| Latent M. tuberculosis infections is a common problem observed in gaining control over tuberculosis world wide. Isocitrate Lyase, 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.In order to obtain recombinant protein with enzymatic activities of Isocitrate Lyase, the key enzyme of the Mycobacterium tuberculosis metabolic Pathway have been chosed as an original target for the new drugs research and development.To obtain recombinant protein with enzymatic activities of isocitrate lyase. The ICL gene was amplified by polymerase chain reaction (PCR) from Mycobacterium tuberculosis H37Rv strain genomic DNA and cloned into pET28-a(+) vector, named pET28-icl. The recombinant protein was expressed in E.coli BL21(DE3). Enzyme activity of the protein was assayed after purifing with Ni-NTA resin.The ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed. The enzymatic properties demonstrated the purified recombinant protein had activities of ICL. To determine the activity of the isocitrate lyase, the glyoxylate formed during catalysis was reduced to glycolate by lactate dehydrogenase with the concomitant oxidation of NADH. and the decrease of the NADH was determined by adding the formazan dye MTS (MTS/PMS ratio of 100:1) and reading the plate at 490nm. The recombinant ICL was purified in a highly active state with a specific activity of about 1.84×102μmol×mg-1×min-1. The pH curve indicated that recombinant ICL activity was optimal at pH 7.4.Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point. This work can facilitate immunologic research and the discovery of novel antimicrobial agents against Mycobacterium tuberculosis. |
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