肠出血性大肠杆菌O157∶H7 LexA蛋白的表达、纯化及鉴定  

Expression,purification and identification of EHEC O157∶H7 LexA protein

作　　者：沙建平[1] 邹全明[1] 张卫军[1] 杨珺[1] 毛旭虎[1] 郭刚[1] 罗萍[1] 刘开云[1] 陈洪章[1] 

机构地区：[1]第三军医大学医学检验系临床微生物学及免疫学教研室,重庆市生物制药工程技术研究中心,重庆400038

出　　处：《第三军医大学学报》2008年第12期1114-1117,共4页Journal of Third Military Medical University

基　　金：国家高技术研究发展计划(“863”计划)(2001AA215161)~~

摘　　要：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 clone lexA gene from enterohemorrhagic Escherichia coli （EHEC） O157： H7, and express and purify it, and measure its immunogenicity. Methods The objective fragment was amplified from standard EHEC O157：H7 cells by PCR, and then linked with the vector pET22b（ ＋ ）. The objective protein was induced to express in the BL21 （ DE3 ） cells after transfection. The expressed protein was purified by 2 steps of Heparin Agrose chelate affinity chromatography and gel filtration chromatography respectively. The purified lexA protein was used to immune the rabbits to prepare the polyclonal antibody. ELISA and Western blotting was used to detect the obtained serum. Results The expressed protein was found in insoluble form, accounted for 40% of the total bacteria protein. The final purity was 98% , which was determined by HPLC. The obtained protein was confirmed to have sound immunogenicity. Conclusion Our obtained recombinant LexA protein expressed in E. coli BL2.1 （DE3） has good immunological activity.

关 键 词：肠出血性大肠杆菌O157 LEXA 纯化 免疫活性 

分 类 号：R341[医药卫生—基础医学] R378.21