ESAT6蛋白与结核菌素纯蛋白衍生物联合ELISA法诊断结核病的研究

Serodiagnosis Study of ESAT6 Antigen Combination with Purified Protein Derivative for Tuberculosis Patients

目的探讨早期分泌性抗原靶6(early secretory antigenic target-6,ESAT-6)、结核菌素纯蛋白衍生物(purified protein derivative,PPD)联合进行血清学诊断结核病的效果。方法 ESAT6基因被插入到原核表达载体pGEX-5T中,与谷胱甘肽S-转移酶(glutathione S-transferase,GST)融合进行表达。表达的GST-ESAT6融合蛋白经亲和层析和分子筛进行纯化。针对64例结核患者血清和40例健康人血清,进行了ESAT6、PPD抗体反应性实验,评估二者血清学诊断的敏感性和特异性。结果融合表达的ESAT6蛋白保持天然的免疫原性,在Western blot实验中,可以被结核病人血清所识别。PPD抗体诊断敏感性是84.4%,特异性是65%。ESAT6抗体诊断敏感性是68.75%,特异性是95%。ESAT6、PPD-酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)联合诊断,敏感性是93.4%,特异性是95%。结论 ESAT6、PPD抗体联合判断可以提高结核病诊断的准确性。

Objective To investigate the strategy of combination of early secretory antigenic target-6（ESAT6）and purified protein derivative（PPD）tuberculin serodiagnosis.Methods In this study,ESAT6 gene of Mycobacterium tuberculosis（M.tuberculosis）was cloned into the prokaryotic expression vector pGEX-5T with the N-terminal of glutathione S-transferase（GST）,and the fusion GST-ESAT6 protein was expressed and purified from Escherichia coli by affinity chromatography and size chromatography.The antibody reactivity against ESAT6 or PPD with sera from tuberculosis（TB）patients（n=64）and healthy controls（n=40）were implemented with enzyme-linked immuno sorbent assays,respectively.The sensitivity and specificity were calculated.Results The fused ESAT6 protein kept the native property because it could be captured by the sera from TB patient by Western-blot.The combination of ESAT6 and PPD antibodies detection presented 93.4% of sensitivity and 95% of specificity,whereas PPD-antibodies detection showed 84.4% of sensitivity and 65% of specificity and ESAT6-antibodies detection showed 68.75% of sensitivity and 95% of specificity.Conclusion The study suggests that the combined evaluation of ESAT6 and PPD antibodies is capable to improve diagnosis accuracy and can be as a candidate strategy for diagnosis of M.tuberculosis.