文章摘要
鲤疱疹病毒Ⅱ型主要免疫原性蛋白的鉴定
Identification of major immunogenic proteins from Cyprinid Herpesvirus 2
投稿时间:2019-06-06  修订日期:2019-09-17
DOI:
中文关键词: 鲤疱疹病毒Ⅱ型  免疫原性  蛋白  质谱鉴定
英文关键词: Cyprinid herpesvirus II (CyHV-2)  immunogenicity  protein  mass spectrometry identification
基金项目:
作者单位E-mail
高娃 上海海洋大学 843285924@qq.com 
温虹 上海海洋大学  
王浩 上海海洋大学  
陆佳荃 上海海洋大学  
吕利群 上海海洋大学  
姜有声 上海海洋大学 ysjiang@shou.edu.cn 
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中文摘要:
      近年来,鲫鱼疱疹病毒造血器官坏死病病毒CyHV-2给我国异育银鲫养殖业带来了巨大的经济损失。为了鉴定CyHV-2的主要免疫原性蛋白,本研究用本实验室分离的CyHV-2-YC-1分离株感染异育银鲫尾鳍细胞系(GiCF),用蔗糖密度梯度超速离心法对细胞感染液中的CyHV-2进行纯化,纯化后的CyHV-2病毒免疫小鼠制备抗CyHV-2多克隆抗体。纯化的病毒颗粒经SDS-PAGE和考马斯亮蓝染色后,用抗CyHV-2多克隆抗体进行Western blotting分析和质谱鉴定。结果显示,透射电镜观察下发现50%-66%的蔗糖梯度多见完整囊膜包裹的CyHV-2病毒颗粒,也有少量囊膜破损的病毒颗粒。Western blotting结果显示抗CyHV-2多克隆抗体与多种病毒蛋白具有特异性免疫反应,质谱鉴定显示其中八种主要免疫原性蛋白分别是ORF92、ORF115、ORF25、ORF57、ORF66、ORF72、ORF131和ORF132。研究表明,通过蔗糖密度梯度超速离心法提纯CyHV-2病毒颗粒后,本研究制备的抗CyHV-2多克隆抗体能够特异性识别CyHV-2病毒的主要免疫原性蛋白。本研究将为CyHV-2免疫学检测方法的建立以及疫苗的研制提供更多的候选抗原。
英文摘要:
      Cyprinid herpesvirus II (CyHV-2) is a newly emerged main pathogen causing severe haematopoietic necrosis disease in Carassius auratus gibelio and has caused huge economic losses in China. As promising tools for improved diagnostics and new vaccine development, we purified CyHV-2 particles and identified more immunologic proteins in CyHV-2 particles by LC-MS. Firstly, The caudal fin of Carassius auratus gibelio (GiCF) cell line was infected with CyHV-2, and viral particles were purified from the cell supernatant by sucrose density gradient in combination with ultracentrifugation. The purified virions were observed by transmission electron microscope. Then the prepared purified virions were immunized mice to prepare an anti-CyHV-2 polyclonal antibody, and at the same time purified virions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with coomassie brilliant blue, and then subjected to western blotting analysis and mass spectrometry identification using anti-CyHV-2 polyclonal antibody. Transmission electron microscope (TEM) results showed that a large amount of intact CyHV-2 virus particles were detected in the 50%-66% sucrose density and virus particles without capsule are occasionally observed. Western blotting results showed that some protein bands had specific immunoreactivity with anti-CyHV-2 polyclonal antibody. 8 clearly immunogenic proteins were further identified as ORF92, ORF115, ORF25, ORF57, ORF66, ORF72, ORF131and ORF132 through mass spectrometry identification. Among them, ORF92, ORF66 and ORF72 are capsid proteins, ORF115, ORF25, ORF131 and ORF132 are membrane proteins. The capsid protein and membrane protein of the virus play an important role for virus infection. In conclusion, the major immunogenic proteins of CyHV-2 virion were identified. This was fundamental and applied research on CyHV-2. Indeed, this knowledge is crucial for understanding the biology and pathogenesis of CyHV-2 infection and for facilitating the development of efficacious protein-based diagnostic methods and vaccine candidates.
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