鼠疫黏膜佐剂疫苗的初步研究

发布时间：2018-01-21 15:52

  本文关键词： 鼠疫F1-V抗原 黏膜佐剂 滴鼻免疫　出处：《西北农林科技大学》2006年硕士论文　论文类型：学位论文

【摘要】： 大多数感染因子是通过黏膜进入动物和人体的。因此,黏膜免疫的预防接种技术显得很重要。研究表明霍乱毒素B亚单位(cholera toxin B,CTB)、蛋白体(proteosomes)和protollin是比较有效的黏膜佐剂。蛋白体中的PorA、PorB和Class 4类蛋白分子佐剂效应的研究对于开发新黏膜佐剂和对蛋白体作用机制的研究有重要的意义。 鼠疫在全世界范围内有死灰复燃之势,近年来中国已暴发数次小规模的人间鼠疫。在各种鼠疫疾病之中,尤以肺鼠疫破坏最大。为了能达到长期起效的目的,对肺鼠疫提供全面和长久的保护,鼠疫黏膜佐剂疫苗的研究是十分必要和重要的。 本研究从B群2b型脑膜炎奈瑟氏双球菌中提取蛋白体佐剂;从福氏2a痢疾杆菌中提取脂多糖(Lipopolysaccharides,LPS),与蛋白体复合制备protollin佐剂;基因重组了CTB佐剂和用于黏膜佐剂研究的PorA,PorB和Class 4蛋白,为制备鼠疫F1-V黏膜佐剂疫苗提供可靠的黏膜佐剂,进一步探讨滴鼻免疫Balb/c小鼠后诱导的免疫效果。通过比较筛选出最有效的鼠疫黏膜佐剂疫苗和佐剂分子。 1.通过不同条件的优化,从B群2b型脑膜炎奈瑟氏双球菌中成功提取了高纯度的蛋白体,经蛋白质指纹图谱(MALDI-TOF-MS)鉴定鉴定为脑膜炎B群2b型奈瑟氏双球菌外膜蛋白的PorA,PorB和Class 4孔道膜蛋白,相对分子量分别为41 ku,38 ku和34 ku。经检测蛋白体中内毒素含量,核酸含量,荚膜多糖的含量都低于样品的1%。鼠疫F-V抗原与蛋白体佐剂分别以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。结果蛋白体佐剂不仅提高鼠疫F1-V抗原的系统免疫应答,而且能诱导小鼠呼吸道、消化道和生殖道等局部黏膜免疫应答。通过比较,抗原和佐剂剂量比例在4∶1时免疫效果最佳。用100 LD50的鼠疫141强毒株进行腹腔攻毒,蛋白体佐剂疫苗获得了67%的保护。 2.从福氏2a痢疾杆菌中提取了LPS,将LPS脱毒,经检测其中的蛋白质含量,核酸含量符合生物制品要求,具有良好的抗原性。将LPS与蛋白体制备成protollin佐剂,与鼠疫F1-V抗原制备疫苗滴鼻免疫小鼠,结果protollin佐剂比蛋白体能显著提高鼠疫F1-V抗原的系统免疫水平和黏膜免疫水平,而单独的LPS不具有免疫增强作用。 3.通过PCR技术从埃尔托霍乱弧菌获得CTB的基因序列,将其克隆入表达质粒pET-32a(+) ,基因测序正确。在IPTG诱导下,在大肠杆菌中表达,获得纯化的CTB蛋白。免疫印迹试验鉴定具有良好的免疫原性。鼠疫F1-V抗原与rCT-B佐剂以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，

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