[Expression and purification of CFP32 of Mycobacterium tuberculosis and its serodiagnostic analysis].
OBJECTIVE
To establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity.


METHODS
Rv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA.


RESULTS
Recombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively.


CONCLUSIONS
The recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.
Bi
Ai-xiao
AX
Department of Microbiology, Medical School, Suzhou University, Suzhou 215123, China.
Ding
Yuan-sheng
YS
Liu
Zhong-hua
ZH
Hu
Zhong-yi
ZY
D000942:Antigens, Bacterial / Q000097:blood; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins; D012698:Serologic Tests; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C480221:Cfp32 protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins