幽门螺杆菌外膜蛋白omp22和黏附素hpaA融合基因原核表达系统的构建与鉴定  

Construction and identification of prokaryotic expression system for omp22-hpaA fusion gene from Helicobacter pylori

作　　者：黄学勇[1] 段广才[1] 范清堂[2] 郗园林[1] 黄志刚[2] 宋春花[1] 

机构地区：[1]郑州大学公共卫生学院流行病学教研室 [2]河南省分子医学重点学科开放实验室,河南郑州450052

出　　处：《第四军医大学学报》2007年第3期225-228,共4页Journal of the Fourth Military Medical University

基　　金：河南省医学创新人才基金(2000-84)

摘　　要：目的:构建表达幽门螺杆菌(Hp)的外膜蛋白omp22和黏附素hpaA融合基因的重组蛋白质候选菌株,为Hp疫苗研究提供依据.方法:用PCR方法扩增出带3个甘氨酸残基柔韧接头的融合基因omp22-hpaA,将其克隆到表达载体pMAL-c2X中转化大肠杆菌(E.coliTB1),用IPTG诱导目的基因表达,SDS-PAGE方法对表达产物进行分析,Western Blot鉴定其免疫原性.结果:测序结果显示,omp22-hpaA融合基因片段由1326bp组成,为编码440个氨基酸残基的多肽,接头序列GGTGGAGGC已成功地插入omp22-hpaA融合基因中;SDS-PAGE结果显示表达产物相对分子质量约为53ku,融合蛋白的表达量约占全菌总蛋白的20%.结论:成功克隆并构建了omp22-hpaA融合基因原核表达系统.AIM： To construct a prokaryotic high expression system which expresses fusion gene of outer membrane protein （omp22） gene and adhesion gene hpaA from Helicobacter pylori. METHODS ： The omp22-hpaA fusion gene with an adapter of 3 glycine residues was amplified by PCR, and cloned to the expres- sion vector pMAL-c2X. The constructed recombinant plasmid was transformed in E. coli TB1 host bacterial strain and was induced by IPTG. The expression products were analyzed by SDS-PAGE and Western Blot. RESULTS： Sequencing results showed that the omp22-hpaA fusion gene consisted of 1326 bp and encoded the polypeptides of 440 amino acids and that the sequence （ GGT- GGAGGC） encoding 3 glycine residues was inserted into omp22- hpaA fusion gene as an adapter. The relative molecular weight of the expression product of omp22-hpaA fusion gene was about 53 ku. The amount of the recombinant gene expression products accounted for 20% of the total proteins of the host bacteria. CONCLUSION： The omp22-hpaA fusion gene has been cloned and a prokaryotic high expression system for omp22-hpaA fusion gene been successfully constructed.

关 键 词：幽门螺杆菌 omp22-hpaA融合基因 克隆 基因表达 

分 类 号：R183.4[医药卫生—流行病学]