Expression And Application Of CFP10-PPE68Fusion Protein Of Mycobacterium Tuberculosis

Objective:To construct a prokaryotic expression plasmid carrying theCFP10-PPE68fusion gene of Mycobacterium tuberculosis, and observe theexpression of CFP10-PPE68fusion gene in E.coli BL21, then purify theCFP10-PPE68protein expressed in E.coli BL21by affinity chromatograph.The purified CFP10-PPE68recombinant protein was taken as antigen todetect healthyand patients, including mycobacterium tuberculosis andextrapulmonary tuberculosis patients (n=66). The sensitivity, specificity andaccuracy were measured. It provides a new idea for the early diagnosis oftuberculosis.Methods:1. The PPE68and CFP10gene were amplified by PCR from thegenome of mycobacterium tuberculosis H37Rv strain, the CFP10-PPE68fusion gene was amplified by Gene SOEing, and cloned into pET32a(+)vector, then transformed into E.coli Top10. After identified by restrictive digestion and DNA sequencing, the constructed recombinant plasmid wastransformed into E.coli BL21, and the recombinant fusion protein wasexpressed with IPTG induction. The CFP10-PPE68recombinant proteinwas identified by SDS-PAGE and Western blotting.2. The purified CFP10-PPE68recombinant protein was taken asantigen to detect healthy (n=52), pulmonary tuberculosis patients (n=251)and extrapulmonary tuberculosis patients (n=66). The specificity, sensitivityand accuracy were measured.Results:1. After identified by restrictive digestion and DNA sequencing, theplasmid pET32a(+)/CFP10-PPE68was constructed successfully. Arecombinant protein Trx-CFP10-PPE68about68000Da was expressed inE.coli BL21, which is a fusion protein of CFP10(10900), PPE68(373300)and Trx-His (20400).2. Take the purified CFP10-PPE68recombinant protein as diagnosticantigen, to detect pulmonary tuberculosis patients and extrapulmonarytuberculosis patients. The result shows that the sensitivity is98.4%and98.3%respectively, specificity is27.6%and85.0%respectively, and theaccuracy is56.1%and91.5%respectively.Conclusions:1. The specific gene CFP10and PPE68of mycobacterium tuberculosisH37Rv strain were cloned into pET32a (+) vector, and then expressed in E. coli BL21successfully, the recombinant CFP10-PPE68protein wasobtained.2. CFP10-PPE68fusion protein as antigen in the diagnosis of MTBinfection has high sensitivity, so it can be used for auxiliary diagnosis oftuberculosis, especially for extrapulmonary tuberculosis which is difficult todiagnosis.