本文選題：重組大腸桿菌ghost + 鞭毛粘附素HpaA　； 參考：《第三軍醫(yī)大學(xué)》2005年碩士論文

【摘要】：幽門螺桿菌(Helicobacter pylori,Hp) 是一種定植于胃內(nèi)的革蘭氏染色陰性、螺桿狀或S 狀的微需氧菌,可引發(fā)慢性胃炎、胃潰瘍、十二指腸潰瘍等上消化道疾病,并與胃癌、胃粘膜相關(guān)淋巴組織淋巴瘤的發(fā)生有關(guān)。由于傳統(tǒng)的抗生素療法面臨著價格高、耐藥性等諸多問題,開發(fā)有效的Hp 疫苗成為防治Hp 感染的研究熱點。目前研發(fā)的Hp 疫苗,多是采用基因工程獲得HP 的有效抗原,再輔以佐劑,如福氏佐劑、CT、LT,脂質(zhì)A,但這些佐劑都只是非特異地加強抗原的免疫效應(yīng)。而傳統(tǒng)的滅活疫苗雖有好的保護作用,但是現(xiàn)用的加熱、照射、化學(xué)處理等滅活方法,常常引起抗原性的降低和改變。 “Bacterial ghost”作為一種新型疫苗或抗原遞送系統(tǒng),是通過控制PhiX174 噬菌體E 裂解基因在細(xì)菌中表達(dá)得以制備的,其實質(zhì)是一種無細(xì)菌胞漿和核酸的空細(xì)菌外膜。據(jù)報道,E 裂解基因編碼的蛋白在細(xì)菌胞壁上可形成穿膜隧道,通過此隧道胞漿成分全部滲出。因為這種形式保留了和活菌一樣完整的細(xì)菌胞膜結(jié)構(gòu)和相關(guān)抗原蛋白,故而能夠攜帶外源抗原靶向黏附于體內(nèi)黏膜組織并易于被相應(yīng)抗原提呈細(xì)胞捕獲。Hp 粘附于胃上皮細(xì)胞是其定植進而致病的前提,有效阻斷Hp 粘附是防治Hp 感染的途徑之一�，F(xiàn)在認(rèn)為幽門螺桿菌黏附素A(Hp adhesion A,HpaA)是Hp 的主要黏附因子,該蛋白為Hp 所特有的抗原成分,其氨基酸序列高度保守,且多數(shù)Hp 感染患者血清中能檢測到HpaA 抗體,因此可作為理想的Hp 疫苗靶抗原。 本課題嘗試用“Bacterial ghost”這一新型疫苗遞送形式和內(nèi)佐劑來傳輸HpaA 抗原,擬構(gòu)建表達(dá)HpaA 的重組大腸桿菌ghost 疫苗,即先把HpaA 錨定表達(dá)于大腸桿菌的胞膜,然后誘導(dǎo)細(xì)菌裂解以制備表達(dá)HpaA 的重組大腸桿菌ghost。本研究中,在成功制備大腸桿菌ghost 的基礎(chǔ)上,采用兩種技術(shù)路線制備表達(dá)HpaA 的重組大腸桿菌ghost:一種為雙質(zhì)粒共表達(dá)法,即將含裂解基因盒的裂解質(zhì)粒pHH43 和含hpaA 表達(dá)盒的表達(dá)質(zhì)粒pBIOEX-E’-hpaA 同時轉(zhuǎn)入大腸桿菌BL21,先IPTG 誘導(dǎo)HpaA 表達(dá),再熱誘導(dǎo)E 裂解蛋白表達(dá)以制備重組ghost;二為單質(zhì)粒雙表達(dá)法,即將裂解基因盒和hpaA 表達(dá)盒構(gòu)建到同一質(zhì)粒pET-28a 上,先IPTG 誘導(dǎo)表達(dá)HpaA 蛋白,再用熱誘導(dǎo)表達(dá)E 裂解蛋白而獲得重組ghost。主要研究內(nèi)容簡述如下:
[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:
【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】：2005
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 謝波;重組雙基因支氣管敗血波氏桿菌幽靈的研究[D];南昌大學(xué);2010年

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本文編號：1785619