森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及其免疫原性研究

发布时间：2018-05-06 10:06

  本文选题：森林脑炎病毒 + prM-E基因　； 参考：《中国人民解放军军事医学科学院》2005年硕士论文

【摘要】：森林脑炎又称蜱传脑炎,是由森林脑炎病毒经硬蜱媒介所致自然疫源性急性中枢神经系统传染病,主要分布于亚洲和欧洲一些国家。我国主要见于东北及西北原始森林地区。近年来随着旅游事业的发展、外来人群的涌入、森林工业生产环境的改变和林区生态环境的破坏,使得森林脑炎流行范围逐渐扩大,病例数逐年上升。因此森林脑炎的防治受到高度重视。 中和试验(NT)和血凝抑制试验(HI)是诊断黄病毒感染的最特异方法,然而中和试验耗时较长,常需要几天,且需要专门进行活病毒操作的封闭实验室,因此应用受到一定限制。现在一般使用ELISA试验检测血清和CSF中特异性IgM和IgG抗体。商品化ELISA试剂盒使用灭活的病毒作为抗原,此种病毒的培养和抗原的纯化需要在生物安全实验室进行,存在许多安全隐患,并且发现完整灭活病毒作为抗原的ELISA试验与其他黄病毒感染有交叉反应,因此许多学者针对替代抗原进行了研究。 M和E蛋白是森林脑炎病毒的结构蛋白,E蛋白能诱导保护性抗体,具有与受体结合、细胞膜融合的功能和血凝活性。prM-E基因在转染细胞中表达时,可牢固地锚定在胞内内质网膜上,形成稳定的空间结构,并保证prM与E间的共价结合。 本研究选择我国森林脑炎病毒MDJ01株的prM-E基因作为靶基因,首先对其进行了克隆表达。从TBE病毒MDJ01株感染的BHK21细胞培养液中提取总RNA,RT-PCR扩增获得含prM-E基因的DNA,利用NotⅠ和HindⅢ双酶切后,将prM-E基因插入供体质粒pFastBacl中,测序证实编码基因插入正确。将该重组质粒转化含有Bacmid DNA和Help质粒的DH10Bac菌株,利用Tn7转座子为工具,使之发生同源转座,通过蓝白斑筛选和PCR扩增,获得含有prM-E基因的重组Bacmid DNA。 将重组Bacmid DNA转染昆虫细胞Sf9后获得重组杆状病毒(rvBACME),将rvBACME感染Sf9细胞,用RT-PCR对其转录产物进行了鉴定,结果表明
[Abstract]:Forest encephalitis, also known as tick-borne encephalitis, is a natural infectious disease of central nervous system caused by tick-borne encephalitis virus. It mainly distributes in some countries in Asia and Europe. China is mainly found in the northeast and northwest of the primeval forest region. In recent years, with the development of tourism, the influx of foreign people, the change of the production environment of forest industry and the destruction of ecological environment in forest area, the epidemic range of forest encephalitis is gradually expanded, and the number of cases increases year by year. Therefore, the prevention and treatment of forest encephalitis is highly valued. Neutralization test (NTT) and hemagglutination inhibition test (HIH) are the most specific methods for the diagnosis of yellow virus infection. However, neutralization test takes a long time and usually takes a few days. ELISA tests are now commonly used to detect specific IgM and IgG antibodies in serum and CSF. Commercial ELISA kit uses inactivated virus as antigen. The culture and purification of this virus need to be carried out in biosafety laboratory. It was also found that the ELISA test of intact inactivated virus as antigen had cross reaction with other yellow virus infections, so many researchers have studied alternative antigens. M and E proteins, the structural proteins of TEV, can induce protective antibody, and have the function of binding to receptor, cell membrane fusion and hemagglutination activity. PrM-E gene is expressed in transfected cells. It can be firmly anchored on the endoplasmic omentum to form a stable spatial structure and to ensure the covalent binding between prM and E. In this study, the prM-E gene of MDJ01 strain of Chinese Forest Encephalitis virus was selected as the target gene. The DNA containing prM-E gene was amplified by RT-PCR from BHK21 cells infected with TBE virus MDJ01 strain. The prM-E gene was inserted into the donor plasmid pFastBacl by double enzyme digestion of Not 鈪，

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