Abstract: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.
许艳, 陈海丽, 刘晓茜, 熊延青, 席秀红, 宰淑蓓, 蔡金凤, 卢水华, 朱召芹. 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[J]. 中国人兽共患病学报, 2015, 31(4): 315-319.
XU Yan, CHEN Hai-li, LIU Xiao-qian, XIONG Yan-qing, XI Xiu-hong, ZAI Shu-bei, CAI Jin-feng, LU Shui-hua, ZHU Zhao-qin. Usage of CFP10-ESAT6 fusing protein as a ELISA diagnostic method for TB. Chinese Journal of Zoonoses, 2015, 31(4): 315-319.
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