Construction Of Prokaryotic Expression Vector On Isocitrate Lyase AceA Gene In Mycobacterium Tuberculosis H37Rv And Studies Of Fusion Protein

Objective Latent M.tuberculosis infections is present one of the major obstacles in gaining control over tuberculosis worldwide.Isocitrate Lyase(ICL), a key enzyme in the glyoxylate cycle encoded by the icl gene of M.tuberculosis plays a pivotal role in sustaining intracellular infection in inflammatory macrophages and the persistence of M.tuberculosis. We chose the key enzyme of the Mycobacterium tuberculosis metabolic pathway as an original target for the new drugs research and development. To amplify and clone aceA gene to pUC18 resulting in pUC18- aceA and to sequence aceA; To construct an expression vector pET28a(+)-aceA; To optimize the express of AceA protein in E.coli BL21(DE3)plysS; To purify AceA protein and identify the function of AceA protein.Methods The aceA gene fragment with Hind III and BamH I sites was amplified by PCR from MTB genome, then cloned into pUC18 plasmid, sequenced and cloned into pET28a(+) plasmid. The latter was transformed into E.coli BL21(DE3)plysS, and the aceA gene was expressed in the presence of IPTG. The AceA was identified by SDS-PAGE and purified with Ni-NTA column. The characteristics of the recombinant enzyme was assayed after it was purified with Ni-NTA resin.Results DNA sequences analysis and double restriction enzyme digestion confirmed expression plasmids successfully constructed. The fusion proteins were expressed in E.coli. The recombinant AceA was purified in a highly active state with a specific activity. The LC/MS spectrometry gave a 89660Da molecular mass of recombinant AceA. The CD spectrum showed that the percentages forα- Helix,β- Turn , and Random coil are 32.4 %, 27.5%,and 40.1 %,respectively.Conclusions In this study, we cloned and expressed aceA gene of M. tuberculosis H37Rv and analyzed enzymology properties research of AceA, supplied a platform that facilitates the discovery of novel antimicrobial agents against Mycobacterium tuberculosis.