The Constructing Of Svv Gene Of Antiapoptosis Factor And Its Prokaryotic Expression

Objective: The aim of this study was to express the antiapoptosis gene Sw and to prepare it's monoclonal antibodies. Methods: (1) The Svv gene was amplified by RT-PCR and purified PCR products were ligated into the sequencing vector pGEM-T-easy. The white clones were selected and the recombinant plasmid was purified, which was further identified by restriction endonucleases and was sequenced. The sequenced gene was subcloned into corresponding restriction MCS of pRSET-c to generate expression vectors pRSET-c-Sw. (2) By the recombinant DNA technology, the expression vectors containing the aimed gene was transfected into E.coli.BL21, and induced by IPTG the Sw protein was expressed. (3) The monoclonal antibodies against Sw protein was produced by hybridom as derived from SP2/0 myeloma cells and spleen cells from Balb/c mice immunized with primary purified Sw fusion protein in E.coli.BL21. The hybridom cellsecreting McAb were selected and verified with ELISA. RESULTS: (1) By sequencing, restriction digestion and PCR we confirmed that Svv gene were correctly insert the expression vectors. Then the expression vector were constructed successfully. (2) By SDS-PAGE , we confirmed that the protein of Svv was fully expressed in E.coli.BL21 which was transfected by recombinant plasmid pRSET-c-Svv. (3) We got four clones secreting McAb against Svv protein verified with indirected ELISA, the further identification need to be done. Conclusion: We successfully constructed expression vectors containing the antiapoptosis gene Svv, got the protein and prepared it's monoclonal antibodies. These results will lay some foundation for the further study of the protein Svv function on apoptosis manipulation, special expression in tumor cell. These McAbs may also be useful in the diagnosis of cancer in future.