Expression Of CFP10-ESAT6-PPE68Fusion Gene Of Mycobacterium Tuberculosis And Its Preliminary Application In The Diagnosis Of Tuberculosis

Objective：To construct a prokaryotic expression vector expressting culturefiltrate protein10(CFP10)-early secreted antigenic target-6kDa Antigen(ESAT6)-pentose-5-Phosphate-3-epimerase68(PPE68) fusion gene ofMycobacterium tuberculosis (M.tb) and induce the expression of fusionprotein in E. coli. After separated and purified by affinity chromatography,the recombinant protein was taken as a specific antigen to react with serumof diagnosed tuberculosis patient (including tuberculosis andextrapulmonary tuberculosis) by indirect ELISA. Specificity, accuracy andsensitivity were calculated by statistical analysis. This laid a foundation forfurther application in clinical.Methods：1. ESAT6and PPE68genes were amplified respectively by PCR fromthe genome of M.tb H37Rv strain. The fusion gene ESAT6-PPE68wasobtained by Gene splicing by over lap extension (Gene-SOEing) and cloned into pET-32a (+) to construct a recombinant plasmid pET-32a (+)-ESAT6-PPE68. CFP10and ESAT6-PPE68genes were amplified from M.tb H37Rvstrain and pET-32a (+)-ESAT6-PPE68and the fusion gene CFP10-ESAT6-PPE68was synthesized by Gene-SOEing as well. After CFP10-ESAT6-PPE68gene was cloned into pET-32a (+), the recombinant plasmid pET-32a(+)-CFP10-ESAT6-PPE68was constructed. Its correctness was identifiedby restrictive digestion and DNA sequencing.2. The constructed recombinant plasmid pET-32a (+)-CFP10-ESAT6-PPE68was transferred into E.coli BL21(DE3) to obtain the recombinantfusion protein after induced by IPTG, then immunogenicity of fusionprotein was detected by Western blot. Fusion protein was separated andpurified by affinity chromatography3. Take purified fusion protein as specific antigen to react with serumof diagnosed tuberculosis patient (including250cases of tuberculosis and66cases of extrapulmonary tuberculosis) and58cases of healthy control byindirect ELISA. Specificity, accuracy and sensitivity were calculated bystatistical analysis.Results：1. After pET-32a (+)-CFP10-ESAT6-PPE68was identified byrestrictive digestion, a visible fragment about1782bp is consistent withanticipated. And the result of DNA sequencing is also consistent withtheoretical analysis. 2. A recombinant protein CFP10-ESAT6-PPE68about77KDa wasexpressed in E.coli BL21, which is a fusion protein of Trx (20KDa), CFP10(11KDa), ESAT6(6KDa) and PPE68(37KDa). Western blot analysisdemonstrated that fusion protein could react specifically with serum ofBALB/c mice infected with M.tb, which appear band of77KDa to thenaked eyes. The purity of fusion protein is87.3%as detected by Bandscanafter separated and purified by affinity chromatography.3. Regarded serum of diagnosed tuberculosis patient as positive controland58cases of healthy person as negative control. Statistical analysisshowed that specificity of fusion protein in detection of tuberculosis andextrapulmonary tuberculosis were94.8%and98.3%respectively, accuracywere63.6%and93.5%respectively, and sensitivity were56.4%and89.3%respectively.Conclusion：1. CFP10, ESAT6and PPE68genes were fused and cloned intopET-32a (+) successfully, and fusion protein was induced by IPTG in E.coli BL21(DE3) and identified.2. CFP10-ESAT6-PPE6fusion protein which acts as antigen indiagnosis of M.tb infection showed a certain reference value, especially fordiagnosis of extrapulmonary tuberculosis.