CN107400676A - Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application - Google Patents

Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application Download PDF

Info

Publication number
CN107400676A
CN107400676A CN201710581129.XA CN201710581129A CN107400676A CN 107400676 A CN107400676 A CN 107400676A CN 201710581129 A CN201710581129 A CN 201710581129A CN 107400676 A CN107400676 A CN 107400676A
Authority
CN
China
Prior art keywords
pedv
expression
blunt
gene
dual
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710581129.XA
Other languages
Chinese (zh)
Inventor
王隆柏
陈秋勇
吴学敏
王晨燕
周伦江
车勇良
刘玉涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Original Assignee
Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences filed Critical Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
Priority to CN201710581129.XA priority Critical patent/CN107400676A/en
Publication of CN107400676A publication Critical patent/CN107400676A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/20011Coronaviridae
    • C12N2770/20051Methods of production or purification of viral material

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to biology field, and in particular to prokaryotic fusion expression vector dual-gene a kind of M+N of expression Porcine epidemic diarrhea virus (PEDV).By PEDV M and N genetic fragments, using Blunt E2 expression vectors, by seamless clone technology, the dual-gene prokaryotic fusion expression vectors of expression PEDV M+N are constructed, and provide its application in engineered protein recombination expression.The present invention is that directly the M genetic fragments for purifying PEDV are connected in Blunt E2 mammal prokaryotic fusion expression vectors, require no connection in monoclonal carrier PMD19 T, then M+Blunt E2 plasmids pass through homologous recombination technique, seamless spliced N genetic fragments, so as to obtain the dual-gene fusion expression vectors of M+N, its plasmid is identified by round pcr, and the expression that succeeded in cell is transfected again after sequencing is accurate.Therefore, other pronucleus expression technologies are compared in the preparation of the prokaryotic fusion expression vector, have the characteristics that operating procedure is simple, the reaction time is short.

Description

Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application
Technical field
The invention belongs to biology field, and in particular to pronucleus table dual-gene a kind of expression PEDV M+N Up to carrier and its application.
Background technology
Pig epidemic diarrhea (PED) causes swinery vomiting, diarrhoea, dehydration and death by Porcine epidemic diarrhea virus (PEDV) The digestive tract disease viral disease being characterized.The disease in 1971 first Britain report, China confirmed in 1984 the disease In the presence of.Since second half 2010, because traditional PEDV is lacked on gene, inserted and made a variation, the disease is caused Pathogenic enhancing to swinery, substantial amounts of swinery morbidity death is caused, is suffered heavy losses.Current pig-raising countries most of in the world There is the report for the PEDV that morphs, it is especially serious with the morbidity of the country such as China, South Korea, Japan, Thailand and U.S., to pig industry Huge economic loss is caused, and has turned into one of important epidemic disease for influenceing global pig industry sound development.PEDV gene Group leader about in 28000bp or so, mainly contains 4 structural proteins, respectively S, M, N and E protein.At present, seen and passed through PEDV individual gene such as N, S and M gene section piece, gives expression to single-gene albumen, but have no and pass through by prokaryotic expression method PEDV M and N genetic fragments, using pEASY-Blunt-E2 prokaryotic expression carriers, by seamless clone technology, construct PEDV M+Blunt-E2+N dual-gene prokaryotic expression carrier.Therefore, a kind of expression PEDV dual-gene prokaryotic expression carriers of M+N Structure, the diagnostic techniques research to carrying out the disease or other cause of diseases from now on, significant and application prospect.
The content of the invention
It is an object of the present invention to provide a kind of preparation of prokaryotic fusion expression vector dual-gene expression PEDV M+N Method, and the carrier prepared by the preparation method.
Recombinantly expressed it is a further object to provide described prokaryotic fusion expression vector in engineered protein In application.
What the present invention was realized in:
Present invention firstly provides a kind of preparation method of prokaryotic fusion expression vector dual-gene expression PEDV M+N, Comprise the following steps:
(1) according to PEDV M, N gene and the fragment sequence of pEASY-Blunt-E2 expression vector genes, design is with synthesizing Specific primer;
(2) using PEDV cDNA as template, M and N genetic fragments are amplified respectively by round pcr, and reclaim pure Change, the M genetic fragments of purifying are connected on pEASY-Blunt-E2 expression vectors, construct M+Blunt-E2 plasmids;
(3) using M+Blunt-E2 plasmids as template, the genetic fragment of plasmid, recovery purifying M+Blunt-E2 gene pieces are expanded Section;
(4) pEASY-Uni Seamless Cloning and Assembly kit are used, will by seamless clone technology M+Blunt-E2 genes carry out restructuring with N genetic fragments and are connected, and connection product are converted again to Trans 1-T1 competent cells In, be coated on amicillin resistance LB culture mediums cultivated after, extraction M+Blunt-E2+N plasmids carry out sequencing identification, Obtain the dual-gene prokaryotic fusion expression vectors of expression PEDV M+N.
The PEDV preferably makes a variation Porcine epidemic diarrhea virus.
Wherein step (1) described specific primer sequence is as follows:
M:
M-F:ATGTCTAACGGTTTTATTCC
M-R:GACTAAATGAAGCACTTTCT
E2+M:
E2+M-F:AAGGGCCAATTCCTCGAGCAC
E2+M-R:GACTAAATGAAGCACTTTCT
N (includes carrier part homology arm):
N-F:GAGAAAGTGCTTCATTTAGTCGCTTCTGTCAGCTTTCAGGAT
N-R:TGCTCGAGGAATTGGCCCTTATTTCCTGTATCGAAGATCTC。
Secondly, the invention provides the dual-gene pronucleus tables of the expression PEDV prepared by the preparation method M+N Up to carrier.
In addition, present invention also offers prokaryotic fusion expression vector dual-gene described expression PEDV M+N in gene Application in engineered protein recombination expression.
The invention has the advantages that:The present invention is that the M genetic fragments for purifying PEDV directly are connected into Blunt-E2 originals In nuclear expression carrier, it is not necessary to be connected in monoclonal carrier PMD19-T, then M+Blunt-E2 plasmids pass through homologous recombination skill Art, seamless spliced N genetic fragments, so as to obtain the dual-gene fusion expression vectors of M+N.Therefore, the prokaryotic fusion expression vector Other pronucleus expression technologies are compared in preparation, have the characteristics that operating procedure is simple, the reaction time is short.
Brief description of the drawings
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is M gene fragment amplification results.
Fig. 2 is N gene fragment amplification electrophoretograms.
Fig. 3 is the seamless spliced bacterium colony PCR qualification results of M+N fragments.
It is Transetta (DE3) in 37 DEG C and 16 DEG C of induction expression protein results that Fig. 4, which is competent cell used,;In figure Label represents:1 does not induce, 2 supernatants (37 DEG C), 3 precipitations (37 DEG C), 4 supernatants (16 DEG C), 5 precipitations (16 DEG C).
It is Transetta (DE3) in 37 DEG C of induced expression purifying proteins that Fig. 5, which is competent cell used,;Label table in figure Show:Wherein 1 does not induce control;2 supernatants;3 precipitations;4PBS inclusion bodys penetrate liquid;Inclusion body after 5PBS washings;6 urea inclusion bodys Penetrate liquid;7 urea wash after inclusion body.
Fig. 6 is albumen WB electrophoretograms.
Embodiment
Embodiment 1
Need to overcome following technical barrier before implementing the technology of the present invention:
Design of primers, according to expressed gene and the gene order of Blunt-E2 carriers, with reference to homologous recombination principle, if Meter and and synthetic primer.
Biological material source:PEASY-Blunt E2 expression vectors, pEASY-Uni Seamless Cloning and Assembly kit are purchased from Beijing Quanshijin Biotechnology Co., Ltd.
First, according to M the and N genes and Blunt-E2+M of the Porcine epidemic diarrhea virus (PEDV) disclosed in GenBank Plasmid sequence, design synthesize related amplimer, sequence such as table 1 below.
The primer sequence of table 1
2nd, RT reacts:With PEDV (lactation piggys of the Strain from the scale pig farm of Fujian in 2013 generation PEDV Small intestine, by small intestine with PBS liquid grind, draw supernatant, use commercial kit extract RNA) RNA for template, carry out instead Transcription, synthesize cDNA, its system such as table 2 below.
The reverse transcription system of table 2
Reaction condition:25℃10min;50℃30min;85℃5s
3rd, the PCR amplifications of M fragments:Using cDNA as template, enter performing PCR amplification, amplification system such as table 3 below.
The PCR amplification system of the M fragments of table 3
Reaction condition is as follows:
PCR primer is detected by 1.5% agarose gel electrophoresis, obtains purpose fragment such as Fig. 1.
4th, M genetic fragments are connected to E2 carriers
1st, linked system is as follows:2.0 μ l M fragments, 1.0 μ l Blunt-E2 carriers, use ddH2O polishings volume to 5.0 μ L, 25 DEG C of connection 10min;
2nd, connection product is converted into Trans1-T1 competent cells, flicks mixing, ice bath 30min;
3rd, 42 DEG C of heat shock 30s, it is immediately placed on 2min on ice;
4th, balance is added to the LB culture mediums of room temperature, 250rpm, 37 DEG C of culture 1h;
5th, 4000rpm centrifuges 1min, discards part supernatant, retains 100 μ l, and Amp+ resistant panels are applied to after flicking resuspension On, 37 DEG C of overnight incubations;
6th, monoclonal bacterial strain is chosen respectively into 10 μ l sterilized waters, is vortexed and is mixed laggard performing PCR identification, reaction system such as following table 4。
The PCR reaction systems of table 4
Reaction condition is as follows:
7th, electrophoresis detection is carried out using 1.5% Ago-Gel, obtains purpose fragment.Positive bacterium colony is taken to send sequencing company to survey Sequence is accurate, then enters performing PCR amplification using purpose plasmid as template, obtains linear carrier.
5th, the PCR amplifications of M+E2 fragments
Using M+E2 plasmids as template, expanded, amplification system such as table 5 below.
The amplification system of table 5
Reaction system is as follows, detects PCR primer using 1.5% agarose gel electrophoresis, obtains purpose band.
6th, the PCR amplifications of N fragments
Using PEDV cDNA as template, expanded, amplification system such as table 6 below.
The amplification system of table 6
Reaction system is as follows, detects PCR primer using 1.5% agarose gel electrophoresis, obtains purpose fragment
1320bp, such as Fig. 2.
7th, purified fragments
The N fragments of glue reclaim purifying PCR amplifications and the M+E2 fragments of PCR amplifications respectively, are carried out pure using purification kit Change.
8th, it is seamless spliced
1. recommend system to carry out recombining reaction with pEASY-Uni Seamless Cloning and Assembly Kit, Linked system is as follows:2×Assembly Mix 5μl;The μ l of purifying recovery N fragments 2;The μ l of purifying recovery E2+M fragments 3;In PCR instrument In 50 DEG C connection 15min;
2. connection product is totally converted in Trans 1-T1 competent cells;
3.42 DEG C of heat shock 30s, 2min is stood on ice, add in 400 μ l LB culture mediums, 37 DEG C of 200rpm shake bacterium 1h;
4. take the bacterium solution 4000rpm that has shaken to abandon part supernatant after centrifuging 1min, take bacterial strain blow it is outstanding after be applied to resistance containing Amp+ LB flat boards on, the overnight incubation in 37 DEG C of incubators;
Flat board, the competence grown after random picking conversion are taken out after 5.18h from incubator, extraction plasmid carries out bacterium colony PCR identifies positive monoclonal, obtains purpose fragment 1998bp, such as Fig. 3;
6. positive colony is sequenced after shaking bacterium, homology illustrates that splicing restructuring is accurate more than 99%.
9th, big upgrading grain
Take the viable bacteria that correct M+E2+N is sequenced to be transferred after overactivation into LB ammonia benzyl resistance culture bases, put
After entering 37 DEG C of shaking table, 200rpm cultures 16h, bacterium solution extraction plasmid is collected.
Tenth, prokaryotic protein expression
1. M+E2+N genetic transformation Transetta (DE3) competent cell built is taken, on ice placement 30 minutes, 42 DEG C heat shock 45 seconds, after placing 2 minutes on ice plus 37 DEG C of shaking tables of non-resistant LB culture mediums are recovered 30 minutes.
2. the competent cell 5000rpm after recovery is centrifuged 2 minutes, 450 μ l culture mediums are discarded, utilize remaining culture medium Thalline is resuspended and is spread evenly across on the flat board of that resistance of card, is positioned over 37 DEG C of overnight incubations.
3. picking Transetta (DE3) is cloned in each LB culture medium to that resistance of 2ml cards, 37 DEG C of shaking tables shook Night.
4. by 1:100 ratios will shake overnight bacterium solution and be inoculated in 2ml cards that resistance LB culture mediums, the pipe of inoculation 9,37 DEG C of shaking table trainings Support.
5. treating that OD600 values reach 0.5 or so, Transetta (DE3) strain (is added into 0.25 μ g/ml according to 37 DEG C respectively IPTG), 16 DEG C (0.25 μ g/ml IPTG) temperature shaking table cultures.
The bacteriums of 6.37 DEG C of cultures collect bacterium solution in 1.5ml EP pipes after IPTG 4.5 hours is added, 16 DEG C of cultures Bacterium overnight incubation after IPTG is added collects bacterium solution for (about 16 hours), and 5000rpm is centrifuged 3 minutes, and 500 μ l are added after abandoning supernatant 1*PBS be resuspended, be positioned on mixture of ice and water, with broken 10 minutes of 25% power ultrasonic (ultrasound 3 seconds interval 3 seconds), make bacterium Liquid is bright.
7. the bacterium solution 12000rpm after ultrasound is centrifuged 5 minutes, supernatant is moved into new EP pipes, 500 μ l of precipitation 1* PBS is resuspended, and takes the μ l of 50 μ l supernatants 50 to precipitate re-suspension liquid respectively, adds 10 μ l albumen sample-loading buffers, boils 10 minutes.
8. protein sample is run into 12%SDS-PAGE (μ l of applied sample amount 5), carry out examining dye afterwards, obtain express express target protein, Clip size is in 75KD or so such as Fig. 4.
11, the expression of WB verifying purpose albumen again
1. determination of protein concentration, rear inclusion body washing methods is taken to carry out after purification, obtaining clip size to extraction albumen In 75KD or so, such as Fig. 5.Measure protein concentration is 0.55mg/ml.
2. carrying out SDS-PAGE electrophoresis using the glue that resolving gel concentration is 10%, upper sample is 12ug, 200V constant pressure electrophoresis 45min。
3. transferring film:Transferring film, 100V constant pressure transferring films 1h are carried out using pvdf membrane.
4. closing:Use 5% skim milk (0.5%TBST dilutions), 37 DEG C of closing 2h.
5. primary antibody is incubated:Use 0.5%TBST dilution primary antibodies (rabbit-anti hyper-immune serum), dilution ratio 1:4000,4 DEG C of mistakes Night is incubated.
6. washing:Washed 4 times using TBST, each 10min.
7. secondary antibody is incubated:Use 0.5%TBST dilution secondary antibodies (anti-rabbit HRP enzymes), dilution ratio 1:4000, room temperature, which is rocked, incubates Educate 2h.
8. colour developing:Washed 4 times, each 10min, detected using chemiluminescence imaging instrument using TBST.As a result mesh is obtained Fragment, for size in 75KD or so such as Fig. 6, expressing protein has reaction to PEDV rabbit-antis hyper-immune serum, illustrates the albumen of expression With immunogenicity.
Although the foregoing describing the embodiment of the present invention, those familiar with the art should manage Solution, the specific embodiment described by us are merely exemplary, rather than for the restriction to the scope of the present invention, are familiar with this The equivalent modification and change that the technical staff in field is made in the spirit according to the present invention, should all cover the present invention's In scope of the claimed protection.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
atgtctaacg gttttattcc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gactaaatga agcactttct 20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
aagggccaat tcctcgagca c 21
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
gactaaatga agcactttct 20
<210> 5
<211> 42
<212> DNA
<213>Artificial sequence
<400> 5
gagaaagtgc ttcatttagt cgcttctgtc agctttcagg at 42
<210> 6
<211> 41
<212> DNA
<213>Artificial sequence
<400> 6
tgctcgagga attggccctt atttcctgta tcgaagatct c 41

Claims (5)

  1. A kind of 1. preparation method of prokaryotic fusion expression vector dual-gene expression PEDV M+N, it is characterised in that:Including as follows Step:
    (1) it is special with synthesis according to PEDV M, N gene and the fragment sequence of pEASY-Blunt-E2 expression vector genes, design Property primer;
    (2) using PEDV cDNA as template, M and N genetic fragments are amplified respectively by round pcr, and carries out recovery purifying, will The M genetic fragments of purifying are connected on pEASY-Blunt-E2 expression vectors, construct M+Blunt-E2 plasmids;
    (3) using M+Blunt-E2 plasmids as template, the genetic fragment of plasmid, recovery purifying M+Blunt-E2 genetic fragments are expanded;
    (4) pEASY-Uni Seamless Cloning and Assembly kit are used, by seamless clone technology by M+ Blunt-E2 genes carry out restructuring with N genetic fragments and are connected, and connection product are converted again into Trans 1-T1 competent cells, Be coated on amicillin resistance LB culture mediums cultivated after, extraction M+Blunt-E2+N plasmids carry out sequencing identification, obtain The prokaryotic fusion expression vector dual-gene to expression PEDV M+N.
  2. 2. the preparation method of prokaryotic fusion expression vector dual-gene expression PEDV according to claim 1 M+N, it is special Sign is:The PEDV is variation Porcine epidemic diarrhea virus.
  3. 3. the preparation method of prokaryotic fusion expression vector dual-gene expression PEDV according to claim 1 M+N, it is special Sign is:Step (1) described specific primer sequence is as follows:
    M:
    M-F:ATGTCTAACGGTTTTATTCC
    M-R:GACTAAATGAAGCACTTTCT
    E2+M:
    E2+M-F:AAGGGCCAATTCCTCGAGCAC
    E2+M-R:GACTAAATGAAGCACTTTCT
    N:
    N-F:GAGAAAGTGCTTCATTTAGTCGCTTCTGTCAGCTTTCAGGATN-R: TGCTCGAGGAATTGGCCCTTATTTCCTGTATCGAAGATCTC。
  4. 4. pronucleus expression dual-gene expression PEDV prepared by the preparation method as described in claim any one of 1-3 M+N Carrier.
  5. 5. prokaryotic fusion expression vector dual-gene expression PEDV M+N is in engineered protein weight as claimed in claim 4 Application in group expression.
CN201710581129.XA 2017-07-17 2017-07-17 Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application Pending CN107400676A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710581129.XA CN107400676A (en) 2017-07-17 2017-07-17 Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710581129.XA CN107400676A (en) 2017-07-17 2017-07-17 Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application

Publications (1)

Publication Number Publication Date
CN107400676A true CN107400676A (en) 2017-11-28

Family

ID=60400726

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710581129.XA Pending CN107400676A (en) 2017-07-17 2017-07-17 Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application

Country Status (1)

Country Link
CN (1) CN107400676A (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105349562A (en) * 2015-12-18 2016-02-24 江苏省农业科学院 Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105349562A (en) * 2015-12-18 2016-02-24 江苏省农业科学院 Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
任玉鹏: "携PEDV M、N基因的减毒沙门氏菌株构建及其免疫原性研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
吴万军等: "日本血吸虫外分泌蛋白、跨膜蛋白基因筛选与无缝克隆", 《湖北医药学院学报》 *
程向朝等编著: "《动物基因工程》", 31 May 2008, 中国农业出版社 *

Similar Documents

Publication Publication Date Title
CN104725515B (en) One species elastin polypeptide and Coxsackie Adenovirus Receptor fusion protein and its preparation method and application
CN112111006B (en) Antibody for resisting bovine sarcoidosis virus, detection test paper and kit
CN108761076A (en) PEDV immune detections chromatograph test strip and its preparation method and application in milk
CN101016541A (en) Method of producing brucella vaccine antigen protein
CN105755118A (en) Method for quickly detecting vibrio parahaemolyticus by aid of immunomagnetic beads and loop-mediated isothermal amplification processes
CN108059684A (en) A kind of bovine viral diarrhoea recombinant protein, its preparation method and application
CN113698475B (en) Monoclonal antibody of anti-porcine delta coronavirus N protein and porcine delta coronavirus colloidal gold rapid detection test strip
CN111560341B (en) Generic inert vector escherichia coli and potential application thereof
CN103837684A (en) Antibody reagent for rapidly detecting salmonellas and detection method thereof
CN108318686A (en) A kind of bovine coronavirus ELISA detection kit
CN104678097B (en) A kind of mycobacterium tuberculosis combined antigen for diagnosis of pulmonary tuberculosis
CN103103209B (en) Expression and purification method for bombyx mori odorant binding protein (BmOBP2)
CN114409804B (en) Escherichia coli enterotoxin multi-epitope fusion protein and preparation method and application thereof
CN107400676A (en) Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application
CN106701687A (en) Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same
CN108251546A (en) A kind of Forecasting Methodology of lactobacillus plantarum endogenous signal peptides and its application
CN107164408A (en) Express the dual-gene carrier for expression of eukaryon of PEDV M+N
CN107253983A (en) A kind of recombinant protein composition for detecting toxoplasma antibody and preparation method thereof
CN107287237A (en) Express the dual-gene carrier for expression of eukaryon of PEDV S+N
CN108588096B (en) Babesia orientalis spheroid protein gene 4 and protein coded by same
CN108264541A (en) A kind of method of high efficient expression dog circovirus Cap protein
CN102993283B (en) Antigen protein for mycobacterium tuberculosis and application
CN116217714B (en) Full porcine PEDV monoclonal antibody and epitope thereof
CN104788543B (en) A kind of zearalenone antibody analog and its application based on polypeptide
CN116925198B (en) Recombinant protein of microsporidian polar tube protein EcPTP and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20171128

RJ01 Rejection of invention patent application after publication