基于幽门螺杆菌UreB优势抗原表位多抗原肽疫苗制备及其免疫保护作用  

Synthesis of multiple antigenic peptide vaccine based on predominant epitopes of Helicobacter pylori UreB protein and immunoprotection of the vaccine

作　　者：王艳芳[1] 王欢[2] 张慧[3] 严杰[1] 阮萍[4] 

机构地区：[1]杭州师范大学基础医学部,310036 [2]浙江大学医学院病原生物学系,杭州310058 [3]浙江树人大学生物工程学院 [4]绍兴文理学院医学院检验系,312000

出　　处：《中华微生物学和免疫学杂志》2012年第3期268-275,共8页Chinese Journal of Microbiology and Immunology

基　　金：浙江省教育厅科研计划项目（Y201016840）

摘　　要：目的构建串联式幽门螺杆菌UreB抗原优势表位UreB322和UreB527原核重组表达系统，制备UreB322和UreB527表位与多肽载体Poly—Asp．Lys的多抗原肽（MAP）疫苗并确定其免疫原性和免疫保护性。方法采用连接引物PCR构建含肠激酶（EK）位点的串联式UreB322和UreB527表位基因及其原核表达系统。表达的目的重组融合蛋白8×[rEK-UreB322-EK-UreB527-EK]经EK水解后用SephadexG-25柱分离rUreB322-EK和rUreB527-EK片段。采用碳二亚胺法将rUreB322-EK、rUreB527-EK与Poly．Asp—Lys载体分子交联，制备出多抗原肽疫苗MAP—rUreB322／B527。采用ELISA和Westernblot分别检测各重组表位肽及MAP-rUreB322／527的抗原性和免疫反应性。采用幽门螺杆菌SS1株感染BALB／c小鼠模型检测MAP—rUreB322／527免疫保护效果。结果获得了8次重复串联的UreB322和UreB527编码基因及其原核表达系统，目的融合蛋白8x[rEK—UreB322-EK—rUreB527-EK]表达量可达细菌总蛋白的48％。肠激酶可将目的融合蛋白完全水解为rUreB322-EK和rUreB527-EK片段。rUreB322-EK和rUreB527-EK与Poly—Asp-Lys交联率高达92．5％。幽门螺杆菌全菌抗体及rUreB．IgG均能识别rUreB322．EK、rUreB527．EK和MAP．rUreB322／527并与之结合。MAP．rUreB322／527免疫小鼠血清抗体水平明显高于rUreB（P〈0．05）。50或100p,gMAP—rUreB322／527对幽门螺杆菌SS1株感染小鼠的保护率（83．3％和91．7％）明显高于等量rUreB（41．7％和50．0％）（P〈0．05）。结论本研究成功地构建了幽门螺杆菌UreB抗原优势表位UreB322和UreB527重复串联式编码基因及其原核表达系统，基于UreB优势抗原表位的多抗原肽疫苗MAP—rUreB322／527能显著提高免疫保护效果。Objective To generate a prokaryotic expression system of series predominant epitopes （UreB322 and UreB527） of Helicobacter pylori UreB protein, and to synthesize a multiple antigenic peptide （MAP） vaccine by linking both the two epitopes with a peptide carrier （Poly-Asp-Lys）, and to determine the immunogenicity and immunoprotection of the MAP vaccine. Methods Linking primer PCR was performed to generate an enterokinase（EK） site-containing series UreB322 and UreB527 epitope encoding gene for construction of its prokaryotic expression system. The expressed target recombinant fusion protein 8 × [ rEK-UreB322-EK-UreB527-EK] was hydrolyzed with EK and then rUreB322-EK and rUreB527-EK epitope peptides were extracted using a Sephadex G-25 column, rUreB322-EK, rUreB527-EK and Poly-Asp-Lys were linked using carbodiimide method to produce a MAP vaccine （MAP-rUreB322/B527）. The antigenicity and immunoreactivity of each of the two epitope peptides and MAP-rUreB322/527 were determined by ELISA and Western blot assay. An animal H. pylori strain SSl-infected model in BALB/c mice was used to detect the immunoprotection of MAP-rUreB322/527. Results An octuple-repeated series UreB322-UreB527 enco- ding gene and its prokaryotic expression system were obtained. The yield of target fusion protein 8 × [ rEK- UreB322-EK-rUreB527-EK] was as high as 48% of the total bacterial proteins. EK hydrolyzed the target fusion protein completely into rUreB322-EK and rUreB527-EK peptides. The linking ratio of rUreB322-EK, rUreB527-EK and Poly-Asp-Lys was as high as 92.5%. The antibody against whole cell of H. pylori and rUreB-IgG could recognize and combine with the rUreB322-EK, rUreB527-EK or MAP-rUreB322/527. The specific serum antibody level in MAP-rUreB322/527-immunized mice was significantly higher than that in rUreB-immunized mice （ P〈0.05 ）. The immunoprotective rates（83.3% and 91.7% ） by immunization with 50 or 100 μg MAP-rUreB322/527 in the H. pylori strain SSl-infected mice were significantly higher that t

关 键 词：幽门螺杆菌 尿素酶B亚单位基因 优势抗原表位 多抗原肽疫苗 免疫保护 

分 类 号：R392[医药卫生—免疫学]