幽门螺杆菌HspA-CtxB口服疫苗的研制及免疫原性研究

Preparation and immunogenicity study of HspA-CtxB fusion protein to Helicobacter pylori in mice

目的 观察幽门螺杆菌融合蛋白质热休克蛋白A 霍乱毒素B(HspA CtxB ,HCT)经口服免疫在小鼠小肠内的吸收情况及免疫后机体产生的免疫应答。方法 用PCR方法扩增hspA和ctxB 2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诱导表达融合蛋白质HCT ;融合蛋白质经镍离子柱纯化、复性后 ,与HspA重组蛋白质共同标记同位素1 2 5I,经小鼠灌胃实验观察其在小鼠小肠内的吸收情况 ;HCT和HspA分别给小鼠灌胃进行免疫 ,每周灌胃 2次 ,共 8次 ,末次免疫后 3d起收集小鼠粪便 ,次日眶后静脉采集静脉血 ,分别对标本进行IgG和IgA检测。结果 经测序 ,HspA CtxB(HCT)融合基因片段由 72 6bp组成 ,为编码 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 (M) r 约为 30× 10 3 。标记同位素1 2 5I的HCT和HspA ,经小鼠灌胃实验观察不同时间段的吸收情况 ,结果发现在 10～ 15min时间段 ,HCT组出现吸收峰值 ,约为对照组的 8倍以上 (P <0 .0 0 1) ,且吸收峰值时间明显提前 ;将HCT口服免疫小鼠 ,检测小鼠血清和粪便中的特异性抗体水平。结果HCT组 (A40 5) ,血清IgA ,2 .

Objective To study the immune response in mice fed orally with the conjugated antigen of HspA-CtxB(HCT) as well as the absorption of the antigen in the small intestine of mice. Methods A recombinant strain which could express bivalent antigen of HspA and CtxB subunit was constructed. hspA and hct gene was amplified by PCR. The DNA products of HspA and CtxB were inserted into a prokaryotic expression vector pET-22b (+) respectively, and then transtected into E.coli strain BL-21(DE3) to express HCT fusion protein. Results hct gene was sequenced as 726 base pairs, the fusion protein encoded polypeptides of 242 amino acid residues, corresponding to calculated molecular masses(M r) of 30×10 3. Western blot analysis of the recombinant protein HCT confirmed that it could be specifically recognized by the serum of H.pylori-infected patients. HspA and HCT labelled 125I were orally deliveried into the stomach of mice respectively, and the radioactivity of 125I in serum of each mouse was detected in different periods: 5 min, 10 min, 15 min, 30 min, 60 min and 90 min. Antibody response in animals immunized with recombinant HCT or HspA was determined by ELISA(A 405). The group of HCT, serum IgA 2.98±0.35, serum IgG 4.38±0.56, fecal IgA 2.89±0.68; HspA groups, serum IgA 0.38±0.15, serum IgG 0.45±0.16; fecal IgA 0.69±0.23; normal control, serum IgA 0.06±0.02, serum IgG 0.09±0.06, fecal IgA 0.12±0.03. Conclusions These results indicate that HCT can be immediately absorbed in a large amount from small intestine of mice, and high value of HspA-specific antibodies are produced by orally immunization with HCT. The recombinant fusion protein HCT can be used as an effective oral vaccine for prevention and treatment of infection of H.pylori.