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．tbrv2352c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Westernblot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Westernblot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Westernblot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Westernblot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。
Objective To express, purify Mycobactefium tuberculosis （M. tb） rv2352c protein in Ecoli and study its antigenicity. Methods The M. tb rv2352c gene was amplified by PCR and then cloned into the expression plasmid pET30a（＋）. The positive clones were analyzed and transformed into E.. coil BL21 （DE3）. The bacteria expressing Rv2352c were cultured in different concentration of IPTG. The fusion protein was purified by Ni＋- NTA column, detected by SDS-PAGE, and reacted with the sera from tuberculosis patients by Western blot. Rabbits were immunized with purified recombinant Rv2352c proteins. The specific antibody of rabbit serum was detected by enzyme-linked immunosorbent assay （ELISA）, and reacted with the purified recombinant Rv2352c proteins by Western blot. Results It is confirmed that the prokaryotic expression plasmid was correctly constructed by enzyme digestion and sequence analysis. The results of SDS-PAGE and Western blot 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 recombinant proteins Rv2352c identified by Western blot had strong antigenicity. Conclusion The prokaryotie expression recombinant plasmids pET30a（＋）： rv2352e was successfully constructed. The recombinant Rv2352c protein could induce high titer of anti-Rv2352c antibody.
出处
《结核病与胸部肿瘤》
2011年第1期14-18,共5页
Tuberculosis and Thoracic Tumor
基金
结核病诊断分子标识研究（项目编号：2008ZX10003-005）：结核病防治关键技术研究（课题编号：D08050700640805）
关键词
分枝杆菌
结核
细菌蛋白质类
抗原
细菌
重组融合蛋白质类
Mycobactcdum tuberculosis
Bacterial proteins： Antigens, bacterial： Recombinant fusion proteins
