Expression, And Characterization Of 38kD Protein Of Mycobacterium Tuberculosis In Escherichia Coli

38kD protein of Mycobacterium tuberculosis is of value for immunodiagnosis of tuberculosis. In this study, we have cloned the 38 kD gene and constructed the expression vector of E. coli, then transformed it into the E. coli BL21(DE3) via trans-gene technology.A pair of primers was designed in accordance with the reported 38kD gene sequence. The 38kD gene was amplified from the total DNA of Mycobacterium tuberculosis H37Rv by PCR. The gene then was cloned into the clone vector pMD18-T simple vector and then sequenced for identification. The correct 38kD gene was inserted into the prokaryotic expression vector pET-30a with the C-terminal 6His-tag. The recombinant fusion expression vector pET-30a-38kD was transformed into BL21(DE3) cells via chemical transformation. The positive clone was screened by means of PCR. The target protein was expressed in E. coli after 4 hours'induction with 1mmol/L IPTG. The induction temperature grades were analyzed by SDS-PAGE.The sequence of the 38kD protein was identical to the one in Genbank thoroughly. The positive clones had been screened via PCR and the protein was expressed after 4 hours'induction with 1mmol/L IPTG. The highest amount was reached at 37℃. The solubility analysis showed that the recombinant protein existed as inclusion body in the E. coli. The fusion protein then was purified by affinity chromatography. Western blot analysis indicated that the recombinant 38kD was reactive to rabbit anti-His-tag polyclonal antibody.After the ELISA and immunoblotting experiments, it was found that 38kD antigen was positive with sera from TB patients. The 38kD protein engineering bacteria and the technique concerned is good foundation for clinical diagnosis research of TB.