结核分枝杆菌甲硫腺苷核苷酶的原核表达及活性分析

Prokaryotic expression and activity analysis of 5＇-methylthioadenosine nucleosidase in Mycobacterium tuberculosis

目的克隆、表达、鉴定结核分枝杆菌（MTB）Rv0091基因编码蛋白，分析酶活性，初步鉴定其功能。方法构建Rv0091基因原核表达质粒，在大肠杆菌BL21trxB进行表达，经IPTG诱导，Ni2＋-NTA柱纯化后获得可溶性蛋白，通过SDS-PAGE分析目的蛋白纯度、Westernblot进行免疫学活性鉴定、质谱分析重组蛋白相对分子质量、酶耦联法检测酶活性，分析酶学性质。结果成功构建Rv0091基因原核表达质粒，建立了可溶性蛋白最佳表达体系，获得纯度在95％以上的可溶性蛋白，Westernblot结果证实重组蛋白为结核分枝杆菌蛋白，质谱鉴定其相对分子质量与理论值基本一致，酶学活性实验证实重组蛋白能够分解底物5’一甲硫腺苷（MTA），酶学性质分析表明最适缓冲液为磷酸盐缓冲液和Hepes，酶的热稳定性较差，37℃为最适反应温度，最适pH为10～12。结论初步证明该重组蛋白在体外能够分解MTA，发挥甲硫腺苷核苷酶（MTAN）的作用，可能在MTB的代谢中起关键作用。

Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuber- culosis, identify and characterize of the enzyme activities. Methods Construct the RvO091 prokaryotic ex- pression plasmid, the vector was transformed into E. coli strain BL21trxB. After induced by IPTG, recombi- nant protein was purified by Ni2＋-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue. Immunological activity was identified by Western blot. The recombinant protein molec- ular weight was identified by Mass spectrometry. The enzyme-coupled assay detectes enzyme activity. Resuits The expression plasmid pET32a-Rv0091 was constructed and expressed in E. coli. BL21trxB, and the optimum expression system was conformed. The purity of the recombinant protein was more than 95%. Western blot analysis confirmed that recombinant protein was one of Mycobacterium tuberculosis proteins. Mass spectrometry identified the relative molecular weight and theoretical molecular weight was basically the same. Enzyme assay showed the recombinant protein able to catalyze the substrate MTA. Enzymatic proper- ties showed that the optimal buffer for the phosphate and Hepes buffer, the poor thermal stability of the en- zyme, the optimal temperature of 37℃, optimal pill0-12, when the pH ≤7 , the protein denaturation and loss of some vitality. Conclusion The recombinant protein methyhhioadenosine nucleosidase （MTAN） was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.