本文關(guān)鍵詞： 鼠疫F1-V抗原 黏膜佐劑 滴鼻免疫　出處：《西北農(nóng)林科技大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 大多數(shù)感染因子是通過黏膜進入動物和人體的。因此,黏膜免疫的預(yù)防接種技術(shù)顯得很重要。研究表明霍亂毒素B亞單位(cholera toxin B,CTB)、蛋白體(proteosomes)和protollin是比較有效的黏膜佐劑。蛋白體中的PorA、PorB和Class 4類蛋白分子佐劑效應(yīng)的研究對于開發(fā)新黏膜佐劑和對蛋白體作用機制的研究有重要的意義。 鼠疫在全世界范圍內(nèi)有死灰復(fù)燃之勢,近年來中國已暴發(fā)數(shù)次小規(guī)模的人間鼠疫。在各種鼠疫疾病之中,尤以肺鼠疫破壞最大。為了能達到長期起效的目的,對肺鼠疫提供全面和長久的保護,鼠疫黏膜佐劑疫苗的研究是十分必要和重要的。 本研究從B群2b型腦膜炎奈瑟氏雙球菌中提取蛋白體佐劑;從福氏2a痢疾桿菌中提取脂多糖(Lipopolysaccharides,LPS),與蛋白體復(fù)合制備protollin佐劑;基因重組了CTB佐劑和用于黏膜佐劑研究的PorA,PorB和Class 4蛋白,為制備鼠疫F1-V黏膜佐劑疫苗提供可靠的黏膜佐劑,進一步探討滴鼻免疫Balb/c小鼠后誘導(dǎo)的免疫效果。通過比較篩選出最有效的鼠疫黏膜佐劑疫苗和佐劑分子。 1.通過不同條件的優(yōu)化,從B群2b型腦膜炎奈瑟氏雙球菌中成功提取了高純度的蛋白體,經(jīng)蛋白質(zhì)指紋圖譜(MALDI-TOF-MS)鑒定鑒定為腦膜炎B群2b型奈瑟氏雙球菌外膜蛋白的PorA,PorB和Class 4孔道膜蛋白,相對分子量分別為41 ku,38 ku和34 ku。經(jīng)檢測蛋白體中內(nèi)毒素含量,核酸含量,莢膜多糖的含量都低于樣品的1%。鼠疫F-V抗原與蛋白體佐劑分別以4∶1,2∶1,1∶1,1∶2和1∶4在4℃疏水合成疫苗,滴鼻免疫小鼠。結(jié)果蛋白體佐劑不僅提高鼠疫F1-V抗原的系統(tǒng)免疫應(yīng)答,而且能誘導(dǎo)小鼠呼吸道、消化道和生殖道等局部黏膜免疫應(yīng)答。通過比較,抗原和佐劑劑量比例在4∶1時免疫效果最佳。用100 LD50的鼠疫141強毒株進行腹腔攻毒,蛋白體佐劑疫苗獲得了67%的保護。 2.從福氏2a痢疾桿菌中提取了LPS,將LPS脫毒,經(jīng)檢測其中的蛋白質(zhì)含量,核酸含量符合生物制品要求,具有良好的抗原性。將LPS與蛋白體制備成protollin佐劑,與鼠疫F1-V抗原制備疫苗滴鼻免疫小鼠,結(jié)果protollin佐劑比蛋白體能顯著提高鼠疫F1-V抗原的系統(tǒng)免疫水平和黏膜免疫水平,而單獨的LPS不具有免疫增強作用。 3.通過PCR技術(shù)從埃爾托霍亂弧菌獲得CTB的基因序列,將其克隆入表達質(zhì)粒pET-32a(+) ,基因測序正確。在IPTG誘導(dǎo)下,在大腸桿菌中表達,獲得純化的CTB蛋白。免疫印跡試驗鑒定具有良好的免疫原性。鼠疫F1-V抗原與rCT-B佐劑以5∶2、5∶1、1∶
[Abstract]:Most of the infection factors enter animal and human body through mucous membrane. Therefore, mucosal immunization is very important. Studies show that cholera toxin B subunit cholera toxin B is very important. CTB, proteosomes) and protollin are effective mucosal adjuvants. PorA in protein bodies. The study of the molecular adjuvant effects of PorB and Class 4 proteins is of great significance for the development of new mucosal adjuvants and the study of the mechanism of protein body action. Plague has a tendency of resurgence in the world. In recent years, there have been several outbreaks of human plague on a small scale in China. Among all kinds of plague diseases, the lung plague is the most destructive. In order to achieve long-term effects. It is necessary and important to study the mucosal adjuvant vaccine for the protection of pneumonic plague. In this study, protein body adjuvants were extracted from Neisseria Neisseria from group B meningococcal meningococci. Lipopolysaccharide lipopolysaccharide (LPS) was extracted from Shigella flexneri 2a to prepare protollin adjuvant. The recombinant CTB adjuvant and Poran Porb and Class 4 proteins used in mucosal adjuvant study provided reliable mucosal adjuvant for preparing Yersinia pestis F1-V mucosal adjuvant vaccine. To further study the immune effect of nasal immunization of Balb/c mice, the most effective adjuvant vaccine and adjuvant molecule of plague mucous membrane were screened by comparison. 1. High purity protein was extracted from Neisseria meningitidis Neisserich from group B meningitis by optimization of different conditions. The porin PorB and Class 4 pore membrane proteins were identified by MALDI-TOF-MS as the outer membrane proteins of Neisseria Neisseri group B in meningitis. The relative molecular weights were 41 kur 38 ku and 34 ku respectively. The contents of endotoxin and nucleic acid in the protein were determined. The content of capsule polysaccharides was lower than that of the sample. The vaccine was synthesized with 4: 1: 2: 1: 1 1: 1 1: 1 1: 2 and 1: 4 at 4 鈩，

本文編號：1451901