Recombinant mistletoe lectin (rml)

 

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes. The use of nucleic acid molecules encoding the polypeptides and polypeptide dimers of mistletoe lectins, allows you to diagnose the presence of the corresponding genes in the organism and their polypeptides and their dimers can be used in the composition of medicines, with immunostimulation and cytotoxic activity. 12 c. and 9 C.p. f-crystals, 15 ill.

The present invention relates to nucleic acid molecules coding for preproprotein, which after maturation exhibit the biological activity of the lectin dimer mistletoe, vectors containing these nucleic acid molecule, transformed by these vectors organisms-owners and to the polypeptides or dimers of the polypeptides encoded by these nucleic acid molecules. The polypeptides according to the invention or the polypeptide dimers are used in a variety of therapeutic purposes. Therefore, the invention further relates to immunotoxins, as well as to medicines, Islam Siam for the diagnosis, containing the nucleic acid molecule according to the invention, the polypeptides or polypeptide dimers according to the invention and/or primers that specifically hybridize with nucleic acid molecules according to the invention. In addition, the invention relates to herbicides containing polypeptides and/or polypeptide dimers according to the invention.

Extracts of mistletoe for many centuries used for therapeutic purposes. Since the beginning of this century the preparations of mistletoe with one or another successfully used in cancer therapy (Bocci, 1993; Gabius and others, 1993; Gabius and Gabius, 1994; Ganguly and Das, 1994). Hajto and others (1989, 1990) were able to show therapeutic effects, in particular, the so-called lectins from mistletoe (viscumin, agglutinin Viscum album, VAA). Currently, in addition to their cytotoxic action is discussed, in particular, non-specific stimulation of the immune response, positive effects of which are used for concomitant therapy and aftercare of patients with tumors. The extended survival of such patients probably occurs due to the selection of homologous endorphin (Heiny and Beuth, 1994).

Numerous in vitro studies (Hajto and others, 1990; Mannel and others, 1991; Beuth and others, 1993) and in vivo (Hajto, 1986; Hajto and d the tion of inflammatory cytokines (factortumor necrosis (TNF-), interleukin-1 (IL-1), interleukin-6 (IL-6)), as well as activation of cellular components of the immune system (THcells, natural killer cells (NK-cells)).

As the active beginning of the extract of mistletoe are currently considering the protein of mistletoe lectin (ML) mass of 60 kDa, which can be obtained by biochemical extracts (Franz and others, 1977; Gabius and others, 1992). Protein ML consists of two covalently connected by a disulfide bridge subunits, whose A-chain is responsible for the enzymatic inactivation of ribosomes (Endo and others, 1988), and the chain is responsible for binding carbohydrates. Biological activity according to the known data is reduced mainly to lectinology activity of the b-chain (Hajto and others, 1990).

However, at present there is insufficient information about the relationship between structure and activity of mistletoe lectin (ML). So, for example, unclear contribution of individual chains and their inherent different biochemical and enzymatic activity in the observed effect or therapeutic effects ML-1. The analysis of the relationship between structure and activity is hampered by contamination of drugs other vegetable substances in status is the composition of the extracts, that, in turn, depends on the type of wood from which the selected extract (e.g., Apple, pine, poplar) (and others, 1986). Viscotoxin (Garcia-Olmedo and others, 1983; Mendez and others, 1990), as well as other viscumin (for example, ML-2, ML-3) have a similar effect (ifler and others, 1994). Even biochemically purified (affinity chromatography) to high purity preparations ML find significant heterogeneity (Fig.8). This heterogeneity refers to the biochemical activity of the circuits and the associated effects that occur in vitro and in vivo, and also the structure of proteins. The literature describes structural variants glycosylation of a - and b-chains ML, as well as variations of the sequences. The Gabius and others (1992) and Dietrich and others (1992) described the variability of the sequences A1 - and A2-chains ML-1.

Order detail to investigate therapeutic effect of mistletoe lectin is desirable to obtain it in pure form as structurally homogeneous substance. Next for professionals interested in the possibility of obtaining ML or its component parts in large quantities in a pure form, to them, for example, can be used as active constituent parts of medicines in industrial receiv who was able to solve these problems. When selecting from vegetable source with current levels of technology always get a heterogeneous mixture of substances.

Heterogeneity of herbal preparations of mistletoe lectin is among others the result of posttranslational processing ML-1 isoforms in ML-2 and ML-3, so that preparations ML depending on the method of allocation or the duration of the fermentation discover different content ML-1, ML-2 and ML-3 (and others, 1995). Each of these isoforms, in addition, has microheterogeneity, which is shown in Fig.8 for example ML-1 using isoelectric chromatofocusing.

The basis of the present invention, therefore, were based on the task to get the mistletoe lectin in pure form and in such quantities that provide industrial use. This problem is solved using the various embodiments disclosed in the claims of the invention.

Thus, the invention relates to a nucleic acid molecule that (a) encodes preproprotein, which after maturation exhibits biological function, characteristic of the dimer of mistletoe lectin, and characterized is shown in Fig.4B nucleotide posledovatelnoy part of the dimer of mistletoe lectin; (C) differs from the nucleic acid molecule according to paragraph (a) or (b) due to the degeneracy of the genetic code, or (g) under strict conditions hybridizes with the nucleic acid molecule according to paragraphs (a), (b) or (C) and encodes a polypeptide specified in paragraph (a) or (b) biological function, activity, respectively.

Based on the gene sequences of mistletoe lectin, first can be obtained high-purity recombinant single chain (rMLA, rMLB), which can be reassociated in vitro, thus will result in getting holoprotein rML, which is chemical, enzymatic and structurally homogeneous. Reassociating recombinant protein does not show variability and microheterogeneity, especially with regard to the primary structure and posttranslational modifications (glycosylation, phosphorylation), and most suitable for therapeutic purposes in the form of holoprotein, and as part of a chain and subfragments.

The term "fragment" preprotein of mistletoe lectin means each fragment according to the invention (not only of natural origin), which is the biologically active part of the dimer of mistletoe lectin. DL the EPA lectin mistletoe means such component parts, which are integral parts of a single-chain dimer. It is obvious also that the individual chains or fragments thereof, which are part shown in Fig.4B sequence, are the object of the invention.

The term "natural" in combination with the expression "part of the mistletoe lectin in the context of the present invention means that so marked a fragment or is a chain dimer of mistletoe lectin, or is subfragments chain, which is naturally present in the circuit. These fragments are biologically active.

The term "biologically active" according to the invention assumes that these fragments have at least one biological function inherent in the circuits or the dimer, as presented herein, or any other biological function of individual circuits or dimer. In addition, the term "biologically active" refers to pharmacological and/or immunological activity.

Using recombinant protein ML for the first time it became possible additional experimental study of the contribution of individual domains or subdomains. Rekombinantnye, containing one of these substances as a replacement drugs representing extracts and standardized extracts.

Unexpectedly it was found that the cloning of the gene encoding the lectin mistletoe can be made on the basis of a new strategy for cloning, after conventional strategies of cloning has not led to success.

There are a number of data about the chemical properties of the protein of mistletoe lectin ML-1. To them along with the molecular weight and structure of the subunits include, in particular, a short N-terminal peptides, the amino acid sequence of which is independently described by Dietrich and others (1992) and Gabius and others (1992) [see also German patent 4221836]. On the basis of N-terminal peptides of a - or b-chain, on the basis of their amino acid composition due to the high degree of innate derivative sequences of nucleic acids is practically impossible to obtain synthetic oligonucleotides, the degree of degeneracy which is sufficiently low so that when the screening genomic libraries to identify fragments of the gene ML. It was also referred to the Bank and genes cDNA obtained by using poly(A+)-RNA from Viscum album.

Polymerase chain reaction (PCR) allows amplitude for N-end MLA and of antisense oligonucleotides bound to the N-Terminus MLB became possible amplification located between the portion of a gene ML, does not contain introns (Fig.1A). In practice, however, analysis of the N-terminal sequence of the b-chain shows that the degree of degeneracy (number of possible combinations of oligonucleotides) is still too high for the success of this process. This, in particular, due to the fact that the sequence of N-end In-circuit unsuitable for construction of the oligonucleotide, resulting in the amplification of sequences of the gene ML, based on the known sites of amino acid sequences, is not practiced (Fig.1B).

For cloning of the gene ML using a modified PCR strategy investigated the possibility of using to build the amplified oligonucleotides additional data about the proteins, in particular, based on the relationship of mistletoe lectin class ribosome inactivating proteins type I and type II (PIR) (Stirpe and others, 1992). Based on numerous structures inherent in (a) pier type I and a-chain of ricin, and (b) In-chains abrin and ricin, was identified in 8 General conservative areas of the sequence. Based on these plots sequences with consideration of codons used in a related species, was built 21 oligonucleotide, and all these oligonucletide spolzli received specific amplification products even though the PCR conditions related to the annealing temperature, the content of Mg2+and the cycle parameters were varied within wide limits.

No screening genomic banks and banks cDNA or PCR technique did not allow taking into account the above reasoning to identify sequence-specific DNA ML.

So started the search for new ways to use additional data about the structure of ricin and abrin to build oligonucleotides as a result of amplification.

Because the enzymatic mechanism inherent in the feast, in particular, the feast of type II ricin-like mechanism ML (Endo and others, a + 19886), could not be ruled out that between them there is a structural similarity in the field of functionally active sites of the primary and tertiary structures. Based on the crystal structure of ricin (Katzin and others, 1991; Rutenberg and Robertus, 1991; Weston and others, 1994), analysis of plasticity in the a-chain of ricin testified low mobility Arg180, which is located within the conservative sequence. Further analysis was made possible in this case, the substitution of amino acids at this site active site on the basis of the steric arrangement of the main circuit, taking into account the interaction with the STI a-chain of ricin and other sequences related to the feast of type I.

By complementing the results of sequence comparison information about the structure of the received data on the likely inclusion of certain amino acid residues at certain positions. This made it possible to determine the number of theoretical amino acid sequence for this region, ML, and use this to build an appropriate, very few degenerate oligonucleotide (RMLA2) (Fig.1B).

Other fragments could be obtained only by the combination RMLA1 (degenerate oligonucleotide derived from the N-terminal amino acid sequence of the a-chain ML; cf. Fig.1B) and is built on the basis of the above reasoning when specific PCR oligonucleotide "active site" RMLA2 based on a complex genomic DNA ML, after all relevant alternative approaches, as described above, were unsuccessful.

For more information about the gene sequence obtained only with the use of specific non-degenerate oligonucleotide primers derived from partially defined sequence MLA gene in the cloning and sequencing of the fragment "a" (Fig.3), and degenerate of Oleg isih PCR-amplification. For construction of degenerate oligonucleotides for In-chains used completing the sequence of the b-chain of ricin and abrin, which also were found in separate areas of higher invariance.

To obtain the 5’- and 3’-ends of holoprotein by reverse transcription were synthesized fragments In-chains, as well as 5’- and 3’-terminal noncoding sites based on selected RNA mistletoe similar to cDNA, and using the method of rapid amplification of mRNA and cDNA using PCR (RACE) (Frohman and others, 1988) received the corresponding parts of the gene. After receiving in each case a large number of overlapping fragments of the gene (Fig.3) then, using specific PCR were obtained full plots of the genes encoding the a-chain and b-chain, based in each case from complex genomic DNA of mistletoe. The sequences of genes rMLA and rMLB end modifications shown in Fig.4A and Fig.4B. In Fig.4B is represented as 5’- and 3’-noncoding sites, and the full sequence of the gene ML, covering the sites of the gene encoding antipeptide and signal peptide.

In a preferred embodiment of the invention the nucleic acid molecule is a fragment of the a-chain lectin of Omel Occitania embodiment of the invention the nucleic acid molecule is a fragment of the b-chain of mistletoe lectin, which is encoded by the nucleotide sequence shown in Fig.4B (MLB).

Another preferred embodiment of the invention relates to a nucleic acid molecule, which is a DNA molecule.

According to the invention, the term "DNA molecule" refers to both genomic and cDNA molecule or semi-synthetic DNA molecule. Methods of obtaining these different DNA molecules known to specialists in this field of technology.

In another preferred embodiment of the invention the nucleic acid molecule is a RNA molecule.

Further, the invention relates to a nucleic acid molecule which is antisense thread to the above nucleic acid molecule according to the invention. Such antisense strand may be, for example, used to inhibit transcription in the case of research on the expression or regulation in the plant.

The invention further relates to a vector which contains at least one molecule of nucleic acid according to the invention.

The vector according to the invention can, for example, contain a single molecule of nucleic acid according to the invention, which encodes the entire preproprotein received in suitable for this purpose transformed host and Monomeric fragments can connect in vivo or in vitro dimer of mistletoe lectin. In another embodiment of the invention, the proposed vector is a vector that is intended solely for playback nucleic acid according to the invention.

In a preferred embodiment of the invention, the proposed vector contains a nucleic acid molecule, which encodes the A-chain of mistletoe lectin or a fragment, but also the nucleic acid molecule encoding a B-chain or fragment. Fragments of the monomers are predominantly biologically active.

In another preferred embodiment of the invention, the vector according to the invention is an expression vector. Specialists in the art will know how to get acceptable expression vectors for different organisms hosts.

According to the invention for heterologous expression based on a comprehensive genomic DNA mistletoe using specific PCR was obtained sequence encoding the a-chain of mistletoe lectin. While using complementary sites built oligonucleotide primers were introduced elements that control tra considered genomic gene reprolatina mistletoe, were possible cloning and differential expression of a-chain of mistletoe lectin.

Site at the 5’end of the coding rMLA sequence corresponding to amino acid residues tyrosine1-tyrosine17(Dietrich and others, 1992; Gabius and others, 1992), was obtained by embedding before codon that initiates translation of the synthetic gene fragment, using hybridization and cloning of double oligonucleotides. Was optimized gene sequence relative to the selector codon, as described for highly expressed in Escherichia coli of the gene (Gribskov and others, 1984). On the 3’-end of the synthetic gene fragment rMLA, as well as at the 5’end of the obtained PCR fragment of the gene rMLA with targeted replacement of the codon of tyrosine17TAS on TAT was introduced restriction site, resulting from the cleavage by the restriction enzyme Ssp I, which made possible the connection of both gene fragments rMLA upon receipt of the vector pML14-17 (Fig.5). Was sequenced coding rMLA sequence (Fig.4A). For the expression of rMLA in Escherichia coli of the gene sequence was isolated from the vector pML14-17 and was built in expressing vector RT-7 (Studier and Moffart, 1986) under the control of T7 promoter-RNA polymerase, and transcription terminator. T is cpressey gene is accompanied by the appearance of protein bands, the respective And-circuit replicationmanager recombinant mistletoe lectin that has a relative molecular mass of 25 kDa. Detection and identification of recombinant expression product was performed using Western blotting using antibodies specific to MLA (Fig.7).

For heterologous expression of the b-chain of mistletoe lectin complete coding MLB sequence of complex genomic DNA Viscum album was amplified using specific PCR, and using complementary sites built oligonucleotide primers were introduced elements that control the transmission, and the sequence recognized by the restriction enzyme (Fig.6). The resulting PCR product with a length of 0.8 T. p. N. after cloning into TA-cloning vector pCRII was built into the expression vector RT-7 under the control elements controlling transcription, and using the resulting expression vector pT7-MLB was transformed E. coli strain BL21.

Correct coding rMLB sequence was confirmed when sequencing (Fig.4B). Expression were confirmed by Western blot using antibodies specific to MLB (TB33, Tonevitsky, etc., Halekulani mass of 31 kDa, the corresponding In-circuit replicationmanager recombinant mistletoe lectin (Fig.7b). Analysis of cell fractions after appropriate treatment of E. coli cells showed a distribution of the synthesized In-circuit rML in the soluble fraction in the supernatant and insoluble fractions in the "inclusion bodies", i.e. in the sediment after obrabotki of E. coli cells, and 4 hours after induction, the proportion of the soluble or insoluble fractions were, respectively, 50% of the total output (Fig.7b).

Further, the invention relates to the body-master, which is transformed by at least one vector according to the invention.

Depending on the purpose of use of the host body according to the invention can be obtained only one of the two monomers, or a combination of both monomers, mainly in the form of the associated dimer. Master-organism according to the invention may be a eukaryotic or prokaryotic cell, a transgenic plant or transgenic animals.

Mostly master-organism according to the invention is a cell of a mammal, plant cell, a bacterium, a cell, a fungus, a yeast cell, an insect cell or a transgenic plant.

In a particularly preferred variantly cell Spodoptera, preferably the cell Spodoptera frugiperda.

Further, the invention relates to a polypeptide that is encoded by a nucleic acid molecule according to the invention or a vector according to the invention and/or produced by an organism of the host according to the invention.

The polypeptide according to the invention has mainly the biological activity characteristic of the a-chain or b-chain of mistletoe lectin. In other embodiments of the invention the polypeptide according to the invention may, however, also be part of the biological activity or not to show biological activity. The term "part of the biological activity according to the invention mean or reduced activity or/and the range of activities of the spectrum of biological activity. The polypeptide according to the invention can also be a fragment of A - or b-chain, which exhibits one of the above-mentioned properties.

The study of the properties of rMLA, rMLB and holoprotein rML

(I) the Relative molecular mass and structure

The relative molecular mass was determined by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (APAG-ordinator) and in the presence of reducing agents and the following is the first detection in the framework of the Western blot.

Unexpectedly it was found that recombinant deglycosylation A-chain of mistletoe lectin has a relative molecular mass of 25 kDa and, thus, differs significantly from the natural A-chain of mistletoe lectin And1(31 kDa) or a2(29 kDa). This difference in relative molecular mass is unexpected, primarily because according to the prior art assumed that the a-chain is not glycosylated. Recombinant B-chain of mistletoe lectin has a relative molecular mass of 31 kDa and is therefore significantly easier than glycosylated natural-chain of mistletoe lectin with a relative molecular mass of 36 kDa (Fig.7).

Inherent in natural proteins ML heterogeneity due to glycosylation and/or sequence variation, which is shown in the gel in the presence of LTOs in the form of a wide strip, is not observed in any of the analyzed cases for recombinant types.

Relative molecular mass reassociating of holoprotein rMLA/rMLB (rML) are summed up to 56 kDa compared to the heavy natural holoprotein (nML) with a relative molecular mass 65-67 kDa.

(II) Electrophoretic homogeneity

rMLA is detected as isoelectrically white, which are divided into four types with isoelectric point of 5.2; 5,4; of 5.7 and 6.2 (Fig.8).

rMLB is detected as isoelectric homogeneous protein with an isoelectric point of 5.1 unlike natural b-chain of mistletoe lectin, which is divided by at least 2 species with isoelectric points 7.1 and 7.3 (Fig.8).

When this natural holoprotein ML detects a large number of variants of molecules and combinations of molecules (Fig.8 below), at the same time for the recombinant protein of mistletoe lectin reveals a homogeneous mobility in chromatofocusing with isoelectrofocusing, which indicates the homogeneity of the rML unlike microheterogeneity natural types of proteins.

(III) the Enzymatic activity of rMLA

When you enable purified using immunoaffinity chromatography of drugs rMLA in experience coupled transcription/translation was discovered inhibiting translation activity rMLA (isolated from the soluble fraction of the product of expression) and rMLA (isolated from the insoluble fraction in the "inclusion bodies").

rMLA when compared with the natural chain of mistletoe lectin shows different inhibitory characteristic concerning the dependence of the degree of inhibition of translation of Otta (Fig.9). Property of the enzyme, underlying the toxic effects of holoprotein ML, significantly less in recombinant forms.

(IV) Activity rMLB in relation to the binding of carbohydrates

In-circuit rML, which are obtained by renaturation and recycline of the primary products of expression, as well as reassociations in vitro rMLA/rMLB, rMLA/MLB and holoprotein MLA/rMLB show activity against the binding of carbohydrates, which can be detected when analysis using legislative enzyme (ELLA) for binding to the carbohydrate asianavenue or petrinovic matrices. Specificity in relation to carbohydrates recombinant b-chain rML can be defined and quantified in ELLA-system in a competitive binding to galactose,-lactose, N-atsetilgalaktozamin (Ga1NAc) and sialic acid (N-acetylneuraminic acid, NANA). When ELLA-test in a competitive environment unexpectedly exhibit different specificity nMLB and rMLB in relation to carbohydrates. The affinity for binding to describe this using system specific values IR50for premaxillae displacement of proteins with immobilized asianavenue ligand galactose (IR50nMLB: 4.5 mm; IR50rMLB: the K50nMLB: 4.9 mm; IR50rMLB: >70 mm), N-atsetilgalaktozamin (IR50nMLB: 20.7 mm; IR50rMLB: 109 mm) or with immobilized petrinovich ligand sialic acid (IR50nMLB: 49,8 mm; IR50rMLB: 47,1 mm).

While natural In-circuit nML described as specific for galactose lectin, as expected, can compete with galactose and-lactose obtained in E. coli recombinant B-chain rML does not detect any detectable interaction with galactose and finds little interaction with-lactose. Recombinant rMLB has a clear affinity to N-atsetilgalaktozamin and sialic acid and unexpectedly discovers when compared with vegetable nMLB a clear shift of the carbohydrate specificity of lectins to specificity in relation to N-atsetilgalaktozamin/sialic acid. As a consequence, taking into account the biological activity of rMLB and rMLB-containing holoprotein, you can use a wider and diverse range of ligands, receptors and target cells compared to vegetable protein lectin mistletoe.

In a preferred embodiment, the polypeptide according to the invention contains less than the change known biological activity of the polypeptide, lower it or raise. Such modification may, for example, be carried out after the broadcast and highlight polypeptide. On the other hand, such a modification can be obtained by chemical or synthetic receipt of the polypeptide according to the invention. These modifications using known methods can be used with the intention to alter, preferably improve, the pharmacological activity of mistletoe lectin.

In another preferred embodiment, the polypeptide according to the invention is fused protein. Fused protein has mainly above a certain biological activity.

This version of the proposed polypeptide is primarily meant to change or preferably improve the pharmacological properties of the polypeptide of mistletoe lectin with respect to its directional effects at the cellular level.

Further, the invention relates to a dimer of a polypeptide with the biological activity of mistletoe lectin, both monomer are encoded by nucleic acid molecules according to the invention.

The term "biological activity of mistletoe lectin" refers to any biological activity of the spectrum vehiclevehicle action of mistletoe lectin.

In many lines of human tumor cells and animal vegetable ML-1 caused cell death through mechanisms of apoptosis (Janssen, 1993). ML-1 or In-circuit induced the secretion of cytokines from peripheral mononuclear cells of healthy individuals served as blood donors (Hajto, 1990). The neutrophilic granulocytes of patients with cancer of ML-1 induced secretion of superoxide ions (Timoshenko, 1993). ML-1 induced the expression of the a-chain of the receptor for interleukin-2 (CD25) or antigen HLA-DQ in peripheral lymphocytes of healthy individuals served as blood donors (Beuth, 1992). After applying ML-1 mice had a marked increase in the number of cells of the thymus, the number of cytotoxic T-lymphocytes (Lyt-2+) and T cells-helper cells (L3T4+) in the thymus and the number of peritoneal macrophages, namely those who tolerated the activation marker MAC-3 (Beuth, 1994). In addition, we have increased the ratio L3N4+/Lyt2+ in the thymus of the investigated animals. In the peripheral blood of mice after treatment ML-1 was generally increased density of leukocytes, lymphocytes, monocytes and that of lymphocytes that Express the receptor of interleukin-2 as a surface marker of cell activation, as well as monocytes, which Express the activation marker MAC-3 (Beuth, 1994). In the blood of patients with cancer use ML-1 was found to increase endogenous opiate mediator-endorphin in plasma of patients with breast cancer (Heiny, 1994). In addition, it was found that ML-1 enhances cytotoxic activity of peripheral cells of natural killer cells against tumor cells K-562 and density of large granular lymphocytes (LGL) in the peripheral blood (Hajto, 1989). Was confirmed antimetastatic activity of ML-1 in the cells of sarcoma of mice (Beuth, 1991).

In another preferred embodiment, a dimer of a polypeptide according to the invention detects the same spectrum of biological activity as the natural dimer of mistletoe lectin.

(V) the Biological activity of recombinant mistletoe lectin

Getting holoprotein rML using independently synthesized by recombinant single chain was carried out on the basis of havingthe structure of the soluble chains or denaturirovannykh a - and b-chains rML in joint formationstructure, and rMLB was mainly reassociation in vitro with a molar excess of rMLA in the presence of a redox system of glutathione and partially in the presence of dyslipidemias. Corresponding to heterodimer holoprotein rML was isolated from reas-agarose to obtain free rMLA and rMLA dimers. A similar approach has been rMLA/rMLB (rML) and holoprotein of heterodimeric rMLA/nMLB.

Cytotoxic activity

Cytotoxic effect, as an example of biological activity reassociating of holoprotein, was studied on cell line monocytic leukemia (MOLT4). In-circuit (surface binding), as well as the a-chain (enzymatic inactivation of ribosomes) make a definite contribution to the observed cytotoxic effect. Reassociating in vitro holoprotein rMLA/rMLB and reassociating in vitro holoprotein rMLA/nMLB was compared with the "double" natural holoprotein nML. Recombinant holoprotein rMLA/rMLB and rMLA/nMLB are characterized by comparable high cytotoxic properties with values IR50about 10-30 PG/ml (Fig.11), indicating that functional integrity and biological activity reassociating in vitro recombinant circuits included in holoprotein rML. To achieve cytotoxic activity must objinit functionally active In-circuit with enzymatically active A-chain, because a dedicated In-circuit rML itself unexpectedly did not show cytotoxic activity. Identified to date cytotoxic activity of herbal medicines In-chains Les nML. When obtaining the individual circuits with the use of recombinant DNA technology for the first time, this results in the possibility of independent study and application of activity inherent mistletoe lectin in relation to the binding of carbohydrates and its enzymatic activity.

The induction of apoptosis

The ability to induction of apoptosis as an example of the biological activity of mistletoe lectin was detected for recombinant holoprotein rML on cell line U937 monocytes. Thus, when processing cells 70 PG/ml rML 24 hours were able to detect the induction of apoptosis under the action of holoprotein rML (Fig.12). In studies of mistletoe lectins on cells MOLT-4 and mononuclear cells of peripheral blood (RVMS) it was shown that the basis of the cytotoxic activity of mistletoe lectin by using low doses is the induction of apoptosis. Because in the area of concentration, low cytotoxicity, is the induction of the cytokine (Fig.13, Fig.14), the correlation of cytotoxicity with inducing apoptosis activity is quite understandable. On the contrary, in high doses and more prolonged incubation, apoptosis is blocked necrotic effects. Due to the fact that the cytotoxicity as a result, Biologicheskaya activity, characterized by the induction apollosa in low doses, can be explained only by the action of holoprotein.

Immunostimulirutuyu activity

Immunostimulating effect as an example of the biological activity of recombinant holoprotein of mistletoe lectin was detected by induction allocation factor-tumor necrosis (TNF-and-interferon (IFN-human mononuclear cells of healthy blood donors (model RVS), as well as by induction of interleukin-1(IL-1) and interleukin-6 (IL-6) cocultures human primary keratinocytes and dermal fibroblasts (skin model2ZK1200). Thus, the model RVS using recombinant holoprotein rML concentration 3-48 ng/ml demonstrated a dose-dependent secretion of TNF-and IFN-and on the model skin2when the concentration of the recombinant holoprotein rML 0.25 to 8 ng/ml shows the dose-dependent secretion of IL-1and IL-6.

All these cytokines I have to activate first of all cellular immune response.

Contrary to the prevailing view, according to which immunostimulirutuyu activity is mainly to lectinology activity of the b-chain (Hajto and others, 1990), using only one recombinant b-chain rML could not induce the above-mentioned release cytokines. When using low doses immunostimulirutuyu activity could be achieved only at the expense of functionally active holoprotein rML. This unexpected result allows us to conclude that the immunostimulatory drugs of plant In-circuit nML contained traces of holoprotein nML and the prescribed In-circuit nML immunostimulatory effects due to residual contents of nML. The previously described methods of obtaining nMLB is not possible to determine the number of holoprotein, while upon receipt of a separate chain of mistletoe lectin by recombinant introduced the ability to research and obtain a homogeneous preparations of b-chain of mistletoe lectin. This allowed for the first time separately to learn and use the biological activity inherent in the a - and b-chains, and also to distinguish between biological activity which is peculiar to the individual circuits and functionally active holoprotein.

(VI) Biological activity, Pris is to investigated the induction of protein cell surface CD69. CD69 acts as one of the first of antigens on the cell surface upon activation of T-cells, b-cells and primarily "natural killer cells" (NK-cells). CD69 is a while the activation token above immunocompetent cell populations, as the protein on the cell surface is not expressed by lymphocytes without their induction. In addition, the induced surface protein CD69 was detected function, providing the cytolytic activity of NK-cells and T-cell TcR/(Floors are only and others, 1991). Using flow cytometry (FACS) using monoclonal antibodies to CD69 when using concentrations of 1-100 ng/ml was able to install the activation of mononuclear cells as the emergence of CD69 on the cell surface, as well as the percentage positive for CD69 cells. This revealed a bell-shaped curve depending on the dose, which indicates that cross-linking of cell receptors with both binding sites of the ligands, which is In-circuit rML. However, the cytotoxic effect of the target in this case RVMS could not be detected when using higher concentrations of rMLB 100 ng/ml.

In the preferred Eski or enzymatically modified polypeptide according to the invention or one fused protein according to the invention.

As a result of modifications can be achieved as the optimal gain activity, and by eliminating the influence of individual characteristics on the activity (for example, at the point of attachment of carbohydrates in In-circuit or on glycosidase activity a-chain) to expand the possibility of therapeutic application, which excluded the possible side effects. Polypeptides with modified properties can also serve as a tool to identify mechanisms of activity. For certain types of therapy are sometimes necessary to decrease the antigenicity and immunogenicity of the polypeptides and/or optimization of their pharmacokinetic properties that can be achieved by targeted replacement of individual amino acids.

In addition, the invention relates to antibodies or their fragments or derivatives that specifically bind the polypeptides according to the invention and/or dimer polypeptide. But they do not recognize the natural mistletoe lectin or a separate circuit. Preferably the antibody according to the invention associated with imitators that are masked behind due to glycosylation of the natural mistletoe lectins. Antibodies may be monoclonal, polyclonal or semi-synthetic antibodies. Fragments ate his slice specifically bind to a polypeptide according to the invention, and/or dimer polypeptide can be obtained by the method described, for example, in the book Harlow and Lane, Antibodies, a Laboratory Manual, CSH Press, Cold Spring Harbor (1988). Monoclonal antibody can be obtained, for example, by the method first described in articles Kohler and Milstein, Nature, 256, 495 (1975), and Galfre, Meth. Enzymol., 73, 3 (1981).

The invention further relates to a method for producing a polypeptide or a dimer of a polypeptide according to the invention, moreover, the body is the master according to the invention are cultured in suitable conditions and produce thus obtained polypeptide or dimer polypeptide.

Specialists in the art known conditions suitable for culturing and separating the polypeptide from the host body. For example, a polypeptide or a dimer of a polypeptide according to the invention can be obtained from the host body with suitable for this purpose system expressii and isolated from the environment. On the other hand, the polypeptides or polypeptide dimers may remain in the cell and to be separated from her. Below is another preferred variant of the method according to the invention.

To highlight rMLA performed a full destruction of E. coli cells, transformed with soo is stripperboy. Analysis of cell fractions showed that depending on the conditions of expression and duration of expression from 5 to 50% of the recombinant a-chain of mistletoe lectin is accumulated in a soluble form or from 50 to 95% is accumulated in the form of insoluble protein accumulations ("Taurus enable").

The presence of soluble and insoluble proteins, when possible destructionstructure or denaturate proteins rMLA, provides at least two ways to select rMLA. Aggregated with calves on" rMLA after washing the precipitate to remove proteins from E. coli (Babbitt and others, 1990) was dissolved in denaturing conditions and were destroyedthe structure by 90-fold dilution in an appropriate buffer (50 mm Tris-HC1, 2 mm dithiothreitol (DTT), 1 mm ethylenediaminetetraacetic acid (edtc), pH 8.0.

After this procedure, receive, on the one hand, soluble with-the structure of proteins, as shown in Fig.9, and showing the full enzymatic activity denaturirovannyj types rMLA that the source was insoluble and denaturirovannyj. Selection denaturirovannah rMLA can be performed by the same method, motorla TA, specific to MLA (Tonevitsky and others, 1995).

Due to the existence of rMLB as in soluble form and in the form of insoluble Taurus enable there are two methods of isolation of recombinant b-chain of mistletoe lectin.

For allocation from the cytoplasm of E. coli soluble b-chain rML, with a strong recovery properties for building internal disulfide bridge, the cells were incubated in the presence of a redox system with the use of reduced and oxidized glutathione, and in the presence of-lactose ligand to stabilize functionally active after formingstructure products. Of a mixture for formingstructure selectively in one stage were isolated, respectively, were purified functionally active, linking the carbohydrate chain rML using affinity chromatography on lactose-agarose or N-atsetilgalaktozamin-agarose.

To highlight rMLB of insoluble presented in the form of "Taurus enable" faction of the expression product after appropriate treatment of E. coli cells, the precipitate was washed to remove proteins from E. coli (Babitt and others, 1990) and was dissolved in the preparatory system, using reduced and oxidized glutathione, and in the presence of-lactose ligand, while functionally active, linking the carbohydrate chain rML was selectively isolated from the mixture after renaturation and purified using affinity chromatography on N-atsetilgalaktozamin-agarose or lactose-agarose.

The invention also relates to a medicinal product containing the polypeptide according to the invention or a dimer of a polypeptide according to the invention and/or below the immunotoxin according to the invention optionally together with a pharmaceutically acceptable carrier.

The proposed polypeptides, their associates or modifications suitable for various uses in the treatment of cancer and infections in accordance with known pharmacological properties inherent in natural mistletoe lectins. Immunostimulating effect is used in the treatment of tumors, when the direct and/or indirect stimulation occurs homologous immune protection, providing effective against tumor and possible metastases. The same thing happens in the case of infections, especially viral diseases. The polypeptides according to the invention may that is stimulating the formation of colonies of bacteria, that allows you to achieve synergistic effects, respectively, to reduce neobhodimo dose combine components and, thereby, reduce side effects.

In combination with cytotoxic drugs or irradiation is achieved by mitigating or reducing side effects of radiation/mielosupression, so with these generally accepted in the present methods of treatment restored purchased the weakening of the immune system.

Direct cytotoxic effect of polypeptides with glycosidase activity leads to apoptosis of tumor cells and can also be used in therapy. The specificity of action of the polypeptides can be achieved through the use of immunotoxins, when the polypeptides according to the invention is associated with the corresponding antibodies. Thus, the invention further relates to immunotoxins that are associated with at least one polypeptide according to the invention or a dimer polypeptide. For example, when using the methods that are used in the chemistry of proteins, can bind functionally active a-chain or holoprotein with the antibody or its fragment. These linking techniques known in the art, with the corresponding the m is a design fused proteins which contain the antigen binding domains, for example, an antibody, and optionally cytotoxic fragments of the polypeptide according to the invention.

Further metastasis can be prevented as a result of inhibition of binding of tumor cells to other cells. This binding can be prevented by using the polypeptides according to the invention through competitive binding lectin.

The invention further relates to a primer and/or a pair of primers that/which specifically hybridizes with the nucleic acid molecule according to the invention or complementary to her thread.

In addition, the invention relates to diagnostic compositions, which contain at least:

a) a nucleic acid molecule according to the invention;

b) primer and/or a pair of primers that/which specifically hybridizes with the nucleic acid molecule according to the invention or her with complementary thread; and/or

C) the polypeptide according to the invention and/or a dimer of a polypeptide according to the invention.

The proposed diagnostic composition comprising in a preferred embodiment of the invention the primer or pair of primers can be used to study the organism to predmestnaya from a pharmacological point of view, the interest of the lectins. Contained in the diagnostic composition according to the invention, the nucleic acid molecule may also be used, for example, southern blotting or Northern-blot to study the organisms on the subject of the existence of the corresponding genes lectin. Then the degree of hybridization of the strands is possible to identify genes related lectin gene. Dimer polypeptide can be, for example, used to produce antibodies or antisera, using known methods in different organisms can be detected, for example, the corresponding lectins from mistletoe.

The invention also relates to the means of protection of plants containing the polypeptide according to the invention and/or a dimer of a polypeptide according to the invention. The proposed polypeptides, their associates or modifications can be used to protect plants in accordance with their discussed below functions peculiar to the plant lectin mistletoe. For the protection of plant feed is taken into account inherent mistletoe lectin function, based on the properties of toxicity, as well as for antiviral protection, based on the property that affect the permeability and structure of the membranes. On the enclosed

in Fig.2 - the primary product of amplification ML Viscum album;

in Fig.3 - cloning strategy to obtain gene ML;

in Fig.4 - insert for rMLA and rMLB in expression vectors and full gene sequence ML;

in Fig.5 is a diagram of the construction of a vector expressing rMLA;

in Fig.6 is a diagram of the construction of a vector expressing rMLB;

in Fig.7 - expression of rMLA, rMLB and immunological detection;

in Fig.8 - chromatofocusing with isoelectrofocusing rMLA and rMLB relatively natural ML;

in Fig.9 - enzymatic activity rMLA (PIR);

in Fig.10 - characteristic in relation to the binding of carbohydrates rMLB;

in Fig.11 - cytotoxicity rML towards MOLT4;

in Fig.12 - the induction of apoptosis using rML;

in Fig.13 - immunostimulatory effects of recombinant mistletoe lectin on the model RVS;

in Fig.14 - immunostimulatory effects of recombinant mistletoe lectin on the model skin2;

in Fig.15 - induction of the cell surface marker CD69 in RWMS.

Below the invention is illustrated by examples.

Example 1

Building primary oligonucleotides by amplification

The mistletoe lectin (ML) belongs to the class of ribosome inactivating proteins (Stirpe and others, 1992), which represent the under activity of its constituent subunits was classified as ribosome inactivating proteins type II (Endo and others, A).

However, to determine the sequence of the gene ML unsuitable frequently used in other cases, the mixture obtained by screening the cDNA library and genomic library Viscum album. So, in the libraries of genes from poly(A+)-RNA Viscum album despite the use of different DNA probes were not able to identify specific ML clones. Based on the assumption that the sequence of the gene ML does not contain introns, was applied PCR strategy. Since N-terminal amino acid sequence of the a-chain ML, and In-circuit ML were known (Dietrich and others, 1992; Gabius and others, 1992), it was possible to amplify the coding MLA plot using degenerate oligonucleotides, the resulting amplification of genes of known peptides (Fig.1a). While suitable oligonucleotide with a slight degree of degeneracy can be derived from the N-terminal a-chain ML (RMLA1, Fig.1B), to build the appropriate oligonucleotides with satisfactory specificity on the basis of the N-end In-circuit ML is impossible (RMLB1, RMLB2, RMLB3, Fig.1B).

Therefore, it was necessary to develop an alternative strategy, which would allow for attraction information related proteins using amplification to get about is Telenesti feast of type I and type II showed a number of conservative sites with high sequence homology. In Fig.1B shows a high degree of affinity feast of type I and type II for example, the active site of ricin. In the literature part A and R180 are inside a sequence MISEAARF, enzymatic mechanism (Kim and others, 1992; Lord and others, 1994). Hence it was concluded that at least these two residue may be present in the sequence ML. As a result of further studies of the structure about the availability of individual residues, which are particularly taken into account the residues having a low degree of degeneracy used codon, were obtained using amplification design of the oligonucleotide RMLA2. The sequence of this oligonucleotide is shown in Fig.1B.

Example 2

Obtaining DNA fragments. specific gene ML

High molecular weight genomic DNA was isolated from fresh leaves of Viscum album (the tree-owner Populus wilsonii) according to the method of Baur and others (1993). To obtain specific gene ML of DNA fragments by PCR in each amplificare mixture was injected at 100 ng genomic DNA. Amplification was performed in a total volume of 50 μl containing buffer for PCR (10 mm Tris-Hcl, 1.5 mm MgCl2, 50 mm KCl, 0.25 mm of deoxynucleosides (dNTP), pH 8.3), 78 pmole primer RMLA1 and 50 pmoles (mix 2) or 100 pmoles (mix 1) RM: denaturation for 1 minute at 90°C annealing for 1 minute at 50°C, elongation for 1 min at 72°With a total of 30 cycles. Amplification products were analyzed by electrophoresis in 5% polyacrylamide gel and staining ethidiumbromid (Fig.2). Obtained in a mixture of 2-specific product amplification length of about 500 base pairs (p. N.) was suirable from the gel and cloned into TA vectors.

Example 3

The strategy of cloning

The origin used for the primary PCR amplification oligonucleotides described in example 1 (Fig.1A), and obtain the initial fragment of the gene ML Viscum album, later identified as a, shown in example 2 (Fig.2). Based on the sequence of the cloned gene fragment and assuming that the gene ML has no introns, you can get 5’-oligonucleotides with specific sequences, with which it is possible to amplify fragments b, C, d and E. while the 3’oligonucleotide for with was also derived from the DNA sequence of the fragment and the construction of degenerate 3’-primer for amplification of gene fragments b, d, e and g was performed using analysis of homologous sites PIR type I (b) and class II (d, e, g). In this case again, based on comparison of sequences within the sustained fashion the degree of degeneracy used codon. In particular, to build about 50 combinations of oligonucleotides specific to ML, were used known cDNA sequence and derived protein sequence of the ricin and abrin. In Fig.3 shows only those fragments of the gene, which could be cloned as specific amplification products and could be presented for further analysis. Based on other combinations of oligonucleotides could not be obtained any specific ML amplification products. Obtaining gene fragments f (encoding A-chain ML) and g (coding In-circuit ML) described in example 5 and example 6.

For analysis of the 5’- and 3’-end sections translated and untranslated regions of the gene sequence of ML have been developed conditions for rapid amplification of 5’- and 3’- ends using PCR (method RACE) (Frohmann and others, 1988), which resulted in the receipt of fragments h, i and j. Amplification of fragment j using (RACE)-PCR is an alternative to obtain fragments of the full gene MLB. Reaction using RACE was performed using cDNA, which was obtained by reverse transcription total RNA Viscum album, isolated from the leaves of mistletoe (wood-owner Populus wilsonii).

Prima pT7-MLA and pT7-MLB, were sequenced by standard methods using the strategy of "wandering seed" (the definition of fully overlapping sequences of both strands) using different specific ML for oligonucleotides (Fig.4A,b). The underlined parts of the sequences indicate restriction sites for cloning into the expression vectors RT. Both fragments of the gene are modified in accordance with the scheme for the construction of expression vectors as described in example 5 and example 6.

In Fig.4B is given on the basis of these fragments of the full sequence of the gene ML. It covers both 5’- and 3’-noncoding sites and plots, encoding antipeptide and signal peptide.

Example 5

Construction of expression vector pT7-MLA

For heterologous expression based on a comprehensive genomic DNA mistletoe using specific PCR was obtained sequence encoding the a-chain of mistletoe lectin, and modified at the ends. Using complementary sites built oligonucleotide primers were attached elements controlling the transmission, and the sequence recognized by restriction endonucleases, resulting from presented in the white crystals.

In Fig.5B presents the receipt using the PCR fragments of the gene encoding the MLA. For amplification of the gene sequence, the coding MLA, used 200 ng of genomic DNA Viscum album, 1.5 mm (mixture 1) or 2.5 mm (mix 2) MgCl240 pmoles each oligonucleotide primer RML16 and RML17 in the buffer for PCR (10 mm Tris-HCl, 50 mm KCl, 0.25 mm each of dNTP, pH 8.3) in a total volume of 50 µl. PCR was performed using Taq polymerase (1,5%/mixture company Boehringer Mannheim) in a total of 30 cycles with a temperature of 94°With denaturation for 1 min, with a temperature of 52°C annealing for 1 min, with a temperature of 72°With elongation for 1.5 minutes. Amplification products were analyzed by electrophoresis in 1% agarose gel and color ethidiumbromid (Fig.5B) and was suirable from the gel and cloned into TA vectors.

5’-terminal site of the coding rMLA sequence corresponding to amino acid residues tyrosine1-tyrosine17(Dietrich and others, 1992; Gabius and others, 1992), was obtained by embedding codon that initiates translation, in the form of a synthetic gene fragment by hybridization and cloning of two oligonucleotides. Thus was obtained the optimum regarding selection of codons sequence gene gene rMLA, as well as the 5’-end of the obtained PCR fragment of the gene rMLA by the directed exchange of the codon for tyrosine17TAS on TAT has introduced a restriction site that is recognizable by the restriction enzyme Ssp I, which contributed to the joint of the two fragments of the gene rMLA upon receipt of the vector pML14-17 (Fig.5).

Received the full sequence encoding rMLA, was sequenced (Fig.4A). For the expression of rMLA in E. coli were isolated gene sequence of the vector pML14-17 and have been built into the expression vector RT-7 under the control of T7 promoter-RNA polymerase, and transcription terminator. Using the resulting vector pT7-MLA expression (Fig.5) transformed E. coli strain BL21.

Example 6

Build expressing vector pT7-MLB

For heterologous expression of the b-chain of mistletoe lectin full sequence encoding MLB was amplified from complex genomic DNA Viscum album by specific PCR, while using complementary part built oligonucleotide primers were introduced elements that control the transmission, and the sequence recognized by restriction endonucleases.

In Fig.6b presents the receipt by PCR only a fragment of the gene encoding full rMLB. Amplification folami oligonucleotide primer RML25 and 30 Polemi (mixture 1) or 10 Ptolemy (mix 2) oligonucleotide primer RML26 in total volume of 50 µl buffer for PCR (10 mm Tris-HCl, 50 mm KCl, 1.5 mm MgCl2, 0.25 mm of each dNTP, pH 8.3). PCR was performed using Taq polymerase (1.5 U/mixture company Boehringer Mannheim) in 30 cycles including denaturation for 1 min at 94°C, annealing at 52°C for 1 min and elongation at 72°C for 1.5 min. Products obtained by PCR were analyzed by electrophoresis in 1% agarose gel and staining ethidiumbromid. The result obtained PCR product with a length of 0.8 T. p. H., which was suirable from the gel and cloned into TA vectors. By inserting the expression vector RT-7 trevali fragment of the gene encoding rMLB, under the control elements controlling transcription, and using the resulting expression vector pT7-MLB transformed E. coli strain BL21. The accuracy obtained using PCR sequence that encodes a full rMLB, was confirmed by sequencing (Fig.4B).

Example 7

Expression, immunological detection, destructionstructure and reassociate rMLA and rMLB in vitro

(I) Expression of rMLA in E. coli

For expression of recombinant a-chain of mistletoe lectin in 1000 ml LBAmpinto a 2-liter flask shaking with baffles sown 5 ml grown in stationary conditions on the LAmp-Sudani by measuring turbidity at 578 nm. Gene expression was induced upon reaching the density of cells corresponding to an optical density of about 0.9-1.0 at 578 nm, the addition of 0.5 mm isopropylthioxanthone (IPTG). To collect product cells 2 hours after induction besieged by centrifugation for 20 minutes at 5000 rpm./min and 4°C in the centrifuge GS-3 (Sorvall) and decantation with culture medium of 1 l volume of culture allocated 3-4 g of wet cell mass.

Cells were processed using a belt filter press French press) (firm SLM Instruments), which precipitate the cells re-suspended in 20 ml of buffer for processing (50 mm Tris-HCl, 100 mm NaC1, 1 mm etc, 5 mm DTT, 1 mm phenylmethylsulfonyl (PMSF), pH 8.0 and twice passed through the French press under pressure 105,45 kg/cm2. Subsequent centrifugation for 30 minutes at 10000 rpm./min and 4°C in the centrifuge, SS-34 (Sorvall) besieged the insoluble components of the cells, and contained "Taurus enable" and was separated from remaining in the supernatant of soluble E. coli proteins or soluble products expression.

For analysis of expression of the same volumes of processed fractions of cells was analyzed by electrophoresis in a 12.5% polyacrylamide gel Pris is specific to MLA antisera TO (Fig.7a). Monoclonal antibodies TO (Tonevitsky and others, 1995) were provided by the author. As used in this case, antibodies, get them in standard ways using appropriate immunogen (if TO this ML-1 or MLA). To determine the expression equal amounts of soluble fraction (track 2, 4, 6, 8) and insoluble fraction with "calves enable (track 1, 3, 5, 7) of the processed mass of E. coli were investigated on the content of the rMLA. To study the nature of the flow expression was injected probes before induction (track 1+2), 2 hours (track 3+4), 4 hours (mark 5+6) and 6 hours (mark 7-8) after induction of gene expression. Expression was observed within 1 hour after induction by the appearance of immunoreactive expression product in 25 kDa, corresponding rMLA, the maximum expression of which is reached in 2 hours after induction. Distribution rMLA in the soluble or insoluble fractions of treated cells every 2 hours after the induction of approximately 50%, with a longer expression leads to increased formation of insoluble Taurus enable".

(II) the Allocation of rMLA of insoluble Taurus enable"

Precipitate all of the treated E. coli cells to remove proteins from E. coli double-according Babitt and other (1990). The remaining residue with calves on" was dissolved in 20 ml of buffer to denature (6M guanidine hydrochloride, 100 mm DTT, 50 mm Tris-HCl, pH 8.0) followed by incubation for 16 hours at room temperature with shaking.

For renaturation of rMLA available protein solution in buffer denaturation was slowly added dropwise at 90-fold volume of buffer for formingstructure (50 mm Tris-HCl, 2 mm DTT, 1 mm add, pH 8.0) and incubated for 16 hours at room temperature and under stirring. Again precipitated precipitated protein was separated by centrifugation for 30 minutes at 6000 rpm./min and 4°C in the centrifuge GS-3 (Sorvall). Containing rMLA supernatant for storing poured in 20% (vol/vol) glycerol and stored at 4°C.

(III) Purification of rMLA using immunoaffinity chromatography

For one-step purification rMLA (soluble part of the expression product, respectively, of the protein obtained after the destruction of the-patterns) using immunoaffinity chromatography 200 µg monoclonal antibodies anti-nMLA-IgG to the a-chain of mistletoe lectin (TA, Tonevitsky and others, 1995) was immobilized on protein-a-sepharose CL4B (Sigma, Deisenhofen) according to the method Hariow and Spur (198 fatny buffer, pH 7.0) in an amount corresponding to 10 volumes of column filler, and a buffer for washing 2 (10 mm phosphate buffer, pH 7.0), corresponding to 10 volumes of column filler, specifically related rMLA was suirable corresponding buffer (0.1 M glycine, pH of 2.5) to remove nonspecific related proteins. Elution was carried out to restore the pH Preboot in 1M phosphate buffer, pH 8.0.

(IV) Expression of rMLB in E. coli

For expression of recombinant b-chain of mistletoe lectin in 1000 ml LBAmpinto a 2-liter flask shaking with baffles sown 5 ml grown in stationary conditions on the LAmpenvironment pre-culture of E. coli BL21/pT7-MLB and cultivated at 37°C and with shaking, the growth was observed by measuring the turbidity at 578 nm. Gene expression was induced upon reaching the density of cells corresponding to an optical density of about 0.9-1.0 at 578 nm, by adding 0.5 mm IPTG. To collect 4 hours after induction besieged by centrifugation for 20 min at 5000 rpm./min and 4°C in the centrifuge GS-3 (Sorvall) and decantation with culture medium.

Treatment of cells was performed using the French pressTM(firm SLM Instruments), which sieges is f, pH 7,2) and double-passed through the French press under pressure 105,45 kg/cm2. Subsequent centrifugation for 30 min at 10000 rpm./min and 4°C in the centrifuge, SS-34 (Sorvall) besieged the insoluble components of the cells and parts, containing "Taurus enable", and was separated from remaining in the supernatant of soluble E. coli proteins or soluble expression product.

To confirm the expression of equal volume fractions of treated cells was analyzed by electrophoresis in a 12.5% polyacrylamide gel in the presence of LTOs and staining Kumasi brilliant blue, and by Western blotting using specific MLB antisera TVSS (Fig.7b). Monoclonal antibodies TV (Tonevitsky and others, 1995) were provided by the author. They were obtained using standard methods. Appropriate antibodies ML-1 and MLB can also be obtained by standard methods with ML-1 or MLB as immunogen. To confirm the expression of equal volumes of soluble fraction (track 2, 4, 6, 8) and insoluble fraction with "calves enable (track 1, 3, 5, 7) product processing E. coli were investigated on the content of rMLB. To study the nature of the flow expression was injected probes to induction of expressi the turn-blotting showed the appearance of bands of immunoreactive protein (31 kDa), the corresponding rMLB, within 1 hour after induction, and the maximum of its expression was achieved after 4 hours after induction. In each case, 4 hours after the induction of approximately 50% rMLB was in soluble and about 50% in the insoluble fraction obtained after treatment of the cells. Longer incubation will lead to greater accumulation expressed rMLB in the form of insoluble Taurus enable".

(V) the Allocation of rMLB insoluble Taurus enable"

Precipitate all of the treated E. coli cells to remove proteins from E. coli was twice washed with 20 ml of STAT-buffer (50 mm Tris-HCl, 8% (weight/volume) sucrose, 50 mm etc, 1,5 vol.% Triton X-100, pH 7.4) according to the method Babbitt and others (1990). The remaining sediment with it contained "calves inclusion was dissolved in 20 ml of buffer to denature (6M guanidine hydrochloride, 100 mm DTT, 50 mm Tris-HCl, pH 8.0) followed by incubation for 16 hours at room temperature with shaking.

For renaturation of rMLB available protein solution in buffer denaturation was slowly added dropwise at 90-fold volume of buffer for formingstructure (20 mm phosphate buffer, 50 mm KCl, 1 mm etc, 100 mm glucose, 10% vol. glycerol, 10 mmstructure, by centrifugation for 30 minutes at 6000 rpm./min and 4°C in the centrifuge GS-3 (Sorvall).

(VI) the Allocation of rMLB by affinity chromatography on N-acetyl-galactosamine-agarose

To select the active linking carbohydrates rMLB affine matrix with N-atsetilgalaktozamin-agarose (PIERCE, USA) was balanced buffer chromatography (50 mm phosphate buffer, 300 mm NaCl, 1 mm add, 10% vol. glycerin, 0,05% vol. Twin®-20, pH 7.0) in a volume corresponding to 10 volumes of column filler. Sample inflicted "portions" by incubation affine matrix with a solution sample containing the rMLB, for at least 2 hours at 4°C. After washing the affinity matrix buffer chromatography to remove nonspecific related proteins associated rMLB was suirable by competitive substitution of 0.3 M N-atsetilgalaktozamin in the buffer for chromatography at pH 4.0.

(VII) Reassociate chain of mistletoe lectin to obtain holoprotein in vitro

The preparation of recombinant holoprotein of mistletoe lectin (rML) can be done using both a - and b-chains, the resulting allocation and formed the simulationstructure denaturirovannykh a - and b-chain of mistletoe lectin. For reassociation separate circuits, the resulting allocation and withstructure allocated In a-chain of mistletoe lectin (nMLB or rMLB) were incubated with a molar excess of rMLA in 20 mm phosphate buffer, 50 mm NaCl, 1 mm add at pH 7,2 within 16-48 hours at 4°C. the Formation of disulfide bonds between the filaments contributes incubation in the presence of a redox system of 6 mm glutathione (the ratio of the recovered oxidized to 5:1 or 10 mm of glutathione (the ratio of the recovered oxidized to 2:1) and 1 µm protein disulfide-isomerase (Boehringer Mannheim).

For reassociation based on denaturirovannykh separate circuits within the joint formationstructure was dissolved And chain rML in the 6M guanidine hydrochloride, 2 mm DTT, 50 mm Tris-HCl, pH 8.0 to a concentration of 2 mg/ml For a full recovery of the cysteine residue In the chain rML was dissolved in the 6M guanidine hydrochloride, 100 mm DTT, 50 mm Tris-HCl, pH 8.0, then after incubation for 20 minutes at room temperature sauterelle 6M guanidine hydrochloride, 50 mm Tris-HCl, pH 8.0, using gel permeation chromatography n the tion formationstructure rMLB with a molar excess of rMLA by incubation for 16 hours at 4°C in the mixture obtained by slow dilution (1:30) a solution of guanidine salt in the buffer for binding (50 mm matrifocality buffer, 50 mm KCl, 1 mm add, 10% vol. glycerol, 100 mm glucose, 20 mm lactose, pH 8.0). For the formation of disulfide bridges between threads incubation was carried out in the presence of a redox system of 2 mm glutathione (the ratio of the recovered oxidized to 1:1).

The mixture resulting from binding, dialyzed in a counter buffer for storing (50 mm matrifocality buffer, 300 mm NaCl, 1 mm add, 10% vol. glycerin, 0,05% vol. Twin®-20, pH 8.0). The definition and identification of the educated heterodimer was performed using electrophoresis in SDS page-ordinator in nevosstanovlenie conditions with subsequent Western blotting using monoclonal antibodies specific for the a-chain of mistletoe lectin (TA) or b-chain of mistletoe lectin (TV). The selection of the formed holoprotein or compartment free rMLA or clusters rMLA was performed using affinity chromatography on N-atsetilgalaktozamin-sepharose or lactose-agarose, as described in section (VI).

Example 8

Isoelectric protein, used as a standard when isoelectrofocusing (IEF) (BioRad, USA) in IEF-gels Servalyt PreNets (pH 3-10, 125×125 mm, 150 μm, the company Serva, Heidelberg). For detection of proteins immobilizerpower by "semi-dry electroblotting" on the membranes of nitrocellulose (0.2 μm, Schleicher &Dassel). Immunological staining was performed using monoclonal antibodies to the MLA (TA5, Tonevitsky and others, 1995), specific rMLA and nMLA, or by using monoclonal antibodies to MLB (TV, Tonevitsky and others, 1995), specific rMLB, nMLB and holoprotein ML. The immune complexes were stained using conjugated with alkaline phosphatase antimisting IgG-IgG (firm Sigma, Deisenhofen) and the conversion of the substrates NBT and BCIP (Fig.8).

While high-purity A-chain of mistletoe lectin, as well as In high-purity-chain of mistletoe lectin represented as isoelectric inhomogeneous proteins with isoelectric points nMLA 5,2:5,4:5,5:6,2, respectively nMLB 7,1:7,3, recombinant A-chain rML with isoelectric point of 6.8, as well as recombinant In-circuit rML with isoelectric point of 5.1 is homogeneous protein (Fig.8). In the case of natural holoprotein ML find a large number of satisfied lectin mistletoe proves the homogeneity of the rML compared with microheterogeneity plant lectin mistletoe.

Example 9

Determination of enzyme activity rMLA associated with the inactivation of ribosomes

The concentration of protein rMLA (after the destruction of the-patterns) and rMLA (soluble), treated with immunoaffinity chromatography, as well as natural a-chain ML (nMLA) was determined according to Bradford (1976) using as a standard of bovine serum albumin (BSA).

To determine and quantify the enzymatic pPHK-N-glycosidase activity MLA has developed a non-radioactive test system using "linked TNT system reticulocytes" (Promega, USA). With each of the investigated mixture is pre-incubated for the same amount (20 μl) TNT system for 15 minutes at 30°C. To quantify inhibition broadcast to the test mixtures were added 2 μl of the appropriate buffer or to the investigated mixtures were added 2 μl of increasing dilutions of the MLA (the range of concentrations 350-0 PM). From each mixture with an interval of 8 minutes was collected 2 samples and for stopping the reaction was frozen in liquid nitrogen. As a measure of translational activity in bioluminescent test using a scintillation counter was determined relative amount of luciferase (kvadratichnogo root of the number of counts per minute for both time samples as a measure of the relative translational activity, moreover, the activity of the control mixture without the feast was taken as 0% and the degree of inactivation (MI).

When building the graph of the dependence of the relative degree of inactivation broadcast from the used concentrations rMLA using non-linear regression determined the protein concentration that leads to 50% increase translational inhibition activity compared with the control mixture. This value IR50is a system-dependent value, which allows to identify and quantify the enzymatic activity of rMLA (soluble), rMLA (after the destruction of the-patterns) compared with nMLA (Fig.9).

In Fig.9 shows the definition associated with the inactivation of ribosomes enzymatic activity of the recombinant a-chain ML, and the enzymatic active expression product receive by separating the soluble portion of the product of expression (rMLA soluble), and by destroyingstructure selected from Taurus inclusion protein (rMLA after the destruction of the-patterns). For rMLA (soluble) and rMLA (after the destruction of the-patterns) was vielen activity, characterized value is compared with the natural A-chain ML (IR501,1±0,7 gr).

Example 10

Activity against binding of carbohydrate-chain of mistletoe lectin

Determination of activity against the binding of carbohydrates recombinant B-chain of mistletoe lectin, as well as a comparison of the activity against the binding of carbohydrates and specificity of recombinant and plant b-chain of mistletoe lectin was performed using analysis of lectin-binding enzyme (ELLA) in the presence of competing hydrocarbons. When using the immobilized asianavenue matrix-chain nML and rML was mainly associated with residues of galactose and N-atsetilgalaktozamin, and in the application of the immobilized petrinovic matrix was associated mainly with residues of sialic acid.

For immobilization of carbohydrate matrix 100 μl of a solution of 1.1 mg asianfetish (firm Sigma, Deisenhofen) or 1.1 mg of fetuin (firm Sigma, Deisenhofen) in 11 ml of phosphate buffered saline (SFR) were placed in the wells of tablets for micrometrology C96 MaxiSorp, Nunc, Wiesbaden) and incubated for 16 hours at room temperature. After triple washing tablets 150 µl/well SFR-T (10 mm matrifocality buffer, 130 mm NaC1, 0,1% vol. Twin®-20, pH 7,2) to block swas mm NaCl, 0,1% vol. Twin®-20, 1% (weight/volume) BSA, pH of 7.2) for 1 hour at 36°C and then washed as described above. For tests used 100 μl of preparations containing In-chain, at a concentration of 100-500 ng/ml, preferably 400 ng/ml, while the concentration for the experiment was determined by dilution of the samples in SR-a 0.05% BSA (10 mm matrifocality buffer, 130 mm NaCl, 0,05% (weight/volume) BSA, pH of 7.2). For pre-concentration and control was performed 2-3 repetitions. Determine the value for "blank" experiment was carried out using SFR-a 0.05% BSA or appropriate preparative buffer. To determine the specificity of the binding incubation of the samples were carried out in the presence of free competing sugar. To displace rMLB, nMLB or holoprotein ML of connections on asianavenue or petrinovic matrix was added mainly galactose at a concentration of 0-280 mm, N-atsetilgalaktozamin concentration 0-280 mm, lactose at a concentration of 0-140 mm or sialic acid concentration 0-140 mm.

Tablets after loading were incubated for 2 h at 36°C and then washed as described above. In the completed well) was added 100 μl/well of pig antisera to mistletoe lectin (dilution 1:19800 pooled serum) in SFR-T-0,1% BSA-TX (10 mm acrivastine 2 h at 36°C and then washed, as explained above. For the detection of immune complexes in each of the filled holes were added 100 μl antisinga IgG-IgG conjugated to horseradish peroxidase (firm Sigma, Deisenhofen), at a dilution of 1:3500 in SFR-1% BSA (10 mm matrifocality buffer, 130 mm NaCl, 1% (weight/volume) BSA, pH of 7.2), and incubated for 1 h at 36°C. Then the wells are washed 6 times SPR-T poliziani 150 µl/well. The developer added 100 μl/well. the substrate solution (1 tablet of o-phenylenediamine (Sigma, Deisenhofen) in 25 ml of 65 mm citric acid, pH 5.0+10 μl of 30% hydrogen peroxide), and incubated for 20 minutes at room temperature in the dark. The reaction was stopped by adding 100 μl of 1M sulfuric acid/well. Quantitative evaluation was performed by measuring the absorption at 450 nm with wavelength for the reference 690 nm and the value IR50expected by describing the measurement results using the approximation in 4-parametric function.

In Fig.10 shows the dependence observed in ELLA-system displacement rMLB and nMLB with immobilized asianavenue ligand with increasing amounts of D-galactose (Fig.10A),-lactose (Fig.106) or N-atsetilgalaktozamin (Fig.10B), as well as the displacement with immobilizovannaya CEM and rMLB describes the value IR50corresponding premaxillae displacement.

While when linking carbohydrates of plant In-circuit nML primarily compete galactose (IR50=4.5 mm) and-lactose (IR50=4.9 mm), recombinant B-chain rML finds unexpectedly clearly modified carbohydrate specificity. Unlike nMLB activity of binding carbohydrates rMLB does not apply to galactose (IR50it is impossible to determine) and only to a small extent, applies to-lactose (IR50>70 mm). In addition to dramatically reduced affinity for galactose and-lactose recombinant B-chain rML finds, however, a clear specificity to N-atsetilgalaktozamin (IR50109 mm). Further, as described for nMLB activity of binding ligands with sialic acid was also detected for recombinant b-chain ML (Fig.10 g). For plant b-chain nML (IR5049,8 mm) and recombinant b-chain rML (IR5047,1 mm) is characterized by consistent affinity for binding.

In contrast to the plant In-circuit nML, primarily specific for galactose/-lactose, recombinant In-sepecifically for carbohydrates.

Example 11

Determination of cytotoxicity reassociating of holoprotein rML in the cells of human leukemia in vitro

The purity of holoprotein of mistletoe lectin was confirmed by quantitative determination of cytotoxicity with respect to the line core (lymph) cells human leukemia MOLT-4 (European Collection of Cell Cultures, Animals, catalogue number 85011413).

Cells MOLT-4 were cultured in not containing serum environment MDC-1 (PAN SYSTEMS, Aidenbach) and used in the test with a density of seeding cells 1,6×105cells MOLT-4/ml for the reproducibility of >98%. To determine the cytotoxicity in each well of 96-well plate to micrometrology contributed 90 μl of the suspension of cells MOLT-4, respectively 18000 cells/well and were mixed with 10 μl of a solution of the sample. For the experiment were injected drugs holoprotein of mistletoe lectin (batch No. 220793 (Madaus) and BRAIN 12/94, which were isolated by standard methods from the leaves of mistletoe or tea mistletoe on lactose-sepharose (Franz and others, 1977), as well as reassociating in vitro holoprotein rML in concentrations of 1-100 PG/ml, which corresponds to 1.6 FM-1,6 PM, when it was preparing a series of dilutions in cell culture medium MDC-1. For each concentration of the sample and control is, asystem gas. Quantitative evaluation of cytotoxic effect was performed by determination of cell viability using soluble formisano dye according to the method of WST-1 (Scudiero and others, 1988) using a suitable reagent, causing cell proliferation WST-1 (Boehringer Mannheim).

Recombinant holoprotein as the chimeric holoprotein rMLB/nMLA are against MOLT-4 cytotoxic activity with IR50about 10-30 PG/ml, coinciding with the biological activity of the natural protein. On the contrary, rMLB in the analyzed concentration range (rMLB to 1 ng/ml) did not show any cytotoxic activity.

Example 12

Induction of apoptosis by the example of a cell line of human U937 monocytes with recombinant mistletoe lectin (rML)

Detecting induction of apoptosis under the influence of recombinant mistletoe lectin (rMLA/rMLB) was performed in this case by staining the cell nuclei of the fluorescent dye 4’,6-diamino-2-phenylindole (DUFFY) (Hotz and others, 1992). Typical apoptotic changes of the morphology of the nuclei can be seen through the microscope and quantified by counting 500-1000 cells per sample. The use does not contain serum environment has op is otherwise available number of lectins (for example, 40 times in the case of 10% amniotic calf serum (OPST), Ribereau-Gayon and others, 1995). The induction time in 24 hours makes a conditional direct correlation with the analysis of MOLT, since the cytotoxic effect when analyzing the viability of a fully visible only after 72 hours, however, apoptosis is an early effect. At longer incubation apoptosis is blocked by secondary necrosis.

In Fig.12 shows a clear increase in the share undergone apoptosis of U937 cells after treatment with recombinant holoprotein ML. At 70 PG/ml, the number of such cells increases 3 times in 24 hours for the two different cultures in media containing no serum. Recombinant mistletoe lectin, as well as natural protein have (Janssen and others, 1993) the ability to induce the death of cells due to apoptosis.

Example 13

Immunostimulatory effects of recombinant mistletoe lectin on the model RVS

In the case of cytokines TNF-(monocytes, macrophages) and-interferon (IFN-) (T-cell helpers) refers to the Central mediators in the complex network of the human immune system.

Human mononuclear cells (RVMS, soderi on the centrifuge FICOLL-PAQUE®in accordance with the manufacturer's instructions (firm Pharmacia, Sweden). For the induction selection tnf-cells (4×106mononuclear cells/ml) were incubated in gas incubator in RPMI medium 1640 with 10% vol. amniotic calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin during the first 18 h only with recombinant proteins of mistletoe lectin, and then over the next 24 h with 1 μg/ml of LPS from Salmonella abortus equi as a companion stimulating factor at 37°C in an atmosphere with 5% CO2at a relative humidity of >95% in the U-shaped tablets for micrometrology cell cultures. Then free from the cells supernatant immediately quantitatively evaluated the concentration of TNF-using enzyme-linked immunosorbent assay (ELISA) (Genzyme Diagnostics, rüsselsheim).

For the induction selection IFN-cells were incubated according to the method described above and in the above environment during the first 1 h only with recombinant proteins of mistletoe lectin, and then in the next 65 hours with 0.5 µl/ml of phytohemagglutinin-L as a companion stimulating factor. Then free from CL/img> using ELISA test (ENDOGEN INC., Cambridge, USA).

Example 14

Immunostimulatory effects of recombinant mistletoe lectin on the model skin2-ZK1200

Used as a biological sample model skin2consists of a three-dimensional containing the fibroblasts of the dermis and structured epidermis from non-squamous keratinocytes in belonging to them, naturally selected matrix (Joller and others, 1996). Pieces of skin tissue (11×11 mm2derived from human progenitor cells; Advanced Tissue Sciences, Inc. (ATS), La JOLLA, USA) in each case was placed on a woven lattice of nylon on the agarose and immediately after the load was transferred to the special environment And (ATS, La JOLLA, USA).

IL-1and IL-6 are important cytokines that stimulate the immune system.

To induce the release of IL-1or IL-6 pieces of cloth in the model skin2incubated in each case with the test substance in 2 ml special environment (ATS, La JOLLA, USA) for 24 hours at 37°C in an atmosphere with 5% CO2and at a relative humidity of >95% in 12-hole microplate for cell culture (Corning Glass Works, Corning, USA). After this supernatant, not soda is f" border="0">(set Quintikine for the quantitative analysis of IL-1man, R & D Systems Inc., Minneapolis, USA) or IL-6 (ELISA test for the human interleukin-6, Boehringer Mannheim GmbH).

On the model skin2it was shown in a dose-dependent induced with 0.25 to 8 ng rML/ml excretion of IL-1and also dose-dependent, induced with 0.5 to 8 ng rML/ml excretion of IL-6 (Fig.14). It has been unexpectedly discovered that by using a single recombinant b-chain rML failed to induce the selection of the aforementioned cytokines, which contradicts known to date information, according to which immunostimulirutuyu activity mainly relates to lectinology activity of the b-chain (Hajto and others, 1990).

Example 15

Activation of immunocompetent cells using recombinant b-chain of mistletoe lectin (rMLB)

Activation of immunocompetent cells was studied through induction of protein cell surface CD69. Protein CD69 appears as one of the first of antigens on the cell surface after activation of T cells, b cells and primarily "natural killer cells" (NK-cells), which, thanks to their ability to recognize and lyse the neoplastic cells acquire boleh immunocompetent populations of cells, because this protein on the cell surface is not expressed untreated lymphocytes. In addition, the induced surface protein CD69 was detected function, contributing cytolytic activity of NK-cells and T-cell TcR/(oretta others, 1991).

For detection of surface markers in single human cells by flow cytometry (FACS) was allocated RVMS using density gradient on hipace (sodium salt of 3,5-bis[acetylamino]-2,4,6-triiodobenzoic acid) (firm Sigma) analogously to example 13. After transfer of cells in medium RPMI 160 with 5% of OPT and planting approximately 250,000 cells/well tablet for micrometrology incubation was performed for 4 hours with 1, 10, 30 and 100 ng of the test substance rMLB. After incubation for 20 minutes in a bath of ice with a monoclonal antibody to CD69 containing fluorescent label, was carried out by washing in a solution of Khanka with 5% of OPT. Precipitated labeled cells were placed in 200 μl of "liquid membrane", and fluorescence was measured using a FACScan instrument (Becton Dickinson). On the appropriate drawing shows the average fluorescence in accordance with the number of markers CD69 cell surface to the cell, as well as on the go activation of mononuclear cells as the emergence of CD69 on the cell surface, as well as the percentage positive in respect of CD69 cells. There was observed a bell-shaped curve depending on the dose. At concentrations of rMLB more than 100 ng/ml cytotoxic effect on the studied RWMS was not found.

Sources of information

Babbitt, P. C., West, B. L., Buechter D. D., Kuntz I. D. and Kenyon, G. L. [1990]: Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme Bio/Technology 8, 945-949.

Baur, A., Buschinger, A. and Zimmermann F. K. (1993): Molecular cloning and sequencing of 18S rDNA fragments of six different ant species. Ins. Soc. 40, 325-335.

Beuth j, Ko H. L., Gabius H.-J. and Pulverer G. [1991]: Influence of treatment with the immunomodulatory effective dose of the-galactoside-specific lectin from Mistletoe on tumor colonization in BALB/c mice for two experimental model systems. in vivo 5, 29-32.

Beuth j, Ko H. L., Gabius H.-J., Burrichter H., K. Oette and Pulverer G. [1992]: Behavior of lymphocyte subsets and expression of activation markers in response to immunotherapy with galactoside-specific lectin from mistletoe in breast cancer patients. Clin. Investig. 70, 658-661.

Beuth j, Ko H. L., Tunggal L., M. K. Steuer, Geisel J., J. Jeljaszewicz and Pulverer G. [1993]: Thymocyte proliferation and maturation in response to galactoside-specific Mistletoe Lectin-1. in vivo 7, 407-410.

Beuth j, Ko H. L., Tunggal L., Geisel J. and Pulverer G. [1993]: Vergleichende Untersuchungen zur immunaktiven Wirkung von Galactosid-spezifischem Mistellektin. Arzneim. Forsch. 43, 166-169.

Bocci V. [1993]: Mistletoe (Viscum album) lectins as cytokine inducers and immunoadjuvant in tumor therapy. J. of Biological Regulators and Homeostatic Agents 7,1-6.

Beuth j, Ko H. L., Tungal L., G. Buss, Jeljaszewicz, J., Steuer, M. K. and Pulverer G. [1994]: Immuntities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Dietrich J. B.,Jung M. L., Franz H., Beck J. P. and Anton R. [1992]: the Identity of the N-terminal sequences of the three A chains of mistletoe (Viscum album L.) lectins: homology with ricin-like plant toxins and single-chain ribosome-inhibiting proteins. Anti-Cancer Drugs 3, 507-511.

Eifler R.,[1994]: Improved procedures for isolation and standardization of mistletoe lectins and their subunits: implications for the analysis of lectin pattern of the european mistletoe. In: Lectins Biology-Biochemistry Clinical Biochemistry (Bog-Hansen, Hrsg.), volume 9, M/S Wiley, New Delhi.

Endo y, Tsurugi K., and Franz H. [1988a]: The site of action of the A-chain of mistletoe lectin I on eukaryotic ribosomes. The RNA N-glycosidase activity of the protein. FEBS Lett. 231, 378-80.

Endo y, Tsurugi K., and Lambert, J. M. [1988b]: The site of action of six different ribosom-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins. Biochem. Biophys. Res. Commun. 150, 1032-1036.

Erlich H. A., Gelfand D. H., and Saiki R. K. [1988]: Specific DNA amplification. Nature 331, 461.

Franz H., Haustein Century, Luther P., Kuropka u and Kindt A. [1977]: Isolierung und Charakterisierung von Inhaltsstoffen der Mistel (Viscum album L.) I. Affinittschromatographie an fixierten Plasmaproteinen. Acta biol. med. germ. 36, 113-117.

Frohman, M. A., Dush, M. K., and Martin, G. R. [1988]: Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002.

Gabius, H.-J., Walzel, H., Joshi, S. S., Kruip, J., Kojima, S., Gerke V., Kratzin, H. and Gabius S. [1992]: The immunomodulatory p-galactoside-specific lectin from Mistletoe: Partial sequence analysis, cell and tissue binding, and impact on intracellu-lar biosignalling of monocytic leukemia cells. Anticancer Res. 12, 669-676.

Gabius H.-J. Gabius, S., Joshi, S. S., Koch, C., Schroeder m, Manzke W. M. us S. [1994]: Die Misteltherapie auf dem naturwissenschaftlichen. PZ 139, 9-16.

Ganguly, S. and Das, S. [1994]: Plant Lectins as inhibitors of tumor growth and modulators of host immune response. Chemotherapy 40, 272-278.

Garcia-Olmedo f, Carbonero P., Hernandez-Lucas C, Paz-Ares j, Ponz F., Vicente O. and Sierra J. M. [1983]: Inhibition of eukaryotic cell-free protein synthesis by thionins from Wheat endosperm. Biochim. Biophys. Acta 740, 52-56.

Gribskov, M., Devereux J. and Burgess R. [1984]: Nucl. Acids Res. 12, 539-549.

Hajto T. [1986]: Immunomodulatory effects of Iscador: A Viscum album preparation. Oncology 43 suppl.l, 51-65.

Hajto T., Hostanska K. and Gabius H.-J. [1989]: Modulatory potency of the-galactoside-specific lectin from Mistletoe extract (Iscador) on the host defense system in vivo in Rabbits and Patients. Cancer Res. 49, 4803-4808.

Hajto T., Hostanska, K., Frei, K., C. Rordorf and Gabius H.-J. [1990]: Increased secretion of Tumor necrosis factor a, Interleukin 1, and Interleukin 6 by Human Mononuclear Cells exposed to-Galactoside-specific lectin from clinically applied Mistletoe extract. Cancer Res. 50, 3322-3326.

Harlowe E. and Spur D. [1988]: In: Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Heiny B.-M. and Beuth J. [1994]: Mistletoe extract standardized for the galactoside-specific lectin (ML-1) dosage-endorphin release and immunopotentiation in breast cancer patients. Anticancer Research 14, 1339-1342.

Hotz M., Del Bino g, Lassota p, Traganos f, Darzynkiewicz Z. [1992]: Cytostatic and cytotoxic effects of fostriecin on human promyelocytic HL-60 and lymphocytic MOLT-4 leukemic cells. Cancer Res. 52, 1530-1535.

H., Doser S. and Mechelke F. [1986]: Differences in the in vitro effectiveness of preparations produced from Mistletoion of lectins in Mistletoe preparations. Arzneim.-Forsch./Drug Res. 45, 905-909.

Janssen 0., Scheffler, A. and Kabelitz D. [1993]: In vitro Effects of Mistletoe Extracts and Mistletoe Lectins. Cytotoxicity towards tumor cells due to the induction of programmed cell death (apoptosis). Arzneim. forsch. 43, 1221-1227.

Joller, P. W., Menrad J. M., Schwarz, T.,U., Parnham, M. L., Weyhenmeyer R and Lentzen, H. [1996]: Stimulation of cytokine production via a special standardized Mistletoe preparation in an in vitro skin bioassay. Arzneim.-Forsch. /Drug Res. 46, 649-653.

Katzin, B. J., Collins, E. J. and J. D. Robertus [1991]: Structure of Ricin A-Chain at 2.5Proteins 10, 251-259.

Kim Y., Misna D., Monzingo, A. F., Ready, M. P., Frankel, A. and Robertus, J. D. [1992]: Structure of a Ricin mutant showing rescue of activity by a noncatalytic residue. Biochemistry, 31, 3294-3296.

Lord J. M., L. M. Roberts, and J. D. Robertus [1994]: Ricin: structure, mode of action, and some current applications. FASEB J. 8, 201-208.

D. N., Becker H., Gundt A., Kist, A. and Franz H. [1991]: Induction of tumor necrosis factor expression by a lectin from Viscum album. Cancer Immunol. Immunother. 33, 177-182.

Minowada J., Ohnuma T., Moore, G. E. [1972]: Rosette-forming human lymphoid cell lines. J. Nat. Cancer Inst. 49, 891-895.

Mendez E., Moreno, A., Colilla f, Pelaez f, Limas G. G., Mendez R., Soriano F., Salinas M. and de Naga S. [1990]: Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, g-hordothionin, from barley endosperm. Eur. J. Biochem. 194, 533-539.

Floors are only A. and others [1991]: J. Exp. Med. 172, 701-707.

Ribereau-Gayon g, Jung M.-L., Beck J.-P., Anton R. [1995]: Effect of fetal calf serum on the cytotoxic activity of Mistletoe (Viscum album L.) lectins in cell culture. Phytotherapy Res. 9,336-339.

Rutenber, E., and Robertus, J. D. [1991]: Structure of Ricin B-chain at 2.5Stirpe F., Barbieri L., Battelli, M. G., Soria M. and D. A. Lappi [1992]: Ribosome-inactivating proteins from plants: Present status and future prospects. Bio/Technology 10, 405-412.

Studier F. W. and Moffart B. A. [1986]: Use of Bacteriophage T7 RNA Polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130.

Tonevitsky, A. G., V. A. Rakhmanova, I. Agapov, Shamshiev, A. T., Usacheva E. A., Prokoph'ev S. A., Denisenko N. O., Y. Alekseev and Pfueller U. [1995]: The interactions of anti-MLI monoclonal antibodies with isoforms of the lectin from Viscum album. Immunol. Lett. 44, 31-4.

Vitetta, E. S., Thorpe, P. E., J. W. Uhr [1993]: Immunotoxins: magic bullets of misguided missiles? Immunol. Today, 14, 252-259.

Weston, S. A., Tucker, A. D., Thatcher, D. R., Derbyshire DJ. and Pauptit, R. A. [1994]: X-ray structure of recombinant Ricin A-Chain at 1.8resolution. J. Mol. Biol. 244, 410-422.

Claims

1. A nucleic acid molecule that (a) encodes preproprotein, which after maturation exhibits biological function, characteristic of the dimer of mistletoe lectin, and is shown in Fig.4B nucleotide posledovatelnoy part of the dimer of mistletoe lectin; (C) differs from the nucleic acid molecule according to (a) or (b) due to the degeneracy of the genetic code; or (g) hybridizes under stringent conditions with the nucleic acid molecule according to (a), (b) or (C) and encodes a polypeptide specified in (a) or (b) biological function or activity.

2. The nucleic acid molecule under item 1, where the fragment is the A-chain of mistletoe lectin, which is encoded by the nucleotide sequence shown in Fig.4A.

3. The nucleic acid molecule under item 1, where the fragment is In-chain of mistletoe lectin, which is encoded by the nucleotide sequence shown in Fig.4B.

4. The nucleic acid molecule according to one of paragraphs.1-3, which is a DNA molecule.

5. The nucleic acid molecule according to any one of paragraphs.1-3, which is a RNA molecule.

6. A nucleic acid molecule which is antisense a strand of nucleic acid molecule according to any one of paragraphs.1-5.

7. A vector that contains at least one molecule of nucleic acid according to any one of paragraphs.1-6.

8. Vector under item 7, which contains a nucleic acid molecule under item 2 or 4, and a nucleic acid molecule under item 3 or 4.

9. Vector under item 7 or 8, which is victory white.

11. The polypeptide with the biological activity of mistletoe lectin, which is encoded by a nucleic acid molecule according to any one of paragraphs.1-5 or a vector according to any one of paragraphs.7-9 and/or is produced by the body-master, transformed the specified molecule or vector.

12. The polypeptide according to p. 11, which contains at least one chemical or enzymatic modification, which is not the glycosylation occurring in Viscum album.

13. Protein containing at least one polypeptide with the biological activity of mistletoe lectin on p. 11 or 12.

14. Dimer polypeptide with biological function, characteristic of the mistletoe lectin, and one of its monomers encoded by the nucleic acid molecule according to any one of paragraphs.2, 4, or 5, and the second monomer is encoded by a nucleic acid molecule according to any one of paragraphs.3-5.

15. Dimer polypeptide under item 14, in which at least one of the monomers is a polypeptide under item 12 or 13.

16. The antibody or its derivative which is a fragment and the antibody is obtained using antigen, which is the polypeptide according to PP.11-13 or its dimer under item 14.

17. A method of obtaining a polypeptide or a dimer of a polypeptide having the activity letterbomb of PP.1-5 or a vector according to any one of paragraphs.7-9, cultivated under appropriate conditions and produce thus obtained polypeptide or dimer polypeptide.

18. Immunotoxin comprising as active compounds polypeptide according to any one of paragraphs.11-13 or one dimer polypeptide under item 14 or 15.

19. Pharmaceutical composition having immunostimulating and cytotoxic activity containing the polypeptide according to any one of paragraphs.11-13, and/or dimer polypeptide under item 14 or 15, and/or immunotoxin on p. 18, optionally together with a pharmaceutically acceptable carrier.

20. Primer or pair of primers designed(th) to obtain a vector containing the gene of mistletoe lectin that/which specifically hybridizes with the nucleic acid molecule according to any one of paragraphs.1-5 or complementary to her thread.

21. The composition is intended for studies of the body on the subject of the existence of lectin genes containing at least (a) a nucleic acid molecule according to any one of paragraphs.1-5; (b) one primer and/or one pair of primers for p. 20; or (b) the polypeptide according to any one of paragraphs.11-13 and/or dimer polypeptide under item 14 or 15.



 

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