结核分枝杆菌H_(37)Rv株Rv0341编码基因iniB的克隆与表达

Cloning and Expression of iniB Gene Encoding Rv0341 Protein of Mycobacterium tuberculosis H_(37)Rv Strain

目的克隆并表达编码结核分枝杆菌H37Rv株Rv0341蛋白的iniB基因。方法利用PCR从结核分枝杆菌H37Rv株中扩增iniB基因,克隆于pET-22b(+)原核表达载体,转化大肠杆菌BL21(DE3),经IPTG诱导表达,镍离子亲和层析柱纯化后,通过SDS-PAGE和Western-blot鉴定目的蛋白的表达及反应原性。结果克隆并表达了iniB基因,表达的蛋白相对分子质量约43000,诱导2h表达量较高,约为25%。目的蛋白主要以包涵体形式存在于超声沉淀中,纯化后蛋白纯度可达98%以上。经Western blot检测,纯化蛋白与依赖利福平结核分枝杆菌(依R菌)肺结核患者血清呈现强阳性反应,而与非依R菌肺结核患者血清不反应。结论已成功克隆并表达了编码结核分枝杆菌H37Rv株Rv0341蛋白的iniB基因,表达的重组蛋白具有反应原性及特异性,为依R菌结核病临床血清学快速诊断方法的建立及试剂盒的研发奠定了基础。

Objective To clone and express the iniB gene encoding iniB protein of Mycobacterium tuberculosis H37Rv strain. Methods Amplify iniB gene from M. tuberculosis H37Rv strain by PCR and clone into prokaryotic expression vector pET-22b （ ＋ ）. Transform the constructed recombinant plasmid pET-22b （＋）-iniB to E. coli BL21 （DE3） for expression under induction of IPTG. Purify the expressed product by nickel ion affinity chromatography and identify by SDS-PAGE and Western blot. Results The iniB gene fragment at a length of 1 440 bp was cloned, and the target protein with a relative molecular mass of about 43 000 was expressed. The expression level of iniB protein in E. coli 2 h after induction reached about 25%. The expressed product mainly existed in a form of inclusion body and reached a purity of more than 98% after purification. Western blot proved that the purified expressed protein showed strong positive reaction with the sera of patients with tuberculosis caused by rifampin-dependent M. tuberculosis, while showed no reaction with those caused by rifampin-independent M. tuberculosis. Conclusion The iniB gene encoding iniB protein of M. tuberculosis H37Rv strain was successfully cloned and expressed, and the expressed protein showed reactogenicity and specificity. It laid a foundation of development of method and kit for rapid serological diagnosis of tuberculosis caused by rifampindependent M. tuberculosis.