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摘要: 为了鉴定CyHV-2的主要免疫原性蛋白，本研究用分离的CyHV-2-YC-1分离株(下简称CyHV-2)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。纯化的病毒颗粒经变性聚丙烯酰胺凝胶电泳 (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，透射电镜下观察发现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8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。Abstract: In order to obtain promising tools for improved diagnostics and new vaccine development, we purified Cyprinid herpesvirus 2 (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 used to immunize 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 at 50%-66% sucrose density and virus particles without capsule were occasionally observed. Western blotting results showed that some protein bands had specific immunoreactivity with anti-CyHV-2 polyclonal antibody. 8 major 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, and ORF115, ORF25, ORF131 and ORF132 are membrane proteins. The capsid protein and membrane protein of the virus play an important role in 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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图 1 CyHV-2蔗糖密度梯度超速离心纯化结果
Figure 1. Results of CyHV-2 purification by sucrose density gradient ultracentrifugation
图 图版Ⅱ 透射电镜下纯化的CyHV-2颗粒
Figure 图版Ⅱ. Purified particles of CyHV-2 under transmission electron microscope
图 2 纯化的CyHV-2病毒SDS-PAGE电泳(a)和Western blotting结果(b)
Figure 2. Results of SDS-PAGE of purified CyHV-2 virions (a) and Western blotting analysis (b)
表 1 CyHV-2主要免疫原性蛋白LC-MS/MS鉴定
Table 1. Identification of major immunogenic proteins from CyHV-2 by LC-MS/MS
序号
no.蛋白
protein蛋白编号
accession分子质量/ku
mass可信度
−10lgP总肽段总数
peptides定位
localization蛋白描述
description1 ORF92 YP_007003911.1 140 447 67 核衣壳
nucleocapsid主要衣壳蛋白
major capsid protein2 ORF115 YP_007003933.1 84 245 13 囊膜
envelope膜蛋白
membrane protein3 ORF25 YP_007003844.1 66 155 5 囊膜
envelope膜蛋白
membrane protein4 ORF57 YP_007003878.1 64 348 32 未知
unknown未知蛋白
unknown protein5 ORF66 YP_007003885.1 45 309 24 核衣壳
nucleocapsid衣壳三重联体蛋白1
putative capsid triplex subunit 16 ORF72 YP_007003891.1 41 324 17 核衣壳
nucleocapsid衣壳三重联体蛋白2
putative capsid triplex subunit 27 ORF131 YP_007003952.1 35 87 2 囊膜
envelope膜蛋白
membrane protein8 ORF132 YP_007003951.1 17 150 4 囊膜
envelope膜蛋白
membrane protein注：蛋白可信度得分，值越大表明该蛋白包含的可信肽段越多
Notes: −10lgP, a larger value indicates that the protein contains more reliable peptides
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