Morihara Fumiko, Fujii Ryoji, Hifumi Emi, Nishizono Akira, Uda Taizo
CREST of JST (Japan Science and Technology Corporation), 4-1-8 Hon-cho, Kawaguchi-shi, Saitama 332-0012, Japan.
Fukuyama Medical Laboratory Co. Ltd, 1-23-21 Kusado-cho, Fukuyama-shi, Hiroshima 720-8510, Japan.
J Med Microbiol. 2007 Jun;56(Pt 6):847-853. doi: 10.1099/jmm.0.47061-0.
Helicobacter pylori has to counteract acidity during colonization in the stomach. The most important region for the enzymic activity of H. pylori urease, consisting of 138 aa (ureB138), was determined by a comparison of the homology of amino acid sequences, and a structural analysis, between urease of H. pylori and various other species. This region was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which was cleaved by PreScission protease between the GST moiety and ureB138. The ureB138 protein was then purified by gel filtration. The polyclonal antibody (pAb) induced by immunization with the purified ureB138 could suppress urease activity by about 50 %, while the pAb against the H. pylori urease did not show any inhibitory effect at all. Immunohistochemical analysis indicated that the ureB138-specific pAb specifically recognized the H. pylori infecting human gastric tissues. The effects of vaccination of recombinant ureB138 against infection by this organism were also examined. Specific IgG and IgA antibodies against H. pylori urease were induced in the serum of mice immunized with ureB138. A reduction in the number of colonizing H. pylori was observed in mice treated with ureB138 compared to ones treated with BSA and infection control mice. In the protected mice, severe gastritis characterized by marked infiltration of mononuclear cells was noted compared with the gastritis observed in unprotected mice. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in ureB138-vaccinated mice than in non-vaccinated mice. This indicates that local IgA antibody and severe post-immunization gastritis correlate well with the protection of mice against H. pylori infection.
幽门螺杆菌在胃内定植过程中必须抵御酸性环境。通过比较幽门螺杆菌脲酶与其他各种物种脲酶的氨基酸序列同源性及结构分析，确定了幽门螺杆菌脲酶由138个氨基酸组成的酶活性最重要区域（ureB138）。该区域在大肠杆菌中作为与谷胱甘肽S-转移酶（GST）的融合蛋白表达，GST部分与ureB138之间被PreScission蛋白酶切割。然后通过凝胶过滤纯化ureB138蛋白。用纯化的ureB138免疫诱导的多克隆抗体（pAb）可使脲酶活性抑制约50%，而针对幽门螺杆菌脲酶的pAb则完全没有抑制作用。免疫组织化学分析表明，ureB138特异性pAb能特异性识别感染人类胃组织的幽门螺杆菌。还检测了重组ureB138疫苗接种对该菌感染的影响。用ureB138免疫的小鼠血清中诱导出了针对幽门螺杆菌脲酶的特异性IgG和IgA抗体。与用牛血清白蛋白处理的小鼠及感染对照小鼠相比，用ureB138处理的小鼠中幽门螺杆菌定植数量减少。在受到保护的小鼠中，与未受保护小鼠中观察到的胃炎相比，可见以单核细胞明显浸润为特征的严重胃炎。胃黏膜中IgA的免疫组织化学染色显示，接种ureB138的小鼠中IgA阳性染色的小鼠数量显著高于未接种小鼠。这表明局部IgA抗体和免疫后严重胃炎与小鼠抵抗幽门螺杆菌感染的保护作用密切相关。
