出血性大肠杆菌O157 eae-stx1/2B融合基因的构建和表达  

Construction and Expression of eae-stx1/2B Fusion Gene from EHEC O157∶H7

作　　者：陈萍[1] 刘军[1] 祝令伟[1] 孙洋[1] 郭学军[1] 齐翀[1] 冯书章[1] 郑明光[1] 

机构地区：[1]解放军军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2005年第3期270-272,共3页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30270985)

摘　　要：构建表达eae和stx1/2B的融合基因,克隆eae基因的C端280个氨基酸残基(Int280)基因部分,以正确地阅读框定向插入到含有stx1/2B融合基因的质粒,构建重组质粒,将其转化于BL21(DE3),用IPTG进行诱导表达,经SDS-PAGE电泳检测,该融合蛋白获得了高效表达。薄层扫描分析表明目的蛋白表达量占菌体总蛋白含量的50.67%。由于该融合蛋白由eae、stx1B、stx2B等三部分抗原组成,可刺激机体产生针对紧密素和StxB的抗体,在E-HECO157亚单位疫苗设计或单克隆抗体抗制备中具有重要价值。To construct eae-stx1/2B fusion gene,the DNA fragment encoding C-terminal 280 amino acids(Int280) of intimin was fused in frame with the stx1/2B gene.We created a recombinant plasmid by insertion of eae-stx1B-stx2B fusion gene into pET-28a,the recombinant plasmid was transformed into BL21(DE_3),and the fusion protein was expressed in E.coli BL21 efficiently with IPTG.Analysis of SDS-PAGE and thin layer scanning results showed that amount of expressed fusion protein was 50.67% in total of bacteria protein.Because the fusion protein was consisted of eae Int280,stx1B and stx2B antigens,it can evoke immune responses against Int280,Stx1B and Stx2B.The eae-stx1/2B fusion protein expressed in E.coli could offer an important ingredient that should be consider in construction of recombinant EHEC O157 vaccine or preparation of experimental material for the monoclonal antibody.

关 键 词：大肠杆菌O157 融合基因 构建 出血性 融合蛋白 氨基酸残基 总蛋白含量 单克隆抗体 亚单位疫苗 重组质粒 诱导表达 IPTG 电泳检测 PAGE 高效表达 目的蛋白 薄层扫描 E基因 SDS 表达量 分析表 C端 

分 类 号：Q78[生物学—分子生物学] TS207.4[轻工技术与工程—食品科学]