Construction And Evaluation Of Dual-functional-expression-vector And Application In Classical Swine Fever Virus Vaccine Research

Construction of a new dual-function gene vaccine expression vector containing hyperparasite intracellular endogenous inducible-promoter enhances the effective protective immunity induced by the DNA vaccine.This adapts to the research and development trend of veterinary gene vaccine carried by attenuated intracellular parasites. Based on the above considerations,this research aims to develop a new type expression vector for gene vaccine,using vaccine strain of Salmonella choleraesuis(S.C500) as the transporter with the endogenous promoter of its own.Thereby to creat swine live recombinant vaccines that can activate mucosal immunity.Therefore,this study first amplified the nirB,pagC promoter gene fragments of Salmonella choleraesuis,and then to carry out systematic analysis of promoters activity.Based on this,constructing the new type expression vector pCB-neo and pCC-neo,and analysising the characteristics of promoting activity of the vectors;in the end,verify the feasibility of gene vaccines deliveried by S.C500 built on pCB-neo,pCC-neo.The main research contents include:â… .Molecular cloning and bioinformatics analysis of the nirB,pagC promoter genes of Salmonella choleraesuisThe nirB,pagC promoter gene fragments were amplified from the genome DNA of the vaccine strain Salmonella choleraesuis S.C500.The recombinant plasmid pUCX-PnirB and pUCX-PpagC were successfully constructed,after TA cloning and sequencing.The sequential analysis showed that the homology between the PnirB,PpagC promoter genes of S.C500 and other Salmonellaes related nucleotide sequence are 96-100%,93-99% respectively.The nucleotide homology between the PnirB,PpagC promoter gene of S.C500 and Salmonella cholera SC-B67 are up to 100%,99%respectively.These revealed that the two promoter gene regions were highly conservative.The bioinformatics software NNPP analysis revealed that the PnirB promoter gene includes three strong promotor sequences,and the PpagC promoter gene includes 12 promotor sequences.It indicated that the two Promotor genes have the basic components to activated gene expression.â…¡.The activity study of nirB,pagC promoter genes of Salmonella choleraesuisIn order to study the activity of PnirB,PpagC promoter,we builted single-promoter expression vectors pPB,pPC with endogenous promoter of Salmonella by using genetic engineering techniques,and further constructed reporter gene vectors pPB-EGFP, pPC-EGFP,and then Transformed them into S.C500 for inducible expression.The results of observing the fluorescence of expression bacilli,detection the fluorescence of bacteria cells,and SDS-PAGE tests confirmed the successful expression of EGFP.The results showed that have promoter activity of the cloned PnirB,PpagC promoter gene fragments of S.CS00.â…¢.Construction and Expression of activity Research of New type dual-functional gene vaccine expression vectors To develop the new type dual-functional gene vaccine expression vector containing endogenous promoter of Salmonella,we subcloned necessary promoter gene core-region fragments of PnirB(CB) 190bp),PpagC(CC)(490bp),and also added SD/Kozak Hybrid sequences to replace the Pcmv promoter downstream Chimeric intron genes of pCI-neo, and then built dual-functional gene vaccine expression vectors pCB-neo and pCC-neo. Further Constructed reporter gene vectors pCB-EGFP and pCC-EGFP.The successful expression of EGFP in S.C500 confirmed that PnirB(CB),PpagC(CC) could drive foreign genes expression.The realization of EGFP expression in Vero cells,confirmed the original promoter vector Pcmv could also drive the expression of foreign genes,and the new inserted promoters don't affect its promoter activity;the realization of EGFP expression in mice,confirmed the feasibility of delivery the gene vaccine based on pCB-neo,pCC-neo by S.C500.â…£.Molecular cloning,expression and the effects of immune adjuvant of the Neijiang porcine interleukin-15 genePorcine interleukin-15 gene fragment was amplified from Neijiang porcine PMBC stimulated by Con A by RT-PCR technology.After the TA cloning to identify the selection and sequencing,the cloned recombinant plasmid pUCX-njpIL-15 was constructed successfully.Prokaryotic expression vector pMAL-pmIL-15 was constructed,and expressed at a high level in E.coli.MTT test showed that after initial purification, renaturation and dialysis,the fusion-protein MBP-pmIL-15 could significantly enhance the proliferation of lymphocyte.Construction of eukaryotic expression vector pCI-pIL-15, combining with pCI-gD(PRV),and to immune BALB/c female mice by intramuscular injection,the result demonstrated that pCI-pIL15 contributed to proliferation of spleen CD4+T,CD8+T cells,strengthened the production of PRV-gD gene vaccine specific neutralizing antibody.The experiment confirmed that Neijiang porcine IL-15 has the immunity adjuvant function.â…¤.Molecule cloning of Classical Swine Fever Virus E2 major-antigen protein coding gene and Prokaryotic expression reserchThe CSFV-E2 complete gene was successfully cloned from CSFV proliferated in PK-15 cells by using RT-PCR technology.The CSFV-E2 gene contains a complete sequence coding E2 protein,includes 1119bp in total,and encodes 373aa.Construction of prokaryotic expression vector pET-mE2(pe),pET-E2(pe).Transformed them into host bacteria E.coli Rosetta-gami-TM(DE3) plysS,and then studied the expression of recombinant bacteria R/pET-mE2(pe) and R/pET-E2(pe).The results of SDS-PAGE electrophoresis and Western blot test show that mE2(pe),E2(pe) were expressed successfully have a certain degree of biological activity of the immune response.The successful prokaryotic expression of the main antigen sites(mE2) of CSFV-E2 lays theoretical basis for the further study of Classical Swine Fever virus gene vaccine.â…¥.Construction and expression of dual-genes fusion expression vectors of Neijiang porcine IL-15 and CSFV-E2 genes Combining Neijiang porcine IL-15 gene with the CSFV-E2 major-antigen protein encding gene by molecular biology technology to build a dual-genes fusion recombinant cloning plasmid pUCX-ME2-IL-15 and plasmid pUCX-dME2-IL-15.Construction of eukaryotic expression vector pEGFP-dME2-IL-15,transfected into Vero cells,The result of fluorescence detection confirmed the expression of enhanced green fluorescent fusion protein dME2-IL-15-EGFP,and the possibility of expression of the double-genes ME2-IL-15 in eukaryotic Vero cells.This provided theoretical basis for further study of the fusion dual-genes ME2-IL-15.Construction of recombinant eukaryotic expression vectors pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15,transformed them into S.C500.The results of SDS-PAGE electrophoresis and Western-blot test showed the successful expression of the fusion dual-genes ME2-IL-15 in S.C500.The experiments confirmed that PnirB(CB),PpagC(CC) promoter in S.C500 could drive the ME2-IL-15 expression. Transfected pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15 into Vero cells,the results of immunofiuorescence and ELISA confirmed that the Pcmv could drive expression of exogenous gene ME2-IL-15 in Vero cells.This provided a experimental basis for further study of the expression of dual-genes vaccines delivering by S.C500.â…¦.Stability and immunizing safety of fusion dual-genes vaccines using Salmonella choleraesuis S.C500 as transporterIn order to investigate the stability of the integration of dual-genes vaccine expression vectors in S.C500 and its impact on invasiveness of the carrying bacteria,we built a systematic research of live recombinant vaccine.The results demonstrated that the vaccine expression vectors pCB-ME2-IL-15,pCC-ME2-IL-15,pCI-ME2-IL-15 didn't affect the biological characteristics and their characteristics of biochemical identification of the carrying bacteria S.C500.The drug-sensitivity of the recombinant bacteria were same to the carrying bacteria except the Amp resistance.The fusion gene ME2-IL-15 that was inserted into the gene vaccine expression vectors increased the stability of the expression vectors in the carrying bacteria S.C500.The gene vaccine declined the adhesion and invasion of the carrying bacteria to ST cell,cut down the proliferative ability of S.C500 in ST cells;and also influenced the immune response of the carrying bacteria.It's stable and safe to immune the 28-day-old BALB/c female mice at a certain degree.It could deliver the gene vaccines effectively to the immune cells of the mice.These studies provided a experimental basis for further study of the immune response of gene vaccine carrying by S.C500.â…§.Preliminary study on immune response of the fusion dual-genes recombinant live vaccines delivered by S.C500 in rabbitsA method of indirect ELISA of Salmonella choleraesuis was established to detect the level of antibodies against the S.C500 in rabbits serum.The experiment Established the optimal antigen concentration was 0.305μg/mL,and the optimal serum dilution was 1:40. Testing the value D_492nm of the serum sample,determined through a standard negative serum in accordance with the P/N ratio,P/N≥2.1 positive,1.5＜P/N≤2.1 as suspicious,1.5 ＜P/N for negative.This indirectly reflected the level of antibody response.The results of testing the rabbits serum CSFV antibody showed that A group(oral PBS buffer) non-CSFV antibodies,but B group(oral S.C500/pCB-ME2-IL-15),C group(oral S.C500/pCC-ME2-IL-15),D group(oral S.C500/pCI-ME2-IL-15),E group(oral S.C500/pCI-ME2) CSFV antibodies emerged after the first 14 days.The level of antibodies were higher after the second and third immunization.The immune effect of B,C group is superior to that of D,E group.But the immune effect of C Group was better than B group, D groups better than E group.The development of general dual-functional gene vaccine expression vectors pCB-neo and pCC-neo have a certain advantage.The results of S.C500 antibody detection showed that the four recombinant strains groups by gavage produced a higher antibody level,while the control buffer PBS gavage group failed to detect specific antibodies against the existence of S.C500.The analysis of T lymphocyte proliferation of the immuned rabbits showed that the control A group of T lymphocyte proliferation in rabbits were lower than 15%after immuning three times,and there were little differences among each time.The T lymphocytes proliferation rate of B,C,D,E oral immunization group were higher than 18%.After 14 days from the last immunization,the proliferation rate of B group declined a little,while the proliferation rate of C,D,E groups enhanced in different levels.After 14d from the last immunization,to infect the rabbits with four copies of Classical Swine Fever Virus of rabbits spleen vaccine by intramuscular injection,to determine the standards for the thermal reaction,the protection rates were 0%,40%,60%,40%,25%.The result demonstrated that the classical swine fever gene vaccine carrying by S.C500 in rabbits could induce some protective anti-CSFV-specific antibodies,the protection rates of the antigen gene carried by the general dual-functional gene vaccine expression vector pCC-neo was higher.So the pCC-neo has certain advantages.The rabbits infected with 4×10¹⁰ CFU/dose of Salmonella paratyphi wild strain by oral inoculation.The results showed that the four recombinant strains were able to induce rabbits to produce a certain level of anti-pig wild strain of Salmonella protective specific antibodies.