结核杆菌hsp70在乙脑基因工程疫苗研制中的免疫增强作用

发布时间：2018-01-03 23:28

本文关键词：结核杆菌hsp70在乙脑基因工程疫苗研制中的免疫增强作用　出处：《南京农业大学》2006年博士论文　论文类型：学位论文

【摘要】：流行性乙型脑炎(简称乙脑)属于黄病毒科，引起人类或马的中枢神经系统感染以及猪的流产，能通过持续感染的蚊子传播给人，并且主要在脊椎动物宿主内存在，特别是具有较高病毒血症的猪和鸟类，在人类乙脑主要引起致死率较高的脑炎，致死率从20-50％，但是绝大多数的幸存者都具有中枢神经系统的后遗症；在家养动物如猪和马，主要引起脑炎，流产甚至死亡。与黄病毒科的其它成员类似，为单链正股RNA，长度为11kb，编码一个含多聚蛋白的开放性阅读框，这个多聚蛋白可通过蛋白酶切割成至少11个蛋白，该病毒的结构蛋白包括衣壳蛋白，膜蛋白和囊膜蛋白，位于5’端三分之一的区域，而非结构蛋白从NS1到NS5位于阅读框其余的区域。目前用于猪的乙脑疫苗包括弱毒苗和灭活苗，但是有关它们的安全性，生产和使用费用等问题越来越突出。WHO正在呼吁改善和研制新型乙脑疫苗，乙脑病毒的E蛋白与病毒和细胞受体结合，膜融合有关，也是引起中和抗体及宿主保护的主要免疫蛋白。结核杆菌热休克蛋白70具有分子佐剂和载体效应，是结核杆菌的主要抗原成分之一，可诱导和增强机体体液免疫和细胞免疫的发生，，因此用该基因与一些传染病或寄生虫病病原体的抗原基因融合表达以制备疫苗，借助它的载体分子效应和佐剂作用，以提高病原体抗原的免疫原性和疫苗的免疫预防效果。本文将结核杆菌热休克蛋白70与乙型脑炎E蛋白主要抗原片段在毕赤酵母中进行融合表达免疫小鼠，用以研究hsp70对免疫反应的影响，此外还对结核杆菌hsp70与抗原肽以何种方式共同作用才能发挥其佐剂效应进行了研究，根据报道结核杆菌hsp70分为两个区域，N末端为ATPase区，C末端为肽连接区，其中肽连接区为hsp70中真正行使佐剂功能的区域，因此本文将E蛋白主要抗原片段与肽连接区进行融合表达并免疫，研究肽连接区的分子佐剂功能；另外，将已报道的E蛋白上的中和表位与结核杆菌hsp70在大肠杆菌中融合表达，将其免疫效果与乙脑弱毒苗SA14-14-2进行比较。实验结果如下： 1，结核杆菌热休克蛋白70及其功能域肽连接区对乙型脑炎E蛋白主要抗原片段引起的免疫反应的影响。在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达，该片段长1113bp，编码371个氨基
[Abstract]:Japanese encephalitis (JE) belongs to the family Flaviviridae, causing infections of the central nervous system and the nerve of human or pig abortion horse, through the persistent infection of mosquitoes to humans, and mainly in the vertebrate host in memory, especially with high viremia in pigs and birds in the human Japanese encephalitis mainly caused a high mortality rate. Encephalitis, the death rate from 20-50%, but most of the survivors have central nervous system sequelae; in the domestic animal such as pigs and horses, mainly caused by encephalitis, abortion and even death. With other members of the Flaviviridae family, single stranded positive strands of RNA, length of 11KB, encoding a polyprotein open reading frame of the multimeric protein can be cut into at least 11 protein by protease, structural proteins of the virus including capsid protein, membrane protein and membrane protein, is located in the 5 'end of 1/3 areas, Non structural proteins, from NS1 to NS5, are located in the rest of the reading frame.
At present for swine JE vaccine including attenuated vaccine and inactivated vaccine, but their safety, production costs and the use of more and more prominent issues such as.WHO are calling for improvement and development of a new type of JE vaccine, Japanese encephalitis virus E binding protein and virus and cellular receptors, membrane fusion, is mainly caused by the immune protein neutralizing antibody and the host protection. Mycobacterium tuberculosis heat shock protein 70 with molecular adjuvant and carrier effect, is one of the major antigens of Mycobacterium tuberculosis, which can induce and enhance the humoral and cellular immunity, so the antigen gene gene and some infectious or parasitic disease pathogen fusion protein to prepare a vaccine, with the help of it the carrier molecule effect and immune adjuvant effect, to improve the primary pathogen antigen and vaccine prevention effect.
This will be the main antigen fragment of Mycobacterium tuberculosis heat shock protein 70 and E protein of Japanese encephalitis in mice immunized with fusion expression in Pichia pastoris, and used to study the effects of HSP70 on immune response, in addition to the Mycobacterium tuberculosis HSP70 antigen peptide and the manner in which the interaction can play its adjuvant effect was studied, according to the reports of Mycobacterium tuberculosis Hsp70 is divided into two regions, N terminal ATPase, C terminal peptide linker region where peptide connection region for regional real exercise adjuvant function in HSP70, so the antigen peptide E protein fragment and the connection region for the fusion expression and immune function, molecular adjuvant peptide of the connecting region; in addition, the E protein has been reported on the neutralizing epitope of Mycobacterium tuberculosis and HSP70 fusion expression in Escherichia coli, the immune effect and Japanese encephalitis attenuated vaccine SA14-14-2 were compared. The experimental results are as follows:
1, the effect of the heat shock protein 70 and its functional domain peptide junction on the immune response caused by the main antigen fragment of E protein in Japanese encephalitis.
On the basis of the prokaryotic expression vector (containing the main antigen fragment of E gene of SA14-14-2 strain of Japanese encephalitis vaccine strain), it was expressed in Pichia pastoris system. The fragment is long 1113bp and encodes 371 amino groups, which is constructed in our laboratory.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

【引证文献】