摘要
【目的】构建表达幽门螺杆菌 (Helicobacterpylori,H .pylori)尿素酶B亚单位 (UreB)减毒鼠伤寒沙门氏疫苗菌 ,研究其对小鼠抗H .pylori的免疫保护作用。【方法】用PCR扩增ureB ,将其克隆入高效原核表达质粒 pTrc99A ,进行基因测序 ,重组质粒鉴定后导入减毒鼠伤寒沙门氏菌SL32 6 1。用SDS聚丙烯酰胺凝胶电泳、Westernblot和薄层扫描进行目的蛋白表达分析。C5 7BL/ 6小鼠用重组菌免疫 ,4周后用H .pyloriSS1攻击 ,再 4周后处死小鼠 ,取胃做快速尿素酶试验和H .pylori定量培养 ,对照观察免疫效果。【结果】构建了携带ureB的重组原核表达质粒pTrc99A ureB ,并将它成功转化了减毒鼠伤寒沙门氏菌SL32 6 1。重组菌SL32 6 1(pTrc99A ureB)表达了约 6 6ku的UreB。与对照组比 ,重组菌免疫组 H .pylori定植水平明显下降 (P <0 0 5 )。【结论】构建了表达H .pyloriUreB的重组减毒鼠伤寒沙门氏疫苗菌 ,该菌株对C5 7BL/ 6有免疫保护作用。
To construct a recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori (H.pylori) urease subunit B (UreB), and to investigate its immunoprotection to mice against H.pylori infection. ureB was amplified by PCR and cloned into a procaryotic expression plasmid pTrc99A, and the identified recombinant plasmid was then used to transform an attenuated Salmonella typhimurium vaccine vector SL3261, and the positive clones were screened by PCR and restriction enzyme digestion. The UreB expressed in the recombinant vaccine strain was analyzed by SDS PAGE, Western blot and optical density scanning. C57BL/6 mice were immunized with recombinant vacine strain, challenged with H.pylori SS1 4 weeks later, and sacrificed for rapid urea test and H.pylori quantitative culture of the gastric mucosa another 4 weeks later. The ureB could be amplified from the plasmid extracted from the recombinant SL3261(pTrc99A ureB) by PCR, and also the ureB flagment could be produced from this plasmid after restriction enzyme digestion. SDS PAGE, Western blot and optical density scanning indicated that UreB was expressed in the recombinant vaccine strain SL3261(pTrc99A ureB) as a 66 ku protein. H.pylori quantitative culture of the gastric mucosa indicated that, the colonized level of H.pylori in immunized mice was lower than that of the control (P<0 05). [Conclusion] A recombinant live attenuated Salmonella typhimurium vaccine strain expressing H.pylori UreB was constructed and identified, and it induced immunoprotective effect against H.pylori in mice.
出处
《中山医科大学学报》
CSCD
北大核心
2001年第6期418-422,共5页
Academic Journal of Sun Yat-sen University of Medical Sciences
基金
卫生部临床学科重点基金资助项目 ( 970 40 2 2 6 )
广东省科技计划重点攻关基金资助项目( 99M 0 480 2G)
广东省自然科学基金资助项目 ( 990 0 77)
广东省高教厅"千百十工程"优秀人才基金资助项目 ( 2 0 0 0 -0 8)
关键词
免疫学
沙门氏菌
疫苗
鼠伤寒
小鼠
幽门螺杆菌
动物实验
Helicobacter pylori/immunology
Salmonella typhimurium
vaccine,attenuated/biosynthesis
mice
