【作者】 陈鸿军；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2006， 博士

【摘要】 血清I型马立克氏病病毒(Marek’s disease virus serotype 1,MDV-1)的UL49h基因与I型单纯疱疹病毒(HSV-1)UL49同源,编码病毒被膜蛋白VP22,分子量约27.6kD,是病毒被膜的主要成分。现已证实,MDV-1 VP22对病毒的复制和传播有着非常重要的作用。Dorange等发现MDV-1 VP22具有与HSV-1相似的蛋白转导功能。Hung等研究发现MDV-1 VP22能增强人16型乳头状瘤病毒(HPV-16)E7抗原融合表达的免疫效果,主要产生CD8+ T细胞介导的细胞免疫应答。本研究利用无致病性的CVI988/Rispens疫苗株扩增VP22基因,结果发现其C端缺失6个氨基酸,即201TKSERT206,对CVI988 VP22蛋白转导功能的研究结果提示:CVI988 VP22可作为一种高效便捷的蛋白转运工具,对于弥补DNA免疫缺陷和提高治疗性物质的转导效率具有非常重要的意义。1不同致病型马立克氏病病毒VP22蛋白的序列比较和分析用MDV-1 CVI988/Rispens株、GA株、RB1B株和648A株,MDV-2 Z4株和FC126株分别感染鸭胚成纤维细胞或鸡胚成纤维细胞,待出现大量空斑时,提取总DNA。根据已发表的MDV GA株VP22序列设计引物,成功扩增出MDV-1不同毒株的VP22基因。将PCR产物克隆T载体并测序分析,结果发现:强毒株GA株、超强毒RB1B株和特超强毒648A株的VP22长度保持750bp,但CVI988弱毒株的VP22基因C端缺失18bp,即存在一个6氨基酸残基的缺失性突变:201TKSERT206。2 CVI988弱毒疫苗株VP22羧基端原核表达及特异性抗体的制备设计特异性引物,扩增全长VP22、CVI988 VP22的羧基端区域(VP22C:aa94~243)、VP22末端区(VP22T:aa207~249)、缺失N端的VP22(VP22H:aa19~243)。将BamHⅠ、EcoRⅠ双酶切PCR产物,克隆入pGEX-6P-1载体中,转化BL21(DE3)细菌,经IPTG诱导表达,结果发现:VP22C获得了高效可溶性表达。SDS-PAGE分离阳性条带,采用切胶免疫或用诱导后的细菌经超声波裂解的更多还原

【Abstract】 The UL49 homolog (UL49h) gene of Marek’s disease virus serotype 1 (MDV-1) encodes the viral protein 22 (VP22) of 27.6kD with a homology of about 25% to the VP22 of Herpes Simplex virus type 1 (HSV-1). In HSV-1, VP22 is a novel translocating protein with a potential protein tranduction domain (PTD) in its cytoplamic region, which has been successfully used to enhance the delivery and therapeutic or immuno-stimulatory effects of several proteins in vitro or in vivo. The VP22 protein in the virulent MDV-1 strain RB1B has an intercellular trafficking function similar to that of HSV-1, which appears to be indispensable for cell-to-cell spread and propagation of MDV-1. A DNA vaccine containing the E7 gene of human papillomavirus type 16 linked to MDV-1 VP22 gene has a significantly increased immunogenicity in terms of stimulating E7-Specific CD8+ T cells, suggesting that the MDV-1 VP22 is an alternative transduction protein for enhancing vaccine potency.Our previous data showed that the VP22 of the attenuated MDV-1 strain CVI988/Rispens had a deletion of 201TKSERT206 compared to that of the oncogenic GA strain and other virulent strains. To investigate the influence of the deletion on the intercellular trafficking of the VP22 protein, antibodies specific for the carboxyl terminus of CVI988 VP22 was prepared by immunizing mice with truncated protein expressed in E. coli, and used to detect the expression and functions of VP22 and its truncated proteins/peptides in different eukocytic systems. It was described that the preliminary applications of VP22 in avirulent vaccine strain CVI988/Rispens and in the strategies for novel vaccines.The sequence comparison of VP22 protein amplified from different pathotypes of Marek’s disease virusSeveral fragments of VP22 gene were amplified from total DNAs, which were extracted更多还原