肠出血型大肠埃希菌O157：H7 Tir的表达、纯化及不同免疫途径对其免疫效价的影响  

Expression, purification and effect of different immunization approaches of Tir from EHEC O157： H7

作　　者：王玲[1] 胡黎平[1] 龙北国[2] 周勇[3] 罗军[2] 袁广明[1] 范宏英[2] 

机构地区：[1]中山大学基础医学实验教学中心,广州510080 [2]南方医科大学公共卫生与热带医学学院微生物学系,广州510515 [3]广州市疾病预防控制中心

出　　处：《中华微生物学和免疫学杂志》2011年第4期335-340,共6页Chinese Journal of Microbiology and Immunology

基　　金：广州市科技攻关项目（200723.E0391）

摘　　要：目的克隆、表达和纯化肠出血型大肠埃希菌（enterohaemorrhagic E．coli，EHEC）O157：H7转位紧密黏附素受体蛋白（Tir），观察不同免疫途径对其免疫效价的影响，为EHEC0157：H7亚单位疫苗的研究提供了实验资料。方法扩增tir基因，克隆到pET．30a（＋）载体上，转化至大肠埃希菌BL21／DE3，诱导表达目的蛋白，通过Ni—IDA亲和层析进行纯化；将重组蛋白Tir免疫小鼠，检测血清和粪便提取物中抗体效价。结果双酶切和测序鉴定结果均显示重组质粒pET-30a（＋）-tir构建成功。SDS-PAGE结果表明，目的蛋白Tir在大肠埃希菌BL21（DB3）中得到表达。皮F免疫和鼻腔免疫小鼠，血清中均能检测到高效价的IgG类抗体，鼻腔免疫组小鼠血清和粪便IgA类抗体效价明显高于皮下免疫组。结论Tir蛋白具有一定的免疫原性：Objective To clone and express translocation intimin receptor（Tir） of enterohemorrhagic Escherichia colt （EHEC）O157： H7, and to analyze the effect of different routes on the induction of immunity to the recombinant protein. Methods The tir encoding genes were amplified from EHEC O157：H7 strain guangzhou 246 genome, and genes were cloned into the vector pET-3Oa（ ＋ ）. The pET-30a （ ＋ ）-tit recombinant was transformed into E. colt BI21, and expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and purified by Ni-IDA affinity chromatography. The immunized mice sera and fecal against the recombinant protein was detected. Results The length of the tir is 1677 bp, with the initiation codon ATG and the termination codon TAA. Double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid pET-30a（ ＋ ）-tir was constructed. The recombinant protein was expressed in Escherichia colt expression system, and was purified by Ni-IDA affinity chromatography. The mice were able to produce a high serum IgG antibody titer after both subcutaneous and intranasal immunizations. Meanwhile, the intranasal immunization induced serum and fecal IgA antibody titer was sig- nificantly higher than that of the subcutaneous immunization group. Conclusion Tit molecule is potential vaccine candidate for preventing EHEC disease.

关 键 词：大肠埃希菌O157:H7 转位紧密黏附素受体 疫苗 

分 类 号：R392[医药卫生—免疫学]