11-gene.

In Fig. 5 illustrates the ligation C1N3-DNA portion and 5'-1L-DNA fragment using polymerase chain reaction.

In Fig. 6 illustrates the construction of plasmid pK SUN 9.

In Fig. 7 shows the restriction map of the plasmid PUCKM31-7.

In Fig. 8a and b shows the comparison of the nucleotide sequences of the 3'-ends in pUCKM31-7 and pcD-31.

In Fig. 9 is a diagram of a design of pSR 31-7.

In Fig. 10 shows a diagram introduction sequence that encodes a his-tag hexamer, plasmid pUCKM31-7.

In Fig. 11 shows the construction of plasmid pMAL31-7.

In Fig. 12 illustrates the analysis dichlorphenol-indophenol-healing activity.

In Fig. 13 illustrates the determination of the activity aimed at restoring oxidized glutathione.

Detailed opisaniya its implementation, however, this description can also be attributed to his second option, except in those cases where it is obvious that this discussion cannot be applied to the specified second option.

It should be noted that the preferred form of the polynucleotide sequences of the present invention is DNA, and therefore, further description will relate primarily to DNA. However, where this is necessary, in the description there are also indications on RNA. RNA is not the preferred form of the nucleotide sequence for the present invention because its use is practically limited. For example, mRNA can be expressed in socito Xenopus (Xenopus) or in the system lysates wheat seedlings, but none of expressing these systems are not able to economically produce large quantities of hybrid protein.

Used in the present description, the term "peptide" refers to any molecule that contains 2 or more amino acids linked by a peptide bond. This term can mean Oligopeptide, polypeptide or protein. The term "hybrid protein" refers to any individual polypeptide, on the same note, the preferred sequence of the present invention is double-stranded sequence; however, the present invention also addresses the antisense sequence corresponding to the sequence of the present invention. Double-stranded (DC) sequence of the present invention can have one or two "sticky" end, and in this case, it is not necessary that the antisense and sense strands closely match each other.

This meant that the protein encoded by the sequence (a), defined above, cleaves the peptide encoded by the sequence (b), defined above, resulting in the released polypeptide(s) encoded by the sequence (C), defined above, provided that the specified sequence of the present invention is expressed in a suitable expressing the system.

Cleavage of the hybrid protein can take place at any time after the broadcast of the sequences (a) and (b), defined above. In this regard, it should be noted that the polypeptide encoded by the sequence (c), defined above, does not have to be fully translated before splitting. For a practitioner who is fully translated before splitting.

If the sequence (c), defined above, encodes more than one polypeptide, in this case, it is necessary that, in addition, was coded split sequence located between each of the encoded polypeptides, except for those cases when you want to get unsplit hybrid of many polypeptides.

If the sequence (c), defined above, encodes more than one polypeptide, that this sequence may be susceptible to attenuation in terms of transcription. When attenuation transcription of the mRNA sequences of the present invention is terminated before you read all of mRNA, resulting in polypeptides encoded closer to the 3'-end of the sequence will be produced in smaller quantities than the polypeptides encoded closer to the 5'-end. However, if not coded several polypeptides and/or if these polypeptides are not very long, any problems associated with attenuation, does not occur.

Mostly it is preferable that the sequence (c), defined above, encode only one polypeptide, except for those occasions when you need to get a few peptides for the case, after splitting, you will need to select each individual polypeptide, and this operation can be very time-consuming, and, in addition, the cleanup can lead to a significant loss of the final product.

However, if the sequence (c), defined above, encodes more than one polypeptide, and if between each of these encoded polypeptide has split the sequence, then it is not necessary that these split sequence is recognized CYVV-Nla. All that is required in this case is to split the sequence between the sequence (a) and 5'-end sequence (b), is recognized by the protease encoded by the sequence (a). However, if necessary or appropriate, to hybrid protein was subjected to samaradarley with the formation of several polypeptides, any other split sequences can be recognized by the protease, encoded by the sequence (a). Moreover, these other sequences can be chosen in such a way that they can be split in other ways. For example, a hybrid protein will be broken down to its Nla-part after transcription education in rezultatele trypsin.

Cleaved peptide encoded by the sequence (b), defined above (hereinafter also referred to as a "split-sequence" and "split peptide") can be a sequence that entirely or partially included in any of the sequences encoded by the sequences (a) and (c) ("Nla" or "protease" and "polypeptide", respectively). So, for example, N-end of the cleaved peptide can also be included in the C-terminal sequence of the protease, and the C-terminal portion of the cleaved peptide may be included in the N-terminal part of the polypeptide. In this case, the cleaved peptide cannot exist independently from the C-Terminus of the protease and to the N-Terminus of the polypeptide must be designed restriction sites for the protease.

Cleaved peptide may be included only in part or protease or a polypeptide. In this case, the N-end of the cleaved peptide is included in the C-terminal part of the protease, and N is the portion of the polypeptide is either directly connected with fissionable sequence, or between the polypeptide and the linker may contain one or more amino acid residues.

If split between sequence and prikatnyh residues, the number and nature of these residues must be such that these residues do not hamper the action of the protease. Excess amino acid residues at the N end of the polypeptide mostly undesirable, as these residues have to be removed to obtain the Mature forms of the polypeptide. It is generally preferable, if possible, and if you do not have any contraindications to construct split the peptide so that the Mature form of the desired protein was formed after cleavage of the hybrid protein. However, it is possible to obtain a protein that has, for example, Gly, Ser or Ala, is attached to the N-end, which can then be removed, if necessary, using the appropriate amino peptidases.

In some cases, it may be preferred between the N end of the polypeptide and the split sequence to encode a Proline. This will allow, for example, by using amino peptidases P (3.4.11.9) to split the remains in front of Proline, but not after it. And then polynomy residue may be removed using a Proline-aminopeptidase with getting Mature protein. This alternative implementation of the present invention is preferred.

Sequence (a) Cody is. the accordance with the present invention, the protease is "nuclear inclusion a (Nla) virus yellow prozhilkovo mosaic clover or a mutant or variant with the same proteolytic specificity, as specified Nla-protease of the virus clover yellow mosaic.

This prosthesis, namely nuclear inclusion a (Nla) virus yellow prozhilkovo mosaic clover (CYVV), encoded by nucleotides 10-1311 sequence ID No. 1 (see below, the list of sequences), and the primary sequence Nla represented by amino acids 4-437 sequence ID No. 2 (see below, the list of sequences). These sequences are new, and therefore, as well as their mutants and variants, they are included in the scope of the present invention.

"Nuclear inclusion and has proteolytic activity, contributing to the specific hydrolysis of the peptide bond between Gln-Ala, Gln-Gly or Gln-Ser peptide substrate. In addition, it was also discovered that the Nla may split Gln-Val. Thus, the authors of this application it was found that in contrast to other protease family potyviruses, Nla CYVV (hereinafter, if it is not specified particularly, the designation Nla will mean Nla CYVV) can split the placenta, what more preferably Gly, Ala or Ser.

It should also be noted that peptides comprising the sequence AsnCys SerPheGlnX can be split Nla-protease, especially if this sequence is subjected to steric effects Nla protease. Thus, the present invention also relates to a system used to obtain the polypeptide, where the precursor polypeptide containing a sequence AsnCys SerPheClnX, split Nla-protease. This system can also include any other stage of processing, carried out, if necessary, before, after or simultaneously with the splitting of the Nla-protease.

Although generally it is preferable to use a sequence encoding a natural Nla (sequence ID No. 2), however, in the present invention with the same success can be used mutants or variants Nla.

As mentioned above, these mutants or variants must have a specificity similar to the specificity of natural Nla. In accordance with this mutant or variant should have the sequence, mainly homologous amino acid sequence 4-437 in SEQ ID N2, except in those cases where, obviously AC is gaudie protease activity below an acceptable level.

In General, it can be noted that the activity of this protein depends on certain conserved regions of this molecule, while other areas do not play such an important role and can be virtually or totally unnecessary. Accordingly, as mentioned above, the present invention includes any variants and mutants, which possess a specificity similar to the specificity of natural Nla protease. These variants or mutants may include, for example, deletions, insertions, additions, inversions, repeats, and substitutions (e.g., one hydrophilic residue on the other hydrophilic balance, but no such substitution, as strongly hydrophobic residue on highly hydrophilic residue). Small changes usually do not have significant effect on the activity of the molecule, if only they do not fall on the main part of the molecule, and such changes can occur as a by-product of genetic manipulation, for example, when designing unnecessary restriction sites, if necessary.

Generally speaking, any compelling reasons to change the structure of Nla usually does not occur, except, may be, separate from the obvious cases. Indeed, most the t in the result of the release of new versions of the natural wild-type virus. Nevertheless, intentional or even accidental modifications should also be included in the present invention, provided that they possess the necessary specificity and sufficient proteolytic activity.

Used in the present description, the term "adverse effect" means any effect on the specificity or activity of the protease, resulting in significant reduction of its efficiency compared to natural Nla (see above), due to a decrease in its activity below the required level.

This can be done many substitutions, insertions, etc., unless they adversely affect the activity of the protease. As a rule, a serious adverse effect on the protease activity takes place only in the case when 3-D (tertiary) structure Nla undergoes significant modification.

Used in the present description, the term "mutant" refers to deletions, additions, insertions and substitutions of amino acid residues in the sequence of the prosthesis, the activity of which is not subject to adverse effects. Used in the present description, the term "variants" refers to natural Nla CYVV, have a major within, expected for this population. The term "variants" includes parallel options, and peptides from those species which exhibit a similar type of activity and have a related sequence

It should be noted that neither the Nla or protein related to the second variant of implementation of the present invention, does not completely correspond to the sequence ID No. 2 and 12, respectively. The only thing that is needed is that each of these polypeptides had the desired activity, regardless of whether the polypeptide of any specified part of the natural sequence, or it is just a mutant or variant of this part of the sequence.

It is believed that the genes of eukaryotes, such as interferon gene, usually detect polymorphism (see T. Nishi et al, 1985), J. Biochem 97, 153 to 159). This polymorphism leads to the fact that in some cases one or several amino acids in the polypeptide are substituted, and in other cases, such substitution is not observed, despite the substitution in the nucleotide sequence.

In accordance with this, it should be noted that the coding sequence of the polynucleotide can also be modi who I protease activity. For example, can be made point mutations and other changes with the addition or deletion of restriction sites, for example, to exercise certain genetic manipulation/expressions or for the more effective carrying out certain modifications Nla-molecules.

The term "mutant" or "variant" as used in the present description, refers to polynucleotide sequences that can be constructed with the appropriate necessary changes. It should be noted that although mutations or variations in the peptide sequence is always reflected in the coding nucleotide sequence, but the converse is not necessarily true. Therefore, it may be that a significant change in the nucleotide sequence (see below the discussion about the degeneracy of the genetic code) will not have any effect on the peptide sequence. In accordance with the above-mentioned mutants and variants of the nucleotide sequence is also included in the scope of the present invention.

For example, it was found that the protein obtained by replacing the cysteine codon in the gene for interleukin (ID-2) serine code capable of what goes any nucleotide sequence provided that it encodes a natural or synthetic protein, suitably Nla-activity.

The gene encoding Nla-protease of the present invention can be easily obtained by any specialist by reverse engineering on the basis of sequence ID N2.

It should be noted that due to the degeneracy of the genetic code, any specifically designed sequence is not necessarily good, but it will not hybridisierung with any of this complementary sequence is constructed on the basis of the same peptide. This factor is known to every specialist, and the degeneracy of any particular sequence is often so obvious that the synthesis of even very short complementary oligonucleotide sequences for use as probes for natural oligonucleotide sequence represents a significant challenge.

For example, the degeneracy of the code may be such that the most frequently occurring amino acids can match 4 or more codons. Hence it is obvious that the number of possible coding sequences for pepti is one of the coding sequences for the Nla of the present invention may be expressed by six-digit numbers or even numbers of higher order. However, it may be desirable to balance the G+C content in accordance with the considered expressing system, and in this case it is necessary to consider other factors such as the frequency of occurrence of the codon for expressing this system.

As mentioned above, hybridization cannot serve as a reliable indicator of homology sequences, but nevertheless mostly preferred are those sequences, the homology of which in relation to sequence ID No. 1 is more than 50%, preferably 70%, and more preferably 80%. But in any case, the protease defined above, must be encoded.

The present invention also relates to the possible use of a leader sequence, coded "higher" than the protease. This sequence facilitates the externalization of the hybrid protein from the host cell and its release into the culture supernatant. Externalised hybrid protein can then be samorasschepleniyu with the formation of the polypeptide. For these signal sequences may be used in any suitable sequence, in particular such sequence, which was spaceram, containing a sequence of the present invention. In accordance with the present invention the nature of the vector is not critical. Suitable expression vectors and constructs that are required for their production, can be selected by a specialist, depending on the objectives pursued, for example, cloning or expression.

Suitable expressing vectors are phage or plasmid vectors, both of which are host-specific, although they are often used for other owners. Other suitable vectors are Comedy and retroviruses or any other media that may be specific or non-specific for this system. Suitable regulatory sequences, such as the recognition sequence, a promoter, operator, inductor, the terminator and the other sequences are typically used for regulating the expression can be easily selected by the specialist, and these sequences can be associated with CYVV or with the vector, or they may occur from any other source. These vectors can be modified or designed accordingly.

It should be noted, is a protease. To facilitate alloying in an appropriate vector or expression or both to this sequence can be added to the terminator, the promoter and any other regulatory sequence.

It should be noted that the DNA fragment encoding Nla of the present invention together with any fragment, encoding the split sequence and the fragment encoding the polypeptide (s) can be easily inserted into any suitable sector. In the ideal case, to facilitate the insertion is desirable to use a vector with appropriate restriction sites, but can also carry out ligation for blunt ends, although it may lead to uncertainty in respect of the open reading frame and the direction of insertion. In such cases, of course, it is desirable to conduct the experimental transformation of the hosts transfected with a vector in the order of selection vectors having the desired fragments, interturbine in the right direction and in the correct reading frame. For full guarantee that these fragments will be in the correct reading frame, it is desirable to create an appropriate design of the sequences (a), (b) and (c), which can then be utilized in the vector. Suitable vectors can be chosen by the expert in accordance with the needs expressed by the system.

The desired protein can be produced, for example, by transformation of E. coli obtained by plasmid followed by selection of transformants using ampicillin or other suitable means, and the addition of tryptophan or other suitable inducer of the promoter (such as intolerability acid). The level of expression can be estimated using LTO-polyacrylamide gel electrophoresis (LTOs-page) (Laemmli et al., Nature, (1970), 227, pp. 680-685).

It should also be noted that when using other vector may be used any other suitable selective marker or markers or an alternative method of selection, and/or, if necessary or convenient, may be used any suitable promoter.

If a hybrid protein must be distinguished from host cells, after culturing the transformed cells are harvested, disintegrate, for example, ultrasonic treatment and centrifuged. The destruction of cells can also be carried out by enzymatic hydrolysis, for example, using the pulp or by mixing with Stekla is the duty to regulate the use of additional ingredients. The resulting supernatant can be analyzed for polypeptide activity, and the cleavage products can be evaluated, for example, using DSP-PAG electrophoresis.

To obtain the expression product are standard protein purification methods.

The DNA of the present invention can be obtained by separating the RNA genome of the virus, the yellow prozhilkovo mosaic clover (Uyeda, 1. et al., (1975) Ann Phytopath. Soc. Japan 41: 92-203). A suitable source CYVV is the American type culture collection (ATCC, N PV 123). Virus yellow prozhilkovo mosaic clover can be defined as the virus that causes necrosis in Vicia faba.

Genomic RNA can be obtained from CYVV-particles isolated from infected plants, and then using the reverse transcription of the indicated RNA and using well-known methods can be obtained double-stranded cDNA.

To determine whether this virus mutant or variant strain CYVV, a good indicator is the homology of the sequences. Specialists known many types of potyviruses, and they all vary in their pathogenicity. The identity of this virus to the family of potyviruses can be established on the basis of Sirolo with these viruses, amino acid sequences which have a 90% homology, are considered as viruses belonging to the same strain, whereas viruses with amino acid sequences which have a homology of less than 70% are considered

as members of different families (Shukla, D. D. & Ward, C. W. (1989), Arch Virol 106: 171-200). Based on the different properties of the protein shell CYVV used in the present invention (Uyeda, 1, et al. (1991), 32: 234-Intervirol 245), CYVV considered as independent member of the family of potyviruses. So any virus that detects 90% or more homology to the primary amino acid sequence of the envelope protein, or any virus, positive on ELISA analysis using antisera against CYVV (for example, ATCC N PVA-123: anticavity to CYVV), is considered as a strain of the virus the yellow prozhilkovo mosaic clover.

Plants that can be cultivated CYVV are Phaseolus vulgaris (common bean), Vicia faba (broad beans) and Pisum sativum (garden pea), it is preferable Vicia faba, grade Wase-soramme.

The preferred method of purification of viral particles involves homogenization of leaves of infected plants and their cultivation in a suitable buffer with Natalini by centrifugation in a density gradient of sucrose.

One way to confirm that this viral particle belongs to the species CYVV, is the analysis of this viral particles under the electron microscope. Another way is to inoculation of Vicia faba this viral particle with subsequent observation of developing symptoms.

Suitable methods of extraction of the genomic RNA of the virus particles is a method of using thiocynate guanidine/phenol method using thiocyanate guanidine/triptonite and method using phenol/LTOs. However, the preferred method is centrifugation in density gradient alkaline sucrose (Dougherty, W. G. & Hiebert, E. (1980), Virology 101: 466-474).

In order to ascertain whether or not the obtained RNA encodes a protease, this RNA is subjected to testing by broadcast in the cell-free translation system. The autolysis (camaraderie) can then be detected in the case where RNA encodes not only one protease in full translational product, by monitoring any changes in molecular weight products, collected from lysates. Such control may be effected, for example, using antibodies against the envelope protein.

If necessary, the period of time using antibodies against the envelope protein, moreover, genomic RNA can be translated by its injection into oocytes Xenopus laevis (clawed frog) (Gurdon, J. B. (1872), Nature 233: 177-182) or by using rabbit reticulocytes or system lysates wheat seedlings (Schleif. P. F. & Wensink, P. C. (1981), "Practical Methods in Molecular Biology; Springer-Verlag, N. Y.).

Single-stranded DNA can be synthesized using the thus obtained PHK as a matrix and using reverse transcriptase, and double-stranded (DC) cDNA can be synthesized from single-stranded(est) DNA in accordance with standard procedures. Suitable for this purpose, methods are method using nuclease s1 (Efstratidiatis, A. et. al. (1976), Cell 7: 279-288: Okayama H. &Berg, P. (1982), Mol. Cell. Biol. 2: 161-170, and others), the method Landa (H. Land et.al., (1981), Nucleis Acid Res, 9, 2251-2266) and method O. Joon Yoo (Yoo O. J. et al., (1983), Proc. Natl. Acao. Sci USA 79, 1049-1053). For the purposes of the present invention, it is preferable to use the method after Gubler-Hoffman (Gubber, U. & Hoffman, B. J. (1983), Gee 25: 263-269).

Thus obtained dzanc can then be introduced into the cloning vector such as a plasmid or lambda phage, and produced, as a result, the recombinant vector can be transformed into a microorganism such as Escherichia coli. Especially preferred is gentam, such as tetracycline or ampicillin, using standard equipment.

The transformation can be carried out, for example, by the method of Hanahan (Hanahan D (1983), J. Mol. Biol. 166: 557-580), namely, that the recombinant DNA vector is introduced into a cell that did pre-competent by treatment with calcium chloride, magnesium chloride or rubidium chloride.

Selection of transformants containing DNA encoding Nla, was carried out by the method described below.

(1) Screening using probes

If as the source of RNA is desirable to use the virus CYVV wild type, the selection of the corresponding RNA must be used cDNA-probe provided that will be pre-identified amino acid sequence Nla (part used sequence may occur from any area Nla). Thus synthesize an oligonucleotide probe corresponding to a specific amino acid sequence. Generally speaking, the selected amino acid sequences must contain, if possible, the minimum number of Varazdinska, otherwise you need to produce multiple probes using, if possible, different codons. In ativn, can be considered a multitude of nucleotide sequences, and the different nucleotides can be replaced by inosine. Then the probes can be subjected to labeling with radioactive isotopes, such as32P,35S, or Biotin. After that transformatie strains can be identified by fixing the denatured plasmid DNA on nitrocellulose filters using radioactively labeled probes, positive clones can be detected using autoradiography.

(2) using a PCR probe

In this way the semantic chain and antisense chain synthesize oligonucleotides corresponding to the amino acid sequence, and carry out polymerase chain reaction (Saiki, P. K. et al. (1988), Science 239, 487-491). These oligonucleotides used for amplification of the DNA fragment encoding Nla. Suitable DNA-matrix is a cDNA obtained by reverse transcription of viral genomic RNA, which is known to encode Nla. Thus obtained DNA fragments are subjected to tagging, for example,32P,35S or Biotin and for selection of the desired clone carry out the hybridization of colonies or plaques.

(3) Skiinstructor strain for amplification of the gene and then the transfection of this gene into a cell of an animal (usually using plasmids with transcriptional competence and containing an area transcriptional promoter, or using plasmids, which can be integrated in the corresponding chromosome), which results in the production of the protein encoded by the specified genome. Transformatsii strain can be selected by evaluating the activity.

(4) Selection using antibodies against Nla

From a plant infected with a virus CYVV produce antibody against nuclear inclusions (Nla and Nlo) or antibody against a protein produced by using expressing the vector in the corresponding system. Right Nla or the desired strain can then be detected using antibodies or intential.

(5) transmission system for selective hybridization

Transformant cDNA was hybridisable with mRNA derived from cells expressing Nla, and described above, mRNA associated with cDNA dissociatively and allocated. Selected mRNA was aired in protein translation system, for example, by injection into Xenopus laevis oocytes or in cell-free systems, such as the lysate is by detection using Nla antibodies against the Nla.

DNA encoding CYVV-Nla, can be obtained from the transformed strains by known methods (see T. Maniatis et al. (1982) in "Molecular cloning A Laboratory Manual" Cold Spring Harbor Laboratory, N. Y.).

For this purpose, for example, from cells secrete fraction corresponding to the vector DNA and the indicated plasmid DNA cut out the region of DNA encoding the desired protein.

The definition of the thus obtained DNA sequences can be performed, for example, by the method of chemical modification of Maxima-Gilbert (Maxam, A. M & Gilbert, W. (1980), "Methods in Enzymology" 65: 499-276) or by the method of dideoxy-termination circuit using phage M13 (Messing, J. & Vieira, J (1982), Gene 19: 269-276).

Recently, for determining DNA sequences have a tendency to use fluorochrome instead of the more dangerous radioactive isotopes. In addition, in the present procedure, dideoxy-termination circuit is performed with the use of robotics with computer control. Widely used systems for reciting sequences after electrophoresis, the examples of such systems can serve as a CATALYST 800 (robot for sequencing and DNA-sequencing machine 373A (Perkin-Elmer Japan Applied Biosystems). These systems allow for the determination of DNA-posledovateli organized they will be expressed in "standard cells", either eukaryotic or prokaryotic. In addition, by introducing the vector of the corresponding promoter sequence them for phenotypic expression of the desired gene can be expressed in the selected cells of the host.

Suitable prokaryotic hosts are E. coli and Bacillus subtilis. For phenotypic expression of the corresponding gene in the vector can be introduced replicon, derived from a species which is compatible with the host. In the case of E. coli plasmid may contain the site of replication initiation and promoter sequence, such as lac or Uv5. While preferred are vectors that on the basis of their phenotypic trait can tell transformed cell selectivity.

In most cases, as the owner using Escherichia coli, and particularly preferred host is a strain JM 109, derived from E. coli strain K12. Vectors for E. coli is usually chosen from the series of plasmids pBP322 or pUC, however, if necessary, can be used and other vectors and strains. Suitable promoters for E. coli are the lactose promoter (lac), tryptophane and promoter Tu-factor elongation of polypeptide chain (tufB), however, the present invention is not limited to these promoters.

Suitable strains of Bacillus subtilis is a strain 207-25. Suitable vectors is pT B228 (K. Ohmura et al (1984), J. Biochem 95: 87-93) and other Suitable promoter is a regulatory sequence of a gene-amylase of Bacillus subtilis. The signal peptide sequence (- amylase) can be used for extracellular secretion.

If you want to Express the hybrid protein in eukaryotic cells, for this purpose can be used in cells of vertebrates, insects, yeast, plants, etc. Preferred vertebrate cells are COS cells, in particular cells COS-1 (see, for example, Y. Gluzman (1981) Cell 23: 175-182), or cell line of Chinese hamster ovary (CHO), in which there is no gidromolot-reductase (Urlaub G & Chasin L. A. (1980), Proc. Natl. Acad Sci. USA, 77, 4216-4220), although the present invention is not limited to only these examples.

As mentioned above, COS cells are appropriate cells of vertebrate hosts, and they can be used as an example. Expressing the vectors containing the SV40 replicon is capable of Autonomous replication and is provided with a transcription promoter, a transcription sledovatelnot, can be used for transformation of COS cells by various methods such as a method using DEAE-dextran (Luthman, H. & Magnusson G (1983), Nucleic Acids Res. 11, 1295-1308), the method of coprecipitation calcium phosphate-DNA (Graham F. L. & Vander Eb. , A. J. (1973), and the method of electroporation (Neumann, E. et. al. (1982) EMBO J. 1, 841-845).

If the host cells are CHO cells, it is desirable to use a vector that can Express the neo gene, indicating G418-resistance. Suitable markers bearing this mark are ppSVneo (Sambrook J. et. al. (1989), "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboramory), u pSV2-neo (Southern, P. J. & Berg P. (1982), J. Mol. Appl. Genet, 1, 327-341).

Transformants can then be selected by their resistance to G418.

Selected transformants can then be cultured by standard methods, as in the case of the implementation of the second variant of the present invention the polypeptide produce both intracellular and extracellular. The culture medium may be selected in accordance with the used cells-owners. For example, COS cells, to such an environment, as PRM1-1640 or modified by the method of Dulbecco environment Needle (DMEM), if necessary, can be added to a medium containing blood components, such as FET is to be used by cells, derived from Spodoptera frugipedra (Smith, G. E. et al. (1983), Mol. Cell. Biol. 3: 2156-2165).

Suitable yeast is Baker's yeast (Saccharomyces cereviside) and fission yeast (Schizosaccharomyces pomle).

Suitable plant cells are cells from Nicotiana tabacum. (tobacco) and Oryza sativa (rice planting).

It should be noted that all of these owners are standard cells-owners commonly used in practice, however, experts can choose other cells suitable for use as hosts.

Suitable expression vectors for vertebrate cells are those vectors that contain a promoter that is located above the expressed gene, together with such sites as the site of the RNA splicing, polyadenylation site, and the site of transcription termination, and who, moreover, if this should contain the site of replication initiation. An example of a suitable expressing vector is pSV2dnfr, which contains the early SV40 promoter (Subramani, S. et al, (1981), Mol. Cell. Biol: 854-864).

A suitable system for expressing insect cells are cultured cells Spodoptera frugipedra. Suitable expressing vectors contain, for example, the promoter of poliedro the ü AcMNPV-Genoa (nuclear polyhedrosis virus Acutographa californica), required for homologous recombination. An example of such a vector is pBacPAK8 (Matuura Y. et al. (1987), J. Gen. Virol 68: 1233-1250).

For eukaryotic expression usually used yeasts such as Baker's yeast (S. cerevisiae). Suitable expressing vectors for yeast may contain alcoholdehydrogenase promoter (Bennetzen J. L & Hall, B. D. (1982), J. Biol. Chem. 257: 3018-3025), the promoters of acid phosphatase (A. Miyahara et al. (1983). Proc. Natl. Acad. Sci USA, 80, 1-5) or the promoter of carboxypeptidase Y (K. Ichikawa et al. (1993), Biosci. Biotech. Biochem. 57: 1686-1690). In this case, for the implementation of the secretion in the extracellular space can be used for the signal peptide sequence of carboxypeptidase Y.

Suitable expressing vector for plants, for example, is pB1121 containing 35S-promoter (derived from the early promoter of cauliflower mosaic virus), the site of polyadenylation of the gene synthesis nopaline from Agrobacterium tumefaciens; and the site of the Agrobacterium tumefaciens gene transfer (Jefferson, P. A. et al. (1987), EMBOJ 6: 3901-3907). Such a vector can be introduced into plant cells, for example, by infection of bacteria Agrobacterium tumefaciens and by electroporation.

Plasmid pKK388-1 (designed Clonetech Co.), containing trc-promoter, a suitable m from strain K12 Escherichia coli such as the strain JM109. this vector can be easily introduced in E. coli are well known methods mentioned above. The resulting strain can be inoculated in the medium, for example, such well-known environment, as LB-medium, and cultivated in this environment for a certain period of time.

trc-Promoter can then be activated by adding, for example, isopropyl- -galactopyranoside (IPTG). After further cultivation produced protein can then be extracted from cells by lysis, for example, by means of an ultrasonic device. If you want to produce a protein having Pro at N-end, in this case, as the owner it is desirable to use E. coli.

If necessary, in accordance with the examples accompanying the present description, the fraction containing the desired polypeptide may be isolated from a culture of transformed cells and purified by the known methods is selected depending on the physical and chemical properties of the polypeptide. Suitable for this purpose methods is the treatment of precipitating agent, ultrafiltration, chromatography of different types (for example, chromatography on a molecular sieve (gel filtration), adsorption tography (URGH), dialysis and combined methods.

In order to ensure that the downregulation of Nla-protein is needed protease activity, purified Nla-protein may be subjected to reaction with a substrate containing a splitting sequence for a protease, such as the expression product of the gene encoding a hybrid protein comprising a protein of the viral envelope (natural substrate for 1a) together "nuclear inclusion b (Nlb) (Dougherty, W. G. at al. (1988), EMBO J, 7: 1281-1287). Specified hybrid protein can be isolated from the viral genomic cDNA and inserted in expressing vector. The resulting expressing the vector is then introduced into an appropriate host cell to produce the specified hybrid protein. The resulting hybrid protein can be isolated and purified by standard methods, for example, by precipitation with a suitable precipitating agent or by using chromatography.

Selected and thus purified substrate protein can then be subjected to reaction with the indicated protease in an appropriate buffer solution at a suitable temperature with the release of the reaction product. The resulting reaction product can be patience by activity, find the difference in mobility of the strip. In this method you can also use synthetic Oligopeptide containing the appropriate split sequence.

Proteolytic activity of the protease of the present invention can also be determined without purification. So, for example, the activity can be determined by attaching protease gene in the sequence and with the preservation of the reading frame so that it was located "below" promoter, for example, the trc-promoter, and "above" split sequence for a protease. If the protease is active in the expression product, when Western-blotting will be observed band with higher mobility than the hybrid protein.

By allocating this band from a gel, in which he observed splitting, and by analysis of its amino acid sequence by standard methods can be set, split whether this protein in the desired peptide bond.

The desired protein can be produced, for example, in the form of a hybrid protein with the protein, such as glutathione-S-transferase, and Nla of the present invention can then be used for cleavage of the linker sequence in v protein together with the indicated protease, after which the desired protein is formed by camerasmobile.

If necessary, using the above methods Nla can be easily produced with high yield and with high purity, and thus obtained Nla (nuclear inclusion a") of the present invention can be used as the protease.

Optionally, using a standard method, for example triethylphosphite method (Hunkapiller, M. et al. (1984) Nature 310: 105-111), or a method of chemical synthesis of nucleic acids (Grantham P. et al. (1981) Nucleic Acids Res 9: g-g) can be carried out chemical synthesis of DNA of the present invention. If necessary, can be carried out a partial modification of the above nucleotide sequences using standard techniques such as site-specific mutagenesis, which uses a primer that contains a synthetic oligonucleotide encoding the desired modification (Mark, D. F. et al. (1984), Proc. Natl. Acad, Sci. 81: 5662-5666).

Hybridization mentioned above, may be installed, for example, using a probe labeled with, for example, [ -32P] oCTP, method scattered priming (Feinberg, A. P. & Vagelstein (1983) Anal. Biochem 132: 6-13), or by nick translation (Maniatis T, et al. (1982) in "Molecular Cl on the nitrocellulose or nylon membrane, and then heated or subjected to UV radiation. Then the solid is immersed in a solution for pre-hybridization, containing 6 x SSC, 5% denhardt's solution and 0.1% LTOs, and incubated at 55oC for 4 hours or more. Then the solution for pre-hybridization add the advance received probe to achieve a final specific activity of 1 to 106imp./min per ml, after which the mixture incubated at 60oC during the night. Then the solid phase is five times at room temperature and for 5 minutes, washed with 6 x SSC, followed by a 20-minute washing with 57oC, and then, in order to determine whether there was a hybridization of DNA or not, spend the autoradiograph. It should be noted that the above example is purely illustrative, and for the purposes of the present invention with the same success can be used in other methods.

The desired protein can be obtained either by direct intracellular cleavage, either by extracellular cleavage.

Method direct intracellular cleavage

DNA encoding Nla, attached to DNA that encodes a desired protein by splitting sequence, preferably Gln-Gly, Gln-Ser and the R, and the specified vector used in the corresponding cells of the host. The result is expressed hybrid protein alone is cleaved due by activity, thus forming a peptide in which Gly, Ser or Ala related peptide bond at the N-Terminus of the desired protein. Especially preferred protein for expression is 1L-11.

If from the N-Terminus of the obtained protein is necessary to split any residue, it can be done, as described above, that is, for example, removal of N-terminal Pro residue can be used aminopeptidase P.

In some systems, the expression already exists in aminopeptidase P. However, if the specified aminopeptidase P is missing, but this requires the N-terminal amino acid residues were derived in situ, in this case, the cell master can be introduced expression vector for amino peptidases P. Aminopeptidase P is a known protein, and the gene sequence for this enzyme is easily accessible for every person.

If you want to get protein, N-end of which begins with a Pro, it is desirable to extend the time of cultivation, to ensure the cellular amino peptidases P.

Thus, thanks to the present invention can be produced proteins having any desired N-terminal residue.

If the sequence of the present invention produces a polypeptide with N-terminal Ala, this alanine residue may be removed by catalytic action of alanine-amino peptidases (3.4.11.14), which is also known enzyme, and gene sequence which is easily accessible for every person. The described technique also allows us to produce a polypeptide having freely chosen N-terminal residue.

Then the desired protein can be isolated and purified in accordance with the standard method.

Method extracellular cleavage.

Nuclear inclusion a (Nla) can be produced by introducing the corresponding D is formirovanii cells, can then be purified by ion-exchange chromatography, gel filtration or reverse-phase chromatography. Alternatively, the Nla can be produced in the form of a hybrid with another polypeptide, such as glutathione-S-transferase or communicating with maltose protein, and specified a hybrid protein may be divided and allocated using a column with glutathione or maltose, respectively. After cleavage of purified product enterokinase or factor Xa Nla can be cleaned and used for appropriate purposes.

Meanwhile, get the precursor protein, which contains the sequence recognition Nla (tsepliaeva sequence). It can be present in a given area, or the protein may form part of a hybrid protein, for example, with protein binding to maltose, or glutathione-S-transferase connected via a suitable linker (tsepliaeva sequence). The reaction of the specified predecessor with the Nla in the appropriate buffer solution contributes to the decomposition of the precursor in vitro. If necessary, the N-terminal amino acid residues may be deleted, as described above.

As mentioned above, received blastoidea of the invention, the authors believe that the natural recovery of the peptide has the sequence shown in the list of sequences (see below) as SEQ ID No. 12, and consists of 526 amino acids Val residue at N-end.

The authors also argue that natural DNA encoding this peptide has a nucleotide sequence consisting of nucleotides 70-1647, and shown in the list of sequences as SEQ ID No. 11.

The peptide of the present invention is able to restore oxidized glutathione and dichloroindophenol. Here in this application the description of the specified peptide is pretty accurate, but somewhat cumbersome, and therefore, hereinafter, the peptide of the present invention will be referred to as KM31-7-peptide or protein.

KM31-7 protein or its mutant, or variant is produced in the body, and therefore the problems associated with toxicity and/or antigenicity of proteins, in this case virtually absent.

In accordance with the second embodiment of the present invention, it is assumed that the polypeptide having the amino acid sequence from -23 to 526 (sequence ID 12), is a precursor KM31-7 protein. In this regard, it should be noted that the present izobreteniyami any of these peptides.

The following discussion refers mainly to the second variant of implementation of the present invention. It should be noted that the procedures described above for CYVV-Nla, can also be used for isolation, cloning and expresii DNA that encodes a peptide of the present invention. The basis of all possible differences is the fact that CY Nla is a protein of the virus in plants, whereas the peptide of the present invention is a mammalian protein. Below will be described the main stages of the specific polypeptide produced from mammalian cells by means of genetic recombination.

mRNA encoding KM31-7-peptide can be obtained, and then transcribed back in dzanc in accordance with well known techniques. As a source of mRNA can be used any suitable cells, cell lines or tissues of mammals, however, it is preferable to use the cell line KM-102, derived from stromal cells of the human brain (K. Harigaya & Handa H. (1985), Proc, Natl, Acad, Sci, USA, 82, 3447-3480).

For the extraction of mRNA from mammalian cells can be used in any suitable methods such as a method using guanidinoacetate the MD preferred method is using guanidinoacetate - cesium chloride.

Because in most cases the mRNA present in the cytoplasm of eukaryotic cells, has, as is known, the 3'end (polyA) sequence purification of mammalian mRNA can be carried out by adsorption on a column of oligo(dT)-cellulose. Allerona mRNA can then be fractionated by centrifugation in a density gradient of sucrose.

Make sure that mRNA really encodes the desired peptide can be achieved by translation of mRNA in the corresponding system, for example in the system of Xenopus laevis oocytes system from rabbit reticulocytes or wheat germ (see above).

The measurement of the reductive activity of the expression product can be carried out in accordance with the following description.

I. Definition dichloroindophenol-healing activity

The methodology for such determination described Beinert H, in "Methods in Enzymology" (1962), 5, 546.

50 μm-drug dichloroindophenol (Sigma) is obtained using 20 mm phosphate buffer and 0.5 M NaCl (pH 7.8), 1 ml of this preparation is then placed in the cuvette (SARSTEDT, HH mm) and then add the sample to measure. 15 microlitres 1 mm NADPH (NADP) (Boeringer-Mannheim) made in them determined by the subsequent decrease in the absorbance of oxidized dichloroindophenol at 600 nm or by a subsequent decrease in the absorbance of A PH at 340 nm.

II) determining the activity recovery of oxidized glutathione.

Methodology this analysis is described Nakajima T and others in the "New Basic Experimental Methods in Biochemistry (6) - Assay Methods Using Biological Activity", 3-34.

The drug 10 mm oxidized glutathione (Boeringer-Manheim) is manufactured using a 20 mm phosphate buffer and 0.5 M NaCl (pH 7.8), 15 μl of this preparation was placed in a cuvette (HH mm) after there was placed a sample for measurement. 15 ál of the drug 1 mm NADPH get in the same buffer and then added to the cuvette at room temperature to initiate the reaction. Glutationreductaza activity can then be determined by a subsequent decrease in absorbance at 340 nm.

To obtain dzanc of mRNA can be used various methods described above. Such methods are S1-nukleazy method, Landa and method O. Joo Yoo (see above), however, in the present work, the authors chose to use the method of Okayama-Bero (Okayama H. & Bero, P. (1982), Mol. Cell. Biol. 2, 161-170).

Thus obtained DC-cDNA can then be introduced into the cloning vector and the resulting recombinant plasmid can be introduced into Escherichia coli, as described above.

Transformatsii strain containing the desired DNA encoding s above in connection with the selection of transformants, containing Nla-DNA, with appropriate modifications. For example, if the receiving probe is used polymerase chain reaction (PCR), then the corresponding DNA-matrix can be cDNA, described above, or genomic DNA.

In the second embodiment of the present invention to reduce the number of strains of transformants under test, can be conducted in primary screening. Holding such primary screening is quite possible, since the peptide of the present invention relates to cytokines, and its mRNA has in common with the mRNA of cytokines element AUUUA (Shaw. G. & Kamen P. (1986), Cell 46, 659-667). Thus, for primary screening can be used a synthetic oligonucleotide probe, complementary element AUUUA. Can then be screened by analysis of the production of an exogenous protein in mammalian cells.

DNA encoding the peptide can then be cut out from the vector and sequenced in accordance with the methodology described above for the Nla-DNA. And as described above, the DNA fragment can then be introduced into an appropriate vector and used to transform prokaryotic or eukaryotic host.

KM31-7 protein may be used sibabalwe, caused or mediated oxidative stress, or any other diseases caused by active oxygen. Such diseases or conditions include, but are not limited to, atherosclerosis, diabetes, ischemic disorders (such as reperfusion injury, ischemic heart disease, cerebral ischemia and ischemic enteritis), edema, vascular sorprendentemente, inflammatory diseases, gastric mucosal injury, acute pancreatitis, Crohn's disease, ulcerative colitis, liver disease, disease Paraguat, emphysema, chemical carcinogenesis, metastasis of cancer, respiratory distress syndrome in adults, disseminated intravascular coagulation (DIC), cataract, petrolatina fibroplasia, autoimmune diseases, porfiria, hemolysis, erythroblastopenia (Mediterranean) anemia, Parkinson's disease, Alzheimer's disease, epilepsy, disorders associated with UV exposure, disorders caused by the radiation (radiation sickness), frostbite and burns.

In accordance with the second embodiment of the present invention the pharmaceutical composition includes a pharmaceutically active if the can be administered orally, for example, in the form of tablets, capsules, granules, powders or syrups, or parenterally in the form of injections, injections and suppositories. If necessary, can be used and other ways of introduction.

If the peptide of the present invention must be entered in the form of injection or infusion, for this purpose can be made pyrogen-free preparation of the indicated peptide, dissolved in a pharmaceutically acceptable aqueous solution suitable for parenteral administration. This drug polypeptide solution should have an appropriate pH, isotonicity and stability, according to the conclusion of technical expertise.

The dose and form of the administered drug can be easily installed by the specialist on the basis of criteria such as the patient's condition, weight, sex, age, and diet, the presence of other infections, the time of administration and other clinically important factors. In General, however, the normal oral dose for an adult is in the order of from about 0.01 mg to about 1000 mg per day. This number can be entered as a single dose or in the form of fractional doses within 24 hours. If the specified peptide must enter parenterally, it can be injected subcutaneously, inside the key KM31-7 protein is very important to produce antibody to this protein. This antibody can be used for analysis of protein functions, its quantitative analysis and purification, as well as to estimate the distribution KM31-7 protein in the tissues.

For this, first, by inoculation of laboratory animals of the polypeptide produced by E. coli transformed with plasmid pMA 31-7, received hybridoma producing antibody against KM31-7, and then this hybridoma formed antibody-producing cells and myeloma cells were skanirovali and cloned. Antibodies produced by the specified hybridomas, possessed the ability to recognize the polypeptide isolated from cell-free supernatant of COS-1 cells transformed by the plasmid pSP 31-7.

Thus, the present invention relates to an antibody, preferably to a monoclonal antibody, or its equivalent, is able to specifically recognize KM31-7 protein or its mutant, or variant.

The antibody of the present invention directed against KM31-7 protein or its mutant, or variant. Such antibody may be polyclonal or monoclonal, although preferred is a monoclonal antibody. The reason for this preference is the uncertainty associated with Hispalis whether this antibody for therapeutic purposes or for cleaning KM31-protein, it is preferable to use monoclonal antibodies.

Antibodies of the present invention can be obtained from any suitable animal. However, if the antibody is not of man, in this case, you may experience problems with antigenicity of the antibody. Therefore, in order to obtain antibodies that are closer to human antibodies, the desired antibody can be constructed. This can be achieved by chemical or genetic modification by methods well known to specialists.

The present invention also addresses antiidiotypic antibodies, i.e. antibodies, website recognition, which recognizes the site of recognition of the above monoclonal antibodies. Such antiidiotypic antibodies can be obtained by introducing a source of antibodies suitable animal. It should be noted that this process can last virtually indefinitely, where each generation will match either the source or antiidiotype. However, if these antibodies will not be selected for correct recognition after each generation, their specificity may be largely lost. These antiidiotypic toadie the invention also relates to fragments of the antibodies of the present invention, able to recognition KM31-7 protein, and to molecules that carry the site of recognition of these antibodies. Next, these fragments will be referred to as "equivalents" of the antibodies of the present invention.

Plasmid pMA 31-7 used to transform E. coli, and the expression profiles were purified and used for immunization of laboratory animals. The spleen cells from immunized animals were used to obtain hybridoma by fusion of these cells with myeloma cells, which produced a clone producing a monoclonal antibody against KM31 with a high concentration and with good stability (this clone was designated MKM150-2 and deposited in the research Institute of fermentation Management of industrial science and technology Japan (Fermentation Research of the Agency of Industrial Science and Technology, Japan) under the incoming number of GERM BP-5086). The result of the cultivation of this clone from the supernatant of the culture was obtained monoclonal antibodies against KM31-7.

The obtained monoclonal antibodies against KM31-7 immunochemical react with a hybrid protein, obtained by the introduction and expression pMA131-7 in E. coli. This antibody also immunochemical reacts with KM31-7 protein, obtained from the supernatant of mammalian cells, Proc. of the conduct in accordance with the following procedures, includes:

(a) cleaning of the biopolymer used as antigen,

(b) immunization of mice by injection of antigen and receipt of splenic antibody-producing cells for an appropriate period of time by sampling and blood tests,

(c) obtaining myeloma cells,

(d) the merger of spleen cells and myeloma cells,

(e) screening for selection of hybridomas producing the desired antibody

(f) obtaining a clone from a single cell (cloning),

(g) culturing hybridoma for large-scale production of monoclonal antibodies, or the breeding of mice infected with hybridomas, depending on the circumstances,

(h) investigation of the physiological activity of the obtained monoclonal antibodies or evaluation of its properties as a reagent for labeling.

Production of monoclonal antibodies against KM31-7 was carried out in accordance with the description below. It should be noted that there is no need to follow this description with absolute precision, and where it is required, you can use any appropriate procedure. For example, antibody-producing cells, instead of spleen cells can IP the listed procedure is the preferred method of obtaining monoclonal antibodies against KM31-7.

(a) Purification of antigen

A hybrid protein obtained by expression pMAL31-7 in E. coli, followed by purification of the expression product is an effective antigen. The expression of E. coli TB-1, transformed with plasmid pMAL31-7, induced with isopropyl-N-thiogalactopyranoside (IPTG). Then expressed hybrid protein was purified using affinity chromatography, using a column with amylose resin (New England Biolabs). KM31-7 protein, purified from serum-free culture supernatant of COS-1 cells transformed by the plasmid pSR 31-7, is also an effective antigen.

(b) Obtaining antibody-producing cells

Purified hybrid protein obtained in (a), was mixed with complete or incomplete adjuvant's adjuvant, or adjuvant, as potassium alum, and the resulting vaccine were immunized laboratory animals. As laboratory animals is preferable to use a mouse BALB/c mice, because in most cases used murine myeloma stem from BALB/c mice. In addition, these mice have been well and thoroughly investigated, and their characteristics are described in detail. Furthermore, if the antibody-producing cells and the myeloma stem from mouse BALB/c, then p is BALB/c mice has significant advantages, making it easy to obtain monoclonal antibodies from ascites without the use of cumbersome and complicated procedures. However, it should be noted that the present invention is not limited to the use of BALB/c mice.

The specified antigen can be introduced in any suitable form, for example, by subcutaneous injection, intraperitoneal injection, intradermal injection, or intramuscular injection, while it is preferable subcutaneous or intraperitoneal injection.

Immunization of mice can be performed using a single injection or multiple injections, administered at appropriate intervals. The preferred scheme immunization provides for the introduction of the first injection of antigen, and then repeated injections, one or more time intervals from 1 to 5 weeks. The efficiency of the subsequent procedures may be increased, if regularly assess the titer of antibodies to a specific antigen in serum of immunized animals, and animals with high titers of antibodies can then be used to obtain antibody-producing cells. Antibody-producing cells used for the subsequent fusion of the cells, preferably vypolzovo various known methods, for example, such as an immunoassay with the use of radioactive isotope (RIA), enzyme-linked immunosorbent assay (ELISA) method using fluorescent antibodies, and passive hemagglutination, however, are preferred analyses of RIA and ELISA because of their high sensitivity, speed, accuracy and automation of the process.

Procedure immunoassay ELISA as follows. Antigen adsorb on a solid phase, and then solid-phase surface exhibiting a protein that is not related antigen, for example, bovine serum albumin (BSA) to block any surface areas, which was not adsorbed antigen. Then the solid phase is washed and processed serially diluted sample of primary antibodies (e.g., mouse serum). Any antibody against KM31-7 in the sample binds to the antigen. After rinsing, add a "second" associated with the enzyme antimurine antibody and leave to associate with the associated mouse antibody. After rinsing, add enzyme substrate, and then can be calculated titer of the antibody by determining the degree of color development resulting from the decomposition of the substrate.

-5M 2-mercaptoethanol, 10 µg/ml gentamicin and 10% fetal calf serum), environment Dulbecco, modified by the method of Claims (IMDM), or modified by way of Dulbecco Wednesday Needle (DMEM). When subcultivation normal environment known as a full GIT medium (5.5 ml MEM solution of amino acids, non-essential (NEAA, Gibco), 27.5 ml CTC109 (Cibco), 6 ml of penicillin-streptomycin (Sigma) and 11 ml of a 200 mm solution of glutamine in 500 ml of GIT medium (Wako Pure Chemical Industry)) for 3-4 days before fusion of cells, number of cells on the day the merger was increased at least up to 2107.

(O) Merge cells

Antibody-producing cells are plasma cells and lymphocytes precursor. They can be obtained

from the respective organiczna used cells of the spleen.

Antibody-producing cells were taken after 3 to 5 days after the final immunization in mice with pre-defined high titer antibodies. Then there was the fusion of antibody-producing cells with myeloma cells, obtained as described above in (c). The fusion of spleen cells with myeloma cells usually conducted by a method using polyethylene glycol, due to the relatively low level of cell toxicity of this compound and the convenience of its use. Procedure merge cells was carried out as follows.

The spleen cells and myeloma thoroughly washed with medium or phosphate buffer solution (PBS), and then mixed so that the ratio of spleen cells to myeloma cells was approximately 5-10:1, after which they were besieged by centrifugation. The supernatant was removed, and the accumulation of cells were thoroughly digested, and then, stirring, was added a mixed solution of polyethylene glycol (PEG), molecular weight: 1000 to 4000. After a few minutes, cells were subjected to sedimentation by centrifugation. Then the supernatant was again discarded, and the precipitated cells are suspended in an appropriate number of full-GIT medium containing 5-10 ng/ml murine 11-6, and the resulting cell suspen is OK Wednesday was replaced with HAT-medium (full GIT-Wednesday, containing 5-10 ng/ml murine 11-6, 10-6- 10-3M gipoksantina, 10-8- 10-7M aminopterin and 10-6- 10-4M thymidine).

(e) Breeding colonies hybrid

Tablet for cultivation incubated in CO2-incubator for 10-14 days at 35-40oC. during this time, every 1-3 days, added fresh HAT-medium in an amount equivalent to half the number of the environment.

Myeloma cells were derived from 8-azaguanine-resistant cell lines and myeloma cells, and cells hybridoma, consisting only of some myeloma cells cannot survive in HAT medium. On the other hand, hybridoma containing antibody-producing cells, i.e. hybridoma consisting of antibody-producing cells and the myeloma cells are able to survive in this environment. Hybridoma containing only the antibody-producing cells, are viable, and therefore hybridoma consisting of myeloma cells and antibody-producing cells can be selected by culturing in HAT-medium.

In those holes, which have seen the development of hybrid colonies, NATI environment was replaced with HT medium (which differed from HAT-environment because it lacked aminopterin). Then part SUSE procedures apply to 8-azaguanine-resistant cell lines, however, for the purposes of the present invention can also be used and other cell lines, provided that such use allows the selection of a hybrid. Needless to say that in this case it is also necessary to use a different medium composition.

(f) Cloning

Hybridoma, skanirovaniya, as described in (e), and analyzed for the production of specific antibodies against KM31-7, transferred to another tablet for cloning. There are many different cloning methods that can be used for the purposes of the present invention, for example sieving method (in which hybridoma subjected to analysis by serial dilution so that each well contained only one hybridoma), method of cloning in soft agar (where planted colonies grown in soft agar medium, the culture method (in which a single cell is removed by means of a micromanipulator) and the cloning method using a cell sorting device (in which individual cells are "sorted" by cell sorting device). The most commonly used analysis method serial dilution due to its simplicity.

When performing analysis by serial dilution cloning is a first antibody against KM31-7, selected as target hybridoma.

(g) Obtaining monoclonal antibodies using cultivation hybridoma

Hybridoma selected as described in (f), then cultured in an ordinary environment. Large scale cultivation is carried out by rotary cultivation or in a large vessel for culturing or in a centrifuge. Monoclonal antibodies to KM31-7 can be obtained by gel filtration of cell supernatant, followed by collection and treatment of IgG-fractions. In addition, hybridoma can also be grown in the abdominal cavity of a mouse of the same strain (e.g., the above-mentioned mouse BALB/c) or, for example, "Nude" mice (NU/NU mice). This method can be very simply implemented, if you use a special set for obtaining monoclonal antibodies (for example, MAbTrap of GlI Pharmacia)

(h) Identification of monoclonal antibodies

Determination of the isotype and subclass of the monoclonal antibody obtained in (g) may be carried out in accordance with the following description. As an example of the techniques of identification, which can be used for the purposes of the present invention can serve as a method of Ouchterlony, ELISA-method and RIA method. Although the method of Ouchterlony is in those cases when its concentration is too low.

If using ELISA or RIA culture supernatant can be directly exposed solid phase, which was adsorbed antigen. To identify the subclass can then be used to "second" antibody, specific for each type of the subclass of IgG. Alternatively, it may be used set to ittipiboon (for example, a set of Amersham for ittipiboon mouse monoclonal antibodies). Quantification of the protein can be carried out by a method Polina-Lowry (Folin-Lowry), or by measuring the optical density at 280 nm of 1.4 (OP280)=1 mg/ml of immunoglobulin).

In the examples below, a monoclonal antibody derived from hybridoma marked MKM150-2, was evaluated as isotel IgG class and identified as the antibody belongs to the IgG1 subclass.

Monoclonal antibody obtained in accordance with the present invention, has a high specificity with respect to protein KM31-7. In addition, since the monoclonal antibody continuously and in large quantities is produced by culturing the above-described hybrid, it can be used for isolation and purification m immune precipitation, where used, the reaction of the "antigen-antibody", is also part of the present invention.

The present invention is illustrated by the following examples, which, however, should not be construed as a limitation of the invention. All solutions are aqueous and obtained using deionized water (their quantitative ratios are given in percent (wt./volume), if it is not specifically mentioned). If the methods are not described specifically, they can be found in "Molecular Cloning-A Laboratory Manual (second edition, Sambrook, J. Fritisch, E. F. &Maniatis (1989), Cold Spring Harbor Laboratory Press). Methods of obtaining solutions and other media, if it is not specified in the examples given in the last section of this specification entitled "Environment". The methods mentioned in the examples without a description of their implementation are explained in the previous examples.

A) the Source material for CYVV

Leaves infected with the virus of yellow prozhilkovo mosaic clover and stored in a frozen state (CY-isolate N 30: Uyeda, 1 and others, (1975), Ann. Phytopath. Soc. Japan 41: 192-203) grinded with 10 volumes of buffer for inoculation.

Reproduction CYVV was performed using Vicia faba (sort Wase-Sora mame) (broad beans) grown developed before the second sheet. Razvedyonnie glass spatula. After 8-10 days after inoculation inoculated leaf as if covered with blocky image mosaics. This sheet was taken out and used as a source for CYVV.

B) Purification of the virus

Leaves, isolated as described in AX, crushed in a machine for cutting containing 3 volumes of buffer for extraction. After cutting the leaves were crushed in a mortar. The drug is slowly stirred for 1 hour at room temperature, using a motor, equipped with paddles for mixing stainless steel. Liquid preparation was then pressed through a double layer of gauze.

All subsequent procedures were performed at 4oC.

The crude liquid was mixed with a half volume of chloroform and the resulting mixture was stirred in the mixer of Waring (Waring), after which the preparation was centrifuged for 15 minutes at 6000 x g. After centrifugation the aqueous layer was collected and to this fraction was added polyethylene glycol (PEG 6000) to a final concentration of 4% vol/vol.

The resulting mixture was stirred on ice for 1 hour, and then left on ice for one hour. Thus obtained liquid was centrifuged for 15 minutes at 6000 x g, and the precipitate was identified as a virus-containing fracc the Ali with an equal volume of carbon tetrachloride and vigorously stirred for 5 minutes, then the preparation was centrifuged for 10 minutes at 3000 x g. Then the aqueous phase was subjected to ultracentrifugation at 30,000 rpm for 90 minutes at 4oC, using2when the rotor Hitachi PP-30. The precipitate was identified as a virus-containing fraction.

The residue is suspended in 10 mm phosphate buffer containing 1% Triton X100 (pH 7,4), and the suspension was centrifuged for 1 minute at 4oC and at 8000 x g. The obtained supernatant was applied layer on the graded column density gradient of sucrose 10-40% (40%:10 ml, 30%:10 ml, 20%:10 ml, 10%: 10 ml and left overnight at 4oC) that was previously obtained using 10 mm phosphate buffer (pH of 7.4). Then the test tube is covered with a layer of supernatant and centrifuged at 23000 rpm and at 4oC for 120 minutes in the rotor 25 PP Hitachi.

After centrifugation column tube with a density gradient of sucrose was fractionally apparatus for fractionation (model UA-2, manufactured by ISCO Co., equipped with OP260nm detector. Fraction having a density of sucrose from about 20% to 30% and adsorbiruyuschee when OPNmcollected as a virus-containing fraction.

The selected virus-containing fraction 2-fold reshenie 90 minutes using a rotor PP-65 Hitachi. The precipitate resuspendable in 10 mm phosphate buffer (pH 7,4), resulting in a received purified virus solution.

C) Isolation of viral RNA

The genomic RNA of the virus CYVV, containing about 10,000 bases were subjected to polyadenylated at its 3'-end. The combination of length and polyadenylation complicates the extraction of the full-sized RNA without any loss, if the extraction is carried out by a standard method using phenol/sodium dodecyl sulfate or a method using guanidinoacetate. Therefore, viral genomic RNA was obtained by centrifuging the density gradient alkaline sucrose.

500 μl of viral solution (which contained 2 mg of the virus, as was calculated assuming that 1 mg/ml of the virus has OPNm= 0,5), was added to 500 μl of a solution to denature and the resulting mixture was left for 20 minutes at room temperature. After that, the mixture was layered on the tube column with a gradient density 0% - 34% sucrose (33,4%:1,4 ml, 30.4 per cent:of 7.6 ml, 27%: 7,0 ml, 23%:6.3 ml, 18.7 percent:5 ml, 12%:3.2 ml, 0%:2.7 ml and left overnight at 4oC), which was obtained using 1SSC. As the buffer is also used 1SSC. Then the tubes coated with the mixture was subjected to ultra is kaliveli for fractionation of density gradients of sucrose. The fraction of viral genomic RNA identified by measuring the optical density of each fraction at 260 nm. Sedimentation of the viral genomic RNA is carried out at concentrations of sucrose from about 20% to 30%. The output of the genomic RNA was approximately 25 mcg.

D) Synthesis of viral cDNA

cDNA was synthesized using as template genomic RNA obtained as described above in stage (C). Synthesis of cDNA was performed using the cDNA Synthesis System Plus (manufactured by Amersham). The obtained cDNA was purified on a column of Sephadex C50 (registered trademark, Pharmacia). And using dCTP and terminal deoxynucleotidyl-transferase (Bethesda Research Laboratories) was added polyG-chain to the 5'-end of the purified cDNA. Plasmid preparation pBP322 (Bethesda Research Laboratories) was obtained by digestion of this plasmid restriction enzyme Pst1 and then adding 3' polyG-circuit both ends. Then this cDNA preparation was added and the mixture incubated for 5 minutes at 65oC, and then incubated for 2 hours at 57oC. the mixture was gradually cooled to hybridization polyG-chain cDNA with polyG-chain plasmids.

E) Transformation

Strain HB 101 E/coli transformed with the new plasmids, recip is made by shaking overnight in liquid B-medium. 0.5 ml of this seed culture was used for inoculation of 50 ml of fresh liquid B-environment, and the resulting inoculum was cultured by shaking at 37oC up until OP550nmdo not become equal to 0.5. Bacterial cells were isolated by centrifugation for 5 minutes at 5000 x g and 4oC, and the resulting precipitate slightly suspended in 25 ml of Tris-calcium buffer and left on ice for 5 minutes. The resulting suspension is centrifuged at 4000 x g for 4 minutes, and the residue is suspended in 5 ml of Tris-calcium buffer and left for 2 hours on ice to obtain competent cells.

It received 200 μl of competent cells were added 100 μl of a new plasmid, obtained in stage (D), and the resulting mixture was left on ice for 30 minutes. After this time the mixture is incubated for 2 minutes at 42oC, after which was added 1 ml of liquid LB-medium and the resulting mixture was cultured by shaking at 37oC for one hour. Then this mixture was applied on solid LB-medium containing 12.5 µg/ml tetracycline hydrochloride and 1.5% agar. Cells were cultured overnight at 37oC to obtain a cDNA library clone.

F) Receiving and breeding PLA the LTOs. More details on this method are described below.

2 ml of liquid LB-medium was inoculable colony of bacteria that are resistant to tetracycline but sensitive to ampicillin and obtained from the solid LB-culture, as described in (E), after which the resulting suspension was cultured by shaking in over night at 37oC. the Cells were besieged by centrifugation at 10,000 x g and suspended in 70 μl of buffer for lysis, containing 10 μl of a solution of lysozyme (10 mg/ml). After stirring for 5 seconds to form a suspension of the resulting suspension was left for 5 minutes at room temperature. Then, to the suspension was added 160 μl of alkaline solution LTOs and the resulting mixture was first mixed by inverting the tubes several times, and then accounted for 5 minutes on ice. To this mixture was added 120 μl of 5 M potassium acetate, and then left for 5 minutes on ice. After this time the mixture was centrifuged at 10000 x g and 4oC for 5 minutes.

After centrifugation the supernatant was transferred into a fresh tube and add 250 ál of isopropanol, and then left on ice for 30 minutes. After this time the mixture is centrifuged for 5 minutes Polom by adding different amounts of a mixture of phenol/chloroform/isoamyl alcohol (25:24: 1) to the mixture obtained above, vigorously stirring at the same time, after which the mixture was centrifuged at 10000 x g and 4oC for 5 minutes (hereinafter, this procedure will be referred to as extraction with phenol).

100 μl of the aqueous layer obtained by centrifugation, was mixed with 10 ál of 3 M sodium acetate (pH 5.2) and 250 μl of ethanol, after which the mixture was left on dry ice for 10 minutes and then centrifuged at 10,000 x g for 5 minutes with the release fraction nucleic acid (hereinafter, this procedure using the same relative amounts of supernatant ethanol and sodium acetate solution will be called "precipitation with ethanol").

The precipitate suspended in 50 ml of RNase A (10 µg/ml in TE-buffer) and then incubated for 1 hour at 37oC. then 30 μl of a solution of polyethylene glycol (2.5 M sodium chloride, 20% polyethylene glycol 8000) was added to the incubated suspension and the resulting mixture was left for one hour on ice. After this time the mixture was centrifuged at 10000 x g and 4oC and was isolated DNA fraction as a precipitate, and then twice conducted the procedure of deposition of ethanol and received purified plasmid DNA.

Purified plasmid DNA peritore. After electrophoresis the gel was subjected to southern blot hybridization (Southern, E. M. (1975), J. Mol. Biol. 89: 503-517). In particular, the gel was shaken for 40 minutes in a solution to denature, and then transferred for 2 hours, shaking in buffer for neutralization.

After shaking the gel was transferred to the polyurethane sponge containing 20 x SSC for DNA transfer on the part of the membrane Hybond-N (registered trademark Amersham), located on the sponge. After transferring the DNA to the membrane this membrane was shaken for 10 minutes in 1 x SSC and then dried for 1 hour at 80oC for fixing DNA. After that, the membrane was placed in the solution for pre-hybridization (5 ml formamide, 1 ml 50 x denhardt's solution, 2.5 ml 20 x SSC, 100 μl of yeast tRNA (50 mg/ml), 100 ál of 10% of the LTO and 1.3 ml bidistilled water) and incubated at 50oC for 3 hours.

Viral genomic RNA, which was split with the help of Mg2+used as a probe. In particular, the solution (16 μl), obtained as described in (C) and containing 1 μg of viral RNA, was added 4 μl of 5x buffer to denature and the resulting mixture was incubated 3 hours at 37oC. 5 μl of the solution of denatured RNA containing 100 ng of RNA (the payment of the criterion by heating at 70oC for 5 minutes and cooled on ice.

4 ál of 5 x buffer for labeling 1 mm 3.3 mm [ -32P]ATP (of 0.37 MBq) and 10 ál of bidistilled water was added to a solution of denatured RNA. Then to the mixture was added a solution of 20 units T4 polynucleotide-kineti (supplied by Takara Shuzo), and incubated for 30 minutes at 37oC. Uninvolved [ -32P]ATP was removed by 5-fold precipitation with ethanol.

The obtained labeled probe was added to the solution for hybridization (5 ml formamide, 2 ml 50% dextran sulfate, 200 μl of 50 x denhardt's solution, 2.5 ml 20 x SSC, 50 μl of yeast tRNA (50 mg/ml), 100 ál of 10% of the LTO, 1.3 ml bidistilled water) to a final concentration of 5 x 105imp. in min/ml, after which this solution was transferred membrane Hybond-N, obtained above, and subjected to hybridization by incubation over night at 50oC, followed by washing with shaking, 2 x SSC containing 0.1% sodium dodecyl sulfate (LTOs). This procedure was repeated three times, after which the membrane Hybond-N again washed with 0.1 x SSC containing 0.1% LTOs, for 1 hour at room temperature with shaking, and then drained.

The autoradiograph showed that plasmid, which was then labeled PNS, 51, had inserted fragment, Opredelenie sequence insertion pNS51

In order to construct a restriction map of cDNA, cloned in pNS51 indicated plasmid was digested each of restricteduse enzymes: BamHI, EcoRI, HindIII, kpni restriction sites, NcoI, RstI, SaII, SphI, SacI, SmaI, XhoI, XbaI, and the resulting map is shown in Fig.1.

Then, each of the fragments obtained by digestion restricteduse enzymes SalI and > PST, inserted in the RF-DNA M13 mp19. This plasmid pNS 51 digested restricteduse enzymes > PST and SaII, and the cleaved products were subjected to electrophoresis on 5% polyacrylamide gel using 1 x TBE. After electrophoresis, the gel was stained in 1 μg/ml of ethidiumbromid to be able to identify bands under the action of UV radiation. Strips of gel containing each DNA band was cut out with a razor and subjected electroelution using the device for electroelution (Amicon) with Method-30 (Amicon).

Then, using the set for ligating DNA (izgotovlivaemoj Taka a Shuzo), each fragment was inserted into M13 mp19, which was previously restrictive any one enzyme SaII or two enzymes > PST and SaII, and which has been treated with alkaline phosphatase.

The obtained recombinant M13mp19-RF-DNA (where the fragments were the. dinoco colony strain JM109, which was cultivated on minimal agar medium M9, inoculable and cultured in liquid SOB-medium overnight with shaking. 0.6 ml overnight culture was inoculable in 50 ml of fresh liquid SOB-medium and cultivated at 37oC and with shaking until, while OP600Nmnot reached 0?5. The cells are then besieged by centrifugation at 5000 x g and 4oC for 10 minutes and the precipitate is slightly suspended in 25 ml TGF buffer, then left on ice for 20 minutes.

After that, the suspension is centrifuged at 3000 x g for 5 minutes and the residue is suspended in 2 ml of TGF-buffer and left on ice for 20 minutes to obtain competent cells.

After ligation of recombinant RF-DNA M13 mp19, obtained as described above was used in an amount of from 1 to 10 ál for mixing with 100 μl of competent cells, after which the mixture was left for one hour on ice. After this time the mixture was incubated at 42oC for 1.5 minutes, and then to the mixture was added 500 μl of liquid SOC environment, after which the resulting mixture was cultured for 1 hour with shaking and at 37oC.

To the resulting culture, ramashiva ál bacteria indicator (overnight culture of strain JM109, cultured with shaking in 2 x YT medium at 37oC), and the resulting mixture was poured on a solid 2 UT-environment, containing 1.2% agar. After curing the mixture was cultured overnight at 37oC. Recombinant phages were then found as white plaques.

Each of the obtained white plaques were inoculable in the liquid 2 x UT-medium containing the bacteria indicator number of 1/100, and cultivated overnight at 37oC with shaking. After centrifugation for 1 minute at 10,000 x g supernatant was frozen or kept at 4oC in the form of phage solution, and the precipitate was treated using standard procedures to obtain plasmids emitting RF-dzanc (also called RF-DNA), which is an intermediate product in the replication phase. The presence of the inserted fragment was confirmed by digestion restriction enzyme.

Thus, the received 51SS/M13mp19 (i.e., M13mp 19 containing SaII/SaII fragment, which, in turn, is part of 51PS5'/M13mp19-genome containing RstI-SaII fragment from the 5'-region CYVV-genome). RF-DNA was purified from phage clones using alkaline LTOs solution and using centrifugation in a gradient of DNA was first digested with the enzyme BamHI to obtain sticky 5'-end, and then were digested with the enzyme kpni restriction sites with getting sticky 3'-end. Thereafter, in accordance with the scheme that shipped with the system Erase-a-base (Promega) was added exonuclease for stepwise deletions of bases from sticky 5'-end of each cDNA linearized plasmids. Treatment with exonuclease was carried out for 10 minutes and samples were removed at various intervals from 1 minute to 10 minutes. Since the vector has a sticky 3'-end, it is not exposed to ectonucleoside. Then various samples obtained in the time interval of 1-10 minutes, were blunt at the ends by treatment with S1-nuclease and legirovanyh in an annular shape using T4 DNA ligase.

Received the ring recombinant M13mp-19-RG-DNA was introduced into the strain JM 109 E. coli method using rubidium chloride described above. In accordance with this method, the recombinant phages with cDNA inserts of various lengths were obtained as white plaques.

RF-DNA was extracted from subclones of recombinant phage obtained as described above, using standard techniques for selection of clones with inserts of different lengths. The selected clones were separately inoculable in the liquid 2 x UT-medium containing the indicator strain of E. coli, or for 5 minutes at 10,000 x g and 4oC. 1 ml of the supernatant was transferred into a fresh tube and this tube, stirring, was added 250 μl of 20% polyethylene glycol (an aqueous solution of 20% peg 8000, 2.5 sodium chloride), after which the mixture was left for 30 minutes on ice. Then the mixture was centrifuged for 5 minutes at 10,000 x g and 4oC, and the resulting residue suspended in 100 μl TE-buffer. Then by extraction with phenol and precipitation with ethanol (as described above) of each drug were isolated phage genomic single-stranded DNA (abbreviated hereinafter referred to IDNC).

After this method dideoxy-termination circuit and using as matrix IDNC was carried out by sequencing of the nucleotides of each of the obtained subclones. In particular, phage IDNC marked [ -32P]dCTP (220 TBq/mm, Amersham) using the set containing 7-deaza-sequenase, version 2.0 dCTP (manufactured USB). The resulting reaction product was subjected to electrophoresis in 5% polyacrylamide gel containing 8 M urea, 1 x TBE buffer. Then the gel was dried, and the nucleotide sequence was determined using autoradiography.

By analyzing the nucleotide sequence 5'-Pst1 SalI fragment and M-SaII-SaII fragment PNS51, the main open reading frame (also called ORF) sequence obtained above, (+)- circuit of the viral genome was detected in one frame readout for the polypeptide. After it was determined the sequence of the polypeptide encoded in the ORF. Research conducted by comparing this polypeptide sequence with known sequences using the gene Bank sequences (GeneBank), revealed homology of this polypeptide with TEV-HAT.

It is known that viruses, like TEV, "smallpox" plum (hidden mosaic plum), or polio virus (animals) ripen under the action of protease, and that this usually protease cleaves the peptide bond containing Gln-Ala, Gln-Ser or Gln-Gly-relationship (J. Willink & Van Kammen, A. (1988), Arch. Virol 98: 1-26). The analysis of the sites of cleavage of the protein shell of the virus CY - N 30 (I. Uyeda et al. (1991), Intervirology 32: 234-245) also managed to find three split sequences derived polypeptide sequence. Based on one of these sites of cleavage, it was found that the polypeptide extending from Gly at the 4 position to Gln in 437 position, is the nuclear inclusion a, derived from virus CYVV.

H) Obtaining a hybrid protein CYVV-Nla/1L-11

DNA identified above (see item (G)) as a sequence: 1L (IL-11 human via a linker sequence that encodes Gln-Ala.

While following studies were conducted:

1) encodes whether identified in paragraph (G) DNA nuclear inclusion a (Nla) having proteolytic activity,

2) splits or not the specified protease (i.e., Nla) of the desired amino acid sequence,

3) does Nla by activity if it is part of a hybrid protein with the desired heterocyclic protein, and whether this activity is the same, what is the activity in the virus-infected cell,

4) does heterocyclic protein, obtained by splitting the specified hybrid protein, its integrity, structure and function.

Since Nla-protein itself is produced by cleavage from the viral polypeptide predecessor, the Nla-cistron is not the initiating codon. Therefore, in the 5'-end of the Nla gene, you need to add the initiating codon ATG. In addition, the 3'-end of the Nla gene must also be attached to the 5'-end of the gene IL-11 with preservation of the reading frame. In order to solve all the tasks, Nla modified using polymerase chain reaction (PCR) using restrita recognition for restrictase NcoI, need to clone the 5'-end of the Nla (Nla5'), were PCR primers. These primers had the following sequences: 5' GTCCATGGGGAAAAGTAAGAGAACA 3' (denoted hereinafter as NSATG, sequence ID No. 3) and 5' ACTCTGAGACCGTGCTCGAG 3' (denoted hereinafter as NSX1, sequence ID No. 4).

0,8 µl dNTR-solution (25 mm of each of the dATA, dTTP, dCTP and dCTP), 10 x Tag-buffer (manufactured Promega) and 1 μg of each of the obtained primers were added to 1 μg pNS51-plasmid DNA, and the resulting mixture was brought up to 100 μl by adding bidistilled water. Then for the implementation of polymerase chain reaction were added 5 units of DNA polymerase 10 x Tag-buffer. PCR was carried out according to the following scheme: cycle 1 min at 92oC, 1 min at 37oC and 2 min at 72oC - repeated 20 times after one cycle of 1 min, 92oC, 1 min, 37oC and 30 min 72oC.

The obtained amplified DNA was subjected to extraction procedures with phenol and precipitation with ethanol, and then electrophoresis on 5% polyacrylamide gel. Using staining with ethidium bromide was found one lane containing DNA, this band was cut out from the gel and subjected to electroelution using the apparatus for electroelution Centreluter (Amicon) with Method-30 (Amicon). After elution loirevalley 4oC for 45 minutes. The DNA concentrate was then purified by extraction with phenol and precipitation with ethanol.

Regardless of this, 1 μg of plasmid pKK388 (Clonetech, in which the SacI site of recognition was replaced by a XhoI site of recognition, were digested restricteduse enzymes NcoI and XhoI, and then dephosphorylated intestinal alkaline phosphatase (calf called CIAR and manufactured by Takara Shuzo. The obtained DNA is ligated by means of a set for alloying Takara Shuzo) with 100 ng plasmid pKK388-1, which was also restriction enzymes NcoI and XhoI and the dephosphorylated as described above, resulting in a received recombinant DNA, which is then cloned into JM 109 E. coli. Thus was obtained a plasmid pKN15', which had an insert in a normal orientation and located "below" from trc-promoter plasmids pKK388-1 (Fig. 2).

Plasmid pCD20-2 (Kawashima 1. et. al., (1991), FEBS L 283: 199-202) contained cDNA encoding the precursor of IL-11 (pre-IL-11) and having a sequence of the secretion signal. This plasmid was digested restricteduse enzymes BamHI and ApaI and cut out the area containing the pre-IL-11 and the SV40 promoter. The resulting fragment ligated into the BamHI and ApaI sites of the vector pBLUESCRIPT 11 SK+, and then were digested restricteduse farm>The obtained Mat-IL-11-fragment (lacking its N-Terminus) was integrated using T4 ligase in pKN15', which was previously restriction enzymes XnoI and kpni restriction sites, and CIAP treated. The resulting construct was designated pKN141 (Fig.3). For the implementation of the ligating specific sequence in the C-end of Nla with a fragment of Mat-IL-11 by Ala and maintaining the reading frame were synthesized four types of PCR primers:

5'AGGAAAAGACTTCCTCGACC 3'

(marked NSX2, sequence ID No. 5),

5' AATTGTTCATTCCAAGCACCTGGGCCACCACCTGGC 3'

(marked NSJ001P, sequence ID No. 6),

5' GCCAGGTGGTGGCCCAGGTGCTTGGAATGAACAATT 3'

(marked NSJ002) sequence ID No. 7), and

5' TTGTCAGCACACCTGGGAGCTGTAGAGCTC 3'

(indicated by the ILSAC sequence ID No. 8).

The first PCR reaction was carried out (using a pair of primers NSX2 and NSJ002N and using DNA pNS51 as matrix) for amplifying the region of the insert that encodes a C-Nla end (marked CN13-region). Then separately performed another PCR reaction (using pairs of primers NSJ001 and 11SAC and using as template DNA pNS51) for amplifying the region of the insert that encodes N-end-IL-11-peptide (labeled 5' IL-scope). PCR was carried out according to the following scheme is C / 1 min at 92oC, 1 min at 37oC and 30 min at 72oC. the Products obtained by polymerase chain reactions were extracted with phenol and precipitated with ethanol, and then subjected to electrophoresis on 5% polyacrylamide gel. The gel strip containing the amplified DNA band was cut out and the DNA was separated from the gel by the method of electroelution described above. The results are shown in Fig. 4.

The two resulting amplified DNA fragment was a region primers NSJ001P and NSJ002N, i.e., the 3'-terminal part of CIN 3-DNA fragment and the 5'-part 5'IL-DNA fragment, nucleotide, the sequence of which was found a partial homology. For example, if re-polymerase chain reaction using primers NSX2 and ILSAC, homologous areas will be hybridisierung with the formation of hybrid DNA containing both genes, while the homologous site will act as cross-linking region (Fig. 5). In line with this, the selected CIN3-DNA fragment and the 5'II - DNA fragment were mixed and again spent PCP using as primers only NSX2 and ILSAC. PCR was carried out according to the following scheme: first spent 10 cycles in mode: 1 min at 92oC, 1 min at 37oC and 2 min at 72oC, followed by 20 cycles in rain at 92oC, 1 min at 55oC and 30 min at 72oC. After treatment with phenol and precipitation with ethanol was carried out by electrophoresis on 5% polyacrylamide gel, which was received CNI3IL-DNA fragment in which CNI3 was attached to the 5'IL. This method is shown in Fig. 5.

CNI3IL-DNA fragment were digested restriction enzyme XhoI, and the resulting fragment using T4-DNA-ligase, inserted in pKN15', which was previously split restriction enzyme XhoI and the dephosphorylated CIAP. The obtained plasmid was cloned into a strain JM 109 E. coli. For selection of a clone in which CNI3IL-fragment was inserted in the correct orientation, plasmids were extracted from the obtained clones using primers NSX2 and ILSAC was carried out by PCR. In accordance with the system used was found only one DNA band from the clone, in which this piece was insertion in the correct orientation. The result was obtained plasmid pKSUN9, in which the Nla and Mat-IL-11 is connected by split sequence Gln-Ala with preservation of the reading frame (Fig. 6).

1) Expression of Ala-IL-11 in the bacterial cell

E. coli carrying plasmid pK 9 (denoted further strain KSUN9), and cultivated with shaking and at 37oC in accordance with the government of ampicillin) was added to 2.5 ml of the obtained KSUN 9 - culture and cultivated with shaking and at 37oC up until OP600Nmreached a value of 1.0. After OP600Nmwas equal to 0.1 mm IPTG was added to a final concentration of 0.1 mm and the mixture was cultured with shaking and the 28oC for 12 hours.

The second cultivation was carried out in full accordance with the above procedure, except that the last cultivation was performed at 28oC for 36 hours or more with IPTG concentration of 1 mm, resulting in a received Mature IL-11 (which had an N-terminal Proline).

(J) Western blot

Western blotting was carried out to determine whether pKSUN 9 functional in E. coli, as well as to determine is whether the constructed recombinant gene.

Each of the 12 - and 36-hour cultures induced with IPTG, were subjected to subsequent processing described below. For this purpose, 2 ml of culture by 5 minutes centrifugation at 3000 x g and 4oC was isolated cells obtained by centrifugation of the precipitate suspended in 100 μl of 20 mm natreporting buffer (brought to pH to 9.0 with NaOH 0.1 N.). The resulting suspension process the minutes when the apparatus is in position 8, for disintegration of cells, and then, within 10 minutes, was carried out by centrifugation at 10,000 x g and 4oC. the Supernatant containing the soluble protein fraction, was isolated. 5 μl of this supernatant was subjected to electrophoresis in 12% polyacrylamide gel with LTOs in accordance with the method Laemmli (Laemmli (1970) Nature 227, 680 - 685).

After electrophoresis the gel for 5 minutes was dissolved in a buffer for transfer (25 mm Tris, 192 mm glycine, 20% methanol) and blokirovala on the part of the PVDF-membrane (Thans-Blot Transfer Medium is a medium for transfer, manufactured by Bio-Rad Laboratories) by processing at 15 V for 1 hour in a special chamber Trans-Blot SD Semi-Dry Transfer Cell (manufactured by Bio-Rad Laboratories). Batrouney PVDF-membrane was washed in PBS-TW-environment for 10 minutes. PVDF-membrane was transferred to PBS-TW-medium containing 5% separated milk (manufactured by Snow Brand Milk Products), and left for 1 hour at 37oC for blokirovaniya.

After blokirovaniya PVDF-membrane was again washed in PBS-TW-environment, once for 10 minutes and twice for 5 minutes and then transferred to rabbit serum against IL-11, pre-10000-fold diluted in PBS-TW and incubated for 20 minutes at 37oC. after this time PVDF-membrane was again washed in PBS-TW, once in the course of producing chemiluminescence (ECL) (manufactured by Amersham) and strip, which react with the antibody against IL-11, find by exposing specified PVDF membrane with x-ray film during the period of time from 30 seconds to 5 minutes

As a result of Western blotting, as described above, the signal band corresponding to a molecular mass of about 50 kDa and about 23 kDa, was detected between 12-hour and 36-hour cultures. 23 kDa band had the mobility is essentially the same as the mobility of the Mature IL-11, used as a control. Thus, it is obvious that the 230-kDa signal bar represents the IL-11, which was derived from Nla/IL-11 hybrid protein Gln-Ala-linker sequence due to the proteolytic activity of Nla. Hence we can conclude that the heavier band (50 kDa) is a hybrid undigested protein.

Next, a 23 kDa protein obtained from a 12-hour culture, will be denoted 23 kDa-ON, and the 23-kDa protein derived from the 36-hour culture, will be denoted 23-kDa-36hr.

K) Purification of protein of 23 kDa-ON and 23 kDa-hr

Protein 23 kDa-ON and 23 kDa-36hr were purified to determine the amino acid sequence of their N-ends. In accordance with the procedure described in paragraph (1), received 250 ml of each of the 12-hour and 36-cash g, at 4oC, and the residue suspended in 10 ml of 20 mm borate buffer (pH 9,0), and then subjected to disintegration by use of a press jacket and apparatus for ultrasonic treatment (both). After centrifugation for 30 minutes at 15000 x g and 4oC soluble protein fraction was isolated in the form of a supernatant.

The obtained soluble protein fraction was subjected to weak ion-exchange column chromatography using FPLC (Pharmacia) under the following conditions:

Column: CM Toyopearl Pack 650 M (2.2 x 20 cm, manufactured by Toso)

Buffers for elution: A = 10 mm boric acid - sodium hydroxide (pH 9,0), 13 mm potassium chloride, B = 10 mm boric acid - sodium hydroxide (pH 9,0), 13 mm KCl, 400 mm sodium chloride.

Flow rate: 2.5 ml/min

Volume fraction: 5 ml per tube

Used the concentration gradient (for 120 min) consisted of a linear gradient eluent A to eluent B"

Then each elyuirovaniya fraction was subjected to enzyme-linked immunosorbent analysis (ELISA) to identify fractions containing IL-11.

ELISA was carried out as follows. Each well of 96-well immunoplate (Max oap, manufactured by Nunc.) download 100 μl of 50 mm nutricare is for 1 hour at 37oC. after this time, each well was washed 4 times with PBS-T-medium (PBS containing 0.1% tween-20).

Each of FPLC fractions obtained as described above was diluted 1:100 PBS-T and were loaded into the wells in the aliquot 100 ál per well. The plates were incubated for 1 hour at 37oC, after which the wells are again washed with PBS-T and to each well was added 100 μl of rabbit IgG against IL-11, diluted with PBS-T to a final concentration of 1 µg/ml.

The tablet then incubated for 1 hour at 37oC and washed with PBS-T, and then each well was added 100 μl of labeled alkaline phosphatase goat antibodies against rabbit IgG (Gibco Bethesda Research Laboratories), which was 3000-fold diluted in PBS-T. the Tablet again incubated for 1 hour at 37oC, and then washed with PBS-T. then each well was added 100 μl of the substrate solution of alkaline phosphatase. Tablet incubated at room temperature for another 30 minutes to 1 hour, after which the coloring of each well was measured as optical density at 450 nm in order to identify the required (or desired) fractions (fractions).

Fractions obtained by FPLC and procedures identified in accordance with the above-described ELISA as fractions, the content is whether using Centprep-10 (Amicon) and subjected LTO-electrophoresis in 12% polyacrylamide gel according to the method Laemmli (see above). Then the gel was subjected to elektrobatareyu on PVDF-membrane in accordance with the above procedure for Western blotting, except that PVDF-membranes used the Problot membrane (manufactured by Applied Biosystems).

After blokirovaniya the membrane was thoroughly washed with bidistilled water, stained, Kumasi brilliant blue P-250, was treated with 50% methanol for washing color and the band containing the protein, which reacts with the antibody against IL-11 in Western blot analysis, cut.

The amino acid sequence of the N-Terminus of this protein were analyzed using protein sequencing machine (Applied-Biosyntems). As a result of this analysis, it was found that the N-terminal sequence of band of 23 kDa-ON reacting with the antibody against IL-11, is:

Ala-Pro-Gly-Pro-Pro-Pro-Gly- (sequence ID No. 9)

This sequence corresponds to the sequence from -1 to +6 amino acid sequence of Mature IL-11 protein. Based on this, we can conclude that the 23 kDa protein obtained from a 12-hour culture and reacts with the antibody against IL-11, is an Ala-IL-11. In accordance with this it is obvious that white is ln-Ala-site specific split sequence.

In addition, it was found that the N-terminal sequence of band of 23 kDa-36hr-reactive antibody against IL-11, is:

Pro-Gly-Pro-Pro-Pro-Gly-Pro- (sequence ID No. 10)

This sequence corresponds to the amino acid sequence from +1 to +7 Mature IL-11 protein. On this basis, the following conclusions were made:

In the induction with IPTG, a hybrid protein Nla/IL-11 is expressed in E. coli.

In the 12-hour cultivation after induction produced Nla/IL-11-hybrid protein is cleaved in Gln-Ala-peptide bond with a specific split

sequence due by activity Nla.

After cleavage of the peptide bond Nla-Mature protein, but with an additional Ala at its N end of IL-11 is produced in E. coli.

Under cultivation even 24 hours after the expression of Ala-IL-11, it was found that Ala-IL-11 "Matures" to IL-11, in which the Ala residue, present in the N-end Ala-IL-11, deleterule.

On the basis of obtained results it was concluded that the 23 kDa protein obtained after 36 hours of cultivation, is a Mature type IL-11 protein in which the N-end is a Pro.

In aktivnosti Nla this protein must be broken down into specific linker sequence, containing Gln-Ala, with the formation of Ala-IL-11. Subsequent cultivation leads to the formation of Mature IL-11, in which the alanine residue deleterule by the factor, is expressed in E. coli, resulting in the N-end is a Pro.

In order to ensure that produced IL-11 and Ala-IL-11 are biologically active, as an indicator of such activity was determined activity adipokines-inhibitory factor (AGIF). The fact that IL-11 is adipokines-inhibitory activity, was installed previously (Kawashima, 1, et al. (1991), FEBSL. 283: 199-202).

Evaluation of inhibitory effect on morphological changes from ZTZ-LI-cells to adipocytes

The following describes the method of determining the AGIF-activity used in the present invention. This method used a cell line ZTZ-L1 mouse embryonic fibroblasts (Green H. & Kehinde (1974), Cell 1: 113-116), available from the ATCC. In all cases, these cells were cultured in a mixed humid atmosphere (10 CO2- 90% air) at 37oC and perseval in environment A. the Induction adipogenic differentiation was carried out in accordance with the procedure described by Rubin and others (Rubin C. S. et al. (1978), J. Biol. Che,. 253: 7570-7578).

Cells ZTZ-L1 suspended in medium A to access unka, made Coaster), and then cultured. After 3 days of culturing cells was achieved continuity. Then the medium was replaced with fresh medium A and after culturing for a further 2 days the medium was replaced with adipogenic-inducing medium (medium B), were simultaneously added 0.5 ml of the test sample. This medium was replaced with fresh medium B with the addition of fresh sample every two days.

At different time intervals for different holes, starting with 4 and day 7 after adding the first test sample, emaciated environment B in the wells was replaced with adipocyte-supportive environment C.

After culturing in medium C for 2 days the cells were fixed with 5% formaldehyde and all fat particles, which were accumulated in the cells and cell nuclei were stained with oil red O and hematoxylin, respectively. On the obtained micrographs were counting the number of cores and the number of cells that contain accumulated painted fat particles. Alipogene ratio (AR) was calculated by the following equation:

< / BR>
Fixing the cells and staining with oil red O and hematoxylin was performed in accordance with the procedures described Yoshio Mitomo and Shojiro Takayama in "Lectures on Clinical Testing is the distribution of the inhibitory activity of lipoprotein lipase was carried out in accordance with the method, described by Beutler and others (Beutler B. A. et al. (1985) J. Immunol 135: 3972-3977). Adipogenesis differentiated cells ZTZ-L1 was obtained as described in paragraph (1), except that the induction of cell differentiation in adipocytes of the test sample was added. Instead, the test sample was added with fresh medium C, after which cells were cultured for 18 hours. After this time the medium was removed, and cells were twice washed in PBS(-) (phosphate buffer solution, made Nissui Seiyaku), and then to each well was added to 3000 ml of medium D and cultivated for another 1 hour. Then from each supernatant culture took 100-Microlitre aliquots for measurement of lipoprotein lipase activity, which was measured in the triple duplicate for each sample to obtain an average value.

The activity of lipoprotein lipase (LPL) was measured as described by Nilsson-Ehle & Schotz (Nilsson-Ehle P. & Schotz, M. C. (1976), J. Lipid Res. 17: 536 - 541). Each aliquot of the supernatant obtained as described above was mixed with an equal volume of substrate solution and left for 120 minutes at 37oC to perform the reaction. The reaction was stopped by addition of 1.05 ml of 0.1 M clickbooth buffer (brought to a pH of 10.5 using 0.1 M potassium borate) and 3, the mixture was centrifuged for 15 minutes at 3000 x g and room temperature. Then using a liquid scintillation counter counted the contents of the3H Vodopyanova phase.

For one unit of LPL activity took such activity, which generates 1 μm fatty acid per minute. 13 mm three[9.10(n)-3H] oleate of glycerol in the substrate solution was obtained by diluting three[9.10(n)-3H] oleate of glycerol (37,0 GBq/M) (manufactured Amersham) triolein (manufactured Sigma), followed by purification using column chromatography on silica gel.

In the following examples illustrates a variant implementation of the present invention, which relates to glutathione-regenerating protein.

Example 1

The extraction of Poly(A)RNA from cells KM-102

Cells KM-102 was grown in 36 plastic cups for cultivation with a diameter of 15 cm, in minimum essential medium modified according to the method of Claims (Boeringer Mannheim) containing 10% fetal bovine serum. After growing the cells to the continuity was added formalistically (PMA) and Infor calcium A23187 (Sigma) to a final concentration of 10 ng/ml and 0.2 μm, respectively, and the cultivation was carried out at a temperature of 37oC. After 3, 6 and 14 hours collecting party from

The allocation of Pol(A)+RNA was carried out basically in accordance with the description given in "Molecular Cloning A Laboratory Manual [Maniatis, T. et al. (1982) pp. 196-198]. Below is a detailed description of this procedure.

Each selected liquid phase was separately treated in the following way. The liquid is re-gathered and poured from the piston 10 ml syringe equipped with a syringe needle (21G. 3-Ml solution of 5.7 M CsCl - 0.1 M EDTA (pH 7.5) was previously added into a test tube centrifuge Polyoromar, selected in accordance with the size of the blades of the rotor of a centrifuge RPS40-T (Hitachi Koki). The obtained cells were applied on top of the solution contained in the tube, as long as the tube does not become full.

After 18-hour centrifugation at 3000 rpm and at a temperature of 20oC the precipitate suspended in 400 μl of distilled water and precipitated by adding ethanol. The precipitate was dissolved in 400 μl of distilled water and the resulting solution, stirring, was added to equal volume mixture of chloroform/1-butanol (4:1, V/V), and then the aqueous layer was collected by centrifugation. Besieged ethanol centrifuged once and the precipitate was dissolved in 600 μl of distilled water, R is lo received approximately 4.5 mg of whole RNA.

600 μg of each of the three types of whole RNA obtained by the method described above, were combined and subjected to chromatography on a column of oligo(dT)-cellulose for the purification of poly(A)+The RNA.

Whole RNA was dissolved in the buffer for adsorption, and then was heated for 5 minutes at 65oC. the resulting solution was applied on a column of oligo(dT)-cellulose (Pharmacia, type 7) loaded adsorption buffer, and suirable eluting solution, resulting in a $ 100 µg poly(A)+The RNA.

Example 2

Obtaining cDNA-libraries

cDNA library was obtained by a method described Okayama Beg.

5 μg poly(A)+RNA and 24 units of reverse transcriptase (Seikagaku Corp.) left for reaction for one hour at 42oC in 20 ál of reaction solution reverse transcriptase.

After that, the reaction was stopped by adding 2 μl of 0.25 M EDTA and 1 μl of 10% LTOs, and then the resulting solution was deproteinization using 20 μl of a mixture of phenol and chloroform (1:1, V/V). After centrifugation to remove fractions containing protein, the supernatant was added to 20 μl of 4 M ammonium acetate and 80 μl of ethanol, and then the mixture was cooled for 15 minutes at -70oC. At the end of this percent the leaders introduce pressure.

The dried precipitate was dissolved in 15 μl of reaction solution terminal transferase and was heated at a temperature of 37oC for 3 minutes. After this time 18 units end deoxynucleotidyltransferase (Pharmacia) was added to the reaction solution and the resulting mixture was left to react for 5 minutes. After that, the reaction was stopped by adding 1 μl of 0.25 M EDTA and 0.5 ál of 10% of the LTO and the resulting solution was deproteinization mixture of phenol/chloroform (as described above), and then centrifuged to remove the fractions containing protein. The supernatant was collected and vigorously mixed with 15 μl and 4 M ammonium acetate and 60 μl of ethanol. Thus obtained mixture was cooled at -70oC for 15 minutes, and the precipitate was collected by centrifugation.

The precipitate was dissolved in 10 μl of buffer for restriction enzyme and to this solution was added 2.5 units of restriction enzyme HindIII, and then the solution was left for 1 hour at a temperature of 37oC for digestion.

Then the reaction solution was deproteinization mixture of phenol/chloroform, then precipitated with ethanol, and the obtained supernatant was cooled for 15 minutes at -70oC. the Obtained obtained solution was added to 10 μl of the reaction solution, containing 10 mm Tris-HCl (pH 7.5), 1 mm EDTA, 100 mm sodium chloride (NaCl), and 10 ng of linker DNA with oligo(dT) tail [pL-1-HindIII-linker to the 3'-oligo(dG) tail, Pharmacia, after which the resulting mixture was heated at a temperature of 65oC for 5 minutes and then was heated at a temperature of 42oC for 30 minutes. This reaction mixture was cooled with ice water, followed by adding 10 ál of 10x ligase buffer, 78 μl of distilled water and 8 units of T4 DNA ligase. The resulting reaction solution was kept overnight at a temperature of 12oC.

The next day the reaction mixture was combined with 10 μl of 1 M KCl, ribonuclease H (1 unit), 33 units of DNA polymerase 1, 4 units of T4 DNA ligase and 0.5 μl of dNTP solution (20 mm dATP, 20 mm dCTP, 20 mm dGTP and 20 mm dTTP), and 0.1 μl of 50 μg/ml of bovine serum albumin (BSA). Thus obtained mixture was heated for one hour at a temperature of 12oC, and then for one hour at a temperature of 25oC. after this time the reaction solution five times diluted with distilled water, and then directly used to transform E. coli DH5 method [Hana-Hanahan D. D. (1983) J. Mol. Biol 166, 557 - 580], resulting in the obtained cDNA library cells KM-102.

Example 3

Getting Oli who was b chemically synthesized oligonucleotide 5'-TAAATAAATAAATAA-3', consisting of 15 bases (sequence ID No. 13) and marked as ATT-3. The synthesis was performed using an automatic DNA synthesizer 380B (Perkin-Elmer Japan Applied Biosystems) in accordance with the instructions supplied in the accompanying instructions. This procedure was carried out vospitannym method described Caruther et al. (Matteucci, M. D. and Caruthers, M. H. (1981) J. Am. Chem. Soc. 103, 3185-2191]. After the synthesis of 15-measure obtained oligonucleotide was separated from the medium, and the protective group was removed. Then the solution of the oligonucleotide was liofilizovane and got the powder, which was dissolved in distilled water and kept at -20oC until its use.

Example 4

Screening cDNA libraries

6500 colonies generated from the cDNA library obtained in example 2 was fixed to the nitrocellulose filter in accordance with the method described by Grunstein &Hogness (Grunstein, M & Hogness, D. S. (1975) Proc. Acad. Sci USA 72, 3691-3695] . ATT-3-probe obtained in example 3 was marked at the 5'-end with32P and in accordance with standard procedures (see "Molecular Cloning-A Laborotory Manual"), and labeled zones used for hybridization of colonies.

Pre-hybridization was carried out at a temperature of 37oC for 3 hours, ispolzuya salmon sperm. Then, during the night, when the temperature of the 31oC hybridization was carried out using the following components: 6 x SSC, 1 x denhardt's solution, 17 μg/ml yeast transfer RNA and 0.05% sodium pyrophosphate containing32P-labeled probe ATTT-3.

The next day, the nitrocellulose filter was washed at the temperature for 2 hours 6 x SSC solution containing 0.05% sodium pyrophosphate. After autoradiography was identified 33 positive clone.

DNA plasmids were extracted from positive clones in accordance with standard procedures. Then randomly selected a few clones and their partial nucleotide sequence of the cDNA were determined using dideoxy method. These partial sequences were evaluated for homology with nucleotide sequences registered in the EMBL data Bank or Geneank, using a personal computer, as a result it was found that some partial sequences of the clones detected using ATT-3, had homology with parts AIu-repeat (Schmid, C. W. & Jelinek, W. R. (1982) Science 216, 1065-1070).

A DNA fragment containing a sequence AIu-repeat, were obtained from human genomic DNA and was tagged with 3 the colonies using the 33 clones, identified above, in the result, it was found that 12 of the clones had AIu-repeat. Then determined the length of the cDNA insert each of the remaining 21 clones, and it was found that the length of the cDNA insert was changed in the range from 50 to 3600 grounds.

Mapping of restriction enzymes was carried out on cDNA inserts of the remaining 21 clone, and partial nucleotide sequences were determined by the method described above. These partial sequences were evaluated for homology (as described above) with nucleotide sequences registered in the data Bank EMB or GenBank, using a personal computer, resulting in the selected clones, with a new sequence.

Example 5

Northern hybridization of clone N 31

One of the clones, namely clone N 31 (labeled pcD-31), had the cDNA insert with approximately 560 base pairs. In accordance with the procedure described in example 1, PST1-Aat1-fragment (292 p. O.), obtained from cDNA inserts pcD-31 and placed32P, served as a probe in Rozemblit-procedure, using poly(A)+RNA obtained from cells KM-102. This hybridization was used to determine the length of the natural mRNA corresponding to su the RNA was obtained from cells KM-102, and then incubated for one hour at a temperature of 50oC in a mixture of 1 M of glycosuria, 50% dimethyl sulfoxide (DMSO) and 0.01 M of dinitromethane (pH 7.0). After this time, 4 μl of pigment for electrophoresis was added to the incubated mixture, which was then subjected to electrophoresis on 1% agarose gel in 1 x TAE-solution.

After electrophoresis, the RNA on an agarose gel, transferred and left overnight to nylon membrane filter (BioPad, Z-probe) (using 20 x SSC) by capillary transfer (see "Molecular Cloning-A Laboratory Manual"). After the procedure, transfer the filter slightly washed with 2 x SSC solution, was dried by air, and then was further dried at a temperature of 80oC for 2 hours to commit mRNA.

> PST -Aat1-fragment of clone pcD-31 was marked with32P using the standard system of labeling DNA (Amersham)

Pre-hybridization was carried out on the filter for 3 hours at a temperature of 37oC in a solution containing 5 × SSCP, 2.5 × denhardt's solution, 50% formamide, 10 mm dinatriumfosfaatti (pH 7.0), 0.5% of LTOs and 100 μg/ml denatured DNA salmon sperm.

Then hybridization was carried out on the filter overnight at a temperature of 42oC in a solution containing32o
C with a solution containing 50% formamide, 5 x SSC and 0.1% LTOs, and then washed for 2 hours at the same temperature a solution containing 40% formamide, 5 x SSC and 0.1% LTOs, and finally washed at room temperature for 15 minutes a solution of 2 x SSC. As a result of autoradiography it was found that the cDNA insert of clone pcD-31 has a limited length, and full length of the corresponding mRNA is 3.9 kb (determined using a calibration curve based on the molecular weight marker).

Example 6

Getting a fresh library for screening cDNA clone pcD-31

Fresh cDNA library was obtained using cDNA synthesis (cDNA Synthesis Systems Plus) and system cloning cDNA ( gt10, adapter method, Amersham

5 μg poly(A)+RNA was extracted from cells KM-102 (in accordance with the procedure described in example 1) and 100) units of reverse transcriptase was subjected to reaction for 40 minutes at a temperature of 42oC in 50 ál of the solution for the reaction of reverse transcriptase. After this time, 20 μl of [ -32P] dCTP, 93,5 μl of buffer 4 units of ribonuclease H and 115 units of DNA polymerase 1 (they were all included in the kit) was added to reactio the e 22oC for one hour, and finally heated for 10 minutes at a temperature of 70oC. After heating to the reaction solution was added 10 units of T4 DNA polymerase (included in the set), and the resulting mixture was left to react for 10 minutes at a temperature of 37oC.

Then the reaction mixture was deproteinization using a mixture of phenol/chloroform. The reaction solution was subjected to centrifugation and the supernatant was collected and thoroughly mixed with 250 μl of 4 M ammonium acetate and 1 ml of ethanol. The mixture was cooled overnight at -20oC, and the resulting precipitate was collected by centrifugation. After that, the precipitate was dissolved in 30 μl of sterilized water. 10 μl of the obtained solution was removed and added to a mixture of 2 μl of ligase/kinase buffer. 250 gr EcoRI-adapter, and 5 units of T4-DNA-ligase (they were all included in the set), after which the resulting mixture was incubated over night at a temperature of 15oC.

The entire reaction solution was applied on the column for fractionation by size, using a set to remove the EcoRI adapter. Then the reaction solution was collected in 120 ál aliquot and each aliquot was mixed with 200 μl of 0.25 x TE buffer. Fractions (10 to the ü volume of concentrated product was mixed with 55 μl of sterilized water, 20 μl of ligase/kinase buffer and 40 units of T4 polynucleotide-kinase (they were all included in the solution), after which the resulting mixture was incubated for 30 minutes at a temperature of 37oC. after this time the reaction mixture was deproteinization 3 times with a mixture of phenol/chloroform and then precipitated with ethanol and cooled overnight at -20oC. the precipitate was collected by centrifugation and dissolved in 10 μl of sterilized water, resulting in a received sample cDNA.

1 μg EcoRI ( gt10), 1 μl of ligase/kinase buffer and 2.5 units of T4 DNA ligase (they were all included in the kit) was added to 2 μl of the above sample cDNA and the mixture incubated overnight at 15oC. the Sample containing 4 μl of sample cDNA, was obtained in the same way. After that, each sample was processed as follows. All the obtained reaction solution was first added to 10 μl of Extract A (included in the set, and then the mixture was added to 15 Extract B (included in kit) and the resulting mixture incubated at 20oC for 20 minutes to perform the reaction in vitro packaging.

After this time the reaction is Tivoli magnesium sulfate (10 mm) and were infected using the above solution, then get gt-10 cDNA library from cells, the KM-102.

Example 7

Screening cDNA libraries

2 105plaques obtained from the United cDNA library (obtained in example 6), was shot on nylon filters (Hyobnd N, Amersham) in accordance with the following procedure.

Infected E. coli, obtained in example 6, were cultured in ten 9 cm-tablets containing solid LB-slice, so that one tablet a number of plaques ranged from 1 to 2 104. After the plaques were transferred to the tablet by a soft pressing of nylon filter into a tablet. The needle of the syringe 18G used to pierce the filter and marking gel in 3 places for comparison. The filter is then left on for 5-10 minutes at a temperature of 4oC, then cleaned and immersed in an alkaline solution (0.1 N. sodium hydroxide, 1.5 M sodium chloride) for 20 seconds. The filter was transferred into a neutral solution [0.2 M Tris-HCl (pH 7.5), 2 x SSCP] for a period of time from 20 seconds to one minute and dried with air at room temperature for 2 hours. Then the filter was dried at a temperature of 80oC within 2 hours.

Example 8

The receiving probe and hybridization

32P-labeled probes consultancies standard system for labeling DNA (Multiprime). The plaque hybridization was carried out on the filter obtained in example 7, using the above probes.

Pre-hybridization was carried out by placing the filter in a bath containing 50% formamide, 5 × SSCP, 2.5 × denhardt's solution, 0.01 M dinatriumfosfaatti (pH 7.0), 0.5% of LTOs, and 100 μg/ml denatured DNA salmon sperm. The resulting mixture was incubated for 2 hours at a temperature of 37oC.

Then hybridization was carried out by placing the filter in the reaction solution, containing32P-labeled probes, obtained as described above, also, 50% formamide, 5 × SSCP, 1 x denhardt's solution, 0,01% M dinatriumfosfaatti (pH 7.0), 0.1% of LTOs and 100 μg/ml denatured DNA salmon sperm, and then the resulting mixture was incubated overnight at 37oC.

The next day, the filter was washed for three hours at room temperature with a solution containing 50% formamide, 5 x SSC solution, and T, 1% LTOs, and then washed for five minutes at room temperature with 2 x SSC solution. As a result of autoradiography revealed 80 positive clones obtained in the primary screening.

Using clones identified as positive (in each case, the positive clones. From each of the 17 clones were isolated cDNA and insertion cut by the enzyme EcoR1. The length of each cDNA insert was analyzed using electrophoresis on agarose gel, after which he was selected clone N 31-7 with cDNA insert, consisting of 3,9 p. O., and corresponding full length of the original mRNA.

Example 9

Restriction mapping of clones N 31-7

Clones N 31-7 digested by the enzyme EcoR1 for isolation and purification of 3.9 kb-framgent containing the cDNA insert. This fragment was inserted into pUC18 using T4 DNA ligase. DH5. E. coli transformed with the new plasmids. Transformed cells were selected by their resistance to ampicillin and clone pUCKM31-7 with the insertion of a cDNA from 3.9 K. O., identified by digestion of DNA with the enzyme EcoRI and the resulting cleaved DNA was subjected to electrophoresis on agarose gel.

pUCKM31-7 were digested with each restriction enzyme HindIII, SacI, XbaI, SmaI, BgIII, EcoT22I and AatI or their parts. The resulting fragments were subjected to electrophoresis on agarose gel, after which the length of each fragment was measured using HindIII/Thai marker that is used as the indicator. The obtained restriction map shown in Fig. 7.

Example 10

ODA is CKM-31-7 was determined by dideoxy method using M13 phage. In addition, part of this sequence was analyzed using DNA-sequenator 373A (Perkin Elmer Japan Applied Biosystems). The obtained nucleotide sequence is illustrated in the List of sequences under number 1D N 11.

cDNA insert of the plasmid pUCKM-31-7 has a length 3815 grounds and, in addition, has, obviously, an open reading frame consisting of 549 amino acids, starting with methionine. poly(A) tail, obviously, no. By comparing the sequence of bases at the 3'-end of the insertion plasmid pcD-31 sequence of clone pUCKM-31-7 it was found that in the insert of clone pUCKM-31-7 missing only part of the poly(A) tail (Fig. 8) and no more.

The nucleotide database EMBL and GenBank and NBRF database and SWISS-PROT allows you to compare the sequence of bases and amino acids, respectively. It was found that the closest match is 35.3% of the homology of this peptide sequence and the sequence of glutathione reductase of human rights. In line with this it was concluded that the ORF cDNA insert of the plasmid pUCKM31-7, it is obvious that encodes a new polypeptide. This new polypeptide shown as a sequence of the following list of sequences under numericseries vector and the expression of it in the cells COS-1.

Plasmid pUCKM31-7 was digested with the enzyme HindIII, and 3003 p. O. fragment containing the cDNA insert was isolated and purified in accordance with standard procedures. The ends of the obtained fragment was a small mistake with a set for blunting DNA (Takara Shuzo).

Meanwhile, vysokodispersnyi vector pcDL-SR 296 [Takabe, Y. et al. (1988) Mol. Cell. Biol. 8, 466-472] digested by enzymes > PST and Cloned, and the ends were a small mistake with the set to break the DNA. After this blunt box was Legerova in blunt plasmid by reaction using T4 DNA ligase. E. coli transformed with the resulting DNA method using calcium chloride and the resulting AmpRthe transformants were selected, and DNA plasmid held by the microorganisms analyzed.

In particular, the strain in which the direction of transcription of the cDNA is identical to the direction of the promoter SR , were selected by digesting the plasmids with the enzymes HindIII and BgIII, followed by electrophoresis on an agarose gel for separation of 800 p. O. fragment and selected this plasmid was designated pUR 31-7 (Fig. 9). The promoter SR contains the original SV40 promoter and a sequence of R-U5 long terminal repeat (LTP) HTLV-1, and has promoter activity, 10 and 100 times previsouly pSR 31-7. Transfection of cells COS-1 was carried out by electroporation using the device for introduction of a gene GTE-1 (Shimadzu).

COS-1 cells were cultured to polyplasdone over the entire surface of the bottom seven 150 cm3-flasks, each of which contained 25 ml of DMEM (containing 10% fetal calf serum). Then the culture was collected and each of them was treated with 3 ml of a solution of Trypsin-EDTA (10 x the solution then was left at room temperature as long as the cells are not separated. Then added 1 ml of inactivated fetal calf serum and 9 ml of fresh solution of trypsin-EDTA and the cells were collected by centrifugation. The collected cells twice washed with PBS (-) buffer and suspended in PBS (-) buffer to achieve a density of 6 to 107cells/ml.

In addition, the method using cesium chloride was obtained plasmid DNA was diluted to a concentration of 200 μg/ml in PBS (-) buffer.

20 µl of each of the above preparations of cells and plasmids were mixed and the resulting mixture was placed in a chamber containing electrodes spaced from each other at a distance of 2 mm, then the mixture was subjected to two pulses 600 B at time intervals of 1 sec and will continue to cells and DNA, inside, was mixed with 10 ml of DMEM containing 10% fetal calf serum. Then this mixture was transferred into Petri dishes and cultured overnight at 37oC in 5% CO2-the atmosphere. The next day, the culture supernatant was discarded, and the cells were washed in serum-free DMEM, suspended in 10 ml DMEM and cultured for 3 days at 37oC. after this time, the cell supernatant was collected.

The culture supernatant was also collected from the negative control. The plasmid used for the negative control, namely plasmid pcDL-SR 296, did not contain cDNA inserts, but otherwise it was received as well as testing culture.

1 ml of each culture supernatant and negative control and the test culture was separately treated in the following way. First, the supernatant was treated trichlorosucrose acid (TCA) to precipitate the protein and the resulting precipitate was collected by centrifugation. This precipitate was washed in ice-acetone, and dried by air, and then was dissolved in LTO-SDS page buffer for sample containing 2-mercaptoethanol. Then spent LTO-SDS page electrophoresis on a 12% gel under reducing conditions.

P is a number of specific bands (molecular weight 60,000) from the culture supernatant of the test sample were stained.

Because the molecular mass of the polypeptide encoded in the pSR 31, amounts to about 60,000, and because, based on their amino acid sequences, it is unlikely that, as posttranscriptional modification helped to add sharidny side chains, it is possible to conclude that these few specific bands pertain to the polypeptide encoded by cDNA pSR 31-7.

Example 12

Getting vysokoaktivnye plasmids to cells COS-1

The following stage was carried out in order to ensure that some specific 60 kDa-bands identified in example 11, the same polypeptide encoded by the insert SR 31-7. It would also be desirable to determine the N-terminal amino acid sequence of this polypeptide. In line with this was the resulting clone, which were coded six additional His residues to the C-Terminus of the polypeptide encoded by the pSR 31-7-insert located directly before the stop codon. His-tag residues have a high degree of affinity for Ni2+and therefore would be desirable to produce a polypeptide containing a his-tag gossamer (6 x His), which could be purified using affinity column loaded Ni2+.

oC, and then to implement annealing was additionally heated for another 30 min at 37oC. After this, the ends were fosforilirovanii using T4 polynucleotide-kinase.

The obtained double-stranded (DC) fragment ligated in pUCKM31-7 using T4 DNA ligase, and the specified pUCKM31-7 pre-digested by the enzyme Eco47III. This design is shown in Fig. 10. Using calcicludine method and the DNA was used to transform DH5 E. coli and the resulting transformed strains were selected and were skanirovali, resulting received pUCKM31-7His. By analyzing the corresponding sequence of bases specified pUCKM31-7His, in which was embedded fragment, it was found that in this part of the pUCKM31-7His any abnormalities are absent.

By sublimirovanny insert pUCKM31-7His in pcDL-SR 296 was obtained vysokoagressivnyh plasmid for COS-1 cells.

pUCKM31-7His was digested with the enzymes XbaI and HindIII, the resulting fragments were purified and the ends of the fragments were small mistake using 1 unit of fragment maple and the presence of 2 nm dATP, 2 mm dCTP, 2 mm dGTP, 2 mm dTTP, 50 mm Tris-HCl (pH of 7.2), 10 mm MgSO4, 0.1 mm of dithiothreitol and 50 μg/ml BSA.

Example 13

Purification and analysis of N-terminal amino acid sequence,

600 ml of the supernatant obtained in example 12 were subjected to dialysis against 17 volumes of buffer for dialysis for 15 hours at 4oC. Then the buffer was replaced with another 17 volumes of buffer for dialysis after dialysis was continued for another 4 hours at 4oC.

Cialisovernight the drug was subjected to affinity chromatography using FPLC (Liquid Express chromatography of proteins - Pharmacia) under the following conditions:

Column: 20 ml of ProBond resinTM(Invitrogen), loaded in a XK16/20 (2.0 x 20 cm, Pharmacia)

Eluting buffer

A) 20 nm phosphate buffer (pH 7.8), containing 200 mm imidazole, 0.5 M NaCl

B) 20 mm phosphate buffer (pH 7.8), SODERZhANIYa elution: after collecting 4 fractions eluting buffer (A) was collected 16 fractions eluting buffer (B) and these fractions were numbered from 1 to 20.

From each of the obtained fractions was taking 300 μl of the sample and each sample was separately treated with TCA, after which the precipitate was subjected LTO-PAG-electrophoresis using a 12.5% gel in terms of recovery, as described above. By silver staining was detected three bands centered fraction N 10. The existence of three bands indicated that the pSR 31-7His-insert encodes a polypeptide having 3 sequences of different lengths with different N-terminal sequences.

The remaining fractions 7-14 concentrated by TCA-precipitation and the precipitate was subjected LTO-PAG-electrophoresis using a 10% gel under reducing conditions. Then the protein bands transferred from the polyacrylamide gel to polyvinylidenedifluoride (PVDF) membrane (ProBlotTM, Applied Biosystems) using the device to transfer the gel onto the membrane (Marisol, KS-8441) operating at 9 B, in the presence of buffer to transfer (0,02% LTOs, 20% methanol, 25 mm Tris-borate (pH 9,5)), for 2.5 hours at 4oC.

After that, the membrane was stained with 0.2% Naftowy dark blue (Sigma), three bands corresponding to the pre-identified bands were cut out of the membrane, and then using a gas-phase Balimela second largest apparent molecular weight (mol. wt. about 60,000), had the following sequence:

Val-Val-Phe-Val-Lys-Gln (amino acids NN 1-6 sequence ID No. 12).

These six amino acids corresponded to the first six amino acids of the ORF of clone 31-7, and corresponded to the sequence of six amino acids, starting with 24 amino acids (VAl) from N-Terminus of the precursor polypeptide encoded by cDNA-insert pSR 31-7H and pSR 31-7. In accordance with this, the deletion of amino acids 1-23 of the N-Terminus of the precursor should lead to the release of the Mature form of the protein, starting with Val-balance.

Example 14

The definition of restorative activity

i) Construction of expression vector of

Polypeptide, purified as described in the previous examples, could be obtained only in extremely small quantities, as it was expressed from COS-1 cells. Therefore, this polypeptide may not be used for other purposes, for example for the analysis activity. Accordingly it would be desirable to find a means of expression of the polypeptide encoded by cDNA-insert pSR 31-7, in the alternative host, which would produce the polypeptide in quantities sufficient for cleaning and analysis. To achieve this goal was carried out by following and cleansed, but the end was a small mistake with the set to break the DNA. Then the fragment was digested with the enzyme XbaI.

Expressing the vector pMAL-c (Guan, C. et al. (1987) Gene 67, 21-30) were digested by enzymes XoaI and StuI, and then the above XbaI-modified HindII fragment ligated into this restrictively plasmid using T4 DNA ligase. The resulting design is shown in Fig. 11. This construction is then used for transformation of UNIT-1 of E. coli and the resulting AmpPthe transformants were selected and were skanirovali. After this selected strain, in which the direction of cDNA transcription was identical to the direction of the promoter, and thus obtained plasmid was designated pMAL31-7.

II) Expression and purification of the hybrid protein.

Seed culture of E. coli containing pMAL31-7, was obtained by culturing overnight, shaking at this at 37oC in 3 ml LB-medium containing 50 μg/ml ampicillin. The next day, 1 ml of seed culture was added to 100 ml of fresh culture of LB-medium containing 50 μg/ml of ampicillin, and cultured with shaking and at 37oC up until OP600Nmreaches the value of 0.5. At this stage, the culture was added IPTG to a final concentration of 0.1 mm, then ku is the tier of the overnight culture was isolated from bacterial cells by centrifugation at 6500 rpm and at 4oC for 20 minutes. The precipitate suspended in 10 ml of column buffer, and the cells in this suspension disintegrable by ultrasonic treatment. Whole cells and cell fragments were removed by centrifugation for 30 minutes at 8800 rpm and at 0oC, after which the soluble protein fraction was isolated in the form of a supernatant. 1 ml of this soluble fraction was subjected to chromatography on a column with amylose resin (New England Biolabs).

Eluting buffer for chromatography were obtained by adding maltose to 10 ml of column buffer to a final concentration of 10 mm.

Sample negative control was also chromatographically. This negative control was obtained in accordance with the same procedure, except that the vector pMAL-c did not contain any cDNA insert. Then were analyzed by restoring the activity of the protein in chromatographic samples.

iii) Determination of restoring activity

The definition of restorative activity carried out in the cuvette (SARSTEDT, 10 x 4 x 45 mm) using dichlorophenol-indophenol (DCIP) and oxidized glutathione.

a) Definition of the healing activity using DCIP

90,4 mcg (as b is obtained from (11) was separately mixed with 1 ml of 50 μm DCIP (Sigma). Then for each of the samples was added 15 μl of 1 mm NADPH (Boehringer-Mannheim) and constantly monitored OP600Nmand OPNm. The resulting decrease in absorbance at both wavelengths is illustrated in Fig. 12, where it is seen that only pMAL31-7-the sample contains a factor that restores DCIP.

b) Determination of the healing activity using oxidized glutathione

15 ml of 10 mm oxidized glutathione (Boerihger-Mannheim) was added to 90,4 μg of each of the chromatographic samples obtained as described in (11), and pre-loaded in a separate cell. To each cuvette was added 15 μl of 1 mm NADPH and watched the change in optical density at 340 nm (OD340) over time. The results are presented in Fig. 13, which shows that the only protein from pMAL31 model has the ability to regenerate oxidized glutathione. According to the observations, it was found that in the case of oxidized glutathione is absent, NADPH absorption does not occur, and on this basis it was concluded that protein from pMAL31-7-sample can restore oxidized glutathione only in the presence of NADPH.

Example 15

Purification and analysis of N-terminal amino acid sequence,

IP is within three types of N-ends.

In a separate experiment, to obtain a preparation of polyclonal antibodies against KM31-7-unit conducted immunization of rabbits with the use of a hybrid protein isolated from E. coli transformed pMAL31-7. Then using this polyclonal antibody was performed by Western blotting, in which these three types of bands were also detected in the serum-free culture supernatant obtained from COS-1 cells transfected with pSR 31-7. These results are similar to results obtained in example 13.

In accordance with this COS-1 cells were transfected with pSR 31-7 in order to obtain a large volume of serum-free culture supernatant for purification and analysis of N-terminal sequence KM31-7.

COS-1 cells were transferrable pSR 31-7 and were cultured for 3 days in 150 mm Petri dishes, each of which contained 30 ml of DMEM. After this time the culture supernatant was collected and in each Cup was added 30 ml of fresh medium, and then the cultivation was continued for another 3 days. Then the culture supernatant was again collected. In example 11 were described other options transfection and cultivation, but regardless it was cultivated 199 cups.

inogo supernatant, then deliberately overnight against 10 mm Tris-HCl (pH 9,0). Received diarizonae drugs eight times subjected to ion exchange chromatography using FPLC (Pharmacia) under the following conditions:

Column: 20 ml DEAE-sepharose to Express chromatography (Pharmacia) loaded in a XK16/20 ( 2.0 x 29 cm, Pharmacia)

Eluting buffers:

A) 10 mm Tris-HCl (pH 9,0)

B) 10 mm Tris-HCl (pH 9,0) and 0.5 M NaCl

Flow rate: 1 ml/min

The solution fractions: 3 ml/vial

The conditions of elution: changing the eluting buffer A on an eluting buffer B in a linear concentration gradient within 60 minutes.

These fractions, erwerbende at each NaCl concentration from 0.1 M to 0.4 M, was collected and combined, and then during the night were dialyzed against buffer for dialysis containing 0.1 M Tris-HCl, 5 mm EDTA (pH 7.6) and 1 mm 2-mercaptoethanol. Cialisovernight the drug was subjected to affinity chromatography using 2', 5', -ADP-Sepharose 4B (Pharmacia) under the following conditions:

Column: 20 ml of 2',5'-ADP-Sepharose loaded in a XK16/20 ( 2.0 x 20 cm, Pharmacia).

A) 0.1 M Tris-HCl, 5 mm EDTA (pH of 7.6), 1 mm 2-mercaptoethanol

B) 0.1 M Tris-HCl, 5 mm EDTA (pH of 7.6), 1 mm 2-mercaptoethanol, 10 mm NADPH.

Flow rate: 0.5 ml/min

The solution fractions: 2 ml/vial
is 120 minutes.

100 μl-aliquots from each of the obtained fractions was besieged with TCA, and the sediments were subjected to DSM-PAG-electrophoresis using 12.5% gel in reducing conditions.

After electrophoresis the gel was stained with silver for the detection of protein bands. Based on the fraction N 11, were obtained three bands.

All other fractions N 11 - N 14 then concentrated by TCA precipitation and the precipitate was subjected LTO-PAG-electrophoresis on a 12.5% gel in terms of recovery. After electrophoresis, protein was transferred from the gel onto PVDF-membrane Problot (Applied Biosystems). After transfer of the protein to the membrane of the membrane were stained with 0.2% Naftowy blue-black and three protein bands were cut out. Analysis of the N-terminal sequence was performed using gas-phase protein sequencing machine.

It was found that the N-end of the band, apparently, with the lowest molecular weight of the three types had the sequence Lys-Leu-Leu-Lys-Met. These five amino acids correspond to five amino acids, beginning with the 49th amino acids from the N-terminal sequence of the polypeptide encoded by cDNA-insert pSR 31-7. In line with this it was concluded that the cleavage of the peptide in the 48th balance leads to the formation of against protein KM31-7

a) Obtaining antigenic protein

Seed culture of E. coli containing pMAL31-7, was obtained by culturing loop cells in 3 ml LB-medium containing 50 μg/ml antipilling, when stahovanie and at 37oC during the night. 1 ml of the obtained seed culture was inoculable in 100 ml of fresh LB-medium containing 50 μg/ml of ampicillin, and cultured with shaking and at 37oC up until OP600Nmhe reached to 0.5. At this stage the culture broth was added IPTG to a final concentration of 0.1 mm, and then was cultured and was dissolved by shaking and at 37oC during the night.

From the obtained overnight culture was isolated cells by centrifugation for 20 minutes at 6500 rpm and at 4oC, and the residue suspended in 10 ml of column buffer. Cells in the resulting suspension getintegervalue using ultrasonic disintegrator, and the resulting liquid was centrifuged for 30 minutes at 8000 rpm and at 0oC. the resulting supernatant containing the soluble protein fraction.

This soluble protein fraction was subjected to chromatography on a column with 1 ml amylose resin. Elution was performed using a 10 ml column buffer containing 10 mustve antigen.

b) Receiving spleen cells from immunized mice

2 ml of complete adjuvant's adjuvant was added to 2 ml of antigen (equivalent to 200 μg), purified as described above in stage (a), in the form of an emulsion. This emulsion was dissolved in 5 ml of the cylinder of the syringe, equipped with a glass piston, and then used for immunization of 8-week-old male BALB/c mice by subcutaneous injection.

In subsequent cycles of immunization was carried out by the same procedure as that used for the first immunization, except that in this case, the antigen was administered in incomplete Freund's adjuvant. Immunization was carried out four times, with each injection was done approximately every 2 weeks.

Since the second immunization, directly turn immunization in mice took the blood from the venous plexus of the fundus and using enzyme-linked immunosorbent assay (ELISA) to determine the titer of antibodies to KM31-7 in the serum.

Solid phase ELISA antibodies against KM31-7

150-200 microlitres (corresponding to approximately 200 ng of the hybrid protein) serum-free supernatant obtained from COS-1 cells transfected with pSR / 31-7, were placed in each well of 96-well ELISA tablet (Costar) and used in casestudy day the tablet was washed 3 times with 0.1% tween 20/phosphate-buffer solution (0.1% tween-20/PBS), 0 and to each well was added 100 μl of BSA, increased to 10 μg/ml of a phosphate buffer solution, after which the tablet was left for 1 hour at room temperature.

After this time the tablet was washed three times with 0.1% tween-20/PBS. Then to each well was added 30-100 ál of primary antibody in the form of a serially diluted sample (e.g., mouse serum, supernatant from a culture of hybridoma or monoclonal antibody), after which the tablet was left for 1 hour at room temperature.

After that, the tablet again three times washed with 0.1% tween /PBS and each well was added 100 μl of the second antibody. The second antibody was obtained in the form of a solution with a 3000-fold dilution of the complex "goat antimachine IgG-peroxidase" (Amersham) or in the form of a solution with a 3000-fold dilution of the complex "goat antimachine IgG-alkaline phosphatase" (B10-RAD). Then this tablet was left for two hours at room temperature.

After this time the tablet again washed three times with 0.1% tween-20/PBS, and then to each well was added 100 μl of either peroxidase substrate solution (B10-RAD, Buffer Substrade Kit ABTC), or 10% diethanolamine containing 0,001% para-nitrophenylphosphate solution. Then the tablet was left for 15 - 30 minutes at Kera (B10-RAD) can be calculated titer of antibodies.

(c) Obtaining cells of mouse myeloma

8-Azaguanine-resistant cells of mouse myeloma P3-X63-Ag8.653 (653) (ATCC N CPD-1580) was cultured in normal medium (full GIT-Wednesday) to get at least 2 of 107cells.

(d) Obtaining hybridoma

1,4 108the spleen cells of immunized mice obtained after immunization carried out according to the scheme described above in (b), thoroughly washed with DMEM-medium (Nissui Pharmaceutical). The washed cells were mixed with 1.5 107cells of mouse myeloma P3-X63-Ag8.653 (653), obtained as described above in stage (c), and the mixture was centrifuged for 69 minutes at 800 rpm

The group of cells consisting of spleen cells and cells P3-X63-Ag8.653 (653), was collected in the form of sediment and getintegervalue. Previously received a 50% solution of polyethylene glycol 4000 (peg 4000 N) and DMEM and this solution was poured into the disintegrated cells at the rate of 2 ml/min for 1 min, stirring. Then in a similar way (i.e., for 1 min at a speed of 2 ml/min) to the cell preparation was added to DMEM. This procedure was repeated one more time for the polyethylene solution and DMEM solution. And finally, for 3 minutes was gradually added 16 ml of DMEM. Then the glue suspended in 35 ml of complete GIT environment, containing 5-10 ng/ml murine 1D-6.

(e) Screening the hybrid

100 μl of the suspension obtained as described above in stage (b) was added to each well of 96-hole tablet (Sumimoto Bakelite), and then cultured in a 7.0% CO2-incubator at 37oC. After 7 days after incubation each well was added 50 μl of HAT. After incubation for 4 days to each well was added 50 μl of HAT. The tablet then incubated for another 3 days. After this time from the wells in which growth was observed colonies of hybrid cells were selected a sample of culture supernatant using enzyme-linked immunosorbent assay (ELISA described in paragraph(o)) were analyzed titer antibodies against KM31-7. Selected for the sample environment immediately replaced the HT environment.

(f) Cloning

Cloning of cells taken from the wells that tested positive were performed three times using the analysis method of limiting dilution. Clones that were found as determined by observation, constant antibody titer were selected for use as cell lines hybridoma producing monoclonal antibody against KM31-7. In particular, at this stage, ELISA was carried out not Tolga supernatant, obtained from COS-1 cells transfected pcDL-pSR 296, to obtain a solid phase. In accordance with this cell line, which are reactive in the case of the first ELISA, but are not reactive in the case of the control ELISA were selected for cloning.

(o) Purification of monoclonal antibodies

Culture supernatant from cell line hybridoma producing monoclonal antibodies against KM37-7, were collected, filter sterilized 0,22 ál of Millipore filter, and then the antibody was purified using MabTrap GII (Pharmacia).

(h) Analysis of monoclonal antibodies

1) Antigenic specificity of monoclonal antibodies

By thus conducted using serum-free culture supernatant obtained from COS-1 cells transfected with pSR 31-7, it was found that monoclonal antibodies had specificity with respect to protein KM31-7.

2) Classification of monoclonal antibodies

This test was performed using kit ittipiboon mouse monoclonal antibodies (Amersham), and the result of this test it was found that this antibody belongs to the IgG1 subclass.

Example 17

Selection procedural, described above in example 16 (h) (1). This test was also repeated using antibodies and serum-free supernatant obtained from COS-1 cells transfected pcDL-pSR 296.

1,4 µg monoclonal antibody was added to 1.7 ml each of serum-free supernatants and left for 1 hour at room temperature to perform the reaction, followed by centrifugation in a microcentrifuge 2.2 ml-tubes at 20 rpm Control was performed using serum-free supernatant obtained from COS-1 cells transfected with pSR 31-7, but without the addition of monoclonal antibodies.

30 µl of protein G-sepharose Fast Flow (Pharmacia), pre-washed with 0.1% tween-20/PBS, was added to each tube for absorption of the antibodies, and then centrifuged for 30 minutes at a speed of 20 rpm at room temperature.

After this time, each mixture was centrifuged for a few seconds in microcentrifuge at 10,000 rpm, after which the supernatant was carefully removed so that it does not lead to any loss of the segment. Then precipitation separately washed with 0.1% tween-20/PBS, and then subjected to microcentrifuge and washed 5 times in the same way.aptechnoe. Each suspension was heated for 2 minutes at 90oC, and then subjected LTO-PAG-electrophoresis using 12.5% gel in reducing conditions. After electrophoresis, the product was transferred from the polyacrylamide gel to a nitrocellulose membrane (B10-RAD). Western blotting was performed using polyclonal antibodies against KM31-7, as described in example 1 (a), and the result of this analysis, it was found that monoclonal antibody against KM31 specifically besieging KM31-protein from the serum-free culture supernatant CO-1/pSR 31-7.

Example 18

Getting CYVV-Nla/KM31-7-hybrid protein

For producing protein KM31-7 using CYVV-Nla protease, it is necessary that the 3'-end of the Nla gene was Legerova with DNA protein KM31 in the same reading frame. This was carried out by the following two-step procedure:

1) Introduction to the 3'side chain (SmaI-XbaI, 1005 p. O.) KM31-7-cDNA in pKSUN9

In order to obtain the SmaI-XbaI fragment (1006 p. O.), containing the 3'end KM32-7-cDNA, 7 µg pSR 31-7 plasmid, DNA was digested restricteduse enzymes SmaI and XbaI and the resulting fragment was isolated and purified using the GENECLEAN 11 (Funakoshi, Japan) with the use of 0.8% agar gel.

Meanwhile, 5 μg DNA plasmid pKSUN 9 similar obrazana phosphatase of E. coli C 75, Takara Shuso, Japan). Received dephosphorylating linearized DNA ligated with SmaI-XbaI-KM-31 - fragment set for ligating (Takara Shuso), and the resulting construct was used to transform strain JM109 E. coli. Transformants were selected and were skanirovali, resulting received clone pU1a31 - 7X containing SmaI-XbaI fragment.

11) Stitching Nla protease and KM31-7

To bind the C-terminal sequence N1a N-terminal sequence of subtype KM31-7 with malinowy N-terminal residue in the same reading frame, designed four types of PCR primers using a DNA synthesizer (Perkin-Elmer Japan Applied Biosystems Model 392). These primers had the following sequences:

5' GGT CAG CAC AAA TTT CCA 3' SEQ 1 N 15 (1)

5' AAA CAC AAC TTG GAA TGA ACA ATT 3' SEQ 1 N 16 (2)

5' TCA TTC CAA GTT GTG TTT GTG AAA 3' SEQ 1 N 17 (3)

5' CAT AGG ATG CTC CAA CAA 3' SEQ 1 N 18 (4)

The first cycle of PCR was performed using 1 μg DNA plasmid pK SUN9 as a matrix. To the reaction solution was added successively with 100 PM of each primers (1) and (2) and 1/10 volume of 10x - concentrated reaction buffer solution for the polymerase Tag, and then 5 units of Tag polymerase (Takara Shuzo). PCP-the reaction was carried out according to the following scheme: first, the reaction was carried out at P>C, followed by treatment with 72oC for 10 minutes. After PCR reaction amplificatory DNA product was subjected to electrophoresis on 8% polyacrylamide gel. Strips of the gel containing DNA, identified by staining with ethidium bromide and were crushed. Then, for each of the fragmented stripes were added 300 μl of buffer for elution (0.5 ammonium acetate, 1 mm EDTA, pH 8.0) and incubated overnight at 37oC. After centrifugation was obtained supernatant containing purified and amplified DNA.

Then PCR was performed again in a similar way, except that this time was used as a matrix 1 MGK DNA plasmids p CKM31, and as primers the primers used were (3) and (4) have been purified DNA, as described above.

Amplificatory fragment from the first PCR reaction contained the sequence encoding residues N-terminal KM31-7 protein, since Val-Val-Phe and forth ( for 31 p. O.) and located "above" from the XhoI site of the Nla. Amplificatory fragment from the second PCP-reactions contained the sequence coding for Asn-Cys-Ser-Phe-Gln - from the C-end of the Nla and forth (over 32 p. O.) and located "below" from the SmaI site KM31-7-cDNA.

In accordance with uh what was alocale hybridisierung thread, consisting of 9 p. O. 3 - terminal Nla and 15 p. O. sequence that encodes the desired end KM31-7. Thus, it is possible to generate DNA sequence containing this part as a link.

On this basis, was carried out the second round of PCR in exactly the same way as the first, then the received amplificatory fragment was isolated from gel.

III) the Introduction of Nla/KM31-7 DNA in PUla31-7X

5 µg pUla31-7 X-plasmid DNA obtained in paragraph (1), was digested by enzymes XhbI and SmaI and the resulting DNA was dephosphorylated by treatment with bovine alkaline phosphatase. The PCR product obtained in part (II) was also digested with the enzymes XhoI and SmaI, and then the resulting fragment ligated with digested dephosphorylating pNla31-7SX using a set of ligation. The resulting structure was used for transformation of strain J M109 E. coli.

Then AmpRthe transformants were selected and were skanirovali.

The screening was carried out by digesting enzyme XhoI, followed by electrophoresis. After that was selected clones, with only a strip of 8.0 p. O. Then the plasmids of selected clones was digested with the enzyme HindIII and was again subjected to electrophoresis. The selected plasmid was 330 p. O.-floor"ptx2">

Then determined the nucleotide sequence of clone pNla31-7V, as a result it was found that the sequence encoding the Nla and KM31-7, ligated with preservation of the ORF, and the necessary fissionable G1n-Na1-sequence is located between the Nla and KM31-7 protein.

IV) producing the protein KM31-7

Western blot analysis confirmed that pNla31-7V functions in E. coli and the protein KM31-7 can be expressed constructed recombinant genome.

Seed culture of E. coli containing plasmid pNla31-7, were cultured overnight with shaking in 3 ml of LB-medium containing 50 μg/ml ampicillin. Then 1 ml of this seed culture was added to 100 ml of fresh LB-medium containing 50 μg/ml of ampicillin, and cultured at 38oC and with shaking until, while op600Nmreached a value of 1.0. At this stage the culture broth was added 1PTG to a final concentration of 1 mm, after which the culture was incubated at 28oC and with shaking for a further two nights.

After this time, 1 ml of culture was transferred into a centrifuge tube and was centrifuged at 15,000 rpm for 5 minutes. Then the supernatant was removed, and the residue was mixed with 300 µl of sterile water and 300 m of the internal inclusions. The resulting suspension was heated for 2 minutes at 95oC, and then 10 μl of this suspension was subjected LTO-PAG-electrophoresis on an 8% gel under reducing conditions.

After electrophoresis, protein was transferred from the gel to nitrocellulose membrane. This procedure was carried out by combining the gel membrane with another 2.5-hour incubation at 4oC in the presence of buffer solution for broadcast (25 mm Tris-HCl, 1.4% of glycine and 20% methanol) at 19 B using the device to transfer the gel onto the membrane (Marisol Japan).

Then, the nitrocellulose membrane was washed with 20 ml PBS-T, then for 1 hour was carried out by blocking in 20 ml of PBS-T containing 5% separated milk (Snow Brand Co., Ltd). After this time, the membrane was washed two parties 20 ml PBS-T and then left for 90 min in 20 ml of PBS-T containing 1 μl of 100 x diluted in sterile water rabbit Mab serum ("first" antibody) against KM31-7, to perform the reaction. Thereafter, the nitrocellulose film was washed once for 15 minutes and twice for 5 minutes each time with 20 ml PBS-T).

Then the washed membrane was placed in a bath with h-divorced and labeled with peroxidase goat antibody against rabbit, immuo time the membrane was washed with 20 ml PBS-T and transferred to a bath with a reagent for ECI-detection (Amersham), and with autoradiography discovered bands reacting with the antibody against KM31-7.

Was performed by Western blotting, in which it was found that molecular weight of the band is around 60,000. This band has found the same mobility as the protein having the second most large molecular masses of the three KM31-7 protein detected in serum-free culture supernatant obtained by transfection of cells COS-1 clone pSR 31-7 used as control.

Environment

x M phosphate buffer

x M solution of Na2HPO4brought to the desired pH value using x M solution of NaH2PO4.

Buffer for inoculation

0.1 M Tris-HCl buffer, pH 7.0 0.05 M EDTA, 1% 2-mercaptoethanol.

Buffer for extraction

0.1 M Tris-HCl buffer, 0.05 M EDTA, 1% 2-mercaptoethanol, pH 7.0.

The solution for degradation

200 mm ammonium carbonate, 2% LTOs, 2 mm EDTA, 400 μg/ml of bentonite and 20 μg/ml protease K (pH 9,0).

1 x SSc

0.15 m NaCl, 0.015 G M trinatriytsitrat, pH 7.0.

Liquid LB-medium

10 g Bacto-Tryptone (Difco) 5 g Bacto-yeast extract (Difco) and 5 g of sodium chloride is brought up to a volume of 1 l using distilled water.

Tr is="ptx2">

Buffer for lysis

0.17 g of sucrose, 250 μl of 1M Tris-HCl buffer (pH 8.0) and 200 ál of 0.5 M EDTA (pH 8.0) brought up to a volume of 20 ml using bidistilled water.

Alkali-LTO-solution

0.2 M sodium hydroxide, 1% - ordinator.

TBE solution

100 mm Tris, 100 mm boric acid, 1 mm EDTA.

The solution to denature

1.5 M sodium chloride, 0.5 M sodium hydroxide.

Buffer to neutralize

0.5 M Tris, 3 M sodium chloride (pH 7,4).

50 x denhardt's Solution

1% polyvinylpyrrolidone, 1% bovine serum albumin and 1% Ficoll 400. This solution was diluted with bidistilled water, respectively, and received the desired concentration.

5x Buffer to denature

125 μl of 1 M glycine (pH 9,0), 25 μl of 1 M magnesium chloride and 850 µl bidistilled water.

5x Buffer for labeling

25 ál of 1 M Tris-HCl buffer (pH 7,9), 5 μl of 1 M magnesium chloride, and 2.5 μl of 1 M dithiothreitol and 9.2 ál of bidistilled water.

10 x M9 Salt solution

0,145 M centripetal, 0,172 M potassium dihydrophosphate, 0,187 M ammonium chloride and 0.137 M sodium chloride (pH 7.0).

M9 Minimal agar

10 ml 10x M9 salt solution, 100 ál of 1 M magnesium sulfate, 1 ml of 20% glucose, 50 μl of 1% cleaners containing hydrochloride salt of thiamine 1 ml of 0.01 M chloroplast directly after adding 50 ml of 3% Bacto-agar.

Liquid SOB-medium

10 g Bacto-Tryptone, 2.5 g Bacto-yeast extract, 100 ml of 5 M sodium chloride and 125 μl of 1 M potassium chloride was altered and brought up to a volume of 500 ml using distilled water. After treatment in the autoclave to the mixture was added 5 ml of 1 M magnesium chloride and 5 ml of 1 M magnesium sulfate.

TFB1 buffer

5 ml of 1 M 2-(N-Morpholino)econsultancy acid (MES) (brought to pH 6,2 using 1 H7 HCl), 6,045 g of rubidium chloride, 0,735 g of dihydrate of calcium chloride and 4.94 g of tetrahydrate of manganese chloride was stirred, brought to pH 5.8 using glacial acetic acid, and then the resulting mixture was brought up to a volume of 500 ml using bidistilled water and sterilized by filtration.

TFB2 buffer

1 ml of 1 M 2-(N-morpholino)propanesulfonic acid (MOPS), 1,102 g bihydrate of calcium chloride, 0.12 rubidium chloride and 15 ml of glycerol were mixed and brought to a pH of 6.5 using glacial acetic acid, after which the resulting mixture was brought up to a volume of 100 ml using bidistilled water and sterilized by filtration.

Liquid SOC-medium

5 ml of liquid SOB environment, 90 μl 20% glucose.

2x UT-environment

16 g Bacto-Tryptone, 5 g Bacto-yeast extract and 5 g of chloride

80 mm dinatriumfosfaatti, 20 mm sodium phosphate, 100 mm sodium chloride and 0.1% Tween 20.

TBS-T environment

4 g of sodium chloride, 0.1 g of potassium phosphate, 1.45 g of dodecahedral of sodium phosphate, 0.1 g KCl, 0.1 g of sodium azide was brought to a volume of 1 l using bidistilled water (pH 7,4).

The solution of alkali phosphatase substrate

0.01% of p-nitrophenylphosphate dissolved in 10% aqueous solution of diethanolamine and brought up to a pH of 9.8 with hydrochloric acid.

Environment A

D MEM (Wednesday Needle, modified by way of Dulbecco, and containing 4.5 g/l glucose), 10% inactivated fetal bovine serum (manufactured by Hyclone company) and 10 mm HEPES (pH 7,2).

Environment B

D MEM (containing 4.5 g/l glucose), 10 mm HEPES (pH 7,2) 3% inactivated fetal bovine serum, 5 μg/ml bovine insulin (manufactured by Sigma) of 8 µg/ml d-Biotin (manufactured by Sigma), 4 mg/ml Pantothenic acid (manufactured by Sigma), 1.0 mm dexamethasone (manufactured by Sigma) and 0.5 mm isobutylmethylxanthine (manufactured by Aldrich).

C

G MEM (containing 4.5 g/l glucose) containing 5% inactivated fetal bovine serum, 10 mm HEPES (pH of 7.2), and 100 ng/ml bovine insulin.

Environment D

D MEM (containing 4.5 is in sodium (manufactured by Novo Industry Co.).

The solution LPL substrate

13 mm glycerol-three 9,10 (n)-3N oleate (51,8 Kwkw./μm, manufactured by Amersham), 1.3 mg/ml diatrol-L - a - phosphatidylcholine (manufactured by Sigma Co.), 20 mg/ml of bovine serum albumin (manufactured by Sigma Co.), 135 mm Tris hydrochloride Tris-HCl, pH 8,1, manufactured by Sigma Co., of 16.5% (V/V) glycerol and 16.5% (V/V) inactivated fetal bovine serum.

A solution of guanidine thiocyanate

4 M guanidine thiocyanate, 1% Sarkosyl, 20 mm ethylenediaminetetraacetic acid (EDTA), 25 mm sodium citrate (pH 7.0), 100 mm 2-mercaptoethanol and 0.1% protivopennaya reagent A(Sigma).

Buffer for adsorption

0.5 m NaCl, 20 mm Tris-HCl (pH 7.5), 1 mm EDTA and 0.1% - ordinator.

An eluting solution

10 mm Tris-HCl (to 7.5), 1 mm EDTA and 0.05% ordinator.

The reaction solution reverse transcriptase example 2

50 ml of Tris-HCl (pH 8,3), 8 mm MgCl2, 30 mm KCl, 0.3 mm of dithiothreitol, 2 mm dATP, 2 mm dGTP, 2 mm dTTP, 10 µg [ -32P] dCTP and 1.4 μg of vector plasmid DNA (pcDV-1 c 3'-oligonucleotide (dT)-tail, Pharmacia).

The reaction solution terminal transferase

140 mm cacodylate potassium, 30 mm Tris-HCl (pH 6,8), 1 mm CoCl2, 0.5 mm baytrail, 0.2 ág of polynucleotide A and 100 mm dCTP.

Buffer for restriktsii

50 mm NaCl, 10 mm Tris-HCl (pH 7.5), 10 m is 2 and 100 mm dithiothreitol.

Pigment for electrophoresis

50% glycerol, 0.01 M dinatriumfosfaatti (pH 7.0) and 0.4% bromophenol blue.

1x TAE

0,014 M Tris-acetate and 0.001 M EDTA

1x SSCP

120 mm NaCl, 15 mm sodium citrate, 13 mm potassium phosphate and 1 mm EDTA.

The reaction solution for reverse transcriptase example 6

1x Buffer for the synthesis of the first chain, 5% sodium pyrophosphate, 100 units of ribonuclease inhibitor, 1 mm dATP, 1 mm dGTP, 1 mm dTTP, 0.5 to dCTP and 3.75 mg of oligonucleotide primers were obtained using Cloned cDNA (Amersham).

SM Buffer

100 mm NaCl, 8 mm MgSO47H2O, 50 mm Tris-HCl (pH 7.5) and 0.01% gelatin.

Buffer for analysis

20 mm phosphate buffer (PH 7.8) and 0.5 M NaCl.

Column buffer

10 mm Tris-HCl (pH of 7.4), 200 mm NaCl and 1 mm EDTA.

Buffer solution for Tag-polymerase reaction

500 mm Tris-HCl (pH 8,3), 500 mm KCl, 15 mm MgCl2, 100 mm dATP, 100 mm dCTP, 100 mm dGTP, 100 mm dTTP and mg/ml gelatin.

1. Polynucleotide sequence encoding a protein and characterized by the General structural formula

A-B-C,

where A is a sequence of nucleotides 10-1311 in SEQ ID NO;

B is a sequence to the target polypeptide.

2. Polynucleotide sequence under item 1, wherein C represents a sequence encoding a polypeptide CMS-7, which includes the sequence of amino acids 1 to 526 in SEQ ID No. 12, provided that the polypeptide encoded by the specified polynucleotide sequence, has the ability to restore dichloroindophenol and oxidized glutathione.

3. The polypeptide of nuclear inclusions, having a sequence represented by residues 4 to 437 in SEQ ID No. 2.

4. Protein encoded by the polynucleotide sequence under item 1.

5. Polynucleotide sequence encoding a polypeptide CMS-7, which represents the amino acid sequence of from 1 to 526 in SEQ ID No. 12, provided that the polypeptide encoded by the specified polynucleotide sequence, has the ability to restore dichloroindophenol and oxidized glutathione.

6. Polynucleotide sequence under item 5, characterized in that the coding sequence is a sequence of nucleotides 70 to 1647 indicated in SEQ ID No. 11.

7. Polypeptide CMS-7, representing a sequence of amino acids 1-526 in SEQ ID No. 12, to the battle sequence of amino acids 1-526 in SEQ ID No. 12, under item 7, having the property to recover the oxidized glutathione and dichloroindophenol.

9. Pharmaceutical composition, comprising a pharmaceutically active amount of a peptide according to p. 7, in combination with a pharmaceutically acceptable carrier for him.

10. Monoclonal antibody specifically interacts with the polypeptide CMS-7 and has the following characteristics: is produced by a strain of hybrid cultivated cells of Mus musculus MKM 150-2 FERM BP-5086; refers to the Ig isotype G1, can be used for purification of the polypeptide CMS-7.

11. The hybrid strain of cultured cells Mus musculus FERM BP-5086 used to obtain monoclonal antibodies, which specifically interacts with the polypeptide CMS-7.

Priority points:

13.07.94 on PP.1, 3, 4;

13.09.94 on PP.2, 5 - 9;

13.07.95 on PP.10 and 11.

 

Same patents:

The invention relates to medicine, in particular to Oncology and immunology, and for the treatment of b-cell lymphoma

The invention relates to the field of biochemistry, in particular to the new fused proteins, and can be used in the treatment of neoplastic diseases

The invention relates to the field of Virology, immunology and biotechnology, namely, hybridoma technology, and represents a new hybrid strain of cultured animal cells Mus musculus L. producing cell cultures and ascitic fluids monoclonal antibody (MAB) to the virus vesicular disease swine (WBS) strain T-75, which can be used for scientific research and preparation of diagnostics, prevention and treatment VBS

The invention relates to hybrid technology and can be used in veterinary medicine and medicine in the diagnosis of brucellosis

The invention relates to hybridoma technology and can be used in the diagnosis of hepatitis b

The invention relates to hybridoma technology and can be used in the diagnosis of hepatitis b

The invention relates to biotechnology, in particular genetic engineering, is a recombinant plasmid pCVA designed for the transcription of the genes of the ribozymes in the composition of sequences of virus-associated PHK (VA PHK) adenovirus birds FAV1 (CELO) in eukaryotic cells
Up!