CN107551265A - A kind of vaccine for pigs trichina disease and its preparation method and application - Google Patents
A kind of vaccine for pigs trichina disease and its preparation method and application Download PDFInfo
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- CN107551265A CN107551265A CN201710685687.0A CN201710685687A CN107551265A CN 107551265 A CN107551265 A CN 107551265A CN 201710685687 A CN201710685687 A CN 201710685687A CN 107551265 A CN107551265 A CN 107551265A
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- tscpb2
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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
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
<400> 1
atgaaaattt tgttgctcag cttgtttgct ttcgctttgg cggaaaatta tgaagaaaat 60
tatgaacgct tgaaagtcga attggaacga caacggcaag ccaatggcaa cactttctct 120
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
<211> 333
<212> PRT
<213>Trichina cathepsin B2
<400> 2
Met Lys Ile Leu Leu Leu Ser Leu Phe Ala Phe Ala Leu Ala Glu Asn
1 5 10 15
Tyr Glu Glu Asn Tyr Glu Arg Leu Lys Val Glu Leu Glu Arg Gln Arg
20 25 30
Gln Ala Asn Gly Asn Thr Phe Ser Trp Lys Phe Gly Arg Asn Ala Tyr
35 40 45
Phe Lys Asn Lys Ser Ile Gly Glu Ile Lys Lys Leu Leu Gly Tyr Arg
50 55 60
Met Leu Pro Lys Thr Val Lys Glu Arg Asn Glu Met Pro Met Pro Glu
65 70 75 80
Asp Leu Leu Asn Leu Glu Asn Phe Asn Tyr Pro Val Glu Phe Asp Ser
85 90 95
Arg Lys His Trp Pro Gln Cys Glu Lys Val Ile Ser Phe Ile Lys Asp
100 105 110
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
130 135 140
Ser Asp Ala Glu Leu Leu Ser Cys Cys Thr Ser Cys Gly Tyr Gly Cys
145 150 155 160
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
290 295 300
Phe Gly Glu Asp Gly Leu Phe Lys Ile Arg Arg Gly Phe Asp Glu Cys
305 310 315 320
Gly Ile Glu Ser Tyr Val Ser Ala Gly Arg Ala Lys Val
325 330
<210> 3
<211> 959
<212> DNA
<213>TsCPB2 genes maturation zone
<400> 3
aaaattatga agaaaattat gaacgcttga aagtcgaatt ggaacgacaa cggcaagcca 60
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 (10)
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.
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Cited By (4)
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CN110734495A (en) * | 2019-09-30 | 2020-01-31 | 吉林大学 | hybridoma cell strains, monoclonal antibody of trichina-resistant serine protease in new larva stage and application |
CN110898216A (en) * | 2019-12-31 | 2020-03-24 | 中国农业科学院兰州兽医研究所 | Application of recombinant trichina thioredoxin peroxidase 2 in preparation of vaccine for resisting trichina infection |
CN111303276A (en) * | 2019-12-20 | 2020-06-19 | 吉林大学 | B cell epitope polypeptide of trichina intestinal cysteine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
CN117721131A (en) * | 2023-12-19 | 2024-03-19 | 吉林大学 | Preparation method and application of recombinant bone morphogenetic protein mutant |
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CN103352047A (en) * | 2013-02-03 | 2013-10-16 | 吉林农业大学 | Trichinella Spiralis larva ES antigen gene vaccine and preparation method |
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CN103352047A (en) * | 2013-02-03 | 2013-10-16 | 吉林农业大学 | Trichinella Spiralis larva ES antigen gene vaccine and preparation method |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110734495A (en) * | 2019-09-30 | 2020-01-31 | 吉林大学 | hybridoma cell strains, monoclonal antibody of trichina-resistant serine protease in new larva stage and application |
CN111303276A (en) * | 2019-12-20 | 2020-06-19 | 吉林大学 | B cell epitope polypeptide of trichina intestinal cysteine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
US11634481B2 (en) | 2019-12-20 | 2023-04-25 | Jilin University | B-cell epitope of Trichinella spiralis cysteine protease inhibitor, hybridoma cell line, monoclonal antibody and uses thereof |
CN110898216A (en) * | 2019-12-31 | 2020-03-24 | 中国农业科学院兰州兽医研究所 | Application of recombinant trichina thioredoxin peroxidase 2 in preparation of vaccine for resisting trichina infection |
CN117721131A (en) * | 2023-12-19 | 2024-03-19 | 吉林大学 | Preparation method and application of recombinant bone morphogenetic protein mutant |
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Application publication date: 20180109 |