结核分枝杆菌rv3873基因原核表达载体的构建、表达和纯化

Construction and expression of prokaryotic expression vector of mycobacterium tuberculosis rv3873 gene and its purification

目的构建结核分枝杆菌（M．tb）rv3873基因原核表达载体并进行表达。方法用聚合酶链反应（PCR）扩增MTBrv3873基因，并克隆入pGEM～TEasy质粒。测序正确后，再亚克隆入pET30a（＋）质粒，构建pET30a（＋）：rv3873重组体。结果以重组体分别转化DH5a和BL21（DE3）菌后，经0．4mmol／L异丙基硫代-β-D-半乳糖苷（IPTG）诱导，pET30a（＋）：rv3873表达出相对分子质量为47000左右的重组蛋白。SDS-PAGE分析显示，IPTG诱导4h重组蛋白的表达量最高。表达蛋白以包涵体形式存在于胞质中，表达量占全菌蛋白质的50％，Westernblot证实其具有良好的抗原性。经Ni-NTA柱纯化，获得纯度为90％的重组蛋白。结论成功地构建原核表达载体pET30a（＋）：rv3873，并获得重组Rv3873蛋白，为辅助诊断结核菌的感染奠定了基础。

Objective To construct prokaryotic expression vector carrying rv3873 gene and express it in Escheridia coli（E, coli）. Methods The mycobacterium tuberculosis（M, tb） rv3873 gene was amplified by polymerase chain reaction（PCR） and then cloned into plasmid pGEM-T Easy. After sequencing, the gene was cloned into plasmid pET30a（＋ ） to construct recombinant prokaryotic expression vector pET30a（ ＋ ）： rv3873. Results After transformation of the E. coli and induction with 0.4 mmol/L of IPTG, recombinant target protein Rv3873 with Mr 47 000 was expressed in pET30a （＋）： rv3873 system, and the expressed protein was the maximum when induced with IPTG for 4 h; The expressed protein existed in cytoplasm in unsoluble form and amounted to 50% of total protein of E. coll. Western blot analysis showed that the protein had good antigenicity. The purity of the protein purified through the Ni-NTA resin reached 90%. Conclusions The prokaryotic expression vector pET30a （＋）： rv3873 was constructed successfully and the recombinant protein Rv3873 was obtained,which laid the foundation for application in the diagnosis of tuberculosis.