CN1746305A - Tissue factor pathway inhibitor-2 gene and protein of Banma fish (zTFP-2) - Google Patents

Abstract

A 2-(zTFP1-2) gene and protein can be used to clone and determine 2-(zTFP1-2) gene, its coding protein have high homology with human TFP1-2, and provide experimental basis for TFP1-2 physiological function, tumor invasion and transfer and wound heal.

Description

Zebrafish tissue factor pathway inhibitor-2's gene and albumen

Technical field

The invention belongs to life science.Relate to (the zebrafishtissue factor pathway inhibitor-2 of zebrafish tissue factor pathway inhibitor-2, zTFPI-2) gene and albumen, its encoded protein matter and people TFPI-2 have very high homology, and in situ hybridization confirms to exist really in the zebra fish body this gene.

Background technology

TFPI-2 is a kind of Kunitz type serpin, three Kunitz type structural domains are arranged, the activity that can suppress multiple serine protease, the mainly synthetic and secretion by endotheliocyte, inoblast, keratinocyte etc. extensively is present in the organs such as pancreas, liver, kidney, lung in the human body.(humanTFPI-2 hTFPI-2) can suppress the Invasion and Metastasis of multiple malignant tumour to people TFPI-2, and comes off relevantly with atherosclerotic plaque, but physiological function it be unclear that.

Zebra fish has become one of best vertebrates model of biological study, and its individuality is little, and oviposition period is short, and egg laying amount is many and ovum is big; The embryo is in external quick growth, and body early embryo is transparent fully, and the acquisition of diplontic making and mutant all is easier to, and sperm can freezingly be preserved.Number gene and people in the zebra fish genome are similar, and there are corresponding relation in its many genes and human body.More rare is that the tumour of zebra fish takes place with the mankind very similar.All these characteristics make zebra fish be very suitable for the research of fetology, epigenetics and function of diseases genomics.Therefore, zebra fish is as a kind of ideal molecular biology model animals, have other vertebratess the advantage that can't compare.

Though the TFPI-2 gene of people, ape, ox, mouse and rat is cloned, which type of physiological function it has actually and does not also have clear and definite answer in the vertebrates body.Studies show that most TFPI-2 are present in the extracellular matrix (ECM), and the multiple protein enzyme is had important regulatory function.Many investigators infer thus, and TFPI-2 relates in the physiological and pathological process that extracellular matrix reinvents at some may play an important role the Invasion and Metastasis of for example fetal development, wound healing, atherosclerosis, malignant tumour etc.In view of the characteristics of model organism zebra fish, the physiological function of research TFPI-2 have the incomparable advantage of other model animals, but prerequisite is at first to be cloned into zebra fish TFPI-2 gene in its growth course.

Summary of the invention

Gene order and its encoded protein matter sequence of the purpose of this invention is to provide zebrafish tissue factor pathway inhibitor-2 (zTFPI-2).

The invention provides gene order and the protein sequence (sequence 1, the nucleotide sequence of sequence 2 and this nucleic acid sequence encoding protein sequence) of zTFPI-2, and application in situ hybridization has confirmed that zTFPI-2 is in the intravital existence of zebra fish.

The invention provides the intravital a kind of kunitz type serpin of model organism zebra fish encoding gene cDNA sequence and thus the prediction aminoacid sequence, with its called after zebra fish TFPI-2 (zebrafish TFPI-2, zTFPI-2).ZTFPI-2 is a kind of typical serpin, is made up of 232 amino acid, and it contains 19 halfcystines, and wherein 18 halfcystines are formed 9 disulfide linkage, form the kunitz structural domain of three arranged in series.Be respectively with the homology of three structural domains of known hTFPI-2: 63%, 59%, 71%.Change on any amount can take place in amino-acid residue quantity before described proteinic the 2nd cysteine residues and the later amino-acid residue quantity of last cysteine residues.

The present invention at first adopts the aminoacid sequence of hTFPI-2 to compare in zebra fish genome and Protein Data Bank, seeks in the zebra fish body and hTFPI-2 homologous protein.Obtain the partial sequence of homologous protein and the gene of zebra fish TFPI-2 according to the result of comparison.Adopt RACE method clone to obtain the coding region sequence and the two ends non-coding area sequence of this gene based on this.

According to the cDNA sequence mark probe that obtains, observe the expression of zTFPI-2 gene in the zebra fish growth course, the result confirms that this gene exists really in the zebra fish body.

According to the cDNA sequence of zTFPI-2, in the genomic library of zebra fish, compare, obtain structure and the base sequence of this gene in the intravital chromosomal localization of zebra fish, coding region.

According to gene order and the aminoacid sequence of zTFPI-2 of the present invention, and the TFPI-2 corresponding sequence of other known species, adopt bioinformatics method can draw evolutionary relationship and the conservative degree of TFPI-2 between different plant species.

The present invention is to the physiological function of research TFPI-2, and TFPI-2 and tumor invasion shift, and the relation of fetal development and wound healing provides experiment basis.

Description of drawings

Fig. 1 has obvious expression for zTFPI-2 in 24 hours embryo of after fertilization growth.

Embodiment

Embodiment 1 sequence alignment

Protein sequence with hTFPI-2 is compared in Trust Sanger Institude zebra fish database, obtain one section the highest peptide section Ensembl Translation of homology, its ID is ENSDARP00000019243, the cDNA sequence is ENSDART00000024355, this sequence is made up of 528bp, and the amino acid quantity of peptide section is 176.

Embodiment 2 obtains zTFPI-2 cDNA sequence

1. the extraction of the total RNA of zebra fish

Adopt the Invitrogen RNA of company organization to extract reagent and extract the total RNA of zebra fish,, do not have DNA to pollute, can satisfy the subsequent experimental requirement through not degraded of denaturing formaldehyde agarose gel electrophoresis proof total RNA that carries.

2. design of primers

Consequence devised following primer according to sequence alignment:

P5’RACE outer：

5’------TCTTCACACTGCTGGCTGAT------3’

P5’RACE inner：

5’------AAAACCGCTGAATGTCGTCA------3’

P3’RACE outer：

5’------CAAAGACATGCGAGGAGTTCA------3’

P3’RACE inner：

5’------TTCATCTCCAAGCAAAGCTG------3’

5’KSMART IIA：

5′------AAGCAGTGGTACCAACGCAGAGTACGCGGG------3’

3′KSMART II A：

5’------GGTTGGTACCGTTTTCCCAGTCACGACT ₍₃₀₎N _-1N-----3’(N＝A，G，C，T；N _-1＝A，G，C)

KM13M4：

5’------GGTTGGTACCGTTTTCCCAGTCACGAC-----3’

Primer all can lottery industry biotech company be synthesized by the Shen, Shanghai.

3.zTFPI-2 the clone of 5 ' and 3 ' unknown nucleotide sequence

The clone of zTPFI-2 unknown nucleotide sequence adopts the SMART technology of Clontech company to carry out, and does corresponding improvement.Concrete steps are as follows:

(1) article one cDNA chain is synthetic

0.5ml the following reagent of mixing in the reaction tubes:

The total RNA:10 μ of zebra fish L (0.5 μ g)

5’KSMART II A(12μM)： 7μL

3′KSMART II A(12μM)： 7μL

Deionized water: 40 μ L

Cumulative volume: 64 μ L

The mixing mentioned reagent, put 65 ℃ 2 minutes, be cooled to 42 ℃, add following reagent:

5 * First-Strand damping fluid: 20 μ L

DTT(100mM)： 2μL

50×dNTP(10mM)： 10μL

RNase Inhibitor(20U/μL)： 5μL

PowerScript reversed transcriptive enzyme: 5 μ L

Cumulative volume: 42 μ L

The mixing mentioned reagent, put 42 ℃ 30 minutes.The disodium ethylene diamine tetraacetate termination reaction that adds 2 μ L 0.5M.Related reagents such as dNTP of the present invention and reversed transcriptive enzyme are available from Clontech company.

(2) purifying of total cDNA

Get the reaction product of above-mentioned reverse transcription, adopt the nucleic acid purification test kit purifying of Shen, Shanghai energy lottery industry biotech company.The concrete operations by specification carries out.

(3) pcr amplification of total cDNA

The cDNA40 μ L that gets above-mentioned purifying adds the deionized water of equal volume, adds following reagent:

Deionized water: 2 μ L

10 * PCR damping fluid: 10 μ L

50×dNTP(10mM)： 2μL

5’KSMART II A(12μM)： 2μL

KM13M4(12μM)： 2μL

Takara HS archaeal dna polymerase (5U/ μ L) 2 μ L

Cumulative volume: 20 μ L

Reaction conditions:

(94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 3 minutes) * 15 circulations, 72 ℃ 7 minutes.

Take out 30 μ L and manage as optimizing to another new reaction tubes from reaction tubes, remaining reaction system places 4 ℃ as experiment tube.

It is standby to take out 5 μ L in the self-optimizing pipe, and the residue reaction system continues 3 circulation totally 18 circulations of reaction by above-mentioned reaction conditions), it is standby to get 5 μ L.Remain 20 μ L and continue by 3 circulations of above-mentioned conditioned response (totally 21 circulations), it is standby to get 5 μ L.Remain 15 μ L and continue by 3 circulations of above-mentioned conditioned response (totally 24 circulations), it is standby to get 5 μ L.

Remain 10 μ L and continue by 3 circulations of above-mentioned conditioned response (totally 27 circulations), it is standby to get 5 μ L.Remaining 5 μ L continues by 3 circulations of above-mentioned conditioned response (30 circulations) totally.The PCR product that at every turn takes out is carried out 1% agarose electrophoresis, observe the result of PCR.Determine that the cycle number when the PCR product does not increase with cycle index is the cycle number of experiment tube.The experiment tube of 4 ℃ of preservations is reacted by above-mentioned reaction conditions, and the reaction cycle number is for optimizing the cycle number that pipe is determined.The present invention tests used archaeal dna polymerase available from Japanese Takara company.

(4) cyclisation of total cDNA:

Get the PCR product 30 μ L of previous step reaction, cut with restriction enzyme KpnI enzyme.Reaction system is as follows:

PCR product: 30 μ L

10 * damping fluid: 10 μ L

100×BSA： 1μL

KpnI(20U/μL)： 10μL

Deionized water: 49 μ L

Cumulative volume: 100 μ L

37 ℃ were reacted 2 hours.

Reaction product adopts the nucleic acid purification test kit purifying of Shen, Shanghai energy lottery industry biotech company.The concrete operations by specification carries out.

With the T4DNA ligase enzyme make enzyme cut and purifying after total cDNA cyclisation, reaction system is as follows:

Total cDNA:30 μ L

10 * T4 dna ligase damping fluid: 10 μ L

T4 dna ligase (6U/ μ L): 2 μ L

Deionized water: 58 μ L

Cumulative volume: 100 μ L

4 ℃ of reactions are spent the night.

Described restriction enzyme, the T4DNA ligase enzyme is available from U.S. NEB company.

(5) clone of zTFPI-2 cDNA unknown nucleotide sequence

With the total cDNA of the zebra fish of cyclisation is that template is done the PCR reaction, and reaction system is as follows:

The total cDNA of cyclisation (about 1.0 μ g/ μ L): 0.5 μ L

10 * damping fluid: 5 μ L

P5’RACE outer(25pmol/μL)：1μL

P3’RACE outer(25pmol/μL)：1μL

dNTP(10mM each)： 1μL

Takara HS DNA polymerase： 0.5μL

Deionized water: 41 μ L

Reaction conditions is as follows:

(94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute) * 72 ℃ of 30 circulations 7 minutes.

Get above-mentioned PCR product and make template, carry out the PCR reaction again, reaction system is as follows:

PCR product: 0.5 μ L

10 * damping fluid: 5 μ L

P5’RACE inner(25pmol/μL)：1μL

P3’RACE inner(25pmol/μL)：1μL

dNTP(10mM each)： 1μL

Takara HS DNA polymerase： 0.5μL

Deionized water: 41 μ L

Reaction conditions is as follows:

(94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute) * 72 ℃ of 30 circulations 7 minutes.

Get the PCR product and carry out 1% agarose gel electrophoresis, the pulsating size of assay products.And with the Shen, Shanghai can lottery industry the above-mentioned PCR product of nucleic acid purification test kit purifying of biotech company.

PCR product behind the purifying and the reorganization of the pGEM-T carrier of Invitrogen company, reaction system is as follows:

2 * ligase enzyme damping fluid: 5 μ L

pGEM-T(50ng)： 1μL

PCR product: 3 μ L

T4 dna ligase: 1 μ L

4 ℃ of connections are spent the night, transformed into escherichia coli competence DH5 α, the order-checking of extracting plasmid, this recombinant plasmid called after rpGEMTzTFPI-2.Used T carrier is available from Invitrogen company.

Embodiment 3 zebra fish in situ hybridizations

1. probe mark

Make the abundant linearizing of rpGEMTzTFPI-2 with restriction enzyme SpeI, reactions steps is as follows:

Reaction system:

rpGEMTzTFPI-2(1μg/μL)： 30μL

10 * reaction buffer: 10 μ L

100 * bovine serum albumin: 1 μ L

SpeI(10U/μL)： 8μL

Water: 51 μ L

Cumulative volume: 100 μ L

37 ℃ of reactions are spent the night.

Linearizing rpGEMTzTFPI-2 adopts the nucleic acid purification test kit purifying of Shen, Shanghai energy lottery industry biotech company, and the concrete steps by specification carries out.

Adopting linearizing rpGEMTzTFPI-2 is template, carries out the rna probe mark with the NTP of digoxigenin labeled.Reaction system is as follows:

10 * T7 RNA polymerase damping fluid: 2 μ L

DTT(50mM)： 2μL

Digoxigenin labeled NTP mixture (every kind of 2.5mM): 4 μ L

RNA enzyme inhibitors (40U/ μ L): 0.5 μ L

rpGEMTzTFPI-2(0.5μg/μL)： 1μL

T7 RNA polymerase (50U/ μ L): 1 μ L

No RNA enzyme water: 9.5 μ L

37 ℃ were reacted 2 hours.

The DNA enzyme that adds 2 μ L (2U/ μ L) was hatched 30 minutes for 37 ℃.

Adding 2ul disodium ethylene diamine tetraacetate (pH8.0,0.2mol/L), 2.5ul lithium chloride (4mol/L), the 75ul dehydrated alcohol spends the night for subzero 20 degrees centigrade.4 ℃ 12000 rev/mins centrifugal 30 minutes, remove supernatant, with the water dissolution of the no RNA enzyme of 50ul.The sex change agarose gel electrophoresis is observed the quality of probe.Add the hybridization buffer of 450ul at last, standby.

2. the embryo fixes

Get after fertilization and grow 24 hours embryos, with 0.5 milliliter of PBST washed twice.0.5 milliliter of the Paraformaldehyde 96 of adding 4%, room temperature left standstill 5 minutes, removed Paraformaldehyde 96.Add 0.5 milliliter of 4% Paraformaldehyde 96 room temperature again and left standstill 5 minutes, place again 4 ℃ 12-15 hour.Remove Paraformaldehyde 96, once with the washing of PBST0.5 milliliter.Use methanol wash again three times, it is frozen in subzero 20 degrees centigrade to add 0.5 ml methanol at last.

3. hybridization

(1) with the solution shown in the table 1 and the time chien shih fixed embryo aquation again:

Table 1

The solution numbering	1	2	3	4
					PBST (milliliter) methyl alcohol (milliliter) washing time	1.5 4.5 5 minutes	3.0 3.0 5 minutes	4.5 1.5 5 minutes	6.0 05 minutes three times

(2) in PBST, remove chorion with the fine needle head;

(3) in room temperature with the protease K digesting embryo of 10 μ g/ml 10 minutes;

(4) room temperature was retightened the embryo 20 minutes with 4% Paraformaldehyde 96;

(5) PBST with 0.5ml washes 5 times, each 5 minutes;

(6) 65 ℃ of prehybridizations 2 hours in 300 μ L hybridization solutions, hybridization finish and placed 65 ℃ of preheatings 30 minutes with hybridization solution 1/20 dilution probe in preceding 30 minutes;

(7) remove prehybridization solution and change the hybridization solution that contains probe into, spend the night 65 ℃ of hybridization;

(8) remove hybridization solution, use table 2 solution 65 ℃ of washings.Solution will be 65 ℃ of preheatings 30 minutes, and every kind of solution washing time is 10 minutes.

Table 2

The solution numbering	1	2	3	4
					2 * SSC (milliliter) hybridization solution (milliliter) time	1.5 (25%) 4.5 (75%) 10 minute	3.0 (50%) 3.0 (50%) 10 minute	4.5 (75%) 1.5 (25%) 10 minute	6.0 (100%) 0 (0%) 10 minute

(9) wash twice with 0.5 milliliter of 0.2 * SSC at 65 ℃, each 30 minutes;

(10) with table 3 solution washing:

Table 3

The solution numbering	1	2	3	4
					PBST (milliliter) 0.2 * SSC (milliliter) time	1.5 (25%) 4.5 (75%) 5 minute	3.0 (50%) 3.0 (50%) 5 minute	4.5 (75%) 1.5 (25%) 5 minute	6.0 (100%) 0 (0%) 5 minute

(11) in PBST, add the BSA of 2mg/ml and 5% sheep serum as confining liquid, 4 ℃ of sealing embryos 1-3 hour;

(12) with the anti digoxin antibody of confining liquid 1/2000 dilution alkali phosphatase enzyme mark, make the embryo in this antibody-solutions 4 ℃ spend the night;

(13) wash each 15 minutes eight times with the PBST that contains 2mg/ml;

(14) with containing the sodium-chlor of 100mM Tris pH=9.5,100mM, magnesium chloride and the 0.1% soil temperature-20 solution equilibria embryo of 50mM;

(15) add alkaline phosphatase colour developing liquid chamber temperature colour developing 30 minutes to 1 hour, observations.

Hybridization related reagent of the present invention is available from Roche diagnostic reagent company.

Experimental result shows that zTFPI-2 has obvious expression (accompanying drawing 1) in 24 hours embryo of after fertilization growth.

Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front should be understood that only to illustrate, but not limits the scope of the invention by any way.

From the above description, those skilled in the art can understand essential characteristic of the present invention at an easy rate, and under the situation that does not depart from its purport and scope, can carry out various changes and improvement to the present invention.

SEQUENCE LISTING

＜110〉Fudan University

＜120〉zebrafish tissue factor pathway inhibitor-2's gene and albumen

<130>Fudan-20050424

<160>2

<170>PatentIn version 3.2

<210>1

<211>1077

<212>DNA

＜213〉zebra fish (Danio rerio)

<220>

<221>CDS

<222>(76)..(777)

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aagtcatatc aggcgagcaa cataacatta acatatcaga cttacacgcg atctcacgac 60

cccaagtcca tcaca atg gcg tgt gat tta ctc gga gct ctg ttg ctt ttc 111

Met Ala Cys Asp Leu Leu Gly Ala Leu Leu Leu Phe

1 5 10

att tct gtg cta gaa agc gcc gtc gga ctg acg tta caa cct aaa gaa 159

Ile Ser Val Leu Glu Ser Ala Val Gly Leu Thr Leu Gln Pro Lys Glu

15 20 25

gtt tgt ctg ttg caa att gaa gaa ggg acg tgt aat gac gac att cag 207

Val Cys Leu Leu Gln Ile Glu Glu Gly Thr Cys Asn Asp Asp Ile Gln

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Arg Phe Tyr Tyr Asn Thr Ile Ser Gln Gln Cys Glu Glu Phe Ser Tyr

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agc ggc tgt gga gga aac caa aac aac ttc agg tct ttc gtg gaa tgt 303

Ser Gly Cys Gly Gly Asn Gln Asn Asn Phe Arg Ser Phe Val Glu Cys

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cag aaa aca tgc ttc agg ata cca aaa atc ccc cag atc tgt cgt ttt 351

Gln Lys Thr Cys Phe Arg Ile Pro Lys Ile Pro Gln Ile Cys Arg Phe

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caa aag aaa gag ggg ccc tgc cgt ggc ctc ttc agc cgt tat ttc ttc 399

Gln Lys Lys Glu Gly Pro Cys Arg Gly Leu Phe Ser Arg Tyr Phe Phe

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aat atg acc tcc atg cag tgt gaa cca ttc act tat ggt ggc tgc caa 447

Asn Met Thr Ser Met Gln Cys Glu Pro Phe Thr Tyr Gly Gly Cys Gln

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ggg aat gag aac aat ttt cga aac cca gag gaa tgc ata gaa tat tgc 495

Gly Asn Glu Asn Asn Phe Arg Asn Pro Glu Glu Cys Ile Glu Tyr Cys

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aaa cct ccg aaa acg atc cct gtg att tgc ctg gat aat ttg gat aaa 543

Lys Pro Pro Lys Thr Ile Pro Val Ile Cys Leu Asp Asn Leu Asp Lys

145 150 155

gga aga tgc tca gca tcc ata cca agg tat tat tac aac tct gcc aca 591

Gly Arg Cys Ser Ala Ser Ile Pro Arg Tyr Tyr Tyr Asn Ser Ala Thr

160 165 170

aag aca tgc gag gag ttc atg tac acc gga tgc gga gga agc aac aat 639

Lys Thr Cys Glu Glu Phe Met Tyr Thr Gly Cys Gly Gly Ser Asn Asn

175 180 185

aac ttc atc tcc aag caa agc tgc gtc gac gtt tgt ggg agc aaa cgt 687

Asn Phe Ile Ser Lys Gln Ser Cys Val Asp Val Cys Gly Ser Lys Arg

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tgg agt ccc act aaa aaa tcg gtg aga gtt tct aag cag tac ctg aga 735

Trp Ser Pro Thr Lys Lys Ser Val Arg Val Ser Lys Gln Tyr Leu Arg

205 210 215 220

cga gtg aaa cct cag cca tcg agg gag aag acg aac aaa taa 777

Arg Val Lys Pro Gln Pro Ser Arg Glu Lys Thr Asn Lys

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tcgtttatga agtcattttc actgttttag ggaacattta catgacaaca atgtagtaat 837

gtagtagttt ttgcttttgg tattttaaca agttatggat accaatgaca atgttggcaa 897

aatgatccct ttacacagat cagcgaacgc agctaaaaag ttggccagta ggtggcgaca 957

taatcgagtt cacaaagaac tttatggtat cgtttacaaa aacttgcact ttgaaacaca 1017

tttgcatgaa aaacgtcagt gttcatcaac agccaagatg cataaagtgt aagtcatatc 1077

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＜213〉zebra fish (Danio rerio)

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Met Ala Cys Asp Leu Leu Gly Ala Leu Leu Leu Phe Ile Ser Val Leu

1 5 10 15

Glu Ser Ala Val Gly Leu Thr Leu Gln Pro Lys Glu Val Cys Leu Leu

20 25 30

Gln Ile Glu Glu Gly Thr Cys Asn Asp Asp Ile Gln Arg Phe Tyr Tyr

35 40 45

Asn Thr Ile Ser Gln Gln Cys Glu Glu Phe Ser Tyr Ser Gly Cys Gly

50 55 60

Gly Asn Gln Asn Asn Phe Arg Ser Phe Val Glu Cys Gln Lys Thr Cys

65 70 75 80

Phe Arg Ile Pro Lys Ile Pro Gln Ile Cys Arg Phe Gln Lys Lys Glu

85 90 95

Gly Pro Cys Arg Gly Leu Phe Ser Arg Tyr Phe Phe Asn Met Thr Ser

100 105 110

Met Gln Cys Glu Pro Phe Thr Tyr Gly Gly Cys Gln Gly Asn Glu Asn

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Asn Phe Arg Asn Pro Glu Glu Cys Ile Glu Tyr Cys Lys Pro Pro Lys

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Thr Ile Pro Val Ile Cys Leu Asp Asn Leu Asp Lys Gly Arg Cys Ser

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Ala Ser Ile Pro Arg Tyr Tyr Tyr Asn Ser Ala Thr Lys Thr Cys Glu

165 170 175

Glu Phe Met Tyr Thr Gly Cys Gly Gly Ser Asn Asn Asn Phe Ile Ser

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Lys Gln Ser Cys Val Asp Val Cys Gly Ser Lys Arg Trp Ser Pro Thr

195 200 205

Lys Lys Ser Val Arg Val Ser Lys Gln Tyr Leu Arg Arg Val Lys Pro

210 215 220

Gln Pro Ser Arg Glu Lys Thr Asn Lys

225 230

Claims (7)

1, zebrafish tissue factor pathway inhibitor-2's gene and albumen is characterized in that having sequence 1, the nucleotide sequence of sequence 2 and this nucleic acid sequence encoding protein sequence.

2, by claim 1 described zebrafish tissue factor pathway inhibitor-2 gene and albumen, it is characterized in that described nucleotide sequence is the cDNA sequence.

3, by claim 1 described zebrafish tissue factor pathway inhibitor-2 gene and albumen, it is characterized in that described nucleotide sequence is the mRNA sequence.

4, a kind of dna vector is characterized in that containing the nucleotide sequence described in the claim 1, called after rpGEMTzTFPI-2, reproducible.

5, by the described dna vector of claim 4, it is characterized in that described carrier is plasmid pGEMT.

6, by claim 1 described zebrafish tissue factor pathway inhibitor-2 gene and albumen, it is characterized in that described protein is made up of 232 amino acid, it contains 19 halfcystines, and wherein 18 halfcystines are formed 9 disulfide linkage, form the kunitz structural domain of three arranged in series.

7, by claim 6 described zebrafish tissue factor pathway inhibitor-2 gene and albumen, the change on any amount can take place in amino-acid residue quantity before wherein said proteinic the 2nd cysteine residues and the later amino-acid residue quantity of last cysteine residues.

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