论文部分内容阅读
目的对EHEC O157∶H7 LexA进行表达、纯化,并检测其免疫活性。方法lexA基因片段插入表达载体pET22b(+),在E.coliBL21(DE3)中表达。包涵体经洗涤并用8 mol/L尿素溶解,Heparin Agrose亲合柱层析为第一步纯化,Superdex75凝胶过滤层析作为第二步精细纯化,HPLC测定LexA蛋白的浓度,将纯化的LexA蛋白经注射途径免疫家兔,制备兔抗lexA血清,采用免疫双扩、ELISA及Western blot分析LexA的免疫活性。结果lexA以包涵体形式表达,表达产量高达总菌体蛋白的40%左右,经Heparin Agrose亲合柱层析和凝胶过滤层析两步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98%,表达及纯化的lexA具有良好的免疫活性。结论在E.coliBL21(DE3)中表达的LexA蛋白具有良好的免疫活性。
Objective To express and purify EHEC O157:H7 LexA and test its immunocompetence. Methods The lexA gene fragment was inserted into the expression vector pET22b (+) and expressed in E. coli BL21 (DE3). Inclusion bodies were washed and dissolved in 8 mol / L urea, Heparin Agrose affinity chromatography was the first purification, Superdex75 gel filtration chromatography as the second step purification, HPLC determination of LexA protein concentration, the purified LexA protein Rabbits were immunized with rabbit anti-lexA serum by immunization. The immunocompetence of LexA was detected by immunoblotting, ELISA and Western blot. Results The lexA was expressed in inclusion bodies and the expression level was up to about 40% of the total bacterial proteins. The recombinant protein was purified by Heparin Agrose affinity chromatography and gel filtration chromatography. The final purity of the target protein was 98%, expressed and purified lexA has good immunocompetence. Conclusion The LexA protein expressed in E. coli BL21 (DE3) has good immunocompetence.