【作者】 曹廷明； 贾红彦； 古淑香； 郑晓静； 李自慧； 杜凤娇； 刘洋； 刘忠泉； 邢爱英； 杜博平； 马玙； 张宗德；

【Author】 Cao tingming,Jia Hongyan,Gu Shuxiang,Zhen xiaojing,Li Zihui,Du fengjiao,Liu yang,Liu Zhongquan,Xing Aiying,Du Boping,Ma Yu,Zhang Zongde Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149

【机构】 北京市结核病胸部肿瘤研究所；

【摘要】 目的构建含结核分枝杆菌(M.tb)rv2352c基因原核表达载体,经转化E.coli以表达Rv2352c融合蛋白,并研究其抗原性。方法用PCR扩增M.tb rv2352c基因,克隆入pET30a(+)质粒,构建pET30a(+):rv2352c重组质粒,阳性克隆测序验证正确后转化入表达宿主大肠杆菌BL21(DE3),经IPTG诱导Rv2352c蛋白表达。经Ni~+-NTA层析柱纯化融合蛋白,通过SDS-PAGE和结核患者血清Western blot进行鉴定。将纯化的重组蛋白分别免疫家兔,制备抗Rv2352c抗血清,抗血清的效价测定采用酶联免疫吸附试验法(ELISA),取兔抗血清与纯化蛋白Rv2352c通过Western blot方法,检测抗体特异性。结果经酶切鉴定和测序分析证实rv2352c原核表达质粒构建正确,SDS-PAGE和Western blot结果显示,在45kD处呈现单一蛋白条带。用重组蛋白Rv2352c免疫接种后可诱导出高滴度的特异性抗体。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(+):rv2352c,制备和纯化的Rv2352c融合蛋白具有较好的纯度和生物学功能,为进一步研究结核病的潜在分子标志物奠定基础。更多还原

【Abstract】 Objective To construct prokaryotic expression vector carrying Mycobacterium tuberculosis(M.tb)rv2352c gene and express it in E.coli and study its antigenicity.Methods The M.tb rv2352c gene was amplified by PCR and then cloned into the expression plasmid pET30a(+),positive clones were analyzed and transfonned into BL21(DE3).The Rv2352c expressing bacteria were cultured in different concentration of IPTG.The fusion protein was purified by Ni~+-NTA column and detected by SDS-PAGE and Western blot with serum from tuberculotic patients.Rabbits were immunized with purified recombinant Rv2352c proteins. Enzyme-Linked Immunosorbent Assay(ELISA)was used to detect the specific antibody titer of rabbit serum.Then the purified recombinant Rv2352c proteins were used to detect antibody in rabbit serum,which had been immunized by Western blot.Results Digestion and sequence analysis confirmed that the identification of prokaryotic expression plasmid was correct.The SDS-PAGE and Western blot results showed that the molecular weight of Rv2352c fusion protein was about 45 kD.The specific IgG antibody was found in serum of rabbit immunized by recombinant Rv2352c protein.The antigenicity of the recombinant proteins Rv2352c was strong, as identified by Western blot.Conclusion The prokaryotie expression recombinant plasmids pET30a(+):rv2352c was successfully constructed,recombinant Rv2352c protein is produced, which induces high titer of anti Rv2352c antibody.All of these results provide a foundation for further studying potential biomarkers of tuberculosis.更多还原