CN101219216B - Method for preparing coexpression PRRSV ORF3 and ORF5 double-gene nucleic acid vaccine - Google Patents
Method for preparing coexpression PRRSV ORF3 and ORF5 double-gene nucleic acid vaccine Download PDFInfo
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- CN101219216B CN101219216B CN 200810013673 CN200810013673A CN101219216B CN 101219216 B CN101219216 B CN 101219216B CN 200810013673 CN200810013673 CN 200810013673 CN 200810013673 A CN200810013673 A CN 200810013673A CN 101219216 B CN101219216 B CN 101219216B
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Abstract
The invention provides a preparation method to co-express a double nucleic acid vaccine of porcine reproductive and respiratory syndrome virus (PRRSV) OEF3 and ORF5. Complete genetic sequences of PRRSV OEF3 and ORF5 genes are amplified respectively by RT-PCR method and two of the genes are inserted into two multiple cloning sites of a mammalian eukaryotic expression vector pIRES, thereby a recombinant plasmid S-W-PIRES can be obtained. COS1 cell is transfected by the recombinant plasmid; transcriptions of PRRSV OEF3 and ORF5 genes are detected by RT-PCR method; a transient expression of the target protein is detected by immunohistochemistry. The DNA vaccine can continually generate GP3 and GP5 proteins in animal bodies; thereby the bodies continually generate antibodies and cellular immune responses of the two proteins to provide long time immunity for pigs. The invention has wide application potential for prevention and control of PRRS.
Description
(1) technical field
The present invention relates to a kind of biotechnology high and new technology field, coexpression porcine reproductive and respiratory syndrome virus (PRRSV) ORF3 and the dual-gene nucleic acid vaccine of ORF5 are used for to pig the immanoprotection action to porcine reproductive and respiratory syndrome virus being provided.
(2) background technology
Porcine reproductive and respiratory syndrome can cause breeding difficulty and piglet respiratory symptoms such as in-pig miscarriage, stillbirth.Since at the beginning of 2006 6 months, the pig that highly pathogenic PRRSV is a main pathogen " innominate high fever syndrome " appears in succession in tens provinces such as China Hubei, Hunan, Jiangxi, Anhui, and caused serious economy loss for the pig industry of China.Porcine reproductive and respiratory syndrome virus is the sub-thread positive chain RNA virus, is about 15kb.ORF3 coding molecule amount is the glycoprotein GP3 of 40～50ku, and GP3 can not stimulate the generation neutralizing antibody in the pig body, but it can be that sow provides certain protection through cellular immunization; The glycoprotein GP5 of the about 25ku of ORF5 coding can induce body to produce neutralizing antibody and specific cellular immunity.The weak malicious Seedling and the inactivated vaccine that are used to prevent PRRS at present are in China's extensive use, but Porcine reproductive and respiratory syndrome is not effectively controlled yet.Nucleic acid vaccine can import animal body with the mode of plasmid with the gene of viral protective antigen; Its expressing protein not only can stimulate and produces humoral immunization but also can produce cellular immunization, among the present invention PRRSV ORF3 and ORF5 gene was inserted the nucleic acid vaccine that recombinant eukaryotic expression plasmid makes up simultaneously and did not appear in the newspapers as yet.
(3) summary of the invention
The objective of the invention is to develop a kind of coexpression PRRSV ORF3 and the dual-gene nucleic acid vaccine of ORF5 that can effectively control Porcine reproductive and respiratory syndrome, the present situation that makes Porcine reproductive and respiratory syndrome be difficult to prevent and treat is able to break through.
The objective of the invention is to realize like this:
In 2 MCSs of mammal carrier for expression of eukaryon, PRRSV ORF3 and ORF5 complete genome have been inserted respectively; Obtain coexpression PRRSV ORF3 and the dual-gene nucleic acid vaccine of ORF5, this nucleic acid vaccine can be expressed GP3 and the GP5 albumen of PRRSV simultaneously in mammalian cell.
Above-mentioned nucleic acid vaccine makes up through following method and forms:
1) amplification of ORF3 and ORF5
According to the gene order of PRRSV America standard strain ATCC VR-2332, designed primer to PRRSV Shandong Province separated strain SD1 strain ORF3 and ORF5, primer sequence is following:
Primer S1:5 '-ATCGCTAGCCGCCACCATGGTTAATAGCTGTA-3 ' of ORF3
S2：5’-GAGGAATTCAGAAGAATGTAAATAATTCTCATCTG-3’
The expection amplification length is 852bp.
Primer W1:5 '-AATCTAGAGCCGCCACCATGTTGGA-3 ' of ORF5
W2：5’-GCGCGGCCGCTCACTGGCGTGTAG-3’
The expection amplification length is 702bp.
CDNA with the SD1 strain is a template, through primer S1 and S2 amplification ORF3, through W1 and W2 amplification ORF5 gene.
2) structure of S-W-PIRES recombinant eukaryotic expression plasmid
The PCR product W fragment of ORF5 and pIRES recombiant plasmid through Xba I and Not I double digestion, are inserted pIRES with ORF5, make up the W-pIRES recombiant plasmid.The PCR product S fragment of W-pIRES and ORF3 behind EcoR I and Nhe I double digestion, is inserted W-pIRES with the PCR product S fragment of ORF3, make up the S-W-PIRES recombinant eukaryotic expression plasmid.
3) transfection of S-W-PIRES eukaryon recombinant plasmid
With S-W-PIRES transfection COS1 cell, through the situation of transcribing of RT-PCR detection ORF3 and ORF5, through the proteic expression of SABC testing goal.Above-mentioned test has all proved the expression of transcribing of genes of interest and destination protein.
4) animal experiment of S-W-PIRES eukaryon recombinant plasmid
With S-W-PIRES plasmid immune mouse and pig, establish empty carrier PIRES and normal saline matched group simultaneously, be directed against ELISA antibody, neutralizing antibody and the lymphproliferation response of GP3 and GP5 behind the detection nucleic acid vaccine immunity in the animal body.Above-mentioned test has proved that all nucleic acid vaccine S-W-PIRES all can stimulate the antibody that produces to GP3 and GP5 in animal body, and NAT can reach more than 1: 18.4, and the lymphopoiesis ability is apparently higher than matched group.
This nucleic acid vaccine has inserted PRRSV ORF3 and ORF5 gene first in the mammalian cell carrier for expression of eukaryon; Broken through the limitation of conventional inactivated vaccine of PRRSV and attenuated vaccine; Avoided attenuated vaccine to use the back virulence to return the generation of strong situation; Remedied the defective that can not produce cellular immunization after the inactivated vaccine immunity, the antibody to GP5 is preferentially produced, changed PRRSV and infected the back and produce slow and the few deficiency of generation to the neutralizing antibody of GP5.This nucleic acid vaccine can continual in animal body generation GP3 albumen and GP5 albumen, makes body constantly produce humoral immunization and cellular immunization to both, to the pig body persistent immune protective efficiency is provided, and in the prevention and control of PRRS, has broad application prospects.
4, embodiment
1) the PCR design of primers is synthetic
Sequence and carrier for expression of eukaryon PIRES MCS according to PRRSV SD1 strain design the primer that is directed against ORF3 and ORF5 respectively, and be synthetic by Shanghai Bo Ya biotech firm.
Primer S1:5 '-ATCGCTAGCCGCCACCATGGTTAATAGCTGTA-3 ' of ORF3
S2：5’-GAGGAATTCAGAAGAATGTAAATAATTCTCATCTG-3’
The expection amplification length is 852bp.
Primer W1:5 '-AATCTAGAGCCGCCACCATGTTGGA-3 ' of ORF5
W2：5’-GCGCGGCCGCTCACTGGCGTGTAG-3’
The expection amplification length is 702bp.
2) extraction of viral RNA
With PRRSV SD1 strain inoculation Marc-145 cell, when cell 75% left and right sides pathological changes occurred and begins to come off, freeze thawing was collected viral liquid 3 times, extracted virus total RNA with the Trizol test kit, and the operational approach by specification carries out.
3) reverse transcription
Get the RNA 7 μ L of extraction, 70 ℃ of effects of Oligo (dT) 1 μ L 10min, the back adds 5 * buffer4 μ L, dNTP Mixture 3 μ L, RNase Inhibitor 1 μ L, ddH on ice
2O 3 μ L, M-MLV 1 μ L, 42 ℃ of water-bath 1h.With this synthetic cDNA is that template is carried out the PCR reaction.
4) pcr amplification
With PRRSV SD1 strain cDNA is that template is carried out pcr amplification, through primer S1 and S2 amplification ORF3, through W1 and W2 amplification ORF5 gene.10 * PCR Buffer, 5 μ L, each 2 μ L of 25pmol and L upstream and downstream primer, 10mmol and L dNTPs 4 μ L, the plasmid template 2 μ L of dilution in 1: 200, ddH
2O 34 μ L, Taq E 1 μ L.95 ℃ of preparatory degeneration 5min, loop parameter is 94 ℃ of 1min, (ORF3, the segmental annealing temperature of ORF5 are respectively 58.7 ℃, 59.6 ℃) 1min under the annealing temperature, 72 ℃ of 1min, 30 circulations are extended 8min for back 72 ℃.After finishing, total overall reaction gets 5 μ L PCR products through the inspection of 1% agarose gel electrophoresis.PCR product difference called after S fragment and W fragment to ORF3 and ORF5.
5) Construction of eukaryotic
Above-mentioned PCR product is reclaimed test kit through UNIQ-10 pillar DNA glue to be reclaimed.With carrier for expression of eukaryon PIRES and W fragment respectively through the XbaI+NotI double digestion.The enzyme action product reclaims test kit through UNIQ-10 pillar DNA glue and reclaims.To reclaim fragment through the PIRES of XbaI+NotI double digestion and be connected through T4 DNA ligase with the W fragment of corresponding enzyme action, 16 ℃ are spent the night.To connect product and transform DH5 α competent cell, bacterium liquid will be coated with on Amp and LB flat board, and cultivate 16h for 37 ℃ and screen.Extract plasmid after the picking clone shakes bacterium, further institute's upgrading grain is carried out enzyme action and PCR evaluation, make up the W-PIRES carrier for expression of eukaryon.Correct W-PIRES recombiant plasmid and the S fragment of evaluation passed through the NheI+EcoRI double digestion more respectively, connect, make up S-W-PIRES eucaryon dual-expression vector by above-mentioned identical method.Identify after correct clone shakes bacterium and serve the order-checking of Hai Boya company.
6) transfection of COS1 cell
With making up successful S-W-PIRES carrier for expression of eukaryon positive colony inoculation Amp and LB, the big extracting of alkaline lysis plasmid, the detection DNA of carrying does not have RNA and superhelix ratio more than being used for transfection at 50% o'clock.The COS1 cell grows to 80% and carries out transfection when converging, and operation is undertaken by transfection reagent box description.72h detects after the transfection.
7) RT-PCR identifies after the transfection PRRSV gene transcription in the cell line
Trizol Reagent with Invitrogen company extracts cell total rna, with 12.5 μ L DEPC water dissolutioies.RNA is used to carry out reverse transcription, and reaction system is 20 μ L, template ribonucleic acid solution 12.5 μ L wherein, 5 * buffer, 4 μ L, 10mmol and L dNTPs Mixture 1 μ L, RNase Inhibitor 0.5 μ L, downstream primer 1 μ L, AMV RNase 1 μ L.Room temperature is placed 10min behind the mixing, changes the last ice bath 2min of insulation 1h in 42 ℃ of temperature chambers then over to.Reverse transcription product is further used for the PCR reaction, and reaction condition is with 4.After finishing, total overall reaction gets 5 μ L PCR products through the inspection of 1% agarose gel electrophoresis.All can amplify specific band with the RT-PCR that primer carried out to ORF3 and ORF5 respectively, size is close with the expection size.
8) SABC detects the moment expression
The cell of suitable concn is applied on the microscope slide, and after drying, 95% ethanol is 10min fixedly, PBS washing 1 time, and it is subsequent use to dry back 4 ℃ of preservations.On the cell sheet, drip during detection and contain 0.3% H
2O
2The 80%-100% methanol solution, room temperature sealing endogenous peroxydase 30min, 0.25%Triton-100 room temperature effect 10min; PBS washes 3 times; With 1%BSA room temperature sealing 20min, add the positive porcine blood serum of 1: 100 PRRSV of 1%BSA dilution, 4 ℃ are spent the night.PBS washes 3 times, drips the anti-pig IgG of rabbit of the HRP labelling of dilution in 1: 100 again, hatches 30min for 37 ℃.PBS washes 3 times, and reuse 50mM Tris-HCl (pH7.5) washes 5min, adds DAB colour developing liquid chamber temperature 3-10min, and background does not have unspecific staining degree of being brown cell to occur, ends colour developing with deionized water rinsing, observes.Can detect and dye brown positive cell, and normal cell and control plasmid PIRES cells transfected become negative, microscopy can't see and dyed brown cell.
9) mouse test of nucleic acid vaccine
With alkaline lysis method of extracting PIRES, S-W-PIRES eukaryon expression plasmid, measure OD
260And OD
280, calculate dna content=OD
260* 50 μ g and mL * extension rates are adjusted into 1 μ g and μ L.Experimental animal is BALB and the c mice of 5 ages in week, body weight 20～22g, is divided into 3 groups at random, 15 every group, carries out injecting immune in the back leg both sides of mice.First group: 100 μ L S-W-PIRES; Second group: injection PIRES contrasts 100 μ L; The 3rd group: inject 100 μ L PBS as blank.Head exempt from and two exempt between 2 weeks at interval, two exempt from and three exempt between 4 weeks at interval, three exempt from the back slaughtered in 4 weeks.Whenever gather serum at a distance from 2 weeks, carry out ELISA with GP3, GP5 as antigen respectively and detect; The serum of getting after slaughtering carries out the neutralizing antibody detection.Two exempt to exempt from 4 weeks of back and three back 4 all every group slaughter 6, separating spleen lymphocyte carries out lymphopoiesis and measures.Two exempt from 2 weeks of back, and anti-GP3 albumen and the proteic serum titer of anti-GP5 begin obvious rising; S-W-PIRES group NAT can reach 1: 32; S-W-PIRES immune group lymphopoiesis ability is apparently higher than PIRES empty carrier immune group and PBS immune group.
10) the pig body of nucleic acid vaccine test
With alkaline lysis method of extracting PIRES, S-W-PIRES eukaryon expression plasmid, measure OD
260And OD
280, calculate dna content=OD
260* 50 μ g and mL * extension rates are adjusted into 1 μ g and μ L.Select for use monthly age serology to detect 24 of the negative DABAI piglets of PRRS, be divided into 3 groups at random, feeding and management condition is identical.Two hindlimb muscle gill back intradermal injection dna vaccinations.The grouping situation is following: inject 400 μ gS-W-PIRES for first group; Inject blank plasmid PIRES contrast for second group; The 3rd group of injection PBS is as blank.Head exempt from and two exempt between 2 weeks at interval, two exempt from and three exempt between 2 weeks at interval.Confess one's crime and exempt from back every interval 2 week blood sampling once, adopt the vena cava anterior blood sampling, totally 4 times.The ELISA that serum carries out PRRSV antibody detects and the neutralizing antibody detection.Exempt to exempt from 2 weeks of back and three back 2 week blood sampling two and separate the peripheral blood splenocyte, carry out lymphocyte proliferation assay.Two exempt from 2 weeks of back, and anti-GP3 albumen and the proteic serum titer of anti-GP5 begin obvious rising; S-W-PIRES group NAT can reach 1: 18.4; S-W-PIRES immune group lymphopoiesis ability is apparently higher than PIRES empty carrier immune group and PBS immune group.
Claims (1)
1. the method for preparing of coexpression PRRSV ORF3 and the dual-gene nucleic acid vaccine of ORF5; It is characterized in that through increase the respectively complete genome sequence of PRRSV ORF3 and ORF5 of RT-PCR method; And 2 genes are inserted 2 MCSs of mammal carrier for expression of eukaryon pIRES; Obtain coexpression PRRSV ORF3 and the dual-gene nucleic acid vaccine of ORF5 of recombiant plasmid S-W-PIRES, this nucleic acid vaccine is expressed GP3 and the GP5 albumen of PRRSV simultaneously in mammalian cell; Preparation process is following:
1) amplification of ORF3 and ORF5:
According to the gene order of PRRSV America standard strain ATCC VR-2332, designed primer to PRRSV Shandong Province separated strain SD1 strain ORF3 and ORF5, primer sequence is following:
Primer S 1:5 '-ATCGCTAGCCGCCACCATGGTTAATAGCTGTA-3 ' of ORF3
S2：5’-GAGGAATTCAGAAGAATGTAAATAATTCTCATCTG-3’
The expection amplification length is 852bp:
Primer W1:5 '-AATCTAGAGCCGCCACCATGTTGGA-3 ' of ORF5
W2：5’-GCGCGGCCGCTCACTGGCGTGTAG-3’
The expection amplification length is 702bp:
5 ' end at every primer all contains restriction enzyme digestion sites, is template with the cDNA of SD1 strain, through primer S1 and S2 amplification ORF3, through W1 and W2 amplification ORF5 gene;
2) structure of S-W-PIRES recombinant eukaryotic expression plasmid:
With the PCR product W fragment of ORF5 and pIRES recombiant plasmid through Xba I and Not I double digestion; ORF5 is inserted pIRES; Make up the W-pIRES recombiant plasmid; The PCR product S fragment of W-pIRES and ORF3 behind EcoR I and Nhe I double digestion, is inserted W-pIRES with the PCR product S fragment of ORF3, make up the S-W-PIRES recombinant eukaryotic expression plasmid;
3) transfection of S-W-PIRES eukaryon recombinant plasmid:
With S-W-PIRES transfection COS1 cell, through the situation of transcribing of RT-PCR detection ORF3 and ORF5, through the proteic expression of SABC testing goal, above-mentioned test has all proved the expression of transcribing of genes of interest and destination protein;
4) animal experiment of S-W-PIRES eukaryon recombinant plasmid:
With S-W-PIRES plasmid immune mouse and pig; Establish empty carrier PIRES and normal saline matched group simultaneously; Be directed against ELISA antibody, neutralizing antibody and the lymphproliferation response of GP3 and GP5 behind the detection nucleic acid vaccine immunity in the animal body; Proof nucleic acid vaccine S-W-PIRES stimulates the antibody that produces to GP3 and GP5 in animal body, and NAT reaches 1: 18.4, and the lymphopoiesis ability is apparently higher than matched group.
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Non-Patent Citations (4)
| Title |
|---|
| JUAN PLANA DURAN et al."Baculovirus Expression of Proteins of Porcine Reproductive and Respiratory Syndrome Virus Strain Olot/91. Involvement of ORF3 and ORF5 Proteins in Protection".《Virus Genes》.1997,(第14期),第19-29页. |
| 沈国顺等."表达PRRS 病毒GP5 、GP3 和猪IL218 的核酸疫苗的构建及实验免疫研究".《免疫学杂志》.2006,第22卷(第6期), |
| 王旭荣等."PRRSV ORF3、ORF5串联基因重组腺病毒的构建".《第一届中国养猪生产和疾病控制技术大会——2005中国畜牧兽医学会学术年会论文集（兽医部分）》.2005,第431-434页. |
| 王旭荣等."PRRSV ORF3、ORF5串联基因重组腺病毒的构建".《第一届中国养猪生产和疾病控制技术大会——2005中国畜牧兽医学会学术年会论文集(兽医部分)》.2005,第431-434页. * |
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