Prokaryotic Expression Of Ⅲa Gene Of Avian Adenovirus Virus JS Strain And Preparation Of The Monoclonal Antibodies Against Ⅲa

Adenovirus was found in 1949, which is tendency to infect epithelial cell. Adenoviridae is divided into two genuses, the Mastadenoviridae and the Aviadenoviridae in the past. However, Atadenoviridae and Siadenoviridae were added to this family in the ICTV 8th report,2005. Avian Adenovirus can be subdivided into three groups: groupâ… , groupâ…¡, groupâ…¢.Based onâ…¢a gene sequence data, a fragment 1728bp was amplified by PCR from the genome of FAVI-JS. The result of PCR showed that the designed fragment was amplified with expected molecular weight and was subcloned into a prokaryotic expression vector pGEX-6p-1, fusion with a gene encoding glutathione S-transferase (GST), respectively named as pGEX-â…¢a. The plasmid was transformed into the competent cells of BL21(DE3) E.coli. After 2.5h incubation,expression ofâ…¢a protein were induced by 0.5mmol IPTG. After brief sonication and centrifugation, the supernatant and precipitation of the cell lysate were separated by 12% SDS-PAGE. Then expressed fusion proteins were identified by Western blot.The spleen cells of Balb/c mice immunized five times with allantoic fluid infected with FAVI-JS were fused with SP2/0 myeloma cells. After 10 days, the cell culture medium of hybridoma cells was detected with pGEX-â…¢a protein expressed in E.coli by ELISA. Six hybridoma cell lines secreting monoclonal antibodies ( McAbs ) against FAV-â…¢a were developed with limited dilution method, respectively named as FAV-IIIa-2E5, FAV-IIIa-3B4, FAV-IIIa-3F8, FAV-IIIa-3E11, FAV-IIIa-4C3, FAV-IIIa-6D3.The preparation of monoclonal antibodies against FAV-â…¢a will be useful materials for studying the biological functions of the protein.