结核分枝杆菌特异性蛋白片段Rv3388和6种特异抗原在结核抗体检测中的应用价值

目的 对结核分枝杆菌( MTB) PE家族的所有成员的结构进行分析,预测其抗原表位,截取Rv3388蛋白的优势抗原片段,对重组Rv3388蛋白及其它6种MTB特异性抗原进行评估,探讨不同结核特异性抗原与血清抗体的反应模式,评价血清学检测在结核病诊断中的价值. 方法 利用基因合成技术重叠延伸PCR扩增Rv3388蛋白637~731位的编码基因片段, 原核表达并纯化重组蛋白pET32a/Rv3388637-731 ,将纯化的重组蛋白免疫BALB/c小鼠,采用间接ELISA法对该抗血清进行免疫原性分析. 同时对这7种MTB特异性蛋白的特异性及敏感性进行评价. 结果 重组蛋白pET32a/Rv3388637-731在大肠杆菌中的表达量占全菌蛋白的80%,ELISA显示有较强的免疫原性. 7 种 MTB特异性抗原具有不同的反应模式,单个抗原检测敏感性较差. 结论 对MTB PE家族的蛋白结构分析,表达并纯化重组蛋白pET32a/ Rv3388637-731 ,7种蛋白在血清抗体检测中具有抗原互补性,不同抗原与机体反应存在不同反应模式,提高结核抗体检测敏感性应多种抗原联合检测.

Assessment of the detection effect of specific protein fragment of Rv3388 and six kinds of antigens in tuberculosis antibody response

Objective To analyze the structure of all members in the PE family of Mycobacterium tuberculosis ( MTB)and predict the antigen epitope, we chose the dominant antigen fragments of Rv3388 protein and estimate the anti-genicity of not only the recombinant Rv3388 protein but aslo the other six kinds of specific antigens of MTB. To in-vestigate the reaction model between the different tuberculosis specific antigen and serum antibody, and to evaluate the value of serological detection in the diagnosis of tuberculosis. Methods The gene coding 637~731 amino acid fragment of Rv3388 was amplified by over-lap extension-PCR. The prokaryotic expressed and purified recombinant protein pET32a/Rv3388637-731 was utilized to immunize BALB/c mice, and the immunogenicity of the antiserum and the specificity and sensitivity of seven kinds specific proteins of MTB were analyzed by indirect ELISA. Results The amount of recombinant protein pET32a/Rv3388637-731 expressed in E. coli was 80% of the total protein. The results of ELISA showed strong immunogenicity. The reaction patterns of antigens were different with each oth-er, and the detection sensitivity of single antigen was poorer. Conclusion The structure of all members in the PE family of MTB is analysed, and the recombinant protein pET32a/Rv3388637-731 is expressed and purified success-fully. Seven kinds of proteins in the serum antibody are antigen complementary in the detection. Different antigen is different reaction pattern. A variety of antigens should be jointly detected to improve the sensitivity of antibody de-tection of tuberculosis.