幽门螺杆菌HspA与大肠杆菌LTB基因融合及表达

Construction and expression of the fusion gene of H pylori HspA subunit and E, coli heat-labile enterotoxin B subunit

目的:构建和表达人幽门螺杆菌(Hpylori)热休克蛋白A亚单位(HspA)与E. coli不耐热肠毒素B亚单位(LTB)的重组融合蛋白,并对其基本的生物学及免疫学特性进行研究.方法:采用PCR技术从人H pylori染色体DNA中扩增出354 bp的HspA基因,并克隆至pPLtB载体中与1tB基因融合,形成含有1tB-H spA融合基因的原核表达载体pPLH,并在工程菌E.coli JM109中诱导表达.结果:经序列分析,1tB-HspA融合基因由684个碱基组成,为编码228个氨基酸残基的多肽.SDS-PAGE和Westernblotting检测发现,融合蛋白的Mr 25×103,并与Hpylori感染的阳性血清发生抗原抗体反应,ELISA检测显示融合蛋白中存在LTB组分,并保持与LT受体-神经节苷脂GM1结合的活性.结论:LTB-HspA融合蛋白有可能用于Hpylori基因工程疫苗的研究.

AIM: To construct and express the fusion gene of H pylori heat shock protein A subunit (HspA) and E. coli heat-labile enterotoxin B subunit (LTB), and analyse the biologic and immunologic characteristics of the fusion protein. METH0DS: HspA gene was amplified from H pylro chro- mosome by PCR. The gene was cloned into plasmid pPLtB and the fusion gene of H pylori urease B subunit (HspA) and E. coli heat-labile enterotoxin B subunit (LTB) was constructed, and then LTB-HspA recombinant protein was expressed in E. coli JM109. ESULTS: LtB-HspA fusion gene was found to be 684 base gairs and encode the recombinant fusion protein, which was composed of 228 amino acid residues. SDS-PAGE and Western blotting analysis showed that the recombinant fusion protein had a molecular weight of 25kD and a positive reaction with the serum from H pylori-infected patients. ELISA analysis showed that LTB protein existed in the fusion protein. At the same time, fusion protein kept the character of binding with LTB receptor-ganglioside GM_1. C0NCLUSION: LTB-HsPA recombinant protein may be used for research of genetically engineered H pylori vaccine.