重组幽门螺杆菌多价疫苗CTB-HUUL免疫治疗效果研究  

Study on Immunological Efficacy of the Reconstructed Multivalent Vaccine CTB-HUUL Against Helicobacter pylori Infection

作　　者：柯鸿 潘龙瑞[2] 牛晓娟 Ke Hong;Pan Longrui;Niu Xiaojuan(Department of Hemotology,People＇s Hospital Affiliated to Hubei University of Medicine,Shiyan 442000,China;Department of Pharmacology,Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]湖北医药学院附属人民医院血液内科,十堰442000 [2]湖北医药学院药理学教研室,十堰442000

出　　处：《成都医学院学报》2018年第4期399-404,共6页Journal of Chengdu Medical College

基　　金：湖北省卫生和计划生育委员会项目(No:WJ2017F075);十堰市科学技术研究与开发项目(No:16Y11)

摘　　要：目的构建口服重组幽门螺杆菌(H.pylori)多价疫苗CTB-HpaA-UreA-UreB-CagL(CTBHUUL),并探究其在BABL/c小鼠体内抗H.pylori SS1感染的免疫治疗效果。方法根据文献资料查找H.pylori相关黏附分子(HpaA、UreA、UreB)抗原的显性表位序列HpaA88-100、HpaA132-141、UreA27-53、UreA183-203、UreB229-251、UreB317-329、UreB321-339、UreB373-385、UreB438-452、UreB546-561,在其C端引入H.pylori黏附分子抗原CagL,再在其N端引入分子内黏膜佐剂CTB,通过Linker(EFM、GS、KK)将其串联,构建重组质粒pET28a(+)/CTB-HapA-UreA-UreB-CagL[pET28a(+)/CTB-HUUL]。将pET28a(+)/CTB-HUUL转化至大肠杆菌BL21(DE3)中,IPTG诱导表达后进行蛋白层析纯化,获得疫苗靶抗原CTB-HUUL,并采用Western Blot和ELISA对其进行免疫反应性和免疫原性鉴定。6周龄BABL/c小鼠,108 CFU,1次/d,进行灌胃,连续感染4次;末次灌胃后4周,灌胃CTB-HUUL重组蛋白200μg·次-1·周-1,连续免疫治疗4次。末次免疫治疗后2周,脱颈处死小鼠,取小鼠胃组织进行H.pylori SS1计数和病理切片HE染色。结果获得了高纯度的CTB-HUUL蛋白,Western Blot结果显示,CTB-HUUL可以与鼠抗H.pylori SS1血清及鼠抗CTB抗体发生特异性反应;ELISA检测结果显示,CTB-HUUL体外可以与重组蛋白rHpaA、rUreA、rUreB、rCagL发生特异性反应。CTB-HUUL组小鼠胃组织H.pylori SS1培养计数结果较CTB组和PBS组低(P<0.01),CTB-HUUL组小鼠胃组织炎症损伤程度及炎症细胞浸润程度较CTB组和PBS组轻微。结论口服重组H.pylori多价疫苗CTB-HUUL对感染H.pylori SS1的BABL/c小鼠具有免疫治疗效果。Objectives To construct the oral recombinant multivalent vaccine CTB HpaA Urea UreB CagL （CTB HUUL） against Helicobacter pylori infection and explore its immunological efficacy on the Helicobacter pylori infection in BABL/c mice. Methods The literature was reviewed to search for the dominant epitope sequences of the antigen of Helicobacter pylori related adhesion molecules （HpaA, Urea and UreB）, and those sequences included HpaA88-100, HpaA132-141, UreA27-53, UreA183-203, UreB229-251, UreB317-329, UreB321-339, UreB373-385, UreB438-452 and UreB546-561. The recombinant plasmid pET28a（＋）/CTB HapA- Urea UreB CagL（CTB HUUL） was constructed by introducing Helicobacter pylori adhesion molecular antigen CagL at C terminal and the intramolecular mucosal adjuvant CTB at N terminal and then linking them by the series of Linkers （EFM, GS, KK）. PET28a（＋）/CTB HUUL was transformed into Escherichia coli BL21 （DE3）. After IPTG induced the expression, the protein chromatographic purification was conducted to obtain the vaccine target antigen CTB HUUL. The immunoreactivity and immunogenicity of the vaccine were identified by Western Blot and ELISA. The 6 week-old BABL/c mice were administered with 108 CFU by lavage once a day and infected four times consecutively. Four weeks after the end of the administration, the mice were administered with the recombinant protein 200 μg CTB-HUUL once each week and then treated 4 times with the consecutive immunotherapy. Two weeks after the last immunotherapy, the mice were sacrificed and the gastric tissues were taken for H. pylori SS1 count and pathological section HE staining. Results The highly purified protein CTB-HUUL was obtained. The results o{ Western Blot showed that CTB-HUUL can react specifically with mouse anti H. pylori SS1 serum and mouse anti CTB antibody. The results of ELISA showed that CTB-HUUL can react specifically with the recombinant proteins rHpaA, rUreA, rUreB and rCagL in vitro. The H. pylori SS1 count of the gastric tissues was significa

关 键 词：幽门螺杆菌 多价疫苗 幽门螺杆菌黏附素A亚单位 尿素酶 细胞毒素相关蛋白L 

分 类 号：R573.9[医药卫生—消化系统]