Cloning and Expression of Mycobacterium bovine Secreted Protein Ag85B in E.coli

The purpose of this study is to clone,identify,and express the mature secreted protein Ag85B from Mycobacterium bovine(MB) and to provide a foundation for diagnosis of MB,for applying MB vaccine into clinic practice,and for detection of immunity effectiveness.The gene encoding Ag85B was amplified from M.bovine Vallee111 chromosomal DNA by using PCR technique and PCR product was approximately 860 bp DNA segment.Clone vector pGEM-T-85B was successfully constructed by the PCR product that was cloned into pGEM-T vector by using T-A clone technique.pGEM-T-85B and pET28a(+) were digested by BamHⅠ and EcoRⅠ double enzymes.The prokaryotic expression vector pET28a-85B was constructed by using the purified Ag85B gene that was subcloned into the expression vector pET28a(+).Plasmid containing pET28a-85B was transformed into competence E.coli BL21(DE3).The bacterium was induced by IPTG and its lysate was loaded directly onto SDS-PAGE.An approximately 30 000 exogenous protein was observed on the SDS-PAGE.The protein was analyzed by using Western-blotting and it had antigenic activity of MB.These results provide the basis for the development of subunit vaccine and DNA vaccine against bovine tuberculosis.