结核分枝杆菌ESXB-ESXA融合蛋白的表达及其作为检测抗原的初步应用

目的:探索一种大量表达结核分枝杆菌ESXB-ESXA融合蛋白的方法,分析其抗原性,评价其在结核抗体检测中的初步应用。方法:采用PCR方法从结核分枝杆菌基因组中分别扩增esxB和esxA基因,用Linker(G4S)3连接2条目的片段,连接pET-32a(+)载体,构建ESXB-ESXA融合蛋白表达载体;重组表达载体转化宿主细胞大肠杆菌BL21(DE3),IPTG诱导表达、纯化,Western印迹分析重组蛋白的抗原性;建立以融合重组蛋白为抗原的ELISA和胶体金检测方法,分析其在结核病抗体检测中的应用。结果:构建了ESXB-ESXA融合蛋白的表达载体,重组ESXB-ESXA在大肠杆菌中得以高效表达,表达量占菌体总蛋白的70%以上;Western印迹分析表明该重组蛋白具有较好的抗原性;ELISA和胶体金检测结果显示,用重组ESXB-ESXA抗原检测临床结核病患者有较好的特异性。结论:重组ESXB-ESXA融合蛋白表达量高并具有较好的抗原性,可作为结核病抗体检测的备选抗原。

Expression and Priliminary Application of Mycobacterium tuberculosis ESXB-ESXA Fusion Proteins

Objective： To recombinantly produce, purify ESXB-ESXA fusion proteins of Mycobacterium tuberculosis, and evaluate its potential values for tuberculosis（TB） serodiagnosis. Methods： esxA and esxB genes were amplified by PCR from M.tuberculosis genome. Linker （G4S）3 was used to link esxA and esxB. The esxB-esxA fusion genes were cloned into pET-32a（＋） vectors and transformed into E.coli BL21（DE3） cells. The recombinant ESXBESXA proteins were expressed by IPTG induction, then purified, denatured and renatured. Its antigenicity was analyzed by Western blotting with TB patient serum. Potential values for TB serodiagnosis were evaluated by ELISA and immunochromatographic assay（ICA）. Results： Results of SDS-PAGE and Western blotting showed that the recombinant ESXB-ESXA fusion proteins are highly expressed in E.coli and have good antigenicity. ELISA and ICA results indicated that the recombinant ESXB-ESXA fusion protein has good antigenicity and specificity. Conclusion： The recombinant ESXB-ESXA fusion protein used in this study can be utilized for TB serodiagnosis.