幽门螺杆菌外膜蛋白双价亚单位融合疫苗的实验研究

发布时间：2018-01-24 09:04

本文关键词： 幽门螺杆菌外膜蛋白 lpp20 ahpC 免疫保护粘膜免疫　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的: 幽门螺杆菌(Helicobacter pylori,Hp)是一种可定植于人胃粘膜表面的革兰氏阴性菌,现已证实Hp感染与慢性胃炎、消化性溃疡和胃粘膜相关淋巴组织淋巴瘤(MALT)发生密切相关。现行推荐的根除Hp感染的治疗方案(抗生素联合质子泵抑制剂或铋剂)虽然有效,但存在耐药菌株增加和药费昂贵等不可避免的问题。据文献报道,免疫接种对Hp感染同时具有预防和治疗的作用,因此,疫苗的研制己成为全球Hp研究的热点。在疫苗研究中,筛选有效的免疫抗原是关键性的环节。目前大多数疫苗采用的是与Hp致病相关的毒力因子作为抗原成分,如尿素酶(urease)、热休克蛋白(heat shock protein, Hsp)、空泡毒素(vacuolating cytotoxin, VacA)、细胞毒素相关抗原(cytotoxin associate antigen,CagA)、中性粒细胞激活蛋白(neutrophil-activating protein, NAP)等。革兰氏阴性细菌外膜蛋白具有良好的抗原活性,这些外膜蛋白作为抗体和免疫细胞攻击的主要靶标,可以介导对细菌最直接有效的杀灭作用,是决定免疫反应是否对人体具有保护性的关键因素。Lpp20、AhpC和UerB都是Hp的外膜蛋白成分,其编码基因具有高度保守性,也是Hp疫苗重要的免疫抗原。但一直令学者们遗憾的是每种抗原单独免疫动物时,获得的保护率均不是很高,而多种抗原联合免疫才能达到较好的效果。因此,本研究拟对Hp外膜蛋白Ahpc和Lpp20基因进行克隆表达,在此基础上,将这两个外膜基因分别与Hp保护性抗原成分尿素酶B亚单位的活性片段(UreB414)和粘膜佐剂-大肠杆菌不耐热肠毒素B亚单位(LTB)构建融合基因。通过不同表达系统得到多亚单位的融合蛋白,分别观察在小鼠体内诱导的不同免疫应答和保护作用,为筛选有效的Hp疫苗抗原奠定基础。方法: 1、采用PCR方法从Hp CQ9803基因组中扩增lpp20、ahpC基因片段并构建在原核表达载体pET-22b(+)中,通过酶切、测序鉴定阳性重组子。应用生物信息学软件DnaStar和Genbank数据库分别对已公布的Hp ahpC和lpp20基因序列进行相似性分
[Abstract]:Objective: Helicobacter pylori (Helicobacter pylori), a gram-negative bacterium that can be colonized on the surface of human gastric mucosa, has been confirmed for HP infection and chronic gastritis. Peptic ulcer is closely associated with gastric mucosa-associated lymphoid tissue lymphoma (malt). The current recommended therapy for the eradication of HP infection (antibiotics combined with proton pump inhibitors or bismuth) is effective. However, there are some unavoidable problems, such as the increase of drug-resistant strains and the high cost of drugs. According to the literature, immunization has both preventive and therapeutic effects on HP infection. The development of vaccine has become a hot spot in the research of HP in the world. Screening of effective immune antigens is a key step in vaccine research. At present, most vaccines use virulence factors associated with HP as antigen components, such as urease (Urease). Heat shock protein (HSP), vacuolating cytotoxin (Vaca). Cytotoxin associated antigen (associate antigena). Neutrophil-activating protein (NAPs). The outer membrane proteins of Gram-negative bacteria have good antigenic activity. These outer membrane proteins, as the main targets of antibody and immune cell attack, can mediate the most direct and effective killing effect on bacteria, and are the key factors to determine whether the immune response is protective to human body. Both AhpC and UerB are outer membrane proteins of HP, and their coding genes are highly conserved. It is also an important immune antigen for HP vaccine. However, it has been regretted by scholars that the protective rate of each antigen alone is not very high, and the combined immunization of multiple antigens can achieve better results. Therefore, the purpose of this study is to clone and express the Ahpc and Lpp20 genes of HP outer membrane protein. The two outer membrane genes were linked with the active fragment of urease B subunit (UreB414) and the mucosal adjuvant-Escherichia coli heat-labile enterotoxin B subunit (LTB), respectively. The fusion gene was constructed. The fusion protein of multiple subunits was obtained by different expression systems. Different immune responses and protective effects were observed in mice, which laid the foundation for screening effective HP vaccine antigens. Methods: 1. The fragment of lpp20ahpC gene was amplified by PCR from the genome of HP CQ9803 and constructed into the prokaryotic expression vector pET-22b (). DnaStar and Genbank databases were used to analyze the similarity of published HP ahpC and lpp20 gene sequences, respectively.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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