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Abstract Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis. It is one of the three major infectious diseases which threaten public health, the other two are AIDS(acquired immune deficiency syndrome) and malaria. Exported repeated protein (Erp) is an important virulence factors of M. tuberculosis. It has potential value in the clinical application. In order to study the function of Erp, short fragments of DomC and Dom8 synthetized by solid-phase phosphoramidite method were assembled by SOE-PCR. M. tuberculosis erp gene PGLTS length of 8 motif, C , N and CN terminal end mutantion of the 8 motif were cloned by overlap extension PCR. The clones in Escherichia coli BL21 (DE3) pLysS strain were expressed. The mutated genes were subcloned into the prokaryotic expression vector pET-28a (+). Positive plasmids were selected by enzyme digestion, PCR, and DNA sequencing. The recombinant protein expression of M. tuberculosis Erp was detected by SDS-PAGE and Western blot. The results showed that the sizes of the amplified fragments areas were as expected. The erp gene PGLTS motif length of 8 motif, C, N and CN terminal end mutant were amplified successfully by overlap extension PCR and built into the prokaryotic expression vector. Recombinant BL21 (DE3)pLysS strains was induced by IPTG. Detected by tricine/SDS-PAGE, the relative molecular weights of the expression products were 13.0, 17.1 and 6.7 kD, respectively. Prepare the Freund's adjuvant vaccine and vaccinate rabbits. The antibody titers were determined with ELISA. When the titer got 1∶512, the heart blood could be drawn and separated. After preparing the antiserum, we used Western-blot to detect the extracted proteins. The primary antibody was l∶200 dilution of rabbit positive sera. The secondary antibody was 1∶800 dilution of HRP-labeled sheep anti- rabbit IgG. The results of Western blot showed that the proteins of MutC8 and MutN8 possessed good antigenicity, which were identified by the rabbit antisera against Erp of M. tuberculosis. The experiment results showed that the target proteins of BL21 (DE3) pLysS (pET28a-C8) and BL21 (DE3) pLysS (pET28a-N8) were expressed specifically, which were identified by the rabbit antisera against Erp in Mycobacterium tuberculosis. Meanwhile, the proteins mentioned above had excellent antigenicity. The experiments provides basic data for the diagnosis of bovine tuberculosis and the recombinant vaccine antigen of the gene in the future
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Received: 13 January 2012
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