摘要
目的构建结核分枝杆菌CFP10-ESAT-6-CFP21(CE21)融合蛋白的重组质粒,通过IFN-γ的检测探讨CE21在结核病检测中的价值。方法通过聚合酶链式反应(PCR)扩增获得CFP21和CFP10-ESAT-6(CE)基因片段,分别连接到pMD18-T,并将两片段2次连接到表达载体pET21a(+)中,获得pET21a-CFP10-ESAT-6-CFP21重组质粒;经表达纯化和复性后,去除内毒素,利用酶联免疫吸附试验检测48例结核病患者、30例非结核肺部疾病患者及32例健康对照者全血中IFN-γ的释放量。结果CE21经SDS-PAGE分析后相对分子质量为45,与预期大小一致。CE21融合蛋白全血标本IFN-γ的检测,结核组阳性率为72.9%,非结核疾病对照组和健康对照组阳性率分别为3.3%和3.1%。结论成功获得CE21融合蛋白,IFN-γ的检测证明该融合抗原具有良好的结核病检测价值。
Objective To construct a recombinant plasmid of CFP10-ESAT-6-CFP21 （CE21） fusion protein of Mycobacterium tuberculosis and evaluate the diagnosis value of CE21 fusion protein to tuberculosis based on IFN-7 detecting. Method The genes encoding CFP21 and CFP10-ESAT- 6（CE） protein were amplified by PCR, cloned into vector pMD18-T respectively and then cloned into expression vector pET21a （＋） twice. Recombinant pET21a-CFP10-ESAT-6-CFP21 plasmid was obtained and transformed into E. coli BL21（DE3）. After expression, purification, refolding and removing endotoxin, CE21 fusion protein was used as a stimulating antigen to detect the concentration of IFN-γ in whole blood samples of TB patients （n=48）, other pulmonary disease patients （n=30） and healthy subjects （n=32） by ELISA. Results The relative molecular weight of the CE21 fusion protein was approximately 45 analyzed by SDS-PAGE, consistent with anticipation.According to the results of IFN-γ detection in whole blood samples, the detecting positive rate of CE21. fusion protein in TB patients was 72.9%, and that in non-TB disease and healthy controls were 3.3% and 3.1%, respectively. Conclusion Recombinant CE21 fusion protein was obtained successfully. It can be concluded that the fusion protein had a promising diagnostic value of tuberculosis based on the results of IFN-γ detection.
出处
《中国防痨杂志》
CAS
2010年第4期211-216,F0003,共7页
Chinese Journal of Antituberculosis
基金
国家科技重大专项课题资助项目(2008ZX10003003-02)
上海市公共卫生重点学科建设项目(08GWZX0104)
上海市科科资助项目(07DZ22012
08DZ2291500
08DZ2271300)
关键词
分枝杆菌
结核
重组融合蛋白质类
抗原
细菌
干扰素Ⅱ型
Mycobacterium tuberculosis
recombinant fusion proteins
antigens, bacterial
interferon type Ⅱ
