DNA Vaccines Co-expressing Containing Hsp70 And E/M Protein Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) Display Enhanced Immunogenicity

The feature of Porcine Reproductive and Respiratory Syndrome (PRRS) is the diseases which can caused the main clinical manifestations are the reproductive disorders and respiratory diseases of piglets. Different species, gender, age, pigs can be infected, and has wide epidemic and high incidence, which seriously affected the development of the pig industry.The research show that the glycoproteins with immunogenicity in the PRRSV structural genes are GP4 (ORF4 coding), E (ORF5 encoding) and M (ORF6 coding). Since the E, M protein have the ability of neutralizing antibody, and the M protein can induce a specific cellular immunity,E,M protein gene are the important candidate genes to develop DNA vaccine against PRRS.Meanwhile,the studies show that the Hsp70 has the characteristics of immunodominantantigens, can induce specific Thl-type cell, and regulate the organism immune response. In this study, we researched on ORF5 and ORF6 gene of PRRSV SCQ strain and Hsp70 gene in the human tuberculosis Tubercle Bacillus (TB), constructed the DNA vaccine containing the Hsp70,ORF5 and ORF6,analyzed of the DNA vaccine-induced immunity level by immunization experiments in mice, and provided the information for further study of PRRS in the clinical application of the DNA vaccines; offered a good vaccine candidate strain for the effective prevention of PRRS.The present research included:I.Construction and Expression of Recombinant Plasmid Containing PRRSV ORF5-ORF6 and Hsp70 MotifsBased on the plasmids pMD19-T-ORF5, pMD19-T-ORF6 that constructed by the lab, and sequence of PRRSV SCQ gene in GenBank,4 pairs of primers were re-designed and expand the sequences of ORF5,ORF6, Hsp70 genes.Then clone goes to the Simple pMD19-T vector,through PCR,double emazy and gene sequences were identified,named them for pMD19-T-ORF5,pMD19-T-ORF6 and pMD19-T-Hsp70.Establish ORF5 and ORF6 genes, then clone goes to the Simple pMD19-T vector, ORF5-ORF6 genes was obtained by tendem of ORF5 and ORF6.After digested by PCR,EcoRI and XbaI emazy,gene sequences, named them for pMD19-T-ORF5-ORF6.Recovered recombinant ORF5-ORF6 plasmid fragment was cloned into the expression vector and the recombinant expression plasmid was constructed:the pCI-ORF5-ORF6A with stop codon and the pCI-ORF5-ORF6B-Hsp70 without termination codon. And then, recombinant plasmid was identified by EcoRI and XbaI, Hsp70 was digested and recoveried by XbaI and SmaI and inserted into fusion expression plasmid pCI-ORF5-ORF6B-Hsp70.The recombinant plasmid was transfected into COS-7 cells, detected by IFA and Western-blotting, the IFA results showed that cell slurry appear particularity green fluorescence of COS-7 cells of the expression plasmid pCI-ORF5-ORF6A and pCI-ORF5-ORF6B-Hsp70; Western-blotting results showed that the recombinant plasmid experess protein appear reaction tape on 42.7Ku and 112.2 Ku,show that ORF5,ORF6 and Hsp70 proteins had biological activity of the immune response.The successful expression of recombinant PRRS DNA vaccine provided the foundation for animals immunization experiments.II.The Effects on DNA Vaccine pCI-ORF5-ORF6A, pCI-ORF5-ORF6B-Hsp70 Immune in MiceThe mice were immunized by the recombinant plasmid, while setting PRRS vaccine, pCI-neo and the PBS control group. Immunization three times, each time interval of 14 days, tailed tip blood and separated of blood serum every 2 weeks, detected the level of cytokines IFN-y, IL-4, splenic T lymphocyte subsets (CD4+ and CD8+) and lymphocyte proliferation (MTT method) and neutralization antibody.The IL-4 and IFN-γresults showed that each immunity group all have obtious change. IL-4 and IFN-γbe induced by pCI-ORF5-ORF6B-Hsp70 vaccine group was higher than pCI-ORF5-ORF6A group induced notability(P<0.01).It provided that Hsp70 can stimulate availably the expression capability of the IL-4 and IFN-γas the immunological adjuvant. Splenic T lymphocyte subsets result show that the number of CD4+ and CD3+ T lymphocyte in pCI-ORF5-ORF6A and pCI-ORF5-ORF6B-Hsp70 vaccine group were higher than pCI-neo and PBS contrast groups,and the same to CD8+T lymphocyte.MTT result show that the ability of lymphocyte multiplication before immunity doesn't difference notability(P>0.05).As the immunity time add to 14d after three immunity, pCI-ORF5-ORF6B-Hsp70 group was notability(P>0.05) compare with the pCI-ORF5-ORF6A and contrast groups. It provided that Hsp70 can stimulate availably the capability of lymphocyte multiplication as the immunological adjuvant.The immunity tests provided the gene co-expression with Hsp70 in ORF5/ORF6 PRRSV DNA vaccine could effectively induce humoral and cellular immune response.