结核分枝杆菌特异性分泌蛋白的原核表达、纯化和免疫原性分析

Prokaryotic Expression, Purification, and Immunogenicity Analysis of Mycobacterium tuberculosis Specific Excretive Proteins

目的构建结核分枝杆菌rv1837c．rv3803c基因原核表达重组质粒，进行表达、纯化，并分析其免疫原性。方法PCR扩增结核分枝杆菌H37Rvrv1837c．rv3803c基因，并克隆入pTA2质粒。测序正确后，再亚克隆入pET30a（＋）质粒，构建pET30a（＋）：rv1837c．pET30a（＋）：rv3803c重组体。然后转化人表达宿主大肠杆菌BL21（DE3），经0．4mmol／L异丙基硫代-β—D-半乳糖苷诱导1分别与组氨酸标签单克隆抗体及结核患者血清进行Western—blot，鉴定Rv1837c、Rv3803c重组蛋白。经镍离子螯和氮川乙酸一组氨酸标签亲和树脂纯化，将纯化的重组蛋白分别免疫家免，取兔血清与纯化蛋白通过Western-blot方法，检测家兔血清中的抗体。结果pET30a（＋）：rv1837c．pET30a（＋）：rv3803c重组体表达相对分子质量为92000及38000的重组蛋白，表达蛋白以包涵体形式存在于胞质中，表达量分别占全菌蛋白质的30％及50％。获得纯度为90％的重组蛋白。纯化蛋白通过Western-blot鉴定证实为目的蛋白，有较强的免疫原性。结论成功构建原核表达重组质粒pET30a（＋）：rv1837c,pET30a（＋）：rv3803c，并获得Rv1837c及Rv3803c重组蛋白，为血清学诊断活动性结核病奠定了基础。

Objective To obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity. Methods The Mycobactetium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction,and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a（＋）. The resulting plasmid, named pET30a（＋）：rv1837c and pET30a（＋）7v3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a（＋）：ra1837c and pET30a（＋）rv3803c were introduced intoE coil BL21 （DE3） by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western-blot with hexa-histidine tag antibody and serum from tuberculosis patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin.Rabbits were immunized with purified recombinant Rv1837c and Rv3803c protein. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbits serum, which had been immunized by Western-blot. Results After transformation of the E. coil and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c （ relative molecular mass： 92 000） and Rv3803c （ relative molecular mass： 38 000） were expressed in pET30a （ ＋ ） ： rv1837c and pET30a （ ＋ ） ： rv3803c system.The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins ofE. coli. The purity of the purified protein reached 90%. The Immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Westernblot. Conclusion The prokaryotic expression recombinant plasmids pET30a（＋）：rv1837c, pET30a（＋）wv3803c was successfully constructed and the recombinant proteins Rv 1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.