The Research To Nonstructural Protein Function Of Infectious Bursal Disease Virus

This reseach is construction of prokaryotic expression vector for VP5 of IBDV, then prepared monoclonal antibody with expression protein of VP5 which can as a detective condn for construct the stable transfected cell line. At the same time, we successfully construction of eukaryotic expression vector for VP5 of IBDV, established Vero E6 cell line which can lasting expressed protein of VP5. These were substantial foundations for us to reseach viral replication and virulence of IBDV. The recombinant plasmid was identified by restricted enzyme digestion, PCR and sequence analysis and named pET30a-Gx(+12aa)VP5,pET28a-GxVP5,pET28a-GtVP5, then was transformed into E.coli BL21 (DE3) and induced by IPTG, respectively. SDS-PAGE results indicated that Gx-VP5 was about 24kDa and Gt-VP5 was about 23kDa, mainly existed as inclusion bodies. High titer anti-VP5 serum was also prepared in BALB/c mice immunized with purified Gx-VP5 fusion protein inclusion. ELISA results showed that titer was above 1:25600 and Western blot analized that Gx-VP5 and Gt-VP5 reacted with polyclonal antibody and possess specific satisfactory immunological reaction. BALB/c mice were immunized with the purified recombinant fusion protein Gx-VP5, SP2/0 myeloma cells and spleen cells of BALB/c mice were fused by PEG(MW1500), three hybridoma cell lines were examinated by indirect ELISA and clone for three times by limited dilution, and were named as 4B4, 6D12 and 3E8. The subtypes of the monoclonal antibodies were IgG1, and light chains were kappa. The ascites titers of monoclonal antibodies were 5×104, 3.5×104 and 3×104 by indirect ELISA, respectively. Indirect ELISA and Western blot results showed that the monoclonal antibodies only acted with VP5 protein, indirect immuno-fluorescent assay (IFA) analysis indicated that three monoclonal antibodies acted with IBDV Gt. There were specific fluorescence in detected Vero E6 cells which transient expressed VP5 protein by IFA. We successfully construction of eukaryotic expression vector named pcDNA3.1-Gt VP5 which was identified by restricted enzyme digestion, PCR and sequence analysis. The recombinant plasmids were transfered into Vero E6 cells introduced by Lipofectamine2000 reagent. The stable transfectants were screened by G418. Cell subclones were isolated by limiting dilution, and were named as C6A8, F12 and D12, the stable expression Vero E6 cell line which could express VP5 is established. We use the cell line to research VP5 protein by TCID50 and other tests, the results veritifed that VP5 protein decreased virulence of IBDV. We also used RNAi technique sequently, and selected one siRNA which could disturbed VP5 protein expression successfully but could not disturbed IBDV replication, so we veritifed VP5 gene is not essential for viral replication in cell culture in another way.