Production Of Monoclonal Antibody Against IBDV VP1 And Establishment Of Vero E6 Cell Line Stably Expressing VP1

Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultryindustry. IBDV destroys the B lymphocytes in the bursa of Fabricius in young chickens, causingimmunodeficiency. The purpose of the reseach is prepare monoclonal antibody against IBDV VP1and establishment Vero E6 cell line stably expressing VP1. Those can be a substantial foundationfor us to reseach viral replication and virulence of IBDV.The following is the main contents of this reseach:1. The VP1 gene of IBDV was cloned into expressing vector pET32a and transformed intoE.coli DH5α. The recombinant plasmid which named pET32a-VP1 was transformed into E.coliBL21 (DE3) and induced by IPTG. SDS-PAGE results indicated that the fusion protein was about115 ku and mainly existed as inclusion bodies. The expressed protein was isolated by electroelutionfollowing size fractionation using SDS-PAGE gel electrophresis. Western blot results showed thatthe expressed protein reacted with the anti-His6 mAb and the anti-chichen body serum. High titeranti-VP1 serum was also prepared in BALB/c mice immunized with purified pET32a-VP1 fusionprotein. ELISA results showed that titer was above 1:12800 and Western blot analized that theexpressed protein pET32a-VP1 reacted with polyclonal antibody and possess specific satisfactoryimmunological reaction.2. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routinemethod and the hybridomas were selected in HAT medium. The hybridoma cells secreting specificantibody were detected by ELISA and cloned by limiting dilution. Two hybridmas producingantibodies against IBDV VP1 were obtained. Experimentation indicated that monoclonal antibodieshave special reaction with the VP1 protein of IBDV. By indirect ELISA, the antibody titers of C4and D4 were 64, 2 in culture supernatants and 1×10~5, 1×10~2 in ascetic fluids. Identification ofsubclass showed that all the produced McAbs belonged to IgG1.3. Eukaryote plasmids pCI-neo/VP1 and pEGFP-N1/VP1 were constructed and transferedinto Vero E6 cells introduced by Lipofectamine2000 reagent. The stable transfectants were screenedby G418. Cell subclones were isolated by limiting dilution. Gene specific RT-PCR showed thatVP1 were transcribted in Vero E6 cells. Indirect immuno-fluorescent assay (IFA) showed that the Vero E6 cell transfected with plasmid pCI-neo/VP1 appeared strong green fluorescence and theexpressed protein was immune-reactivity with monoclonal antibody against IBDV VP1. It provedthe stable expression Vero E6 cell line which could express VP1 is established, and it can be usedfor the further biological study of IBDV.