表达幽门螺杆菌鞭毛粘附素的重组大肠杆菌ghost型疫苗的制备及鉴定

发布时间：2018-04-22 04:29

本文选题：重组大肠杆菌ghost + 鞭毛粘附素HpaA　；参考：《第三军医大学》2005年硕士论文

【摘要】：幽门螺杆菌(Helicobacter pylori,Hp) 是一种定植于胃内的革兰氏染色阴性、螺杆状或S 状的微需氧菌,可引发慢性胃炎、胃溃疡、十二指肠溃疡等上消化道疾病,并与胃癌、胃粘膜相关淋巴组织淋巴瘤的发生有关。由于传统的抗生素疗法面临着价格高、耐药性等诸多问题,开发有效的Hp 疫苗成为防治Hp 感染的研究热点。目前研发的Hp 疫苗,多是采用基因工程获得HP 的有效抗原,再辅以佐剂,如福氏佐剂、CT、LT,脂质A,但这些佐剂都只是非特异地加强抗原的免疫效应。而传统的灭活疫苗虽有好的保护作用,但是现用的加热、照射、化学处理等灭活方法,常常引起抗原性的降低和改变。 “Bacterial ghost”作为一种新型疫苗或抗原递送系统,是通过控制PhiX174 噬菌体E 裂解基因在细菌中表达得以制备的,其实质是一种无细菌胞浆和核酸的空细菌外膜。据报道,E 裂解基因编码的蛋白在细菌胞壁上可形成穿膜隧道,通过此隧道胞浆成分全部渗出。因为这种形式保留了和活菌一样完整的细菌胞膜结构和相关抗原蛋白,故而能够携带外源抗原靶向黏附于体内黏膜组织并易于被相应抗原提呈细胞捕获。Hp 粘附于胃上皮细胞是其定植进而致病的前提,有效阻断Hp 粘附是防治Hp 感染的途径之一。现在认为幽门螺杆菌黏附素A(Hp adhesion A,HpaA)是Hp 的主要黏附因子,该蛋白为Hp 所特有的抗原成分,其氨基酸序列高度保守,且多数Hp 感染患者血清中能检测到HpaA 抗体,因此可作为理想的Hp 疫苗靶抗原。本课题尝试用“Bacterial ghost”这一新型疫苗递送形式和内佐剂来传输HpaA 抗原,拟构建表达HpaA 的重组大肠杆菌ghost 疫苗,即先把HpaA 锚定表达于大肠杆菌的胞膜,然后诱导细菌裂解以制备表达HpaA 的重组大肠杆菌ghost。本研究中,在成功制备大肠杆菌ghost 的基础上,采用两种技术路线制备表达HpaA 的重组大肠杆菌ghost:一种为双质粒共表达法,即将含裂解基因盒的裂解质粒pHH43 和含hpaA 表达盒的表达质粒pBIOEX-E’-hpaA 同时转入大肠杆菌BL21,先IPTG 诱导HpaA 表达,再热诱导E 裂解蛋白表达以制备重组ghost;二为单质粒双表达法,即将裂解基因盒和hpaA 表达盒构建到同一质粒pET-28a 上,先IPTG 诱导表达HpaA 蛋白,再用热诱导表达E 裂解蛋白而获得重组ghost。主要研究内容简述如下:
[Abstract]:Helicobacter pylori (Helicobacter pylori) is a gram-negative, screw or S-shaped microaerobic bacterium found in the stomach that causes chronic gastritis, gastric ulcers, duodenal ulcers, and other upper gastrointestinal diseases, and is associated with gastric cancer. Gastric mucosa-associated lymphoid tissue lymphoma is associated with the development of lymphomas. Because the traditional antibiotic therapy is faced with many problems such as high price, drug resistance and so on, the development of effective HP vaccine has become a research hotspot in the prevention and treatment of HP infection. At present, most of the HP vaccines are obtained by genetic engineering and supplemented with adjuvants, such as Freund's adjuvant CTL, lipid A. however, these adjuvants only enhance the immune effect of the antigen in a nonspecific way. Although the traditional inactivated vaccine has a good protective effect, the current inactivation methods such as heating, irradiation and chemical treatment often cause the antigenicity to decrease and change. As a new vaccine or antigen delivery system, "Bacterial ghost" was prepared by controlling the expression of PhiX174 phage E cleavage gene in bacteria. In essence, it is an empty bacterial outer membrane without bacterial cytoplasm and nucleic acid. It is reported that the protein encoded by the E cleavage gene can form a membranous tunnel on the wall of the bacterial cell, through which all the cytoplasmic components are exudated. Because this form retains a bacterial membrane structure and associated antigen proteins that are as complete as living bacteria. Therefore, it is a prerequisite for the gastric epithelial cells to be able to carry foreign antigen to target adhesion to the mucosal tissue and be easily captured by the corresponding antigen-presenting cells. Effectively blocking the HP adhesion is one of the ways to prevent and treat HP infection. It is now considered that Helicobacter pylori adhesion factor A(Hp adhesion A (HPA) is the main adhesion factor of HP. The protein is a unique antigen of HP, its amino acid sequence is highly conserved, and HpaA antibodies can be detected in the sera of most HP infected patients. Therefore, it can be used as an ideal target antigen of HP vaccine. In this paper, we try to transfer HpaA antigen with "Bacterial ghost", a novel vaccine delivery form and internal adjuvant. We intend to construct recombinant E. coli ghost vaccine expressing HpaA, that is, to anchor HpaA in the cell membrane of Escherichia coli. Then the bacteria were induced to cleavage to prepare recombinant Escherichia coli ghost. expressing HpaA. In this study, on the basis of the successful preparation of Escherichia coli ghost, two technical routes were used to prepare recombinant Escherichia coli ghost expressing HpaA. The lytic plasmid pHH43 containing the lytic gene cassette and the expression plasmid pBIOEX-E'-hpaA containing the hpaA expression cassette were transferred into E. coli BL21 at the same time. The expression of HpaA was induced by IPTG first, then the expression of E lytic protein was induced by heat to prepare recombinant ghost. the second was the double expression of single plasmid. The cleavage gene box and the hpaA expression box were constructed into the same plasmid pET-28a. The HpaA protein was first induced by IPTG, and then the recombinant ghostwas obtained by heat induced expression of E cleavage protein. The main contents of the study are summarized as follows:
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【引证文献】