结核分枝杆菌组合蛋白的表达及血清学诊断价值的评价

目的取得能用于结核病血清学诊断的敏感性、高特异性强的组合蛋白。方法用DNA双链全合成的方法合成ESAT6，MPT64，38KD，Ag85B等结核分枝杆菌特异性抗原的抗原表位基因，用PCR法将这些基因片段连接起来成为新型抗原的编码基因。将该编码基因克隆于pET24b载体，经测序鉴定后转化于大肠杆菌BL21菌株用IPTG进行诱导表达。组合蛋白经镍柱纯化后用Western印迹法和酶联免疫实验（ELISA）对其抗原性和特异性进行检测。结果携带重组质粒的菌株经IPTG诱导后以包涵体的形式大量表达组合蛋白，经镍柱纯化后蛋白质浓度达到0．5mg／ml，western实验表明：该组合蛋白能与结核病人血清特异性结合，而不与健康人血清结合。以该组合蛋白为包被抗原采用ELISA法检测结核抗体的灵敏度为61％，特异性为97．3％。PPD检测的灵敏度和特异性分别为73．7％和91．8％。该组合蛋白检测卡介苗接种阳转血清的阳性率为2．1％，特异性为97．9％，而PPD检测卡介苗接种阳转血清的阳性率为22．3％，特异性为79．7％。结论该组合蛋白用于结核病血清学诊断具有较高的灵敏度和特异性，能够为结核病的临床诊断提供一定的指导。

The study on the Expression of recombination protein of M.tuberculosis and the evaluation on its serological diagnostic value

Objective to obtain a recombinant protein that can be used for TB serological diagnosis with high sensitivity and high specificity. Methods we synthesized the Mycobacterium tuberculosisspecific antigen esat6 ,MPT64 , 38KDa, Ag85B epitope coding genes , these gene fragments were connected to a new antigen coding genes by PCR. The PCR products was cloned intopET24b expression vector correctly, and can express recombinant protein by IPTG induction in E. coli BL21. The recombinant protein was by purified through nickel column, the sensitivity and specificity of the recombinant protein in tuberculosis serological diagnosis was evaluated by Western blot and enzyme - linked immunosorbent assay （ELISA）. Results The E. coli Strain carrying the recombinant plasmid express the recombinant protein in the form of inclusion bodies after IPTG induction , the concentration of purified recombinant protein can achieve 0. 5 mg/ml through nickel column . The western experiments show that： the recombinant protein reacted with tuberculosis serum specifically, can not react with healthy human serum. The ELISA results were ： the sensitivity was 61% and specificity was 97.3% in detectig tuberculosis antibodies in tuberculosis patients when the recombinant protein was used as coating antigen , otherwise PPD detection sensitivity and specificity were 73.7% and 91.8% respectively. In detecting serum of BCG vaccination skin test conversion positive, The sensitivity and specificity of recombinant protein were 2. 1% and 97.9%, but The sensitivity and specificity of PPD were 22.3% and 79. 7% respectivly. Conclusion The recombinant protein in TB serological diagnosis with high sensitivity and specificity, can provide some guidance for the clinical diagnosis of TB.