幽门螺杆菌粘附素hpa A和霍乱毒素B亚单位ctx B融合基因的原核表达

Prokaryotic Expression of Helicobacter pylori Adhesion A (hpa A) and Cholerae Toxin B(ctx B)Fusion Gene

目的 构建幽门螺杆菌粘附素 (hpaA)和霍乱毒素B亚单位 (ctxB)融合基因的原核表达载体 ,诱导表达并进行纯化。方法 用PCR扩增hpaA和ctxB两个目的基因片段 ,克隆至同一pQE 30表达载体中 ,构建含双基因的表达质粒pQE hct,转化E .coliDH5α ,经IPTG诱导表达融合蛋白HCT ;经Westernblot分析其免疫原性 ,采用镍离子柱进行纯化。结果 经测序HCT融合基因片段由 116 1bp组成 ,为编码 387个氨基酸残基的多肽。经SDS PAGE分析相对分子质量约为 4 0 0 0 0。可溶性蛋白占菌体总蛋白的 2 5 %以上 ,经亲和层析后可获得纯度为 92 %以上的重组融合蛋白。经Westernblot检测可被Hp全菌抗血清和CT抗血清识别。结论 已成功构建融合蛋白HCT的原核表达载体并能高效表达 。

Objective To construct a prokaryotic vector for expressing Helicobacter pylori adhesion A(hpa A) and cholerae toxin B(ctx B)fusion gene.Methods Amplify hpa A and ctx B genes by PCR and clone into expression vector pQE-30 to construct a recombinant plasmid pQE-hct,then transform to E.coli DH5α for expression of fusion protein HCT under induction of IPTG.Analyze the expressed product by Western blot and purify by nickel ion affinity column chromatography.Results The fusion gene fragment consisted of (1 161 bp) encoding 387 amino acid residues.SDS-PAGE showed that the expressed fusion protein, with a relative molecular weight of 40 000,contained more than 25% of total somatic protein.The purity of expressed protein after nickel ion affinity column chromatography reached more than 92%. Western blot showed specific recognition of expressed product with antisera against whole cell Hp and CT.Conclusion A prokaryotic vector for high expression of fusion protein HCT was successfully constructed.The study provided a basis for developing oral Hp vaccine.