Mycobacterium tuberculosis strain H37 Rv genomic DNA was adopted as a template to establish a screening model targeting to isocitrate lyase (ICL) and glyoxylate pathway for anti-TB drugs at enzymatic and cellular levels.ICL gene was amplified by polymerase chain reaction ( PCR ) and constructed an expression vector and highly expressed in E. coli BL21 (DE3). The recombinant ICL was purified with Ni2+-NTA resin affinity chromatography and detected for its activity. The ICL enzymatic reaction conditions were optimized, and observed the effects of solvents on the enzymatic activity of the samples of going to screen to establish a screening model at the level of inhibition enzyme;observed and optimized the growth conditions ofM. smegmatis(Ms ) in medium with acetate as a sole carbon source,to establish a screening model at cellular level based on inhibitor of glyoxylate pathway of Ms. The results showed that using the two screening models above-mentioned 1 060 kinds of microbial metabolite samples in the lab possibly possessing antagonistic activity were screened initially and rescreened, and both the screened results had fairly good positive reciprocity.%为建立基于酶水平和细胞水平的新型抗结核分枝杆菌(Mycobacterium tuberculosis)药物的筛选模型,以M.tuberculosis H37Rv基因组DNA为模板,PCR特异性扩增异柠檬酸裂解酶(ICL)基因,构建表达载体,在E.coli BL21(DE3)中高效表达,使用Ni2+亲和层析柱纯化重组ICL,检测其活性.优化ICL酶反应条件,考察待筛选样品溶剂对酶活性的影响,建立ICL抑制剂酶水平筛选模型;考察与优化耻垢分枝杆菌(Mycobacterium smegma)在以乙酸盐为唯一碳源的培养基中的生长状况,建立基于M.smegma的乙醛酸代谢途径抑制剂的细胞水平筛选模型;利用上述2种筛选模型对1060种可能具有拮抗活性的微生物代谢样品进行初筛和复筛,两者筛选结果正相关性较好.
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