日本脑炎病毒核酸疫苗的构建及鉴定

发布时间：2018-02-23 19:42

本文关键词： 日本脑炎病毒 GM-CSF 佐剂 pCAG-JEGM 共表达　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 日本脑炎病毒(Japaneses encephalitis virus, JEV)又称乙型脑炎病毒,为黄病毒科单股正链RNA病毒,基因组约为11 kb,仅有一个开放阅读框(open reading frame, ORF ),从5’到3’端排列着3个结构基因(C、prM和E)和7个非结构基因(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5),分别编码相应的蛋白。JEV是流行性乙型脑炎(简称乙脑)的病原体,经蚊虫叮咬传播。乙脑主要流行于亚洲东南亚各国及远东地区。JEV感染后往往引发严重的中枢神经系统的症状,死亡率较高,幸存者亦有半数以上发展为永久的神经系统后遗症。乙脑尚无有效的治疗手段,因此免疫接种是控制其流行、保护易感人群的重要措施,并且预防接种对于人类对抗JEV感染已发挥了良好的保护作用。目前使用的JEV疫苗主要有三种:一种是由日本研发的灭活疫苗,采用JEV感染的成年鼠脑经福尔马林灭活纯化制成;另外两种由我国开发研制,分别为原代地鼠肾细胞灭活疫苗和经SA14-14-2减毒株感染地鼠肾细胞反复传代研制而成的减毒活疫苗,由于生产标准的原因,后两者仅局限在中国境内使用。然而三种疫苗均有其局限性:灭活疫苗存在高生产成本、免疫力不持久、需多次注射及存在变态反应等缺陷;而减毒活疫苗则存在毒力回复的危险。因此亟待发展一种安全、有效、质优价廉的新型疫苗用于对抗JEV引发的世界范围内的感染。近年来,核酸DNA疫苗的出现的是替代传统疫苗的新兴策略,与传统疫苗相比,DNA疫苗具有较多的优点,主要在于:(1)在机体内以类似病毒感染的方式合成抗原,从而介导持久的细胞免疫和体液免疫;(2)DNA疫苗安全稳定,易于制备,成本低廉。业已证明JEV的结构蛋白prM-E和非结构蛋白NS1是保护性抗原位点集中的部位,将表达JEV的prM、E或是NS1的质粒免疫小鼠后,能够产生特异性的保护性免疫,因此认为prM-E-NS1基因是JEV DNA疫苗首选靶点。但是单纯的DNA疫苗尚不足以引发良好的免疫效果,为此近年来出现一个较新的策略,即在基因水平将细胞因子与核酸疫苗共同表达,利用细胞因子上调机体免疫水平的作用,进而显著增强核酸疫苗的效果。粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating
[Abstract]:Japanese encephalitis virus (Japaneses encephalitis, virus, JEV) and Japanese encephalitis virus family, single strand RNA virus genome was about 11 KB, only one open reading frame (open reading frame, ORF), from the 5 'to 3' end is lined with 3 structural genes (C, prM and E) and 7 non structural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), respectively, the corresponding encoding protein.JEV is Japanese encephalitis (JE) pathogens, the spread of mosquito bites. JE is popular mainly in Southeast Asia and the Far East Asian.JEV infection often causes central nervous serious symptoms, high mortality, there are more than half of survivors for permanent neurological sequelae.
There is no effective treatment for JE vaccination, so is the control of the epidemic, the important measures to protect vulnerable populations, and vaccination against JEV infection in humans has played a good protective effect. There are three main types of currently used JEV vaccine: a vaccine by the Japanese research, formalin inactivated live purification made of JEV infected adult rat brain; the other two by China's development, respectively, primary hamster kidney cell inactivated vaccine and virus infection of hamster kidney cell passaged repeatedly to develop into a live attenuated vaccine by SA14-14-2 reduction, due to production standards, which are only used in the territory of China however. Three kinds of vaccine has its limitations: the inactivated vaccine has high production cost, the lack of long-term immunity, multiple injections and the risk of allergic reactions.; and the live attenuated vaccine is dangerous because of virulence reversion. This is an urgent need to develop a safe, effective, quality and inexpensive new vaccine to fight the worldwide infection caused by JEV.
In recent years, there is a new nucleic acid vaccine DNA strategy to replace the traditional vaccine, compared with the traditional vaccines, DNA vaccines have many advantages, mainly lies in: (1) infection in a similar virus in vivo synthesis of antigen, which mediates cellular immunity and humoral immunity lasting stability; (2) DNA vaccine safety, easy preparation and low cost. It has been proved that the structure of JEV prM-E protein and non structural protein NS1 is a protective antigen site concentration sites, JEV expression of prM, E or immunized mice were NS1, can produce specific protective immunity, so that the prM-E-NS1 gene is JEV DNA the preferred vaccine target. But it is not enough to trigger a DNA vaccine immune effect is good, so in recent years the emergence of a new strategy, which is in the level of gene expression of cytokines and the common use of nucleic acid vaccine, cytokine upregulated immune The effect of the nucleic acid vaccine was significantly enhanced by the effect of the level, and the granulocyte macrophage colony stimulating factor (granulocyte-macrophage colony-stimulating)

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q789;R392

【引证文献】