结核分支杆菌ESAT6蛋白的表达、纯化及抗原性研究

目的：获得重组ESAT6蛋白，研究其免疫学特性，评价其在结核病血清学诊断中的价值。方法：分别用生理盐水和卡介苗免疫小鼠8周后，以结核分支杆菌接种小鼠。应用基因工程技术表达、纯化ESAT6蛋白，分别以结核分支杆菌PPD或纯化的rESAT6蛋白为抗原，通过ELISA方法及结核病一步法检测小鼠或人血清中抗结核抗体。结果：重组质粒pET24b-ESAT6在大肠杆菌BL2（DE3）细胞内以包涵体形式存在，分子量约6kDa，每100ml培养菌可获得约18mg纯化的重组蛋白。生理盐水和卡介苗免疫小鼠血后血清中抗ESAT6抗体无区别。结论分支杆菌攻击后，两组小鼠血清中抗ESAT6抗体均明显升高，但只有卡介苗组小鼠抗体水平超过平均值＋2标准误。以33例正常人血清的OD值＋2S为正常界限值，33例正常人和32例结核病人血清一步法和PDD、rESAT6 ELISA检测抗结核抗体的特异性和敏感性分别为：一步法87.9%(29/33),65.6%(21/32),PPD93.9%(31/33),62.5%(20/32);rESAT6 97%(32/33),18.2%(4/32)。结论：pET24b-ESAT6大肠杆菌工程菌能以包涵形式高效表达重组ESAT6蛋白，卡介苗免疫对血清中ESAT6抗体无影响，结核病人血清中抗ESAT6抗体阳性率低，rESAT6纯化蛋白可作为结核病血清学诊断的混合抗原之一。

Study on Expression,Purifing and antigenicity of Esat6 Protein of Tuberculosis Mycobacterium

Objective:To obtain the recombinant ESAT6 protein,to study its immunological characteristics,and to evaluate its potential value for serodiagnosis of tuberculosis.Methods:The mice were immunized with the saline or M.bovis BCG ,and were then infected by intraperitoneal injection M.tuberculosi strain H 37 Rv.The gene coding ESAT6 protein inserted into a expression vector pET-24b,and then transferred to E.coli BL21(DE3).The expressed product was purified by metal chelation chromatography.Mouse and human serum was detected the antibodies against M.tuberculosis rESAT6 and PPD antigens by ELISA,and antibodies against M.tuberculosis antigens by tuberculosis one-step test.Results:The recombinant ESAT6 protein existed in inclusion bodies of E.coli. Its molecular weight was about 6 kilodalton.18mg of rESAT6 protein per 100ml culture could be obtained.The level of ESAT6-specific antibodies between the mouse group saline and group BCG had no difference.After the mice were infected,ESAT6-specific antibodies incresed,but the level of antibodies in gropu BCG was more higher than that in group saline.The positive cutoff values were OD 492 plus 2 standard deviation of 33 negative serum detected by ELISA.Of 33 serum from tuberculosis patients,the specificity and sensitivity of PPD as antigen of ELISA were 93.9% and 62.5%.That of rESAT6 were 97% and 18.2%,and that of tuberculosis one-step test were 87.9% and 65.6%,respectively.Conclusions:The recombinant ESAT6 protein could be highly expressed in the form of inclusion bodies in E.coli.M.bovis BCG vaccination did not effected on ESAT6-specific antibodies in serum.The positive rate of antibodies aganist M.tuberculosis rESA6 in the tuberculosis serum was lower.It might be used as one of serodignostic antigen of tuberculosis.