Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli.
The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using TA cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M.bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.
Jiang
Xin-yun
XY
College of Animal Science, Jilin Agricultural University, Changchun 130118, China.
Wang
Chun-feng
CF
Wang
Chun-fang
CF
Zhang
Peng-ju
PJ
He
Zhao-yang
ZY
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism*; D014376:Tuberculosis / Q000517:prevention & control; D032581:Tuberculosis Vaccines; D019444:Vaccines, DNA
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C094904:MPB83 protein, Mycobacterium; D008565:Membrane Proteins; D032581:Tuberculosis Vaccines; D019444:Vaccines, DNA