表达幽门螺杆菌尿素酶B亚单位的减毒鼠伤寒杆菌口服疫苗的制备

目的制备抗幽门螺杆菌(Hp)尿素酶B亚单位(UreB)减毒鼠伤寒杆菌活菌疫苗,观察其免疫效果.方法构建表达UreB的原核表达载体PTc01-UreB并转化减毒鼠伤寒杆菌SL3261,得到重组菌SL3261/PTc01-UreB.应用抗Hp菌体蛋白兔血清行Western-blot检测UreB在SL3261中的表达.将SL3261/PTc01-UreB口服免疫Balb/c小鼠,12周后应用ELISA检测肠液和血清中的特异性抗体反应.SL3261/PTc01-UreB在Luria-Bertani培养液中连续传代60代以确定其稳定性.结果成功构建PTc01-UreB原核表达载体.Western-blot显示,其转化减毒鼠伤寒杆菌SL3261后能表达相对分子质量约61×103的蛋白,与HpUreB亚单位相符,具有抗原性.口服免疫小鼠后,在肠液和血清中可分别检测到针对UreB的特异性IgA和IgG抗体.体外连续培养60代未见PTc01-UreB质粒丢失及对宿主细胞毒性.结论表达HpUreB的减毒鼠伤寒杆菌SL3261/PTc01-UreB可用作抗Hp感染口服疫苗.

Oral immunization of mice with attenuated Salmonella typhimurium expressing Helicobacter pylori urease B subunit

Objective To establish attenuated S. typhimurium expressing Helicobacter pylori (H.pylori) urease subunit B (UreB) and determine whether it could be used as oral vaccine against H.pylori Methods H.pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector PTc 01 after sequencing, then transformed into attenuated S.typhimurium SL3261 to acquire SL3261/PTc 01 UreB. The expression of H.pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA. In vitro stability of PTc 01 UreB plasmid in S. typhimurium SL3261 was confirmed by growing in Luria Bertani medium to 60 generations. Results The UreB gene fragment amplified by PCR was consistent with the sequence of the H.pylori UreB by sequence analysis. Enzyme digestion revealed that the correct PTc 01 UreB was obtained. Western blot showed that 61kD protein was expressed in SL3261/PTc 01 UreB which could be recognized by anti H.pylori UreB antiserum. Anti UreB IgA antibodies in mouse intestinal fluid and IgG antibodies in serum were determined by ELISA. After 60 generations of continuous culture, the recombinant plasmid PTc 01 UreB was stable in SL3261 and had no obvious toxicity. Conclusion The attenuated Salmonella typhimurium expressing H.pylori UreB may be used as oral vaccine against H.pylori infection.