重组Hp疫苗CTB-HUUC对BALB/c小鼠Hp感染的预防效果  

Preventive Effect of Recombinant Hp Vaccine CTB-HUUC on Helicobacter Pylori Infection in BALB/c Mice Model

作　　者：柯鸿 潘兴 牛晓娟 李莉 张友旺 潘龙瑞 KE Hong;PAN Xing;NIU Xiao-juan;LI Li;ZHANG You-wang;PAN Long-rui(Department of Hemotology, Renmin Hospital;Institute of Infection and Immunity, Taihe Hospital;School of Basic Medical Sciences, Hu- bei University of Medicine, Shiyan, Hubei 442000, China)

机构地区：[1]湖北医药学院附属人民医院血液内科,湖北十堰442000 [2]湖北医药学院附属太和医院感染与免疫研究所,湖北十堰442000 [3]湖北医药学院基础医学院,湖北十堰442000

出　　处：《湖北医药学院学报》2018年第3期203-207,212,F0002,共7页Journal of Hubei University of Medicine

基　　金：湖北省卫生和计划生育委员会项目(WJ2017F075);十堰市科学技术研究与开发项目(16Y11)

摘　　要：目的:构建口服幽门螺杆菌(Helicobacter pylori,Hp)多价表位疫苗CTB-Hap A-Ure A-Ure B-CagA(CTB-HUUC),观察其在BABL/c小鼠体内抗Hp SS1感染的免疫预防效果。方法:文献查找Hp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Hp黏附分子抗原CagA,再在其N端引入分子内黏膜佐剂霍乱毒素B亚基(cholera toxin B,CTB),通过Linker(EFM、GS、KK)将其串联,构建重组质粒pET28a(+)/ctB-hpa A-ureA-ureB-CagA[pET28a(+)/ctB-HUUC]。将pET28a(+)/ctB-HUUC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-HUUC,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,分别用CTB-HUUC重组蛋白、CTB、PBS 200μg,每周一次灌胃,连续免疫治疗4次。末次免疫治疗后2周,用10~8CFU的幽门螺杆菌悉尼株(Helieobacter pylori Sydney Strain1,Hp SS1),每天一次灌胃,每次50μL,连续感染4次;两周后脱颈处死小鼠,取小鼠的胃窦部组织进行快速尿素酶试验,其余胃组织进行Hp SS1计数和病理切片HE染色。结果:获得了高纯度的CTB-HUUC蛋白,Western Blot结果显示CTB-HUUC可以与鼠抗Hp SS1血清及鼠抗CTB抗体发生特异性反应,人抗神经节苷脂抗体酶联免疫吸附法(GM1-ELISA)检测重组蛋白CTB-HUUC体外可结合GM1,具有良好的佐剂活性。CTB-HUUC组小鼠胃组织快速尿素酶试验阳性明显比CTB组和PBS组少(P<0.01),Hp SS1培养计数结果较CTB组和PBS组显著减少(P<0.01),炎性损伤程度及炎性细胞浸润程度较CTB组和PBS组轻微。结论:口服重组幽门螺杆菌多价疫苗CTB-HUUC对BALB/c小鼠的Hp感染预防有效。Objective To construct an oral Helicobacter pylori（ Hp） polyvalent epitope vaccine CTB-Hap A-UreA-UreB-CagA（ CTB-HUUC） and observe the immunity against Hp SS1 infection and preventive effect in BALB/c mice. Methods The dominant epitope sequences were found through literature searching for Hp associated adhesion molecule（ HpaA,UreA,UreB） antigens HpaA88-100,HpaA132-141,UreA27-53,UreA183-203,UreB229-251,UreB317-329,UreB438-452 and UreB546-561. The Hp adhesion molecule antigen CagA was introduced at their C-terminus,and then the intramolecular mucosal adjuvant（ choleratoxin B,CTB） was introduced into their N-terminus. The recombinant plasmid pET28 a（ ＋）/ctB-hpa A-ureA-ureB-CagA[pET28 a（ ＋）/ctB-HUUC]was constructed through Linker（ EFM,GS,KK）. pET28 a（ ＋）/ctB-HUUC was transformed into Escherichia coli BL21（ DE3）,and the expression was induced by IPTG. The vaccine target antigen CTB-HUUC was obtained through protein chromatography. The immunogenicity of the target antigen was identified by Western Blot and ELISA. Six weeks old BABL/c mice were treated with CTB-HUUC recombinant protein,CTB（ 200 μg,intragastric administration） or PBS four times a week for 4 consecutive times. Two weeks after the last immunotherapy,10^8 CFU of the H. pylori strain of Sydney（ Hp SS1） was given once a day,50 μL each,for 4 consecutive infections; mice were sacrificed by cervical dislocation two weeks later. The urethral tissue of the mice was taken for rapid urease test,and the remaining gastric tissue was counted for Hp SS1 and pathological section with HE staining. Results High-purity CTB-HUUC protein was obtained. Western Blot showed that CTB-HUUC can specifically react with mouse anti-Hp SS1 serum and mouse anti-CTB antibody,and human anti-ganglioside antibody enzyme-linked immunosorbent assay（ GM1-ELISA） result indicated that the recombinant protein CTB-HUUC can bind to GM1 in vitro and has good adjuvant activity. The positive rate of rapid urease test in CTB-HUUC group was less th

关 键 词：CTB-HUUC 幽门螺杆菌黏附素A亚单位 尿素酶 细胞毒素相关蛋白A 预防性接种 

分 类 号：R392[医药卫生—免疫学]