幽门螺杆菌GroEL蛋白重组表达及表达产物小鼠免疫保护作用  

Recombinant expression of Helicobacter pylori GroEL protein and immunoprotection of the expression product in mice

作　　者：王艳芳[1] 张慧[2] 王欢[3] 孙萍萍[3] 严杰[3] 孙爱华[4] 

机构地区：[1]杭州师范大学基础医学部,杭州310036 [2]浙江树人大学生物工程学院,杭州310015 [3]浙江大学医学院病原生物学系 [4]浙江医学高等专科学校基础医学部,杭州310053

出　　处：《中华微生物学和免疫学杂志》2012年第4期340-345,共6页Chinese Journal of Microbiology and Immunology

基　　金：浙江省教育厅科技计划项目（Y201016840）

摘　　要：目的检测幽门螺杆菌临床菌株groEL基因序列差异，确定重组表达的GroEL蛋白（rGroEL）抗原性、免疫反应性以及对幽门螺杆菌感染小鼠的免疫保护性。方法采用PCR及测序法了解幽门螺杆菌临床菌株groEL基因序列。构建幽门螺杆菌groEL基因pET42a-E．coli BL21DE3原核表达系统，采用SDS-PAGE及凝胶图像分析系统确定rGroEL表达量，Ni-NTA亲和层析法提纯rGroEL。采用ELISA和Western blot检测rGroEL的抗原性和免疫反应性，采用幽门螺杆菌全菌为包被抗原的ELISA确定GruEL膜定位。采用幽门螺杆菌小鼠感染模型了解rGroEL免疫保护作用。结果35株幽门螺杆菌临床菌株groEL基因序列相似性高达99．4％～100％。rGroEL表达量可达细菌总蛋白的56％，SDS—PAGE后提纯的rGmEL在凝胶中显示为单一的条带。rGroEL可诱导家兔和小鼠产生高效价IgG抗体，同时也能被幽门螺杆菌全菌抗体（Hp-IgG）或rGroEL-IgG识别并与之结合。GroEL是幽门螺杆菌表面蛋白抗原。50、100或200μg rGroEL免疫可分别使50．0％（6／12）、75．0％（9／12）和91．7％（11／12）小鼠免于幽门螺杆菌SS1株的感染，200μg rGroEL免疫保护率（91．7％）显著高于50μg rGroEL（50．0％）（P〈0．05）。结论幽门螺杆菌GroEL蛋白是序列保守、具有良好免疫原性和免疫保护性的表面抗原，可作为基因工程疫苗候选抗原。Objective To determine the sequence diversity in groEL gene of Helicobacter pylori isolates, and the antigenieity, immunoreactivity and immunoprotection of recombinant expressed GroEL （rGroEL） against H. pylori-infection in mice. Methods The sequences of groEL genes from H. pylori isolates were detected by PCR and sequencing. A prokaryotie expression system of tl. pylori groEL gene using pET42a and E. coli BL21DE3 was generated. SDS-PAGE and Gel Image Analyzer were used to measure the yield of rGroEL and Ni-NTA affinity chromatography was applied to extract the expressed rGroEL. The antigenicity and immunoreaetivity of rGroEL were examined using ELISA and Western blot assay. The membrane location of GroEL was determined by ELISA using whole ceils of H. pylori as the coated antigen. The immunoprotection of rGroEL was detected in H. pylori-infected mouse model. Results All the 35 tested H. pylori isolates possess the groEL gene with 99.4% to 100% sequence identities. The yield of rGroEL expression occupied 56% of the total baeterlal proteins and the extracted rGroEL showed a single band in gel after SDS- PAGE. rGroEL could induce the production of high-titer IgG in rabbits or mice. The IgG against whole cell of H.pylori （Hp-IgG） and rGroEL-IgG could recognize as well as combine with rGroEL protein. GroEL is a superficial protein antigen of H. pylori. The immunization with 50, 100 or 200 μg rGroEL prevented 50.0% （6/12）, 75.0% （9/12） or 91.7% （11/12） mice from infection of H. pylori strain SS1. The immunoprotective rate （91. 7% ） due to the immunization of 200 Ixg rGroEL was significantly higher than that （50.0%） of 50 μg rGroEL （P〈0.05）. Conclusion The GroEL protein is a sequence-conserved, immunogenic and immunoprotective superficial antigen of H. pylori, which can be used as the immunogen candi- date for developing genetically engineering vaccines.

关 键 词：幽门螺杆菌 GroEL蛋白 重组表达 免疫保护作用 

分 类 号：R378[医药卫生—病原生物学]