夏远志, 王雪枝, 刘海灿, 李马超, 赵秀芹, 楼永良, 吕建新, 万康林. 结核分枝杆菌抗原Rv2654c、Rv1985c和Rv3868的克隆表达及血清诊断学初步评价[J]. 疾病监测, 2016, 31(4): 272-277. DOI: 10.3784/j.issn.1003-9961.2016.04.004
引用本文: 夏远志, 王雪枝, 刘海灿, 李马超, 赵秀芹, 楼永良, 吕建新, 万康林. 结核分枝杆菌抗原Rv2654c、Rv1985c和Rv3868的克隆表达及血清诊断学初步评价[J]. 疾病监测, 2016, 31(4): 272-277. DOI: 10.3784/j.issn.1003-9961.2016.04.004
XIA Yuan-zhi, WANG Xue-zhi, LIU Hai-can, LI Ma-chao, ZHAO Xiu-qin, LOU Yong-liang, LYU Jian-xin, WANG Kang-lin. Cloning expression of antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis and value in serological diagnosis of tuberculosis[J]. Disease Surveillance, 2016, 31(4): 272-277. DOI: 10.3784/j.issn.1003-9961.2016.04.004
Citation: XIA Yuan-zhi, WANG Xue-zhi, LIU Hai-can, LI Ma-chao, ZHAO Xiu-qin, LOU Yong-liang, LYU Jian-xin, WANG Kang-lin. Cloning expression of antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis and value in serological diagnosis of tuberculosis[J]. Disease Surveillance, 2016, 31(4): 272-277. DOI: 10.3784/j.issn.1003-9961.2016.04.004

结核分枝杆菌抗原Rv2654c、Rv1985c和Rv3868的克隆表达及血清诊断学初步评价

Cloning expression of antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis and value in serological diagnosis of tuberculosis

  • 摘要: 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pET-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IgG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。

     

    Abstract: Objective To evaluate the value of three antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis in the serological diagnosis of tuberculosis (TB). Methods The whole genes of Rv2654c, Rv1985c and Rv3868 were amplified from the genomic DNA of M. tuberculosis strain H37Rv by using PCR and the recombinant plasmid was constructed with expression vector pET-32a. The proteins were expressed by prokaryotic expression and purified by affinity chromatography. The best reactive conditions of enzyme linked immunosorbent assay (ELISA) of each antigen were determined by chessboard titration method. The antibodies IgG against M. tuberculosis were tested with 190 serum samples by using ELISA and the diagnostic efficiency of each antigen was analyzed by means of receiver operating characteristic curve (ROC). Results The results showed that the recombinant antigens were cloned and expressed stably and the DNA sequences of the protein antigens were completely consistent with the sequences in Genbank of NCBI respectively by BLAST alignment. The serological results of the three antigens which had been purified were analyzed with statistical software. The diagnostic efficiency of Rv2654c, Rv1985c and Rv3868 was 73.16%, 56.84% and 71.05% respectively. The best combination from the three antigens was Rv2654c+Rv3868, the sensitivity, specificity and efficiency were 78.95%, 72.63% and 75.79%. Conclusion The recombinant antigens Rv2654c, Rv1985c and Rv3868 have good diagnostic value and can be applied as candidate antigen for TB diagnosis. The combination of antigens Rv2654c and Rv3868 has good performance in TB diagnosis and possesses a certain application value in clinical practice.

     

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