Study On Molecule CD154 Enhancing Immunity Of Nucleic Acid Vaccines Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs, is really a new viral disease of swine. Since it was first described in 1987 in the United States, and in 1990 in Europe, PRRS has been one of the most economically important diseases of the global swine industry. The ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and is the leading target for the development of the new generation of vaccines against PRRS. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing PRRSV GP5.CD154 (CD40L, gp39) is a typeⅡintergrel membrane protein on the surface of activated CD4+T cells. CD154 belongs to tumor necrosis factor(TNF) superfamily. The interaction of CD154 and CD40 plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. More and more studys show that CD 154 can enhance the immunized effect of vaccine significantly.In this study, we choosed PRRSV ORF5 gene as target gene, constructed ORF5 DNA vaccine contain CD154 gene, studied the immunized effect of porcine CD 154 enhancing the PRRSV ORF5 DNA vaccine.1. Cloning, Sequence Analysis and Expression of CD154 Gene of swineIn this study, the full length of CD154 molecule was amplified from swine peripheral blood lymphocytes via RT-PCR, and The fragment of swine CD154 (tCD 154) with absence of signal peptide(1-22aa) and transmembrance region(23-46aa) was amplified via PCR and cloned into pronucleus expressive vector pQE80-L, Expression of a fusion protein (6×His-tCD154) with 29kD molecular mass was observed in E. coli DH5a after IPTG induction, and western-blot testing indicated it can be recognized by anti-6×His monoclonal antibody. In addition, the full-length cDNA fragment of swine CD154 was inserted into downstream of EGFP gene of eurokaryotic expression vector pEGFP-Cl, resulting in the expression plasmid pEGFP-Cl-CD154. After transfection in Hela cells, the fluorescence of the fusion protein EGFP-CD154 was observed on cell membrane by fluorescent microscope. In addition, the CD154 antibody was produced using pronucleus expressive plasmid pQE80-CD154 and its biological activity was proved by indirect immunofluorescence test.2. Construction and immune effect of new DNA vaccines against PRRSV In order to determine the function of CD154 gene improving the immune effect of PRRSV ORF5 DNA vaccine, we choosed PRRSV ORF5 gene as target gene, pCI-neo as vector, constructed two ORF5 DNA vaccines contain CD154 gene: pCI-ORF5-CD154 co-expressing ORF5 gene and CD154 gene, pCI-ORF5n60-tCD154 expressing fusing the CD154 gene and ORF5 gene. Immunological activeness of ORF5 protein were detected by IFA experiment (Indirect Fluorescence Assay) and the immune effect of two ORF5 DNA vaccines contain CD154 gene were investigated in mouse model. The results showed that all immunized mice developed ELISA antibodies at 2 weeks after primary immunization and achieved the peak at 6 weeks after primary immunization. The ELISA antibodies level induced by co-expression of ORF5 and CD154 and fusion-exprssion genes were higher than the DNA vaccine pCI-ORF5 singly expressing of ORF5 gene, in addion, the antibodies level of co-expression of ORF5 and CD154 was higher than fusion-exprssion ORF5n60 and tCD154. And Lymphocyte Proliferation Induced Test indicated the same result. These results showed that PRRSV ORF5 DNA vaccine contain CD 154 gene can give swine more efficient immune protection.