The Diagnostic Value Of Mycobacterium Tuberculosis Protein ESAT-6, CFP-10 And RCFP10-ESAT6 In HIV With TB Coninfected Patients

Objective To obtain tuberculosis ESAT-6, CFP-10 recombinant proteins and rCFP10-ESAT6 fusionprotein, prokaryotic expression vector of ESAT-6, CFP-10 and rCFP10-ESAT6 were constructed. The recombinant proteins were expressed and used as stimulus to detect the specific T cell responses in PBMC of HIV infected individuals, and set up a method to facilitate to diagnose potential TB infection in HIV positive population in China.Methods The gene ESAT-6, CFP-10, rCFP10-ESAT6 were obtained from HB37v, using the Fusion PCR, and cloned into inducible prokaryotic expression vector pET-32a (+) and pET-28a (+). The recombinant rectors were transfected into E.coli BL21(DE3), the product were verified with SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blot hybridization. Using the recombinant protein ESAT-6, CFP-10 and rCFP10-ESAT6 as stimulus to detect TB specific T cell responses of HIV(+) patients/HIV and TB coinfected patients, and HIV(-) control.Results The recombinant protein ESAT-6, CFP-10 and rCFP10-ESAT6 was highly expressed. The CFP-10, rCFP10-ESAT6 fusion protein exist in cytoplasm in soluble form and take about 73%,48% of total bacterial protein of E.coli respectively, and the molecular weight is 33kD,28kD respectively. The ESAT-6 recombination protein expressed in bodies form, represented 42% and the molecular weight is 31kD. The expression of recombinant ESAT-6, CFP-10, rCFP10-ESAT6 was verified by SDS-PAGE and Western blotting. ELISA and Elispot were performed and the antigenicity were confirmed. Our in-house ELISPOT–IFN-γassay using recombinant antigen derived from ESAT-6, CFP-10 and rCFP10-ESAT6 proteins showed higher frequencies in both of TB patients with or without HIV infection than that in the healthy control and HIV(+) only group.Conclusions The recombinant ESAT-6, CFP-10 and rCFP10-ESAT6 genes can be expressed in prokaryotic expression system of E.coli successfully and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that some of HIV positive patient indeed have high frequencies of TB specific T cell responses, it maybe a clue to find potential TB infect patients in this population..