CN103784969A - Swine transmissible gastroenteritis nucleic acid vaccine and immuno-enhancer and preparation method of vaccine - Google Patents
Swine transmissible gastroenteritis nucleic acid vaccine and immuno-enhancer and preparation method of vaccine Download PDFInfo
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Abstract
The invention relates to a swine transmissible gastroenteritis nucleic acid vaccine and an immuno-enhancer and a preparation method of the vaccine. The swine transmissible gastroenteritis nucleic acid vaccine is shown as a sequence table SEQ ID No. 1. The sequence of the immuno-enhancer for the swine transmissible gastroenteritis nucleic acid vaccine is shown as a sequence table SEQ ID No. 2. Immunization is implemented by adopting the nucleic acid vaccine, the nucleic acid vaccine is simple to prepare, easy to store and transport, low in cost and suitable for large-scale production, and the safety and stability of the vaccine are ensured. The main antigen site of an immunogen is TGEV-S1 (transmissible gastroenteritis virtus-S1), so that the nucleic acid vaccine is more specific. Moreover, pIL12 is used as the immuno-enhancer, so that the immune level of CD4 and CD8 of the nucleic acid vaccine are improved, the titer of antibodies is improved, meanwhile, the duration of the antibodies is also prolonged and the immune effect of the TGEV-S1 nucleic acid vaccine is also improved.
Description
Technical field
The present invention relates to technical field of molecular biology, be specifically related to a kind of transmissible gastroenteritis of swine nucleic acid vaccine and immunostimulant and preparation method.
Background technology
Nucleic acid vaccine (nucleic acid vaccine), also claim gene vaccine (genetic vaccine), refer to the plasmid vector of the protein gene sequence that contains coding, in the methods such as intramuscular injection import host, by the anti-source protein of host cell expression, induction host cell produces the immunne response to this anti-source protein, to reach the object of prevention and treatment disease.
Nucleic acid vaccine utilizes modern biotechnology immunology, biochemistry, molecular biology etc. to be developed into, and is divided into two kinds of DNA vaccination and RNA vaccines.But at present to the research of nucleic acid Seedling take DNA vaccination as main.After DNA vaccination imports in host, absorbed by cell (histiocyte, antigen presenting cell or other inflammatory cell), and at the proteantigen of cell inner expression pathogen, produce cellular immunization and humoral immunization by a series of response stimulus body.
Due to the development of biomolecular science, make immunology enter the epoch of molecular immune in recent years, it indicates the research and development of various immunostimulants.The immune effect that how further to improve traditional vaccine is the direction of effort from now on.Immunological adjuvant refers to apply with antigen simultaneously or in advance, energy enhancing body is for the immunne response ability of antigen, or the material of change type of immune response, comprise that inorganic adjuvant (as aluminium hydroxide), organic adjuvant (as lipopolysaccharide, mycobacterium) and synthetic adjuvant cave are as double-stranded poly, cytidylic acid eye.In recent years along with cytokine progress of research, find that many cytokines also have obvious immunological adjuvant effect, immunogenicity or the reactivity of enhancing body to antigen that can strengthen specific antigen, these cytokines comprise IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12 etc.
At present, transmissible gastroenteritis of swine vaccine mostly on the market is inactivated vaccine or live vaccine, and it has defect separately.Nucleic acid vaccine, also in conceptual phase, reports, and the spike protein (TGEV S gene) of transmissible gastro-enteritis virus has main antigenicity.Wherein, S gene has A, B, C, tetra-main antigen sites of D.Have report, the first half section of S gene comprises main antigen site, and immune effect is better than total length S gene, still, so immune effect is generally necessary to study for improving its immune effect.
Summary of the invention
Based on not knowing above part, the object of the invention is to, by immunoadjuvant function and transmissible gastro-enteritis virus nucleic acid vaccine, improve the immune effect of transmissible gastroenteritis of swine nucleic acid vaccine, extend the persistent period of immunne response.
Object of the present invention is achieved through the following technical solutions:
1, a transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1, sequence is as shown in sequence table Seq No.1.
2, a kind of immunostimulant pVAX-pIL12p (40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine, sequence is as shown in sequence table Seq No.2.
The present invention also has following technical characterictic:
1, a kind of transmissible gastroenteritis of swine nucleic acid vaccine described above, for the primer pair of the transmissible gastro-enteritis virus TGEV S1 that increases, upper primer P1: as shown in sequence table Seq No.3, lower primer P2: as shown in sequence table SeqNo.4.
2, a kind of immunostimulant pVAX-pIL12p (40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine described above, the primer pair of pIL12p (40) is used for increasing, upper primer P3: as shown in sequence table Seq No.5, lower primer P4: as shown in sequence table Seq No.6; Be used for the increasing primer pair of pIL12p (35), is characterized in that upper primer P5: as shown in sequence table Seq No.7, and lower primer P6: as shown in sequence table SeqNo.8.
3, a construction method of transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1, comprises the steps:
(1) according to the nucleotide sequence design primer pair of plasmid PMD-18-T-TGEV-S gene, upper primer P1, sequence is as shown in sequence table Seq No.3, and lower primer P2, as shown in sequence table Seq No.4;
(2) carry out conventional chain polymerization enzyme reaction amplification TGEV S1 gene;
(3) construction recombination plasmid pVAX-TGEV S1.
4, the construction method of a kind of transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1 as described in 3, described step (2) adopts the concrete steps of conventional chain polymerization enzyme reaction amplification TGEV S1 gene as follows: by mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 1min; 45.7 ℃ 1min; 72 ℃ of 2min; 30 circulations, last 72 ℃ of 10min.
Described mixed liquor is composed as follows:
5, the construction method of a kind of transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1 as described in 3, the construction method concrete steps of described step (3) restructuring matter pVAX-TGEV S1 are as follows: first by PCR product TGEV S1 gene through HindIII and EcoR I enzyme action, utilize glue to reclaim test kit and reclaim purification genes of interest fragment, genes of interest after purification is cloned into HindIII and the EcoR I site of expression vector pVAX1, will identifies correct recombiant plasmid called after pVAX-TGEV S1.
6, a construction method of immunostimulant pVAX-pIL12p (the 40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine, comprises the steps:
(1) design respectively primer pair according to the nucleotide sequence of plasmid PUC-18-IL-12 (p40) and PUC-18-IL-12 (p35), upper primer P3, sequence is as shown in sequence table Seq No.5, and lower primer P4, as shown in sequence table Seq No.6; Upper primer P5, sequence is as shown in sequence table Seq No.7, and lower primer P6, as shown in sequence table SeqNo.8;
(2) carry out conventional chain polymerization enzyme reaction amplification pIL12p (40)-linker-pIL12p (35) gene;
(3) construction recombination plasmid pVAX-pIL12p (40)-linker-pIL12p (35).
7, the construction method of a kind of immunostimulant pVAX-pIL12p (the 40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine as described in 6, described step (2) adopts the concrete steps of conventional chain polymerization enzyme reaction amplification pIL12p (40)-linker-pIL12p (35) gene as follows:
(a) pcr amplification of pIL-12 (p40) gene
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 30s; 55.7 ℃ 30s; 72 ℃ of 1min; 30 circulations, last 72 ℃ of 10min.
Described mixed liquor is composed as follows:
(b) pcr amplification of pIL-12 (p35) gene
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 30s; 50.5 ℃ 30s; 72 ℃ of 1min; 30 circulations, last 72 ℃ of 10min.
Described mixed liquor is composed as follows:
(c) SOE-PCR of pIL-12 (p40) and pIL-12 (p35) genetic fragment
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 50s; 51.6 ℃ 50s; 72 ℃ of 1min30s; 16 circulations, last 72 ℃ of 10min.
Described mixed liquor is composed as follows:
(d) amplification of pIL-12 (p40)-linker-pIL-12 (p35) genetic fragment
By mixed liquor 95 ℃ of 5min denaturations; 94 ℃ of 50s; 51.6 ℃ 50s; 72 ℃ of 1min30s; 30 circulations, 72 ℃ of 10min.
8, the construction method of a kind of immunostimulant pVAX-pIL12p (40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine as described in 6, the construction method concrete steps of described step (3) restructuring matter pVAX-pIL12p (40)-linker-pIL12p (35) are as follows: first by PCR product pIL12p (40)-linker-pIL12p (35) gene through Xho I and Xba I enzyme action, utilize glue to reclaim test kit and reclaim purification genes of interest fragment, genes of interest after purification is cloned into Xho I and the Xba I site of expression vector pVAX1, correct recombiant plasmid called after pVAX-pIL12p (40)-linker-pIL12p (35) will be identified.
The present invention adopts the mode of nucleic acid vaccine to carry out immunity, and preparation is simple, is easy to preserve and transportation large-scale production, the safety of the vaccine of assurance, the stability of being suitable for low cost.Immunogen is main antigen site TGEV-S1, makes this nucleic acid vaccine more special.And pIL12 is as immunostimulant in application, and it has improved the CD4 of this nucleic acid vaccine, the immune level of CD8, has improved tiring of antibody and has also extended the lasting time of antibody simultaneously, all improves in every respect the immune effect of TGEV-S1 nucleic acid vaccine.
Accompanying drawing explanation
Fig. 1 is TGEVS1 gene PCR amplification,
Wherein: M:DL2000Marker; 1:pVAX-TGEVS1PCR amplification.
Fig. 2 is the qualification result of pVAX-TGEVS1 recombiant plasmid list double digestion and PCR,
Wherein, M:DL8000Marker; 1: single endonuclease digestion qualification result; 2:HindIII, EcoR I double digestion qualification result; 3:PCR qualification result.
Fig. 3 is pIL12p (40) gene, pIL12p (35) gene PCR amplification and pIL12p (40)-linker-pIL12p (35) gene SOE-PCR amplification,
Wherein, M:DL8000Marker; 1:pIL12p (40) pcr amplification result; 3:pIL12p (35) pcr amplification result; 4:pIL12p (40)-linker-pIL12p (35) SOE-PCR amplification.
Fig. 4 is single double digestion of pVAX-pIL12 (p40)-linker-pIL12 (p35) recombiant plasmid and the qualification result of PCR
Wherein, M:DL8000Marker; 1: single endonuclease digestion qualification result; 2:Xho I, Xba I double digestion qualification result; 3:PCR qualification result; 4: plasmid.
Fig. 5 is blood CD4+T lymphocyte dynamic change result after mouse immune.
Fig. 6 is blood CD8+T lymphocyte dynamic change result after mouse immune.
Fig. 7 is spleen CD4+T lymphocyte dynamic change result after mouse immune.
Fig. 8 is spleen CD8+T lymphocyte dynamic change result after mouse immune.
Fig. 9 is in external virus and experimental result.
The specific embodiment
The structure of embodiment 1pVAX1-TGEV-S1 recombiant plasmid
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail:
1, the amplification of TGEV-S1 gene order
(1) design primer pair P1 and P2 with the nucleotide sequence of existing plasmid PMD-18-T-TGEV-S gene, the TGEV-S1 gene of 2106bp before amplification TGEV-S sequence:
P1:GGGG
aAGCTTaTGAAAAAACTATTTGTG (underscore represents that restriction enzyme site is HindIII)
P2:CCCC
gAATTCtTAGTTAGTTTGTCTAATA (underscore represents that restriction enzyme site is EcoR I)
(2) carry out conventional chain polymerization enzyme reaction amplification TGEV-S1 gene
Take PMD-18-T-TGEV S gene plasmid as template, carry out take P1/P2 as primer TGEV S1 genetic fragment carry out pcr amplification by mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 1min; 45.7 ℃ 1min; 72 ℃ of 2min; 30 circulations, last 72 ℃ of 10min.Gained pcr amplification result as shown in Figure 1.
Described mixed liquor composition is as follows:
2, the Construction and identification of recombiant plasmid pVAX1-TGEV-S1
(1) structure of recombiant plasmid pVAX1-TGEV-S1
Carrier pVAX1 and TGEV-S1 gene amplification product are carried out to double digestion with HindIII, EcoR I respectively, and enzyme action condition is 37 ℃, and object fragment action time is 8～10h, and pVAX1 carrier segments action time is 2～4h.
Object fragment enzyme action system is as follows:
PVAX1 carrier segments enzyme action system is as follows:
TGEV-S1 gene amplification fragment double digestion is reclaimed to product and mix with the double digestion recovery product of pVAX1, carry out the connection of recombiant plasmid, reaction condition is 16 ℃ of connection 8-10h, and linked system is as follows:
(2) evaluation of recombiant plasmid pVAX1-TGEV-S1
10 μ L are connected to products, join in 100 μ L JM109 competence escherichia coli, flick that tube wall makes to connect product and competent cell mixes.After ice bath 30min, 42 ℃ of water-bath heat shock 90s, put into rapidly the cooling 2min of ice bath (noting not shake), under aseptic condition, add the SOC culture medium 500 μ L that have been preheated to 37 ℃, 37 ℃ of 200rpm jolting incubation 1h, get appropriate bacterium liquid, coat agar plate (containing 60 μ g/mL Kan+) upper, after Liquid Absorption is complete, is inverted and is put in 37 ℃, cultivate 12h～16h, occur that white transforms bacterium colony.Single bacterium colony of growing on picking LB solid medium, is inoculated in 2mL LB (60 μ g/mL Kan+) fluid medium, and 37 ℃, 200rpm shaken cultivation spend the night.Adopt alkaline lysis to prepare in a small amount plasmid DNA.Identify recombiant plasmid with Hind III, EcoR I double digestion, reaction system is as follows:
37 ℃ of enzyme action effect 3h.Enzyme action product is analyzed with 10mg/L agarose gel electrophoresis, and result as shown in Figure 2.Send Hua Da gene sequencing by the plasmid of the doubtful positive, order-checking is accredited as to positive recombiant plasmid called after pVAX1-TGEV-S1.
The structure of embodiment 2pVAX1-pIL-12 (p40)-linker-pIL-12 (p35) recombiant plasmid
1, the pcr amplification of pIL-12 (p40) gene
(1) with nucleotide sequence design primer pair P3 and the P4 of existing plasmid PUC-18-IL-12 (p40) gene, pIL-12 (p40) genetic fragment increases
P3:GGGG
cTCGAGaTGCACCTTCAGCAGCTG (underscore represents that restriction enzyme site is Xho I)
P4：CCCGACCCACCACCGCCCGAGCCACCGCCACCATTGCAGGACACAGATGC
(2) carry out conventional chain polymerization enzyme reaction amplification pIL-12 (p40) gene
Take existing plasmid PUC-18-IL-12 (p40) as template, take P3 and P4 as primer, by mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 30s; 55.7 ℃ 30s; 72 ℃ of 1min; 30 circulations, last 72 ℃ of 10min.Amplification as shown in Figure 3.
Described mixed liquor is composed as follows:
2, the pcr amplification of pIL-12 (p35) gene
(1) with nucleotide sequence design primer pair P5 and the P6 of plasmid PUC-18-IL-12 (p35) gene, pIL-12 (p35) genetic fragment increases
P5：CGGGCGGTGGTGGGTCGGGTGGGGGCGGATCTATGTGTCCGCTGCGCAAC
P6:CCCC
tCTAGAtTAGGAAGAATTCAGATA (underscore represents that restriction enzyme site is Xba I)
(2) carry out conventional chain polymerization enzyme reaction amplification pIL-12 (p35) gene
Take existing plasmid PUC-18-IL-12 (p35) as template, take P5 and P6 as primer, by mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 30s; 50.5 ℃ 30s; 72 ℃ of 1min; 30 circulations, last 72 ℃ of 10min.Pcr amplification result as shown in Figure 3.
Described mixed liquor is composed as follows:
3, the structure of pIL-12 (p40)-linker-pIL-12 (p35) genetic fragment
(1) SOE-PCR of pIL-12 (p40) and pIL-12 (p35) genetic fragment
With step 1 and step 2 gained pcr amplified fragment template each other, carry out the structure of pIL-12 (p40)-linker-pIL-12 (p35) genetic fragment.In primer, introduce the flexible aminoacid sequence of one section of (Gly4Ser) 3 as linker, by SOE-PCR, pIL-12 (p40) and pIL-12 (p35) are coupled together, before heavy chain pIL-12 (p40) being placed on according to report, and remove its 3 ' termination codon of holding.
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 50s; 51
-6 ℃ of 50s; 72 ℃ of 1min30s; 16 circulations, last 72 ℃ of 10min.
Described mixed liquor is composed as follows:
(2) amplification of pIL-12 (p40)-linker-pIL-12 (p35) genetic fragment
Take the product of above-mentioned SOE-PCR as template, carry out pcr amplification take P3, P6 as primer.By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 50s; 51.6 ℃ 50s; 72 ℃ of 1min30s; 30 circulations, last 72 ℃ of 10min.Reaction finishes to carry out electrophoresis with 10mg/L agarose gel afterwards.Result as shown in Figure 3.
Described mixed liquor is composed as follows:
4, the Construction and identification of pVAX1-pIL-12 (p40)-linker-pIL-12 (p35) recombiant plasmid
(1) structure of pVAX1-pIL-12 (p40)-linker-pIL-12 (p35) recombiant plasmid
Carrier pVAX1 and pIL-12 (p40)-linker-pIL-12 (p35) gene amplification product are carried out to double digestion with Xho I/Xba I respectively, and enzyme action system is as follows, and enzyme action condition is 37 ℃ of effect 8-10h.
Object fragment endonuclease reaction system:
PVAX1 carrier endonuclease reaction system:
PIL-12 (p40)-linker-pIL-12 (p35) gene amplification fragment double digestion is reclaimed to product to be mixed with the double digestion recovery product of pVAX1 carrier, carry out the connection of recombiant plasmid, reaction condition is 16 ℃ of connection 8-10h, and linked system is as follows:
(2) evaluation of recombiant plasmid pVAX1-pIL-12 (p40)-linker-pIL-12 (p35)
10 μ L are connected to products, join in 100 μ L JM109 competence escherichia coli, flick that tube wall makes to connect product and competent cell mixes.After ice bath 30min, 42 ℃ of water-bath heat shock 90s, put into rapidly the cooling 2min of ice bath (noting not shake), under aseptic condition, add the SO ℃ of culture medium 500 μ L that have been preheated to 37 ℃, 37 ℃ of 200rpm jolting incubation 1h, get appropriate bacterium liquid, coat agar plate (containing 60 μ g/mL Kan+) upper, after Liquid Absorption is complete, is inverted and is put in 37 ℃, cultivate 12h～16h, occur that white transforms bacterium colony.Single bacterium colony of growing on picking LB solid medium, is inoculated in 2mL LB (60 μ g/mL Kan+) fluid medium, and 37 ℃, 200rpm shaken cultivation spend the night.Adopt alkaline lysis to prepare in a small amount plasmid DNA.With Xho I, Xba I double digestion evaluation recombiant plasmid.
Reaction system is as follows:
37 ℃ of enzyme action 3h.Enzyme action product carries out electrophoresis with 10mg/L agarose gel, and result as shown in Figure 4.Send Hua Da gene sequencing by the plasmid of the doubtful positive, order-checking is accredited as to positive recombiant plasmid called after pVAX1-pIL-12 (p40)-linker-pIL-12 (p35).
1, according to " molecular cloning test guide ", prepare in a large number recombiant plasmid pVAX1-pIL-12 (p40)-linker-pIL-12 (p35) and pVAX1-TGEV-S1 with SDS alkaline lysis, and by polyethylene glycol precipitation purification of Recombinant plasmid DNA, carry out DNA concentration determination with ultraviolet spectrophotometer, final concentration is adjusted to 1ug/ul with the 0.1M PBS of appropriate sterilizing.
2, get 18-25g, the kunming mice in age in 6-8 week, is divided into 6 groups at random: blank group, and PBS group, pVAX group, pVAX-pIL-12 group, pVAX-TGEV group, pVAX-TGEV+pVAX-pIL-12 group, 28 every group.The first inject salt lidocaine hydrochloride of leg muscle 50 μ L/ only, are inoculating recombiant plasmid after 15min.At interval of immunity in two weeks 1 time, immunity 3 times altogether, respectively at 7d before immunity and after immunity, 14d, 21d, 28d, 35d, every group of 42d gets 3 mices, and eyeball is got blood and prepared serum and detect antibody, gets splenocyte and blood lymphocytes and detect t lymphocyte subset group's quantity.
The detection of embodiment 4T lymphocyte subgroup quantity
1, the separation of peripheral blood lymphocyte
Eyeball of mouse blood sampling, a part does not add anticoagulant separation of serum, another part adds 3.8% sodium citrate anticoagulant and prepares anticoagulation, dilute with PBS (pH7.4) diluent 1:1, slowly add in the test tube that 2mL lymphocyte separation medium is housed, now upper strata is blood, and lower floor is lymphocyte separation medium, with 2000rpm horizontal centrifugal 20min.With 2000rpm horizontal centrifugal 20min.Collect the nebulous buffy coat that is between plasma layer and lymphocyte separation medium layer, put into the test tube containing incomplete RPMI1640 (not containing hyclone and dual anti-) culture fluid 3ml, after fully mixing, with the centrifugal 15min of 1500rpm.Abandon the supernatant precipitation incomplete RPMI1640 culture fluid of 3mL cleaning mixture and wash twice with the centrifugal 15min of 1500rpm, gained precipitation is lymphocyte.Cell precipitation suspends with the nutritional solution of the complete RPMI1640 of 1mL, and the vigor that carries out detects and counting.
2, the detection of lymphocyte subpopulation quantity
Press the blood lymphocytes suspension of preparation in 1, and by its concentration furnishing 1 × 10
7every milliliter, individual cell, every duplicate samples is got 500 μ L, the centrifugal 5min precipitation of 3000 × g lymphocyte.Wash 2 times the centrifugal 5min of each 3000 × g with cold PBS buffer (pH7.4).Then add respectively the CD4+-FITC labelling of 1:1000 dilution, the pre-cooling PBS100 μ L of CD8+-FITC traget antibody to react, hatch after 30min for 4 ℃, wash 2 times with cold PBS buffer (pH7.4), add 500 μ L buffer resuspended, flow cytometer detects.Testing result as shown in Figure 5,6.
3, the detection of splenic T-lymphocyte subsets quantity
The aseptic spleen of taking fast, grind and be crushed to single cell suspension with copper mesh (200 order), cell suspension (is referred to as below RPMI1640 culture fluid, includes 200IU/mL penicillin, 200 μ g/mL chain syphilis, 10% inactivated fetal bovine serum, 20mmol/LHepes, 2mmol/L L-glutaminate, 5 × 10 in RPMI1640 complete culture solution
-5in mol/L2-mercaptoethanol).Lymphocyte separation medium density－gradient centrifuga－tion method (400 × g, 20min) separating spleen lymphocyte, again with RPMI1640 medium centrifugal washing (350 × g, 15min) 3 times, with trypan blue staining detection cell viability >95%, carry out cell counting simultaneously, adjust cell concentration to 2 × 10
7/ mL, in 96 hole microtest plates, adds lymphocyte suspension 50 μ L in every hole.Afterwards, add respectively 50 μ L only to add RPMI1640 culture fluid containing RPMI1640 culture fluid, the 50 μ L of 20 μ g/mL ℃ on A containing RPMI1640 culture fluid, the control wells of 20 μ g/mL TGEV-S1 albumen.Culture cumulative volume is 100 μ L, and final concentration of cells is 1 × 10
6/ mL, Con A final concentration is 10 μ g/mL, TGEV-S1 final concentration of protein is 10 μ g/mL, establishes three repeating holes.Cell is put 37 ℃, 5%CO
2, cultivate under saturated humidity condition.Spleen lymphocyte is cultivated 48h.Every hole adds 5mg/mL MTT solution 10 μ L, then cultivates after 4h, then adds 100 μ L DMSO solution, is placed in incubator isothermal reaction 15min, with enzyme-linked immunosorbent assay instrument detection, with the zeroing of blank hole, detects the absorbance value (A under 490nm wavelength
490nm), result represents with three repeating hole meansigma methodss.Testing result as shown in Figure 7,8.
The detection of embodiment 5 serum neutralizing antibodies
Extract eyeball and gather immune mouse whole blood, place 2h for 4 ℃, 3000rpm, 4 ℃ of centrifugal 15min, collect supernatant and are serum.Then carry out serum neutralization test, concrete grammar is as follows:
1, pass the 24 porocyte plates that cover with cell monolayer, 5 × 10 with ST cell
4cells/well.
2, virus titer is 3.16 × 10
6tCID
50/ mL, we use 100TCID
50/ 0.1mL carries out virus neutralization tests, does 10
3doubly dilution.
3, with the virus liquid having diluted, dilute each serum.From 1:10 doubling dilution to 1:20,1:40,1:80,1:160.
4, shake up 37 ℃ of effect 1h, inoculating cell.After 48h～60h, treat that virus control pitting speckle number reaches (100-200/ hole), observed result, calculates plaque number by CPE method.
5, result is calculated, and calculates serum to viral suppression ratio, not dead serum dilution and the NAT of protection 50% cell.Testing result as shown in Figure 9.Wherein, TGEV group antibody titer is 1:35, and TGEV+IL12 group antibody titer is 1:75.
Claims (10)
1. a transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1, is characterized in that, sequence is as shown in sequence table SeqNo.1.
2. immunostimulant pVAX-pIL12p (the 40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine, is characterized in that, sequence is as shown in sequence table Seq No.2.
3. a kind of transmissible gastroenteritis of swine nucleic acid vaccine according to claim 1, for the primer pair of the transmissible gastro-enteritis virus TGEV S1 that increases, it is characterized in that upper primer P1: as shown in sequence table Seq No.3, lower primer P2: as shown in sequence table Seq No.4.
4. a kind of immunostimulant pVAX-pIL12p (40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine according to claim 2, the primer pair of pIL12p (40) is used for increasing, it is characterized in that, upper primer P3: as shown in sequence table Seq No.5, lower primer P4: as shown in sequence table Seq No.6; Be used for the increasing primer pair of pIL12p (35), is characterized in that upper primer P5: as shown in sequence table Seq No.7, and lower primer P6: as shown in sequence table Seq No.8.
5. a construction method of transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1, is characterized in that, comprises the steps:
(1) according to the nucleotide sequence design primer pair of plasmid PMD-18-T-TGEV-S gene, upper primer P1, sequence is as shown in sequence table Seq No.3, and lower primer P2, as shown in sequence table Seq No.4;
(2) carry out conventional chain polymerization enzyme reaction amplification TGEV S1 gene;
(3) construction recombination plasmid pVAX-TGEV S1.
6. the construction method of a kind of transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1 according to claim 5, is characterized in that: described step (2) adopts the concrete steps of conventional chain polymerization enzyme reaction amplification TGEV S1 gene as follows: by mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 1min; 45.7 ℃ 1min; 72 ℃ of 2min; 30 circulations, last 72 ℃ of 10min.
Described mixed liquor is composed as follows:
7. the construction method of a kind of transmissible gastroenteritis of swine nucleic acid vaccine pVAX-TGEV S1 according to claim 5, it is characterized in that: the construction method concrete steps of described step (3) restructuring matter pVAX-TGEV S1 are as follows: first by PCR product TGEV S1 gene through HindIII and EcoR I enzyme action, utilize glue to reclaim test kit and reclaim purification genes of interest fragment, genes of interest after purification is cloned into HindIII and the EcoR I site of expression vector pVAX1, will identifies correct recombiant plasmid called after pVAX-TGEV S1.
8. a construction method of immunostimulant pVAX-pIL12p (the 40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine, is characterized in that, comprises the steps:
(1) design respectively primer pair according to the nucleotide sequence of plasmid PUC-18-IL-12 (p40) and PUC-18-IL-12 (p35), upper primer P3, sequence is as shown in sequence table Seq No.5, and lower primer P4, as shown in sequence table Seq No.6; Upper primer P5, sequence is as shown in sequence table SeqNo.7, and lower primer P6, as shown in sequence table Seq No.8;
(2) carry out conventional chain polymerization enzyme reaction amplification pIL12p (40)-linker-pIL12p (35) gene;
(3) construction recombination plasmid pVAX-pIL12p (40)-linker-pIL12p (35).
9. the construction method of a kind of immunostimulant pVAX-pIL12p (40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine according to claim 8, is characterized in that: described step (2) adopts the concrete steps of conventional chain polymerization enzyme reaction amplification pIL12p (40)-linker-pIL12p (35) gene as follows:
(a) pcr amplification of pIL-12 (p40) gene
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 30s; 55.7 ℃ 30s; 72 ℃ of 1min;
30 circulations, last 72 ℃ of 10min,
Described mixed liquor is composed as follows:
(b) pcr amplification of pIL-12 (p35) gene
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 30s; 50.5 ℃ 30s; 72 ℃ of 1min; 30 circulations, last 72 ℃ of 10min,
Described mixed liquor is composed as follows:
(c) SOE-PCR of pIL-12 (p40) and pIL-12 (p35) genetic fragment
By mixed liquor 95 ℃ of 5min denaturations; Then 94 ℃ of 50s; 51.6 ℃ 50s; 72 ℃ of 1min30s; 16 circulations, last 72 ℃ of 10min,
Described mixed liquor is composed as follows:
(d) amplification of pIL-12 (p40)-linker-pIL-12 (p35) genetic fragment
By mixed liquor 95 ℃ of 5min denaturations; 94 ℃ of 50s; 51.6 ℃ 50s; 72 ℃ of 1min30s; 30 circulations, 72 ℃ of 10min, described mixed liquor is composed as follows:
10. the construction method of a kind of immunostimulant pVAX-pIL12p (40)-linker-pIL12p (35) to transmissible gastroenteritis of swine nucleic acid vaccine according to claim 8, it is characterized in that: the construction method concrete steps of described step (3) restructuring matter pVAX-pIL12p (40)-linker-pIL12p (35) are as follows: first by PCR product pIL12p (40)-linker-pIL12p (35) gene through Xho I and Xba I enzyme action, utilize glue to reclaim test kit and reclaim purification genes of interest fragment, genes of interest after purification is cloned into Xho I and the Xba I site of expression vector pVAX1, correct recombiant plasmid called after pVAX-pIL12p (40)-linker-pIL12p (35) will be identified.
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