Method for making recombinant heterocarpine

FIELD: genetic engineering.

SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.

EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.

9 cl, 6 ex

 

The invention relates to a method for producing recombinant heterocarpon.

Heterocarpon is a protein with anti-cancer properties, which were first described by the applicant in the patent application PCT WO 02/068461. This isolated protein has a molecular mass of about 90,9 kDa, contains fragments from the peptide sequence SEQ.ID.NO.1, SEQ.ID.NO.2 and SEQ.ID.NO.3 (see Appendix for the list of sequences) and can be obtained by extraction from the cells of the plant Pilocarpus heterophyllus, cultured in vitro in accordance with the description above of the application. However, the full sequence of this protein remained unknown up to the present time, as his clone was not implemented.

In the present application describes polynucleotide, which can serve as a seed for cloning heterocarpon, also described DNA encoding heterocarpon, mRNA corresponding to heterocarpon, the expression vectors containing the named mRNA, cell host transformed or transfetsirovannyh using these vectors and the method of obtaining recombinant heterocarpon.

Therefore, the first object of the invention is selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.8. Preferably specified selected polynucleotide presents polynucleotide sequence SEQ.ID.NO.8.

what bhakta invention is also " antisense polynucleotide, comprising a sequence complementary to the sequence specified selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.8. Preferably specified " antisense polynucleotide represented by a sequence, a complementary polynucleotide sequence SEQ.ID.NO.8.

The invention relates also to a selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.8 or one of the fragments of this sequence, with a specified polynucleotide is such that it encodes a polypeptide having at least immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation. Preferably specified selected polynucleotide presents polynucleotide sequence SEQ.ID.NO.8 or a fragment of this sequence.

On this basis, the invention applies in particular to the selected polynucleotide with nucleotide sequence SEQ.ID.NO.9 or to selected polynucleotide with a nucleotide sequence complementary to the nucleotide sequence SEQ.ID.NO.9.

Heterocarpon, or, in other words, a protein with sequence SEQ.ID.NO.10, is encoded by a fragment of polynucleotide with polynucleotide sequence is lnasty SEQ.ID.NO.8, located between the bases at positions 115 (the initiating ATG codon encoding methionine) and 2437 (stop codon UAA), i.e. the polynucleotide sequence SEQ.ID.NO.9.

The invention relates also to an expression vector containing the selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.8 or one of the fragments of this sequence, or a sequence complementary polynucleotide sequence SEQ.ID.NO.8 or one of the fragments of this sequence, and the polypeptide encoded by the specified selected polynucleotide has, at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation. Preferably the specified expression vector includes a polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9.

The invention relates also to the cell host transformed or transtitional specified expression vector.

The invention relates also to a selected polypeptide containing a polypeptide sequence SEQ.ID.NO.14 or one of its fragments, and the selected polypeptide has at least, immunological and/or biological activity of ha is Acterna for protein, binding human GHRH and is associated with modulation of cell proliferation. Preferably specified selected polypeptide represented by the polypeptide sequence SEQ.ID.NO.14 or one of its fragments. In particular, the polypeptide has the sequence SEQ.ID.NO.14.

The object of the invention is a monoclonal antibody or fragment carrying the binding of an antigen which specifically binds the selected polypeptide described above, in particular the object of the invention is a monoclonal antibody or fragment carrying the binding of an antigen which specifically binds the protein sequence SEQ.ID.NO.14, but does not bind the protein sequence SEQ.ID.NO.10.

The invention relates also to a selected polynucleotide containing

- polynucleotide sequence SEQ.ID.NO.8 or one of its fragments, or

- polynucleotide sequence SEQ.ID.NO.9 or one of its fragments, as a medicine,

moreover, the specified selected polynucleotide is such that it encodes a selected polypeptide having at least immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation.

Preferably, the separation of the hydrated polynucleotide represented by polynucleotides with polynucleotide sequence SEQ.ID.NO.8 or one of its fragments, or polynucleotides with polynucleotide sequence SEQ.ID.NO.9 or one of its fragments.

In particular, the selected polynucleotide represented by polynucleotides with polynucleotide sequence SEQ.ID.NO.8 or polynucleotides with polynucleotide sequence SEQ.ID.NO.9.

When this is specified, the selected polynucleotide used as a drug, preferably is a viral vector, while the viral vector may be selected, for example, from the group consisting of adenovirus, adeno-associated virus, retrovirus and poxvirus.

The invention relates also to a selected polypeptide containing a polypeptide sequence SEQ.ID.NO.14 or one of its fragments, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, as a medicinal product. Preferably specified selected polypeptide represented by the polypeptide sequence SEQ.ID.NO.14 or one of its fragments. In particular, the polypeptide has the sequence SEQ.ID.NO.14.

The invention relates to a monoclonal antibody or its fragment carrying the binding is nigena, which specifically binds the selected polypeptide containing the protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or one of its fragments or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments, as a drug, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation. Preferably specified monoclonal antibody or specified fragment carrying the binding with the antigen specifically binds the selected polypeptide represented by protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or one of its fragments or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments. Even more preferably the specified monoclonal antibody or specified fragment carrying the binding with the antigen specifically binds the selected polypeptide represented by protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 (in other words, a protein with n is a sequence of SEQ.ID.NO.10).

Another object of the invention is a pharmaceutical composition containing as active principle selected polynucleotide containing

- polynucleotide sequence SEQ.ID.NO.8 or one of its fragments, or

- polynucleotide sequence SEQ.ID.NO.9 or one of its fragments,

moreover, the specified selected polynucleotide is such that it encodes a selected polypeptide having at least immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation,

and pharmaceutically acceptable excipient or excipients.

Preferably the selected polynucleotide, which is an active early, represented by polynucleotides with polynucleotide sequence SEQ.ID.NO.8 or one of its fragments, or polynucleotides with polynucleotide sequence SEQ.ID.NO.9 or one of its fragments.

In particular, the selected polynucleotide, which is an active early, represented by polynucleotides with polynucleotide sequence SEQ.ID.NO.8 or polynucleotides with polynucleotide sequence SEQ.ID.NO.9.

The specified selected polynucleotide entered in the pharmaceutical composition according to the invention, preferably is a viral vector, while the VI is just the vector is chosen, for example, from the group consisting of adenovirus, adeno-associated virus, retrovirus and poxvirus.

The invention relates also to pharmaceutical compositions containing the selected polypeptide containing a polypeptide sequence SEQ.ID.NO.14 or one of its fragments, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein protein binding human GHRH and is associated with modulation of cell proliferation. Preferably specified selected polypeptide represented by the polypeptide sequence SEQ.ID.NO.14 or one of its fragments. In particular, the polypeptide has the sequence SEQ.ID.NO.14.

In addition, the invention relates to pharmaceutical compositions containing the monoclonal antibody or its fragment carrying the binding of an antigen which specifically binds the selected polypeptide containing at least a fragment of a protein encoded by a polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and associated the Yu with modulation of cell proliferation, while this composition includes a pharmaceutically acceptable excipient or excipients. Preferably specified pharmaceutical composition according to the invention is a composition comprising a monoclonal antibody or fragment carrying the binding of an antigen which specifically binds the selected polypeptide represented by protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or its fragment, or a sequence complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments.

In particular, the invention relates to pharmaceutical compositions containing the monoclonal antibody or its fragment carrying the binding of an antigen which specifically binds the protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 (in other words, a protein with sequence SEQ.ID.NO.10), and pharmaceutically acceptable excipient or excipients.

Another object of the invention is the use of selected polynucleotide containing

- polynucleotide sequence SEQ.ID.NO.8 or one of its fragments, or

- polynucleotide sequence SEQ.ID.NO.9 or one of its fragments,

moreover, the specified selected olignucleotides is that it encodes a selected polypeptide having at least immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation,

for obtaining a medicinal product intended for the treatment of proliferative diseases.

Preferably used are selected polynucleotide represented by polynucleotides with polynucleotide sequence SEQ.ID.NO.8 or one of its fragments or polynucleotides with polynucleotide sequence SEQ.ID.NO.9 or one of its fragments.

In particular, the selected polynucleotide represented by polynucleotides with polynucleotide sequence SEQ.ID.NO.8 or polynucleotides with polynucleotide sequence SEQ.ID.NO.9.

Specified used a dedicated polynucleotide preferably is a viral vector, and the viral vector is chosen, for example, from the group consisting of adenovirus, adeno-associated virus, retrovirus and poxvirus.

The present invention relates also to the use of the selected polypeptide containing a polypeptide sequence SEQ.ID.NO.14 or one of its fragments, and the selected polypeptide has at least, immunological and/or biological activity, the characteristic is kterou for protein, binding human GHRH and is associated with modulation of cell proliferation, for obtaining a medicinal product intended for the treatment of proliferative diseases. Preferably specified selected polypeptide represented by the polypeptide sequence SEQ.ID.NO.14 or one of its fragments. In particular, the polypeptide has the sequence SEQ.ID.NO.14.

Alternatively, according to the invention the monoclonal antibody or its fragment carrying the binding of an antigen which specifically binds the selected polypeptide containing the protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or one of its fragments or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, can be used to produce a medicinal product intended for the treatment of proliferative diseases. Preferably specified monoclonal antibody or specified fragment carrying the binding with the antigen specifically binds to the floor of the peptide, which is represented by the protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or one of its fragments or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments.

In particular, the monoclonal antibody or its fragment carrying the binding of an antigen which specifically binds the selected polypeptide encoded by the polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 (in other words, the monoclonal antibody or its fragment carrying the binding of an antigen which specifically binds the protein sequence SEQ.ID.NO.10), can be used to produce a medicinal product intended for the treatment of proliferative diseases.

In accordance with the above preferred applications of the proliferative disease to be treated with the polypeptide or polynucleotide described above, is cancer. In accordance with the more preferred variants of cancer belongs to the group, including prostate cancer, breast cancer, lung cancer (and in particular small cell lung cancer) and cancer of the rectum. Even more preferably the disease is cancer, breast cancer and melcochita the hydrated lung cancer.

In addition, the invention relates to a method of obtaining the selected polypeptide containing the protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13 or one of its fragments or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, while this method includes the following successive stages:

(a) culturing, under conditions suitable for expression of the specified polypeptide, the host cell transformed or transtitional expression vector comprising the selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13, a sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13 or one of the fragments of these sequences, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, and

(b) isolation of the polypeptide from the cell culture-economy is s.

Preferably this method of obtaining relates to the production of the selected polypeptide represented by protein encoded by the polynucleotide sequence SEQ.ID.NO.9 or one of its fragments or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or one of its fragments, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, while this method includes the following successive stages:

(a) culturing, under conditions suitable for expression of the specified polypeptide, the host cell transformed or transtitional expression vector comprising the selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13, a sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13 or one of the fragments of these sequences, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, and

(b) isolation of the polypeptide from the culture glue is OK-hosts.

In particular, the method relates to the production of the selected polypeptide encoded by the polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13 (in other words, to obtain the protein sequence SEQ.ID.NO.10).

According to a further preferred variant of this method it relates to the production of a protein sequence SEQ.ID.NO.10 and includes the following stages:

(a) culturing, under conditions suitable for expression of the specified polypeptide, the host cell transformed or transtitional expression vector comprising the selected polynucleotide containing a polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13, and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, and

(b) isolation of the polypeptide from the culture of host cells.

The present invention relates also to a method of identifying compounds capable of binding human GHRH and to modulate cell proliferation, the method includes ewusie successive stages:

(a) contacting each test compound for a time and under conditions sufficient for binding of the test compound with the polypeptide, with the selected polypeptide containing

any fragment of a protein encoded by a polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9,

any fragment of a protein encoded by a polynucleotide sequence SEQ.ID.NO.13 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.13,

and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, and

(b) detection of binding of each of the tested compounds with the specified polypeptide and identification among the tested compounds, those compounds which are capable of binding human GHRH and to modulate cell proliferation.

In particular, the method for identifying compounds capable of binding human GHRH and to modulate cell proliferation, includes at the stage of (a) contacting each test compound for a time and under conditions sufficient for binding of the test compound with the polypeptide, with the kind of the slow polypeptide, encoded by a polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 (in other words, with protein sequence SEQ.ID.NO.10).

An alternative method of identifying compounds capable of binding human GHRH and to modulate cell proliferation, comprises the following successive stages:

(a) contacting each test compound for a time and under conditions sufficient for the interaction of cells with the test compound with a cell that can Express the selected polypeptide containing

any fragment of a protein encoded by a polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9,

any fragment of a protein encoded by a polynucleotide sequence SEQ.ID.NO.13 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.13,

and specified the selected polypeptide has at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation, and

(b) determining the effect of each test compound concentration in the cell polypeptide and identification of cf is di test compounds those compounds, which are capable of binding human GHRH and to modulate cell proliferation.

In particular, the alternative variant of the method includes at the stage of (a) contacting each test compound for a time and under conditions sufficient for interaction with the cell under test compound with a cell that can Express the selected polypeptide encoded by the polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 (in other words, the cell is able to Express the protein sequence SEQ.ID.NO.10).

According to preferred variants of the implementation of the methods of identifying compounds capable of binding human GHRH and to modulate cell proliferation, as described above, the test compound is derived from libraries of small molecules resulting from the application of combinatorial chemistry.

Pharmacological properties inherent in polynucleotides and polypeptides according to the invention, make possible their use for pharmaceutical purposes. So, isolated polypeptides, including

any fragment of a protein encoded by a polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9,

any fragment b is the left main coronary artery encoded by a polynucleotide sequence SEQ.ID.NO.13 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.13,

and which have, at least, immunological and/or biological activity characteristic of a protein binding human GHRH and is associated with modulation of cell proliferation and polynucleotide encoding these polypeptides according to the invention can be administered to cancer patients to ease their tumor growth or regression of these tumors.

In the above-described methods, protein binding human GHRH and associated with the modulation of cell proliferation, may be, in particular, highlighted the polypeptide encoded by the polynucleotide sequence SEQ.ID.NO.9 or sequence, a complementary polynucleotide sequence SEQ.ID.NO.9 (in other words, the protein sequence SEQ.ID.NO.10).

Finally, the invention relates to polynucleotide sequence SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.11 and SEQ.ID.NO.12, which can be used, in particular, diluted in PCR reactions for cloning of heterocarpon.

Various of the above features will become apparent to a person skilled in reading a more detailed description of various aspects of the invention.

A detailed description of the discrepancies between the different aspects of the inventions

As indicated above, the present invention is mainly directed at the products and techniques to modulate cell growth and cancer treatment. The present invention is based, in part, on the identification of sequences that are associated with the modulation of cell proliferation, which are polypeptide and polynucleotide sequences associated with the modulation of cell proliferation. Such cDNA molecules can be obtained from drugs RNA or mRNA using standard techniques, such as reverse transcription. Similarly protein or polypeptide associated with differentiation, has sequence encoded by the mRNA associated with cell differentiation.

Described in the present invention the pharmaceutical composition may include one or more polypeptides, nucleic acids sequences and/or antibodies. The polypeptides according to the invention contain at least a portion of the protein or variant protein binding human GHRH and associated with the modulation of cell proliferation. The nucleic acid sequences according to the invention contain a sequence of DNA or RNA that encodes at least a portion of such a polypeptide or which is complementary to this codereuse the sequence.

Antibodies are proteins of the immune system or fragments thereof, engaged in binding to antigen, which is capable of binding a portion of the polypeptides described above.

Polynucleotide, polypeptides and proteins according to the invention

The present invention relates more specifically to polynucleotide sequence SEQ.ID.NO.8, SEQ.ID.NO.9 or SEQ.ID.NO.13, as well as polypeptide or protein sequence SEQ.ID.NO.14.

The invention also includes polynucleotide containing a polynucleotide sequence that is at least 75%, preferably at least 85%, even more preferably at least 90-95% are homologous polynucleotide sequences described above, in particular the sequences SEQ.ID.NO.8, SEQ.ID.NO.9 and SEQ.ID.NO.13. This applies equally to mutatis mutandis to other polynucleotides, polypeptides and proteins that make up part of the invention, in particular to a protein sequence SEQ.ID.NO.14.

The degree of gomologichnosti, expressed in %, calculated as follows:

100-100×(N'/N)

where N' denotes the number of nucleotides or amino acids, modified relative to the sequence SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.13 or SEQ.ID.NO.14, and N represents the number of nucleotides in the sequence SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.13 or SEQ.ID.NO.14.

According to the invention Pauline is Letenye sequence, which encode polypeptides or proteins according to the invention, and fragments or fused proteins of these polypeptides or proteins can be used to generate recombinant DNA molecules that control the expression of these polypeptides or proteins, or active fragments in the appropriate cell hosts. Alternatively, polynucleotide sequences that hybridize to fragments of sequences polynucleotides according to the invention can also be used in the tests hybridization of nucleic acids, such as blotting for the Southern, Northern blotting, etc.

Due to the degeneracy of the genetic code, other DNA sequences encoding mainly polypeptide or protein amino acid sequence according to the invention, can be used for cloning and expression of these polypeptides or proteins. These DNA sequences include sequences that are capable of hybridizing polynucleotide sequence polynucleotides according to the invention in certain harsh environments that can be created in various ways. For example, in the process of polymerase chain reaction (PCR) can withstand the temperature at which hybridize the seed matrix, or the concentration of MgCl2in the reaction booth is Pnom solution. When using DNA fragments with radioactive labels or oligonucleotides to probe membranes strict conditions set by adjusting the ionic strength of the wash solutions or strictly controlling the temperature of washing.

Preferably specified homologous nucleotide sequence specifically hybridizes with a sequence complementary to the sequence of SEQ.ID.NO.8, SEQ.ID.NO.9 or SEQ.ID.NO.13 in severe conditions (or very hard). The parameters characterizing the harsh conditions, are determined by the temperature at which diverge 50% of paired threads (Tm).

For sequences with more than 30 grounds and in accordance with Sambrook et coll. (Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989), Tmdetermined according to the equation

Tm=81,5+of 0.41×(% G+C)of 16.6×log[cation]-0,63×(% formamide)-(600/number of bases).

In accordance with the invention "very strict conditions are conditions when used, the hybridization temperature by 10°C below Tmand the hybridization buffer contains 6-fold SSC solution (0.9 M solution of sodium chloride and 0.09 M solution of sodium citrate). Under such conditions, polynucleotide with nonspecific sequences do not hybridize with polynucleotides having a sequence complementary to the sequence of SEQ.ID.NO.8, SE.ID.NO.9 or SEQ.ID.NO.13.

The modified DNA sequence, which can be used in accordance with the present invention include deletions, additions or substitutions of different nucleotide residues and lead to a sequence that encodes the same gene product or its functional equivalent. Gene product may contain deletions, additions or substitutions of amino acid residues in protein sequences according to the invention, which lead to the so-called silent change and receive, thus, polypeptides and proteins with equivalent function.

Such replacement of amino acid residues can be made on the basis of polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of this balance.

For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine; amino acids with polar groups that have close values of hydrophobicity, include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

For a number of reasons the DNA sequence according to the present invention can be modified to change the polynucleotide sequences according to the invention, including the traveler changes but not limited to, which modify the expression of the gene product. For example, mutations can be carried out using well-known specialist methods, for example, using directed mutagenesis, to insert new restriction sites, changes in glycosylation, phosphorylation, etc.

In particular, in some expression systems, such as yeast, a host cell can sverkhvinoslivie gene product. In such a system, prefer to change the polynucleotide sequence in order to eliminate glycosylation sites. In the scope of the present invention also includes modified polynucleotide sequence associated with heterologous sequences, encoding the fused protein. Fused protein (which may be, for example, the protein sequence SEQ.ID.NO.14) can be modified to establish the site of cleavage, localized between the protein sequence according to the invention (for example, the sequence SEQ.ID.NO.10) and the heterologous protein sequence, so that the protein sequence according to the invention could be derived from heterologous part.

Polynucleotide encoding polypeptides associated with the modulation of cell proliferation

Any polynucleotide encoding the polyp is ptid or part or its variant, which, as described in the present invention binds human GHRH and is associated with modulation of cell proliferation, is included in the scope of the invention. Such polynucleotide can be single-stranded (coding or antimuslim) or double-stranded and may be DNA (genomic, cDNA or synthetic) or RNA molecules.

Polynucleotide encoding the polypeptides, binding human GHRH and associated with the modulation of cell proliferation, can be obtained using any method available to the specialist. For example, polynucleotide may be amplified by polymerase chain reaction (PCR)based on cDNA derived from the cells. For this specific priming can be set or synthesized, or they must be commercially available; these seed are based on the sequence specified polynucleotide. Amplificatory plot can then be used to highlight the complete gene from the Bank genomic DNA or from cDNA Bank of any cell or any fabric with well-known specialist of ways, outlined below. Alternatively, the complete gene can be constructed from several fragments of PCR product. The cDNA molecule encoding the protein, the locking GHRH person and associated with the modulation of cell prolif the radio or part thereof, can be obtained by screening cDNA Bank, obtained for example from mRNA of cells or tissues. Such libraries are commercially available or can be obtained according to the classical methods (see Sambrook et coll., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989).

According to the alternative can also be applied to other screening methods, well known to the specialist.

The cDNA molecule encoding a polypeptide, the locking GHRH person and associated with the modulation of cell proliferation, can be sequenced classical methods using enzymes, such as fragment maple DNA polymerase I, sequenase X (US Biochemical Corp., Cleveland, OH, Etats-Unis), Taq polymerase (Perkin Elmer, Foster City, CA, Etats-Unis), thermostable T7 polymerase (Amersham, Chicago, IL, Etats-Unis), or by combining recombinant polymerase and ectonucleoside having the ability to activate re-read, as, for example, amplification of Elongase (Gibco BRL, Gaithersburg, MD, Etats-Unis). Automatic sequencing can be used with tools, commercially available from suppliers such as Perkin Elmer and Pharmacia.

Partial sequence of cDNA can be used to identify polynucleotide sequence that encodes complete protein associated with the modulation of the cell proliferation, using classical methods, well known to the specialist. Such methods include a method of screening the cDNA library using one or more polynucleotide probes, using recombinant properties of RecA (ClonCapture cDNA Selection Kit, Clontech Laboratories, Etats-Unis).

To carry out hybridization radioactive tagging partial sequence (e.g., by publishing a gap or by tagging all using32P or33P) according to the classical methods. Library of bacteria or bacteriophages are then subjected to screening by hybridization on filters containing colonies denaturirovannykh bacteria (or prints containing phage plaques) with the labeled probe (see Sambrook et coll., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Positive colonies or plaques are then selected, amplified and produce DNA for future analysis.

The full sequence can then be determined according to standard methods. Overlapping sequences are then combined into one continuous sequence. The complete cDNA molecule can be obtained by ligating the fragments in accordance with the classical methods.

As an alternative there are many techniques based on the amplification, to obtain the full coding for the sledovatelnot from a partial cDNA sequence. These include amplification carried out usually by means of PCR. A collection of sets, commercially available, can be used for the implementation of stages of amplification. The seed can be set using, for example, software systems are known to the specialist. Preferably nucleotide blades are molecules with 20-30 nucleotides containing guanidine and cytosine in the amount of not less than 50% and which hybridize with a sequence of a target at temperatures from 50 to 72°C. Amplificatory plot can be sequenced, as described above, and overlapping sequences together in a continuous sequence.

As an alternative solution it is possible to obtain sequences edge with partial sequence by amplification with the seed of a binding sequence specific priming of known area. Amplificatoare sequence is then sent to the second cycle of amplification.

Additional methods include PCR with capture (Lagestrom et coll., PCT Methods Applic. (1991), 1, 111-19) and progressive PCR (Parker et coll., Nucl. Acids. Res. (1991), 19, 3055-60). Can be applied in other ways, using amplification, to obtain the complete cDNA sequence.

You can obtain the complete cDNA sequence in the analysis of the reproduction is of telestai, deposited in the public database of "Expressed Sequence Tags" (EST), located in the Bank of Genes (GenBank). Search overlapping EST can be carried out using computer programs known to the expert (for example, NCBI BLAST), and such EST can be used to generate full continuous sequence.

In the scope of the invention also includes variants of the polynucleotide sequences described above (in particular, sequences SEQ.ID.NO.8, SEQ.ID.NO.9 and SEQ.ID.NO.13). Options polynucleotides may contain one or more substitutions, deletions or insertions (see also above Chapter titled "Polynucleotide, polypeptides and proteins according to the invention").

Part of the sequence, complementary to the coding sequence (i.e. the antisense polynucleotide) can also be used as a probe or a modulator of gene expression. Construction of cDNA that can be transcribed into antisense RNA may be introduced into cells or tissues to facilitate the production of antisense RNA. As described in the present invention, the antisense polynucleotide can be used to inhibit gene expression associated with the modulation of cell proliferation. Method antimyeloma polynucleotide used to regulate gene expression with the education of triple helix, which reduces the ability of the double helix to open sufficiently for the binding of the polymerase, transcription factors, or regulatory molecules (see Gee et coll., Huber et Carr, Molecular and Immunologic Approaches (1994), Futura Publishing Co., Mt. Kisco, NY). According to alternative antisense molecule may be used for hybridization with a plot of the control gene (e.g., promoter, or site of transcription initiation) and block transcription of the gene or block translation by inhibiting the binding of ribosomes with the transcript.

Polynucleotide can then be modified to increase their stability in vivo. Possible modifications include, but are not limited to) adding sequences to the ends 5' and/or 3'; the preferred use of phosphorothioate or 2 O-methyl, and not binding phosphodiesterase in the skeleton; and/or insertion of bases, such as inosine, Tosin and wybutosine and acetylation, methylthioadenosine and other modified forms of adenine, citizen, guanine, thymine and uridine.

Other changes polynucleotides according to the invention have been described above in the Chapter titled "Polynucleotide, polypeptides and proteins according to the invention".

Of a nucleotide sequence as described in the present invention, can join other nucleotide sequences in the use of the implement known methods with recombinant DNA. For example, polynucleotide can be cloned in a wide range of expression vectors, including plasmids, family derived lambda phage and Comedy. Vectors of particular interest include expression vectors, the vectors are replication and sequencing vectors. Typically, the vector contains a source of functional replication, at least in the body, appropriate sites, their endonuclease enzyme and one or more markers for selection. The presence of other elements depending on the use desired by the specialist, which selects the characteristics of the expression vector, depending on their needs and available funds.

Polynucleotide can be included in the compositions for injection into the cell and expression of the corresponding polypeptide. Such compositions are particularly suitable for therapeutic purposes, as described below.

Experts are well aware that there are several methods for the expression of polynucleotide in the target cell and that can be used by any adequate technology. For example, polynucleotide may be injected into a viral vector such as adenovirus or retrovirus (but also others). The technology of introducing DNA into such vectors are well known to the specialist. The retroviral vector can transfer or to introduce a gene in marker selection and/or ele is UNT, serves as a target, as, for example, the gene encoding the ligand-specific receptor of the target cell to make the vector specific for the target.

Other compositions of polynucleotides include disperse colloidal systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and systems based on the use of lipids, including emulsion oil/water, micelles, mixed micelles, and liposomes. The preferred colloidal system used for delivery of the product in vitro and in vivo, is a liposome (i.e. artificial membrane vesicles).

Polypeptides, binding human GHRH and modulating cell proliferation

In the scope of the invention include polypeptides comprising at least part of the protein is associated with modulation of cell proliferation, or its variant, and this part is immunologically and/or biologically active. Such polypeptides can be of any length, including full protein, Oligopeptide (i.e. consisting of a relatively small number of amino acids, as, for example, 8-10 residues, connected by peptide bonds) or the peptide intermediate length. The polypeptide may contain additional sequences.

Similarly, the polypeptide is biologically active if it has one or more structural, regulatory ሺ/or biochemical functions, inherent in the native protein, associated with binding human GHRH and modulation of cell proliferation.

The presence of biological activity can be determined according to well known specialist methods. However, in accordance with the definition in the framework of the present invention the polypeptide is recognized as "having at least, immunological and/or biological activity characteristic of a protein binding human GHRH and associated with the modulation of cell proliferation", if it is inhibitory concentration CI50measured in conditions described in example 6 of the present application, will be less than or equal to 10 nm (preferably less than or equal to 1 nm).

For example, comparative studies of the sequences indicate the specific biological activity of the protein. Experiments aimed at measuring the specified activity can be carried out on the basis of experiments, known for this area. Some portions and other variants of these proteins must also demonstrate that the activity test in vitro or in vivo.

As mentioned, the polypeptides according to the present invention may contain one or more parts of a variant of the endogenous protein in which this part is immunologically and/or biologically active (i.e., the part has one or more antigenic, IMM is noennig and/or biological characteristics of complete protein). Preferably the portion is at least as active as the whole protein, if experience allows us to detect these properties. "Variant" polypeptide is a polypeptide that differs from the native protein by the presence of substitutions, insertions, deletions and/or modifications of amino acids. Some options include conservative substitutions. "Conservative substitution" means the replacement, in which one amino acid is replaced with another amino acid having the same properties as those identified by a specialist who does not expect any changes in the secondary structure, as well as in hydropathical nature of the polypeptide. Replacement of amino acids is usually carried out on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipatic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; amino acids with a positive charge include lysine and arginine; uncharged polar amino acids having similar hydrophobicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may indicate conservative substitutions are the following: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5)Phe, Tyr, Trp, His. Option may also contain non-conservative changes.

Options that are part of the invention also include polypeptides in which the primary structure of a native protein modified with the formation of covalent or non-covalent conjugates with other polypeptides or chemical structures, such as, for example, lipid group or glucosamine group or azetilirovanie group.

The present invention also includes polypeptides, whether or not containing glycosylated links. Polypeptides expressed in the expression system of yeast or in mammalian cells, from the point of view of molecular weight or schema of glycosylation can be similar or have mild differences from the native molecules, depending on the system used in the expression.

Expression of DNA in bacteria, such as E. coli, leads to deglycosylated molecules. Sites of N-glycosylation sites in eukaryotic proteins are characterized by a triplet of amino acids Asn-A1-Z, where A1 stands for any amino acid except Pro, and Z denotes serine or threonine.

Other modifications of polypeptides and proteins according to the invention described in the above section entitled "Polypeptides and polynucleotide according to the invention".

To obtain variants of the polypeptide can be used by standard mutagenesis techniques, such ka is directed mutagenesis using the guide oligonucleotide.

Usually use any well-known specialist of the expression vector for ekspressirovali recombinant polypeptides according to the invention. The expression can be accomplished in any suitable cell host, which is transformed or transliterowany expression vector containing a DNA sequence which encodes a recombinant polypeptide. Suitable cells prokaryotic hosts include cells of higher eukaryotic cells or yeast cells. Preferably used cells hosts are E. coli, yeast cells, or mammalian cells such as COS, CHO, HEK-293, MCF7 (tumor human cells isolated from breast cancer) or DU 145 (tumor human cells isolated from cancer of the prostate).

Some parts and other options can be obtained by the methods of synthesis are well known to the person skilled in the art. For example, portions and variants containing less than 500 amino acids, preferably less than 100 amino acids, more preferably less than 50 amino acids can be synthesized by chemical means. Polypeptides can be synthesized using the methods of solid-phase synthesis, commercially available as, for example, the method of synthesis on the Merrifield resin, which consistently join amino acids to the amino acid chain in the synthesis process (the m Merrifield, J. Am. Chem. Soc. (1963), 85, 2146-2149). There are also numerous other methods of solid-phase synthesis (for example, a method Roberge et coll., Science (1995), 269, 202-204). Equipment for automated synthesis of polypeptides is marketed in such a provider, such as Applied Biosystems, Inc. (Foster City, CA, Etats-Unis); in this case, the synthesis of polypeptides is carried out in accordance with the recommendations of the developer.

Selected polynucleotide or polypeptides.

Usually described in the present invention polypeptides and polynucleotide emit. "Isolated" polypeptide or polynucleotide is polynucleotide or peptide, separated from its original environment. For example, natural protein selected if it is separated from the biological material with which it co-existed in the natural system. Polynucleotide is selected, for example, if it is cloned in the vector, which is not part of the natural environment.

Antibodies and fragments thereof

The present invention provides binding agents, such as antibodies that specifically bind to the protein associated with binding human GHRH and modulation of cell proliferation. Such an agent is referred to as "specifically bind" to a protein modulation of cell proliferation, if its interaction with the protein associated with the modulation of cell is roliferation, or any part thereof, or its variant is at a level sufficient for detection (for example, using ELISA test), and not detected by its interaction with other proteins. "Link" means a non-covalent bond between two separate molecules, which form a complex. The ability to bind can be measured, for example, by using the binding constants for the complex formation. The binding constant of the mean value obtained by dividing the concentration of the complex to the product of the values of the concentrations of the components. Usually two products called "related"if the binding constant of the reaches 103 1/mole. The binding constant determined by methods known to the expert.

Any agent that meets the above criteria, can be considered as a binding agent.

In the present invention the binding agent preferably is an antibody or its fragment. Antibodies can be obtained by any method available to the specialist (see Harlow et Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Usually receive antibodies according to the method of culturing cells, including the production of monoclonal antibodies, or by transpency antibody genes in the cells of the host bacteria or mammals for the production of recombinant antibodies.

Among the other m the methods they prefer to use the methods described below. Immunogen containing polypeptide, is administered to the group of mammals (e.g. mice, rats, sheep or goats). At this stage, the polypeptides according to the invention can perform the role of immunogen without modification. In another embodiment, in particular, for peptides of the small size of the largest immune response can be initiated, if the polypeptide is joined to the transport protein, such as bovine serum albumin or hemocyanin mollusk. The immunogen is injected animal host, preferably in accordance with the above-described scheme, and the animals are periodically taken away the blood. Specific polypeptide polyclonal antibodies can be purified from antiabortion elements, for example, by affinity chromatography using the peptide associated with the corresponding solid substrate.

To obtain antibodies specifically binding the protein sequence, but not binding protein sequence, protein sequence And introduce the animal to the owner, preferably in accordance with the above-described scheme, and the animals are periodically taken away the blood. Specific polypeptide polyclonal antibody sequence And can be purified from antiabortion elements, for example, by affinity chromatography using Belko the second sequence, paired with an appropriate solid substrate. The appropriate eluate contains the antibody specifically binding to the protein sequence, but does not bind to the protein sequence of C.

Slit proteins

Any fused gene can be obtained by a specialist to determine the subcellular localization of the protein according to the invention, in particular subcellular localization of the protein having the sequence SEQ.ID.NO.10. Commercially available are numerous plasmid constructs, such as protein Glutathione S Transférase (GST) or fluorescent proteins such as Green Fluorescent Protein (GFP) or, but not limited to it, the label polyhistidine.

Eukaryotic human cell-hosts (for example, SOME 293) subcultured within 24 hours before transfection procedure that ensures the normal metabolism of cells and the best transfection efficiency. Increasing concentrations (1, 5 and 10 μg) of one vector containing protein tag (GFP, GST or Histidine Tag), or the vector containing polynucleotide sequence SEQ.ID.NO.8 or polynucleotide sequence SEQ.ID.NO.9, fused with a protein tag, create when using Effectene reagent®in accordance with the manufacturer's recommendations (Qiagen).

Cells are then analyzed under satkunam a microscope, for example, to determine localizar and protein. If it is assumed that protein, for example, secreted, supernatant collect, lyophilized, put on acrylamide gel and analyzed by the method of Western blotting using antibodies directed against a protein tag.

The pharmaceutical composition

In accordance with some aspects of the invention products such as polypeptides, antibodies and/or nucleic acids can be administered in pharmaceutical compositions or vaccines. The pharmaceutical compositions contain one or more of such products and one or more pharmaceutically acceptable excipients (carriers). Some pharmaceutical compositions may be used as vaccines, may include one or more polypeptides and activator of the immune response, such as an adjuvant or a liposome (into which the entered product). Pharmaceutical compositions and vaccines may further comprise a system for administration, such as, for example, biodegradable microspheres (and, for example, microspheres consisting of copolymers of lactic and glycolic acid, or PLGA). Pharmaceutical compositions and vaccines included in the scope of the invention may also contain other products that may be biologically active or inactive.

The pharmaceutical composition or vaccine may contain DNA encoding one or more polypeptide is, as described above, so that the polypeptide produced in situ. As noted above, the DNA may be in any form suitable for introduction into the body and are known to the specialist, including systems for the expression of nucleic acids, bacterial or viral system. Suitable expression system, a nucleic acid containing DNA sequences necessary for expression in patients.

System based introduction bacteria require the introduction of bacteria as Bacillus-Calmette-Guerrin)that expresses the immunogenic portion of the polypeptide on its surface. Preferably, the DNA may be introduced in the form of a viral expression system (e.g., using poxvirus, retrovirus, or adenovirus), requiring the use of non-pathogenic agents (defective).

Although it may be used in any media, well-known specialist in the pharmaceutical compositions according to the invention, the media type is chosen depending on the method of administration. The composition according to the invention may be prepared for each relevant method of administration, including, for example, topical, nasal, intravenous, intracranial, intraperitoneally, subcutaneous and intramuscular route.

When parenteral, such as subcutaneous injection, the carrier preferably contains water, salt, alcohol, a fat, a wax or a buffer. When PE is oral administration can be used any medium, above, or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, talcum, cellulose, glucose, sucrose, and magnesium carbonate. In the pharmaceutical compositions according to the invention as a carrier can also be used biodegradable microspheres. In some forms of topical application of preferred compounds in the form of creams or lotions.

Such compositions may also comprise buffers (e.g., salt solutions containing neutral or phosphate buffers), carbohydrates (e.g. glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents, such as, for example, EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or protective agents. According to an alternative variant of the composition according to the invention can be in the form of a lyophilisate. Products can also be encapsulated in liposomes using classical technologies.

According to the invention can be used in any of a variety of adjuvants in vaccines for the induction of immune responses. Most adjuvants contain a substance that protects the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, FR the ins, derived from Bordetella Pertussis or Mycobacterium tyberculosis. Similar adjuvants are commercially available, such as beta-blockers and complete adjuvant (Difco Laboratories, Detroit, MIY, Etats-Unis; Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ, Etat-Unis)), biodegradable microspheres; monophosphoryl lipid A; and cytokines, such as GM-CSF or interleukin-2, -7, or-12.

The above-described compositions can be administered in the form of compositions with delayed action (i.e. compositions in the form of capsules or sponge, which slowly release the product after its admission). Such compositions are usually obtained with the use of well-known specialists and technologies are introduced, for example, oral, rectal route or by subcutaneous implantation, or by implantation at the desired site. The delayed action composition can contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained in the vessel, protected diffuse membrane. The media used in these compositions are biocompatible and must be, in addition, biodegradable. Preferably the composition creates a relatively constant release rate of the active component. The amount of the active substance contained in the composition in slow motion, depends on the location of implantation.

Anticancer therapy

According to friends the aspects of the invention described products can be used in anticancer therapy. In particular, polynucleotide or polypeptides associated with the fixation of GHRH person and modulation of cell proliferation, can be used for growth inhibition and modulation of cell proliferation in specific tumors of breast, prostate or lung cancer.

These polypeptides or polynucleotide can also be used in the treatment of many carcinomas, including melanoma, multiple forms of glioblastoma, carcinoma of the lung, and cancer of the rectum. Agents that activate the expression of these polypeptides or polynucleotides can also be used in the framework of this therapy.

According to these aspects of the invention products (which can be a polypeptide or nucleic acid) is preferably introduced in the pharmaceutical compositions mentioned above.

Patients that may benefit from this therapy are all warm-blooded animals, preferably humans. The patient is directed to therapy according to the invention, can be diagnosed as a patient ill with cancer. In other words, the pharmaceutical compositions described above can be used to inhibit the development of cancer at different stages of the disease (how to prevent cancer and for the treatment of a patient who has cancer).

Farmace the political composition according to the invention is administered in accordance with the method, corresponding to each specific occasion of the cancer to be treated.

Path, the duration and frequency of injection is determined based on the status of the patient, type and severity of the disease, as well as a way of introduction. Route and frequency of injection may vary from one patient to another. Typically, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intradermal, intravenous, intramuscular or subcutaneous route), nazalnam route (e.g. inhalation) or through the mouth. Preferably from 1 to 10 doses can be administered for 52 weeks. Alternative methods can be set individually for each patient.

Usually the appropriate dose and treatment regimen include the number of active product, sufficient to achieve therapeutic and/or prophylactic success. Such a response may be received as a result of the establishment of improved clinical status (e.g., more frequent remission, life in the absence of full, partial, or longer disease) in treated patients compared with patients not treated or treated with lower doses.

According to other aspects of the present invention, the polypeptide may be administered in doses of 100 μg to 5 mg of DNA Molecules encoding these polypeptides, can,as a rule, enter in sufficient quantity to produce polypeptides on a comparable level. The required dose is usually determined by experimental models and/or in clinical trials. Usually prefer to use the minimum dose sufficient to achieve a therapeutic effect. As a rule, to assess the effectiveness of treatment for patients see using tests appropriate to the conditions of treatment or prevention, which appear to be well-known to the specialist.

If not otherwise defined, all technical and scientific terms used in this description, have the same meaning, which is widely recognized as an ordinary specialist in this field, to which the invention relates. In addition, all listed here publications, patent applications, all patents and other references included in the present application by reference.

The following examples are presented to illustrate the above procedures and should not be construed as limiting the scope of invention.

EXAMPLES

Example 1: cloning of cDNA encoding heterocarpon

1.1) Extraction of RNA from cells Pilocarpus Heterophyllus

Cultured cells stored at -80°C before extraction stages all RNA. Extraction of all RNA carried out on the basis of the method described in the scientific literature (Chomczynski et Sacchi, Anal. Biochem. (1987), 162, 56) using the reagent Trizol (Gibco/BRL). The quantity of extracted RNA analyzed on 1%agarose gel in the presence of ethidium bromide.

1.2) Synthesis of cDNA by reverse transcription

Are retrotranscription all DNA according to two different working methods in order to separate reverse transcription and favoring the reverse transcription of the parts 5' and 3' of all RNA using a set of SMART™ RACE cDNA Amplification Kit (Clontech).

1.3) Design and synthesis of seed for polymerase chain reaction (PCR)

The 2 amplification of specific cDNA sequences of heterocarpon carried out by polymerase chain reaction (PCR) products of reverse transcription, using the seed Rev1 for products, specific 5 cdnc, and seed Fwd1 for products, specific 3 kDNK having the sequence respectively SEQ.ID.NO.4 and SEQ.ID.NO.5.

Sequence SEQ.ID.NO.4 and SEQ.ID.NO.5 are as follows:

- SEQ.ID.NO.4:

5'-TCC AAG GAG CAA AAA CTA GTG ACC CAG GGG CCA TTA TAT CT-3'

- SEQ.ID.NO.5:

5'-CGG TAT GGA CGC GGC TAT TGC TAT TGC TGA TGG TGT TGA TGT AA-3'

1.4) Polymerase chain reaction (PCR) and its results

Reaction conditions include 0.2 μm Fwd1 for products, specific 3 cdnc and 0.2 μm Rev1 for products, specific 5 kDNK, 200 μm dNTP, 40 mm Tricine-KOH (pH 8,7), 15 mm COAs, 3.5 mm Mg(OAc)2, of 3.75 μg/ml BSA, 0.005% of Tween-20, 0.005% of Nonidet-P40 and 0.5 U Taq DNA polymerase in a final volume of 50 MK is. The PCR reactions carried out on a thermal Cycling device Perkin-Elmer 9700 with the following parameters of thermal cycles: 5 cycles including denaturation at 94°C for 5 seconds, hybridization nucleating at 72°C, 5 cycles including denaturation at 94°C for 5 seconds, hybridization seed at 70°C for 10 seconds, and continuing the polymerization at 72°C for 3 minutes and finally 25 cycles comprising denaturation at 94°C for 5 seconds, hybridization of seeds at 68°C for 10 seconds and continue polymerization at 72°C for 3 minutes.

The products obtained by PCR, separated on 1%agarose gel and assessed visually by staining with ethidium bromide.

The nucleic acid sequences in the PCR products, specific 5 cdnc and 3 kDNK, determined using an automatic sequencer. It is accordingly about sequences SEQ.ID.NO.6 and SEQ.ID.NO.7 presented below:

Overlapping sequence SEQ.ID.NO.6 and SEQ.ID.NO.7 allow you to isolate a complete cDNA sequence with the sequence SEQ.ID.NO.8, coding heterocarpon. The sequence SEQ.ID.NO.8 presented below:

In the sequence SEQ.ID.NO.8 observed open frame please take the cation in the presence of the initiation codon (ATG), encoding the initiating methionine at position 115, and a stop codon (UAA) in position 2437. Polynucleotide containing the sequence encoding heterocarpon, corresponds to the sequence SEQ.ID.NO.9 presented below:

This translated to polynucleotide corresponds to protein sequence SEQ.ID.NO.10 consisting of 774 amino acids and are presented below:

Example 2. Obtain a complete cDNA encoding heterocarpon to produce recombinant heterocarpon

2.1) Obtaining RNA from cell culture Pilocarpus Heterophyllus

Cultured cells stored at -80°C at the stage of extraction of all RNA. Extraction of all RNA carried out on the basis of the method described in the scientific literature (Chomczynski et Sacchi, Anal. Biochem. (1987), 162, 156) using the reagent Trizol (Gibco/BRL). The quantity of extracted RNA analyzed on 1%agarose gel in the presence of ethidium bromide.

2.2) Reverse transcription of the RNA

All RNA is subjected to reverse transcription using nucleating Oligo (dT) using reverse transcriptase Superscript®as described in the manufacturer's instructions (Gibco/BRL).

2.3) Polymerase chain reaction (PCR) products obtained after reverse transcription

Amplification of cDNA Goethe is of Ocarina carried out by polymerase chain reaction (PCR) products of reverse transcription using nucleating Fwd2 and Rev2 sequences respectively SEQ.ID.NO.11 and SEQ.ID.NO.12.

Sequence SEQ.ID.NO.11 and SEQ.ID.NO.12 are as follows:

SEQ.ID.NO.11:

5'-GGG GGA TCC GAG GTC TAG GAA TGG TGT TCT TCA-3'

SEQ.ID.NO.12:

5'-GGG CTC GAG TTG TGT ACC CAT AAA CAC AAA GTT ACT CAT GG-3'

The seed Fwd2 corresponds to nucleotides 118-140 sequence SEQ.ID.NO.8 and includes a BamH1 site (underlined) and three additional nucleotides in the region 5' to facilitate cloning. The seed Rev2 corresponds to the sequence complementary to the plot containing the nucleotide 2405-2436 sequence SEQ.ID.NO.8 and includes the Xho1 site (underlined) and three additional nucleotides in the region 5' to facilitate cloning.

Reaction conditions include 50 ng cDNA obtained by reverse transcription reaction described above, 0.2 μm Fwd2 (SEQ.ID.NO.11) and Rev2 (SEQ.ID.NO.12), 200 μm dNTP, 40 mm Tricine-KOH (pH 8,7), 15 mm COAs, 3.5 mm Mg(OAc)2, of 3.75 μg/ml BSA, 0.005% of Tween-20, 0.005% of Nonidet-P40 and 0.5 U Taq DNA polymerase in a final volume of 50 µl. The PCR reactions carried out on a thermal Cycling device Perkin-Elmer 9700 with the following parameters of thermal cycles: 5 cycles including denaturation at 94°C for 5 seconds, hybridization nucleating at 72°C, 5 cycles including denaturation at 94°C for 5 seconds, hybridization seed at 70°C for 10 seconds, and continuing the polymerization at 72°C for 3 minutes, and finally 25 cycles comprising denaturation at 94°C for 5 seconds, hybridization of seeds at 68°C. within 10 seconds and continued polymerization at 72°C for 3 minutes.

The products obtained by PCR, separated on 1%agarose gel and assessed visually by staining with ethidium bromide. Get a band of about 2.3 KB.

The sequence of the nucleic acid of PCR product determined using an automatic sequencer, it corresponds to the sequence SEQ.ID.NO.9 after artificial deletions of the initiating codon ATG to implement the expression of recombinant heterocarpon using vector pQE-TriSystem (Qiagen) in phase with BamH1 site, and deletion of the stop codon to save the translation phase of protein and, thus, to carry out the synthesis sequence 8×His at the C-terminal segment of heterocarpon. This sequence corresponds to the sequence SEQ.ID.NO.13 presented below:

This sequence encodes a protein with sequence SEQ.ID.NO.14 presented below:

Example 3. Production of recombinant heterocarpon bacteria

Part of the cDNA encoding heterocarpon inserted into the sites BamH1/Xho1 the expression vector pQE-TriSystem (Qiagen) and Express using the bacteria E. coli M15, as the bacterial hosts. Inoculant 20 ml of LB medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin, and bacteria TSA is ahavat at 37°C for 12 hours. On the basis of this culture inoculant 1 liter of LB medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin, and bacteria shaken at 37°C until reaching an optical density at 600 nm, equal to 0.6.

Expression of recombinant heterocarpon carried out by adding IPTG to a final concentration of 1 mm in a period of time from 4 to 5 hours. Bacteria are then separated by centrifugation at a speed of 4000×g for 20 minutes, then frozen in liquid nitrogen. The precipitate is then thawed on ice for 15 minutes and suspension on ice to lyse buffer with pH 8.0, consisting of 50 mm

NaH2PO4, 300 mm NaCl and 10 mm imidazole, in the presence of 1 mg/ml lysozyme for 30 minutes. After lysis carry out stage ultrasonic treatment and wastes are removed by centrifugation. The clarified lysate (4 ml) is mixed with 1 ml suspension of the Nickel matrix and utrachivayut at 4°C for 60 minutes. The reaction medium is transferred into a column, washed in the presence of 50 mm NaH2PO4, 300 mm NaCl and 20 mm imidazole. In conclusion, heterocarpon elute using 4×0.5 ml buffer consisting of 50 mm NaH2PO4, 300 mm NaCl and 250 mm imidazole.

Example 4. Production of recombinant heterocarpon by insect cells infected with baculovirus

Part of the cDNA encoding heterocarpon inserted into the sites BamH1/Xho1 vector expr the hurt pQE-TriSystem (Qiagen). The vector pQE-TriSystem contains sequences of the virus Autographa california nuclear polyhedrosis virus (AcNPV), which creates homologous recombination. Recombinant baculovirus containing the sequence of heterocarpon produced by cotransfection vector pQE-TriSystem with linearized genomic DNA of the baculovirus in insect cells sf9 or sf21, taken from the ovarian tissue of larvae of Spodoptera frugiperda. Transfetsirovannyh cells washed with phosphate buffer and separated by centrifugation at 1000×g for 5 minutes. The precipitate is then suspension in lytic buffer with pH 8.0, consisting of 50 mm NaH2PO4, 300 mm NaCl and 10 mm imidazole. After lysis carry out stage ultrasonic treatment and wastes are removed by centrifugation. The clarified lysate (4 ml) is mixed with 200 μl of the suspension of the Nickel matrix and shaken at 4°C for 60 minutes. The reaction medium is transferred into a column, washed in the presence of 50 mm NaH2PO4, 300 mm NaCl and 20 mm imidazole. In conclusion, heterocarpon elute using 4×0.5 ml buffer consisting of 50 mm NaH2PO4, 300 mm NaCl and 250 mm imidazole.

Example 5. Production of recombinant heterocarpon cells of a mammal

Part of the cDNA encoding heterocarpon inserted into the sites BamH1/Xho1 the expression vector pQE-TriSystem (Qiagen). The vector pQE-TriSystem contains an activating sequence cytomegalo the virus (CMV), fused with the promoter of the beta-actin chicken, enabling a very important stage of heterologous expression. Kidney cells human embryo (SOME 293) were cultured in DMEM (Wednesday Needle, modified, Dulbecco)containing 100 U/ml penicillin and 100 μg/ml streptomycin sulfate, supplemented with 10%amniotic calf serum. Cells will subcultured 24 hours before transfection in order to carry out normal metabolism and high efficiency transfection. Transfection of 1 μg of the vector pQE-TriSystem containing cDNA encoding heterocarpon, carried out using Effectene reagent®in accordance with rekomendacijami manufacturer (Qiagen).

Transfetsirovannyh cells washed with phosphate buffer and separated by centrifugation at 1000×g for 5 minutes. Sediment suspension in lytic buffer with pH 8.0, consisting of 50 mm NaH2PO4, 300 mm NaCl and 10 mm imidazole in the presence of 0.05% Tween®20. After lysis carry out stage ultrasonic treatment and wastes are removed by centrifugation. The clarified lysate (4 ml) is mixed with 200 μl of the suspension of the Nickel matrix and shaken at 4°C for 60 minutes. The reaction medium is transferred into a column, washed in the presence of 50 mm NaH2PO4, 300 mm NaCl and 20 mm imidazole. In conclusion, heterocarpon elute through 4 is 0.5 ml buffer, consisting of 50 mm NaH2PO4, 300 mm NaCl and 250 mm imidazole.

Example 6. Measurement communication with the receptor of the person to GHRH

Stable transfection of receptor human GHRH (hGHRH-R)

Kidney cells human embryo, SOME 293 (cell line, developed by Dr. Stuart Sealfon, Mount Sinai Medical School, New York), stably expressing the receptor for GHRH man, were obtained from Dr. Kelly Mayo, Northwestern University, Chicago, IL).

Cell culture and membrane preparation

Cells of SOME 293 stably transfetsirovannyh GHRH receptor of man, described above, were cultured in DMEM (Wednesday Needle, modified, Dulbecco, with a high glucose content; comes with firm Life technologies), supplemented with 0.4 mm G418 (Life technologies), in the presence of 10%amniotic calf serum and 4 mm L-glutamine (Life technologies). Cells are homogenized in buffer containing 50 mm HEPES (pH 7.4), 5 mm of magnesium chloride (MgCl2), 2 mm ethylene glycol-bis-(2-amino-ethyl)-N,N,N',N'-tetraoxane acid (EGTA) and 50 μg/ml bacitracin, and then subjected to ultrasonic treatment in the same buffer A. homogenized thus the cells are centrifuged at 4°C with a speed 39000×g for 10 minutes, suspended in buffer a and centrifuged again at 4°C at the rate of 40,000×g for 10 minutes. The total content of membrane proteins is determined by the method of Bradford. Membrane precipitation stored at -80°C for further use of the Finance.

Test for competitive binding with the receptor hGHRH-R

The cell membranes of SOME 293 stably transfetsirovannyh GHRH receptor human, dilute to a concentration of 100 μg/ml in reaction buffer containing 50 mm HEPES (pH 7.4), 5 mm MgCl2, 2 mm EGTA, 50 μg/ml bacitracin, and 0.5% bovine serum albumin (BSA). Membranes incubated with the help of 0.05 nm [125I]GHRH(1-44 amide) (Amersham) in a final volume of 200 ál with increasing concentrations of heterocarpon for 2 hours at 23°C. the Reaction is stopped by rapid filtration onto 96-well filters GF/C, pre-filled with 0.1% polyethylenimine. The filters are then washed three times at 4°C with wash buffer containing 50 mm Tris (pH of 7.4) using a filtration unit Packard with 96 wells. Dried filters immersed in 20 µl scintillare mixture (Microscint O, Packard) and perform the calculation using counter Topcount (Packard). Non-specific activity determined in the presence of 100 nm hGHRH. Draw a curve dose-response for hGHRH (0.001 nm - 100 nm) and define it inhibitory concentration

CI50protein/polypeptide, in which 50% of GHRH person not associated with the receptor for GHRH person.

1. Selected polynucleotide, which includes a sequence encoding a polypeptide heterocarpon, possessing the ability to bind GHRH and to modulate cell proliferation, and is characterized by the nucleotide sequence shown in SEQ ID NO:8, or a fragment of the sequence shown in SEQ ID NO:9 and the corresponding coding of the full sequence.

2. Selected polynucleotide according to claim 1, characterized in that it is polynucleotide with the sequence SEQ ID NO:8.

3. Selected polynucleotide according to claim 1, characterized in that it is polynucleotide with the sequence SEQ ID NO:9.

4. Primer sequence SEQ ID NO:4 or SEQ ID NO:5 to obtain polynucleotide encoding the polypeptide heterocarpon.

5. Polynucleotide with the sequence SEQ ID NO:13 encoding a protein comprising a polypeptide heterocarpon.

6. Protein that includes the amino acid sequence of heterocarpon and is characterized by the sequence given in SEQ ID NO:14.

7. The expression vector pQE containing Pauline is looted with the sequence SEQ ID NO:13.

8. A host cell transformed or transfusiona expression vector of claim 8, which is the producer of fused protein with SEQ ID NO:14.

9. A method of obtaining a fused protein containing the polypeptide heterocarpon encoded by polynucleotide sequence SEQ ID NO:9, which includes the following stages:
(a) culturing under conditions suitable for expression of the indicated protein, host cell transformed or traditioanal expression vector functionally active in the cell containing the polynucleotide sequence of SEQ ID NO:13, and
(b) isolation of the protein from the culture of host cells.



 

Same patents:

FIELD: medicine.

SUBSTANCE: peptide is obtained from phage peptide library including set of static peptides with length of 12 aminoacid residues, by affine selection of phage clones containing peptide capable of specific linking to antibodies of benzo[α]pyrene and benzo[α]anthracene. Peptide displays specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene and features molecular weight of 1.3 kDa and registered aminoacid sequence LHLPHHDGVGWG encoded by nucleotide sequence SEQ ID NO:1.

EFFECT: application in medicine as a base for peptide medicine development for immunologic prevention of malignant tumours of humans.

4 dwg, 3 ex

FIELD: biology.

SUBSTANCE: present invention relates to genetic engineering, more specifically to obtaining anticoagulative protein extracted from nematodes (NAP) and can be used in medicine. To obtain a medicinal preparation based on NAP, methanotrophic yeast host cells are cultured, encoding rNAPc2 or rNAPc2/praline, until attaining the desired cell density. NAP is then extracted from the said yeast host cells through cation-exchange chromatography on an expanding layer. To purify the NAP medicinal preparation, hydrophobic-interaction chromatography is used. NAP is extracted and purified at pH levels below 4.

EFFECT: simple and more efficient method of obtaining anticoagulation proteins from nematodes.

25 cl, 8 dwg, 7 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: claimed are expression vectors for obtaining IL-21 in E.coli cells. IL-21 coding nucleotide sequence, included in composition of novel vectors, contains modifications aimed at optimisation of codons and secondary mRNA structure for translation in E.coli. As a result of transformation with claimed vector structures E.coli strains suitable for industrial scale application have been obtained. Methods of wide scale IL-21 production which use said strains have been elaborated, allowing to obtain more than 1g/l of recombinant cytokine.

EFFECT: novel compounds possess useful biological properties.

14 cl, 1 dwg, 12 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.

EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.

6 cl, 11 dwg, 9 ex

FIELD: biotechnology.

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4 cl, 8 dwg, 1 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

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60 cl, 16 dwg

FIELD: medicine; biotechnologies.

SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.

EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.

FIELD: medicine, biotechnologies.

SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.

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20 cl, 2 dwg, 8 ex

FIELD: chemistry, biotechnology.

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23 cl, 15 dwg, 3 tbl

FIELD: biology.

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37 cl, 7 dwg, 6 tbl, 8 ex

FIELD: medicine; biology.

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EFFECT: hypertrophy of specific plant organ, male sterility or stress resistance.

12 cl, 33 dwg, 26 ex

FIELD: chemistry; biochemistry.

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EFFECT: production of transgenic plants with enhanced pathogene immunity due to reduced expression of RacB protein or functional equivalent thereof.

23 cl, 8 dwg, 7 tbl, 12 ex

FIELD: genetic engineering.

SUBSTANCE: provides genetic sequence of grass pollen main allergen Phi p 4. Fragments of this sequence, combinations of its partial sequences and point mutants have hypoallergenic action. Recombinant molecules of DNA and derivative polypeptides, fragments, new combination of partial sequences and versions can be applied for treatment of diseases associated with grass pollen allergy.

EFFECT: proteins produced by recombinant methods can be applied for diagnostics of grass pollen allergosis.

16 cl, 8 dwg, 6 tbl

FIELD: medicine; pharmacology.

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29 cl, 3 dwg, 2 tbl, 3 ex

FIELD: gene engineering.

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EFFECT: plants with increased yield and heat stability.

86 cl, 9 dwg, 4 tbl, 2 ex

FIELD: genetic engineering, agriculture.

SUBSTANCE: invention relates to a method for preparing transgenic plants. Method involves selection of the strain of the required plant, preparing mother plants and carrying out the multiplication of the vegetable material for preparing leaf explants. Then method involves making a vector for transfer of genetic material expressing the end protein that enhances resistance of plants against phytopathogens followed by the agrobacterial transformation of explants with agrobacterium Agrobacterium tumifaciens and the following selection of transgenic tissue and multiplication of transformants. At the transformation stage method involves stage-by-stage co-cultivation of explants with steps amount taken in the range from 2 to 5 and wherein leaf disks with the ratio of cut length to the area disk is in the range 0.1-0.2 mm/mm2 are used as explants. Invention provides preparing transgenic plants showing the enhanced resistance against phytopathogens and also to enhance frequency of the transient expression, frequency in formation of transgenic tissues, frequency in regeneration of transgenic sprouts, part of direct transformants and to reduce frequency of somaclonal alterations.

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5 cl, 9 dwg, 12 tbl, 16 ex

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9 cl, 37 dwg, 18 tbl, 10 ex

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes

FIELD: medicine.

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EFFECT: obtainment of polypeptides for treatment of diseases requiring modulation of thrombocyte-mediated aggregation.

40 cl, 69 ex, 30 dwg, 32 tbl

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