Construction of the recombinant expression plasmid encoding ESAT6 gene of the tuberculosis bacterium

Objective To clone and construct a expression plasmid containing ESAT6 gene of tuberculosis bacterium.Methods According to the nucleic acid sequence of the ESAT6 gene from Genbank,a pair of primer was synthesized and ESAT6 gene was amplified (PCR method) from the H37Rv,then cloned into pGEM T easy vector,finally,subcloned into pGEX 4T 2 according to its special orientation.The positive clone was identified by restriction endoenzyme analysis and DNA sequencing.Results The fragment digested by endoenzymes was as large as the predicted result.The sequence was the same as the sequence reported on the literatures.Conclusion The ESAT6 gene was successfully cloned into pGEX 4T 2 pronucleus expression vector.