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目的评价结核分枝杆菌蛋白抗原Rv2654c、Rv1985c和Rv3868的血清诊断价值和潜在应用前景。方法以结核分枝杆菌H37Rv标准株全基因组DNA为模板扩增得到Rv2654c、Rv1985c和Rv3868基因的完整序列,并与表达载体p ET-32a构建重组质粒。原核表达Rv2654c、Rv1985c和Rv3868蛋白,利用亲和层析的方法进行纯化。采用棋盘滴定的方法,确定各抗原的酶联免疫吸附试验(ELISA)最佳反应条件,并应用190份血清进行血清Ig G抗体检测,结合受试者工作特征曲线(ROC)对其诊断效能进行分析和评价。通过ROC计算各抗原组合的诊断效能,确定最佳组合方案。结果成功构建了重组蛋白,对重组后的片段进行测序,经BLAST比对与目的基因完全一致,且能够稳定表达。对经纯化后的3种蛋白进行ELISA检测,经统计学分析,Rv2654c、Rv1985c和Rv3868抗原的诊断效能分别达到73.16%、56.84%和71.05%。结合ROC得到最佳的抗原组合方案为Rv2654c+Rv3868,其敏感性、特异性和诊断效能分别达到78.95%、72.63%和75.79%。结论结核分枝杆菌重组抗原Rv2654c、Rv1985c和Rv3868具有作为结核病诊断抗原的潜力,可以作为结核病免疫学快速诊断的候选蛋白。抗原组合Rv2654c+Rv3868具有较高的诊断效能,有较好的潜在应用价值。
Objective To evaluate the serum diagnostic value and potential application of Mycobacterium tuberculosis protein Rv2654c, Rv1985c and Rv3868. Methods The complete sequence of Rv2654c, Rv1985c and Rv3868 genes was amplified by using the complete genomic DNA of Mycobacterium tuberculosis H37Rv as a template and the recombinant plasmid was constructed with the expression vector p ET-32a. Prokaryotic expression of Rv2654c, Rv1985c and Rv3868 protein, using affinity chromatography purification. The optimal reaction conditions of enzyme-linked immunosorbent assay (ELISA) for each antigen were determined by chessboard titration. Serum Ig G antibody was detected by using 190 serum samples. Combined with the receiver operating characteristic curve (ROC), its diagnostic efficacy Analysis and evaluation. Calculate the diagnostic efficiency of each antigen combination by ROC to determine the optimal combination plan. Results The recombinant protein was successfully constructed, and the recombinant fragment was sequenced. The result of BLAST analysis was identical with that of the target gene and was stably expressed. The purified three proteins were detected by ELISA. The statistical analysis showed that the diagnostic efficiency of Rv2654c, Rv1985c and Rv3868 antigen were 73.16%, 56.84% and 71.05% respectively. The optimal combination of antigen and ROC for ROC was Rv2654c + Rv3868, with the sensitivity, specificity and diagnostic efficiency of 78.95%, 72.63% and 75.79% respectively. Conclusion Mycobacterium tuberculosis recombinant antigens Rv2654c, Rv1985c and Rv3868 have potential as diagnostic antigen for tuberculosis and can be used as candidate proteins for rapid diagnosis of tuberculosis immunology. Antigen combination Rv2654c + Rv3868 has high diagnostic efficacy, has a good potential value.