结核分支杆菌14KDa蛋白的基因克隆、表达、纯化及其抗原性的研究

Gene cloning, expression, purification and antigenicity analysis of 14 KDa protein of mycobacterium tuberculosis

目的基因克隆、表达、纯化结核分枝杆菌14KDa蛋白,研究其抗原性,评价其在血清学诊断中的价值。方法以结核分枝杆菌H37Rv基因组DNA为模板,应用PCR技术扩增14KDa蛋白基因片断,将其插入到高效表达载体PET-22b(+)上,构建重组质粒PET-22b(+)/14KDa,重组质粒在大肠杆菌中由IPTG诱导表达,表达产物经金属离子鳌合亲和层析方法纯化,免疫印迹和酶联免疫吸附(ELISA)试验分析重组蛋白的抗原性。结果构建了具有正确基因序列的14KDa蛋白重组质粒,在大肠杆菌BL21(DE3)中以可溶性蛋白形式表达,重组蛋白的表达量占菌体蛋白的40.8%,经过一步金属离子鳌合亲和层析后得到纯度为94.3%的目的蛋白。免疫印迹试验结果表明该蛋白能与羊抗结核血清发生特异免疫结合反应。应用ELISA方法对结核血清参考品进行检测,敏感性和特异性分别为75.6%和96.0%。结论获得了能高效表达14KDa蛋白的大肠杆菌工程菌,目的蛋白以可溶性形式表达,该重组蛋白具有良好的免疫原性,可望成为结核血清学的诊断抗原之一。

[Objective] To Clone, express, purify 14 KDa protein of mycobacterium tuberculosis, study its immunological characteristics, and evaluate its potenial value as serological diagnostic reagent of tuberculosis. [Methods] The Gene Coding 14 KDa protein was amplified by polymerase chain reaction （PCR）. The gene was inserted with an high-exprssion vector PET-22 b（＋） to construct the recombinant plasmid PET-22 b（＋）/14 KDa. The recombinant plasmid was transformed into expressive strain E.coli BL21（DE3）. The E.coli BL21（DE3） carrying recombinant plasmid was induced with IPTG. The present form of the recombinant protein in expressive strain was analyzed by SDS-PAGE. The recombinant protein was purified by Nickel affinity chromatography, its immunological characteristics was analyzed by Western blotting and ELISA technology. [Results] The clone was analyzed at the nucleotide lever and showed the same DNA sequence coding for natural 14 KDa protein. The recombinant protein expressed in solubility in E.coli BL21（DE3）. The expressed protein accounted for 40.8% of total bacteria protein. The purity of terget protein was 94.3% by Nickel affinity chromatography, Western blotting assays indicated that the recombinant protein had satisfactory antigenicity. 14 KDa protein detected TB postive and negative refference serumbased on the mechanism of indirect ELISA. Results showed that the sensitivity was 75.6% and the specificity reached to 96%. [Conclusions] The recombinant protein express of solubility in E.coli BL21（DE3） and have satisfactory antigenicity, and may become one of the immunodiagnostic reagents.