表达幽门螺杆菌hpaA基因的减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用

Immunoprotection of mice against Helicobacter pylori infection by oral immunization with attenuated Salmonella typhimurium expressing hpaA gene of Helicobacter pylori

目的 克隆幽门螺杆菌 (H pylori)全长hpaA基因 ,构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并研究其对小鼠的免疫保护作用。方法 用PCR扩增全长hpaA基因 ,经适当的酶切 -连接反应将其连入原核表达质粒 pTrc99A ,并进行了基因测序。重组质粒经鉴定后再导入减毒鼠伤寒沙门氏菌SL32 6 1,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS -PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌 3× 10 8CFU/ 0 4ml/只免疫C5 7BL/ 6小鼠 ,4周后H pyloriSS110 5CFU/只攻击小鼠 ,再 5周后处死小鼠 ,取腺胃做快速尿素酶试验和Giemsa染色 ,以明确H pylori定植情况 ,对照观察免疫保护效果。 结果 经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 pTrc99A -hpaA ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约 30kDaHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 38 9% ,Westernblot证实其有免疫反应性。重组菌对小鼠免疫保护率为 4 3 4 8% (10 / 2 3) ,与空白对照组比统计差异显著 (P =0 0 1)。结论 构建了表达H pyloriHpaA的重组减毒鼠伤寒沙门氏菌 ,该菌株对C5 7BL/ 6小鼠有免疫保护作用。

Aim To construct a recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori hpaA gene,and to investigate its immunoprotection of mice against H pylori infection Methods By genetic engineering methods,783bp hpaA gene was cloned into a procaryotic expression plasmid pTrc99A,and the identified recombinant plasmid was then used to transform an attenuated Salmonella typhimurium vaccine strain SL3261,and the positive clones were screened by PCR and restriction enzyme digestion HpaA expression was analyzed by SDS-PAGE and Western blot C57BL/6 mice were immunized with 3×10 8CFU/0 4ml/mouse of recombinant vaccine strains,challenged with 10 5 CFU/mouse of H pylori SS1 4 weeks later,and sacrificed for rapid urease test and histopathological examination of the gastric mucosa 5 weeks later Results Confirmed by PCR and restriction enzyme digestion,a recombinant procaryotic expression plasmid pTrc99A hpaA was constructed,and the recombinant plasmid was then introduced into an attenuated Salmonella typhimurium vaccine strain SL3261 successfully HpaA was expressed in the recombinant strains as a 30kDa protein,and also its immunogenicity was confirmed by Western blot 43 48% (10/23) of mice were completely protected,and the protective rate was higher than that of the control (P=0 01) Conclusion A recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori hpaA gene has been constructed and identified,and it inducs immunoprotective effect against Helicobacter pylori challenge in mice