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

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0.002 μg/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,ELISPOT>ELISA(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。

Usage of CFP10-ESAT6 fusing protein as a ELISA diagnostic method for TB

In this study, we prepared the Mycobacterium tuberculosis CFP10-ESAT6 fusion protein and evaluate its potential value for immunodiagnosis of tuberculosis. The cfp-10 and esat6 gene fragments were amplified respectively and cloned into the pET-28a expression vector. Optimized the expression conditions, His-tagged recombinant CFP10-ESAT6 protein (rCFP10-ESAT6) was purified using nickel affinity chromatography. Rabbit polyclonal antibody was prepared to establish rCFP10-ESAT6 antigen enzyme-linked immunosorbent assay (ELISA) method. We detected 170 tuberculosis sera antibodies and 60 health sera as negative control. Results showed that the recombinant plasmid pET28a-CFP10-ESAT6 target gene sequencing results consisted with the expected design. Soluble expression CFP10-ESAT6 was accounted for 40% of the total bacterial protein in E. coli BL21 (DE3). We used metal chelate affinity chromatography to purify the recombinant proteins directly, which was over 90% purity. Western blot analysis showed the recombinant protein in TB patient sera with specific immune response. CFP10-ESAT6 rabbit polyclonal antibody was in a titer of 1∶8 000. The optimal coating concentration of CFP10-ESAT6 as coating antigen-specific antibodies in serum indirect ELISA was 0.002 μg/mL and the optimal serum dilution was 1∶500. Results' consistency with clinical diagnosis of three methods was ELISPOT>ELISA (rCFP10-ESAT6)>colloidal gold. We established an indirect ELISA method that CFP10-ESAT6 as a coating antigen for TB antibodies in human serum. CFP10-ESAT6 fusion protein was efficiently expressed in pET28a-CFP10-ESAT6/BL21 (DE3), which may be a potential immunological diagnosis of tuberculosis-specific antigens.