Cloning and expression of Mycobacterium bovis secreted protein MPB51 in Escherichia coli

Abstract

The purpose of this study is to clone, identify, and express the mature secreted protein MPB51 from Mycobacterium bovis and to lay a good foundation for the diagnosis of M. bovis, for applying M. bovis vaccine into clinical practice, and for detection of immunity effectiveness. The gene encoding MPB51 was amplified from M. bovis Vallee111 chromosomal DNA by using PCR technique, PCR product was approximately 800 bp DNA segment. Clone vector pGEM-T-51 was successfully constructed by the PCR product that was cloned into pGEM-T vector by using T-A clone technique. pGEM-T-51 and pET28a(+) were digested by BamH I and EcoR I double enzymes. The prokaryotic expression vector pET28a-51 was constructed by using the purified MPB51 gene that was subcloned into the expression vector pET28a(+). Plasmid containing pET28a-51 was transformed into competence E. coli BL21 (DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. An approximately 30 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting and it had the antigenic activity of M. bovis. These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.

Key words： Mycobacterium bovis; MPB51 gene; cloning; prokaryotic expression

Acknowledgements

This project was supported by grants from the National Natural Science Foundation of China (No. 30270986) and the Chinese National Programs for High Technology Research and Development (No. 2003AA 241121002) and Jilin Agricultural University Science Foundation.

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