CAJ | 학술논문

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、Western blot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,Western blot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′-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.
목적 극륭、표체、감정결핵분지간균(MTB) Rv0091기인편마단백,분석매활성,초보감정기공능.방법 구건Rv0091기인원핵표체질립,재대장간균BL21trxB진행표체,경IPTG유도,Ni2+-NTA주순화후획득가용성단백,통과SDS-PAGE분석목적단백순도、Western blot진행면역학활성감정、질보분석중조단백상대분자질량、매우련법검측매활성,분석매학성질.결과 성공구건Rv0091기인원핵표체질립,건립료가용성단백최가표체체계,획득순도재95％이상적가용성단백,Western blot결과증실중조단백위결핵분지간균단백,질보감정기상대분자질량여이론치기본일치,매학활성실험증실중조단백능구분해저물5′-갑류선감(MTA),매학성질분석표명최괄완충액위린산염완충액화Hepes,매적열은정성교차,37℃위최괄반응온도,최괄pH위10 ～ 12.결론 초보증명해중조단백재체외능구분해MTA,발휘갑류선감핵감매(MTAN)적작용,가능재MTB적대사중기관건작용.
Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuberculosis,identify and characterize of the enzyme activities.Methods Construct the Rv0091 prokaryotic expression plasmid,the vector was transformed into E.coli strain BL21trxB.After induced by IPTG,recombinant 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 molecular weight was identified by Mass spectrometry.The enzyme-coupled assay detectes enzyme activity.Results 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 properties showed that the optimal buffer for the phosphate and Hepes buffer,the poor thermal stability of the enzyme,the optimal temperature of 37℃,optimal pH10-12,when the pH ≤7,the protein denaturation and loss of some vitality.Conclusion The recombinant protein methylthioadenosine nucleosidase(MTAN) was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.