出血性大肠杆菌O157:H7的Tir与stx1/2B融合基因的构建和表达  

Construction and Expression of Recombinant Gene Containing Tir and Stx1/2B of Enterohemorrhagic Escherichia Coli O157:H7

作　　者：张雪寒[1] 何孔旺[1] 卢维彩[1] 俞正玉[1] 茅爱华[1] 刘亚栋[1] 

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014

出　　处：《内蒙古农业科技》2009年第3期59-61,共3页Inner Mongolia Agricultural Science and Technology

基　　金：国家支撑计划(2007BAD40B01)

摘　　要：为了更好防治由出血性大肠杆菌O157:H7引起的疾病,尝试研制基因工程疫苗,其中亚单位疫苗具有很大优势。方法:构建表达tir和stxb的融合基因,将tir基因中间295个氨基酸残基(Tir295)与stxB亚基基因72个氨基酸串联,构建pET28a-stx2b-Tir295-stx1b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。结果:薄层扫描分析表明,目的蛋白表达量占菌体总蛋白含量的30%左右。结论:由于该融合蛋白由Tir、stx1b和stx2b三部分抗原组成,可刺激机体产生针对转位紧密素受体(Tir)和志贺毒素(stx)的抗体,在EHECO157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 tir-stx fusion gene, the DNA fragments encoding middle 295 amino acids （Tir295）of tir and 72 amino acids （Stx72）of Stxlb and Stx2b were fused in turn in frame with the multiple cloning sites of pET-28a, and the recombinant plasmid of pET28a- Stx2b-Tir-Stxlb was transferred to host cells E.coli BL21 strain （DE3）. Then, the protein was induced and expressed with induction of IPTG. The expression quantities and style of fusion protein were determined by SDS-PAGE and thin layer scanning results showed that amount of expressed fusion protein was 30% in total of bacteria protein. Because the fusion protein was consisted of tir295, and stxb72 antigens, it can evoke immune responses against tir295 and stxb72. The tir-stxb 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：EHEC O157:H7 转位紧密素受体 志贺毒素 融合基因 

分 类 号：Q78[生物学—分子生物学]