OBJECTIVETo construct esat6-ppe68 fusion gene and its prokaryotic expression vector for expression in E. coli.

METHODSWith GeneSOEing method, a fusion gene was constructed by splicing esat6 gene and ppe68 gene and cloned into pGEX-4T-1 plasmid to construct the recombinant prokaryotic expression plasmid pGesat6-ppe68. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis of the plasmid, E. coli BL21 was transformed with the recombinant plasmid and induced with IPTG to obtain the expression of the fusion protein ESAT6-PPE68 with GST-tag (about 69 kD), which were purified with GST-fusion protein purification kit. The expression of esat6-ppe68 fusion gene was subsequently detected by SDS-PAGE and Western blot analysis.

RESULTSThe sequence of esat6 and ppe68 in the recombinant plasmid was consistent with that in GenBank report. The fusion protein was detected in the cytoplasm in soluble form and represented approximately 40% of the total bacterial protein of E. coli. After purification, the purity of the fusion protein reached 90%, and its antigenicity was confirmed by Western blotting.

CONCLUSIONThe prokaryotic expression vector pGesat6-ppe68 has been constructed and the fusion protein ESAT6-PPE68 obtained successfully, which provides an experimental basis for potential application of the recombinant ESAT6-PPE68 in the diagnosis of tuberculosis.