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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front. Agric. China    2007, Vol. 1 Issue (1) : 101-104     DOI: 10.1007/s11703-007-0019-6
Research article |
Cloning and expression of Mycobacterium bovis secreted protein MPB51 in Escherichia coli
Xiuyun JIANG1(),Chunfeng WANG2,Chunfang WANG2,Zhaoyang HE2()
1 College of Biotechnology, Jilin Agricultural University, Changchun 130118, China
2 College of Animal Science, Jilin Agricultural University, Changchun 130118
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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.

Keywords Mycobacterium bovis      MPB51 gene      cloning      prokaryotic expression     
Issue Date: 22 February 2016
 Cite this article:   
Xiuyun JIANG,Chunfeng WANG,Chunfang WANG, et al. Cloning and expression of Mycobacterium bovis secreted protein MPB51 in Escherichia coli [J]. Front. Agric. China, 2007, 1(1): 101-104.
 URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-007-0019-6
http://academic.hep.com.cn/fag/EN/Y2007/V1/I1/101
Fig. 1  pET28a-51 expression in E. coli BL21

1: pET28a expression result in E. coli BL21 with IPTG induced 10 h, as control; 2: pET28a-51 expression result in E. coli BL21 with non IPTG induced; 3-9: pET28a-51 expression results in E. coli BL21 with IPTG induced 2 h, 6 h, 7 h, 3 h, 4 h, 5 h, 1 h, respectively; M: low molecular weight protein Marker.

Fig. 2  Western-blot analysis of the expression product of the recombinant plasmid pET28a-51

1-4: expression product of the recombinant plasmid pET28a-51 in E. coli BL21; M: low molecular weight protein Marker.

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