Construction And Immunity Of The Recombinant DNA Vaccine Containing GP5, N Protein And Hsp70 Genes For Porcine Reproductive And Respiratory Syndrome

In this study according to Genbank porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene, ORF7 genes and Mycobacterium tuberculosis Hsp70 gene specific,primers were designed and synthesized in order to save our laboratory strain PRRSV-SCQ total extracted RNA as a template, RT-PCR amplification of a 369bp target fragment ORF7A (including terminator) and ORF7B;To build the laboratory pMD18-T-ORF5 and pMD18-T-Hsp70 as a template, PCR expansion by the target fragment of 516bp and 1878bp ORF5 and Hsp70, the 4 gene was cloned into pMD19-T, the amplified strains of PRRSV-SCQ ORF7A and ORF7B gene and insert the pMD19-T-ORF5,get T-ORF5-ORF7A and T-ORF5-ORF7B.The two cloned into the pCI-neo to obtain pCI-ORF5-ORF7A and pCI-ORF5-ORF7B.Finally, Hsp70 was inserted into pCI-ORF5-ORF7B by pCI-ORF5-ORF7B-Hsp70.Healthy mice to pCI-ORF5-ORF7B-Hsp70 immunization, while the establishment of pCI-ORF5-ORF7A DNA, pCI-neo DNA, PRRSV attenuated vaccine, saline four control group. Tail blood collection and testing of blood mRNA expression ORF5,ORF7 and Hsp70 cases, PRRSV antibody levels, IFN-y and IL-4 levels and T lymphocyte subsets.The results showed:1,Eukaryotic expression vector pCI-ORF5-ORF7A and pCI-ORF5-ORF7B-Hsp70 was successfully constructed.2,Mice'peripheral blood(after treatment by DNase) inoculated with two recombinant plasmids after 14d were able to extract ORF5 and ORF7 genes in inoculated pCI-ORF5-ORF7B-Hsp70 in the mouse peripheral blood can be extracted Hsp70 gene, showed that the recombinant DNA in mice have been effectively transcription and expression.3,After 14d,21d,28d,35d,42d, of mice immunized by recombinant plasmid pCI-ORF5-ORF7B-Hsp70,the PRRSV-specific antibody levels of the serum detected by ELISA kits were higher than of pCI-ORF5-ORF7A group and PRRS attenuated vaccine group. The antibody levels (OD630 value of 0.875) of immunized by recombinant plasmid pCI-ORF5-ORF7B-Hsp70 peak at 28d, but fell down after 42d (OD630 value of 0.782);The antibody levels of this treatment significantly larger than pCI-ORF5-ORF7A (p<0.01),PRRS attenuated vaccine group (p<0.05).4,Detected by ELISA test kit, the secretion level of IFN-γ(196.35pg/mL) and IL-4 (53.27pg/mL) in pCI-ORF5-ORF7B-Hsp70 inoculated mice reached the highest level at 35d, and was significantly larger than pCI-ORF5-ORF7A (p<0.01)and PRRS attenuated vaccine group (p<0.05).IFN-γand IL-4 levels of pCI-neo group and the saline group were very low.5,Detecting peripheral blood T lymphocyte subsets through the flow cytometry, the result showed:the level of peripheral blood CD3+T cells (78.39%) of pCI-ORF5-ORF7B-Hsp70 group mice was the highest, and was significantly high than level of pCI-ORF5-ORF7A group and PRRS attenuated (p<0.01);The level of CD4+ (51.72%) was also the highest, and was significantly high than level of pCI-ORF5-ORF7A group (p<0.01)and PRRS attenuated vaccine (p<0.05).The level of CD8+ (24.66%) showed no significant difference with the control group (p>0.05);CD4+/CD8+ratio of pCI-ORF5-ORF7B-Hsp70 group was the highest, but had no significant difference compared with control group (p>0.05).The results show that Hsp70 as a molecular adjuvant can significantly enhance the DNA vaccine ORF5-ORF7 DNA Vaccine-induced immune response, as PRRS new recombinant vaccine to provide reference data.