CFP10-ESAT6融合蛋白用于结核分枝杆菌感染诊断ELISA方法的初步研究

发布时间：2018-06-23 21:05

本文选题：结核分枝杆菌 + CFP-ESAT　；参考：《中国人兽共患病学报》2015年04期

【摘要】：目的制备结核分枝杆菌CFP10-ESAT6融合蛋白,探讨CFP10-ESAT6融合蛋白在结核分枝杆菌免疫诊断中的应用价值。方法 PCR扩增得到CFP-10、ESAT6基因片断,将其先后克隆入pET-28a表达载体。优化表达条件、用镍亲和层析的方法纯化带His标签的重组CFP10-ESAT6融合蛋白(rCFP10-ESAT6)。制备兔多克隆抗体,建立rCFP10-ESAT6为抗原的酶联免疫吸附试验(ELISA)方法,以健康者血清为对照,检测170例结核患者血清的抗体。结果重组质粒pET28a-CFP10-ESAT6靶基因测序结果与预计设计完全一致。融合蛋白在E.coli BL21(DE3)工程菌中均为可溶性表达,表达量占菌体总蛋白的40%。利用金属螯合亲和层析直接纯化重组蛋白,纯度达到95%以上。Western blot分析表明,重组蛋白能与结核患者血清发生特异性免疫结合反应,与健康者血清不发生反应。CFP10-ESAT6的兔多克隆抗体的效价为1∶8 000,CFP10-ESAT6作为包被抗原的检测结核病人血清中特异性抗体的间接ELISA最佳包被浓度为0.002μg/mL,患者血清的最佳稀释度为1∶500。检测结果与临床诊断的符合率,ELISPOTELISA(rCFP10-ESAT6)ELISA(kit)。结论CFP10-ESAT6融合蛋白具有较高的抗原特异性和免疫反应性,有可能成为结核病免疫学诊断的特异抗原,对结核的诊断具有重要参考价值。
[Abstract]:Objective to prepare the CFP10-ESAT6 fusion protein of Mycobacterium tuberculosis and to explore the value of CFP10-ESAT6 fusion protein in the immunological diagnosis of Mycobacterium tuberculosis. Methods CFP-10 ESAT6 gene fragment was amplified by PCR and cloned into pET-28a expression vector. The recombinant CFP10-ESAT6 fusion protein (rCFP10-ESAT6) with his label was purified by nickel affinity chromatography. An enzyme-linked immunosorbent assay (Elisa) with rCFP10-ESAT6 as antigen was developed to detect antibodies in 170 tuberculosis patients. Results the sequencing results of the recombinant plasmid pET28a-CFP10-ESAT6 were in good agreement with the predicted design. The fusion protein was soluble expressed in E. coli BL21 (DE3) engineering strain and accounted for 40% of the total protein in E. coli BL21 (DE3). The recombinant protein was purified directly by metal chelate affinity chromatography. The purity of the recombinant protein was over 95%. Western blot analysis showed that the recombinant protein could react specifically with the serum of tuberculosis patients. The titer of rabbit polyclonal antibody against CFP10-ESAT6 was 1:8, 000 CFP10-ESAT6 as coating antigen. The best indirect Elisa coating concentration of specific antibody in TB patients was 0.002 渭 g / mL, and the optimal dilution of patient serum was 1: 500. The coincidence rate of the results with clinical diagnosis: ELISPOTELISA (rCFP10-ESAT6) Elisa (kit). Conclusion CFP10-ESAT6 fusion protein has high antigen-specificity and immunoreactivity, which may be a specific antigen for the immunological diagnosis of tuberculosis and has important reference value for the diagnosis of tuberculosis.
【作者单位】：上海市公共卫生临床中心;
【基金】：上海市卫生局面上基金(No.20124467) 国家自然科学基金青年基金(No.31200108) 十二五科技重大专项(No.2013ZX10004-221和2013ZX10004221-004)~~
【分类号】：R446.6;R52

【参考文献】