摘要目的制备表达幽门螺杆菌 (H. pylori) 尿素酶B亚单位(UreB)的减毒鼠伤寒杆菌活菌重组疫苗,并观测Balb/c小鼠口服该重组疫苗后的免疫效果. 方法构建表达UreB的重组减毒鼠伤寒杆菌的疫苗株,SL3261/pTC01-UreB ,并用Western-blot检测UreB的表达.将SL3261/pTC01-UreB口服免疫Balb/c小鼠,12周后检测肠液和血清中的特异性抗体反应及脾细胞培养液上清中的IFN-γ和IL-10含量.结果 Western-blot显示减毒鼠伤寒杆菌株SL3261/ pTC01-UreB能表达约 61KD的蛋白,与H. pylori UreB亚单位蛋白分子量相符,具有抗原性.口服免疫小鼠后,疫苗组小鼠的肠液和血清中可分别检测到针对UreB的特异性IgA和IgG抗体,小鼠脾细胞培养上清中的IFN-γ和IL-10的含量亦明显升高.病理学检查显示疫苗组小鼠与对照组小鼠胃粘膜炎症指数无差异.结论表达H. pylori UreB的减毒鼠伤寒杆菌SL3261/pTC01-UreB可用作抗H. pylori感染的口服疫苗.

abstractsObjective To establish attenuated Salmonella typhimu rium producing Helicobacter pylori (H. pylori) urease subunit B (UreB) and determine whether it could be used as an oral vaccine against H. pylori.Methods H. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL32 61 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL32 61 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) in the supernatant of spleen cells culture were also assessed by ELISA. Invitro stability of pTC01-UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medi um to 80 generations. Results The UreB gene fragment amplified by PCR was co nsistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01-UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01-UreB, wh ich could be recognized by anti-H. pylori UreB antiserum. After 80 genera tions of continuous culture, the recombinant plasmid pTC01-UreB was stable in S L3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC0 1-UreB could significantly induce H. pylori-specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN-γand IL-10 in the SL3261/pTC01-UreB group. Finally, no obvious side eff ects for mice and no change in gastric inflammation were observed. Conclusion Attenuated Salmonella typhimurium expre ssing H. pylori UreB may be used as oral vaccine against H. pylori inf ection.