CN1952158A - Pig A group rotavirus Vp4 whole gene eukaryotic expression plasmid - Google Patents

Pig A group rotavirus Vp4 whole gene eukaryotic expression plasmid Download PDF

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CN1952158A
CN1952158A CNA2005100171985A CN200510017198A CN1952158A CN 1952158 A CN1952158 A CN 1952158A CN A2005100171985 A CNA2005100171985 A CN A2005100171985A CN 200510017198 A CN200510017198 A CN 200510017198A CN 1952158 A CN1952158 A CN 1952158A
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王春凤
王会岩
李玉
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Jilin Agricultural University
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Jilin Agricultural University
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Abstract

The invention belongs to the field of biological gene, disclosing holo-gene eukaryotic expression vector of swine A group rotavirus Vp4, and it is characterized in: amplifying the Vp4 holo-gene using reverse transcription polymerase chain reaction (RT-PCR)RV from cultured RV, open reading frames containing 2331 bp, encoding 776 amino acids, subcloning to pGEM-T vector through T-A cloning technology to obtain pGEM-T-Vp4, recovering it and pVAX1-Vp4 after digesting by KpnI and BamHI,linking using T4 ligase and transforming to construct eukaryotic recombinant expression vector pVAX1-Vp4. Beneficial effects are: injecting the vector into the body through intramuscular injection to induce humoral immunity and cell immunity. Cell immunity submitting the protein expressed intra-cellular through MHC I class to activate CTL and kill target cell, which is very important to rotavirus. The vector is safe and has high efficiency of inducing overall immune reponse. In addition, it is also easy to produce and stable and secure.

Description

The pig A group rotavirus Vp 4 whole gene eukaryotic expression plasmid
Technical field
The invention belongs to field of biological genes, be specifically related to the gene order of pig A group rotavirus Vp 4 whole gene, clone, structure eukaryon expression plasmid and the expression in eukaryotic cell of the full gene of Vp4.
Background technology
(Rotavirus is to cause one of main pathogen that infant and various young animal non-bacterial are suffered from diarrhoea RV) to rotavirus.RV diarrhoea is a kind of Pandemic infection disease.From Mebus in 1969 etc. isolated rotavirus (RV) first from calf diarrhea since, people found that gradually this virus is to cause infant and young animal diarrhoea and the main pathogens that causes death and die.The infection that rotavirus causes causes nearly 1,000,000 infant's death every year in developing country.A group RV causes the cacatory most important cause of disease of whole world infant, in developing country, and annual generation moderate and about 18000000 people of cacatory children, 350000～390000 people that surpass that die from rotavirus infection; In developed country, be children's common disease of being in hospital because of, medical expense height because RV diarrhoea causes dewatering, the annual directly medicine of the U.S. is with a toll of 200,000,000 6 thousand ten thousand dollars according to estimates.In November, 2004, Guangzhou rotavirus day is infected nearly 1000 people.In February, 2005, Bangladesh has 190000 people and infects rotavirus, wherein 32 people's death.In animality diarrhoea, the normal infection morbidity of birth animal such as piglet, hoggerel, horse, dog, cat, rabbit, ox, chicken, squab turkey, Yokogawa monkey, mouse [10]After nineteen eighty-two, China was separated to the buffalo rotavirus first, find that in succession the RV of pig, ox, sheep, dog, rabbit and multiple bird infects report, and found or isolation identification virus.According to the investigation to rotavirus antibody in East China ox, the porcine blood serum, the antibody positive rate of milk cow, ox and swinery reaches 85.4%, 83.0%, 68.3% respectively.Show that China's animal rotavirus infection is more general, infection rate is also very high.Rotavirus is mainly encroached on children livestock and poultry in age, and the piglet rotavirus infection of 15 ~ 30 ages in days is the most general; The age of onset of ox does not wait to 60 ages in days from being born, but the highest with 15 ~ 40 age in days sickness rate; The vulnerable period of rabbit and chicken is 30 ~ 70 ages in days and 7 ~ 30 ages in days.Adult animals also has susceptibility to rotavirus.Piglet sharply descends because of diarrhoea makes immunity of organisms after infecting PRV, and easily the secondary various bacterial infects, and causes the piglet survival rate to descend survivor's poor growth or stagnation.In recent years, along with the increase of the quantity of raising pigs, the popular scope of this disease constantly enlarges, and sickness rate constantly increases.Its sickness rate reaches as high as 80%, 14 age in days can reach 100% with the case fatality rate of interior newborn piglet, and China is the big country of raising pigs, and the piglet survival rate is low to have caused serious economy loss to pig industry.It is generally acknowledged that RV only causes the Digestive tract infringement, but the RV infection that studies show that in recent years can cause many organ injuries, viremia is the approach that the RV multisystem is sent out.Behind the zoogenetic infection RV production and growth there are considerable influence, cause serious economy loss.The improvement of environmental health and water sanitary condition can not reduce the frequency of disease development of rotavirus.Owing to still there is not the specific medicament of treatment rotavirus at present, therefore prevention seems particularly important.For this reason, the scientist that repeatedly calls upon States of the World Health Organization develops effective Rotavirus Vaccine, and it is classified as one of vaccine project of override development.Point out that vaccine prevention is only the unique effective means of containment rotavirus diarrhea.Therefore think the generation of control rotavirus disease, must set about from the development of vaccine.Longitudinal research shows, no matter developed country or developing country, the superinfection of infant's rotavirus is all very common, especially in postnatal initial several years.Compare with primary infection, infection symptoms is generally gentle once more, perhaps is symptomless infection, and this result of study has been affirmed with the vaccine immunity baby can prevent severe rotavirus disease.According to estimates, if use effective vaccine, the annual life that can save 500000 ~ 1000000 children in the whole world.Vaccine prevention is this sick optimal selection of control.Though current attenuated vaccine plays an important role in the generation of this disease of prevention, to compare with nucleic acid vaccine, it still exists the unstable relatively of attenuated vaccine, easily reversion, defectives such as latent infection.Along with molecular biological further investigation, the appearance of Novel DNA vaccine provides a brand-new approach for the diagnosis and the prevention of porcine rotavirus.Dna vaccination is a kind of new generation vaccine that began to develop rapidly from 1992, is considered to third generation vaccine.The advantage of dna vaccination is that dna vaccination can stimulate body to produce persistent humoral immunization and cellular immunization effectively, and demonstrate it as the 3rd generation vaccine superiority.Traditional subunit vaccine can not induce body to produce cellular immunization.And dna vaccination passes through MHCI quasi-molecule antigen-presenting at the proteantigen of cell inner expression, thereby activates CTL and kill target cell.This point is particularly important to rotavirus.Dna vaccination also has the security of recombinant subunit vaccine and the high efficiency of inducing comprehensive immunne response of attenuated live seedling concurrently simultaneously.In addition, dna vaccination also is easy to produce, and stability is strong and safe and reliable.
Summary of the invention
The objective of the invention is:
A kind of genetic engineering technique that utilizes is provided, the coat protein VP4 genetic expression of RV is come out to do antigen, produce the pig A group rotavirus Vp 4 whole gene eukaryotic expression plasmid.
Technical scheme of the present invention is:
With the full gene of inverse transcription polymerase chain reaction (RT-PCR) technology amplification Vp4, open reading frame contains 2331bp, 776 amino acid of encoding from the RV that cultivates.By the T-A clone technology, the PCR product cloning to cloning vector pGEM-T carrier, is obtained recombinant clone plasmid pGEM-T-Vp4.Recombinant clone plasmid pGEM-T-Vp4 and expression vector pVAX1 all with KpnI and BamHI double digestion, after purifying reclaims, are connected, transform with the T4DNA ligase enzyme, make up eukaryotic expression recombination plasmid pVAX1-Vp4.
From the RV that cultivates,,, the PCR product cloning to cloning vector pGEM-T carrier, is obtained recombinant clone plasmid pGEM-T-Vp4 by the T-A clone technology with the full gene of inverse transcription polymerase chain reaction (RT-PCR) technology amplification Vp4;
Through DNASTAR software analysis nucleotide sequence and aminoacid sequence be:
1 atg gct tcg ctc att tat aga caa cta ctt act aat tca tac aca gtc
1 Met Ala Ser Leu Ile Tyr Arg gln Leu Leu Thr Asn Ser Tyr Thr Val
49 aat ctt tct gac gaa att caa gag att gga tca gct aag tca cag gat
17 Asn Leu Ser Asp Glu Ile Gln Glu Ile Gly Ser Ala Lys Ser Gln Asp
97 gtt act ata aat cct ggt cca ttc gca cga acs ggt tat gca cca gtt
33 Val Thr Ile Asn Pro Gly Pro Phe Ala Arg Thr Gly Tyr Ala Pro Val
145 aat tgg gga gca ggt gag act aat gac tcc aca act gtc gag ccg tta
49 Asn Trp Gly Ala Gly Glu Thr Asn Asp Ser Thr Thr Val Glu Pro Leu
193 tta gat ggt cca tac caa cca acc act ttc aat cca cca aca agc tat
65 Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Asn Pro Pro Thr Ser Tyr
241 tgg gta cta ctt gcg cca act gta gag ggc gta att att caa gga aca
81 Trp Val Leu Leu Ala Pro Thr Val Glu Gly Val Ile Ile Gln Gly Thr
289 aac aat acc gat aga tgg ttg gcc act ata cta att gaa cca aac gta
97 Asn Asn Thr Asp Arg Trp Leu Ala Thr Ile Leu Ile Glu Pro Asn Val
337 caa aca act aac aga ata tac aat ctt ttt ggt cag caa gta act tta
113 Gln Thr Thr Asn Arg Ile Tyr Asn Leu Phe Gly Gln Gln Val Thr Leu
385 tcg gtg gag aat acg tca cag aca caa tgg aag ttc att gat gtg agt
129 Ser Val Glu Asn Thr Ser Gln Thr Gln Trp Lys Phe Ile Asp Val Ser
433 aca act acg cca aca gga agt tat acg cag cac gga cca ttg ttc tct
145 Thr Thr Thr Pro Thr Gly Ser Tyr Thr Gln His Gly Pro Leu Phe Ser
481 aca cca aaa tta tac gct gta atg aaa ttc agt ggt aga ata tat aca
161 Thr Pro Lys Leu Tyr Ala Val Met Lys Phe Ser Gly Arg Ile Tyr Thr
529 tat aat gga acc aca cca aac gca aca aca gga tac tat tca act act
177 Tyr Asn Gly Thr Thr Pro Asn Ala Thr Thr Gly Tyr Tyr Ser Thr Thr
577 aat tat gac aca gta aat atg aca tca ttt tgt gat ttt tat att ata
193 Asn Tyr Asp Thr Val Asn Met Thr Ser Phe Cys Asp Phe Tyr Ile Ile
625 cca aga aat caa gaa gaa aaa tgt act gag tat atc aat cat gga tta
209 Pro Arg Asn Gln Glu Glu Lys Cys Thr Glu Tyr Ile Asn His Gly Leu
673 cct cct ata caa aat aca agg aat gtt gtg cca gta tct tta tcg gct
225 Pro Pro Ile Gln Asn Thr Arg Asn Val Val Pro Val Ser Leu Ser Ala
721 aga gag ata gtg cac aca aga gct caa gtt aat gag gat att gtt gtt
241 Arg Glu Ile Val His Thr Arg Ala gln Val Asn glu Asp Ile Val Val
769 tca aaa act tca ctt tgg aaa gaa atg caa tgc aac aga gac ata acc
257 Ser Lys Thr Ser Leu Trp Lys Glu Met Gln Cys Asn Arg Asp Ile Thr
817 ata aga ttc aaa ttt gat aga aca att att aaa gct gga gga tta gga
273 Ile Arg Phe Lys Phe Asp Arg Thr Ile Ile Lys Ala Gly Gly Leu Gly
865 tac aaa tgg tca gaa ata tct ttt aag cca att act tat cag tac aca
289 Tyr Lys Trp Ser Glu Ile Ser Phe Lys Pro Ile Thr Tyr Gln Tyr Thr
913 tac gct aga gat gga gaa caa att aca gcg cac acc aca tgc tca gtt
305 Tyr Ala Arg Asp Gly Glu Gln Ile Thr Ala His Thr Thr Cys Ser Val
961 aat gga gtt aac aat ttt agt tac aat ggc ggt tcg ctg ccg acg gac
321 Asn Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp
1009 ttc gcc ata tca aga tat gaa gtg att aaa gaa aat tca ttt gtt tac
337 Phe Ala Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Phe Val Tyr
1057 att gat tac tgg gat gat tca caa gca ttc aga aac atg gta tat gtt
353 Ile Asp Tyr Trp Asp Asp Ser Gln Ala Phe Arg Asn Met Val Tyr Val
1105 cgg tca ctt gct gct aat ttg aat aca gta acg tgt act ggt ggt agt
369 Arg Ser Leu Ala Ala Asn Leu Asn Thr Val Thr Cys Thr Gly Gly Ser
1153 tac agc ttc gtg ttg cct tta ggt ggt tat cca gtt atg act ggt ggt
385 Tyr Ser Phe Val Leu Pro Leu Gly Gly Tyr Pro Val Met Thr Gly Gly
1201 aca gtt tca cta cac cca gct gga gtt aca tta tct acc caa ttc act
401 Thr Val Ser Leu His Pro Ala Gly Val Thr Leu Ser Thr Gln Phe Thr
1249 gat ttc gta tct ctt aat tca tta cgc ttt aga ttc agg tta act gta
417 Asp Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Arg Leu Thr Val
1297 gga aga acc ttc att ttc tat aac gag gac tag ggt aag tag tta tac
433 Gly Glu Pro Ser Phe Ser Ile Thr Arg Thr Arg Val Ser Arg Leu Tyr
1345 gga ctt cca gca gct aat cca aac aac caa aga gaa tat tat gaa ata
449 Gly Leu Pro Ala Ala Asn Pro Asn Asn Gln Arg Glu Tyr Tyr Glu Ile
1393 agc ggt agg ttt tca tta ata tca tta gtg cca tca aat gat gat atg
465 Ser Gly Arg Phe Ser Leu Ile Ser Leu Val Pro Ser Asn Asp Asp Tyr
1441 caa aca cca att atg aat tca gtt act gtg agg caa gat cta gag aga
481 Gln Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg
1489 caa ttg gga gag cta cga gat gag ttc aat tca ttg tca cag caa ata
497 Gln Leu Gly Glu Leu Arg Asp Glu Phe Asn Ser Leu Ser Gln Gln Ile
1537 gcg ata tca caa ttg att gat ctg gct cta ttg cca tta gat atg ttt
513 Ala Ile Ser Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe
1585 tca atg ttc tca gga att aaa agt aca ata gat gct gcg aaa tcc atg
529 Ser Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Ala Ala Lys Ser Met
1633 gca acg aac gtg atg aaa aga ttt aaa cgg tca aac cta gcc agc tca
545 Ala Thr Asn Val Met Lys Arg Phe Lys Arg Ser Asn Leu Ala Ser Ser
1681 gtt tct aca tta act gat gca atg tct gat gca gca tcg tct att tca
561 Val Ser Thr Leu Thr Asp Ala Met Ser Asp Ala Ala Ser Ser Ile Ser
1729 aga agt tca tca ata cga tca ata gga tca tca gca tcc gct tgg acg
577 Arg Ser Ser Ser Ile Arg Ser Ile Gly Ser Ser Ala Ser Ala Trp Thr
1777 gaa gtg tca aat tcg atc gca gat gtt tct acg aca gtt gat aca gta
593 Glu Val Ser Asn Ser Ile Ala Asp Val Ser Thr Thr Val Asp Thr Val
1825 tca acg caa act gct act att gct aag cgg tta cga ttg aaa gag att
609 Ser Thr Gln Thr Ala Thr Ile Ala Lys Arg Leu Arg Leu Lys Glu Ile
1873 gca act caa acc gat ggt atg aat ttt gat gac ata tct gca gca gtg
625 Ala Thr Gln Thr Asp Gly Met Asn Phe Asp Asp Ile Ser Ala Ala Val
192l tta aaa acg aaa att gac aaa tcc gtg caa ata aca cca aat aca tta
641 Leu Lys Thr Lys Ile Asp Lys Ser Val Gln Ile Thr Pro Asn Thr Leu
1969 ccg gaa ata gtt act gaa gct tct gag aaa ttt atc cca aat aga aca
657 Pro Glu Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Asn Arg Thr
2017 tat agg gtt att aac aat gat gaa gta ttt gaa gct gga atg gat ggt
673 Tyr Arg Val Ile Asn Asn Asp Glu Val Phe Glu Ala Gly Met Asp Gly
2065 aag ttt ttc gca tat cgt gta gac aca ttc gat gaa ata cca ttt gat
689 Lys Phe Phe AlT yrA rgV alA spT hrP heA spG luI leP roP heg Asp
2113 gta cag aaa ttc gca gat tta gtt aca gat tca ccg tca ttt cta gca
705 Val Gln Lys Phe Ala Asp Leu Val Thr Asp Ser Pro Val Ile Ser Ala
2161 ata att gat ctt aag acg tta aaa aat ctg aaa gat aat tat gga ata
72l Ile Ile Asp Leu Lys Thr Leu Lys Asn Leu Lys Asp Asn Tyr Gly Ile
2209 agt aag cag caa gct ttt ggc tta cta cga tca gat cca agg gta tta
737 Ser Lys Gln Gln Ala Phe Gly Leu Leu Arg Ser Asp Pro Arg Val Leu
2257 cgt gaa ttt atc aat cag aat aat cca ata ata cga aat aga att gaa
753 Arg Glu Phe Ile Asn Gln Asn Asn Pro Ile Ile Arg Asn Arg Ile Glu
2305 aac tta atc atg caa tgt aga ctg taa
769 Asn Leu Ile Met Gln Cys Arg Leu ***
Concrete steps are as follows:
Target gene PCR amplification and clone
According to the gene order of (X13190) porcine rotavirus Wa strain Vp4 among the GenBank, design the PCR primer of a pair of this gene of amplification: P 1: 5 '-GTA GTCGACATGGCTTCGCTCATTTATAGA-3 '; P 2: 5 '-CCT GGTACCTTACAGTCTACATTGCATGATTA-3 ' (the line sequence is respectively SaLI, KpnI restriction enzyme site).Press the Trizol kit method and extract the total RNA of porcine rotavirus; With total RNA is template, with P 1, P 2Be primer, the Vp4 gene is carried out the RT-PCR amplification.50 μ l reverse transcription reaction systems and reaction conditions are as follows: get 10 μ l mRNA, 75C heats 5min, makes the double-stranded RNA sex change, place cooled on ice rapidly after, add 5 * AMV Buffer, 10.0 μ l again; DNTPs 4.0 μ l (2.5mM each); RNasin 1 μ l; Downstream primer 0.5 μ l; AMV (5Units/ μ l) 0.5 μ l; Olig (dt) 1.0 μ l; MgCl 2(25mM) 4.0 μ l; Add DEPC water 19.0 μ l.42C reverse transcription 1h, 95C boil 5min deactivation AMV ThermoScript II.50 μ l PCR reaction systems and reaction conditions are as follows: the 0.5ml EP pipe that the DEPC that learns from else's experience handles adds 10 * PCR Buffer, 5.0 μ l in 50 μ l reaction systems; DNTP (2.5mM) 8.0 μ l; Upstream primer 1 μ l; Downstream primer 1 μ l; CDNA 5 μ l; RNasin 1 μ l; MgCl 22.0 μ l; EX-Taq 0.5 μ l; DEPC treating water 26.5 μ l.Reaction conditions is the pre-sex change 8min of 94C; 94C sex change 1.5min, the 45C 1min that anneals, 72C extends 3.5mins, totally 35 cycles; 72C extends 10min.After the PCR product is purified, be connected among the pGEM-T Vector, and be converted among the competence E.coli JM109 with the T4 dna ligase.Identify recombinant clone by plasmid extraction, restriction analysis and pcr amplification, obtain recombinant plasmid pGEM-T-Vp4.
1, the structure of recombinant expression plasmid
Recombinant clone plasmid pGEM-T-Vp4 and expression vector pVAX1 are all with KpnI and BamHI double digestion, after purifying reclaims, with T4DNA ligase after room temperature connects 2h, 4 ℃ are spent the night, 10ul ligation system is as follows: pVAX1 1.0ul, Vp4 2.0ul, 2 * ligation Buffer 5.0ul, T4DNAligase 1.0ul, D.D.W 1.0ul.
2, the screening of recombinant expression plasmid
5ul is connected product be transformed among the competence E.coli DH5a, converted product is coated LB, and (Kan is 50ug/ml) in the agar plate.Extract plasmid in a small amount with alkaline lysis, carry out 1% agarose gel electrophoresis analysis.The plasmid of choosing hysteresis is with KpnI, and BamHI double digestion and PCR identify recombinant expression plasmid.
Eukaryotic expression recombination plasmid pVAX1-Vp4 implementation step is as follows:
1, a large amount of extractions of recombinant expression plasmid
The positive strain that is accredited as recombinant expression plasmid is inoculated in 150ml contains in the LB substratum of Kan, 37 ℃ of shaken overnight are extracted recombinant expression plasmid in a large number with alkaline lysis, and carry out purifying with the PEG method.
2, the evaluation of the quantitative and purity of plasmid
Measure 260nm and the 280nm optical density value of extracting plasmid with ultraviolet spectrophotometer.By experimental formula dna content (ug/ml)=OD 260* 50ug/ml calculates the content of plasmid DNA, with D 260/ OD 280Ratio in the 1.8-2.0 scope, and agarose gel electrophoresis detects and not see the RNA band, shows that the plasmid purity of being extracted is higher, can be used for cell transfecting.
3, eukaryotic expression recombination plasmid transfection MA-104 cell
Eukaryotic expression recombination plasmid transfection MA-104 cell is pressed Lipofectamine TM2000 working instructions carry out, concrete operations are as follows: (1) in transfection the day before yesterday with the MA-104 cell with under the trysinization, insert in the Tissue Culture Plate (6 orifice plate), every hole adds 2mlRPMI1640 growth media (not adding two anti-), and 5% CO2gas incubator is cultivated 24h for 37 ℃.Set up the recombinant expression plasmid group separately, pVAXI control group and normal cell control group.Attached cell accounted for surface-area about 80% in the culture dish bottom when (2) cell concentration of Jie Ruing can make transfection, a general available cover glass (use in advance soaked in absolute ethyl alcohol a couple of days) is put at the bottom of the cell cultures plate hole, need to shine a couple of days under ultraviolet, the purpose strictness is aseptic, and the general cell that inserts is 1 * 10 5-2 * 10 5Individual cell.(3) add the 10ul liposome in the unparalleled anti-RPMI1640 growth media of 250ul serum-free, mixing acts on 5min gently.(4) get adding 4ug recombinant plasmid in the unparalleled anti-RPMI1640 growth media of 250ul serum-free in addition, mixing acts on 5min gently.(5) be added drop-wise in 3 mixing slowly, room temperature 30min with 4.(6) add in the cultured Tissue Culture Dish cotransfection with 5.Add 1.5ml after 6 hours and contain 8% serum, 2% pair of anti-RPMI1640 growth media.5% CO2gas incubator is cultivated 18-42h for 37 ℃.
The invention has the beneficial effects as follows:
It can stimulate body to produce persistent humoral immunization and cellular immunization by intramuscular injection by certain amount in body effectively, and cellular immunization is in cell expressed proteins antigen to be passed through MHCI quasi-molecule antigen-presenting, thereby activates CTL and kill target cell.This point is particularly important to rotavirus.Its not only safety but also the high efficiency of inducing comprehensive immunne response is arranged simultaneously.In addition, it also is easy to produce, and stability is strong and safe and reliable.The research of dna vaccination of the present invention is by with pVAXI-Vp4 immunity BALB/c mouse, detects lymphopoiesis situation and CD4 in its serum antibody and the spleen +, CD8 +The number change situation of T cell, result show that pig A group rotavirus Vp 4 whole gene has not only obtained expression in mammalian cell, and after giving animal immune with eukaryotic expression recombination plasmid pVAXI-Vp4, have obtained immune effect.This provides scientific basis for further applying of pVAXI-Vp4 DNA nucleic acid vaccine.
The present invention is to the laboratory animal immunity, blood specimen collection, and splenocyte is handled: the BLAB/c mouse is divided into A at random, B, three groups of C, 30 every group.To prepare in a large number and process satisfactory eukaryotic expression recombination plasmid pVAXI-Vp4 of detection and carrier for expression of eukaryon pVAXI dilution with sterile saline, every kind of plasmid final concentration is 1ug/ul.PROCAINE HCL, PHARMA GRADE joins in the plasmid by final concentration 0.5%.Each group mouse is carried out injecting immune, every pleural muscle meat injection 50ul.A group injection pVAX1-Vp4; B group injection pVAX1; C organizes injecting normal saline.By with quadrat method interval two all booster immunizations, be total to immunity three times later on.Get 5 mouse orbit veins before the immunity at random and get blood, collect blood sample, extracting spleen cell is put to death in dislocation, carries out CD4 in serum antibody and the splenocyte respectively +, CD8 +T cell relative populations detects.The immunity back is in the 14th, 28, and each group was got 5 mouse respectively at random in 42 days, uses with quadrat method and collects blood sample and handle splenocyte.
The OD of the anti-RV antibody of serum to be checked is surveyed in antibody test with the ELISA method 492Value.Being cushioned liquid with bag wraps RV antigen by Sptting plate by the dilution of 160ug/ hole.After serum to be checked done dilution in 1: 200 with PBS, every hole 100ul, the HRP sheep anti-mouse igg is diluted to working concentration with PBS, every hole 100ul.Operation according to a conventional method.The rotavirus liquid of 200ml with the MA-104 cell cultures is got in the preparation of wheel virus antigen, and 5000 leave heart 30min, get supernatant 20000 and leave heart 1h.Precipitation is dissolved in the physiological saline of 0.5ml.
Content with the spectrophotometric determination rotavirus protein calculates OD by following formula 280* 1.45-OD 260* 0.74=protein content (mg/ml)
Being cushioned liquid with bag wraps RV antigen by Sptting plate by the dilution of 160ug/ hole.After serum to be checked done dilution in 1: 200 with PBS, every hole 100ul, the HRP sheep anti-mouse igg is diluted to working concentration with PBS, every hole 100ul.Operation according to a conventional method.
Measure the absorbance value (OD in each hole at wavelength 492 places with automatic enzyme mark determinator 492Value).With the SAS statistical software data are handled.
Aseptic separation splenic lymphocyte is tested in the T lymphocyte transformation, measures proliferation of lymphocytes with mtt assay, with the intravital cellular immune level of reflection immunized mice.
Mouse is put to death in dislocation in 42 days, puts and soaks 1-2min in 75% ethanolic soln with sterilization skin.Asepticly get the gauze that spleen places sterilization, rub gently, make splenocyte suspension.
Splenocyte counting, viable count are more than 90%, and adjusting cell concn is 5 * 10 6Individual/ml.Every hole adds 100ul splenocyte suspension, totally 8 holes.Wherein 4 holes add 100ulConA, and other 4 holes add the 100ulRPMI1640 growth media and do blank.
Tissue Culture Plate is put into 37 ℃, 5%CO 2Hatch 68h in the incubator.
Every hole adds 20ul MTT, continues to cultivate 4h.
The careful suction removed nutrient solution, and every hole adds 100ulDMSO, and vibration 10min measures OD on enzyme-linked immunosorbent assay instrument 570, the calculating stimulation index (stimulation index, SI), SI=OD 570Experimental group/OD 570Control group.Analyze with Spss 11.0.
CD4 +CD8 +T cell detection splenocyte sample send basic courses department of No.1 Hospital of Jilin Univ. in the 2h after collection, detect CD4 in 10000 splenocytes with flow cytometer (FACSCalibur) +, CD8 +The relative populations of T cell (%).With the SAS statistical software data are handled.
Antibody test
ELISA antibody test result after the recombinant expression plasmid pVAXI-Vp4 immunity shows, A group mouse serum is exempted from the back at head and can be detected anti-RV positive antibody (P/N＞2.0) on the 14th day, detected anti-RV positive antibody (P/N＞2.0) later on respectively at the 28th, 42 day, illustrate that immunity back antigen gene has obtained expression in BLAB/c mouse body, the antigen gene of expression induces mouse to produce humoral immunoresponse(HI).The results are shown in Table 1.
Antibody response after the table 1 recombinant expression plasmid pVAX1-Vp4 immunity
Table1：Antibody responses in mice after vaccination withrecombinant plasmid pVAXI-Vp4
Detection time (Days) Group (Groups)
Group A Group B Group C
0 14 28 42 0.118±0.008 A 0.498±0.012 A 0.642±0.026 A 0.825±0.018 A 0.118±0.008 A 0.240±0.027 B 0.270±0.016 B 0.210±0.014 B 0.118±0.008 A 0.230±0.024 B 0.260±0.008 B 0.220±0.012 B
Annotate: there were significant differences for A and B, and B and B difference are not remarkable.
The lymphocyte transformation experiment
Lymphocyte transformation test is a machines somatocyte immune functional state index commonly used.Aseptic separating immune mouse spleen lymphocyte stimulates lymphopoiesis with ConA as stimulant.Experimental result shows that there is significant difference (p＜0.05) in the lymphproliferation response of Vp4 group and pVAXI and physiological saline control group. this point and flow cytometry are to the CD4 of experimental group +The detected result of T cell quantity is the same.
Table 2 immunized mice lymphocyte transformation result and analysis
Table 2：Results and analysis of lymphocyte transformation test of
the immunized mice
Immune animal Group Sample number Stimulation index (SI)
Mouse mouse mouse Vp4 pVAX I physiological saline 10 10 10 1.426±0.0894 b 1.185±0.0603 cd 0.9476±0.1892 d
Annotate: a, b, c, the different person's significant differences of d letter, identical person is not remarkable.CD4+, the CD8+T cell detection
After the first immunisation, CD4 in the A group mouse spleen +, CD8 +T cell quantity and B, C group compares that all there were significant differences (P＜0.05), and apparently higher than B, C group, illustrate immunity afterwards antigen gene in the mouse body, obtained expression, CD4 +T cell and CD8 +Amplification has taken place in the T cell after effectively activating.See Table 3 and table 4.
CD4 in the splenocyte after the table 3 recombinant expression plasmid pVAXI-Vp4 immunity +The quantity of T cell (%) changes
Table 3 Alteration of CD4 +T Lymphocytes in Spleen of mice after
vaccination with recombinant plasmid pVAXI-Vp4
Detection time Days Group Groups
Group A Group B Group C
0 14 28 42 23.452±0.925 A 40.368±0.687 A 42.834±0.832 BC 49.322±3.854 A 23.452±0.925 A 21.796±1.125 B 30.596±1.412 C 30.124±3.246 B 23.452±0.925 A 21.832±1.264 B 31.082±1.316 C 31.686±1.436 B
(annotate: there were significant differences for A and B in the table, and B, C and C do not have significant difference)
CD8 in the splenocyte after the table 4 recombinant expression plasmid pVAXI-Vp4 immunity +The quantity of T cell (%) changes
Table 4：Alteration of CD8 +T Lymphocytes in Spleen of mice after
vaccination with recombinant plasmid pVAX1-Vp4
Detection time Days Group Groups
Group A Group B Group C
0 14 28 42 11.232±0.865 A 17.813±0.237 A 18.363±1.422 A 19.145±0.364 A 11.232±0.865 A11.486±1.254 B12.374±1.062 A12.556±0.876 A 11.232±0.865 A11.396±1.326 B12.706±0.654 A12.412±0.932 A
(annotate: there were significant differences for A and B in the table, and B, C and C do not have significant difference)
Description of drawings
Fig. 1 is a construction of eukaryon expression plasmid for expressing collection of illustrative plates of the present invention.
Embodiment
Embodiment 1:
One, the clone and the sequential analysis of the full gene of pig A group rotavirus Wa strain Vp4
1.Vp4 the clone and the amplification of full gene
According to the gene order of (X13190) porcine rotavirus Wa strain Vp4 among the GenBank, design the PCR primer (synthetic) of a pair of this gene of amplification: P by Dalian TaKaRa company 1: 5 '-GTAGTCGACATGGCTTCGCTCATTTATAGA-3 '; P 2: 5 '-CCTGGTACCTTACAGTCTACATTGCATGATTA-3 ' (the line sequence is respectively SaLI, KpnI restriction enzyme site).
Press the Trizol kit method and extract the total RNA of porcine rotavirus; With total RNA is template, with P 1, P 2Be primer, the Vp4 gene is carried out the RT-PCR amplification.50 μ l reverse transcription reaction systems and reaction conditions are as follows: get 10 μ l mRNA, 75C heats 5min, makes the double-stranded RNA sex change, place cooled on ice rapidly after, add 5 * AMV Buffer, 10.0 μ l again; DNTPs 4.0 μ l (2.5mM each); RNasin 1 μ l; Downstream primer 0.5 μ l; AMV (5Units/ μ l) 0.5 μ l; Olig (dt) 1.0 μ l; MgCl 2(25mM) 4.0 μ l; Add DEPC water 19.0 μ l.42C reverse transcription 1h, 95C boil 5min deactivation AMV ThermoScript II.50 μ l PCR reaction systems and reaction conditions are as follows: the 0.5ml EP pipe that the DEPC that learns from else's experience handles adds 10 * PCR Buffer, 5.0 μ l in 50 μ l reaction systems; DNTP (2.5mM) 8.0 μ l; Upstream primer 1 μ l; Downstream primer 1 μ l; CDNA 5 μ l; RNasin 1 μ l; MgCl 22.0 μ l; EX-Taq 0.5 μ l; DEPC water 26.5 μ l.Reaction conditions is the pre-sex change 8min of 94C; 94C sex change 1.5min, the 45C 1min that anneals, 72C extends 3.5mins, totally 35 cycles; 72C extends 10min.After the PCR reaction finishes, get 5 μ lPCR amplified production electrophoretic analysiss on 1% sepharose.
2. sequential analysis
After the PCR product is purified, be connected among the pGEM-T Vector, and be converted among the competence E.coli JM109 with the T4 dna ligase.Identify recombinant clone by plasmid extraction, restriction analysis and pcr amplification, obtain recombinant plasmid pGEM-T-Vp4.Deliver to Dalian TaKaRa company and check order, and use DNASTAR software and analyze.
Two, the structure of eukaryon expression plasmid, the expression in the eukaryotic cell
1. the structure of eukaryon expression plasmid and evaluation
Recombinant clone plasmid pGEM-T-Vp4 and expression vector pVAX1 are all with KpnI and BamHI double digestion, after purifying reclaims, with T4DNA ligase after room temperature connects 2h, 4 ℃ are spent the night, 10ul ligation system is as follows: pVAX1 1.0ul, Vp4 2.0ul, 2 * ligation Buffer 5.0ul, T4DNAligase 1.0ul, D.D.W 1.0ul.
5ul is connected product be transformed among the competence E.coli DH5a, converted product is coated LB, and (Kan is 50ug/ml) in the agar plate.Extract plasmid in a small amount with alkaline lysis, carry out 1% agarose gel electrophoresis analysis.The plasmid of choosing hysteresis is with KpnI, and BamHI double digestion and PCR identify recombinant expression plasmid.
The qualification result of recombinant expression plasmid
Extract plasmid in a small amount with alkaline lysis, analyze by the plasmid size, restriction enzyme KpnI/BamHI restriction analysis, pcr amplification is identified positive recombinant expression plasmid.The result has obtained the pVAX1 carrier segments of about 3000bp and the Vp4 target gene fragment of 2331bp.Pcr amplification has also obtained the Vp4 target gene fragment of 2331bp.Successfully constructed the eukaryon expression plasmid of Vp4 gene.
2. the purifying of eukaryon expression plasmid is with quantitative
The positive strain that is accredited as recombinant expression plasmid is inoculated in 150ml contains in the LB substratum of Kan, 37 ℃ of shaken overnight are extracted recombinant expression plasmid in a large number with alkaline lysis, and carry out purifying with the PEG method.
Measure 260nm and the 280nm optical density value of extracting plasmid with ultraviolet spectrophotometer.By experimental formula dna content (ug/ml)=OD 260* 50ug/ml calculates the content of plasmid DNA, with OD 260/ OD 280Ratio in the 1.8-2.0 scope, and agarose gel electrophoresis detects and not see the RNA band, shows that the plasmid purity of being extracted is higher, can be used for transfection and immunity.
3.MA-104 expression in the cell and evaluation
Eukaryotic expression recombination plasmid transfection MA-104 cell is pressed Lipofectamine TM2000 working instructions carry out, concrete operations are as follows: (1) in transfection the day before yesterday with the MA-104 cell with under the trysinization, insert in the Tissue Culture Plate (6 orifice plate), every hole adds 2mlRPMI1640 growth media (not adding two anti-), and 5% CO2gas incubator is cultivated 24h for 37 ℃.Set up the recombinant expression plasmid group separately, pVAXI control group and normal cell control group.Attached cell accounted for surface-area about 80% in the culture dish bottom when (2) cell concentration of Jie Ruing can make transfection, a general available cover glass (use in advance soaked in absolute ethyl alcohol a couple of days) is put at the bottom of the cell cultures plate hole, need to shine a couple of days under ultraviolet, the purpose strictness is aseptic, and the general cell that inserts is 1 * 10 5-2 * 10 5Individual cell.(3) add the 10ul liposome in the unparalleled anti-RPMI1640 growth media of 250ul serum-free, mixing acts on 5min gently.(4) get adding 4ug recombinant plasmid in the unparalleled anti-RPMI1640 growth media of 250ul serum-free in addition, mixing acts on 5min gently.(5) be added drop-wise in 3 mixing slowly, room temperature 30min with 4.(6) add in the cultured Tissue Culture Dish cotransfection with 5.Add 1.5ml after 6 hours and contain 8% serum, 2% pair of anti-RPMI1640 growth media.5% CO2gas incubator is cultivated 18-42h. for 37 ℃ and is collected transfectional cell, extracts total RNA, carries out RT-PCR and identifies.Concrete steps are as follows:
(1) with physiological saline washing transfectional cell, in cell precipitation, add 0.5mlTRIzol reagent, piping and druming repeatedly, room temperature is placed 5min, makes protein dissolution.(2) add the 0.1ml chloroform, firmly shake up 15sec, room temperature leaves standstill 3min, and 4 ℃ 12000 leaves heart 15min.(3) with the upper water phase transition to the new centrifuge tube, add Virahol 0.25ml, room temperature leaves standstill 3min, 4 ℃ 12000 leaves heart 10min.(4) abandon supernatant, add 0.5ml 75% washing with alcohol precipitation, 8000 leave heart 5min.
(5) abandon supernatant, dry air 10min is with the total RNA of 15ul DEPC water dissolution, 55 ℃ of water-bath 10min ,-70 ℃ of preservations.
(6) be template with total RNA, cDNA is synthesized in reverse transcription.Complete sequence according to the rotavirus Vp 4 gene of having delivered in the world designs a pair of primer, and adds the KpnI restriction enzyme site in upstream primer, adds the BamHI restriction enzyme site in the downstream primer, and is synthetic by TaKaRa company.The 25ul reaction system is as follows: Oligod (T) 1.0ul, Rnase Inhibitor 1.0ul, AMV buffer 5.0ul, AMV 1.0ul, dNTP 2.0ul, RNA 3.0ul, 12.0ul.42 ℃ of water-bath 1h of DEPC water, 95 ℃ of 5min deactivation ThermoScript II.With the reverse transcription product is template, and p1, p2 are primer, carry out pcr amplification, and 25ul PCR reaction system is as follows: ExTaq Buffer 2.5ul, p10.5ul, p2 0.5ul, dNTP 4.0ul, Mgcl 22.5ul, ExTaq 1.0ul, cDNA 4.0ul, D.D.W10.0ul adds whiteruss 25.0ul.
Reaction conditions: 94 ℃ of pre-sex change 8min, 94 ℃ of sex change 90sec, 56 ℃ of annealing 1min, 72 ℃ are extended 3min30sec.35 circulations, 72 ℃ are extended 10min eventually.
The RT-PCR qualification result
Adopt Lipofectamine TM2000 with recombinant expression plasmid pVAXI-Vp4 transfection to the MA-104 cell, simultaneously do blank and normal cell is done negative control with the MA-104 cell of pVAXI transfection, behind the transfection 48h, extract total RNA of transfectional cell, meet eukaryote RNA feature; Be that template is carried out RT-PCR with this RNA again, the MA-104 cell amplification of the visible recombinant expression plasmid pVAX1-Vp4 of electrophoresis result transfection goes out a target DNA band of the same size, and the MA-104 cell of empty carrier pVAX1 transfection and normal MA-104 cell there is no the purpose band.
4. in the intravital expression of BLAB/c mouse
The BLAB/c mouse is divided into A at random, B, three groups of C, 30 every group.To prepare in a large number and process satisfactory eukaryotic expression recombination plasmid pVAXI-Vp4 of detection and carrier for expression of eukaryon pVAXI dilution with sterile saline, every kind of plasmid final concentration is 1ug/ul.PROCAINE HCL, PHARMA GRADE joins in the plasmid by final concentration 0.5%.Each group mouse is carried out injecting immune, every pleural muscle meat injection 50ul.A group injection pVAX1-Vp4; B group injection pVAX1; C organizes injecting normal saline.By with quadrat method interval two all booster immunizations, be total to immunity three times later on.Get 5 mouse orbit veins before the immunity at random and get blood, collect blood sample, extracting spleen cell is put to death in dislocation, carries out CD4 in serum antibody and the splenocyte respectively +, CD8 +T cell relative populations detects.The immunity back is in the 14th, 28, and each group was got 5 mouse respectively at random in 42 days, uses with quadrat method and collects blood sample and handle splenocyte.
The OD of the anti-RV antibody of serum to be checked is surveyed in antibody test with the ELISA method 492Value.Being cushioned liquid with bag wraps RV antigen by Sptting plate by the dilution of 160ug/ hole.After serum to be checked done dilution in 1: 200 with PBS, every hole 100ul, the HRP sheep anti-mouse igg is diluted to working concentration with PBS, every hole 100ul.Operation according to a conventional method.
(1) the rotavirus liquid of 200ml with the MA-104 cell cultures is got in the preparation of wheel virus antigen, and 5000 leave heart 30min, get supernatant 20000 and leave heart 1h.Precipitation is dissolved in the physiological saline of 0.5ml.
(2) content of usefulness spectrophotometric determination rotavirus protein.Calculate protein content with following formula.OD 280* 1.45-OD 260* 0.74=protein content (mg/ml)
Being cushioned liquid with bag wraps RV antigen by Sptting plate by the dilution of 160ug/ hole.After serum to be checked done dilution in 1: 200 with PBS, every hole 100ul, the HRP sheep anti-mouse igg is diluted to working concentration with PBS, every hole 100ul.Operation according to a conventional method.
Measure the absorbance value (OD in each hole at wavelength 492 places with automatic enzyme mark determinator 492Value).With the SAS statistical software data are handled.
Aseptic separation splenic lymphocyte is tested in the T lymphocyte transformation, measures proliferation of lymphocytes with mtt assay, with the intravital cellular immune level of reflection immunized mice.
(1) mouse is put to death in dislocation in 42 days, puts and soaks 1-2min in 75% ethanolic soln with sterilization skin.Asepticly get the gauze that spleen places sterilization, rub gently, make splenocyte suspension.
(2) splenocyte counting, viable count is more than 90%, and adjusting cell concn is 5 * 10 6Individual/ml.Every hole adds 100ul splenocyte suspension, totally 8 holes.Wherein 4 holes add 100ulConA, and other 4 holes add the 100ul splenocyte suspension again and do blank.
(3) Tissue Culture Plate is put into 37 ℃, 5%CO 2Hatch 68h in the incubator.
(4) every hole adds 20ul MTT, continues to cultivate 4h.
(5) the careful suction removed nutrient solution, and every hole adds 100ulDMSO, and vibration 10min measures OD on enzyme-linked immunosorbent assay instrument 570, the calculating stimulation index (stimulation index, SI), SI=OD 570Experimental group/OD 570Control group.Analyze with Spss 11.0.
CD4 +CD8 +T cell detection splenocyte sample send basic courses department of No.1 Hospital of Jilin Univ. in the 2h after collection, detect CD4 in 10000 splenocytes with flow cytometer (FACSCalibur) +, CD8 +The relative populations of T cell (%).With the SAS statistical software data are handled.
The antibody test result
ELISA antibody test result after the recombinant expression plasmid pVAXI-Vp4 immunity shows, A group mouse serum is exempted from the back at head and can be detected anti-RV positive antibody (P/N＞2.0) on the 14th day, detected anti-RV positive antibody (P/N＞2.0) later on respectively at the 28th, 42 day, illustrate that immunity back antigen gene has obtained expression in BLAB/c mouse body, the antigen gene of expression induces mouse to produce humoral immunoresponse(HI).The results are shown in Table 1.
Antibody response after the table 1 recombinant expression plasmid pVAX1-Vp4 immunity
Detection time (Days) Group (Groups)
Group A Group B Group C
0 14 28 0.118±0.008 A 0.498±0.012 A 0.642±0.026 A 0.118±0.008 A 0.240±0.027 B 0.270±0.016 B 0.118±0.008 A 0.250±0.024 B 0.260±0.008 B
42 0.825±0.018 A 0.210±0.014 B 0.220±0.012 B
Annotate: there were significant differences for A and B, and B and B difference are not remarkable.
The lymphocyte transformation experimental result
Lymphocyte transformation test is to produce a cellular immunization index commonly used after detecting the antigenic stimulation body.Aseptic separating immune mouse spleen lymphocyte, with ConA as the antigenic stimulation lymphopoiesis.Experimental result shows that there is significant difference (p＜0.05) in the lymphproliferation response of Vp4 group and pVAXI and physiological saline control group. this point and flow cytometry are to the CD4 of experimental group +The detected result of T cell quantity is the same.
Table 2 immunized mice lymphocyte transformation result and analysis
Immune animal Group Sample number Stimulation index (SI)
Mouse mouse mouse Vp4 pVAX I physiological saline 10 10 10 1.426±0.0894 b 1.185±0.0603 cd 0.9476±0.1892 d
Annotate: a, b, c, the different person's significant differences of d letter, identical person is not remarkable.
CD4+, the CD8+T cell detection results
After the first immunisation, CD4 in the A group mouse spleen +, CD8 +T cell quantity and B, C group compares that all there were significant differences (P＜0.05), and apparently higher than B, C group, illustrate immunity afterwards antigen gene in the mouse body, obtained expression, CD4 +T cell and CD8 +Amplification has taken place in the T cell after effectively activating.See Table 3 and table 4.
CD4 in the splenocyte after the table 3 recombinant expression plasmid pVAXI-Vp4 immunity +The quantity of T cell (%) changes
Detection time Days Group Groups
Group A Group B Group C
0 14 28 42 23.452±0.925 A 40.368±0.687 A 42.834±0.832 BC 49.322±3.854 A 23.452±0.925 A21.796±1.125 B30.596±1.412 C30.124±3.246 B 23.452±0.925 A21.832±1.264 B31.082±1.316 C31.686±1.436 B
(annotate: there were significant differences for A and B in the table, and B, C and C do not have significant difference)
CD8 in the splenocyte after the table 4 recombinant expression plasmid pVAXI-Vp4 immunity +The quantity of T cell (%) changes
Detection time Days Group Groups
Group A Group B Group C
0 14 28 42 11.232±0.865 A17.813±0.237 A18.363±1.422 A19.145±0.364 A 11.232±0.865 A 11.486±1.254 B 12.374±1.062 A 12.556±0.876 A 11.232±0.865 A 11.396±1.326 B 12.706±0.654 A 12.412±0.932 A
(annotate: there were significant differences for A and B in the table, and B, C and C do not have significant difference)
The present invention detects lymphopoiesis situation and CD4 in its serum antibody and the spleen with eukaryotic expression recombination plasmid pVAXI-Vp4 immunity BALB/c mouse +, CD8 +The number change situation of T cell, result show that pig A group rotavirus Vp 4 whole gene has not only obtained expression in mammalian cell, and after giving animal immune with eukaryotic expression recombination plasmid pVAXI-Vp4, have obtained immune effect.This provides scientific basis for pVAXI-Vp4 as further applying of dna vaccination.

Claims (2)

1, a kind of pig A group rotavirus Vp 4 whole gene eukaryotic expression plasmid, it is characterized in that: from the RV that cultivates, use the full gene of inverse transcription polymerase chain reaction (RT-PCR) technology amplification Vp4, open reading frame contains 2331bp, 776 amino acid of encoding, by the T-A clone technology, with the PCR product cloning to cloning vector pGEM-T carrier, obtain recombinant clone plasmid pGEM-T-Vp4, with recombinant clone plasmid pGEM-T-Vp4 and expression vector pVAX1 all with KpnI and BamHI double digestion, after purifying reclaims, connect with the T4DNA ligase enzyme, transform, make up eukaryotic expression recombination plasmid pVAX1-Vp4.
2, a kind of pig A group rotavirus Vp 4 whole gene eukaryotic expression plasmid according to claim 1, it is characterized in that: from the RV that cultivates, use the full gene of inverse transcription polymerase chain reaction RT-PCR technology amplification Vp4, by the T-A clone technology, the PCR product cloning to cloning vector pGEM-T carrier, is obtained recombinant clone plasmid pGEM-T-Vp4;
Through DNASTAR software analysis nucleotide sequence and aminoacid sequence be:
1 atg gct tcg ctc att tat aga caa cta ctt act aat tca tac aca gtc
1 Met Ala Ser Leu Ile Tyr Arg gln Leu Leu Thr Asn Ser Tyr Thr Val
49 aat ctt tct gac gaa att caa gag att gga tca gct aag tca cag gat
17 Asn Leu Ser Asp Glu Ile Gln Glu Ile Gly Ser Ala Lys Ser Gln Asp
97 gtt act ata aat cct ggt cca ttc gca cga aca ggt tat gca cca gtt
33 Val Thr Ile Asn Pro Gly Pro Phe Ala Arg Thr Gly Tyr Ala Pro Val
145 aat tgg gga gca ggt gag act aat gac tcc aca act gtc gag ccg tta
49 Asn Trp Gly Ala Gly Glu Thr Asn Asp Ser Thr Thr Val Glu Pro Leu
193 tta gat ggt cca tac caa cca acc act ttc aat cca cca aca agc tat
65 Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Asn Pro Pro Thr Ser Tyr
241 tgg gta cta ctt gcg cca act gta gag ggc gta att att caa gga aca
81 Trp Val Leu Leu Ala Pro Thr Val Glu Gly Val Ile Ile Gln Gly Thr
289 aac aat acc gat aga tgg ttg gcc act ata cta att gaa cca aac gta
97 Asn Asn Thr Asp Arg Trp Leu Ala Thr Ile Leu Ile Glu Pro Asn Val
337 caa aca act aac aga ata tac aat ctt ttt ggt cag caa gta act tta
113 Gln Thr Thr Asn Arg Ile Tyr Asn Leu Phe Gly Gln Gln Val Thr Leu
385 tcg gtg gag aat acg tca cag aca caa tgg aag ttc att gat gtg agt
129 Ser Val Glu Asn Thr Ser Gln Thr Gln Trp Lys Phe Ile Asp Val Ser
433 aca act acg cca aca gga agt tat acg cag cac gga cca ttg ttc tct
145 Thr Thr Thr Pro Thr Gly Ser Tyr Thr Gln His Gly Pro Leu Phe Ser
481 aca cca aaa tta tac gct gta atg aaa ttc agt ggt aga ata tat aca
161 Thr Pro Lys Leu Tyr Ala Val Met Lys Phe Ser Gly Arg Ile Tyr Thr
529 tat aat gga acc aca cca aac gca aca aca gga tac tat tca act act
177 Tyr Asn Gly Thr Thr Pro Asn Ala Thr Thr Gly Tyr Tyr Ser Thr Thr
577 aat tat gac aca gta aat atg aca tca ttt tgt gat ttt tat att ata
193 Asn Tyr Asp Thr Val Asn Met Thr Ser Phe Cys Asp Phe Tyr Ile Ile
625 cca aga aat caa gaa gaa aaa tgt act gag tat atc aat cat gga tta
209 Pro Arg Asn Gln Glu Glu Lys Cys Thr Glu Tyr Ile Asn His Gly Leu
673 cct cct ata caa aat aca agg aat gtt gtg cca gta tct tta tcg gct
225 Pro Pro Ile Gln Asn Thr Arg Asn Val Val Pro Val Ser Leu Ser Ala
721 aga gag ata gtg cac aca aga gct caa gtt aat gag gat att gtt gtt
241 Arg Glu Ile Val His Thr Arg Ala gln Val Asn glu Asp Ile Val Val
769 tca aaa act tca ctt tgg aaa gaa atg caa tgc aac aga gac ata acc
257 Ser Lys Thr Ser Leu Trp Lys Glu Met Gln Cys Asn Arg Asp Ile Thr
817 ata aga ttc aaa ttt gat aga aca att att aaa gct gga gga tta gga
273 Ile Arg Phe Lys Phe Asp Arg Thr Ile Ile Lys Ala Gly Gly Leu Gly
865 tac aaa tgg tca gaa ata tct ttt aag cca att act tat cag tac aca
289 Tyr Lys Trp Ser Glu Ile Ser Phe Lys Pro Ile Thr Tyr Gln Tyr Thr
913 tac gct aga gat gga gaa caa att aca gcg cac acc aca tgc tca gtt
305 Tyr Ala Arg Asp Gly Glu Gln Ile Thr Ala His Thr Thr Cys Ser Val
961 aat gga gtt aac aat ttt agt tac aat ggc ggt tcg ctg ccg acg gac
321 Asn Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp
1009 ttc gcc ata tca aga tat gaa gtg att aaa gaa aat tca ttt gtt tac
337 Phe Ala Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Phe Val Tyr
1057 att gat tac tgg gat gat tca caa gca ttc aga aac atg gta tat gtt
353 Ile Asp Tyr Trp Asp Asp Ser Gln Ala Phe Arg Asn Met Val Tyr Val
1105 cgg tca ctt gct gct aat ttg aat aca gta acg tgt act ggt ggt agt
369 Arg Ser Leu Ala Ala Asn Leu Asn Thr Val Thr Cys Thr Gly Gly Ser
1153 tac agc ttc gtg ttg cct tta ggt ggt tat cca gtt atg act ggt ggt
385 Tyr Ser Phe Val Leu Pro Leu Gly Gly Tyr Pro Val Met Thr Gly Gly
1201 aca gtt tca cta cac cca gct gga gtt aca tta tct acc caa ttc act
401 Thr Val Ser Leu His Pro Ala Gly Val Thr Leu Ser Thr Gln Phe Thr
1249 gat ttc gta tct ctt aat tca tta cgc ttt aga ttc agg tta act gta
417 Asp Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Arg Leu Thr Val
1297 gga aga acc ttc att ttc tat aac gag gac tag ggt aag tag tta tac
433 Gly Glu Pro Ser Phe Ser Ile Thr Arg Thr Arg Val Ser Arg Leu Tyr
1345 gga ctt cca gca gct aat cca aac aac caa aga gaa tat tat gaa ata
449 Gly Leu Pro Ala Ala Asn Pro Asn Asn Gln Arg Glu Tyr Tyr Glu Ile
1393 agc ggt agg ttt tca tta ata tca tta gtg cca tca aat gat gat atg
465 Ser Gly Arg Phe Ser Leu Ile Ser Leu Val Pro Ser Asn Asp Asp Tyr
1441 caa aca cca att atg aat tca gtt act gtg agg caa gat cta gag aga
481 Gln Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg
1489 caa ttg gga gag cta cga gat gag ttc aat tca ttg tca cag caa ata
497 Gln Leu Gly Glu Leu Arg Asp Glu Phe Asn Ser Leu Ser Gln Gln Ile
1537 gcg ata tca caa ttg att gat ctg gct cta ttg cca tta gat atg ttt
513 Ala Ile Ser Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe
1585 tca atg ttc tca gga att aaa agt aca ata gat gct gcg aaa tcc atg
529 Ser Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Ala Ala Lys Ser Met
1633 gca acg aac gtg atg aaa aga ttt aaa cgg tca aac cta gcc agc tca
545 Ala Thr Asn Val Met Lys Arg Phe Lys Arg Ser Asn Leu Ala Ser Ser
1681 gtt tct aca tta act gat gca atg tct gat gca gca tcg tct att tca
561 Val Ser Thr Leu Thr Asp Ala Met Ser Asp Ala Ala Ser Ser Ile Ser
1729 aga agt tca tca ata cga tca ata gga tca tca gca tcc gct tgg acg
577 Arg Ser Ser Ser Ile Arg Ser Ile Gly Ser Ser Ala Ser Ala Trp Thr
1777 gaa gtg tca aat tcg atc gca gat gtt tct acg aca gtt gat aca gta
593 Glu Val Ser Asn Ser Ile Ala Asp Val Ser Thr Thr Val Asp Thr Val
1825 tca acg caa act gct act att gct aag cgg tta cga ttg aaa gag att
609 Ser Thr Gln Thr Ala Thr Ile Ala Lys Arg Leu Arg Leu Lys Glu Ile
1873 gca act caa acc gat ggt atg aat ttt gat gac ata tct gca gca gtg
625 Ala Thr Gln Thr Asp Gly Met Asn Phe Asp Asp Ile Ser Ala Ala Val
1921 tta aaa acg aaa att gac aaa tcc gtg caa ata aca cca aat aca tta
641 Leu Lys Thr Lys Ile Asp Lys Ser Val Gln Ile Thr Pro Asn Thr Leu
1969 ccg gaa ata gtt act gaa gct tct gag aaa ttt atc cca aat aga aca
657 Pro Glu Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Asn Arg Thr
2017 tat agg gtt att aac aat gat gaa gta ttt gaa gct gga atg gat ggt
673 Tyr Arg Val Ile Asn Asn Asp Glu Val Phe Glu Ala Gly Met Asp Gly
2065 aag ttt ttc gca tat cgt gta gac aca ttc gat gaa ata cca ttt gat
689 Lys Phe Phe AlT yrA rgV alA spT hrP heA spG luI leP roP heg Asp
2113 gta cag aaa ttc gca gat tta gtt aca gat tca ccg tca ttt cta gca
705 Val Gln Lys Phe Ala Asp Leu Val Thr Asp Ser Pro Val Ile Ser Ala
2161 ata att gat ctt aag acg tta aaa aat ctg aaa gat aat tat gga ata
721 Ile Ile Asp Leu Lys Thr Leu Lys Asn Leu Lys Asp Asn Tyr Gly Ile
2209 agt aag cag caa gct ttt ggc tta cta cga tca gat cca agg gta tta
737 Ser Lys Gln Gln Ala Phe Gly Leu Leu Arg Ser Asp Pro Arg Val Leu
2257 cgt gaa ttt atc aat cag aat aat cca ata ata cga aat aga att gaa
753 Arg Glu Phe Ile Asn Gln Asn Asn Pro Ile Ile Arg Asn Arg Ile Glu
2305 aac tta atc atg caa tgt aga ctg taa
769 Asn Leu Ile Met Gln Cys Arg Leu ***
CNA2005100171985A 2005-10-19 2005-10-19 Pig A group rotavirus Vp4 whole gene eukaryotic expression plasmid Pending CN1952158A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102321654A (en) * 2011-08-31 2012-01-18 华南农业大学 Colon specific expression vector and construction method and application thereof
CN103305546A (en) * 2011-04-20 2013-09-18 上海交通大学 pGreen Max1 eukaryotic expression vector and preparation method thereof
CN113186170A (en) * 2021-05-26 2021-07-30 西南民族大学 Porcine rotavirus strain and inactivated vaccine prepared from same and application of porcine rotavirus strain
CN114875047A (en) * 2022-05-27 2022-08-09 江苏三仪生物工程有限公司 Recombinant expression and application of optimized porcine rotavirus outer capsid protein VP4

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103305546A (en) * 2011-04-20 2013-09-18 上海交通大学 pGreen Max1 eukaryotic expression vector and preparation method thereof
CN103305546B (en) * 2011-04-20 2014-08-27 上海交通大学 pGreen Max1 eukaryotic expression vector and preparation method thereof
CN102321654A (en) * 2011-08-31 2012-01-18 华南农业大学 Colon specific expression vector and construction method and application thereof
CN113186170A (en) * 2021-05-26 2021-07-30 西南民族大学 Porcine rotavirus strain and inactivated vaccine prepared from same and application of porcine rotavirus strain
CN114875047A (en) * 2022-05-27 2022-08-09 江苏三仪生物工程有限公司 Recombinant expression and application of optimized porcine rotavirus outer capsid protein VP4

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