幽门螺杆菌外膜蛋白GroEL优势T-B联合抗原表位的鉴定

发布时间：2018-04-09 10:44

  本文选题：幽门螺杆菌　切入点：外膜蛋白　出处：《浙江大学》2014年硕士论文

【摘要】：目的筛选并鉴定幽门螺杆菌外膜蛋白GroEL的优势T细胞和B细胞(T-B)联合抗原表位。 方法采用PCR扩增幽门螺杆菌NCTC11637株groEL基因并构建其原核表达系统,SDS-PAGE检查目的重组蛋白rGroEL表达情况。Ni-NTA亲和层析法提纯rGroEL。采用激光共聚焦显微镜法检测幽门螺杆菌NCTC11637和SS1株中GroEL分布。Triton X-114法提取上述菌株外膜蛋白样本,采用Western Blot法检测外膜蛋白样本中的GroEL。采用专业生物信息学软件预测GroEL的T-B联合抗原表位并构建其噬菌体展示系统。采用Western Blot法和ELISA分别检测含T-B联合表位肽的重组噬菌体PⅢ蛋白和人工合成的T-B联合表位肽的免疫反应性。 结果幽门螺杆菌NCTC11637和SSl株groEL基因核苷酸和氨基酸序列相似性高达97.68-99.63%。所构建原核表达系统能有效表达可溶性rGroEL。GroEL定位于幽门螺杆菌NCTC11637和SS1株外膜。GroEL168、GroEL258、GroEL288. GroEL365.GroEL396和GroEL438六个主要的预测T-B联合抗原表位中,GroEL168显示了很强的阳性杂交信号。ELISA结果显示, GroEL168免疫反应性最强(P0.01),其次为GroEL258和GroEL288(P0.05)。 结论GroEL是幽门螺杆菌外膜蛋白。GroEL168是GroEL的优势T-B联合抗原表位,可用于制备幽门螺杆菌多抗原肽疫苗。
[Abstract]:Objective to screen and identify the dominant T cells and B cell T-B associated epitopes of Helicobacter pylori outer membrane protein (GroEL).Methods the groEL gene of Helicobacter pylori NCTC11637 strain was amplified by PCR and its prokaryotic expression system was constructed to detect the rGroEL expression of the recombinant protein. Ni-NTA affinity chromatography was used to purify rGroelle.The distribution of GroEL in Helicobacter pylori NCTC11637 and SS1 strains was detected by laser confocal microscopy. Triton X-114 was used to extract the outer membrane protein samples from the above strains, and Western Blot method was used to detect the outer membrane protein samples.The T-B antigen epitopes of GroEL were predicted by bioinformatics software and its phage display system was constructed.The immunoreactivity of recombinant bacteriophage P 鈪，

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