本文關(guān)鍵詞： 幽門螺桿菌 外膜蛋白 lpp20 ahpC 免疫保護(hù) 粘膜免疫　出處：《第三軍醫(yī)大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 幽門螺桿菌(Helicobacter pylori,Hp)是一種可定植于人胃粘膜表面的革蘭氏陰性菌,現(xiàn)已證實(shí)Hp感染與慢性胃炎、消化性潰瘍和胃粘膜相關(guān)淋巴組織淋巴瘤(MALT)發(fā)生密切相關(guān)�，F(xiàn)行推薦的根除Hp感染的治療方案(抗生素聯(lián)合質(zhì)子泵抑制劑或鉍劑)雖然有效,但存在耐藥菌株增加和藥費(fèi)昂貴等不可避免的問題。據(jù)文獻(xiàn)報道,免疫接種對Hp感染同時具有預(yù)防和治療的作用,因此,疫苗的研制己成為全球Hp研究的熱點(diǎn)。 在疫苗研究中,篩選有效的免疫抗原是關(guān)鍵性的環(huán)節(jié)。目前大多數(shù)疫苗采用的是與Hp致病相關(guān)的毒力因子作為抗原成分,如尿素酶(urease)、熱休克蛋白(heat shock protein, Hsp)、空泡毒素(vacuolating cytotoxin, VacA)、細(xì)胞毒素相關(guān)抗原(cytotoxin associate antigen,CagA)、中性粒細(xì)胞激活蛋白(neutrophil-activating protein, NAP)等。革蘭氏陰性細(xì)菌外膜蛋白具有良好的抗原活性,這些外膜蛋白作為抗體和免疫細(xì)胞攻擊的主要靶標(biāo),可以介導(dǎo)對細(xì)菌最直接有效的殺滅作用,是決定免疫反應(yīng)是否對人體具有保護(hù)性的關(guān)鍵因素。Lpp20、AhpC和UerB都是Hp的外膜蛋白成分,其編碼基因具有高度保守性,也是Hp疫苗重要的免疫抗原。但一直令學(xué)者們遺憾的是每種抗原單獨(dú)免疫動物時,獲得的保護(hù)率均不是很高,而多種抗原聯(lián)合免疫才能達(dá)到較好的效果。 因此,本研究擬對Hp外膜蛋白Ahpc和Lpp20基因進(jìn)行克隆表達(dá),在此基礎(chǔ)上,將這兩個外膜基因分別與Hp保護(hù)性抗原成分尿素酶B亞單位的活性片段(UreB414)和粘膜佐劑-大腸桿菌不耐熱腸毒素B亞單位(LTB)構(gòu)建融合基因。通過不同表達(dá)系統(tǒng)得到多亞單位的融合蛋白,分別觀察在小鼠體內(nèi)誘導(dǎo)的不同免疫應(yīng)答和保護(hù)作用,為篩選有效的Hp疫苗抗原奠定基礎(chǔ)。 方法: 1、采用PCR方法從Hp CQ9803基因組中擴(kuò)增lpp20、ahpC基因片段并構(gòu)建在原核表達(dá)載體pET-22b(+)中,通過酶切、測序鑒定陽性重組子。應(yīng)用生物信息學(xué)軟件DnaStar和Genbank數(shù)據(jù)庫分別對已公布的Hp ahpC和lpp20基因序列進(jìn)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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