CN107551265A

CN107551265A - A kind of vaccine for pigs trichina disease and its preparation method and application

Info

Publication number: CN107551265A
Application number: CN201710685687.0A
Authority: CN
Inventors: 廖婉琴; 周兴旺
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2017-08-11
Filing date: 2017-08-11
Publication date: 2018-01-09

Abstract

The invention discloses a kind of vaccine for pigs trichina disease and its preparation method and application.Expand firstTsCPB2The maturation zone of gene is simultaneously cloned into the expression vector of His labels, then converts Escherichia coli, after IPTG induced expressions, using Ni affinitive layer purification TsCPB2 recombinant proteins, obtains trichina excretion secretion recombinant antigen；Again by TsCPB2 recombinant proteins and immunologic adjuvant mixing and emulsifying, TsCPB2 recombinant antigen vaccines are obtained；The vaccine immune mouse being prepared using the present invention or pig, host can be induced to produce immune response, good immanoprotection action is played to trichinzation, there is larger application prospect.

Description

A kind of vaccine for pigs trichina disease and its preparation method and application

Technical field

The invention belongs to biological technical field.More particularly, to a kind of vaccine for pigs trichina disease and preparation method thereof and Using.

Background technology

Trichina（Trichinella spiralis）A kind of parasitic nematode of infecting both domestic animals and human, can parasitize including More than the 150 kinds of mammal such as people, pig, dog, mouse, cat, causes trichinosis.The mutual infection of pig and mouse is mankind's trichinosis stream Capable important sources, wherein pig are main animal reservoir.The pork eaten raw or partly eat the trichinella larvae Nang Bao that is contaminted raw is The major way of population infection trichina.Trichinosis is in worldwide distribution, is mainly shown as the symptoms such as heating, oedema, then Death can be caused by symptom, the severe patients such as myalgia, limbs fatigue occur.In recent years, there is the disease in many areas in the world Prevalence with outburst.In China, it is raw or not due to eating that resident occurred in the ground such as Yunnan, Henan, Guangxi, Tibet and Sichuan Cooked pork and dog meats and fall ill and dead case occur.In animal husbandry, pigs trichina disease is meat product safety One major issue, huge economic loss is caused to pig industry and meat trade.So far, trichinosis, which there is no, quickly has The diagnostic method of effect.Therefore develop vaccine, be the Critical policies for preventing the disease development.In Trichinella Spiralis Vaccination technical research Aspect, for trichinous all kinds of vaccines all in starting conceptual phase, such as attenuated live vaccine, native antigen vaccine, synthesis The polytypes such as peptide vaccine, recombinant antigen vaccine, nucleic acid vaccine.However, effective Trichinella Spiralis Vaccination is there is no at present.

In the whole life cycle of trichina, antigen plays important work in host environment and host immune response With.Surface antigen, excretion-secretion are classified as according to the trichina region of anatomy（ES）Antigen, somatic antigen and rod cell particle Related antigen.Due to trichina polypide crude antigen and surface antigen complicated component, and trichina can not be completed by vitro culture The whole history of life, it is difficult to which the method produced in batches by artificial challenge's minimal standardsization prepares such antigen, can not meet to be immunized Diagnosis and the needs of immunoprophylaxis antigen.The anti-larva rhabodoids by reaching maturity of ES are secreted, and are directly exposed to exempting from for host Epidemic disease system and with preferably specificity, is the main target antigen for inducing host to produce immune response, and important epidemic disease Seedling candidate molecules.Due to can not still carry out Secondary Culture before Spirotricha in vitro, the ES antigens obtained are difficult to ensure that its is steady It is qualitative, and ES antigens are the mixtures of a variety of antigens, it is difficult to distinguish Effective Antigens composition or purification process.With genetic engineering Technology reaches its maturity and diversified development, gene recombinant antigens have the advantages that largely to prepare, specificity is good and increasingly by To the attention of people.Therefore restructuring ES antigens turn into the focus of trichinosis immune prevention.

Research finds that the nutrient solution of cultivation of larvae of Trichinella spiralis from muscle ES products has immunogenicity.In recent years proteome analysis and Mass spectral analysis finds to contain a certain amount of cysteine proteinase in cultivation of larvae of Trichinella spiralis from muscle ES albumen.Cysteine proteinase bag Include cathepsin（cathepsin）B, H, L, X etc..Research report intestinal nematodes, the cysteine proteinase of intestinal worms exist The enteron aisle of parasite is invaded, divided a word with a hyphen at the end of a line, reproductive development, nutrition intake, play during intramuscular encystation, and immune invasion etc. it is important Effect, it is the important candidate molecules of anti parasitic vaccine.

The content of the invention

The technical problems to be solved by the invention are the defects of overcoming above-mentioned prior art and deficiency, pass through 3 ' RACE-PCR Technology expands to have obtained a brand-new trichina cathepsin B geneTsCPB2, using trichinaTsCPB2Gene structure The recombinant antigen built has good immunogenicity, available for the vaccine for preparing prevention trichinzation, the vaccine being prepared With good immune protective effect, available for prophylactic treatment pigs trichina disease.

It is an object of the invention to provide a kind of trichinosis vaccine.

It is a further object of the present invention to provide the preparation method of the trichinosis vaccine.

The above-mentioned purpose of the present invention is to give realization by the following technical programs：

The present invention withTsCPB2Est sequence（Accession No. EX501780）Gene-specific amplification primer is designed, is utilized 3 ' RACE-PCR technologies expand to have obtained a brand-new trichina cathepsin B geneTsCPB2, its nucleotide sequence Such as SEQ ID NO：Shown in 1, the amino acid sequence such as SEQ ID coded by it：Shown in 2.

Preferably, the specificity amplification primer sequence is as follows：

TsCPB2-5’GSP outer primer：5’-CGGATCTGTTGTCCTGGTTGG-3’（SEQ ID：4）

TsCPB2-5’GSP inner primer：5’-CTGTTGTCCTGGTTGGATAAA-3’（SEQ ID NO：5）

First, the present invention providesTsCPB2Gene and its coded amino acid application as described below：

SEQ ID NO：Trichina cathepsin B gene shown in 1TsCPB2Answering in trichina excretory-secretory antigen is prepared With.

SEQ ID NO：The answering in trichina excretory-secretory antigen is prepared of trichina cathepsin B2 shown in 2 With.

SEQ ID NO：Trichina cathepsin B gene shown in 1TsCPB2Application in trichinosis vaccine is prepared.

SEQ ID NO：Application of trichina cathepsin B2 shown in 2 in trichinosis vaccine is prepared.

Preferably, it is the application in vaccine for pigs trichina disease is prepared.

A kind of preparation method of trichina excretory-secretory antigen, methods described are amplificationTsCPB2The maturation zone of gene is simultaneously It is cloned into the expression vector of His labels, conversion Escherichia coli, after induced expression, Ni- affinitive layer purifications TsCPB2 restructuring eggs In vain, trichina excretory-secretory antigen is obtained；It is describedTsCPB2The sequence of gene such as SEQ ID NO：Shown in 1, its ripe region sequence Such as SEQ ID NO：Shown in 3.

Preferably, the expression vector is pET30a（+）.

Preferably, the induced expression is 37 DEG C of h of Fiber differentiation 4 in final concentration of 1 mM IPTG derivants.

Preferably, thalline is collected by centrifugation after induced expression, after thalline is resuspended, freeze thawing, adds lysozyme lysis, then ultrasound Broken, centrifugation obtains inclusion body protein.

The inclusion body protein need to be washed with the denaturant of detergent and low concentration, remove lipid and memebrane protein, be redissolved Obtain soluble protein.

Preferably, the detergent is Triton X-100, and the denaturant is urea.

It is highly preferred that inclusion body protein respectively washed once with following solutions：

A.50 mM Tris/HCl, 500 mM NaCl, pH 8.0 contain 1% Triton X-100（200 ml/500 ml bacterium Liquid）；

B.50 mM Tris/HCl, 500 mM NaCl, pH 8.0 contain 4M urea（100 ml/500 ml bacterium solutions）；

C.50 mM Tris/HCl, 500 mM NaCl, pH 8.0 contain 2M urea（100 ml/500 ml bacterium solutions）；Then 4 DEG C, 6000 g centrifuge 15 min；The inclusion body protein in precipitation is dissolved in following solution again：50mM Tris/HCl, pH 7.9th, 500 mM NaCl, 6 M urea, room temperature are acutely shaken up overnight so that inclusion body protein is completely dissolved.

Preferably, the Ni- affinitive layer purifications is gently add protein sample Ni posts, coutroi velocity in 5 ml/h, Collect efflux；10 times of 1 × Binding of volume Buffer（Urea containing 6M）Cross post, then 20 times of volume Wash Buffer（Containing 6M Urea）Rinse foreign protein；Finally with the Elute buffer containing imidazoles（Urea containing 6M）The destination protein containing elution.

Meanwhile trichina excretion-secretion that any of the above-described method is prepared（ES）Antigen is also in the scope of the present invention It is interior.

In addition, the trichina excretion-secretion（ES）Antigen is in trichinosis vaccine or trichinosis antibody is prepared Using also in the scope of the present invention.

A kind of trichinosis vaccine, it is by the trichina excretion-secretion（ES）Antigen is TsCPB2 recombinant proteins with exempting from Epidemic disease adjuvant mixing and emulsifying, obtains TsCPB2 recombinant antigen vaccines.

Preferably, the immune emulsification adjuvant is Freund's adjuvant.

It is highly preferred that first immunisation uses Freund's complete adjuvant, two exempt from, three exempt to use incomplete Freund's adjuvant.

In addition, application of the trichinosis vaccine of the invention being prepared in prevention and control pigs trichina disease is also protected in the present invention In the range of shield.

Compared with prior art, the invention has the advantages that：

（1）A kind of trichina restructuring ES antigens have been prepared in the present invention, and the recombinant antigen has good immunogenicity, can For preparing the trichinous vaccine of prevention and control.

（2）A kind of trichinosis vaccine, the vaccine immune mouse or pig has been prepared in the present invention, host can be induced to produce Immune response, and good immanoprotection action is played to trichinzation, there is larger application prospect.

Brief description of the drawings

Fig. 1 is the present inventionTsCPB2The full length nucleotide sequence of gene and the amino acid sequence derived；Wherein, TsCPB2 Maturation zone shadow representation.

Fig. 2 is the expression and purification SDS-PAGE analytical electrophoresis figures of TsCPB2 recombinant proteins.A, SDS-PAGE are analyzed TsCPB2 expression and purification；M, marker；Land 1, empty carrier/bacterium do not induce；Land 2, empty carrier/bacterium IPTG are lured Lead；Land 3, TsCPB2/ bacterium do not induce；Land 4, TsCPB2/ bacterium IPTG are induced；Land 5, TsCPB2/ bacteria lysis Thing supernatant；Land 6, TsCPB2/ bacterial lysate precipitate；Land 7, TsCPB2 albumen after purification.B, Western blot Analyze TsCPB2 expression.

Fig. 3 is TsCPB2 immunoreactivity and the testing result figure of secreting, expressing.A, Western blot are analyzed TsCPB2 recombinant proteins can be by trichinzation different time（3rd, 6,10,16,20,44 days）Mice serum identification；B, Western blot analyze the ES products of muscle larvae（Anti- TsCPB2 recombinant proteins rabbit antibody is primary antibody）.

Fig. 4 is the immune response of induction output after TsCPB2 mice immunized with antigen.Respectively with TsCPB2+FA, TsCPB+FA, BABL/c mouse are immunized in FA, and ELISA detects the total IgG in immune serum（A）、IgG1（B）、 IgG2（C）、IgE（D）Water It is flat.

Fig. 5 is immanoprotection action of the TsCPB2 antigen immunes to mouse trichinzation.A, trichina challenge infection The adult of TsCPB2 vaccine immune mouses counts；B, the muscle larvae of trichina challenge infection TsCPB2 vaccine immune mouses count.

Embodiment

The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art Agent, method and apparatus.

Unless stated otherwise, following examples agents useful for same and material are purchased in market.

A kind of preparation method of 1 Trichinella Spiralis Vaccination of embodiment

1、TsCPB2The clone of gene

Utilize TRIZOL（Invitrogen, USA）Reagent extracts total serum IgE from the muscle larvae after infecting mouse 35 days.Using reversion It is the chains of cDNA first that enzyme (M-MLV RT, Promega), which is recorded, by total serum IgE reverse transcription.WithTsCPB2Est sequence (Accession No. EX501780) design gene-specific amplification primer, TsCPB2-5 ' GSP outer primer: 5’- CGGATCTGTTGTCCTGGTTGG-3’；TsCPB2-5’GSP inner primer: 5’-CTGTTGTCCTGGTTGGATAAA- 3’.TsCPB2 complete sequences are expanded using 3 '-RACE-PCR (TaKaRa, Japan), operation recommends method to carry out by kit. 1% agarose gel electrophoresis of PCR primer, reclaimed using QIAquick gel extraction kit (QIAGEN, USA) PCR primer.PCR primer is cloned into pMD19-T carriers, sequencing obtainsTsCPB2Nucleotide sequence（Fig. 1）, its sequence such as SEQ ID NO：Shown in 1, the amino acid sequence such as SEQ ID NO of its encoding proteins：Shown in 2.

2nd, the expression of TsCPB2 recombinant proteins

WillTsCPB2Maturation zone（SEQ ID：The sequence of coded amino acid 15~333 shown in 3）It is cloned into pET30a（+）Carrier structure Build pET30a-TsCPB2Recombinant expression plasmid.Recombinant expression plasmid is convertedE. coliBL21 competent cells.Picking converts Single bacterium colony afterwards, add in the LB culture mediums containing 100 μ g/mL ampicillins, 37 DEG C of cultures to OD₆₀₀For 0.6~1.0, receive Collect bacterium solution.50 μ L restructuring bacterium solution is taken to be added in LB culture mediums of 5 mL containing 100 μ g/mL ampicillins, 37 DEG C of trainings overnight Support.Next day is by the bacterium solution being incubated overnight by 1:100 ratios are forwarded in the fresh culture containing 100 μ g/mL ampicillins, Continue culture to OD₆₀₀For 0.6~1.0, derivant IPTG to final concentration of 1 mM is added, after 37 DEG C of h of Fiber differentiation 4,4 DEG C, 6000g centrifuges 15 min and collects thalline.Thalline is resuspended in 50mM Tris/HCl, the mM NaCl of pH 8.0,500 solution In（According to the ratio of 30 ml solution/1L bacterium solutions）, freeze thawing is once.Lysozyme is added to the mg/ml of final concentration 1, reacts 30 on ice Minute.Ultrasonication 15 min, ultrasonic 2s stop 3s, and 4 DEG C, 10000 g are centrifuged 20~30 minutes.Then by precipitation following solutions Respectively it washed once：A, 50 mM Tris/HCl, 500 mM NaCl, pH 8.0 contain 1% Triton X-100（200 ml/ 500 ml bacterium solutions）;B, 50 mM Tris/HCl, 500 mM NaCl, pH 8.0 contain 4M urea（100 ml/500 ml bacterium Liquid）;C, 50 mM Tris/HCl, 500 mM NaCl, pH 8.0 contain 2M urea（100 ml/500 ml bacterium solutions）, then 4 DEG C, 6000 g centrifuge 15 min：.Inclusion body protein in precipitation is dissolved in following solution：50mM Tris/HCl, pH 7.9th, 500 mM NaCl, 6 M urea, room temperature are acutely shaken up overnight so that inclusion body protein is completely dissolved.After sample dissolving, 4 DEG C, 16000 g centrifuge 30 min, collect supernatant, 0.45 μm of membrane filtration, prepare purifying.

3rd, the purifying of TsCPB2 recombinant proteins（Under Denaturing containing 6M urea）

（1）1 ml 50%Ni-NTA His-Bind resin suspensions are added in sky chromatographic column, treat resin natural subsidence.With 3 times The distilled water of resin bed volume crosses post, 5 times of 1 × Binding of volume Buffer（Urea containing 6M）Balance resin.By protein sample Chromatographic column is gently added, coutroi velocity is in 5 ml/h, collection efflux.10 times of 1 × Binding of volume Buffer（Urinated containing 6M Element）Cross post.20 times of volume Wash Buffer（Urea containing 6M）Rinse foreign protein.Finally with the Elute buffer containing imidazoles （Urea containing 6M）Elute destination protein.SDS-PAGE electrophoresis and Western blot analysis purifying proteins.

（2）As a result

SDS-PAGE electrophoresis results, which are shown at 37 kDa, obvious band（Fig. 2A）, it is consistent with destination protein prediction size.Profit Western blot inspections are carried out with anti-TsCPB2 recombinant proteins antibody, being as a result also shown at 37 kDa has single band（Figure 2B）, show the success of TsCPB2 recombinant protein purifications.

4th, the detection of TsCPB2 immunoreactivities and secreting, expressing

（1）200 trichinas of every mouse infection, the 3rd after infection, 6,10,16,20,44d collect serum.Hair is revolved with infection The mice serum of worm detects recombinant protein TsCPB2 as primary antibody using Western blot.As a result 37 kDa are shown in （TsCPB2 recombinant protein sizes）Place, there is single band in 10~44d after infection, and band brightness is in increasing trend（Figure 3A）.This shows that TsCPB2 has good immunoreactivity.

（2）Collect the ml of muscle larvae 0.1（About 170,000）, with the 1640 culture medium of serum-free（Containing dual anti-）Rinse 3 times.With The 1640 culture medium of 5000/ml culture density serum-free（0.25% glucose, 2mM glutamine, 100 U/ml's It is dual anti-）24 h are cultivated in 37 DEG C.Culture medium is collected, sieved filter is considered with 300 mesh are sterile, reclaims filtrate.Filtrate is in sterile physiological salt After 4 DEG C of 48 h of dialysis of water, with Amicon Ultra-15 ultra-filtration centrifuge tubes（Millipore）Ultrafiltration concentration obtains muscle larvae secretion Albumen.Muscle larvae secretory protein is separated by electrophoresis with 10% SDS-PAGE, is carried out by primary antibody of anti-TsCPB2 recombinant proteins antibody Western blot are analyzed, and are as a result shown at 34 kDa and single band occur, it is a kind of excretion-secretion to show TsCPB2（ES） Albumen（Fig. 3 B）.

5th, the immune response of induction output after mouse is immunized in TsCPB2

（1）TsCPB2 recombinant proteins remove endotoxin

Recombinant protein is with 1% TritonX-114 is added after PBS, uninterruptedly stirring makes into homogeneous to 4 DEG C of 30 min, and then 37 DEG C water-bath 10 min, 25 DEG C of 14000 g centrifuges 15 min, careful to shift upper strata aqueous phase albumen.It is complete to repeat this extraction steps 2 times It is complete to remove endotoxin.

（2）Antigen and adjuvant emulsion

Recombinant protein after purification and isometric adjuvant are mixed and made into antigen emulsion（First immunisation uses Freund's complete adjuvant （FA, Sigma, USA）, two exempt from, three exempt to use incomplete Freund's adjuvant（Sigma, USA）, vortex concussion 1 hour.By made of Emulsion drop one drops in water surface, is emulsified if instilling the water surface and keeping tear drop completely without disperseing complete.

（3）Mouse is immunized in TsCPB2 recombinant proteins

Totally 3 groups of BALB/c mouse：TsCPB2+FA、TsCPB+FA、FA.40 μ g are subcutaneously injected with completely in two groups of experimental mices The restructuring TsCPB2 albumen or TsCPB albumen of FA emulsifications.Control group only injects the FA of PBS preparations.Then, to inject phase between 10 days With the recombinant protein for cannoing be used up full FA emulsifications of dosage, and control group injects the incomplete FA that PBS is prepared.0,10 after immune, Collect within 20 and 30 days immune mousetail blood.

（4）The antibody level change of ELISA detection TsCPB2 immune serums

It is primary antibody with immune mouse 0, the serum of 10,20,30 days, IgG, IgG1, IgG2, IgE bis- of sheep anti mouse HRP- marks It is anti-, according to indirect ELISA detection method detection antibody level change.After experimental result shows that TsCPB2 and TsCPB tri- exempts from, mouse Total IgG level is significantly raised in serum, and the immune elevation amplitudes of TsCPB2 are noticeably greater than TsCPB and are immunized（Fig. 4 A）. After the immune mouse of TsCPB2 third times, serum IgG 1, IgG2a significantly increase, and after the immune mouse of TsCPB1 third times, IgG1 Horizontal also significantly rise, but IgG2a not significant changes（Fig. 4 B and 4C）.These results show the immune rear inducing mouses of TsCPB2 Produce Th1/Th2 mixed immunities.In addition, after mouse is immunized in TsCPB2, significantly raise, count after IgE levels are exempted from three in serum With statistics sex differernce（Fig. 4 D）.

6th, immanoprotection action of the TsCPB2 vaccine immunities to mouse trichinzation

2 weeks after final immunization mouse, every mouse is with 400 cultivation of larvae of Trichinella spiralis from muscle through challenge infection.6 days Computation immunities after attack Adult number in mouse small intestine.As a result find, after TsCPB2 is immune, adult number significantly reduces in mouse small intestine, reduced rate Up to 52.3%（Fig. 5 A）.42 days Computation immunity mouse muscle larvae numbers after attack.As a result show, after TsCPB2 is immune, mouse small intestine Middle muscle larvae number significantly reduces, reduced rate 51.2%（Fig. 5 B）.

SEQUENCE LISTING

<110>Zhongshan University

<120>A kind of vaccine for pigs trichina disease and its preparation method and application

<130> 2017

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1002

<212> DNA

<213>TsCPB2 genes

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atgaaaattt tgttgctcag cttgtttgct ttcgctttgg cggaaaatta tgaagaaaat 60

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tggaaatttg gacgaaatgc atatttcaaa aataagtcaa ttggtgaaat caaaaagctg 180

ttgggctaca ggatgttgcc taagacggtg aaagagcgaa atgaaatgcc aatgccggaa 240

gatttattaa atttggaaaa tttcaattat ccggtggaat ttgactccag aaaacattgg 300

cctcagtgtg aaaaagtgat cagcttcata aaagatcaag caaactgtgg aagctgttgg 360

gccgtatcca gcgcgtccgt catgtccgac cggacgtgca ttgctactga tggtcaattt 420

acgacattgc tttccgatgc cgaacttctt tcctgttgta catcatgcgg ttatggttgt 480

aacggtggat atcctcagag aactttcaaa tattgggtgt acagcggtat gccaactggt 540

ggaccgtacg gctcgaatga cacctgcaaa ccatacccaa ttccaccctg cagtaactgc 600

tctgaaacaa ggactccaaa atgttccaaa agttgcatct ccacttatcc actttctttg 660

aatgaagatc gacattatgg aagcacatat tatcaatttt ggcttggtga aaaatcaatg 720

atgaaagata tttcactata cggacccatt gttgcaggaa tgtcagttta tgaagacttt 780

ttgcattaca aagaaggtgt ttatacacaa gagagtggaa tgtttcttgg aggacacgcc 840

gtaagaatta ttggatgggg tgaacaagac aacattccgt attggcttgt agccaactca 900

tggaatacta catttggtga agacggatta ttcaaaataa gaagaggctt tgacgaatgt 960

ggcattgagt cgtatgtgag tgctggacgt gcaaaagtat aa 1002

<210> 2

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<213>Trichina cathepsin B2

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Met Lys Ile Leu Leu Leu Ser Leu Phe Ala Phe Ala Leu Ala Glu Asn

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Tyr Glu Glu Asn Tyr Glu Arg Leu Lys Val Glu Leu Glu Arg Gln Arg

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Gln Ala Asn Gly Asn Thr Phe Ser Trp Lys Phe Gly Arg Asn Ala Tyr

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Phe Lys Asn Lys Ser Ile Gly Glu Ile Lys Lys Leu Leu Gly Tyr Arg

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Met Leu Pro Lys Thr Val Lys Glu Arg Asn Glu Met Pro Met Pro Glu

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Asp Leu Leu Asn Leu Glu Asn Phe Asn Tyr Pro Val Glu Phe Asp Ser

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Arg Lys His Trp Pro Gln Cys Glu Lys Val Ile Ser Phe Ile Lys Asp

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Gln Ala Asn Cys Gly Ser Cys Trp Ala Val Ser Ser Ala Ser Val Met

115 120 125

Ser Asp Arg Thr Cys Ile Ala Thr Asp Gly Gln Phe Thr Thr Leu Leu

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Ser Asp Ala Glu Leu Leu Ser Cys Cys Thr Ser Cys Gly Tyr Gly Cys

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Asn Gly Gly Tyr Pro Gln Arg Thr Phe Lys Tyr Trp Val Tyr Ser Gly

165 170 175

Met Pro Thr Gly Gly Pro Tyr Gly Ser Asn Asp Thr Cys Lys Pro Tyr

180 185 190

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

195 200 205

Ser Lys Ser Cys Ile Ser Thr Tyr Pro Leu Ser Leu Asn Glu Asp Arg

210 215 220

His Tyr Gly Ser Thr Tyr Tyr Gln Phe Trp Leu Gly Glu Lys Ser Met

225 230 235 240

Met Lys Asp Ile Ser Leu Tyr Gly Pro Ile Val Ala Gly Met Ser Val

245 250 255

Tyr Glu Asp Phe Leu His Tyr Lys Glu Gly Val Tyr Thr Gln Glu Ser

260 265 270

Gly Met Phe Leu Gly Gly His Ala Val Arg Ile Ile Gly Trp Gly Glu

275 280 285

Gln Asp Asn Ile Pro Tyr Trp Leu Val Ala Asn Ser Trp Asn Thr Thr

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Phe Gly Glu Asp Gly Leu Phe Lys Ile Arg Arg Gly Phe Asp Glu Cys

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Gly Ile Glu Ser Tyr Val Ser Ala Gly Arg Ala Lys Val

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<213>TsCPB2 genes maturation zone

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atggcaacac tttctcttgg aaatttggac gaaatgcata tttcaaaaat aagtcaattg 120

gtgaaatcaa aaagctgttg ggctacagga tgttgcctaa gacggtgaaa gagcgaaatg 180

aaatgccaat gccggaagat ttattaaatt tggaaaattt caattatccg gtggaatttg 240

actccagaaa acattggcct cagtgtgaaa aagtgatcag cttcataaaa gatcaagcaa 300

actgtggaag ctgttgggcc gtatccagcg cgtccgtcat gtccgaccgg acgtgcattg 360

ctactgatgg tcaatttacg acattgcttt ccgatgccga acttctttcc tgttgtacat 420

catgcggtta tggttgtaac ggtggatatc ctcagagaac tttcaaatat tgggtgtaca 480

gcggtatgcc aactggtgga ccgtacggct cgaatgacac ctgcaaacca tacccaattc 540

caccctgcag taactgctct gaaacaagga ctccaaaatg ttccaaaagt tgcatctcca 600

cttatccact ttctttgaat gaagatcgac attatggaag cacatattat caattttggc 660

ttggtgaaaa atcaatgatg aaagatattt cactatacgg acccattgtt gcaggaatgt 720

cagtttatga agactttttg cattacaaag aaggtgttta tacacaagag agtggaatgt 780

ttcttggagg acacgccgta agaattattg gatggggtga acaagacaac attccgtatt 840

ggcttgtagc caactcatgg aatactacat ttggtgaaga cggattattc aaaataagaa 900

gaggctttga cgaatgtggc attgagtcgt atgtgagtgc tggacgtgca aaagtataa 959

<210> 4

<211> 21

<212> DNA

<213> TsCPB2-5'GSP outer primer

<400> 4

cggatctgtt gtcctggttg g 21

<210> 5

<211> 21

<212> DNA

<213> TsCPB2-5'GSP inner primer

<400> 5

ctgttgtcct ggttggataa a 21

Claims

1. SEQ ID NO：Trichina cathepsin B gene shown in 1TsCPB2In trichina excretory-secretory antigen is prepared Application.

2. SEQ ID NO：Application of trichina cathepsin B2 shown in 2 in trichina excretory-secretory antigen is prepared.

3. SEQ ID NO：Trichina cathepsin B gene shown in 1TsCPB2Application in trichinosis vaccine is prepared.

4. SEQ ID NO：Application of trichina cathepsin B2 shown in 2 in trichinosis vaccine is prepared.

5. the application according to claim 3 or 4, it is characterised in that be the application in vaccine for pigs trichina disease is prepared.

A kind of 6. preparation method of trichina excretory-secretory antigen, it is characterised in that amplificationTsCPB2The maturation zone of gene is simultaneously It is cloned into the expression vector of His labels, conversion Escherichia coli, after IPTG induced expressions, Ni- affinitive layer purifications TsCPB2 weights Histone, obtain trichina excretory-secretory antigen；It is describedTsCPB2The sequence of gene such as SEQ ID NO：Shown in 1, its maturation zone Sequence such as SEQ ID NO：Shown in 3.

7. the trichina excretory-secretory antigen that claim 6 is prepared.

8. the answering in trichinosis vaccine or trichinosis antibody is prepared of trichina excretory-secretory antigen described in claim 7 With.

A kind of 9. trichinosis vaccine, it is characterised in that be by trichina excretory-secretory antigen described in claim 7 i.e. TsCPB2 recombinant proteins and immunologic adjuvant mixing and emulsifying, obtain TsCPB2 recombinant antigen vaccines.

10. application of the trichinosis vaccine in prevention and control pigs trichina disease described in claim 9.