Cloning And Expression Of Six Mycobacterium Tuberculosis Specific Antigens And Serodiagnostic Value Of 16 KD-38 KD And Ag85A

Special primers was designed according to the gene sequence of Mycobacterium tuberculosis H37Rv strain published in the GenBank which was used to applify the six target genes, fbpA (Ag85A),fbpB (Ag85B), mpt64 (MPT64), ppe57 (Rv3425), esxB (CFP10), hspX(16KD) and pstSl (38KD) from the total DNA of H37Rv. The purified gene fragments were cloned into pMD18-T. Sequenced correctly genes were cloned into the expression vector pET30a, the PCR and double digestion ensured properly recombinant plasmids were transformed into BL21(DE3) to construct the prokaryotic expression recombinant bacteria. The recombinant BL21(DE3) strain was induced by IPTG, the forms of recombinant proteins was identified by SDS-PAGE: Ag85B and CFP10 were soluble proteins; Ag85A, MPT64 and Rv3425 mainly were inclusion proteins. The hspX-pstS1 fusion gene was amplified by over-lap PCR from the purified gene fragments hspX and pstS1. The fusion gene was cloned into pMD18-T vector, and then connected with pET30a expression vector after sequenced correctly. The recombinant plasmid ensured by PCR and double digestion was transformed into BL21(DE3) to construct the prokaryotic expression recombinant bacteria. The recombinant BL21(DE3) strain was induced by IPTG, and 16KD-38KD was expressed as inclusion protein.The Ag85A and 16KD-38KD protein was used to coated microtiter plates as a serological diagnostic antigen respectively,427 positive sera and 356 negative sera were detected by indirect ELISA, and the experimental results showed that:72 positive and 188 negative sera were identified by Ag85A, the antigen’s sensitivity was 16.86%, specificity was 52.81%, accuracy was 33.21%; 394 positive sera and 315 negative sera were identified by 16KD-38KD, the antigen’s sensitivity was 92.27%, specificity was 88.48%, accuracy was 90.55%.Six MTB-specific antigens were successfully connected with prokaryotic expression plasmid, the open reading frame is correct and no mutation, and a lot of recombinant proteins can be expressed.16KD-38KD has a high serology diagnostic value showed by the ELISA results, they also can be used as candidate antigens for the clinical testing of tuberculosis.