JP2003116574A - Ti-2 PROTEIN DERIVED FROM TRIATOMA INFESTANS HAVING BLOOD CLOTTING INHIBITION ACTIVITY - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new protein having blood clotting inhibition activity. SOLUTION: The present invention provides a Ti-2 protein which is a new protein derived from Triatoma infestans and provide a Ti-2 gene encoding the protein. The Ti-2 protein has blood clotting inhibition activity and, accordingly, pharmaceuticals containing the Ti-2 protein as an active component are useful as a blood clotting inhibition agent for the treatment and prevention of myocardial infarction, pulmonary infarction and cerebral infarction. The Ti-2 protein is highly expectable also in the field for the development of pharmaceuticals as a leading compound of a blood clotting inhibitor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Ti-2 protein having a blood coagulation-inhibiting activity, which is derived from the salivary glands of the Brazilian blood turtle (Triatoma infestans), which is a blood-sucking insect.
-2 Regarding a gene encoding a protein.

[0002]

2. Description of the Related Art With the advent of an aging society, adult diseases have become an increasingly important social problem. Symptoms caused by adult diseases, especially blood-related diseases such as hypertension, pulmonary hypertension, myocardial infarction, cerebral infarction, pulmonary infarction, vasospasm after subarachnoid hemorrhage, etc. Treating and preventing the diseases caused by the disease is an important issue in the society of the elderly. These diseases can be treated or prevented by a vasorelaxant and a blood coagulation inhibitor. Hirudin derived from the salivary gland of leech has been known as a peptide having anticoagulant activity that can be used for such purposes. Hirudin is an anticoagulant peptide identified from the salivary glands of sucking worms and has thrombin inhibitory activity.

[0003]

However, the above-mentioned hirudin has the drawback that it is difficult to synthesize and has side effects. Therefore, in order to obtain a large amount and use it as a safe medicine, it is necessary to solve these problems. Therefore,
It has been required to collect a protein having an anticoagulant activity which is easy to synthesize and has no side effect. Such proteins are useful as lead compounds for antithrombotic agents and are considered to have high utility in the field of drug discovery.

Therefore, an object of the present invention is to provide a protein isolated from the salivary glands of the Brazilian blood turtle (Triatoma infestans), which is a blood-sucking insect, and has a blood coagulation-inhibiting activity. Furthermore, it is to enable large-scale supply of such proteins using the baculovirus system.

[0005]

In order to solve the above problems, the present invention provides the following inventions. The present invention is a Ti-2 protein derived from Brazilian tortoises, which consists of the amino acid sequence represented by amino acid number (-17) -189 shown in SEQ ID NO: 1 in the sequence listing.
A protein having blood coagulation-inhibiting activity, a part of which is deleted, substituted or added is also within the scope of the present invention.

Further, the present invention is a Ti-2 protein derived from Brazilian tortoises, which is characterized by comprising the amino acid sequence represented by amino acid numbers 1-189 shown in SEQ ID NO: 1 of the sequence listing. Has blood clotting inhibitory activity, some of which are defective,
Substituted or added proteins are also within the scope of the invention.

The present invention further relates to a Ti-2 gene derived from Brazilian tortoises, which is characterized by comprising the nucleotide sequence represented by nucleotide numbers 1-699 shown in SEQ ID NO: 2 of the sequence listing. A gene encoding a polypeptide having blood coagulation-inhibiting activity and a part of which is deleted, substituted or added is also within the scope of the present invention.

Further, the present invention is a blood coagulation inhibitor containing the above protein as an active ingredient.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The salivary glands of blood-sucking insects and mites contain substances having specific activity on blood and blood vessels of animals. The present inventors identified active substances having anticoagulant activity from salivary glands, isolated and purified them, analyzed the properties of their active ingredients, and cloned the gene cDNA to obtain baculovirus. The present invention has been completed by clarifying that a protein having a vasorelaxant function can be produced in a large amount by a virus expression system.

That is, the present inventors paid attention to the Brazilian blood turtle (Triatoma infestans: Ti), which is a blood-sucking insect, and attempted to collect a protein having anticoagulant activity derived from the Brazilian water turtle. Salivary glands were excised from about 25 Brazilian tortoises, total RNA was extracted, dsDNA was synthesized by reverse transcriptase as poly (A) + RNA, and incorporated into a transfer vector to prepare a salivary gland cDNA library. Colonies were randomly picked up from the Ti salivary gland cDNA library and the nucleotide sequence was analyzed.
550 sequences were determined and, except for duplication, 44 cDNAs having a secretory signal were obtained. Of these, 16 total nucleotide sequences were determined.

For 15 of them, transfer vector constructs for expression in a protein expression system using baculovirus (AcNPV) were prepared and transfected into the virus, and polynuclear bodies were not formed, that is, the inserted protein was expressed. Isolated virus clones.
The Ti-2 protein of the present invention was collected by confirming the protein expression by SDS-PAGE and purifying by gel filtration / ion exchange chromatography by HPLC. Then, the effect of the protein of the present invention collected by the above method on blood coagulation was examined.

By the way, the process of blood coagulation depends on the initiation mechanism, and therefore, there are two types of coagulation reactions, intrinsic coagulation reaction and extrinsic coagulation reaction.
Two routes are known. The intrinsic coagulation reaction is a reaction caused by the contact of blood with the surface of a foreign substance. The cascade system of blood coagulation is shown in FIG. As shown in Fig. 1, activated No. XI produced by contact with the foreign surface
factor a activates factor IX in the presence of calcium
It produces IXa, which triggers the production of fibrin and the formation of a thrombus. On the other hand, the extrinsic coagulation reaction is initiated when tissue factor (TF) forms a complex with factor VIIa, and activation of both factor IX and factor X triggers the formation of a thrombus. It

It is a common practice to examine the inhibitory effect on blood coagulation by adding a target substance to human plasma and measuring the time until coagulation. But,
Since this method observes the final coagulation due to fibrin formation, it is not possible to judge the specific point of action and mechanism of action of the blood coagulation inhibitor. That is, since the blood coagulation reaction is a complicated chain reaction, if a part of the reaction pathway is blocked, the subsequent reaction will not proceed and the coagulation will not be completed.

Therefore, in the present invention, in order to evaluate the blood coagulation ability, the activated partial thromboplastin time (ac
Tivated partial thromboplastin time (APTT) and prothrombin time (PT) were measured. The former reflects the intrinsic coagulation time, and the latter reflects the extrinsic coagulation time. As a result, Ti-2 protein prolonged the intrinsic coagulation time in a concentration-dependent manner. Therefore, it was shown that the Ti-2 protein of the present invention has an action of suppressing blood coagulation and is effective as a blood coagulation inhibitor. Therefore, the Ti-2 protein is considered to be effective as a therapeutic or preventive drug for myocardial infarction, pulmonary infarction, cerebral infarction and the like.

The Ti-2 protein is specified by the amino acid sequence represented by amino acid numbers (-17) -189 shown in SEQ ID NO: 1 in the sequence listing. In the present specification, a protein in which a part of the protein shown in SEQ ID NO: 1 is deleted, substituted or added is 20 or less, preferably 10 or less, more preferably 5 in the amino acid sequence shown in SEQ ID NO: 1. It is a protein in which no more than 4 amino acids have been substituted. Further, such protein and the amino acid sequence shown in SEQ ID NO: 1 have 95% or more homology, preferably 97% or more homology, and more preferably 99% or more homology. Such a protein is also within the scope of the present invention as long as it has a function as a Ti-2 protein that inhibits blood coagulation. In the SEQ ID NO: 1 of the sequence listing, the portion from -17 to -1 represents a signal peptide, and as a result of processing, the mature protein is represented by amino acid number 1-189.
It consists of individual amino acids.

The Ti-2 gene encodes the above-mentioned Ti-2 protein and has the base number 1-shown in SEQ ID NO: 2 in the sequence listing.
It is characterized by comprising the nucleotide sequence shown by 699. The portion corresponding to base numbers 10-627 in the base sequence is the reading frame and encodes the above protein. According to the gene recombination technology, it is possible to artificially mutate a specific site of the basic DNA without changing the basic characteristics of the DNA or improving the characteristics. A gene having a natural base sequence provided by the present invention or a gene having a base sequence different from that of the natural gene is equivalent to the natural gene by artificially inserting, deleting, or substituting. Or with improved properties, and the invention includes such mutated genes.

That is, a gene in which a part of the gene shown in SEQ ID NO: 2 in the sequence listing is deleted, substituted or added is 20 or less, preferably 20 or less in the nucleotide sequence shown in SEQ ID NO: 2.
It is a gene in which 10 or less, more preferably 5 or less bases are substituted. Further, such gene and the nucleotide sequence shown in SEQ ID NO: 2 have 95% or more homology, preferably 97% or more homology, more preferably 99% or more homology. Such a gene is also within the scope of the present invention as long as it encodes a protein having a function as a Ti-2 protein that inhibits blood coagulation. In addition, such a gene forms a hybrid with the gene shown in SEQ ID NO: 2 in the sequence listing under stringent conditions.

The Ti-2 protein of the present invention can be produced in a large amount in a baculovirus expression system incorporating the cDNA of the Ti-2 protein. Examples of these are given below. Bombyx mori nuclear polyhedrosis virus (BmNPV) can be used to express BmN4 in silkworm culture cells or larvae in silkworm, and can be isolated from the culture medium or silkworm body fluid by chromatography. In addition, the cDNA of Ti-2 protein was cloned from Autographa California (Autograp
ha californica) nuclear polyhedrosis virus (AcNPV), SF9 cells of armyworm (Spodoptera frugiperda), or nettle herb (Trichoplusia ni)
Can be expressed in Tn5 cells and purified from the culture supernatant in the same manner by chromatography.

The Ti-2 protein in the present invention can also be produced in large amounts by using an expression system using Escherichia coli in which the cDNA of the Ti-2 protein is incorporated. For such purpose, the cDNA of Ti-2 protein was amplified to obtain pGEX6P-1, pGE
Glutathione-incorporated into plasmids such as X-2T and pGEX-3X
An S transferase (GST) fusion protein expression vector can be prepared. Then, Escherichia coli is transformed with the expression vector and cultured in a medium containing IPTG to induce the expression of the GST fused Ti-2 protein in E. coli. Examples of Escherichia coli strains that can be used for such purposes include BL21 strain, DH5α strain, and NM522 strain. Then, the GST-fused Ti-2 protein induced in E. coli can be recovered by disrupting E. coli. GS collected in this way
The T-fusion Ti-2 protein can be purified by affinity chromatography using glutathione beads.

The Ti-2 protein of the present invention is a baculovirus expression system by identifying an active substance having a blood coagulation-inhibiting action from the salivary gland of a blood-sucking insect, isolating and purifying it, and cloning the gene cDNA thereof. It is possible to produce a large amount of a protein having an inhibitory effect on blood coagulation. In addition, the active site due to the blood coagulation inhibitory effect of the protein was investigated,
If structural analysis progresses, it is also possible to manufacture the active substance by a general chemical synthesis method by a molecular design method.

Further, the Ti-2 protein of the present invention is considered to be highly useful as a lead compound for a drug having a blood coagulation inhibitory action. That is, it is possible that a substance having a higher blood coagulation inhibitory effect can be obtained by making various modifications to the Ti-2 protein. The Ti-2 protein of the present invention provides a physiologically active substance which is the basis of such studies, and has great utility as a lead compound for obtaining a further novel anticoagulant substance. .

[0022]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited to these examples.

(Method of collecting genes) A salivary gland was excised from the chest of a Brazilian tortoise (Triatoma infestans). From this, MIicroprep mRNA Purification kit (Amersham
The salivary gland mRNA was extracted and purified using Pharmacia). Next, using this mRNA as a template, Superscript
Salivary gland cD using Plasmid system (Life technologies)
NA library was constructed. Randomly picked clones (550 clones in total) from this library, QIA
The plasmid was extracted and purified with Prep Spin miniprep (manufactured by QIAGEN). The nucleotide sequence of the cDNA incorporated in this plasmid was analyzed using the ABI PRISM 310 Genetic analyzer (PE biosy
stems). The decoded cDNA nucleotide sequence is Genetyx ver8.5 (manufactured by Software development)
Was analyzed. As a result, they have the same base sequence
Four cDNA clones were found and named Ti-2.

(Large-scale expression and purification of recombinant protein) The full-length Ti-2 cDNA amplified by the PCR method was transformed with Bam of the plasmid pAcYMl.
A transfer hector incorporated into the HI restriction enzyme site was prepared and purified using a Plasmid mini kit (QIAGEN). This plasmid hector and Baculo Gold Linerli
a mixture of zed Baculovirus DNA (Pharmingen)
1 ipofectin reagent (manufactured by life technogies) was used to introduce into insect culture cells Sf-9 to prepare recombinant baculovirus. This recombinant virus was transformed into another insect culture cell Tn-5.
The recombinant Ti-2 protein was expressed in large amounts. Recombinant Ti-2 protein secreted into the culture medium
Purification was carried out by performing two-step purification by cation exchange chromatography using mersham Pharmacia) and gel filtration chromatography using TSK2000SW (manufactured by Tosoh), and used for the following experiments.

(Measurement of activated partial thromboplastin time) Human plasma preparation (trade name: Kaliplasma Index
100, manufactured by bioMerieux) 20 μl against 50 mM TrisHCl
, PH7.0, 20 μl of purified Ti-2 dissolved in 150 mM NaCl was added and incubated at 37 ° C. After 5 minutes, actin (trade name: Dataphi APTT, manufactured by CYSMEX) diluted 1/10 was added in an amount of 35 μl and further incubated for 2 minutes. Finally 25 mM CaCl ₂ to 25μ1 added, was measured by the time the Amelung KC-10A micro until the freezing is this complete (manufactured by MC Medical Inc.).

The effect of Ti-2 on APTT is shown in FIG. From this result, Ti-2 showed the activity of extending APTT only in a concentration-dependent manner. That is, it was revealed that the protein Ti-2 derived from Brazilian tortoise salivary gland is a protein that inhibits the endogenous Sego reaction.

[0027]

INDUSTRIAL APPLICABILITY According to the present invention, a Ti-2 protein, which is a novel protein derived from Brazilian tortoises, and a Ti-2 gene encoding the protein are provided. Since the Ti-2 protein has blood coagulation inhibitory activity, a drug containing the Ti-2 protein as an active ingredient is effective as a blood coagulation inhibitor for treating and preventing myocardial infarction, pulmonary infarction, and cerebral infarction. In addition, Ti-2 protein also has great potential as a lead compound for blood coagulation inhibitors in the field of drug development.

[0028]

[Sequence list] <110> Mie University President <120> Ti-2 protein derived from Brazilian tortoise having blood coagulation inhibitory activity <160> 2 <210> 1 <211> 206 <212> amino acids <213> Triatoma infestans <400> 1   -17   Met Lys Thr Ile Leu Ala Val Ile Phe Phe Gly Ile Leu Ala Phe Ala -2   Phe Ala Asp Tyr Pro Ser Ile Pro Lys Cys Thr His Pro Pro Ala Met 15   Ala Asn Phe Asn Gln Lys Lys Phe Leu Glu Gly Lys Trp Tyr Val Thr 31   Lys Ala Lys His Gly Ser Asn Ser Thr Val Cys Arg Glu Tyr Arg Ala 47   Lys Thr Lys Gly Asn Asp Gln Ile Leu Val Gly Asp Gly Tyr Tyr Ser 63   Phe Asn Gly Gly Thr Phe Tyr Phe Thr Val Arg Cys Lys Arg Leu Pro 79   Asn Lys Glu Val Gln Lys Pro Leu Gln Phe Thr Cys Thr Gln Lys Ser 95   Pro Asp Asp Pro Ser Lys Met Phe Lys Phe Gln Leu Glu Val Thr Ile 111   Leu Asp Thr Asp Tyr Ala Asn Tyr Ala Val Met Tyr Arg Cys Val Gln 127   Phe Pro Glu Glu Leu Gly Ser His Phe Glu Asp Asn Thr Leu Leu Leu 143   His Arg Lys Leu Asp Gln Leu Val Asp Glu Asn Leu Ile Glu Arg Lys 159   Leu Lys Leu Ser Leu Pro Ser Phe Lys Ser Arg Asp Asp Val Val Glu 175   Gly Cys Arg Glu Leu Pro Ser Lys Lys Lys Lys Thr Lys Pro 189 <210> 2 <211> 699 <212> nucleic acid <213> Triatoma infestans <400> 2  TTCGCCAATA TGAAGACGAT TTTGGCCGTG ATTTTTTTTG GAATTTTGGC GTTTGCATTT 60  GCTGATTATC CATCAATTCC AAAATGCACT CACCCTCCAG CTATGGCAAA CTTTAATCAA 120  AAAAAATTTT TAGAAGGAAA ATGGTATGTA ACAAAAGCAA AACATGGATC AAATTCAACT 180  GTTTGTCGAG AATACAGAGC CAAAACTAAG GGTAACGATC AAATACTTGT CGGTGACGGA 240  TATTACTCGT TTAATGGTGG AACATTCTAC TTTACAGTTC GTTGTAAGAG GCTGCCAAAT 300  AAGGAAGTTC AAAAACCACT GCAATTTACC TGCACTCAAA AAAGTCCTGA CGACCCGAGC 360  AAGATGTTTA AATTTCAACT TGAGGTTACT ATTCTTGACA CAGACTATGC TAACTATGCT 420  GTAATGTATA GATGTGTCCA GTTTCCCGAG GAACTTGGGT CACATTTTGA AGATAATACT 480  TTGCTATTAC ACCGGAAACT AGATCAACTA GTTGACGAAA ATCTAATTGA AAGAAAACTC 540  AAGTTGTCAT TACCTTCATT TAAATCCAGG GATGATGTTG TAGAAGGTTG TCGAGAACTT 600  CCATCAAAAA AGAAAAAGAC AAAGCCATAA ACCTAGTGTT ATAAATAAAT TGACATTTGC 660  AAAAATATAA AACTTTGCGA AACGAAAAAA AAAAAAAAA 699

[Brief description of drawings]

FIG. 1 is a diagram showing a cascade system of blood coagulation leading to thrombus formation.

FIG. 2 is a diagram showing the sequences of the Ti-2 protein and the gene encoding it.

FIG. 3 is a graph showing the effect of Ti-2 protein on activated partial thromboplastin time (APTT).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Haruhiko Izawa             76-1 Isshin Tanakano, Tsu City, Mie Prefecture             Nakano 201 F term (reference) 4B024 AA01 BA80 CA04 DA02 DA05                       EA02 EA04 FA02 GA11 HA01                       HA03                 4C084 AA02 AA06 AA07 BA01 BA22                       CA49 CA53 DC16 DC32 NA06                       ZA542 ZC202                 4H045 AA10 AA20 AA30 BA10 CA51                       DA00 EA20 FA72 FA74

Claims (5)

[Claims] 1. A protein derived from Brazilian tortoises,
A protein comprising the amino acid sequence shown in (a) or (b) below. (A) Amino acid number (-17) -shown in SEQ ID NO: 1 in the sequence listing
A protein comprising the amino acid sequence represented by 189. (B) has blood coagulation-inhibiting activity, part of (a) is defective,
A protein that has been replaced or added. 2. A gene encoding the protein according to claim 1. 3. A protein derived from Brazilian tortoises,
A protein comprising the amino acid sequence shown in (c) or (d) below. (C) Amino acid number 1-189 shown in SEQ ID NO: 1 in the sequence listing
A protein comprising an amino acid sequence represented by: (D) has blood coagulation-inhibiting activity, part of (c) is defective,
A protein that has been replaced or added. 4. A gene encoding a protein derived from Brazilian tortoise and comprising the nucleotide sequence shown in (e) or (f) below. (E) A gene comprising a base sequence represented by base numbers 1-699 shown in SEQ ID NO: 2 of the sequence listing. (F) encodes a protein having blood coagulation inhibitory activity,
A gene in which a part of (e) is deleted, substituted or added. 5. A blood coagulation inhibitor containing the protein according to claim 3 as an active ingredient.