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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 UreB融合蛋白有可能用于幽门螺杆菌基因工程疫苗的研究
Objective To construct and express recombinant fusion protein of Helicobacter pylori urease B subunit (UreB) and Escherichia coli heat - labile enterotoxin subunit (LTB), and to study its basic biological and immunological properties. Methods A 1713bp ureB gene was amplified from Helicobacter pylori chromosome DNA by PCR and cloned into pFS2. 2 vector and fused with ltB gene. The gene of ltB ureB was inserted into the modified prokaryotic expression vector PinPointTMXa Ⅱ. Expression was induced in E. coli JM109. Results By sequence analysis, the ltB ureB fusion gene consisted of 2 10 3 bases, encoding 701 amino acid residues. SDS PAGE and Western blot showed that the relative molecular mass (Mr) of the fusion protein was about 75 × 10 3, and antigen-antibody reaction with Helicobacter pylori-positive sera. ELISA assay showed that there was LTB in the fusion protein. At the same time, it was found that the expressed fusion protein had no urease activity, but retained the activity of binding to LT receptor ganglioside GM1. Conclusion LTB UreB fusion protein may be used in the study of Helicobacter pylori genetic engineering vaccine