JPH05255395A - Polypeptide and its production - Google Patents

Abstract

PURPOSE:To produce a new polypeptide having inhibitory action on blood platelet aggregation by a genetic engineering method. CONSTITUTION:A cDNA encoding a new polypeptide having inhibitory action on blood platelet aggregation in snake poison obtained from a poisonous liquid secretion gland of Agkistrodon blomhoffi is cloned to elucidate the gene structure of the polypeptide. A recombinant DNA containing the cDNA or its variant is constituted, a transformant is transduced, the transformant is cultured and a polypeptide having inhibitory action on blood platelet aggregation is collected from the culture solution to give the polypeptide. A gene encoding the polypeptide having inhibitory action on blood platelet aggregation is manifested by genetic engineering method to give a large amount of the high-purity polypeptide and the polypeptide is useful as an antithrombotic agent, a cancer metastasis inhibitor and a bone resorption inhibitor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention uses a recombinant DNA technique to eukaryote a polypeptide gene encoded by a snake venom gene having a platelet aggregation inhibitory activity, a cancer cell metastasis inhibitory activity and a bone resorption inhibitory activity. Alternatively, it relates to a technique for producing a novel polypeptide having a platelet aggregation inhibitory activity, a cancer cell metastasis inhibitory activity and a bone resorption inhibitory activity by expressing it in a prokaryote.

[0002]

2. Description of the Related Art Recently, a polypeptide having a platelet aggregation inhibitory activity has been isolated from a certain snake venom. T.-F.H
uang, et al. [Journal of Biological Chemistry (J. Biol. Chem.), 262 , 16157 (1987);
-250399] is trimereslus gramineus ( T
A polypeptide of about 9 kDa having the activity of inhibiting platelet aggregation (named Trigramin) was isolated from the snake venom of Rimeresurus gramineus ). Trigramin is
It has been demonstrated to inhibit fibrinogen-induced platelet aggregation of platelets stimulated with adenosine diphosphate (hereinafter sometimes abbreviated as ADP) and chymotrypsin-treated platelets. The binding of ¹²⁵ I-fibrinogen to ADP-stimulated platelets was competitively inhibited by trigramin. These results are
It suggests the interaction of trigramin with the fibrinogen receptor on the platelet membrane. KA Knudsen et al. [Experimental Cell Research (Exp. Cell Re
s.), 179 , 42 (1988)] reported that trigramin inhibits adhesion of human melanoma cells to fibronectin and fibrinogen. Later, the structure of trigramin was determined by T. -F. Huang et al.
mistry), 28 , 661 (1989)], and proved that it is a single-chain polypeptide consisting of 72 amino acid residues and has 6 disulfide bonds. In addition, ¹²⁵ I-trigramin binds to platelets due to GPIIb-IIIa binding to platelets.
Antibody against Arg-Gly-Asp-S
It was proved to be inhibited by er. It was speculated that these biological activities of trigramin depend on the presence of the Arg-Gly-Asp (RGD) sequence, the secondary structure of the molecule and the presence of sequences common to the adhesion proteins.

Z. -R. Gan et al. [J. Biol. Chem., 263 , 198].
27 (1988); JP-A-2-138298] discloses a polypeptide having an inhibitory activity on platelet aggregation from the snake venom of Echis carinatus [Echistatin].
Named] was purified. Exstatin consists of 49 amino acid residues including 8 cysteine residues, GPIIb-III
a common RGD (Arg
-Gly-Asp) sequence. Z.-R. Gan et al.
ene), 79 , 159 (1989); JP-A-2-138298],
A gene encoding a mutant echistatin in which exatin Met ²⁸ was converted to Leu was chemically synthesized, and a fusion gene in which this gene was linked to the che gene was prepared and expressed in Escherichia coli. After reacting the obtained fusion protein with CNBr, mutant echistatin was separated. MA Jacob
Son et al. [Gene, 85 , 511 (1989)] ligated the above extatin synthesis gene downstream of the pre-pro sequence of the α-mate factor to express it in yeast, and extract extatin from the culture supernatant of yeast. Purified. M. Sato et al.
J. Cell Biol., 111 , 1
713 (1990)] reported that echistatin inhibits bone resorption by osteoclasts. RJ Shebuski et al. [J. Bio
L. Chem., 264 , 21550 (1989)] isolated a polypeptide having a platelet aggregation inhibitory activity (named Bitistatin) from the snake venom of Bitis arietans . Bistatin is a single-chain polypeptide consisting of 83 amino acid residues containing 7 disulfide bonds, contains an RGD sequence, and shows high homology with trigramin and etistatin. Bistatin is ex v
It was demonstrated to inhibit both iv platelet aggregation and platelet-dependent reduction of perfusion in a dose-dependent manner. BH Ch
ao et al. (Proc. Natl. Acad. Sci. USA, 86 , 8050 (198
9)] is a North American viper water moccasin ( Agkistrodon
piscivorus piscivorus snake venom isolated an apragin consisting of 71 amino acid residues (containing RGD sequence and high Cys content) (Applaggin). Apragin is ADP,
It was reported to inhibit platelet aggregation induced by collagen, thrombin and arachidonic acid. M.
S. Dennis and others [Procedure of the National Academy of Sciences of the United States of America (Proc. Natl.
Acad. Sci. USA), 87 , 2471 (1989)] have isolated analogs of snake venom polypeptides that inhibit platelet aggregation. From the snake venom of Agkistrodon rhodostoma , there is a kistrin consisting of 68 amino acid residues.
in), venom-α (Bitan-α) consisting of 83 amino acid residues from the venom of Vitis arietans, and three isoforms of trigramin (trigramin-α) from the venom of Trimereslus gramineus [Trigramin-β1].
(72 amino acid residues); Trigramin-β2 (73 amino acid residues); Trigramin-γ (73 amino acid residues)],
From the snake venom of extract Karinatus, the isoform of etistatin (ethistatin-α1) [ethistatin-α2
(47 amino acid residues)] were isolated. These polypeptides have RGD sequences and were shown to have high cysteine content (17 ± 1%). Each polypeptide was purified GPIIb-IIIa on immobilized fibrinogen.
Binding of the pentapeptide Gly-Arg-Gly-Asp-Ser (GRG
DS; RGD-containing peptide that binds to GPIIb-IIIa) was inhibited about 100 times more effectively. Moreover, each peptide inhibited human platelet aggregation stimulated by ADP about 1000 times more effectively than GRGDS. Kistrin showed high affinity for resting and ADP-activated human platelets and also bound reversibly. Single-bolus administration (1 mg / kg) of kistrin to rabbits resulted in ex
It was shown that in vivo collagen-stimulated platelet aggregation was inhibited by 70% after 5 minutes and returned to normal values within 30 minutes.

In JP-A-2-138298, from the snake venom of Agkistrodon piscivorus, Agkistrosta-tin (amino acid sequence is partially unknown), from the snake venom of Vitis allietans, vitistatin 1 (83 amino acid residues) ), Bistatin 2 (83 amino acid residues), Bistatin 3 (84 amino acid residues) and Bistatin 4 (partially elucidated amino acid sequence), Eristocophis McMahoney ( Eristocophis
Isolation of eristostantin consisting of 49 amino acid residues from the snake venom of Macmahanii) and chemical synthesis of echistatin analogs have been reported. J. William
s et al. (Biochimika & Biophysica Actor
m. Biophys.Acta), 1039 , 81 (1990)] is Trimeresurus elegans and Trimeresurus albola.
A polypeptide consisting of 73 amino acid residues having an inhibitory activity on platelet aggregation was isolated from a snake venom of Bris ) and isolated from each of them by using Elegantin and Albolabrin.
in). B. Rucinski et al. [Biochim. Biophys. A
cta, 1054 , 257 (1990)] isolated Batroxostatin consisting of 71 amino acid residues from the snake venom of Bothrops atrox ,
It was further shown that the polypeptide inhibits platelet aggregation and adhesion of melanoma cells to fibronectin. JL Seymour et al. (J. Biol. Chem.,
265 , 10143 (1990)] is a North American hill [Macro Budera.
A polypeptide that inhibits platelet aggregation and acts as an antagonist of the platelet glycoprotein GPIIb-IIIa (named Decorsin) was isolated from Macrobdella decora . Decorsin contains 6 cysteine residues and 6 proline residues with the RGD sequence 39
It was composed of amino acid residues. Also, an N-3 isoform in which the amino-terminal 3 residues of decorsin were deleted was found. Decorsin and N-3 isoforms were shown to inhibit GPIIb-IIIa binding to immobilized fibrinogen, and decorsin was shown to inhibit ADP-induced human platelet aggregation. All of the above-mentioned polypeptides were purified from commercially available snake venom or leech extract, and cloning of these genes is not known due to restrictions on material supply.

[0005]

As described above, various polypeptides having an inhibitory activity on platelet aggregation have been isolated using snake venom or leech as a starting material, and the genes of such peptides have been sufficiently elucidated. It has not been. Therefore, a snake gene library was prepared, a cDNA encoding a novel polypeptide having platelet aggregation inhibitory activity, cancer cell metastasis inhibitory activity and bone resorption inhibitory activity was cloned to elucidate the gene structure of the polypeptide. It has been desired to express the coding region of the polypeptide in eukaryotes and prokaryotes to produce a large amount of a novel polypeptide having the activity.

[0006]

[Means for Solving the Problems] The present inventors have conducted research aiming at elucidating the gene of the platelet aggregation inhibitory polypeptide present in snake venom, and as a result, the viper snake, which is a poisonous snake that lives in Japan ( CD encoding a novel platelet aggregation-inhibiting polypeptide from a cDNA library prepared from the venom gland of Agkistrodon halys blomhoffii )
Successfully cloned NA. Furthermore, the c
Recombinant DNA containing DNA (gene) is constructed and
By culturing the transformant transformed with A,
Enables the production of novel platelet aggregation-inhibiting polypeptides,
The present invention has been completed.

That is, the present invention provides (1) extraction of mRNA from a venom secretory gland of the viper pit viper and synthesis of cDNA by reverse transcribase using the mRNA.
(2) Antagonist of previously reported snake venom GPIIb-IIIa [MS Dennis et al., Proc. Natl. Acad. Sci. USA, 87 ,
2471 (1989)] using the amino acid conserved region of Polymerase Chain React.
(ion: PCR) synthesis of primer and preparation of cDNA library by PCR using this primer, (3)
Consensus sequence of known platelet aggregation-inhibiting polypeptides [MS
Dennis et al., Proc. Natl. Acad. Sci. USA, 87 , 2471 (198
9)] preparation of a primer, and radiolabeling of the 5 ′ end of the primer using T4 polynucleotide kinase and [γ- ³² P] -ATP, (4) this labeled probe and the cDNA library Separation of positive clones by hybridization, (5) identification of the coding region of a novel platelet aggregation-inhibiting polypeptide, (6) preparation of recombinant DNA containing a nucleotide sequence encoding this novel polypeptide, (7) above (6) Recombinant DNA
(8) culturing the transformant described in (7) above, allowing a new polypeptide having a platelet aggregation inhibitory activity to be produced and accumulated in the culture, and collecting this. The present invention relates to a characteristic method for producing the polypeptide. The present inventors have now elucidated this novel platelet aggregation-inhibiting polypeptide and the nucleotide sequence encoding it. Specifically, the pit viper cDN
It has become possible to clone genes encoding a large number of analogs of platelet aggregation-inhibiting polypeptides from the A library and use them to produce the polypeptides. Further, it became possible to produce various mutants of cloned genes and to produce mutant polypeptides using them. This mutant was created for the following reason. That is, in order to make the expressed polypeptide resistant to cyanogen bromide treatment, the methionine residue present in the polypeptide was replaced with an amino acid residue generally found in snake venom instead of the methionine residue. It is a mutant.

The present invention has an amino acid sequence: Phe-A ₁ -L
_{ys-A 2 -Gly-Thr-} A 3 -Cys-Arg-A
_{4 -Ala-Arg-Gly-Asp} -Asp-A 5 -A
A polypeptide containing sp-Asp-Tyr-Cys-Asn-Gly [wherein A ₁ is Met, Leu or Lys; A ₂ is Lys or Glu; A ₃ is Val or Ile; A ₄ is Ile or Arg; A ₅ is Me
Representing t, Leu, Val, Pro or Asn], specific examples of this polypeptide include the following. (1) Amino acid sequence: Phe-Met-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-
Polypeptide (I) containing Asn-Gly (SEQ ID NO: 1), (2) Amino acid sequence: Phe-Met-Lys-Glu-Gly-Thr-Ile-Cy
s-Arg-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-
Polypeptide (II) containing Asn-Gly (SEQ ID NO: 2), (3) Amino acid sequence: Phe-Leu-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-
Polypeptide (III) containing Asn-Gly (SEQ ID NO: 3), (4) Amino acid sequence: Phe-Lys-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-
Polypeptide (IV) containing Asn-Gly (SEQ ID NO: 4), (5) Amino acid sequence: Phe-Leu-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-
Polypeptide (V) containing Asn-Gly (SEQ ID NO: 5), (6) Amino acid sequence: Phe-Leu-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Val-Asp-Asp-Tyr-Cys-
Polypeptide (VI) containing Asn-Gly (SEQ ID NO: 6), (7) Amino acid sequence: Phe-Leu-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Pro-Asp-Asp-Tyr-Cys-
Polypeptide (VII) containing Asn-Gly (SEQ ID NO: 7), (8) Amino acid sequence: Phe-Leu-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Asn-Asp-Asp-Tyr-Cys-
Asn-Gly-containing polypeptide (VIII) (SEQ ID NO: 8), (9) Amino acid sequence: Phe-Lys-Lys-Lys-Gly-Thr-Val-Cy
s-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-
Asn-Gly-containing polypeptide (IX) (SEQ ID NO: 9), (10) Amino acid sequence: Phe-Lys-Lys-Lys-Gly-Thr-Val-
Cys-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Val-Asp-Asp-Tyr-Cy
Polypeptide (X) containing s-Asn-Gly (SEQ ID NO: 1
0), (11) Amino acid sequence: Phe-Lys-Lys-Lys-Gly-Thr-Val-
Cys-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Pro-Asp-Asp-Tyr-Cy
A polypeptide (XI) containing s-Asn-Gly (SEQ ID NO: 1
1), (12) Amino acid sequence: Phe-Lys-Lys-Lys-Gly-Thr-Val-
Cys-Arg-Ile-Ala-Arg-Gly-Asp-Asp-Asn-Asp-Asp-Tyr-Cy
A polypeptide (XII) containing s-Asn-Gly (SEQ ID NO: 1
2), (13) Amino acid sequence: Phe-Leu-Lys-Glu-Gly-Thr-Ile-
Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cy
A polypeptide (XIII) containing s-Asn-Gly (SEQ ID NO: 1
3), (14) Amino acid sequence: Phe-Lys-Lys-Glu-Gly-Thr-Ile-
Cys-Arg-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cy
A polypeptide (XIV) containing s-Asn-Gly (SEQ ID NO: 1
4), (15) Amino acid sequence: Gly-Leu-Cys-Cys-Gly-Gln-Cys-
Arg-Phe-Met-Lys-Lys-Gly-Thr-Val-Cys-Arg-Ile-Ala-Ar
Polypeptide (XV) containing g-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly (SEQ ID NO: 15), (16) Amino acid sequence: Gly-Ser-Cys-Cys-Gly -Gln-Cys-
Arg-Phe-Met-Lys-Glu-Gly-Thr-Ile-Cys-Arg-Arg-Ala-Ar
Polypeptide (XVI) containing g-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-Asn-Gly (SEQ ID NO: 16), (17) Amino acid sequence: Gly-Leu-Cys-Cys-Asp -Gln-Cys-
Arg-Phe-Met-Lys-Lys-Gly-Thr-Val-Cys-Arg-Ile-Ala-Ar
Polypeptide (XVII) containing g-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly (SEQ ID NO: 17), (18) Amino acid sequence: Glu-Asp-Cys-Asp-Cys -Gly-Ser-
Pro-Gly-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Le
u-Arg-Gln-Gly-Ala-Gln-Cys-Ala-Glu-Gly-Leu-Cys-Cys-
Asp-Gln-Cys-Arg-Phe-Met-Lys-Lys-Gly-Thr-Val-Cys-Ar
g-Ile-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-Asn-
Polypeptide (XVIII) containing Gly (SEQ ID NO: 18), (19) 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 above
Recombinant DNA containing a nucleotide sequence encoding the amino acid sequence of 0, 11, 12, 13, 14, 15, 16, 17 or 18
(These DNAs are sequentially labeled as DNA (A), (B), (C),
(D), (E), (F), (G), (H), (I), (J), (K),
(L), (M), (N), (O), (P), (Q) and (R)), (20) DNA (A), (B), (C), (D). ,
(E), (F), (G), (H), (I), (J),
(K), (L), (M), (N), (O), (P),
A transformant transformed with a vector containing (Q) or (R), and (21) DNA (A), (B), (C), (D),
(E), (F), (G), (H), (I), (J),
(K), (L), (M), (N), (O), (P),
A transformant transformed with a vector containing (Q) or (R) is cultured in a medium, and the polypeptides (I), (II), (III), (IV) and (V) are added to the culture. , (V
I), (VII), (VIII), (XI), (X), (XI),
(XII), (XIII), (XIV), (XV), (XV
I), (XVII) or (XVIII) is produced and accumulated, and this is collected, which is a method for producing the polypeptide.

The recombinant DNA (A) containing the base sequence encoding the above-mentioned polypeptide (I) is, for example, the base sequence: 5'-TTTTATGAAAAAAGGAAC.
AGTATGCCGGATAGCAAGGGGTGAT
GACATGGATGAACTACTCAATGGGG-
3 '(SEQ ID NO: 19) and the like. As the recombinant DNA (B) containing the base sequence encoding the above polypeptide (II), for example, the base sequence: 5 '
-TTTATGAAAGAAGGAACAATATGCCGGAGAGCAAGGGGTGATGACCTGG
ATGATTACTGCAATGGG-3 '(SEQ ID NO: 20) and the like. Examples of the recombinant DNA (C) containing the nucleotide sequence encoding the above polypeptide (III) include, for example, the nucleotide sequence:
5'-TTTTTAAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACAT
GGATGATTACTGCAATGGC-3 '(SEQ ID NO: 21) and the like. The recombinant DNA (D) containing the base sequence encoding the above-mentioned polypeptide (IV) is, for example, base sequence: 5 '.
-TTTAAGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACATGG
ATGATTACTGCAATGGC-3 '(SEQ ID NO: 22) and the like. The recombinant DNA (E) containing the base sequence encoding the above-mentioned polypeptide (V) is, for example, base sequence: 5 '.
-TTTTTAAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACCTGG
ATGATTACTGCAATGGC-3 '(SEQ ID NO: 23) and the like. The recombinant DNA (F) containing the nucleotide sequence encoding the above-mentioned polypeptide (VI) is, for example, the nucleotide sequence: 5 '.
-TTTTTAAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACGTCG
ATGATTACTGCAATGGC-3 '(SEQ ID NO: 24) and the like. Examples of the recombinant DNA (G) containing the base sequence encoding the above polypeptide (VII) include, for example, the base sequence:
5'-TTTTTAAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACCC
GGATGATTACTGCAATGGC-3 '(SEQ ID NO: 25) and the like. The recombinant DNA (H) containing the nucleotide sequence encoding the above-mentioned polypeptide (VIII) is, for example, the nucleotide sequence:
5'-TTTTTAAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACAA
CGATGATTACTGCAATGGC-3 '(SEQ ID NO: 26) and the like. The recombinant DNA (I) containing the nucleotide sequence encoding the above-mentioned polypeptide (IX) is, for example, the nucleotide sequence: 5 '
-TTTAAGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACCTGG
ATGATTACTGCAATGGC-3 '(SEQ ID NO: 27) and the like. Examples of the recombinant DNA (J) containing the nucleotide sequence encoding the above polypeptide (X) include, for example, the nucleotide sequence: 5 '.
-TTTAAGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACGTCG
ATGATTACTGCAATGGC-3 '(SEQ ID NO: 28) and the like. The recombinant DNA (K) containing the base sequence encoding the above-mentioned polypeptide (XI) is, for example, the base sequence:
5'-TTTAAGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACCC
GGATGATTACTGCAATGGC-3 '(SEQ ID NO: 29) and the like. The recombinant DNA (L) containing the nucleotide sequence encoding the above-mentioned polypeptide (XII) is, for example, the nucleotide sequence:
5'-TTTAAGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACAA
CGATGATTACTGCAATGGC-3 '(SEQ ID NO: 30) and the like. As the recombinant DNA (M) containing a base sequence encoding the above polypeptide (XIII), for example, the base sequence: 5'-TTTTTAAAAGAAGGAACAATATGCCGGAGAGCAAGGGGTGATG
ACCTGGATGATTACTGCAACGGC-3 '(SEQ ID NO: 31) and the like. The recombinant DNA (N) containing the nucleotide sequence encoding the above-mentioned polypeptide (XIV) is, for example, the nucleotide sequence:
5'-TTTAAGAAAGAAGGAACAATATGCCGGAGAGCAAGGGGTGATGACCT
GGATGATTACTGCAACGGC-3 '(SEQ ID NO: 32) and the like. Examples of the recombinant DNA (O) containing the nucleotide sequence encoding the above polypeptide (XV) include the nucleotide sequence:
5'-GGGCTGTGTTGTGGGCAGTGCAGATTTATGAAAAAAGGAACAGTATG
CCGGATAGCAAGGGGTGATGACATGGATGACTACTGCAATGGG-3 '(SEQ ID NO: 33) and the like. The recombinant DNA (P) containing the nucleotide sequence encoding the above-mentioned polypeptide (XVI) is, for example, the nucleotide sequence:
5'-GGGTCGTGTTGTGGGCAGTGCAGATTTATGAAAGAAGGAACAATATG
CCGGAGAGCAAGGGGTGATGACCTGGATGATTACTGCAATGGG-3 '(SEQ ID NO: 34) and the like. The recombinant DNA (Q) containing the nucleotide sequence encoding the above polypeptide (XVII) is, for example, a nucleotide sequence
5'-GGACTGTGTTGTGACCAGTGCAGATTTATGAAAAAAGGAACAGTATG
CCGGATAGCAAGGGGTGATGACATGGATGATTACTGCAATGGC-3 '(SEQ ID NO: 35) and the like. Examples of the recombinant DNA (R) containing a base sequence encoding the above polypeptide (XVIII) include, for example, base sequence: 5'-GAGGACTGCGACTGTGGCTCTCCTGGAAATCCGTGCTGTGATG.
CTGCAACCTGTAAACTGAGACAAGGAGCACAGTGTGCAGAAGGACTGTGT
TGTGACCAGTGCAGATTTATGAAAAAAGGAACAGTATGCCGGATAGCAAG
GGGTGATGACATGGATGATTACTGCAATGGC-3 '(SEQ ID NO: 36) and the like.

The RNA encoding the novel platelet aggregation-inhibiting polypeptide obtained in the present invention can be obtained from the venom-secreting gland or the liver of the viper venom viper living in mainland Japan. As a method for preparing RNA from these tissues, the guanidine thiocyanate method [J. M. Chirgwin
Et al., Biochemistry, 18 , 5294 (1
979)] and the like. RN thus obtained
A conserved consensus sequence at the C-terminus of the platelet aggregation inhibitory polypeptide previously reported in A [MS Dennis et al., Proc. Natl. Aca
d. Sci. USA, 87 , 2471 (1989)], and then cDNA can be synthesized by reverse transcriptase after addition of antisense primer (A). To this cDNA fraction, a sense primer (B) corresponding to the conserved consensus sequence existing at the N-terminus and an antisense primer (C) corresponding to the conserved consensus sequence present at the C-terminus were added, and the manufacturer (eg, Cetus / PCR is performed according to the kit instruction of Perkin-Elmer), and the obtained product can be used as a cDNA library. After the cDNA library is separated by a method known per se, for example, agarose electrophoresis, the electrophoresed DNA fragment can be transferred to a nitrocellulose filter using a part of the gel [T. Maniatis et al., Molecular. Cloning (Mole
cular Cloning), Cold Spring Harbor Laboratory, 382 pages.
(1982)]. On the other hand, a sense probe (D) corresponding to a peptide containing the conserved amino acid sequence RGD of a known platelet aggregation-inhibiting polypeptide derived from snake venom was synthesized, and the 5'end was labeled with T4.
By labeling with polynucleotide kinase and [γ- ³² P] -ATP and hybridizing with the cDNA on the above filter, the position of the DNA band that strongly reacts with the probe can be determined. The DNA fragment can be recovered from the remaining agarose gel corresponding to this position. Collected DNA fragment is T4
After phosphorylating the 5'end with polynucleotide kinase and [γ- ³² P] -ATP, the plasmid pUCl
It can be inserted at 18 specific sites, such as the Hinc II site. The nucleotide sequence of the cDNA portion was determined by the dideoxynucleotide synthesis chain termination method [T. Messing et al., Nucleic Acids Res.], 9 , 30.
9 (1981)].

The plasmid having the cloned cDNA can be used as it is, or can be cut out with an appropriate restriction enzyme and inserted into another vector if desired. Any vector may be used as long as it can be replicated according to the host. The host is Escherichia (Escher
ichia coli , a plasmid derived from E. coli, such as pBR322 [F. Bolivar et al., Gene, 2 , 9
5 (1979)], pBR325, pUCl2, pUCl3 and the like. When the host is a bacterium of the genus Bacillus, a Staphylococcus- derived plasmid such as pUB110 (TJ Gryczan and D. Dub
nau, Proc. Natl. Acad. Sci. USA, 75 , 128 (1978)],
pTP5 [N. Noguchi, Gene, 2 , 95 (1979)], pCl
94 [D. Dubnau, Experimental Manipulation of Gene Expression (Experime
ntal Manipulation of Gene Expression; ed. M. Inouy
e), p. 83, Academic Press, (1
983)] and the like. If the host is yeast,
Yeast-derived plasmids such as pSH19 [S. Harashima
Et al., Molecular and Cellular Biology (Mol. Cell. Biol.), 4 , 771 (1984)] pSHl9-
1 (European Patent Application Publication EP-A-023543
0) and the like. When the host is an animal cell, for example, pSV2-X [R. C. Mulliga
n and P.N. Berg, Proc. Natl, Acad. Sci.
USA, 78 , 2072 (1981)], pcD-X [H. Okayamaand
P. Berg, Mol. Cell. Biol., 3 , 280 (1983)] and the like.

The cloned cDNA may have a translation initiation codon (ATG) at the 5'end and a translation termination codon (TAG, TGA or TAA) at the 3'end. Further, in order to express the cDNA, a promoter sequence, a promoter-prepro sequence or a promoter-signal sequence is connected upstream thereof. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. If the host is E. coli, tr
p promoter, lac promoter, λPL promoter (L is L with subscript), T7 promoter, t
Examples thereof include ac promoter, lpp promoter and recA promoter, and T7 promoter is particularly preferable. When the host is Bacillus, SPO
1 promoter, SPO2 promoter, penP promoter, MWP promoter, etc. are mentioned, and MWP promoter is particularly preferable. When the host is yeast, GAPDH promoter, PGK promoter, PHO5 promoter, ADH promoter, PH
O81 promoter and the like, and particularly GAPD
The H promoter is preferred. When the host is an animal cell, SV40-derived promoter, retrovirus promoter and the like can be mentioned. The promoter can be prepared from the corresponding gene. It can also be chemically synthesized. The signal sequence and prepro sequence may be anything that functions in the host. In the case of Escherichia coli, the signal sequence of β-lactamase gene, the signal sequence of enterotoxin gene, the signal sequence of alkaline phosphatase gene, the signal sequence of OmpA gene and the like can be mentioned. In the case of Bacillus, a signal sequence of α-amylase gene, a signal sequence of neutral protease gene, a signal sequence of MWP gene and the like can be mentioned. In the case of yeast, egg white lysozyme gene signal sequence, human lysozyme gene signal sequence, invertase gene signal sequence, α
-Examples include prepro sequences of factor genes. In the case of animal cells, the signal sequence of interleukin 2 gene and the like can be mentioned.

A transformant is produced using the vector containing any of the DNAs (A) to (R) constructed as described above. Examples of the host include Escherichia, Bacillus, yeast, and animal cells. For Escherichia, Escherichia coli K
12DH1 [B. Low, Proc. Natl. Acad. Sci. USA, 6
0 , 160 (1968)], C600 [RK Appleyard, Genetics, 39, 440 (1954)], MM29
4 [K. Backman et al., Proc. Natl. Acad. Sci. USA, 73 ,
4174 (1976)] and the like. Examples of the genus Bacillus include, for example, Baccillus subtilis M
Ill4 [K. Yoshimura et al., Gene, 24 , 255 (1983)],
207-21 [K. Ohmura et al., Journal of Biochemistry (J. Biochem.), 95 , 87 (1984)], Bacillus brevis 47 [S. Udaka, Agricultural Biological Chemistry (Agric. Biol. . Che
m.), 40 , 523 (1976)] and the like. Examples of yeast include Saccharomyces cerevisiae
ces cerevisiae) AH22R ~ [A. Miyanohara et al., Proc.
Natl. Acad. Sci. USA, 80 , 1 (1983)], NA87-
11A, DKD-5D, NA74-3A, NA74-3
Aρ ~ [Y. Kaisho et al., Yeast, 5 , 91 (198
9)] and Schizosaccharomys pombe
ces pombe ) ATCC 38399 (h ~ leu1-32), TH16
8 (h ⁹⁰ ade6-M210 ural leul) [M. Kishida and C. Sh
imada, Current Genetics
s), 10 , 443 (1986)]. Examples of animal cells include monkey COS-7 cells, monkey Vero cells, and Chinese hamster ovary (CH) that are adherent cells.
O) cells, mouse L cells, human FL cells, and floating myeloma cells (SP2 / 0 etc.), mouse YAC-1 cells, mouse MethA cells, mouse P
Examples include 388 cells and mouse EL-4 cells. For transformation of Escherichia, for example, T. Maniati
s et al., Molecular Cloning, Cold Spring Harbor Laborato
ry, page 249 (1982) and the like. As a method for transforming Bacillus, S. Cha
ng and SN Cohen, Molecular and General Genetics (Mol. Gen. Genet.), 168 , 111 (1
979) and the like. To transform yeast, for example, A. Hinnen et al., Proc. Natl. Acad.
Sci. USA, 75 , 1929 (1978). To transform animal cells, for example, M. Wigle
r et al., Cell, 14 , 725 (1978). The transformant thus obtained is cultured by a method known per se. When culturing a transformant whose host is a bacterium of the genus Escherichia, examples of the medium include M9 medium containing glucose and casamino acid [JH Mille
r, Experiments in Molecular Genetics, p. 431,
Cold Spring Harbor Laboratory, (1972)] is preferable. If necessary, a drug such as isopropylthiogalactoside or indolyl-3-acrylic acid can be added to the repromoter to work efficiently. Culturing is usually performed at about 15 to 43 ° C for about 3 to 24 hours,
If necessary, aeration and stirring can be added.

When a transformant whose host is a bacterium of the genus Bacillus is cultured, examples of the medium include L-broth medium and T2.
Medium (S. Udaka, Agric. Biol. Chem., 40 , 523 (197
6)] and the like. Culturing is usually carried out at about 15 to 37 ° C. for about 6 to 96 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder.
r) Minimal medium [KL Bostain et al., Proc. Natl. Acad. Sc
i. USA, 77 , 4504 (1980)]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually carried out at about 20 to 35 ° C. for about 24 to 72 hours, and aeration and stirring are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [H. Eagle, Science (Scien
ce), 130 , 432 (1959)], DMEM medium [R. Dulbecco
and G. Freeman, Virology, 8 , 396 (1
959)], RPMI-1640 medium [GE More et al., Journal of the American Medical Association (J. Am. Med. Assoc.), 199 , 519 (1967)],
199 medium [JF Morgan et al., Procedure of
The Society for Experimental Biology and Medicine (Proc. Soc. Exp. Biol.
Med.), 73 , 1 (1950)]. Culturing is usually performed at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and stirring are added if necessary.

According to the present invention, a novel platelet aggregation-inhibiting polypeptide can be isolated from the above-mentioned culture by appropriately combining separation and purification methods known per se.
Known separation and purification methods for these include methods utilizing solubility such as salting out and solvent precipitation, dialysis methods, ultrafiltration methods, gel filtration methods, and SDS-polyacrylamide gel electrophoresis methods. Method utilizing difference, method utilizing difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography , A method of utilizing a difference in isoelectric points, such as an isoelectric focusing method.

[0016]

The action and effect of the novel polypeptide obtained by the present invention include adenosine diphosphate (ADP), collagen, thrombin, arachidonic acid, and platelet activating factor (P
AF) and other factors that inhibit platelet aggregation. Further, the polypeptide inhibits platelet aggregation by antagonism (as a fibrinogen receptor antagonist) to the interaction between GPIIb-IIIa and fibrinogen, and acts as a potent antiplatelet agent or antithrombotic agent by itself. Furthermore, since the polypeptide has cancer cell metastasis inhibitory activity and bone resorption inhibitory activity, it can be used as a cancer metastasis inhibitor or bone resorption inhibitor. Furthermore, by expressing a gene encoding a polypeptide having a platelet aggregation inhibitory activity in the present invention by genetic engineering, it became possible to mass produce a peptide having such an activity in high purity.

In the present specification and drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB is used.
Based on the abbreviations by Commision on Biochemical Nomenclature or abbreviations commonly used in this field,
An example is given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: Deoxyribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine SDS: Sodium dodecyl sulfate Gly: Glycine (G) Ala: Alanine (A) Val: Valine (V) Leu: Leucine (L) Ile: Isoleucine (I) ) Ser: Serine (S) Thr: Threonine (T) Cys: Cysteine (C) 1/2 Cys: Half cystine Met: Methionine (M) Glu: Glutamic acid (E) Asp: Aspartic acid (D) Lys: Lysine (K) ) Arg: arginine (R) His: histidine (H) Phe: phenylalanine (F) Tyr: tyrosine (Y) Trp: tryptophan (W) Pro: proline (P) Asn: aspartic acid (N) Gln: glutamine (Q) ) Apr: Ampicillin resistance Gene (r represents superscript r) Tcr: tetracycline resistance gene (r represents superscript r) In the polypeptide of the present invention, a part of its amino acid sequence is modified (addition, removal, etc.) Substituting with amino acid).

[0018] The following examples the present invention will be explained more concretely,
The present invention is not limited to these. Example 1 Preparation of cDNA library derived from RNA of venom secretory glands of pit viper (1.5 g of venom glands of pit viper ( Agkistrodon halys blomhoffii ))
RNA (560 μg) was extracted by M. Chirgwin et al., Biochemistry, 18 , 5294, (1979)].

On the other hand, a common amino acid sequence of a platelet aggregation inhibitory polypeptide derived from snake venom [MS Dennis et al., Proc. Natl.
Acad. Sci. USA, 87 , 2471 (1989)] C-terminal sequence (A
sp / Gly-Cys-Pro-Arg / Trp-Asn / Tyr-Pro / His) antisense primer No. 9: (SEQ ID NO: 37) Antisense primer No. 3 corresponding to the C-terminal side sequence (Asp-Asp-Tyr-Cys-Asn-Gly): (SEQ ID NO: 38) Antisense primer No. 10 corresponding to the same C-terminal sequence (Asp-Asp-Tyr-Cys-Thr / Asn-Gly): (SEQ ID NO: 3
9) Central sequence (Gly-Pro / Leu-Cys-Cys-Asp / Arg-Gln / Asn-Cy
s) corresponding to the sense primer No. 6: (SEQ ID NO: 4
0) And N-terminal side sequence (Lys / Glu-Asp-Cys-Asp-Cys-Gly)
Sense primer No.4 corresponding to: (SEQ ID NO: 41) Was synthesized and used as a primer for preparing cDNA.

100 μmol of primer No. 9 was added to 3 μg of the above RNA, and the mixture was kept at 70 ° C. for 10 minutes and then rapidly cooled in ice. Next, 50 mM Tris-HCl (pH 8.
3), 75 mM KCl, 3 mM MgCl ₂ , 0.4 mM
CDNA synthesis reaction was carried out at 42 ° C. for 1 hour in a solution containing each dNTP, 10 mM DTT, 200 units reversal transcriptase [SuperScript (registered trademark) RT (Life Technologies, Inc.)], and 70 ° C.
The reaction was stopped by heating for 10 minutes.

Two kinds of primers (No. 3 and 6 or No. 4 and 10: 100 for each) were added to 10% of the obtained cDNA fraction.
pmol), and according to the kit instruction supplied by the manufacturer (Cetus / Perkin-Elmer), PCR at 94 ° C., 1 minute, 50 ° C., 2 minutes, 72 ° C., 3 minutes is repeated 50 times, and PCR is performed. The PCR products obtained were each labeled with cDNA.
It was designated as A library I and II.

Example 2 Cloning of Platelet Aggregation Inhibitory Polypeptide cDNA Derived from Mammus Venom Secretory Gland and Determination of Its Nucleotide Sequence (1) After the above cDNA libraries I and II were separated by 1.2% agarose electrophoresis, respectively. A part of the gel was transferred to a nitrocellulose filter (Schleicher & Schuell, BA85) [T. Maniatis et al., Molecular Cloning, Cold S.
pring Harbor Laboratory, p. 383 (1982)]. On the other hand, a common amino acid sequence of platelet aggregation inhibitory polypeptides derived from snake venom [MS Dennis et al., Proc. Natl. Acad. Sci. USA, 87 ,
2471 (1989)], the central sequence toward the C-terminal side (Gly-Thr / Ly
s-Ile / Val-Cys-Arg / Lys-Arg / Ile-Ala / Pro-Arg-Gly-Asp-
Sense probe No. 7 corresponding to Asp): (SEQ ID NO: 4
2) Was synthesized and labeled at the 5'end with T4 polynucleotide kinase and [γ- ³² P] -ATP, and used as a probe for screening platelet aggregation inhibitory polypeptide cDNA derived from the venom of the pit viper. The labeled probe No. 7 was associated with the filter on which the cDNA library I was immobilized. The association reaction involves 10 μCi of labeled probe 6
X SSC (0.18M NaCl, 0.018M sodium citrate), 5x Denhardt's, 1.0%
SDS, 100 μg / ml denatured salmon sperm DNA in 10 ml of solution containing 45 ° C. for 16 hours. After the reaction
6 × SSC, solution containing 0.1% SDS at 40 ° C., 30
Wash for minutes (T. Maniatis et al., Molecular Cloning, Co
ld Spring Harbor Laboratory, p. 309, 1982). Autoradiography of the washed filter was performed to fix the DNA band that strongly reacts with the probe, and the DNA was recovered from the remaining agarose gel corresponding to this position.
At this time, the DNA fragment obtained from the cDNA library I was 90 bp. The resulting DNA fragment was phosphorylated at the 5 'end with T4 polynucleotide kinase and ATP, the T4DNA ligase and ATP was inserted into the Hin cII site of plasmid PUCl18 (Takara Shuzo), dideoxynucleotide synthetic nucleotide sequence of the cDNA portion Chain termination method [J. Messing et al., Nucleic Acids Res., 9 ,
309, (1981)]. As a result, 90bp
Was found to contain two types of cDNA (α type and β type). The plasmids containing the respective cDNA fragments are designated as pAGα101 and pAGβ101, and the nucleotide sequence of the cDNA portion and the amino acid sequence predicted from the nucleotide sequence are shown in FIG. 1 (SEQ ID NO: 33 and SEQ ID NO: 15) and FIG. 2 (SEQ ID NO: 34). And SEQ ID NO: 1
6).

CDNA library II is also used for cDNA
A library I was performed in the same manner. That is, after fixing the library II to the filter, an association reaction with the probe No. 7 was carried out, and after washing, it was subjected to autoradiography. Then, DNA fragments (174 bp) was recovered from the position of the agarose gel which reacts strongly with the probe, after insertion into Hin cII site of plasmid PUCl18, c
The base sequence of the DNA part was determined. As a result, the plasmid pAGα2 containing the upstream portion of the above α-type cDNA
I got 01. The base sequence and the amino acid sequence predicted from the base sequence are shown in Fig. 3 (SEQ ID NO: 36 and SEQ ID NO: 1).
8).

The amino acid sequences predicted from these α-type and β-type cDNAs are the common amino acid sequences of conventionally known venom-derived platelet aggregation-inhibiting polypeptides [M. S. Denn
Is et al., Proc. Natl. Acad. Sci. USA, 87 , 2471, (198
9)] and showed a high homology, but there was no match with the known inhibitory polypeptide, and it was found to be a novel polypeptide.

Example 3 Cloning of cDNA of platelet aggregation-inhibiting polypeptide derived from venom of ginkgo venom and determination of its nucleotide sequence (2) Two kinds of primer No. 6 were added to 10% of the cDNA fraction described in Example 1. And 9 were added at 100 pmol each and according to the kit instructions supplied by the manufacturer (Cetus / Perkin-Elmer), 94 ° C, 1 min, 50 ° C, 2 min, 72
PCR was carried out by repeating the reaction for 3 minutes at 50 ° C. 50 times, and the obtained PCR product was used as a cDNA library. C above
After separating the DNA library by 1.2% agarose electrophoresis, Southern hybridization was carried out according to the method described in Example 2 using a part of the gel. The ³² P-labeled probe No. prepared in Example 2 was used. A 117 bp DNA fragment that hybridizes with 7 was recovered from the remaining agarose gel and was isolated from T4 polynucleotide kinase and A
After phosphorylating the 5'end with TP, M13mp19 RF DNA (Takara Shuzo) was digested with T4 ligase and ATP.
It was inserted into the HincII site. The nucleotide sequence of the cDNA portion was determined by the dideoxynucleotide synthetic chain termination method [J. Messing
Et al., Nucleic Acids Res., 9, 309 (1981)]. As a result, the 117 bp DNA fragment was the cDNA fragment (90 b contained in the above plasmid pAGβ101.
It was found to be a mixture of at least 6 kinds of DNA fragments having high homology with p). Plasmids containing these DNA fragments were designated pAGβ102 and pAGβ10, respectively.
3, pAGβ104, pAGβ105, pAGβ106
And pAGβ107, and the nucleotide sequence of the cDNA portion and the amino acid sequence predicted from the nucleotide sequence are shown in FIG. 4 (SEQ ID NO: 43), FIG. 5 (SEQ ID NO: 44), FIG. 6 (SEQ ID NO: 45), and FIG. (SEQ ID NO: 46), FIG. 8 (SEQ ID NO: 47) and FIG. 9 (SEQ ID NO: 48).

Example 4 Preparation of Variant of Platelet Aggregation Inhibiting Polypeptide Gene (β Type) The plasmid pAGβ102 shown in SEQ ID NO: 43 was transformed into Escherichia coli JM109 strain (Takara Shuzo), and the obtained plaque was cloned by a conventional method. Double-stranded DNA (ssDNA)
Was prepared. On the other hand, the 10th Met codon (ATG) of the polypeptide gene (β-type) of SEQ ID NO: 43 is set to L
Sense primer N that converts to eu codon (TTA)
o. 101 (* indicates a mutation site): (SEQ ID NO: 49) And sense primer No. that converts the same codon to Lys codon (AAG). 102: (SEQ ID NO: 50) Was synthesized.

The ecsteine method [J. W. Taylor et al., Nucleic Acids Res., 13, 8749 (1
985): ibid, 8674 (1985)], a kit [Oligonucl
eotide-directed in vitro mutagenesis system versio
n 2 (code RPN 1523)] was used. Manufacturer (Amersh
am) and follow the kit instructions supplied by
DNA 5 μg and primer (No. 101 and 10
2) Platelet aggregation inhibitory polypeptide gene (β type) mutants (β101 and β102) were prepared from 4 pmol. The nucleotide sequence after the introduction of the mutation is the dideoxynucleotide synthetic chain termination method [J. Messing et al., Nucleic Acids Res. ，
9 , 309 (1981)]. Each mutant DNA
Fragment-containing plasmids pAGβ1-101 and pAGβ2
It was named -101 and the nucleotide sequence of the mutant DNA portion and the amino acid sequence predicted by the nucleotide sequence are shown in FIG. 10 (SEQ ID NO: 51) and FIG. 11 (SEQ ID NO: 52), respectively.

The digested mutant produced (1) plasmid described in Example 2 PAGarufa201 Example 5 platelet aggregation inhibitor polypeptide gene (alpha type) (FIG. 3) in the Eco RI and Hin dIII, the DNA fragment of 230bp after recovered, the Eco RI of M13 mp18 RF DNA (Takara Shuzo) using T4 ligase and ATP
It was inserted into the HindIII site. Escherichia coli JM109 strain was transformed with this plasmid, and ssDNA was prepared from the obtained plaque according to a conventional method. On the other hand, the sense primer No. for converting the 38th Met codon (ATG) of the polypeptide gene (α type) shown in FIG. 3 into a Leu codon (TTA). 103 (* indicates a mutation site):
(SEQ ID NO: 53) And sense primer No. which converts the same codon to Lys codon (AAG). 104: (SEQ ID NO: 54) Was synthesized. Mutagenesis to the gene was performed in the same manner as in Example 4. From the ssDNA and the primers (Nos. 103 and 104), the platelet aggregation-inhibiting polypeptide gene (α
Type) variants (α101 and α102) were made.
The nucleotide sequence after the introduction of the mutation was pAGα1-101 (Fig. 12, SEQ ID NO: 5) obtained by confirming the plasmid containing each mutant DNA fragment confirmed by the dideoxynucleotide synthetic chain termination method.
5) and pAGα2-101 (FIG. 13, SEQ ID NO: 56). Escherichia coli JM109 strain was transformed with these plasmids, and ssD was obtained from the obtained plaques by a conventional method.
Each NA was prepared.

(2) The Met codon (ATG) at the 52nd position of the polypeptide gene (α type) shown in FIG. 3 was added to Leu.
Sense primer No. that converts to codon (CTG)
105 (SEQ ID NO: 57): And sense primer No. that converts the codon to Val codon (GTC). 106 (SEQ ID NO: 58): Was synthesized. Then, similarly, the above pAGα1-101
(FIG. 12, SEQ ID NO: 55) derived from ssDNA and primers (No. 105 and 106), a variant (α103 and α10) of the platelet aggregation inhibitory polypeptide gene (α type).
4) was produced. The nucleotide sequence after the introduction of the mutation was confirmed by the dideoxynucleotide synthetic chain termination method. Plasmids containing the respective mutant DNA fragments were designated as pAGα3-101 (FIG. 14, SEQ ID NO: 59) and pAGα4-101 (FIG. 15,
It was named as SEQ ID NO: 60).

The 52nd Met codon (ATG) of the polypeptide gene (α type) shown in FIG.
Sense primer No. that converts to codon (CCG)
107 (SEQ ID NO: 61): And a sense primer No. that converts the codon into an Asn codon (AAC). 108 (SEQ ID NO: 62): Was synthesized. Then, similarly, the above pAGα1-101
(FIG. 12, SEQ ID NO: 55) derived ssDNA and primers (Nos. 107 and 108) were used to transform the platelet aggregation-inhibiting polypeptide gene (α type) mutants (α105 and α10).
6) was produced. The nucleotide sequence after the introduction of the mutation was confirmed by the dideoxynucleotide synthetic chain termination method. Plasmids containing the respective mutant DNA fragments were designated pAGα5-101 (FIG. 16, SEQ ID NO: 63) and pAGα6-101 (FIG. 17,
It was named as SEQ ID NO: 64).

(3) The above pAGα2-101 (FIG. 13,
SEQ ID NO: 56) -derived ssDNA and primer (No. 1)
05 and 106), mutants (α107 and α108) of the platelet aggregation inhibitory polypeptide gene (α type) were prepared. Plasmids containing the respective mutant DNA fragments were cloned into pAGα7-101 (FIG. 18, SEQ ID NO: 65) and pAG.
It was named α8-101 (FIG. 19, SEQ ID NO: 66).

Similarly, the above pAGα2-101 (FIG. 13,
SEQ ID NO: 56) -derived ssDNA and primer (No. 1)
07 and 108), mutants (α109 and α110) of the platelet aggregation inhibitory polypeptide gene (α type) were prepared. Plasmids containing the respective mutant DNA fragments were cloned into pAGα9-101 (FIG. 20, SEQ ID NO: 67) and pAG.
It was named α10-101 (FIG. 21, SEQ ID NO: 68).

The nucleotide sequence of the mutant DNA portion obtained as described above and the amino acid sequence predicted from the nucleotide sequence are shown in FIG. 12 (SEQ ID NO: 55), FIG. 13 (SEQ ID NO: 56),
FIG. 14 (SEQ ID NO: 59), FIG. 15 (SEQ ID NO: 60), FIG. 16 (SEQ ID NO: 63), FIG. 17 (SEQ ID NO: 64), FIG.
8 (SEQ ID NO: 65), FIG. 19 (SEQ ID NO: 66), FIG.
(SEQ ID NO: 67) and FIG. 21 (SEQ ID NO: 68).

Example 6 Purification of Platelet Aggregation Inhibitor (Halistatin) from Mamushi Venom 5 g of Mamushi venom was saturated with 40% saturated ammonium sulfate-20 mM Tris.
After diluting with -HCl buffer (pH 8.0), centrifugation was performed at 6000 rpm for 10 minutes using a GSA rotor with a serval centrifuge (manufactured by DuPont) to obtain a supernatant. Butyl-Toyopearl 650M equilibrated with 40% saturated ammonium sulfate-20 mM Tris-HCl buffer (pH 8.0).
After loading onto a column (3.5 x 20 cm, manufactured by Tosoh Corporation), a platelet aggregation inhibitor protein named halistatin was eluted with a concentration gradient of 40% to 0% saturated ammonium sulfate. The elution fraction of halistatin was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) and then concentrated by ultrafiltration (YM-2 manufactured by Amicon). 20m of concentrated liquid
Sephacryl S-100 (manufactured by Pharmacia) column equilibrated with M Tris-HCl buffer (pH 8.0) (2.
0 × 100 cm). The elution fraction of halistatin was loaded on a Microbonder Sphere C4 column (Waters) equilibrated with 0.1% TFA, and a concentration gradient of 0-70% acetonitrile (flow rate: 1 ml / min) for the first 5 minutes. , Followed by 70-85 for 30 minutes
Halistatin was eluted with a concentration gradient of acetonitrile (flow rate: 1 ml / min). The obtained halistatin fractions were collected (15 mg) and freeze-dried to obtain a purified sample. Rabbit platelet aggregation [10 ~ ⁸ M concentration PAF aggregation induced (platelet activating factor)] IC ₅₀ of the purified halistatin respect was 10 ~ ⁵ ~10 ~ ⁶ M (Figure 22).

Example 7 Primary structure of halistatin (1) The purified halistatin preparation obtained in Example 6 was subjected to reverse phase high performance liquid chromatography using the Microbonder Sphere C4 column (produced by Waters) of Example 6 It eluted as a single symmetrical peak (Figure 23).

(2) Purified halistatin preparation, about 1 nmo
1, the amino terminal amino acid sequence was analyzed by a gas phase protein sequencer 473A (manufactured by Applied Biosystems). As a result, the amino terminal amino acid sequence of halistatin was GEECDCGSPGNPCCDAATCKLRQG.
Determined as AQCAEG (SEQ ID NO: 69).

(3) 100 μg of purified halistatin preparation
Was reduced in the presence of 8 M urea and 5 mM dithiothreitol, and SH group of cysteine reduced with iodoacetamide was carboxylmethylated. Then, after hydrolyzing with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), the sample was loaded on a Micro Bonder Pack C18 column (manufactured by Waters) equilibrated with 0.1% TFA, and the sample was loaded for 5 minutes in the first 5 minutes. % Acetonitrile concentration (flow rate: 1 ml / min), followed by 5-7 over 35 min.
0% acetonitrile gradient (flow rate: 1 ml / min),
Elution with a 70-100% acetonitrile concentration gradient (flow rate: 1 ml / min) for a further 15 minutes gave a degradation product consisting of four peptides (Fig. 24). Peaks 1, 2, 3
After isolating each of peptides 4 and 4, the amino acid sequence was analyzed using a gas phase peptide sequencer, and the following results were obtained. Peak 1 GEECDCGSPGNPCCDAATCK (SEQ ID NO: 70) Peak 2 EAGEECDCGSPGNPCCDAATCK (SEQ ID NO: 71) Peak 3 LRQGAQCAEGLCCDQCRFMK (SEQ ID NO: 72) Peak 4 GTVCRIARGDDMDDYCNGISAGCPRNPF (SEQ ID NO: 7)
3).

Example 8 Cloning of Halistatin Precursor cDNA from Mammoth Venom Secretory Gland and Determination of Its Nucleotide Sequence Trigramin Precursor cDNA Nucleotide Sequence [MP Neeper
& MA Jacobson, Nucleic Acids Research, 18, 425
5 (1990)], the primer N corresponding to the 5'untranslated region
o. T1: (SEQ ID NO: 74) And N-terminal side sequence (Met-Ile-Gln-Val-Leu-Leu-Ile)
Primer No. corresponding to T2: (SEQ ID NO: 75) Were synthesized respectively.

Next, chromosome D encoding halistatin
C-terminal side sequence of NA base sequence (Gly-Ile-Ser-Ala-Gly-Cy
s-Pro) antisense primer No. T
3: (SEQ ID NO: 76) Antisense primer No. 3 corresponding to the 3'terminal untranslated region. T4: (SEQ ID NO: 77) And C-terminal sequence (Arg-Asn-Pro-Phe-His-Ala-STOP)
Corresponding to the antisense primer No. T5: (SEQ ID NO: 78) Were synthesized respectively.

On the other hand, the following operation was performed according to the procedure described in Example 1. RNA derived from pit viper venom was prepared and the primer No. A cDNA synthesis reaction was performed using T4.
Next, 10% of the obtained cDNA fraction and primer No. PCR was performed using T1 and T5. Furthermore, 10% of the obtained PCR product and primer No. After performing PCR using T2 and T3, the DNA of the expected size was obtained by separation by 1.2% agarose gel electrophoresis.
A fragment (about 1440 base pairs) was amplified. The DNA fragment was cut out from an agarose gel, collected, and purified, and then prime-it DNA labeling kit (Stratagene) and [α-
It was labeled with 32 P] -CTP (Amersham) and used as a screening probe for halistatin cDNA. Rhizoma derived from pit viper venom secretory gland extracted by the method described in Example 1.
NA was purified with a column packed with oligo (dT) cellulose type 7 (Pharmacia LKB) to obtain poly (A) R.
I got NA. Next, cDNA synthesis system plus (Amer
poly (A) RNA (5 μg) to cDNA using sham)
NA (about 1 μg) was synthesized. The obtained cDNA fragment was inserted into λgt10 vector DNA using the cDNA cloning system λgt10 and the adapter method (Amersham), and in vitro packaging was performed to prepare a cDNA library.

The plate on which the cDNA library was developed was placed on a nitrocellulose filter (Schleicher & Schuel).
l, BA85) and denatured with alkali, then 2 at 78 ℃
Heated for hours. Labeled probe (about 1440 base pairs)
Was allowed to associate with a filter to which the cDNA library was immobilized. The association reaction is 6x with 10 μCi of labeled probe
SSC, 5xDenhardt's, 1.0% SDS,
It was carried out at 68 ° C. for 16 hours in 10 ml of 100 μg / ml denatured salmon sperm DNA solution. After the reaction, 0.1xSSC,
Washing with 0.1% SDS solution at 65 ° C for 30 minutes (T. Ma
niatis et al., Molecular Cloning, Cold Spring Harbor La
boratory, p. 309, 1982). Autoradiography of the washed filter was performed, and plaques that strongly reacted with the probe were collected from the plate. Once again, the recovered phages were developed on the plate and autoradiography was performed in the same procedure.
-75). DNA was extracted from the phage according to a conventional method, digested with EcoRI to recover the cDNA portion, and then pUC was performed with T4 DNA ligase and ATP.
The nucleotide sequence was determined by inserting it into the EcoRI site of 118 plasmid (Takara Shuzo). As a result, the plasmid pAGγ101 containing the precursor cDNA encoding halistatin described in Example 7 was obtained. The nucleotide sequence and the amino acid sequence predicted from the nucleotide sequence are shown in Fig. 25 (SEQ ID NO: 79). Escherichia coli JM109 / pAGγ10 transformed with pAGγ101
1) was established by the Fermentation Research Institute (IFO) as IFO 15222 on September 5, 1991, and by the Institute of Microbiology and Industrial Technology (FRI) of the Ministry of International Trade and Industry in September 1991.
Deposited as FERM P-12529 from 20th of March.

[0042]

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Feature of the sequence Characteristic symbol: domain Method of determining feature: S sequence Phe Met Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Met 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 2 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence features Characteristic symbol: domain Method for determining features: S sequence Phe Met Lys Glu Gly Thr Ile Cys Arg Arg Ala Arg Gly Asp Asp Leu 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 3 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristics Characteristic symbol: domain Method for determining characteristics: S sequence Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Met 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 4 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Met 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 5 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining feature: S sequence Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Leu 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 6 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristics Characteristic symbol: domain Method for determining characteristics: S sequence Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Val 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 7 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Pro 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 8 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence features Characteristic symbols: domain Method for determining features: S sequence Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Asn 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 9 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Leu 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 10 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Val 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 11 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Pro 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 12 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Asn 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 13 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Fragment type: Intermediate sequence Sequence characteristic feature symbol: domain Method for determining characteristic: S sequence Phe Leu Lys Glu Gly Thr Ile Cys Arg Arg Ala Arg Gly Asp Asp Leu 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 14 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: Yes Fragment type: Intermediate sequence Sequence features Characteristic symbol: domain Method for determining features: S Sequence Phe Lys Lys Glu Gly Thr Ile Cys Arg Arg Ala Arg Gly Asp Asp Leu 1 5 10 15 Asp Asp Tyr Cys Asn Gly 20.

SEQ ID NO: 15 Sequence length: 30 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: Yes Fragment type: Intermediate sequence Sequence features Signature symbol: domain Method for determining features: S Sequence Gly Leu Cys Cys Gly Gln Cys Arg Phe Met Lys Lys Gly Thr Val Cys 1 5 10 15 Arg Ile Ala Arg Gly Asp Asp Met Asp Asp Tyr Cys Asn Gly 20 25 30.

SEQ ID NO: 16 Sequence length: 30 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: Yes Fragment type: Intermediate sequence Sequence features Characteristic symbol: domain Method for determining features: S Sequence Gly Ser Cys Cys Gly Gln Cys Arg Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly 20 25 30.

SEQ ID NO: 17 Sequence length: 30 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: Yes Fragment type: Intermediate sequence Sequence features Characteristic symbol: domain Method for determining features: S Sequence Gly Leu Cys Cys Asp Gln Cys Arg Phe Met Lys Lys Gly Thr Val Cys 1 5 10 15 Arg Ile Ala Arg Gly Asp Asp Met Asp Asp Tyr Cys Asn Gly 20 25 30.

SEQ ID NO: 18 Sequence length: 58 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: Yes Fragment type: Intermediate sequence Sequence features Characteristic symbol: domain Method for determining features: S Sequence Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 Asp Gln Cys Arg Phe Met Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 Gly Asp Asp Met Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 19 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Library name: Mamushi Venom secretory gland cDNA library Clone name: pAGα101 Sequence features Characteristic symbols: domain Method for determining features: E sequence TTTATGAAAA ATGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACATGGA TGACTACTGC 60 AATGGG 66.

SEQ ID NO: 20 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGβ101 Sequence features Characteristic symbols: domain Method of determining features: E sequence TTTATGAAAG AAGGAACAAT ATGCCGGAGA GCAAGGGGTG ATGACCTGGA TGATTACTGC 60 AATGGG 66.

SEQ ID NO: 21 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGα1-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTTTAAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACATGGA TGATTACTGC 60 AATGGC 66 .

SEQ ID NO: 22 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGα2-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTAAGAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACATGGA TGATTACTGC 60 AATGGC 66 .

SEQ ID NO: 23 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mammus strain name: Agkistrodon halys blomhoffii organelle name : Venom secretory gland Direct source Library name: Mamushi Venom secretory gland cDNA library Clone name: pAGα3-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTTTAAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACCTGGA TGATTACTGC 60 AATGGC 66 ..

SEQ ID NO: 24 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGα4-101 Sequence features Characteristic symbols: modified base Method of determining features: E sequence TTTTTAAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACGTCGA TGATTACTGC 60 AATGGC 66 ..

SEQ ID NO: 25 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin library name: pit viper venom secretory gland cDNA library clone name: pAGα5-101 sequence feature symbol: modified base method of determining feature: E sequence TTTTTAAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACCCGGA TGATTACTGC 60 AATGGC 66 ..

SEQ ID NO: 26 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGα6-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTTTAAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACAACGA TGATTACTGC 60 AATGGC 66 .

SEQ ID NO: 27 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi strain name: Agkistrodon hays blom
hoffii Organelle name: Venom secretory gland Direct origin Library name: Mammus Venom secretory gland cDNA library Clone name: pAGα7-101 Sequence features Characteristic signature: modified base Method of determining features: E sequence TTTAAGAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACCTGGATGATTACT 60 AATGGC 66.

SEQ ID NO: 28 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct source Library name: Mammoth Venom Secretory gland cDNA library Clone name: pAGα8-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTAAGAAAAAAGGAACAGT ATGCCGGATA GCA
AGGGGTG ATGACGTCGA TGATTACTGC 60 AATGGC 66.

SEQ ID NO: 29 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Library name: Mammoth Venom Secretory gland cDNA library Clone name: pAGα9-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTAAGAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACCCGGA TGATTACTGC 60 AATGGC 66 .

SEQ ID NO: 30 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct source Library name: Mamushi Venom secretory gland cDNA library Clone name: pAGα10-101 Sequence features Characteristic symbols: modified base Method for determining features: E sequence TTTAAGAAAA AAGGAACAGT ATGCCGGATA GCAAGGGGTG ATGACAACGA TGATTACTGC 60 AATGGC 66 SEQ ID NO: 31 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretion Gland Direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGβ1-101 Sequence features Characteristic symbols modified base Characterization method: E sequence TTTTTAAAAG AAGGAACAAT ATGCCGGAGA GCAAGGGGTG ATGACCTGGA TGATTACTGC 60 AACGGC 66 SEQ ID NO: 32 sequence length: 66 sequence type: nucleic acid strand number: double strand topology: linear sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland Direct origin Library name: Mammushi venom secretory gland cDNA library Clone name: pAGβ2-101 Sequence features Characteristic symbols: modified base feature The E sequence TTTAAGAAAG AAGGAACAAT ATGCCGGAGA GCAAGGGGTG ATGACCTGGA TGATTACTGC 60 AACGGC 66.

SEQ ID NO: 33 Sequence length: 90 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Library name: Mammoth Venom secretory gland cDNA library Clone name: pAGα101 Sequence features Characteristic symbols: domain Method for determining features: E sequence GGGCTGTGTT GTGGGCAGTG CAGATTTATG AAAAAAGGAA CAGTATGCCG GATAGCAAGG 60 GGTGATGACA TGGATGGGACTA CT.

SEQ ID NO: 34 Sequence length: 90 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper venom gland cDNA library Clone name: pAGβ101 Sequence features Characteristic symbols: domain Method of determining features: E sequence GGGTCGTGTT GTGGGCAGTG CAGATTTATG AAAGAAGGAA CAATATGCCG GAGAGCAAGG 60 GGTGATGACC TGGATGATTA CTGC.

SEQ ID NO: 35 Sequence length: 90 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland direct origin Library name: pit viper secretory gland cDNA library Clone name: pAGα201 Sequence features Characteristic symbols: domain Method of determining features: E sequence GGACTGTGTT GTGACCAGTG CAGATTTATG AAAAAAGGAA CAGTATGCCG GATAGCAAGG 60 GGTGATGACA TGAATGGCATTA CT.

SEQ ID NO: 36 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Library name: Mamushi Venom secretory gland cDNA library Clone name: pAGα201 Sequence features Characteristic symbols: domain Method of determining features: E sequence GAGGACTGCG ACTGTGGCTC TCCTGGAAAT CCGTGCTGTG ATGCTGCAAC CTGTAAACTG 60 AGACAAGGAG CACAGTGTGCTGTGAAGGACT TATGAAAAAA 120 GGAACAGTAT GCCGGATAGC AAGGGGTGAT GACATGGATG ATTACTGCAA TGGC 174.

SEQ ID NO: 37 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: Antisense sequence G A A A T I / G / T / ICII GG / CAI / C 18 T G T G C.

SEQ ID NO: 38 Sequence length: 17 Sequence type: Nucleic acid Number of strands: 1 Strand Sequence type: Antisense sequence A A A A A CC / TT / CA / T A / TC / TC 17 G G G G G.

SEQ ID NO: 39 Sequence length: 18 Sequence type: Nucleic acid Number of strands: 1 Strand Sequence type: Antisense sequence T A A A A ICCI / T / CA / TA / TC / TC 18 G G G G G.

SEQ ID NO: 40 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA sequence CC CCACC GGI // ITG / TG / I / I / AITG / A 22 TT TTGAT.

SEQ ID NO: 41 Sequence length: 17 Sequence type: Nucleic acid Number of strands: 1 Strand Sequence type: cDNA to mRNA Sequence A AT T T T / A / GA / TG / G A / TG / GG 17 G G C C C C.

SEQ ID NO: 42 Sequence length: 33 Sequence type: Nucleic acid Number of strands: 1 Strand Sequence type: cDNA to mRNA sequence CA CCG AGCTT GGIA / I / TIT G /// I / II / CI / GIGGIGA / GA / 33 AG TAA CC ACC.

SEQ ID NO: 43 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double strand Sequence type: cDNA to mRNA Origin Biological name: Mammushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Library name: Mamushi venom secretory gland cDNA library Clone name: pAGβ102 Sequence features Characteristic symbols: domain Method of determining features: E sequence GGG TCG TGT TGT GGG CAG TGC AGA TTT ATG AAA GAA GGA ACA ATA TGC 48 Gly Ser Cys Cys Gly Gln Cys Arg Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 GCT GGC TGC CCC CGC AAT CAC 117 Ala Gly Cys Pro Arg Asn His 35.

SEQ ID NO: 44 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Library name: pit viper gland secretory gland cDNA library Clone name: pAGβ103 Sequence features Characteristic signature: domain Method for determining features: E sequence GGG CCG TGC TGT GAG CAG TGC AGA TTT ATG AAA GAA GGA ACA ATA TGC 48 Gly Pro Cys Cys Glu Gln Cys Arg Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 GCT GGC TGC CCC CGC TAT CAC 117 Ala Gly Cys Pro Arg Tyr His 35.

SEQ ID NO: 45 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Biological name: Mamushi Strain name: Agkistrodon hays blom
hoffii Organelle name: Venom secretory gland Direct origin Library name: Mammus Venom secretory gland cDNA library Clone name: pAGβ104 Sequence features Characteristic symbols: domain Method of determining features: E sequence GGG CCG TGT TGT GGG CAG TGT AGA TTT ATG AAA GAA GGA ACA ATA TGC 48 Gly Pro Cys Cys Gly Gln Cys Arg Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 GCT GCC TGC CCC CGC TAT CCC 117 Ala Ala Cys Pro Arg Tyr Pro 35.

SEQ ID NO: 46 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Library name: Mamushi venom secretory gland cDNA library Clone name: pAGβ105 Sequence features Characteristic symbols: domain Method for determining features: E sequence GGG TTG TTT TGC AAG ACG TGC AGA TTT ATG AAA GAG GGA ACA ATA TGC 48 Gly Leu Phe Cys Lys Thr Cys Arg Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG CGT CAC GAC CTG CCT GAT TAC TGC AAC GCC ATA TCT 96 Arg Arg Ala Arg Arg His Asp Leu Pro Asp Tyr Cys Asn Ala Ile Ser 20 25 30 CCT GGT TGT CCG GGC AAT AAC 117 Pro Gly Cys Pro Gly Asn Asn 35.

SEQ ID NO: 47 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland Direct Origin Library name: Mamushi venom secretory gland cDNA library Clone name: pAGβ106 Sequence characteristics Symbol: domain Method for determining characteristics: E sequence GGG CTG TTT TGT GGG CAG TGC AGT TTT ATG AAA GAA GGA ACA ATA TGC 48 Gly Leu Phe Cys Gly Gln Cys Ser Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 GCT GGC TGT CCC CGC AAT AAC 117 Ala Gly Cys Pro Arg Asn Asn 35.

SEQ ID NO: 48 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Library name: pit viper venom secretory gland cDNA library Clone name: pAGβ10 7 Sequence features Characteristic symbol: domain Method of determining features: E sequence GGG CTG TCC TGC GGG CAG TGC AGA TTT ATG AAA GAA GGA ACA ATA TGC 48 Gly Leu Ser Cys Gly Gln Cys Arg Phe Met Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 CCT GGC TGC CCC CGC AAT CAC 117 Pro Gly Cys Pro Arg Asn His 35.

SEQ ID NO: 49 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 16 ・ 18 Method of characterizing: E sequence GGGCAGTGCA GATTTTTAAA AGAAGGAACA ATA 33.

SEQ ID NO: 50 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 16, 17 Method of characterizing: E sequence GGGCAGTGCA GATTTAAGAA AGAAGGAACA ATA 33.

SEQ ID NO: 51 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA to mRNA fragment type Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretion Gland Direct origin Clone name: pAGβ1-101 Sequence features Characteristic symbols: modified base Location: 28 ・ 30 Method of determining features: E sequence GGG TCG TGT TGT GGG CAG TGC AGA TTT TTA AAA GAA GGA ACA ATA TGC 48 Gly Ser Cys Cys Gly Gln Cys Arg Phe Leu Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 GCT GGC TGC CCC CGC AAT CAC 117 Ala Gly Cys Pro Arg Asn His 35.

SEQ ID NO: 52 Sequence length: 117 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA to mRNA fragment type Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretion Gland Direct origin Clone name: pAGβ2-101 Sequence features Characteristic symbols: modified base Location: 28, 29 Method of determining features: E sequence GGG TCG TGT TGT GGG CAG TGC AGA TTT AAG AAA GAA GGA ACA ATA TGC 48 Gly Ser Cys Cys Gly Gln Cys Arg Phe Lys Lys Glu Gly Thr Ile Cys 1 5 10 15 CGG AGA GCA AGG GGT GAT GAC CTG GAT GAT TAC TGC AAC GGC ATA TCT 96 Arg Arg Ala Arg Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly Ile Ser 20 25 30 GCT GGC TGC CCC CGC AAT CAC 117 Ala Gly Cys Pro Arg Asn His 35.

SEQ ID NO: 53 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 16 ・ 18 Method of characterization: E sequence GACCAGTGCA GATTTTTAAA AAAAGGAACA GTA 33.

SEQ ID NO: 54 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 17 Features Method determined: E sequence GACCAGTGCA GATTTAAGAA AAAAGGAACA GTA 33.

SEQ ID NO: 55 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Clone name: pAGα1-101 Sequence features Characteristic symbols: modified base Location: 112, 114 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT TTA AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC ATG GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Met Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 56 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Clone name: pAGα2-101 Sequence characteristics Symbol: modified base Location: 113 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT AAG AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC ATG GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Met Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 57 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 16 ・ 18 Method of characterization: E sequence GCAAGGGGTG ATGACCTGGA TGATTACTGC AAT 33.

SEQ ID NO: 58 Sequence length: 33 Sequence type: Nucleic acid Number of strands: 1 Strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 16 ・ 18 Method of characterization: E sequence GCAAGGGGTG ATGACGTCGA TGATTACTGC AAT 33.

SEQ ID NO: 59 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland Direct Origin Clone name: pAGα3-101 Sequence features Characteristic symbols: modified base Location: 154/156 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT TTA AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC CTG GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 60 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland Direct Origin Clone name: pAGα4-101 Sequence features Characteristic symbols: modified base Location: 154/156 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT TTA AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC GTC GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Val Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 61 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Sequence features Characteristic symbols: primer, modified base Location: 16, 17 Method of characterizing: E sequence GCAAGGGGTG ATGACCCGGA TGATTACTGC AAT 33.

SEQ ID NO: 62 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Sequence type: cDNA to mRNA Characteristic symbols: primer, modified base Location: 17, 18 Characterize Method: E sequence GCAAGGGGGTG ATGACAACGA TGATTTACGC AAT
33.

SEQ ID NO: 63 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double stranded Sequence type: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland Direct Origin Clone name: pAGα5-101 Sequence features Characteristic symbols: modified base Location: 154,155 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT TTA AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC CCG GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Pro Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 64 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double-stranded Type of sequence: cDNA to mRNA Origin Organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name: Venom secretory gland direct Origin Clone name: pAGα6-101 Sequence features Characteristic symbols: modified base Location: 155,156 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT TTA AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Leu Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC AAC GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Asn Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 65 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Clone name: pAGα7-101 Sequence features Characteristic symbols: modified base Location: 154/156 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT AAG AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC CTG GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Leu Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 66 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Clone name: pAGα8-101 Sequence features Characteristic symbols: modified base Location: 154/156 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT AAG AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC GTC GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Val Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 67 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Clone name: pAGα9-101 Sequence features Symbols: modified base Location: 154,155 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT AAG AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC CCG GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Pro Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 68 Sequence length: 174 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mamushi Strain name: Agkistrodon halys blomhoffii Organelle name : Venom secretory gland Direct origin Clone name: pAGα10-101 Sequence features Characteristic symbols: modified base Location: 155,156 Method of determining features: E sequence GAG GAC TGC GAC TGT GGC TCT CCT GGA AAT CCG TGC TGT GAT GCT GCA 48 Glu Asp Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala Ala 1 5 10 15 ACC TGT AAA CTG AGA CAA GGA GCA CAG TGT GCA GAA GGA CTG TGT TGT 96 Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys 20 25 30 GAC CAG TGC AGA TTT AAG AAA AAA GGA ACA GTA TGC CGG ATA GCA AGG 144 Asp Gln Cys Arg Phe Lys Lys Lys Gly Thr Val Cys Arg Ile Ala Arg 35 40 45 GGT GAT GAC AAC GAT GAT TAC TGC AAT GGC 174 Gly Asp Asp Asn Asp Asp Tyr Cys Asn Gly 50 55.

SEQ ID NO: 69 Sequence length: 30 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: No Fragment type: N Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Tissue type: Venom secretion Venom secreted from the gland Features of the sequence Characteristic symbols: domain Method of determining the feature: E sequence Gly Glu Glu Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala 1 5 10 15 Ala Thr Cys Lys Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly 20 25 30.

SEQ ID NO: 70 Sequence length: 20 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: No Fragment type: N Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Tissue type: Venom secretion Venom secreted from the gland Characteristic of sequence Characteristic symbol: domain Method for determining characteristic: E sequence Gly Glu Glu Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys Asp Ala 1 5 10 15 Ala Thr Cys Lys 20.

SEQ ID NO: 71 Sequence length: 22 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: No Fragment type: N Origin Biological name: Mamushi Strain name: Agkistrodon hays blom
hoffii type of tissue: venom secreted from venom-secreting gland Character of sequence: domain showing method of characterizing: E sequence Glu Ala Gly Glu Glu Cys Asp Cys Gly Ser Pro Gly Asn Pro Cys Cys 1 5 10 15 Asp Ala Ala Thr Cys Lys 20.

SEQ ID NO: 72 Sequence length: 20 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: No Fragment type: Intermediate origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Tissue type: Venom Venom secreted from the secretory gland Characteristic of sequence Characteristic symbol: domain Method for determining characteristic: E sequence Leu Arg Gln Gly Ala Gln Cys Ala Glu Gly Leu Cys Cys Asp Gln Cys 1 5 10 15 Arg Phe Met Lys 20.

SEQ ID NO: 73 Sequence length: 28 Sequence type: Amino acid Sequence type: Peptide Hypothetical sequence: No Fragment type: C Origin Biological name: Mamushi Strain name: Agkistrodon halys blomhoffii Tissue type: Venom secretion Venom secreted by the gland Features of the sequence Characteristic symbol: domain Method of determining the feature: E sequence Gly Thr Val Cys Arg Ile Ala Arg Gly Asp Asp Met Asp Asp Tyr Cys 1 5 10 15 Asn Gly Ile Ser Ala Gly Cys Pro Arg Asn Pro Phe 20 25.

SEQ ID NO: 74 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: Yes Antisense: No sequence TCCAGCCAAA TCCAGTCTCC 20.

SEQ ID NO: 75 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: Yes Antisense: No sequence ATGATCCAAG TTCTTTTGAT 20.

SEQ ID NO: 76 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Hypothetical Sequence: Yes Antisense: Yes Sequence TGGGACAGCC AGCAGATATG 20.

SEQ ID NO: 77 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Hypothetical Sequence: Yes Antisense: Yes Sequence AGACCATTCC AGCTCCATTG 20.

SEQ ID NO: 78 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Hypothetical Sequence: Yes Antisense: Yes Sequence TTAGGCATGG AAGGGATTTC 20.

SEQ ID NO: 79 Array length: 1558 Array type: Number of nucleic acid strands: Double-stranded topology: Linear array types: cDNA to mRNA hypothetical sequence: No Antisense: No   Array   GTCTCCAAA   ATG   ATA   CAG   GTT   CTG   TTG   ATA   ACT   ATA   TGC   TTG   GCA   GTT   TTT   51   Met   Ile   Gln   Val   Leu   Leu   Ile   Thr   Ile   Cys   Leu   Ala   Val   Phe   1   Five   Ten   CCC   TTC   CAG   GGA   AGC   TCC   ATA   GTT   CTG   GAT   TCC   GGA   AAT   CTT   AAT   GAA   99   Pro   Phe   Gln   Gly   Ser   Ser   Ile   Val   Leu   Asp   Ser   Gly   Asn   Leu   Asn   Glu   15   20   twenty five   30   TTC   GAG   GTT   GTT   TAT   CCA   GAA   AAA   GTT   ACT   GCA   TTG   CCC   AGA   GCA   GCT   147   Phe   Glu   Val   Val   Tyr   Pro   Glu   Lys   Val   Thr   Ala   Leu   Pro   Arg   Ala   Ala   35   40   45   GTT   AAA   AAT   AAG   TAT   GAG   GAC   GCC   ATG   CAA   TAT   GAA   TTT   AAA   GTT   AAT   195   Val   Lys   Asn   Lys   Tyr   Glu   Asp   Ala   Met   Gln   Tyr   Glu   Phe   Lys   Val   Asn   Fifty   55   60   GGA   GAG   CCA   CTG   CTT   CTT   CAT   CTG   GAA   CGG   AAT   AAA   GGA   CTT   TTT   TCC   243   Gly   Glu   Pro   Leu   Leu   Leu   His   Leu   Glu   Arg   Asn   Lys   Gly   Leu   Phe   Ser   65   70   75   GAT   GAT   TAC   AGC   GAG   ATT   CAT   TAT   TCC   CCC   GAT   GCA   AGA   GAA   ATA   TCC   291   Asp   Asp   Tyr   Ser   Glu   Ile   His   Tyr   Ser   Pro   Asp   Ala   Arg   Glu   Ile   Ser   80   85   90   GCA   TAC   CCA   TCC   GTT   GAA   GAT   CAT   TGC   TTC   TAT   CAT   GGA   CGG   GTT   GAG   339   Ala   Tyr   Pro   Ser   Val   Glu   Asp   His   Cys   Phe   Tyr   His   Gly   Arg   Val   Glu   95   100   105   110   AAT   GAT   GCT   GAC   TCA   ACT   GCA   AGT   TTG   AGT   GCA   TGT   GAC   GGA   TTG   AAA   387   Asn   Asp   Ala   Asp   Ser   Thr   Ala   Ser   Leu   Ser   Ala   Cys   Asp   Gly   Leu   Lys   115   120   125   GCA   CAT   TTC   AAA   ATT   CAG   GGG   GAG   ATG   TAT   CTG   ATA   GAA   CCC   TTG   GAG   435   Ala   His   Phe   Lys   Ile   Gln   Gly   Glu   Met   Tyr   Leu   Ile   Glu   Pro   Leu   Glu   130   135   140   GTT   TCC   GAC   ACT   GAT   GCA   CAT   GCA   GTC   TTC   AAA   TAT   GAA   AAT   GTT   GAA   483   Val   Ser   Asp   Thr   Asp   Ala   His   Ala   Val   Phe   Lys   Tyr   Glu   Asn   Val   Glu   145   150   155   AAA   GAG   GAC   GAG   CCC   CCC   AAA   ATG   TGT   GGA   GTT   ACC   CAG   AAT   TGG   GAA   531   Lys   Glu   Asp   Glu   Pro   Pro   Lys   Met   Cys   Gly   Val   Thr   Gln   Asn   Trp   Glu   160   165   170   TCA   TAT   GAG   TCT   ACT   AAA   AAA   GCA   TCC   CAG   TTG   AAT   GTT   TCT   CCC   GAC   579   Ser   Tyr   Glu   Ser   Thr   Lys   Lys   Ala   Ser   Gln   Leu   Asn   Val   Ser   Pro   Asp   175   180   185   190   CAG   CAA   AGA   TTC   CCA   CAG   AGA   TTT   ATT   AAG   CTT   GCA   ATC   TAT   GTG   GAC   627   Gln   Gln   Arg   Phe   Pro   Gln   Arg   Phe   Ile   Lys   Leu   Ala   Ile   Tyr   Val   Asp   195   200   205   CAC   GGA   ATG   TAC   ACA   AAA   TAC   GCA   GGC   AAT   TCT   GAA   AGG   ATA   ACA   AAA   675   His   Gly   Met   Tyr   Thr   Lys   Tyr   Ala   Gly   Asn   Ser   Glu   Arg   Ile   Thr   Lys   210   215   220   AGG   GTA   CAT   CAA   ATG   ATC   AAC   AAT   ATC   AAT   ATG   ATG   TGC   AGA   GCT   CTG   723   Arg   Val   His   Gln   Met   Ile   Asn   Asn   Ile   Asn   Met   Met   Cys   Arg   Ala   Leu   225   230   235   AAT   ATA   GTT   ACT   AGC   CTG   AGT   GTC   CTA   AGG   ATT   TGG   TCC   GAA   AAA   GAT   771   Asn   Ile   Val   Thr   Ser   Leu   Ser   Val   Leu   Arg   Ile   Trp   Ser   Glu   Lys   Asp   240   245   250   TTG   ATA   ACT   GTG   AAT   GCA   TCC   GCA   CCC   AGC   AGT   TTG   ACC   TTA   TTT   GGA   819   Leu   Ile   Thr   Val   Asn   Ala   Ser   Ala   Pro   Ser   Ser   Leu   Thr   Leu   Phe   Gly   255   260   265   270   GCC   TGG   AGA   GAG   ACA   GTC   TTG   CTG   AAT   CGC   ACC   AGT   CAT   GAT   CAT   GCT   867   Ala   Trp   Arg   Glu   Thr   Val   Leu   Leu   Asn   Arg   Thr   Ser   His   Asp   His   Ala   275   280   285   CAG   TTA   ATG   ACG   GCC   ACT   ATC   TTC   AAT   GGA   AAC   GTT   ATA   GGA   AGA   GCT   915   Gln   Leu   Met   Thr   Ala   Thr   Ile   Phe   Asn   Gly   Asn   Val   Ile   Gly   Arg   Ala   290   295   300     CCC   GTG   GGC   GGT   ATG   TGT   GAC   CCG   AAG   CGT   TCT   GTA   GCA   ATA   GTT   CGG   963   Pro   Val   Gly   Gly   Mer   Cys   Asp   Pro   Lys   Arg   Ser   Val   Ala   Ile   Val   Arg   305   310   315   GAT   CAT   AAT   GCA   ATA   TTG   TTC   ATA   GTT   GCA   GTT   ACA   ATG   ACC   CAT   GAG   1011   Asp   His   Asn   Ala   Ile   Leu   Phe   Ile   Val   Ala   Val   Thr   Met   Thr   His   Glu   320   325   330   ATG   GGT   CAT   AAT   CTG   GGC   ATG   CAT   CAT   GAT   GAA   GAT   AAA   TGT   AAT   TGT   1059   Met   Gly   His   Asn   Leu   Gly   Met   His   His   Asp   Glu   Asp   Lys   Cys   Asn   Cys   335   340   345   350   AAC   ACA   TGC   ATT   ATG   TCT   AAA   GTG   TTA   AGC   CGC   CAA   CCT   TCC   TAT   GAG   1107   Asn   Thr   Cys   Ile   Met   Ser   Lys   Val   Leu   Ser   Arg   Gln   Pro   Ser   Tyr   Glu   355   360   365   TTC   AGT   GAT   TGT   AAT   GAG   AAT   GAA   TAT   CAG   ACG   TAT   GTT   ACT   GAT   CAT   1155   Phe   Ser   Asp   Cys   Asn   Glu   Asn   Glu   Tyr   Gln   Thr   Tyr   Val   Thr   Asp   His   370   375   380   AGC   CCA   CAA   TGC   ATT   CTC   AAT   GAC   CCC   TTG   AGA   CCA   GAT   ACT   GTT   TCA   1203   Ser   Pro   Gln   Cys   Ile   Leu   Asn   Asp   Pro   Leu   Arg   Pro   Asp   Thr   Val   Ser   385   390   395   ACT   CCA   GTT   TCT   GGA   AAT   GAA   CTT   TTG   GAG   GCC   GGA   GAA   GAG   TGT   GAC   1251   Thr   Pro   Val   Ser   Gly   Asn   Glu   Leu   Leu   Glu   Ala   Gly   Glu   Glu   Cys   Asp   400   405   410   TGT   GGC   TCT   CCT   GGA   AAT   CCG   TGC   TGC   GAT   GCT   GCA   ACC   TGT   AAA   CTG   1299   Cys   Gly   Ser   Pro   Gly   Asn   Pro   Cys   Cys   Asp   Ala   Ala   Thr   Cys   Lys   Leu   415   420   425   430   AGA   CAG   GGG   GCA   CAG   TGT   GCA   GAA   GGA   CTG   TGT   TGT   GAC   CAG   TGC   AGA   1347   Arg   Gln   Gly   Ala   Gln   Cys   Ala   Glu   Gly   Leu   Cys   Cys   Asp   Gln   Cys   Arg   435   440   445   TTT   ATG   AAA   AAA   GGA   ACA   GTA   TGC   CGG   ATA   GCA   AGG   GGT   GAT   GAC   ATG   1395   Phe   Met   Lys   Lys   Gly   Thr   Val   Cys   Arg   Ile   Ala   Arg   Gly   Asp   Asp   Met   450   455   460   GAT   GAT   TAC   TGC   AAT   GGC   ATA   TCT   GCT   GGC   TGT   CCC   AGA   AAT   CCC   TTC   1443   Asp   Asp   Tyr   Cys   Asn   Gly   Ile   Ser   Ala   Gly   Cys   Pro   Arg   Asn   Pro   Phe   465   470   475   CAT   GCC   TAACCAACAA   TGGAGCTGGA   ATGGTCTGCA   GCAACAGGCA   GTGTGTTGAT   1499   His   Ala   480   GTGACTACAG   CCTACTAATC   AACCTCTGGC   TTCTCTCAGA   TTTGATTTTG   GAGATCCGT   1558

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence and predicted amino acid sequence of the α-type cDNA contained in pAGα101, which is one fragment of the platelet aggregation inhibitory polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 2 is a diagram showing the nucleotide sequence and predicted amino acid sequence of β-type cDNA contained in pAGβ101, which is one fragment of the platelet aggregation inhibitory polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 3 shows the nucleotide sequence and predicted amino acid sequence of the α-type cDNA contained in pAGα201, which is a fragment of the platelet aggregation-inhibiting polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 4 is a diagram showing the nucleotide sequence and predicted amino acid sequence of β-type cDNA contained in pAGβ102, which is one fragment of the platelet aggregation inhibitory polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 5 is a diagram showing the nucleotide sequence and predicted amino acid sequence of β-type cDNA contained in pAGβ103, which is one fragment of the platelet aggregation-inhibiting polypeptide cDNA derived from venom of pit viper venom.

FIG. 6 is a diagram showing the nucleotide sequence and predicted amino acid sequence of β-type cDNA contained in pAGβ104, which is one fragment of the platelet aggregation inhibitory polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 7 shows the nucleotide sequence and predicted amino acid sequence of the β-type cDNA contained in pAGβ105, which is one fragment of the platelet aggregation inhibitory polypeptide cDNA derived from venom of venom secretory glands.

FIG. 8 shows the nucleotide sequence and predicted amino acid sequence of β-type cDNA contained in pAGβ106, which is one fragment of the platelet aggregation inhibitory polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 9 is a diagram showing the nucleotide sequence and predicted amino acid sequence of β-type cDNA contained in pAGβ107, which is one fragment of the platelet aggregation-inhibiting polypeptide cDNA derived from venom secretory glands of pit viper.

FIG. 10: pAGβ1--a mutant of pAGβ102
It is a figure which shows the base sequence of the mutant DNA contained in 101, and the predicted amino acid sequence.

FIG. 11: pAGβ2- which is a mutant of pAGβ102
It is a figure which shows the base sequence of the mutant DNA contained in 101, and the predicted amino acid sequence.

FIG. 12: pAGα1-a mutant of pAGα201
It is a figure which shows the base sequence of the mutant DNA contained in 101, and the predicted amino acid sequence.

FIG. 13: pAGα2-a mutant of pAGα201
It is a figure which shows the base sequence of the mutant DNA contained in 101, and the predicted amino acid sequence.

FIG. 14: pAGα which is a mutant of pAGα1-101
It is a figure which shows the base sequence of the mutant DNA contained in 3-101, and the predicted amino acid sequence.

FIG. 15. pAGα, a mutant of pAGα1-101.
It is a figure which shows the base sequence of the mutant DNA contained in 4-101, and the predicted amino acid sequence.

FIG. 16: pAGα which is a mutant of pAGα1-101
It is a figure which shows the base sequence of the mutant DNA contained in 5-101, and the predicted amino acid sequence.

FIG. 17: pAGα which is a mutant of pAGα1-101
It is a figure which shows the base sequence of the mutant DNA contained in 6-101, and the predicted amino acid sequence.

FIG. 18: pAGα which is a mutant of pAGα2-101
It is a figure which shows the base sequence of the mutant DNA contained in 7-101, and the predicted amino acid sequence.

FIG. 19: pAGα which is a mutant of pAGα2-101
It is a figure which shows the base sequence of the mutant DNA contained in 8-101, and the predicted amino acid sequence.

FIG. 20: pAGα which is a mutant of pAGα2-101
It is a figure which shows the base sequence of the mutant DNA contained in 9-101, and the predicted amino acid sequence.

FIG. 21: pAGα which is a mutant of pAGα2-101
It is a figure which shows the base sequence of the mutant DNA contained in 10-101, and the predicted amino acid sequence.

FIG. 22 is a graph showing the rabbit platelet aggregation inhibitory activity of the purified halistatin obtained in Example 6.

FIG. 23 is a reverse phase high performance liquid chromatogram of purified halistatin obtained in Example 6.

FIG. 24 is a reverse-phase high performance liquid chromatogram of the degradation product of halistatin by lysyl endopeptidase.

FIG. 25 shows the nucleotide sequence of the halistatin precursor cDNA contained in the plasmid pAGγ101 and the amino acid sequence predicted therefrom.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // A61K 37/02 ACA 8314-4C ADS

Claims (43)

[Claims] 1. Amino acid sequence: Phe-A ₁ -Lys-A _{2
-Gly-Thr-A 3 -Cys-} Arg-A 4 -Ala
-Arg-Gly-Asp-Asp- A 5 -Asp-A
A polypeptide containing sp-Tyr-Cys-Asn-Gly [wherein A ₁ is Met, Leu or Ly.
s; A ₂ is Lys or Glu; A ₃ is Val or Ile; A ₄ is Ile or Arg; A ₅ is Met, L
represents eu, Val, Pro or Asn]. 2. Amino acid sequence: Phe-Met-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-Me
t-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 3. Amino acid sequence: Phe-Met-Lys-
Glu-Gly-Thr-Ile-Cys-Arg-A
rg-Ala-Arg-Gly-Asp-Asp-Le
u-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 4. Amino acid sequence: Phe-Leu-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-Me
t-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 5. Amino acid sequence: Phe-Lys-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-Me
t-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 6. Amino acid sequence: Phe-Leu-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-Le
u-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 7. Amino acid sequence: Phe-Leu-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-Va
l-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 8. Amino acid sequence: Phe-Leu-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-Pr
o-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 9. Amino acid sequence: Phe-Leu-Lys-
Lys-Gly-Thr-Val-Cys-Arg-I
le-Ala-Arg-Gly-Asp-Asp-As
n-Asp-Asp-Tyr-Cys-Asn-Gly
The polypeptide according to claim 1, which comprises: 10. Amino acid sequence: Phe-Lys-Lys
-Lys-Gly-Thr-Val-Cys-Arg-
Ile-Ala-Arg-Gly-Asp-Asp-L
eu-Asp-Asp-Tyr-Cys-Asn-Gl
The polypeptide of claim 1 containing y. 11. Amino acid sequence: Phe-Lys-Lys
-Lys-Gly-Thr-Val-Cys-Arg-
Ile-Ala-Arg-Gly-Asp-Asp-V
al-Asp-Asp-Tyr-Cys-Asn-Gl
The polypeptide of claim 1 containing y. 12. Amino acid sequence: Phe-Lys-Lys
-Lys-Gly-Thr-Val-Cys-Arg-
Ile-Ala-Arg-Gly-Asp-Asp-P
ro-Asp-Asp-Tyr-Cys-Asn-Gl
The polypeptide of claim 1 containing y. 13. Amino acid sequence: Phe-Lys-Lys
-Lys-Gly-Thr-Val-Cys-Arg-
Ile-Ala-Arg-Gly-Asp-Asp-A
sn-Asp-Asp-Tyr-Cys-Asn-Gl
The polypeptide of claim 1 containing y. 14. Amino acid sequence: Phe-Leu-Lys
-Glu-Gly-Thr-Ile-Cys-Arg-
Arg-Ala-Arg-Gly-Asp-Asp-L
eu-Asp-Asp-Tyr-Cys-Asn-Gl
The polypeptide of claim 1 containing y. 15. Amino acid sequence: Phe-Lys-Lys
-Glu-Gly-Thr-Ile-Cys-Arg-
Arg-Ala-Arg-Gly-Asp-Asp-L
eu-Asp-Asp-Tyr-Cys-Asn-Gl
The polypeptide of claim 1 containing y. 16. Amino acid sequence: Gly-Leu-Cys
-Cys-Gly-Gln-Cys-Arg-Phe-
Met-Lys-Lys-Gly-Thr-Val-C
ys-Arg-Ile-Ala-Arg-Gly-As
p-Asp-Met-Asp-Asp-Tyr-Cys
The polypeptide according to claim 1, which comprises -Asn-Gly. 17. Amino acid sequence: Gly-Ser-Cys
-Cys-Gly-Gln-Cys-Arg-Phe-
Met-Lys-Glu-Gly-Thr-Ile-C
ys-Arg-Arg-Ala-Arg-Gly-As
p-Asp-Leu-Asp-Asp-Tyr-Cys
The polypeptide according to claim 1, which comprises -Asn-Gly. 18. Amino acid sequence: Gly-Leu-Cys
-Cys-Asp-Gln-Cys-Arg-Phe-
Met-Lys-Lys-Gly-Thr-Val-C
ys-Arg-Ile-Ala-Arg-Gly-As
p-Asp-Met-Asp-Asp-Tyr-Cys
The polypeptide according to claim 1, which comprises -Asn-Gly. 19. Amino acid sequence: Glu-Asp-Cys
-Asp-Cys-Gly-Ser-Pro-Gly-
Asn-Pro-Cys-Cys-Asp-Ala-A
la-Thr-Cys-Lys-Leu-Arg-Gl
n-Gly-Ala-Gln-Cys-Ala-Glu
-Gly-Leu-Cys-Cys-Asp-Gln-
Cys-Arg-Phe-Met-Lys-Lys-G
ly-Thr-Val-Cys-Arg-Ile-Al
a-Arg-Gly-Asp-Asp-Met-Asp
The polypeptide according to claim 1, which comprises -Asp-Tyr-Cys-Asn-Gly. 20. A recombinant DNA containing a base sequence encoding the amino acid sequence according to claim 1. 21. Claims 2, 3, 4, 5, 6, 7, 8,
A recombinant DNA containing a nucleotide sequence encoding any of the amino acid sequences of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. 22. Base sequence: TTTATGAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACATGGATGACTACTGCAATGGG. 23. Base sequence: TTTATGAAAGAAGGAACAATATGCCG
The recombinant DNA according to claim 21, which has a nucleotide sequence containing GAGAGCAAGGGGTGATGACCTGGATGATTACTGCAATGGG. 24. Nucleotide sequence: TTTTTAAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which has a base sequence containing GATAGCAAGGGGTGATGACATGGATGATTACTGCAATGGC. 25. A nucleotide sequence: TTTAAGAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which has a base sequence containing GATAGCAAGGGGTGATGACATGGATGATTACTGCAATGGC. 26. Base sequence: TTTTTAAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACCTGGATGATTACTGCAATGGC. 27. A nucleotide sequence: TTTTTAAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which is a nucleotide sequence containing GATAGCAAGGGGTGATGACGTCGATGATTACTGCAATGGC. 28. Base sequence: TTTTTAAAAAAAGGAACAGTATGCCG
The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACCCGGATGATTACTGCAATGGC. 29. Nucleotide sequence: TTTTTAAAAAAAGGAACAGTATGCCG
The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACAACGATGATTACTGCAATGGC. 30. Base sequence: TTTAAGAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACCTGGATGATTACTGCAATGGC. 31. A nucleotide sequence: TTTAAGAAAAAAGGAACAGTATGCCG
22. The recombinant DNA according to claim 21, which is a nucleotide sequence containing GATAGCAAGGGGTGATGACGTCGATGATTACTGCAATGGC. 32. A base sequence: TTTAAGAAAAAAGGAACAGTATGCCG
The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACCCGGATGATTACTGCAATGGC. 33. A base sequence: TTTAAGAAAAAAGGAACAGTATGCCG
The recombinant DNA according to claim 21, which has a nucleotide sequence containing GATAGCAAGGGGTGATGACAACGATGATTACTGCAATGGC. 34. A nucleotide sequence: TTTTTAAAAGAAGGAACAATATGCCG
22. The recombinant DNA according to claim 21, which is a nucleotide sequence containing GAGAGCAAGGGGTGATGACCTGGATGATTACTGCAACGGC. 35. Base sequence: TTTAAGAAAGAAGGAACAATATGCCG
22. The recombinant DNA according to claim 21, which is a nucleotide sequence containing GAGAGCAAGGGGTGATGACCTGGATGATTACTGCAACGGC. 36. Nucleotide sequence: GGGCTGTGTTGTGGGCAGTGCAGATT
TATGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACATGGATG
The recombinant DNA according to claim 21, which has a base sequence containing ACTACTGCAATGGG. 37. A base sequence: GGTTCGTGTTGTGGGCAGTGCAGATT
TATGAAAGAAGGAACAATATGCCGGAGAGCAAGGGGTGATGACCTGGATG
The recombinant DNA according to claim 21, which has a nucleotide sequence containing ATTACTGCAATGGG. 38. Nucleotide sequence: GGACTGTGTTGTGACCAGTGCAGATT
TATGAAAAAAGGAACAGTATGCCGGATAGCAAGGGGTGATGACATGGATG
22. The recombinant DNA according to claim 21, which has a nucleotide sequence containing ATTACTGCAATGGC. 39. Base sequence: GAGGACTGCGACTGTGGCTCTCCTGG
AAATCCGTGCTGTGATGCTGCAACCTGTAAACTGAGACAAGGAGCACAGT
GTGCAGAAGGACTGTGTTGTGACCAGTGCAGATTTATGAAAAAAGGAACA
The recombinant DN according to claim 21, which is a base sequence containing GTATGCCGGATAGCAAGGGGTGATGACATGGATGATTACTGCAATGGC.
A. 40. A transformant carrying the recombinant DNA according to claim 20. 41. The recombinant DN according to claim 21.
The transformant according to claim 40, which retains A. 42. The transformant according to claim 40 is cultured,
A method for producing the polypeptide, which comprises producing and accumulating the polypeptide according to claim 1 in a culture, and collecting the polypeptide. 43. A transformant according to claim 41 is cultivated, and the transformant is cultivated in the culture.
8, 9, 10, 11, 12, 13, 14, 15, 16,
43. The method for producing the polypeptide according to claim 42, wherein the polypeptide according to 17, 18, or 19 is produced and accumulated, and this is collected.