Generation Of Monoclonal Antibodies Against H Protein Of PPRV And Its Expression In Baculovirus System

Peste des petits ruminants(PPR) which is an acute, highly contagious and fatal disease is caused by peste des petits ruminants virus(PPRV)that belongs to the family Paramyxoviridae. PPR is an OIE(Office International Des Epizooties)-listed disease, it is also classified as the first animal disease in China. As a structural protein of PPRV,Haemagglutinin(H) protein which can induce neutralizing antibody has both hemagglutinin and neuraminidase activities. Therefore, it is an important target of the development of vaccines and diagnostic reagents. In this study, PPRV H specific monoclonal antibodies were produced and the PPRV H protein was also expressed in the Bac-to-Bac Baculovirus Expression System. The immunogenicity of H protein was also investigated through animal experiment. All these results might provide new ideas to the development of vaccines and diagnostic reagents. The content of this study is as follows:1.Preparation and identification of monoclonal antibodies against PPRV HPPRV H gene was constructed into the prokaryotic expression vector pGEX-KG.After getting the purified recombinant pGEX-KG-PPRV-H protein, BALB/c mice were immunized with the recombinant protein for three times. With the techniques of cell fusion and indirect enzyme-linked immunosorbent assay( ELISA), four clones of monoclonal antibodies(MAbs) against PPRV H protein were generated, named 1C4, 1D8,2A1 and 2E1 which can stably secret antibody. The IgG subtype of these MAbs was IgG1 and the the light chain was Kappa. Besides, the specificity of the four MAbs were verified by IFA and Western blot.2. The expression of PPRV H protein in Baculovirus Expression System and its immunogenicityH gene of PPRV was cloned into donor plasmid Pfast Bac HTB. After being sequenced, the recombinant plasmid Pfast Bac HT B-PPRV-H was transformed into competent cells of DHl0 Bac. After being verified by PCR, the recombinant bacmid-PPRV-H was transfected into sf9 cell.The recombinant baculoviruses were successfully harvested after 5 days. Through the analysis of IFA and Western blot, ThePPRV H protein was successfully expressed in the Baculovirus Expression System. Then BALB/c mice were immunized with the recombinant bacmid-PPRV-H protein in different concentrations. The results showed that the recombinant bacmid-PPRV-H protein can induce neutralizing antibody against PPRV.