幽门螺杆菌粘附素与大肠杆菌LtB融合疫苗的口服免疫与预防试验

目的　观察幽门螺杆菌粘附素HpaA与大肠杆菌LtB融合蛋白质经口服免疫BALB/c小鼠后机体产生的免疫应答和蒙古沙鼠免疫预防作用。方法　用PCR方法扩增HpaA和LtB目的基因片段 ,将LtB与 pPIM质粒连接后 ,再与HpaA基因连接 ,转化大肠杆菌DH5α ,经酶切获得含有ltB -hpaA的pPIM -ltB/hpaA融合质粒。将重组质粒转化E coliBL2 1(DE3) ,筛选获得重组工程菌 ,经发酵、包涵体提取 ,复性 ,纯化获得LtB -HpaA。BABL/c小鼠设正常对照组、单独rHpaA对照组、CT +HpaA实验组、融合蛋白LtB -HpaA组和rLtB +rHpaA实验组 ,免疫 4周后检测血清抗原特异性IgG、IgA和胃冲洗液、肠冲洗液中sIgA。蒙古沙鼠分 3个实验组 ,正常对照 (PBS)组 ,单独HpaA免疫组和LtB -HpaA组 ,免疫 4周 ,于末次免疫后 14天灌活力良好的H pyloriSS1活菌 ,1个月处死动物 ,采集标本 ,用细菌培养和病理切片检查评价蒙古沙鼠的免疫保护作用。结果　融合蛋白LtB -HpaA组及将CT和LtB作为外粘膜佐剂的CT +HpaA组和LtB +HpaA组中血清IgA和IgG、胃冲洗液和肠冲洗液中sIgA含量明显高于单独HpaA组和对照组 ,差异非常显著 (P <0 0 0 1)。蒙古沙鼠融合蛋白rLtB-HpaA组比单纯rHpaA组效果明显 (rLtB -HpaA组保护率为 80 % ,单独rHpaA为 2 0 % )。 结论　融合蛋白质LtB

Evaluation of immune prophylaxis of adhesin protein of Helicobacter pylori genetically linked to E.coli heat-labile enterotoxin B subunit

To evaluate the immune prophylaxis of adhesin protein of Helicobacter pylori genetically linked to E.coli heat-labile enterotoxin B subunit,the fusion protein LtB-HpaA,derived from H.pylori adhesion genetically coupled to E.coli heat-labile enterotoxin B subunit,was expressed in E.coli as an insoluble recombinant fusion protein,and the protein was purified by renaturation,ion exchange and size-exclusion chromatography,The BALB/c mice were divided into five groups:negative control group,control group with rHpaA,experimental group with rHpaA+rLtB,experimental group with rHpaA + CTB and experimental group with LtB-HpaA,which were gastrically inoculated with 150μl PBS,150μg rHpaA,150μg rHpaA+1μg CT,150μgHpaA+10μgrLtB and 150μgLtB-HpaA,respectively.Antibody response in mice was determined by ELISA(A 492).Mongolian gerbil were divided into three groups:negative control group,control group with rHpaA and experimental group with LtB-HpaA,which were gastrically inoculated as BALB/c mice.Two weeks later,Mongolian gerbil were challenged with H.pylori SS1 (1×108 CFU per time) and sacrificed 4 weeks after the challenge.Their stomachs were collected for rapid urease test,histologic examination and bacteria culture to observe the gastric H.pylori densities.The experimental results showed that oral immunization of mice with LtB-HpaA induced higher levels of mucosal IgA and serum IgG antibodies to H.pylori HpaA than in mice immunized with HpaA alone.The immunization of Mongolian gerbil with LtB-HpaA protected 80% of the Mongolian gerbil infected with H.pylori ss1 strain,whereas HpaA immunization was not able to confer protection to mongolian gerbil.If condudes that recombinant fusion protein LtB-HpaA can be used as an effective oral vaccine for prevention and treatment of infection of H.pylori.