The protein, inhibiting caused by collagen platelet aggregation mammals, recombinant protein, which inhibits caused by collagen platelet aggregation, the dna fragment encoding the recombinant protein, the expression vector (options), strain cultured animal cells kss - producer of recombinant protein (options), a method of obtaining a recombinant protein, a pharmaceutical composition

 

(57) Abstract:

The invention relates to biotechnology, genetic engineering. Purified natural protein, which inhibits caused by collagen platelet aggregation mammals of saliva Friatoma and set its N-terminal amino acid sequence. Obtained by recombinant protein with activity similar to the natural protein. Its amino acid sequence. Also determined the nucleotide sequence of the DNA fragment encoding the recombinant protein. The constructed expression vectors SV /CMV - ingitor - I (DSM 7223) and SV/V - inhibitor-II containing the DNA fragments encoding the recombinant protein. A method of obtaining a recombinant protein is performed by culturing strains of cultured animal cells KSS transformed mentioned expression vectors, and are able to produce recombinant protein. Then hold its secretion and purification by gel-filtration on "Superose 12" and in the system of the electrophoresis chromatography. The pharmaceutical composition inhibiting induced by collagen platelet aggregation contains natural or recombinant protein as an active substance in efficiency is as a drug for the inhibition caused by collagen aggregation of human platelets or treatment of cancer with metastatic tumor cells. 9 C. and 6 C.p. f-crystals, 29 ill., 3 table.

Collagen is the most effective of the known inducers of platelet aggregation of human rights. For example, after injury to the vessel walls and exhibiting their collagen platelets rapidly adhere to this place and activated /Baumgaptner/ 1977/, Thromb, Haemostas. 37, 1 - 16, Hauriger /1987/ Human Pathol. 18.111-122/.

Thus, caused by collagen platelet aggregation in humans is a risk factor for patients undergoing procedures that affect the blood vessels, for example, plastic operations on vessels or sepsis for patients with myocardial infarction and for recovering after a heart attack, and other

Inhibitors caused by collagen platelet aggregation include synthetic oligopeptides corresponding to the collagenous sequence, a snake venom proteins and Chalin, protein medical leeches. Synthetic oligopeptides, inhibit caused by collagen platelet aggregation due to the fact that bind platelets. Their disadvantage is that they affect platelet aggregation only at relatively high doses (about 70 μg peptide/ ml provide 65% inhibition/. Cm. Bevers et al. /1985/ "Sex is a result of collagen and thrombin Thrombosis Research, 37, 365 - 370, Karniguian et al. - 1983/ "the Influence derived from oktapeptid collagen at different stages of the interaction of the platelet/collagen", Thrombosis Research, 32: 593-604 and Caen et al. /1981/ "Oligopeptides with any abscopal specific properties caused by collagen aggregation, the method of receiving and containing pharmaceutical composition" EPA 0040149. The inhibitory mechanism of snake venom protein is still not known. Cm. Smith et al. "Identification of 50 KD proteins from snake venom, which specifically inhibit the adhesion of platelets to collagen". Thrombosis and Haemostasis /1991, 65, 678/ proceedings of the XIII Congress of the International society on thrombosis and hemostasis, June 30 - July 6, 1991, Amsterdam/. Chalin reacts with collagen, but does not react with platelets. Thus, it is not specific to the interaction of the platelet-collagen, but he interacts with collagen and also in the absence of platelets. Cm. Munro et al. /1991/ "Hallin inhibitor of adhesion of platelets obtained from the saliva of medicinal leeches, Blood Coagulation and Tebrinolysis 2, 179 - 184.

In the published European patent application EP 0480651 "Mags and Co., Inc. published April 15, 1992/, revealed a protein with a molecular weight of about 16 KD and its ability to inhibit induced collagen platelet aggregation is inhibitors such aggregation, possessing or other advanced characteristics.

In the present invention is proposed natural selected, a synthetically derived or recombinant protein that inhibits caused by collagen platelet aggregation, and which emit, or which can be isolated from the salivary glands of insects, sucking blood from mammals.

The preferred platelet primates, and most preferred human platelets. Sensitive and platelets other species such as horses, sheep, cattle, pigs, dogs and cats.

Synthetic proteins can be obtained by methods J. M. Steward and J. D. Young, 1984 /, Solid-phase synthesis of peptides Rehse Chem. Company, Fockford III and Methods der Organischen Chemie /Houben/ Weyl/. vol. 15, No. 1 and 2, E. Wunsch /ed./, Thieme Verlag Stuttgart 1974.

Preferred proteins of the present invention, which are isolated from the saliva of the subfamily /Pat. Faminia /Tratomae and, most preferably, Triatoma pallidipennis.

The present invention includes natural selected, a synthetically derived or recombinant protein that inhibits caused by collagen platelet aggregation mammals, and which has the following N-terminal amino acid posledovatelem platelet aggregation mammals, and moreover, this protein has the following amino acid sequence: a/ sequence represented in aa/ sequence Id No. 1; bb/ sequence Id No. 2 or cc/ sequence Id No. 3, or b/ allelic modification or mutation of sequences in any of sequence Id No. 1 - 3, and these modifications or mutations do not have a significant impact on the activity of the protein, or c/ protein according to any one of sequence Id No. 1, 3 or modifications or mutations specified in paragraph (b/ containing post-translational modifications not have a material effect on the activity of the Mature protein.

The most preferable of the above protein is a recombinant protein.

The present invention includes proteins that are not glycosylated.

The following variant of the invention are cDNA, or a DNA/ encoding a protein with the following amino acid sequence: a/ sequence represented in aa/ sequence Id No. 1, bb/ sequence Id No. 2 or cc/ sequence Id No. 3, or b/ encoding a protein that has an amino acid sequence according to any of sequences of N 1 - 3, with at least teatina, encoded by the corresponding cDNA or DNA sequence.

The protein of the present invention includes the Mature protein and the protein that contains a signal sequence preceding the N-terminal sequence of the Mature protein. The signal sequence is shown in Fig. 13a and 13b, and they correspond to negative numbers. It starts with Met. Lys. Val.IIe.IIe and ends with His. Ala, Phe. Ala. This signal sequence is responsible for penetration through the membrane after protein biosynthesis. The secretory protein is a Mature protein that starts with Glu.Cys.Glu.Leu -- This signal sequence is cleaved prior to secretion.

The present invention preferably contains a cDNA or DNA with the following nucleotide sequence:

a/ nucleotide sequence represented in

aa/ sequence I d N 4, bb/ sequence I d N 5, or cc/ sequence I d N 6, or b/ a nucleotide sequence according to any of sequences of N 4 - 6, with at least one allelic alteration or mutation, which did not significantly affect the activity of the Mature protein, which is encoded corresponding nucleotidesequence earlier, which, in addition, contains a suitable signal peptide, a suitable promoter and, optionally, suitable enhancer. The vectors described in the examples, as well as in European patent application EP 0480651; 0463632 and 0173177.

The following variant of the invention is cell eukaryotic or prokaryotic hosts, transformed previously specified vector.

The most preferred cells-owners are cells baby hamster kidney. As legal requirements make the Deposit of such cells is impossible, the plasmid expressing the construct containing the DNA sequence Id No. 1 was deposited on 2 September 1992 with registration number DSM...

Additionally, the present invention includes a method of obtaining a protein, characterized in that it comprises culturing the host cell transformed by a vector containing the gene encoding the protein, and the isolation and purification of the protein. Specific options are described in the examples of the invention, and the General ways you can see from the description of work and especially of the examples of the invention.

Industrial application of the protein of the present invention is the use of these proteins in farmac the Telem or diluent. Details can be found in the section of the Application description.

Allelic modifications mentioned previously, include changes in the sequence of nucleotides or amino acids, changes in genotype or phenotype. At least one nucleotide or amino acid can be substituted, deleted or inserted.

The majority of deletions, insertions and semadeni should not lead to radical changes in the characteristics of the protein of the present invention. As it is difficult to predict the exact effect of the substitutions, deletions or insertions in advance, compares the features of the Mature protein with the characteristic functions of the protein of the present invention may clarify whether the modified protein is comparable activity.

The genetic code is degenerate; that is, most amino acids are encoded by more than one codon of three nucleotides. Accordingly, allelic variations in the nucleotide sequence can alter or not to alter the amino acid sequence. Therefore, allelic variations occur initially at the DNA level and can also exist as a secondary level of amino acid sequence.

The DNA sequence encoding proceratiinae of the invention, which retains substantially the same activity as the protein of the invention. The activity determined by the method of example 1. Thus, one or more of the amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.. . up to 15 amino acids, you can add, replace or delete, practically without affecting the activity of the protein of the present invention. Replacement is usually done in accordance with table 1, if you want to slightly change the amino acid sequence of the protein of the present invention.

Substantial changes in function or immunological identity can be accomplished by selecting substitutions that are less conservative than shown in table 1, i.e., selecting residues that differ by greater its effect on the /a/ the structure of the polypeptide main chain in the field of such changes, as, for example, flat or spiral conforma or hydrophobicity of the molecule, or /c/ is the volume of the side chains.

Table 1.

Normal replacement of amino acids in protein

The original remains are Examples of substitutions

Ala - Gly, Ser

Arg - Lys

Asn, Gln, His,

Asp Glu

Cys - Ser

Gln - Asn

Glu - Asp

Gly - Ala, Pro

His - Asn, Gln

lle - Leu, Val

Leu - lle, Val

Lys - Arg, Gln, Glu

Met - Leu, Tyr, lle

the AI between the two compared proteins. Expression homology includes the similarity of amino acids and gaps in the sequences for both the compared sequences. The similarity of amino acids determine, for example, in table 1.

Preferably, the protein had an amino acid sequence with a homology of at least 60%, more preferably at least 80%, and even more preferably at least 90%, and most preferably at least 95% homology to one of the sequences Id N 1-3.

As indicated previously, the present invention includes variants of DNA. These sequences hybridize in stringent conditions with the DNA sequence defined in one of the sequences Id 4 - 6. In sequence to cDNA and DNA had the nucleotide sequence with a homology of at least 60%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the sequence represented in one of the sequences Id N 4 - 6. Homology can be determined by hybridization as described in R. Knippers, Moleculare Genetic. 1982, 3ed., Georg Thme Verlag Stuttgart, New York.

Under "post-translation the licensing, the formation of disulfide bonds and chemical modification of amino acids.

Glycosylation is one of the major biosynthetic functions of the endoplasmic reticulum and/or Golgi complex. Sequence and branching of the oligosaccharide, which are formed in the reticulum can be modified in the Golgi complex, lysosomes, or the plasma membrane. Oligosaccharides can be N-linked oligosaccharides /aspartic communication/ or O-linked oligosaccharides /serine, threonine or hydroxylysine link/. Glycosylation depends on the type producing cells and species, of which these cells are obtained. The degree and type of glycosylation may depend on the compounds as described in European patent application EP 0222313. Changes in glycosylation can change the function of the protein.

Usually the Mature protein of the present invention is glycosylated.

Proteins often form covalent cross-links links. These disulfide bonds are formed between the SH-groups cysteine protein stacked circuit or when laying chain protein during translation. These relationships stabilize the three-dimensional structure of the protein. These disulfide bonds are rarely formed in vosstanavlivajusa agent glutathione destroys most of these links.

Because proteins are outside the cytoplasm and are secreted or located on the cell surface, they often form additional covalent nutricare connection.

In addition, amino acids can be changed by the method described in PCT application WO 91/10684. Other modifications of the side chains of amino acids are also possible.

Preferably, gomologichnosti was at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably, the changes related to the one or two amino acids.

The protein of the invention has a purity of at least 40% preferably at least 60%, more preferably at least 80%; and most preferably, at least 90%. The degree of purity is determined by the amount of protein of the present invention with respect to the total amount of protein. Using a clean on two stages, first by gel-filtration on Sepharose /see example 2/, and then use HPEC /see example 15/ get the protein of the present invention, in addition to which no other protein is not detected by the methods of example 15.

Further, the present invention includes swazilan the protein of the invention.

Using the purified protein of the invention (see for example, sequence Id No. 1 - 3/ produce monoclonal antibodies are well known method of Koehler and Milstein, which, in particular, includes the usual immunization of mice with purified protein of the invention as an immunogen.

The best option of the present invention is a protein that is listed as sequence Id No. 1, expressed in transfected cells of the kidney of a baby hamster.

In addition, the invention includes a method of purification of the protein of the present invention, comprising a stage of gel filtration on "Superose 12" /see example 2/, and /b/ stage of processing in a highly efficient system electrophoresis-chromatography /Cm. example 15/.

The use of connections.

The proteins of the present invention exhibit pharmacological activity and can therefore be used as medicines. They can be used in pharmaceutical compositions containing the protein of the present invention together with a pharmaceutically acceptable carrier or diluent. In addition, the present invention includes pharmaceutical compositions containing a pharmaceutically active protein of the present invention, and its fataawaa invention demonstrates the inhibition of collagen-induced aggregation, platelets and inhibition of adhesion of tumor cells, preferably cells of metastatic tumors to the collagen.

Drugs against platelet aggregation

The proteins of the present invention disassemble the inhibition of platelet aggregation. The test system described in example 1. The proteins of the present invention demonstrate a significant inhibition of platelet aggregation in a concentration of 0.5 - 50 μg proteins of saliva in 0.5 ml or 0.5 - 250 μg of protein in 0.7 ml of purified protein /only cleaning by the method of example 2/.

Testing the most preferred protein, protein with sequence Id No. 1, gives the value of IR5050 nmole/l protein with a high degree of purification according to the method of examples 2 and 15. The proteins of the present invention exhibit inhibition of platelet aggregation at concentrations of 5 nmole/l to about 1000 nmole/l

The results obtained for in vitro test systems indicate that the proteins of the present invention can be used as medicines, or can be used for medical applications. The test results can be transferred from the system in vitro system in vitro, as is customary in this field of knowledge. R. J. Shebuski et al. /1990/ Thombosis and Haemostasis, 64:5 is. If you type animals daily to achieve blood concentrations of 100 nmole/l, is the reduction of platelet aggregation. In these conditions not observed serious side effects.

The proteins of the present invention show such inhibition of platelet aggregation in mice at daily doses to achieve blood concentrations from about 10 to 1000 nmole/L.

Therefore, the proteins of the present invention can be used for the treatment of atherosclerosis or thrombotic lesions, or to prevent re-occlusion after treatment of myocardial infarction. In other words, the proteins of the present invention can be used as protivoateroskleroticheskim and antithrombotic agents for mammals, including humans, for example, for the treatment of ateroskleroticheskih/thrombotic lesions, for example, associated with the destruction of atherosclerotic plaques or related to the breach or removal of the endothelium, for example, as a result of sepsis or organ transplant, or for the treatment of unstable angina. It can also be used to prevent re-occlusion after treatment of myocardial infarction and fibrinolysis or antiplastic /PTCA/. If treatment is and activators/, then the proteins of the present invention can be used as an auxiliary agent to prevent re-occlusion of the blood vessels. Treatment of myocardial infarction catheter-balloon /RTSA/ also leads to the damage of blood vessel walls, which can lead to the formation of new blood clots. This can be prevented by introducing the proteins of the invention during or after the procedure. These proteins cannot only be used in coronary angioplasty, but can be used in other embodiments angioplasty.

The present invention provides:

a/ the use of the protein of the present invention for the preparation of drugs for the treatment of atherosclerosis or thrombosis, or to prevent re-occlusion after treatment of myocardial infarction; /these proteins suitable for medication prevention/.

b/ a method of treating atherosclerosis or thrombosis, or to prevent re-occlusion after treatment of myocardial infarction, which includes the introduction of effectively suppressing disease number of protein of the present invention to a patient in need of such treatment;

c/ pharmaceutical composition for the treatment of atherosclerosis or thrombosis, or is subramania and a pharmaceutically acceptable carrier or diluent.

These indications dose, of course, depend on, for example, the compounds of the present invention the host, the route of administration and the nature and extent of the subject to treatment of the disease. However, in General, shows the animals that satisfactory results are achieved with daily doses that achieve blood concentrations from 10 to 1000 nmol/l, preferably at daily doses of from 30 to 300 nmole/L.

The proteins of the present invention can be entered in any conventional way, in particular, enterline, orally, for example in the form of tablets or capsules, or parenteral, for example, in the form of solutions or suspensions for injection.

The preferred protein is a protein with sequence Id No. 1.

The present invention provides pharmaceutical compositions containing compounds of the present invention together with at least one pharmaceutical carrier or diluent. Such compositions compounds can be obtained in the usual way. Cm. Remingtonis Pharmaceutical Science, 15th ed, Mack Publishing Company, Easton Pemsylwana /1980/.

Drug against metastatic tumor cells.

The proteins of the present invention exhibit I. rotini of the present invention demonstrate significantly inhibition of adhesion of metastatic tumor cells to collagen at concentrations from 1 to 100 µg protein in saliva in 0.5 ml or 1 to 500 μg of protein in 0.5 ml of purified protein /cleared only by the method of example 2/.

Testing the most preferred protein, protein with sequence Id No. 1, gives the value IR50100 nmol/l of protein with a high degree of purification according to the methods of examples 2 and 15. The proteins of the present invention exhibit inhibition of adhesion of tumor cells to collagen at concentrations from 10 to 2000 nmol/L.

The results obtained in the test system in vitro, show that proteins of the present invention can be used as medicines or medical treatment. The test results obtained in vitro can be transferred to the system as is common in this field of medicine. Chan et al. /1990/ Science 2:1600 - 1602.

The proteins of the present invention can be added during or after surgery of the primary tumor to prevent the formation of metastases due to the separated tumor cells that may get into the blood stream during surgery. Such antimetastatic is 2:1600 - 1602. The proteins of the invention is administered intraperitoneal injections daily or 2-3 times per week. If animals injected daily dose to achieve blood concentrations of 200 nmol/l, there is a reduced adhesion of metastatic tumor cells, according to determine the number of centers settled metastatic cells. Under such conditions is not observed serious side effects.

The proteins of the present invention can demonstrate the same adhesion inhibitory efficacy by inhibition of metastatic tumor cells to collagen in mice at daily doses that achieve blood concentrations from 20 to 2000 nmol/l, preferably, concentrations of from 60 to 600 nmol/L.

Therefore, the proteins of the present invention is suitable for treating cancer, preferably cancer with metastatic tumor cells, and most preferably, the cancer with a large number of metastatic tumor cells.

The present invention provides

a/ the use of the protein of the present invention to obtain drugs for the treatment of cancer with metastatic tumor cells. /These proteins are suitable for preventive medicine, administered, for example, to chirurgery includes the protein of the present invention and a pharmaceutically acceptable carrier or diluent.

With such indications, the appropriate dosage will, of course, depend, for example, the use of compounds of the invention, the patient, the route of administration and the nature and extent of the disease to be treated. However, in General, satisfactory results in animal experiments achieved at daily doses that achieve blood concentrations from 20 to 2000 nmol/l, preferably at daily doses of from 60 to 600 nmol/L.

The proteins of the present invention can be entered in any convenient way, in particular, enterline, orally, for example in the form of tablets or capsules, or parenteral, for example, in the form of solutions or suspensions for injection.

The preferred compound is a protein with sequence ID No. 1.

The present invention provides pharmaceutical compositions containing a compound of the invention together with at least one pharmaceutical carrier or diluent. Such compositions can be prepared by conventional means. Cm. Pemington Pharmaceutical Science, 15th ed, Mack Publishing Company, Easton Pennsylvania /1980/.

Summary of the invention

In the present invention is proposed inhibitor induced collagen platelet aggregation che also inhibits the interaction of tumor cells with collagen. Such an inhibitor is present in the saliva of blood-sucking insect Triatoma pallidipennis. Cm. example 1.

Thus, the present invention relates to a purified and selected protein, which inhibits induced by collagen aggregation of human platelets. This protein can be isolated from Triatoma pallidipenis. It also relates to pharmaceutical compositions containing the protein, and to methods of use of the latter for the treatment of thrombotic lesions, or to prevent re-occlusion after treatment of myocardial infarction and for the treatment of, among others, the development of metastases.

Thus, the present invention provides a valuable pharmacologically active substance, such as a new protein that specifically inhibits caused by collagen platelet aggregation, with a high degree of specific activity; new protein, which specifically antagonizes the interaction of the platelet-collagen, causing release vnutritrombozitarnyi components, such as the APG, which has the undesirable side effect in itself; a new protein for pharmaceutical use in the treatment of atherosclerosis and thrombosis or to prevent re-occl the tissue collagen and which can be used for example, to prevent metastasis of tumor cells.

Study of the characteristics and properties of the inhibitor led to the following results:

1/ the Inhibitor is not a receptor antagonist of fibrinogen, as experiments with increasing concentrations of fibrinogen did not reveal any effect on the inhibitory activity. Cm. Example 3.

2/ He is not an antagonist of thromboxane, as it prevents the called collagen platelet aggregation, pre-treated with aspirin and not aggregation caused U 46619 /simulator thromboxane/. Cm. example 4.

3/ apparently, it is not an inhibitor direction of the signal transduction carried out through protein kinase C, the aggregation induced by phorbolester /phorbol-12-myristyl-13-acetate/ not inhibited. Cm. example 4.

4/ It inhibits the reaction of the release-treated collagen platelets. Cm. example 5.

5/ It did not inhibit platelet aggregation induced by thrombin or ADRs. Cm. example 6.

6/ the Inhibitor does not react with the collagen. While pre-incubation of the inhibitor with collagen and does not increase inhibitory activity, prolonged and/P> 7/ Inhibition of platelet aggregation is reversible when adding large amounts of collagen. Cm. example 7.

8/ protease Inhibitors do not have a noticeable influence on the activity. Example 8.

9/ Inhibiting activity increases in the presence of ions of Mg2+. Cm. example 9.

10/ the Inhibitor prevents the adhesion of platelets to collagen matrix dependent on the dose way. Cm. example 10.

These results suggest that the inhibitor is an antagonist of the collagen receptor with high specific activity, for example, IR50= 2.5 µg/ml team faction "Superose", described next on the basis of the partially purified inhibitor/, and IR50=50 nmol/l to a high degree of purification protein /cleaned on a successive stages described in examples 2 and 15/.

11/ Inhibitor were incubated with trypsin related to sepharose matrix. The inhibitor completely lost activity due to proteolytic cleavage. Cm. example 11 in which it is shown that the inhibitor is a protein.

12/ the Inhibitor is not cleaved by collagenase. Cm. example 12.

13/ According to gel-filtration chromatography in prysutstsvenni protein /see sequence Id No. 1/ calculated for the cDNA sequence is 18923 Dalton.

14/ the Inhibitor prevents the adhesion of highly metastatic tumor cells /MT Zn 3/ to the collagen-dependent dose-way /Cm. example 14/.

The inhibitor of the present invention is not bound with /or does not react with/ collagen is not associated with the platelets and does not cause flocculation of collagen.

Inhibitor of collagen of the present invention can in the usual way to allocate from the saliva of blood-sucking insect Triatoma pallidipennis, for example, as described in the examples. Conventional methods of collection of saliva is quite applicable to obtain the source material of saliva. Insect Triatoma pallidipennis predominantly prevalent, and therefore easily accessible, Central and North America. They are known as the vector for Trypanosoma Cryi.

In another aspect of the present invention proposed a DNA sequence, vectors containing this sequence of cells containing these vectors, recombinant methods of obtaining protein and antibodies to the proteins of the present invention. Also proposed isolated and/or recombinant DNA sequence /for example, genomic or cDNA/ encoding protein /for example, the present invention provides recombinante derived proteins of the invention, for example, having disclosed the invention in the sequence.

The term "isolated" means that the inhibitor of the present invention or another agent is present in a form selected from purified from components with which it is natural merged, or with which he received recombinante or synthetically.

Generally includes all the extent of separation or purification. Preferred such degrees of separation or purification, after which the inhibitor can be used for pharmaceutical purposes. For example, such a level of separation /for example, activity or purity can be obtained as a result of such chromatographic methods, which are described in the examples. Further purification, for example, to homogeneity, can be conveniently achieved using conventional techniques, for example, disclosed in the following sources:

Methods of Enzymology, Volume 182, Guide to Protein Purification, ed.

Murray P. DEUTSCHER, Academic Press 1999;

Protein Purification Applications - A Practical Approach, ed. E. L. V. Harris and S. ANGEl, IRL-Press, 1990; Prorin Purification, Principles and Practice, Robert SCOPES, Springer-Verlag 1982; and Protein Purification, Principles, High Resolution Methods and Applications, ed. J.-C. JANSON and L. RYDEN, VCH publishers, 1989.

The degree of purity can be determined by any of several known methods, for example, SDS - El is determining the amino acid sequence of the protein according to the method completely well-known to specialists. Hewick, R. m. et al. /1981/, J. Biol. Chem 256. 7990-7997.

The proteins of the present invention include not only proteins extracted from the sample insect species, but also from any other organisms that may contain an inhibitor. In addition, the inhibitor of the present invention includes inhibitors related structures, for example, the inhibitor caused by collagen aggregation of platelets isolated from another organism, which has almost the same amino acid sequence.

Since this protein is selected from stinging insects, and its natural purpose is to prevent clogging of the blood of the wound formed after the bite, for a sufficiently long period of time, so that the insect could suck blood, it is obvious that other inhibitors of platelet aggregation under the influence of collagen could be detected in the saliva of other blood-sucking organisms, especially insects, for example, other Reduviit bugs with tapered noses of the subfamily. Triatomina, such as Triatoma infestans, T, dimidiata, T. maculata, Rhodmius prolixus, Panstrongylus megistus and P. infestans.

The proteins of the present invention include Monomeric single-chain molecules, i.e. such that covaleskie shape of protein molecules, for example, dimers or other oligomers, tertiary structures formed with other polypeptides, fragments of the protein, etc. Included as glycosylated and deglycosylated forms, and both forms can be obtained in the usual way by the expression of, for example, mammals /glycosylated/ or bacterial cells /deglycosylation/, respectively.

Amino acid sequence of the inhibitor of the present invention can be used to determine the sequence of a suitable DNA probe that can be used to find new inhibitors, for example, in other types. Such probes can be synthesized in the usual way, for example using an automatic DNA synthesizers and scrinia genomic or DNA library is similar to methods known in the art (see international publication WO 90/07861 dated July 26, 1990/

For example, the present invention relates to a DNA sequence as disclosed in Fig. 12a-c, 13 a, and b and 22-24. In addition, the present invention relates to a DNA sequence that encodes a mutation as described previously. The sequence for -18 to +5 plot of Fig. 18, 21 and 24 are obtained from the corresponding full length cDNA posledovatelnostyu/ natural DNA sequence /gene/ inhibitor, when her evolve from the natural environment, for example, in solution or on the vector, as well as their mutants and naturally occurring, for example, in other types, the selected form or synthetic, for example, obtained by site-directed mutagenesis. Methods for screening genetic libraries of various types suitable conventional probe for professionals. Methods of obtaining mutants also routine and familiar to specialists, as well as the methods for testing the effectiveness of these new proteins, for example, as described.

Suitable mutants, either synthetic or natural /in a highlighted form/ are those that contain at least part of, for example, 5%, preferably at least 50%, and more preferably at least 90% of the biological activity, such as inhibitor-induced collagen, platelet aggregation, natural, isolated from T. pallidipennis inhibitor, as indicated.

Suitable mutants may differ from the natural protein of any possible modifications, including deletions, additions and/or replacement of one or more of the amino acids, provided that remains the main biological activity; preferably, Biologicheskaya, equivalents of the DNA sequences disclosed here include allelic variants, sequences encoding equivalent mutants disclosed here, and DNA sequences that are homologous.

In addition, the present invention includes thrombolytic fragments of the polypeptide, for example, with similar functions or allocated to the sub function, for example, the selected epitopes, active sites, fibrin and/or FibroGen binding sites, etc., the Preferred single-stranded form of the inhibitor.

The present invention also relates to antibodies and producing antibodies cell lines that can be obtained in the usual way, using purified proteins of the present invention, for example, a common method of Kohler and Milstein, which provides for regular immunization of mice with protein of the present invention as an immunogen. The present invention also relates to fragments of these antibodies, such as fragments that contain domains that bind to the epitopes of inhibitory protein and synthetic binding domains, such as mimotope, specifically recognizing domains of proteins of the present invention.

Inhibitor but to use as protivoateroskleroticheskim and antithrombotic agent for mammals, including humans, for example, for the treatment of atherosclerotic/thrombotic lesions associated with, for example, rupture of atherosclerotic plaques resulting from operations on vessels /PTA/TPCA/ or related to the breach or removal of the endothelium, for example, as a result of sepsis or transplant. It can also be used for the treatment of unstable angina and/or prevent re-occlusion after treatment of myocardial infarction. The details of this application, for example, intervals of doses, modes of introduction /preferably oral or parenteral/ and so forth, can be defined in the usual way, for example, similar and/or when compared with other antithrombotic agents, such as t-PA, streptokinase, or other platelet aggregation inhibitors.

The inhibitor of the present invention can also be used to prevent metastasis of tumor cells by blocking their passage through the connective tissue. They are applicable for preventing metastasis of all invasive tumors such as melanoma. Further features without the intention to provide any theory. During the formation of metastatic tumor cells must penetrate osnovnaya tumor cells requires interaction with these proteins. Evidence of the role of the receptor for collagen /VLA 2/ in this interaction are presented, for example, Chan et al. /1990, 2, 1600 - 1602/, who cloned VLA 2 positive tumor cells, which formed much more metastatic tumor colonies (Kramer and Marks /J. Bid. Chem. 1989, 264, 4684-4688), were able to block the accession of melanoma cells to collagen by antibodies to VLA 2. See also PA US 73234708-A" Monoclonal antibodies against platelets, which inhibit the reaction of platelets with collagen and is used for the detection and treatment of cancer" US Dept. Helthah and Human Services. The inhibitor of the present invention, as a receptor antagonist of collagen, prevents the interaction of tumor cells with the surrounding matrix and, thus, inhibits the formation of metastases.

It can also be used as a standard to determine the effectiveness of new inhibitors that can be created, for example, modifying the structure of this inhibitor standard mutagenesis, directed mutagenesis, for example, deletions and/or insertions of sequences, etc., the Inhibitor of the present invention can also be used as an antithrombotic agent in the screening procedures, which determine the division of the effectiveness of compounds, which block the effects of such induced collagen aggregation of platelets, for example, in patients with insufficient blood clotting.

Without further description, it is believed that the specialist using the above, can make full use of the present invention. It is further preferred options should be considered only as illustrative and in no way limiting the present invention.

Other objectives, features and corresponding advantages of the present invention are more fully disclosed and will become clearer when considering in conjunction with the accompanying drawings, in which:

Fig. 1 represents the dependence of the dose-dependent inhibition of aggregation of human platelets by the saliva of Triatoma;

Fig. 2 represents the dependence of the dose-dependent inhibition of aggregation of human platelets "Superose"-a pool of saliva of Triatoma;

Fig. 3 presents a picture of the gel filtration on "Superose 12".

Fig. 4 represents the dependence of the inhibition of aggregation on the concentration of fibrinogen;

Fig. 5 is preventing induced by collagen platelet aggregation, pre-treated with aspirin;

Fig. 6 demons inhibitor;

Fig. 8 represents the stability of the inhibitor under the action of collagenase;

Fig. 9 represents the determination of molecular weight inhibitor;

Fig. 10 represents the purification of the inhibitor to homogeneity;

Fig. 11 represents the sizes of the PCR products;

Fig. 12a represents the DNA sequence of the subcloned PCR product type 1 and a defined sequence of amino acids;

Fig. 12b represents the DNA sequence of the subcloned PCR product type 2 and installed the amino acid sequence.

Fig. 12c represents the DNA sequence of the subcloned PCR product type 3 and installed amino acid sequence;

Fig. 13a represents the full DNA sequence of the cloned cDNA inhibitor 1 and the corresponding amino acid sequence;

Fig. 13b represents the full DNA sequence of the cloned cDNA inhibitor 2 and the corresponding amino acid sequence;

Fig. 14 represents the expression plasmid containing DNA encoding the inhibitor;

Fig. 15 represents the molecular weight of the Mature recombinant protein specific antibody that specifically recognizes the Mature protein;

Fig. 18 represents the sequence of the Mature protein inhibitor-3;

Fig. 19 represents DNA coding sequence of the Mature protein inhibitor-1;

Fig. 20 represents DNA coding sequence of the Mature protein inhibitor-2;

Fig. 21 represents DNA coding sequence of the Mature protein inhibitor-3;

Fig. 22 represents DNA coding sequence preprotein inhibitor-1;

Fig. 23 is a DNA coding sequence preprotein inhibitor-2;

Fig. 24 represents DNA coding sequence preprotein inhibitor-3;

Fig. 25 is a scheme to obtain cDNA of the present invention and

Fig. 26 represents the N-terminal amino acids of the protein of the invention in sequence Id No. 10.

The list of identified sequences:

The list of sequences:

Sequence 1: sequence of the Mature protein inhibitor - 1.

Sequence 2: sequence of the Mature protein inhibitor - 2.

Sequence 3: sequence of the Mature protein inhibitor - 3.

Sequence 4: DNA the coding sequence of the protein inhibitor - 2.

Sequence 6: DNA the coding sequence of the Mature protein inhibitor - 3.

Sequence 7: the DNA coding sequence of preprotein inhibitor - 1.

Sequence 8: the DNA coding sequence of preprotein inhibitor - 2.

Sequence 9: the DNA coding sequence of preprotein inhibitor - 3, and

Sequence 10: T-Terminalia amino acids of the Mature protein.

In the preceding and following examples, all temperatures are given without amendments inoCelsius and unless otherwise indicated, all parts and percentages are indicated by weight. All full disclosure of all applications, patents and publications, cited previously and hereinafter included here by reference.

Example 1. The activity of the protein of the present invention.

Aggregation of human platelets in the presence of collagen inhibited, if you add the saliva of Triatoma pallidipennis or purified protein. The degree of inhibition correlated with the concentration of saliva protein.

Insects stimulate the secretion of saliva on siliconized glass plates with mechanical stimulation. Separated material is collected, sucking siliconize varying amounts of saliva /1,25 - 20 μg protein in 20 μl/ or with different amounts of "Superose"-pool /0,5 - 10 µg protein in 200 µl/ from example 2 at 37oC aggregometry. After 1 minute, add 1 ág of collagen and control the increase in light transmittance /aggregation/. Cm. Fig. 1 and 2.

Example 2. Purification of the protein.

An important stage of purification of the protein using gel filtration on "Superose 12". 2 ml /5 mg protein/ saliva process chromatographic column Superose 12 HR 16/50 /Pharmacia/ 10 mm T /HCl, pH 7,4; 0,0001% "Pluronic F68". Then inhibitors elute 10 mm Tris /HCl, pH 7,4; 0,0001% "Pluronic F68", 200 mm NaCl. Cm. Fig. 3. Pool inhibitor contains 25 μg/ml of protein. 5 µg of protein demonstrate a 70% inhibition of aggregation. Platelet aggregation without inhibitor was defined as 100% aggregation and 0% inhibition. The rest of the data, respectively, are calculated.

Example 3. The inhibition is not dependent on fibrinogen.

Activity inhibitor represented by the protein of the present invention, does not depend on the concentration of added fibrinogen, 500 µg filtered gel platelets combined with fibrinogen and 50 μg of saliva. After incubation for 1 min at 37oC add 1 μg of collagen and determine the aggregation. Received by the Oia, represented by rectangles 1 and 3 /without saliva/ correlated with the concentration of fibrinogen. Cm. Fig. 4. The test to determine the mechanism of platelet aggregation induced by proteins of the invention (example 4).

To study the mechanism of action of the protein of the present invention some of the standard compound is added to the test system, which in another embodiment, present or protein of the invention, or a buffer. The activity of the protein of the invention does not appreciably affected by the addition of aspirin /rectangle 4/. The influence of compounds U46619 and PMA does not change under the action of the protein of the invention. Therefore, the protein of the invention is an antagonist of thromboxane and probably not an inhibitor of protein kinase C.

The rectangles 1 and 2: 500 μl of platelet-rich plasma /300,000 platelets/μl/ incubated with the protein of the present invention /rectangle 2/ /200 ál pool "Superose"/ or buffer /rectangle 1/, respectively, at 37oC for 1 minute. Then add 1 μg of collagen and aggregation control aggregometer.

Rectangles 3 and 4: 500 μl of platelet-rich plasma is incubated with 1 mm aspirin for 20 minutes at room temperature. After that day at 37oC add 1 μg of collagen, and determine the aggregation.

Rectangles 5 and 6: 500 μl of platelet-rich plasma is incubated with the protein of the invention /rectangle 6/ or buffer /rectangle 5/, respectively, for 1 minute at 37oC. Then add U46619 /1 μm/ and define the aggregation.

Rectangles 7 and 8: 500 μl of platelet-rich plasma is incubated with the protein of the invention /rectangle 8/ or buffer /7/, respectively, at 37oC aggregometry. After 1 minute add 10 ng PMA /phorbol-12-myristate-13-acetate/ and define the aggregation.

The results obtained are shown in Fig. 5.

The reaction of the release of platelet /ATP measurement/ /example 5/. If there are platelets and collagen, the protein of the invention can inhibit the activation of platelets. ATP is used as an indicator of activation. 500 μl of platelet-rich plasma is incubated with 200 μl of the protein of the invention /Supersky or H2O, respectively, at 37oC for 1 minutes Then add 1 μg of collagen. Aggregation control within 10 minutes. After that, 200 μl of the suspension combined with 250 μl of Hepes buffer pH of 7.4, 100 mm luciferin and 5 μg/ml luciferase. Then measure the SS="ptx2">

Example 6. Inhibition of platelet aggregation caused by different substances.

The protein of the invention-specific induced by collagen platelet aggregation. 500 ál of filtered platelets /300,000 platelets/μl/ incubated with the protein of the invention for 1 min at 37oC. Then induce aggregation by collagen /2 μg/ml, thrombin /a 0.06 U/ml and/ or ADP /1 10-5M/, respectively, and measure the aggregation.

Example 7. The inhibition induced by collagen aggregation.

The protein of the present invention does not react with the collagen. The inhibition induced by collagen platelet aggregation in the presence of protein can be neutralized with an excess of added collagen. Induced by collagen aggregation /2 μg/ml to 500 μl of platelet-rich plasma determine the following modifications:

1: control without inhibitor;

2: the protein of the invention /100 µg saliva/ 10 minutes pre-incubated with human platelets before adding collagen;

3: inhibitor /100 µg saliva/ 10 minutes pre-incubated with collagen before adding platelet-rich plasma.

4, 5, 6: after the measurement, aggregation, 2, 5 and 10 μg of the protease inhibitors for inhibitory activity.

The protein of the invention is not a protease. Protease inhibitors /2 mm PMSF, 2 mm leupeptin, 2 mm Aprotinin/ or buffers incubated for 15 minutes with "Superslim" - pool /200 μl/ well. Platelet-rich plasma added, and the aggregation stimulated by collagen /2 ág/ml/

The inhibitor depends on the presence of Mg2+(example 9).

Cations of Mg2+increase the inhibition of platelet aggregation due to the protein of the invention. Cation Ca2+no significant effect on inhibition of platelet aggregation, 500 μl of platelet-rich plasma combined with 2 mm Mg2+Ca2+and 40 µg of saliva or buffer. After incubation for 1 minute at 37oC 1 μg of collagen type and measure aggregation.

Supplements - Maximum aggregation, %

--- - 77

Saliva - 33

2 mm Mg2+- 75

Saliva + 2 mm Mg2+- 19

2 mm Ca2+- 63

Saliva + 2 mm Ca2+- 39

Example 10. Incubation of platelets with the protein of the invention shows reduced adhesion to collagen.

If platelets are incubated with the protein of the invention, they partially lose their ability to bind collagen. Plate with 96 cells coated with collagen /type 1 at 40o for 20 min at 37oC under stirring. They are washed with PBS and sticky platelets fixed with 2.5% glutaraldehyde for 2 h at 37oC. Then the platelets are removed from the cells and is considered under the microscope.

Protein*The number of adhered platelets / mm2< / BR>
--- - 13500

10 ál - 12000

50 ál - 6500

*Protein = "Superose" - pooled, concentrated to 0.5 mg protein/ml

Example 11. Incubation of the inhibitor with trypsin-Separate.

The protein of the invention digested by trypsin related Separate. The digestion of the protein of the invention leads to a complete loss of activity of the protein of the invention. 200 µg of saliva combine with trypsin-related Separate or buffer, respectively, with Separate and trypsin-Separate, combined with the buffer. All portions containing 150 mm NaCl to prevent nonspecific adsorption on the matrix. After incubation overnight at room temperature under stirring, the samples are centrifuged and the supernatant added to the analysis environment on the aggregation /500 μl of platelet-rich plasma, 1 µg collagen/. Proteolytic digestion of control on SDS-polyacrylamide gel. Cm. Fig. 7.

Example 12. Inky. Collagenase /Clostridium Ristolyticum/ associated with Separate and used in the following amounts:

1. 100 μl of collagenase-Sepharose + 60 μl of the protein of the invention + 140 μl of H2O + 10 μl of the buffer.

2. 100 μl of 150 mm NaCl, 50 mm Tris /HCl pH 7,4 + 60 μl of the protein of the invention + 140 μl of H2O + 10 μl of the buffer.

3. 100 μl of collagenase-Sepharose 200 ál of H2O + 10 μl of buffer. As a control, bovine serum connect with Separate and used in the following amounts:

4. 100 μl of BSA-Sepharose + 60 μl of the protein of the invention + 140 μl of H2O + 10 μl of the buffer.

5. 100 μl of BSA-Sepharose 200 ál of H2O + 10 μl of the buffer.

protein: "Superose" pool

buffer: 140 mm Tris /HCl, pH of 7.4, 100 mm CaCl2.

Portions centrifuged and 200 μl of each supernatant fluids incubated with 500 μl of platelet-rich plasma for 1 min at 37oC. Then, 1 μg of collagen type and define the aggregation. The activity of collagenase-Sepharose control incubare with collagen and carry out electrophoresis on SDS-polyacrylamide gel. Cm. Fig. 8.

Example 13. The molecular weight determined for the inhibitor by gel filtration.

The purified protein of the invention has a molecular weight of 20000 5000 daltons; defined on the s /HCl pH 7,4, 150 mm NaCl, 0.0001% per "P1" F68. Bovine serum albumin /Mb 67 kDa/ dimitrivegas /Mb 25 kDa/, ribonuclease /Mb 14 kDa/ used as a marker of molecular weight. Cm. Fig. 9. Adhesion of tumor cells to collagen is reduced in the presence of the protein of the invention (example 14).

The protein of the invention inhibits the adhesion of tumor cells to the collagen matrix. Therefore, the migration of tumor cells can be prevented partially or completely from sedimentation in the organs or blood vessels, if the protein of the present invention is in the blood or plasma of the patient. Cells MT Zn 3 /tumor cells of the mammary gland of rats/ mark51Cr. Cell plates coated with collagen /type III/ 4oC during the night. First 2 of 104labeled cells in 500 ál DMEM F12 medium, 20 mm ner, 1 mm bicarbonate, 1% BSA incubated with 0, 2, 5 or 10 μl of the protein of the invention /"Superose" pool, 0.5 mg protein /ml, respectively, for 10 min at 37oC. Then, the suspension is transferred onto coated with collagen cells and incubated for 12 hours at 37oC. thereafter, the cells washed, and adherent cells are removed 1 M NaOH. Determine the radioactivity of the stuck cells.

The number of added inhibitor, ml - Attaching cells /CPM/
theine invention allocate according to the method of example 2, purified to homogeneity using a system of high-performance electrophoresis chromatography.

Partially purified inhibitor is introduced into the system high-performance electrophoresis chromatography /HPEC/ from Applied Biosystems, Inc./ Foster City, CA). Electrophoresis is performed on a 7.5% polyacrylamide gel in a buffer system Tris /glycine in accordance with the manufacturer's instructions. A sample buffer containing SDS, but not reducing agent (for example, DTT/ and aliquots are not heated before putting on the gel. Protein sequentially elute from the gel, detected by measuring the absorbance at 230 nm, and fractionary. Fractions with inhibitory activity, analyzed by SDS-polyacrylamide gel electrophoresis /a 12.5% SDS-polyacrylamide gel, Coomassie stained Biolliant Blue/. Cm. Fig. 10.

Example 16. Analysis of amino acids.

Samples of proteins is evaporated to dryness and hydrolyzing 6N. HCl containing 2% phenol for 24, 48 and 72 hours the cysteine Content is defined as cysteine acid after oxidation permorming acid /Moore, J. Biol. Chem. 238, 235 - 27 /1963//. The content of tryptophan was determined after hydrolysis in 4 N. N-methansulfonate within 24 hours /Simpson et al., J. Biol. Chem. 251, 1936 - 1940 /1976//. Then the samples will be analyzed by amino acid Ana is Ser = 8,9, Thr = 10,7, Val = 8,7, Leu = 6,6, Ile = 2,5, Pro = 4,5, Cys = 3,0, Met = 1,0, His = 2,3, Tyr = 4,3, Asp = 6,2, Glu = 7,2, Lys = 11,0, Arg = 1,8, Asn = 5,7, Gln = 2,3, Phe = 2.5 and Trp = 1,1.

Example 17. Determination of amino acid sequences

The protein is sequenced by automated amino acid sequencing machine Applied Biosystems, Inc. /IBI/ /Foster City, CA) according to the manufacturer's instructions. The sequence of amino acids 1 - 20 /N-end/ is:

< / BR>
Example 18. PCR amplification, subclavian and DNA sequencing of the main fragments of the three forms of the inhibitor cDNA from cDNA salivary glands Friatoma pallidipennis.

Part 1. Obtaining RNA salivary glands Friatoma pallidipennis and synthesis of the first cDNA strands.

Starting with full of purified RNA from salivary glands, RNA sequences transcribers reverse transcriptase to obtain the first strands of cDNA. For the synthesis of the first cDNA strands using specific oligonucleotide as a seed. Total RNA isolated from the salivary glands Friatoma pallidipennis method, which involves the dissolution of tissue in the guanidine-thiocyanate followed by ultracentrifugation of the lysate in the gradient of cesium chloride /Sambrook, J, Fritsh, E. T. Maniatis, T.: Molecular Cloning of the main 18 - 22, Cold Spring Harbor Laboratory Press 1989/. 10 µg total RNA in the salivary glands, thus obtained, use DL is Noah Moloney leukemia, the appropriate reaction buffer deoxynucleotide and block II RNase, commercially available from Ist Strand Synthesis kit" /Stratagene Cloning Systems, La Yolla, California, USA/ in accordance with the manufacturer's instructions. Oligodeoxynucleotide included in stage annealing reaction premirovany the first strands of cDNA synthesis, is not a nucleotide, included in "Ist Strand Synthesis kit", but is the linker-primer /1,4 µg/ taken from a commercially available kit "ZAP-CDNATMSynthesis kit" / Stratagene Cloning systems/. Its sequence is presented next /and Xh ol sequence that recognizes restrictional endonuclease, emphasized: see also Fig. 25/.

< / BR>
Part 2. PCR amplification of cDNA fragments of an inhibitor of the cDNA first strands of the salivary glands.

On the basis of amino acid sequence, which is defined by splitting Adminu purified protein of the present invention, develop and synthesize three oligodeoxynucleotide and one linker oligodeoxynucleotide. Based on the selected fragments of the amino acid sequence for the N-Terminus of purified inhibitor /ex 17/ three degenerative oligodeoxynucleotide develop and synthesized to amplify the core h is by the bar, indicate the position where the embed two different deoxynucleotide, and the corresponding amino acid sequence is indicated by the three-letter code under deoxynucleotide sequence, a sequence that recognizes Sphl restriction endonuclease, stressed/:

< / BR>
< / BR>
< / BR>
These oligodeoxynucleotide partially overlapping parts found by splitting Adminu amino acid sequence. Oligodeoxynucleotide N 3 contains oligodeoxynucleotide N 1 in the beginning, and oligodeoxynucleotide N 2 at the end.

Additional oligonucleotide is received with the sequence obtained from the linker-primer used for priming the synthesis of the first cDNA strands /part 1/:

Oligonucleotide N 4

< / BR>
After synthesis on a DNA synthesizer, Applied Biosystems PCR-MateTM391 four deoxyoligonucleotide purified by gel - filtration on commercially available NaP-5 columns /Pharmacia Biosystems/ and used as primers in three separate polymerase chain reactions PCR, U.S. patent 4800159/ as described below. Reagents obtained from commercial set "Cepe-ApmTMDNA Amplification kit "c AmplitagTMrecombinant Tag DNA polymerase from Perkin-Elmer, Sanity cDNA, synthesized from total RNA salivary glands Friatoma /part 1/, serves as a matrix in each of the three reactions. Oligodeoxynucleotide primers unite as follows: oligodeoxynucleotide N 1 and N 4 in PCR reaction # 1, oligodeoxynucleotide N 2 and N 4 in PCR reaction # 2, oligodeoxynucleotide N 3 and N 4 in a PCP response N 3. Three PCR reactions are incubated in a Perkin-Elmer Cetus DNA Thermal Cycler using the following program reactions with 38 cycles including phase reactions of N 1 - 3:

* initial stage: 3 min at 94oC

* program response:

* reaction stage N 1 : 1 min 30 s at 94oC;

* stage reaction N 2 : 2 min at 40oC;

* reaction stage N 3 : 3 min at 72oC;

/Sequence phase reactions of N 1 to 3 is repeated 38 times/

* Final stage: 10 min at 72oC

5% /5 full ál reaction volume separated by electrophoresis on a 1.5% agarose gel, using as a standard size ladder DNA from 123 base pairs /Gibco-BPL Life Technlogies, Jaithersburg, MD USA/. After staining the gel ethidiumbromid, strips of single-stranded DNA found in each of the three PCR reactions, and their apparent sizes in accordance with standard DNA size approximately 530 - 560 base pairs /Fig. 11 from left to right; the ladder of 123 the Torah.

The DNA fragment contained in the remaining volume of the PCP response N 1 /part 2/, isolated after electrophoresis on a 1.5% agarose gel and the method including the binding and elution of DNA from NA-45 DEAE membranes /Schleicher and Schuell D-3354 Dassel Germany/ followed by extraction with n-butanol and precipitation with ethanol /Sambrook, J. Fritsch, E. F., Maniatis, T." Molecular Cloning, chief 6: 24-27, Cold Spring Harbor Laboratory Press, 1989/. The ends of selected DNA fragments prepared for ligation to the vector by double digestion of restriction enzymes Sph 1 and Xhol /Boehringer Mannheim GmbH. D-6800 Mannheim Germany/, and then extracted twice with a mixture of phenol/chloroform /1: 1/ and then twice with chloroform. 3 μg DNA plasmid vector pGEMR-5 zf/ /Promega, Madison, W 1, USA/ linearities due to double digestion using restriction enzymes Sphl and SalI/ Boehringer Mannheim GmbH/ and then separate and isolated from a 1.5% agarose gel as described previously. Combine 50% split and extracted amplified DNA fragment and 20% digested and gel purified vector DNA, precipitated with ethanol and are ligated, using the reagents and Protocol commercial set "DNA Ligation Kit /Stratagene Cloning Systems. Complete ligation reaction is used to transform commercially available "Epicurian ColiRXLI-Blue S is then lead to B agar plates, containing ampicillin /100 mcg/ml/ 20 colonies E. Li, resistant to ampicillin, find after incubation and grown in LB broth containing ampicillin /100 μg/ml, and their plasmid DNA secrete using the procedure of alkaline lisica "miniprep" Sambrook Fritseb, E. F., Maniates T.: Molecular Cloning, Chapter I, 25-28, Cold Spring Harbour Laboratory Press, 1989/.

After double digestion of the plasmid DNA from 20 clones with restriction endonuclease Sph I and Sac I /Boehringer Mannheim GmbH/ and electrophoresis on a 1.5% agarose gel, it turns out that 13 of them contain a DNA insert of approximately 580 bp /positive clones/. DNA sequencing to produce extracted with a mixture of phenol/chloroform plasmid DNA 5 of these positive clones using a commercial kit "SeguenaseRVersion 2.0 of DNA Seguencing kit /United state of Biochemical Corporation, Cleveland. Ohio, USA/ after premirovany as T7 and SP6 primers /Promega/. The complete sequences of the inserts of various plasmids is determined in the following way. A separate reading frame can be identified in each of the five sequences of the inserts. Three types of sequences of the inserts was found relative to the amino acid sequence derived from the open reading frame, and three of seacology sequences. The complete DNA sequence of the inserts of each representing three types of plasmids N 1 - 3 shown in Fig. 12 together with the amino acid sequences translated from the open reading frame. The sequence of the first 15 amino acids derived from each type of plasmid inserts identical to the sequence defined for the position of amino acids 6-20 N-Terminus of the inhibitor isolated from the saliva of Triatoma /ex 17/.

Example 19. Position, selection and cloning of the gene encoding the inhibitor of platelet aggregation

Genomic library containing the cloning of restriction fragments from restriction endonuclease Perevalov T. pallidipennis DNA sceneroot probes on the replicative filters in accordance with standard methods. Clones that hybridize with the probes are selected. Then the DNA inserts of these clones subcloning in accordance with standard methods up until not allocate the minimum amount of DNA that bind to these probes.

These fragments is sequenced, and then transferred into a suitable eukaryotic vector expression, embedded coding the site in the expression vector by incorporating the coding of the site in the expression vector, spoligopatterns and the source replication all of which are operable associated with PAI gene /PAI = inhibitor of platelet aggregation, as well as marker selection for allocation thus obtained the expression vector. The expression vector transform then in eukaryotic host for which it is designed, and produce product EAI expression.

Example 20. Sequencing of the gene encoding the inhibitor of platelet aggregation /PAI/

Sequencing of single-strand and double strand DNA is done using the method of termination dideoxynucleotide circuits according to the method of Sanger et al., The OEWG. Natl. Aad. Sci. USA/1977//, 74, 5463 - 5467.

Screening of genomic libraries and clones mutant for new sequences, related sequence of T. pallidipennis /Example 21/. As before, the probes derived from the amino acid sequence of the inhibitor isolated from T. pallidipennis, used for other screening genomic libraries for sequences related to the inhibitors. Similarly, libraries of mutant inhibitors obtained by conventional mutagenesis vectors containing the gene for T. pallidipennis inhibitor, obtained in the way described earlier. NNMG and site-directed mutagenesis sceneroot on their activestrategy, as isoforms.

a General approach.

A cDNA library derived from the poly/+/RNA extracted from T. pallidipennis sceneroot two probes of example 18 on the replicative filter standard ways. Positive clones cleanse separate cultivation and repeat hybridization plaques on the filter. cDNA longest cDNA clones is sequenced dideoxynucleotide method of Sanger.

b/ Detailed description of the results.

Part 1. Construction of cDNA library from RNA salivary glands of Triatoma pallidipennis.

Approximately 500 μg total RNA isolated from the salivary glands of Triatoma pallidipennis? as indicated previously /Example 18, part 1/ use to highlight the poly a+mRNA using dual affinity chromatography on oligo/d T/-cellulose/. For this purpose use the set "mRNA Purification Ket"/Pharmacia Biosystems. GmbH, W - 7800 Freiburg, Germany for two consecutive cycles of enrichment, as indicated in the instructions of the manufacture. The final output after the second stage of purification is 13 μg of poly a+mRNA. 5 mg of this drug is used to construct a cDNA library in Lambda ZAPII vector bacteriophage with reagents and procedures commercial set "ZAP-cDNATM- GigapackII Gold Cloning Kit" /Ligiruwk 2 μg of vector DNA bacteriophage. After the packing is complete ligation reaction in 7 separate aliquot get reamplification cDNA library 20106independent recombinant phage.

Part 2. Selection of clones inhibitor cDNA from cDNA libraries of the salivary glands of Triatoma pallidipennis.

Just 5105recombinant phages from the cDNA library described previously, /1/, sceneroot using DNA-DNA hybridization dual plaques on nylon membranes BiogeneA /Pall Biosupport, East Hills, NY, USA/. Hybridization is carried out in the solution containing 5SSC, 5 x Denhardt solution, and 0.2% SDS and 100 μg/ml sperm DNA denatured salmon, extracted with phenol and processed by the ultrasound /Sigma chemical company, St Louis, MO, USA/ radiocanada DNA probes obtained as described previously. Insert DNA plasmid clone type N 1 of example 18 emit after double digestion using restriction endonucleases SphI and SacI, as indicated previously. Approximately 25 ng of the selected DNA inserts of radiometer using a set of "Prime-ITTMRandom Primer Labeling Kit" /Stratagene Cloning systems/ in the presence of /32P/ d CTP /3000 curies/mmol; Amersham Buchler, W-3300 Germany/. Labeled DNA fragment isolated from the rest of radioactive substances chromatography on a column of N Are leaching takes place in 2SSC of 0.2% SDS. After autoradiography at -70oC for 48 h find over 300 plaques that give signals on both filters. Bacteriophages with 80 sites around such positive signals elute from the source-the overlapping plates and re-cultured separately at a density that allows the purification of individual phage clones. The hybridization procedure plaques, previously described, is repeated, using the same probe DNA, and with plates allocate 76 independent phage clones giving a positive signal.

Part 3. Characterization and sequencing of cDNA clones inhibitor

Phage clones separately subjected in vivo to escanio according to the method described in the Protocol set to construct a cDNA library Stratagene described earlier. "Miniprep" plasmid DNA from 76 different plasmid clones pAluescript SR, isolated after in vivo excision, split in a double digestion with restriction endonucleases EcoRI and XboI (Bochringer Maunheim), separated on a 1.5% agarose gel and stained with ethidiumbromid. Several inserts of various sizes up to about 620 base pairs is obtained. DNA sequencing T3 and T7 primers /Stratagene Cloning systems/ performed as described previously on the square is zavisimyh clones. Thus were discovered cDNA clones that belong to 2 classes in accordance with the amino acid sequence translated from the open reading frame, and one class corresponds exactly with the type N 1 plasmid insert/ clones, referred to as "inhibitor-1 described in example 18/3/, and another class of type N 2 plasmid inserts /2 clone, referred to as "inhibitor-2"/. Further experiments on DNA sequencing is performed to confirm installed to this point sequences using additional oligodeoxynucleotide the founding of the famous sequences:

Oligonucleotide N 5:

5'-TATCACTCTGAACTCAAAGTG-3'.

Oligonucleotide N 6:

5'-TTACCGCCGTTTCCATTTGG-3'.

Oligonucleotide N 7;

5'-TTACTTCAAAGTTGCACC-3';

Oligonucleotide N 8:

5'-GCAACATGAAGGTGATCATTGCAGCAAC-3',

the 5'ends of most of the longest independent clones were found that were identical, and 5'-netransliruemye plot of 5 base pairs, suggesting that the full cDNA of mRNA transcripts, including the sites of initiation of transcription, were cloned. Full DNA and derived amino acid sequences of cDNA clones for inhibitor 1 and inhibitor-2 is shown in Fig. 13 sequence, described in example 17.

Example 23. Expression and secretion of recombinant inhibitor in stable transfection the kidney cells baby hamster /KSS/.

a General approach.

Then, the coding sequence of the cDNA clones are transferred into stable the eukaryotic expression vector, including coding the site in the expression vector, containing all the elements necessary for the expression of, for example, a promoter sequence, termination sequence and the source of replication, all of which are operable associated with PAI gene, and marker selection for allocation thus obtained the expression vector. Then the vector of the transform expression in eukaryotic host for which it is designed, and PAI expression product they release.

b/ the Specific description of the results.

Part 1. Construction of expression plasmids for inhibitor using pMPS V/CMV vector.

Two oligodeoxynucleotide synthesized for PCR amplification of the coding inhibitor sequences as of inhibitor-1 and inhibitor-2 plasmid cDNA clones. One of them /N 9/ make-coding strands of the area around the ATG initiation codon, elongated 5'-end of the t sequence, limiting AG initiation codon that modulates translation by eukaryotic ribosomes. Cell. 44, 283-292, 1986/, while the other /N 10/ derived from non-coding strands of the area around the TAA termination codon of the open reading frame, elongated 5'-tail, including Hind III recognition sequence /recognition sequence of restriction endonuclease Hind III underlined, optimized "Kozak Site or efficient initiation of translation is indicated by asterisks, lots of sequences that are compatible with one or the other thread source sequences of cDNA clones, marked by italics/

< / BR>
< / BR>
Using approximately 3 μg of each cDNA cloned plasmid as template, conduct two separate PCR amplification in the presence of two oligonucleotide primers N 9 and 10, as indicated previously /example 18 part 2/, however, with 18 instead of 38 cycles, including No. 1 - 3. Amplificatoare coding sequences inhibitor-1 and inhibitor-2, which contain optimized website Koz, but do not contain the full polyadenylation signal /'... . AATAAA... -3'/ located immediately after the 3'termination codon, the original cDNA clones, and then allocate and make suitable for ligation by digestion plays the guitar and sings the µg plasmid DNA vector pMP SV/CMV - HE/ With, M., Schumacher. L., Hauser, H.: Construction of new expression vectors for mammalian cells, using immediate early enhancer of human cytomegalovirus to improve expression of heterologous enhancers/ promoters. B Conradt, H./ Eo/ Protein Glycosylation: Cellular, Biotechnical and Analytical Aspects, t. 15, 49-52, VCH publishers Weinhein 1991; Kratzschmar, J., Haendler, B., Bringmann, P. , Dinter, H., Hess, H., Donner, P., Schleuning W-d: Secretion with a high level four plasminogen activators of ordinary vampire Desmodus rotundus stable transfectional cells baby hamster kidney, Gene /1992/ 116, 281-284 linearized by digestion with restriction endonuclease Hind III and emit in the way described earlier. The selected plasmid DNA diphosphorous using 1 unit of alkaline phosphatase, calf intestine/ Boehringer Mannheim/, extragere, and then using sublimirovanny encoding the inhibitor of PCR fragments by the method of example 18, part 3.

DNA obtained pMPSV/CM - inhibitor -1 or -2 constructs containing Hind III insert, digested with restriction endonuclease EcoR1/Boehringer Mannheim/ and in about half of cases, EoRI restriction fragment of about 580 base pairs determines that indicates that these constructs encoding the inhibitor of the insert is gerousia inhibitors insert such pMPSV/CMV - inhibitor and -2 structures then is sequenced using oligodeoxynucleotide N 5 - 8, and two additional primer /oligodeoxynucleotide N 12 and 11/ receive from along the edges of the insertion sites of the expression vector to control for mutations that could occur during PCR amplification:

Oligonucleotide N 11:

5'-ACCAGAAAGTTAACTGG-3',

Oligonucleotide # 12:

5'-CCTAGTTTGTGGTTGTCC-3'.

So get two constructs for the expression of inhibitor-1 and inhibitor-2 in mammalian cells, coding for proteins that are identical in their amino-acid sequences of proteins, is shown in Fig. 13. Schematic map of the structure shown in Fig. 14/ "AMR": resistant to ampicillin marker; "MPSV" promoter: the promoter of the virus, myeloproliferative sarcoma, "SY" : SV40 intron, including the boundary splicing, "peby A region" : SV 40 polyadenylation site, "CMV enhancer": enhancer of cytomegalovirus, "ori" : pBR 322 source replication/.

Part 2. Transfection and selection of BHK cells.

Plasmid DNA two pMPSV/CMV - inhibitor-1 and -2 allocate structures using columns Qiagen=lip 100"/ Qiagen Inc. Chatworth, California, USA/ Similarly, two plasmids, which contains a token resistance genes, one for gidromasazhni with genome hygromycin B kinase in Bluescript Sk, moreover, this fragment is taken from pPMP272 vector described: Bernard, H. U., Krammer. J. Rowekamp. W. G. Constructing gene fusions, which confers resistance against hygromycin B to mammalian cells in culture, Experimental Cell Research, 158, 237-243, 1985/, and the other for puromycin - N-acetyltransferase/ pSV of racer; DeLaLuna, S., Soria. J., Pulido, D., Ortin. J., Jimenez. A.: Efficient transformation of mammalian cells with constructs that contain a token resistance to puromycin, Gene 62, 121-126, 1988/ get the result. Approximately 20 μg of design expression of inhibitor-1 - or -2,6 µg puromycin-resistant plasmid and 2 μg of hygromycin-resistant plasmids used for transfection of kidney cells baby hamster /BHK/ as described in Kratzshmar. J. , Handler, B. , Bringmann,P., Dinter, H., Hess, H., Douner. P., Schleuning, W.-D. 1992. Efficient secretion of the four salivary plasminogen activators of vampire Desmodus rotundus due to stable transfection of kidney cells baby hamster /Gene, 116 - 281-284/ using LipofectinTMReagent" /Gibco-BRL Life Technologies/. The dual procedure of selection is used, using DMEM /10% FCS/Gibco BRL Life Technologies/ containing 0.7 mg/ml hygromycin B/ Calbiochem Corporation, La Golla California, USA/ 5 μg/ml puromycin /Sigma chemical company/. A mixture of BHK cells with double the resistance transcode OPTI-MEM /Gibco-BRL Life Technologies /as described Kratschmar, J. , Haendler, B. , Bringmann, P., Dinter, H, Hess, H., Douner, P., Schleuning W-d : high-performance four secretion of the salivary plasminogen activators of vampire Desmodus rotundus due to stable transfection kidney cells baby hamster. Gene /1992/ 116, 281-284/. Conditioned medium collected after 24 h, released from the cell debris by centrifugation at 2000 g and stored in frozen form.

Part 3. Determination of recombinant inhibitor in the supernatant fluid of the culture of BHK cells.

Aliquots of conditioned medium are tested for the production of the inhibitor in Western-blotting (see example 24/. Antiegalitarian anticavity reacts specifically with 19 kDa protein present in conditioned medium from pMPSV/CMV - inhibitor-1 transfection BHK cells from pMPSV/CMV - inhibitor-2 - transfection BHK cells. Not observed reaction with the control environment of the cells, transfection pMPSV/CMV design containing inhibitory paste, or fresh control medium (see Fig. 15/. Extracts transfection cells provide only a weak signal, indicating that the recombinant proteins are secreted into the environment. In addition to the immunological detection of two recombinant inhibitory forms, the supernatant of the LM which has been created by the collagen platelet aggregation can be measured in the aggregation analysis by the method of example 1.

Example 24. Obtaining antibodies.

About 100 μg of inhibitor, purified by the methods of examples 2 and 15, is added to 0.5 ml of complete adjuvant Freund, and the resulting emulsion is injected subcutaneously rabbit. 2 weeks later administered a second injection of about 80 µg of purified inhibitor and 0.5 ml incomplete adjuvant's adjuvant. After injection select multiple serum samples to test the production of antibodies. They are analysed by Western-blotting. 20 ng of purified inhibitor is applied to 12.5% SDS acrylamide gel and carry out electrophoresis, blotting and detection by standard methods described Harlowe, D., Lane /1988/ Antibodies: a laboratory manual, Cold Spring Harbour Laboratory /dilution of the test serum 1: 500, goat anti-rabbit HRP conjugated IgG as the second antibody, detection ECL-kit from Amersham International, Amersham, velicer. The blot shows that anticavity specifically reacts with purified inhibitor.

1. The protein, inhibiting caused by collagen platelet aggregation mammals, isolated from the saliva of Triatoma pallidipennis, having the following N-terminal amino acid sequence: Glu Glu Cys Glu Leu Met Pro Pro Gly Asp Asn Phe Asp Leu Glu Lys Tyr Phe Ser Ile.

2. Recombinant protein, ing islotes sequence:

where X is Pro or Gln;

Y - Gly, Z - Gly or Val.

3. The protein in p. 2, characterized in that it has the amino acid sequence in which X - Pro-Y - Gly, Z - Gly.

4. The protein in p. 2, characterized in that it has the amino acid sequence in which Thr at position 53 is absent, X - Pro-Y - Gly, Z - Val.

5. The protein in p. 2, characterized in that it has the amino acid sequence in which X is Gln, Y - Gly, Z - Gly.

6. The DNA fragment encoding the recombinant protein, which inhibits caused by collagen platelet aggregation mammals, with the following nucleotide sequence:

where X is a CCG or CAG;

Y - GGT or GTT;

Z - ACA or ACG;

R - TCG or TCA.

7. The DNA fragment under item 6, characterized in that has a nucleotide sequence in which the X - CCG, Y - GGT, Z - ACA, R - TCG.

8. The DNA fragment under item 6, characterized in that has a nucleotide sequence in which the nucleotides at position 148, 149, 150 missing, X - CCG, Y - GTT, Z - ACA, R - TCG.

9. The DNA fragment under item 6, characterized in that has a nucleotide sequence in which the X - CAG, Y - GGT, Z - ACG, R - TCA.

10. The expression vector pMPSV/CMV - inhibitor-I (DSM 7223) containing the DNA fragment under item 7, the area polyacene the>11. The expression vector pMPSV/CMV - inhibitor-II containing the DNA fragment under item 8, the region polyadenylation (poly A), the gene of resistance to ampicillin, CMV enhancer, the mPSV promoter, the site of splicing (SJ), ori pBR 322.

12. Strain cultured animal cells (EGC pMPSV) CMV DSM 7223/ - inhibitor - I - producer of recombinant protein, enzyme inhibition caused by collagen platelet aggregation mammals.

13. Strain cultured animal cells (EGC pMPSV) CMV - inhibitor - II - producer of recombinant protein, enzyme inhibition caused by collagen platelet aggregation mammals.

14. A method of obtaining a recombinant protein, enzyme inhibition caused by collagen platelet aggregation mammals, involving the cultivation of the producer strain, isolation and purification of the target product, characterized in that as a producer uses a strain on p. 12 or 13, and the cleaning is carried gelfiltration "Superose 12" and in the system of the electrophoresis chromatography.

15. The pharmaceutical composition inhibiting induced by collagen platelet aggregation, containing the active ingredient and pharmaceutically acceptable carrier or diluent, characterized in that as the asset is

 

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