幽门螺杆菌尿素酶B抗原表位的鉴定

发布时间：2018-03-25 06:34

本文选题：幽门螺杆菌　切入点：尿素酶B　出处：《华中农业大学》2010年硕士论文

【摘要】： 幽门螺杆菌(Helicobacter pylori, H.pylori)是一种螺旋状、微需氧的革兰氏阴性杆菌,被世界卫生组织列为Ⅰ类致癌因子(TypeⅠcarcinogen)。该菌与人的慢性活动性胃炎、胃和十二指肠消化性溃疡、胃黏膜相关性淋巴组织淋巴瘤(MALT)以及胃腺癌的发生密切相关。目前临床上针对H.pylori使用的抗生素治疗方法并不理想,有着诸多缺点,因此利用疫苗进行预防接种和治疗有望成为控制H.pylori感染行之有效方法。尿素酶B亚单位(UreB)是H.pylori的一个重要保护性抗原,动物模型中也已经验证其作为疫苗抗原的安全性和有效性,寻找鉴定其有效的抗原表位对于H.pylori的诊断、治疗和疫苗开发具有重要的意义。本研究的目的是要筛选出单克隆抗体A1H10、B3D9、A3C10所对应的B细胞抗原表位,并对其免疫学功能进行初步研究。本课题共分为以下三部分： 1.抗幽门螺杆菌UreB单克隆抗体A1H10、B3D9、A3C10抗原识别表位的鉴定通过多次截短法分段表达19段UreB片段,并在此基础上通过免疫印迹方法逐步确定单克隆抗体A1H10、B3D9、A3C10所识别的抗原表位。我们成功构建了19个表达载体且均实现了表达。免疫印迹方法确定了单克隆抗体A1H10、B3D9、A3C10所对应的包含15个氨基酸残基的抗原表位,名称分别为UP32(GGGTGPADGTNATTI)、UP35 (WMLRAAEEYSMNLGF)、UP38 (TLHDMGIFSITSSDS),分别位于H.pylori尿素酶B亚单位(UreB)上第158-172、181-195、349-363位氨基酸残基。所鉴定出来的3个表位通过ELISA得到进一步地证实,纯化的融合表位蛋白能与相对应的单抗及临床感染H.pylori的病人阳性血清发生特异性的结合反应。以鉴定出来的抗原表位核苷酸序列为依据,采用化学合成法合成表位多肽,分别命名为UP32、UP35、UP38,纯度为90%以上。经ELISA或Dot blot方法鉴定,ELISA结果显示单克隆抗体A1H10、B3D9、A3C10分别与合成肽UP32、UP35、UP38发生了特异性抗原抗体反应,说明单抗A1H10、B3D9、A3C10所识别的抗原表位分别为UP32、UP35、UP38。Dot blot方法鉴定结果表明单抗B3D9所针对的抗原表位为UP35。ELISA结果显示合成肽与H.pylori的病人阳性血清也发生了结合反应,说明病人血清中产生了针对这些表位的抗体。 2.幽门螺杆菌尿素酶B亚单位(UreB)B细胞抗原表位的免疫学功能研究将纯化后的融合表位蛋白免疫小鼠,获得了较高效价的抗血清,其效价高达1：51200,说明我们构建的融合表位蛋白能够很好地激发机体的体液免疫应答。免疫印迹试验证实融合表位蛋白产生的抗血清能够与重组的截短片段UreBM及天然的幽门螺杆菌UreB发生特异性抗原抗体反应。ELISA结果发现,免疫的鼠抗血清能够很好地识别相应的表位合成肽,同时也能与天然UreB发生反应。尿素酶活性抑制试验表明,单抗A1H10、B3D9、A3C10对尿素酶的活性具有较好抑制作用,抑制率可达70%,多抗血清对尿素酶的活性也有一定的抑制作用,抑制率可达50%。 3.呈现表达串联表位及其免疫学评价通过基因重组的方法,将串连的表位基因片段敲入到本实验室构建好的减毒沙门菌株S.ty2ΔrfaHΔssaV染色体上,与外膜蛋白A (OmpA)的C-端融合表达于载体菌表面,得到重组菌S. ty2ΔrfaHΔssaV-U258,通过免疫印迹分析证实目标蛋白获得了表达。经口胃途径将表达系统S.ty2ΔrfaHΔssaV-U258免疫小鼠,高、低剂量组都有效地激发了机体产生针对外源抗原UreB的免疫反应,抗外源抗原的IgG滴度高达1：3200,抗载体菌的IgG滴度为1：800-1：3200,而且也能够检测到抗原特异性的分泌型IgA的产生,表明口服免疫的两组均能刺激机体产生有效的黏膜免疫反应。总之,本研究通过分子生物学与免疫学结合的方法鉴定出了单克隆抗体A1H10、B3D9、A3C10所对应的抗原表位,经动物实验验证具有良好的免疫原性,为新型多联多聚表位疫苗的开发提供了重要的理论基础和实验依据。
[Abstract]:Helicobacter pylori (Helicobacter pylori H.pylori) is a spiral, microaerophilic gram negative bacilli, WHO was listed as a class I carcinogen (Type I carcinogen). The pathogen of chronic active gastritis and stomach and duodenum, peptic ulcer, gastric mucosa associated lymphoid tissue lymphoma (MALT) and gastric cancer is closely related to the current clinical use of H.pylori for antibiotic therapy is not ideal, there are many shortcomings, therefore the use of vaccine and treatment of H.pylori infection control is expected to become effective methods. Urease B subunit (UreB) is an important protective antigen of H.pylori, animal model also has been verified as a safe and effective vaccine antigens, to identify the effective epitopes for H.pylori diagnosis, treatment and vaccine development has important significance The purpose of this study is to screen out the B cell epitopes corresponding to monoclonal antibodies A1H10, B3D9 and A3C10, and to preliminarily study their immunological functions. The research is divided into three parts.
Identification of 1. anti Helicobacter pylori UreB monoclonal antibody A1H10, B3D9, A3C10 antigen recognition epitope
Through several truncated fractional expression of 19 UreB fragments, and on this basis by immunoblotting method identified by monoclonal antibody A1H10, B3D9 epitope recognized by A3C10. We successfully constructed 19 expression vector and have achieved expression. Western blot confirmed A1H10 monoclonal antibody, B3D9, corresponding to A3C10 contains 15 amino acid epitopes, the name was UP32 (GGGTGPADGTNATTI), UP35 (WMLRAAEEYSMNLGF), UP38 (TLHDMGIFSITSSDS), located at H.pylori urease B subunit (UreB) on the 158-172181-195349-363 amino acid residues. The 3 epitopes were further confirmed by ELISA identified, combined with patient reaction positive serum purified fusion protein with the corresponding epitope of monoclonal antibody and clinical H.pylori infection specific. To identify the anti epitope nucleotide sequence according to the According to the synthetic epitope peptides by chemical synthesis, which were named UP32, UP35, UP38, the purity was more than 90%. By ELISA or Dot blot identification methods, ELISA results showed that the monoclonal antibody A1H10, B3D9, A3C10 respectively with the synthetic peptide UP32, UP35, the specificity of antigen antibody reaction UP38 that mAb A1H10 B3D9 A3C10, the identified epitopes were UP32, UP35, blot method identification results of UP38.Dot revealed that B3D9 antibody against epitope UP35.ELISA results showed that patients with H.pylori positive serum peptide has the binding reaction to that in serum of patients with the antibodies against these epitopes.
Immunological function of 2. Helicobacter pylori urease B subunit (UreB) B cell antigen epitopes
The purified fusion epitope protein immunized mice obtained high titer antiserum. The titer of 1:51200, we construct the table shows the fusion of humoral immune response protein is able to stimulate the body. Western blot showed that antiserum epitope fusion protein can produce truncated fragments of UreBM and recombinant and the natural UreB of Helicobacter pylori specific antigen antibody reaction.ELISA results showed that mouse antiserum immune to identify the epitopes of the corresponding synthetic peptides, also can react with natural UreB. Urease activity inhibition test showed that the monoclonal antibody A1H10, B3D9, A3C10 on the activity of urease has good inhibition, inhibition the rate of up to 70%, the activity of urease antiserum also has certain inhibitory effect, the inhibition rate is 50%.
3. presentation of expression in series epitopes and their immunological evaluation
By the method of gene recombination, the tandem epitope gene knock in the laboratory to construct the attenuated Salmonella strain S.ty2 Delta rfaH Delta ssaV chromosome, and outer membrane protein A (OmpA) C- terminal fusion expression on the carrier surface to obtain the recombinant bacteria S. bacteria, Ty2 Delta rfaH Delta ssaV-U258, through Western blot analysis confirmed that the target protein was expressed. The oral route expression system S.ty2 Delta rfaH Delta ssaV-U258 immunized mice, high, low dose group can effectively stimulate the immune response to exogenous antigen UreB, IgG titers of anti exogenous antigen up to 1:3200, IgG titers of anti bacteria was 1:800-1:3200 carrier, but also can detection of antigen-specific secretory IgA, indicating that the two groups of oral immunization can elicit mucosal immune response effectively.
In conclusion, this study through the identification of molecular biology and immunology with a monoclonal antibody A1H10, B3D9 epitope corresponding to A3C10, the animal experiment has good immunogenicity, provides an important theoretical basis and experimental basis for the new multi poly epitope vaccine development.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【引证文献】