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

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.coli TB1),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1326 bp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53 ku,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.

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

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 expression 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 (GGTGGAGGC) 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.