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构建表达幽门螺杆菌的保护性抗原成分热休克蛋白A亚单位 (HspA)和霍乱毒素B亚单位 (CtxB)的重组融合蛋白的生物工程菌株 ,以此制备幽门螺杆菌的口服疫苗。用PCR方法扩增hspA和ctxB两个目的基因片段 ,将它们分别克隆至 pSK(+ )质粒上 ,然后插入含T7启动子pET 2 2b(+ )的表达载体中 ,构建含双基因的表达质粒 pET hct,转化E .coliBL2 1(DE3) ,经IPTG诱导表达融合蛋白HCT。经测序 ,hspA ctxB(hct)融合基因片段由 72 6bp组成 ,可以编码 2 42个氨基酸残基的多肽。经SDS PAGE和免疫印迹分析检测发现 ,融合基因表达的蛋白质相对分子质量约为 30kD。融合蛋白经镍离子柱纯化、复性后 ,和HspA共同标记同位素12 5I ,然后给小鼠灌胃 ,结果观察到HCT组小鼠血清中的12 5I的放射量要明显高于HspA组 (P <0 .0 0 1) ,且吸收峰值时间明显提前。融合蛋白中的CtxB可明显促进小鼠对HspA的吸收 ,HCT融合蛋白可以作为预防和治疗幽门螺杆菌感染的候选口服疫苗
An oral vaccine for H. pylori was prepared by constructing a bioengineered strain of recombinant fusion protein expressing the protective antigenic components of Helicobacter pylori, heat shock protein A subunit (HspA) and cholera toxin B subunit (CtxB). Two target genes of hspA and ctxB were amplified by PCR and cloned into pSK (+) plasmid respectively. Then inserted into the expression vector containing the T7 promoter pET 2 2b (+) to construct a double gene expression plasmid pET hct and transformed into E.coli BL21 (DE3). The fusion protein HCT was induced by IPTG. After sequencing, the hspA ctxB (hct) fusion gene fragment consists of 72 6bp and can encode a polypeptide of 422 amino acid residues. SDS PAGE and Western blot analysis showed that the relative molecular mass of fusion protein was about 30kD. The fusion protein was purified by nickel ion column, renaturation, and HspA co-labeled isotope 125I, and then intragastric administration, the results observed HCT group of mice serum 125I emission was significantly higher than the HspA group (P <0 0 01), and the peak absorption time was significantly earlier. CtxB in the fusion protein can significantly promote the absorption of HspA in mice, and the HCT fusion protein can be used as a candidate oral vaccine for preventing and treating Helicobacter pylori infection