结核分枝杆菌PE35在大肠杆菌中的可溶性表达及产物纯化  

Expression and purification of Mycobacterium tuberculosis PE35

作　　者：熊志红[1] 朱琰[1] 孙昌文[1] 庄玉辉[1] 程小星[1] 

机构地区：[1]解放军第309医院结核病研究室,北京100091

出　　处：《临床肺科杂志》2010年第12期1709-1711,共3页Journal of Clinical Pulmonary Medicine

基　　金：国家重大传染病专项基金资助项目(编号:2008ZX10003-012)

摘　　要：目的利用大肠杆菌表达系统大量获得重组的结核分枝杆菌PE35蛋白,并初步评估其在结核病血清学诊断方面的价值。方法用聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv全基因组中获得PE35基因克隆到pET24b中,转化BL21(DE3)构建大肠杆菌表达株;聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定重组蛋白及其表达方式;使用Ni-sepharose纯化蛋白;Western-blot检测结核病人血清的抗结核抗体。结果酶切鉴定和DNA序列分析显示构建了正确的pET-PE35原核表达载体,并能在大肠杆菌BL21(DE3)中可溶性地表达分子量大小相符的重组蛋白。纯化后的蛋白纯度达到90%以上,能与结核病人血清抗体结合。结论成功地构建了pET-PE35原核表达载体,并获得较纯的重组PE35蛋白,此蛋白能检测结核病人血清抗体。为探讨PE35蛋白在结核病的基础研究与诊断运用奠定了基础。Objective To gain the recombinant PE35 protein of Mycobacterium tuberculosis and evaluate its potential value for TB diagnosis.Methods Standard PCR was used to amplify the PE35 gene from H37Rv genome.The amplified gene was cloned into pET24b,generating the expression vector pET-PE35.The expression in E.coli BL21（DE3） was detected by SDS-PAGE.The recombinant PE35 protein was purified using Ni-sepharose.The immunological characteristics of the recombinant PE35 was analyzed by Western-blot.Results The result of restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmids were correct.The recombinant PE35 expression was in the form of solution in E.coli,and the purity of the PE35 was more than 90% after purified by Nisepharose.The purified PE35 was immunologically active and reacted with antibodies in sera from TB patients.Conclusion The recombinant PE35 proteins were successfully expressed and purified,which laid foundation for further study of PE35 in diagnotise and base research of Mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 PE35 可溶性表达 纯化 

分 类 号：R346[医药卫生—基础医学]