幽门螺杆菌UreB抗原表位预测及其有效表位噬菌体展示的筛选

发布时间：2018-07-18 12:26

【摘要】： 背景和目的幽门螺杆菌(Helicobacter pylori，Hp)是微需氧的革兰阴性杆菌，呈螺旋状。多数国家和地区Hp感染率高达50％以上，许多感染者都是无症状的，但是有部分感染者可出现急慢性胃炎和消化性溃疡，Hp的持续性感染还与胃腺癌和胃淋巴瘤的发生密切相关。Hp已被世界卫生组织列为Ⅰ类致癌因子。近年流行病学资料还发现，幽门螺杆菌感染还与冠心病、动脉硬化、血小板减少性紫癜、糖尿病、缺铁性贫血、慢性特发性荨麻疹、支气管炎等发病有关。尿素酶是Hp的一种重要毒力因子，它能够将尿素分解成为氨气和二氧化碳，以中和胃部的酸性环境，有利于Hp的定植。尿素酶占细菌总蛋白的10％左右。尿素酶B亚基(UreB)是尿素酶的大亚基，且是尿素酶催化位点所在亚基。UreB在各螺杆菌属间序列保守，且具有较强的抗原性，故吸引了较多学者研究基于UreB的疫苗。国内外利用重组尿素酶B亚基研制疫苗均在一定程度上能引起免疫应答，但无法在清除Hp效果上达成一致。只包含一种抗原蛋白的疫苗所诱导的免疫保护作用通常较弱，实际应用可用性不高。近年发展起来的含多个和多种抗原表位(epitope)的多抗原肽(multipleantigenic peptide，MAP)研究策略，极有可能成为解决亚基问题有效途径。寻找有效的抗原表位是研制MAP的第一步。研究抗原表位可深层次地揭示蛋白抗原分子诱导免疫应答的分子机制及其特点，并在研制新型分子疫苗、新型血清学检测方法等方面有重要应用前景。本研究拟筛选幽门螺杆菌尿素酶B亚单位(UreB)分子中的B以及Th细胞抗原表位，为研制多抗原肽(MAP)疫苗奠定基础。实验方法Ni-NTA亲和层析法提纯目的重组表达产物rUreB，，常规皮内免疫法制备兔抗血清。采用生物信息学技术对UreB的B细胞和Th细胞表位进行预测和分析。选择UreB主要B细胞和Th细胞联合表位肽并融合表达于M13KE噬菌体PⅢ蛋白上，PEG／NaCl沉淀法提纯重组噬菌体，SDS-PAGE鉴定目的重组PⅢ蛋白(rPⅢ)。分别以自制备rUreB抗血清和商品化抗Hp全菌IgG为一抗，采用Western blot对上述表位肽进行鉴定和筛选。结果rUreB表达量约为细菌总蛋白的30％，提纯后仅见单一蛋白条带。免疫家兔后获得抗血清。与GenBank中相关序列比较，本实验室的ureB基因核苷酸和氨基酸相似性分别为96％～99.5％和96％～100％。综合预测结果，选择4个同时含有Th细胞和B细胞表位肽段UreB230、UreB322、UreB479和UreB527，并在M13噬菌体中获得成功表达，采用不同一抗的Western blot均显示相似的阳性结果，但UreB322和UreB527反应强度明显高于UreB230和UreB479。结论本研究成功获得重组UreB蛋白，并得到兔抗血清。构建了Th细胞和B细胞联合表位肽段的噬菌体展示系统。所采用的生物信息学技术可有效地预测抗原表位。实验室鉴定UreB322和UreB527为UreB有效抗原表位，可作为幽门螺杆菌MAP疫苗的候选表位。
[Abstract]:Background and objective Helicobacter pylori (Hp) is a micro aerobic gram-negative bacilli, which is spiral. In most countries and regions, the rate of Hp infection is more than 50%. Many infected people are asymptomatic, but some infected people can appear acute and chronic gastritis and peptic ulcers. The persistent infection of Hp is also associated with gastric adenocarcinoma and gastric lymph nodes. .Hp has been listed as a class I carcinogenic factor by WHO. In recent years, the epidemiological data also found that Helicobacter pylori infection is associated with coronary heart disease, arteriosclerosis, thrombocytopenic purpura, diabetes, iron deficiency anemia, chronic idiopathic urticaria, bronchitis, and so on.
Urease is an important virulence factor of Hp, which can decompose urea into ammonia and carbon dioxide to neutralize the acid environment of the stomach and facilitate the colonization of Hp. The urease is about 10% of the total bacterial protein. The urease B subunit (UreB) is the large subunit of the urease, and the subunit of the urease catalyzed site.UreB is in the genus Helicobacter. It is conservative and has strong antigenicity, which attracts many scholars to study the vaccine based on UreB. The use of recombinant urease B subunit to produce vaccines to some extent can cause immune response, but can not achieve agreement on the effect of eliminating Hp. The immunization induced by a vaccine containing only one antigen protein is effective. It is often weak, and the practical application is not high.
The research strategy of multiple antigenic epitopes (epitope) with multiple antigen epitopes (multipleantigenic peptide, MAP), which has been developed in recent years, is very likely to be an effective way to solve subunit problems. Finding effective antigen epitopes is the first step in the development of MAP. The study of antigen epitopes can deeply reveal the immune response induced by protein antigen molecules. The molecular mechanism and its characteristics have an important application prospect in the development of new molecular vaccines and new serological detection methods. This study intends to screen B and Th cell antigen epitopes of Helicobacter pylori urease B subunit (UreB), laying a foundation for the development of polyantigenic peptide (MAP) vaccine.
The recombinant expression product rUreB was purified by Ni-NTA affinity chromatography. The rabbit antiserum was prepared by routine intradermal immunization. Bioinformatics was used to predict and analyze the epitopes of B and Th cells of UreB. The combined epitopes of the UreB main B cells and Th cells were selected and the fusion table was reached on the M13KE phage P III protein, PEG / NaCl. The recombinant phage was purified by precipitation method, and the recombinant P III protein (rP III) was identified by SDS-PAGE. RUreB antiserum and commercialized anti Hp whole bacteria IgG were used as one antibody, respectively, and the above epitope peptides were identified and screened by Western blot.
Results the expression of rUreB was about 30% of the total bacterial protein. After purification, only a single protein band was found. After immunizing the rabbit, the antiserum was obtained. Compared with the related sequences in GenBank, the nucleotide and amino acid similarity of the ureB gene in our laboratory was 96% to 99.5% and 96% to 100%., respectively, and 4 had Th cells and B. The cell epitope peptide segments UreB230, UreB322, UreB479 and UreB527 were successfully expressed in the M13 phage. The use of different Western blot showed similar positive results, but the reaction intensity of UreB322 and UreB527 was significantly higher than that of UreB230 and UreB479..
Conclusion the recombinant UreB protein was successfully obtained and rabbit antiserum was obtained. The phage display system of the joint epitope of Th cells and B cells was constructed. The bioinformatics techniques used can effectively predict the epitopes of the antigen. The effective antigen epitopes of UreB322 and UreB527 are identified in the laboratory and can be used as the MAP vaccine of Helicobacter pylori. Select the table.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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