幽门螺杆菌UreB与大肠杆菌LTB基因融合及表达的研究

Construction and expression of the fusion gene of Helicobacter pylori urease B subunit and E.coli heat-labile enterotoxin B subunit

目的 构建和表达幽门螺杆菌尿素酶B亚单位 (UreB)与大肠杆菌不耐热肠毒素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技术从幽门螺杆菌染色体DNA中扩增出 1713bp的ureB基因 ,并克隆至pFS2 .2载体中与ltB基因融合 ,将ltB ureB融合基因插入改造后的原核表达载体PinPointTMXa Ⅱ ,并在工程菌E .coliJM10 9中诱导表达。结果 经序列分析 ,ltB ureB融合基因由 2 10 3个碱基组成 ,为编码 70 1个氨基酸残基的多肽。SDS PAGE和Westernblot检测发现 ,融合蛋白的相对分子质量 (Mr)约为 75× 10 3 ,并与幽门螺杆菌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。同时发现所表达的融合蛋白无尿素酶活性 ,但保持与LT受体 神经节苷脂GM1结合的活性。结论 LTB

Objective To construct and express the fusion gene of H.pylori urease B subunit (UreB) and E.coli heat-labile enterotoxin B subunit (LTB), and to analyzed the biological or immunological characteristics of the fusion protein. Methods The ureB gene was amplified from H.pylori chromosome by PCR. The gene was cloned into the plasmid pFS2.2 and the fusion gene of H.pylori urease B subunit (UreB) and E.coli heat-labile enterotoxin B subunit (LTB) was constructed. This fusion gene was inserted into the prokaryotic expression vector PinPoint TMXa-Ⅱ, and then LTB-UreB recombinant protein was expressed in E.coli JM109. Results ltB-ureB fusion gene was found as a 2103 base pairs molecules and encoded the recombinant fusion protein with 701 amino acid residues. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight(M r) 75×10 3 and a positive reaction with the serum from H.pylori-infected patients. ELISA analysis showed that LTB protein existed in the fusion protein. Meanwhile, the fusion protein is lack of urease activity but keep the character of binding LTB receptor-ganglioside GM1. Conclusion The results suggested that LTB-UreB recombinant protein may be used for development of H.pylori genetic engineering vaccine.