一株大黄鱼虹彩病毒的分离鉴定及多克隆抗体的制备

Isolation and Identification of Iridovirus and Preparation of Its Polyclonal Antibody in Diseased Large Yellow Croaker

2021年7月,浙江省象山某养殖场养殖的大黄鱼(Larimichthys crocea)出现类似大黄鱼虹彩病毒引起的疾病。采用鲤上皮瘤细胞培养和病毒主要衣壳蛋白测序分析的方法,从患病的大黄鱼中分离到一株病毒。该病毒接种到鲤上皮瘤细胞(EPC)后出现空斑、脱落的细胞病变症状。根据虹彩病毒MCP和ATPase基因保守序列设计特异性引物对病毒组织样本进行PCR扩增,得到分别为1 367 bp和740 bp的目的基因片段。将MCP基因扩增片段测序,经BLAST对比及系统发育树聚类分析,确定该分离的病毒属虹彩病毒科细胞肿大病毒属。通过蔗糖密度梯度离心纯化,用透射电镜观察该病毒粒子呈正六边形,直径为120~150 nm。用纯化病毒作为抗原免疫小鼠获得抗大黄鱼虹彩病毒的多克隆抗体,效价为1∶7 000;通过SDS-PAGE和Western blotting初步确定3个免疫蛋白。本研究为大黄鱼虹彩病毒纯化提供一种新方法,并初步分离出免疫蛋白,为该病毒相关分子生物学研究、蛋白研究以及疫苗制备等提供理论依据。

A virus was isolated and identified in diseased large yellow croaker out broken in a fish farm in Xiangshan,Zhejiang Province,in July 2021 by epithlioma papulosum cyprinid(EPC)culture,and major capsid protein(MCP)cloning and sequencing.The tissue homogenate of diseased fish caused typical cytopathic effect including plaque,exfoliation in EPC cells.The amplification of conserved region of ATPase and major capsid protein(MCP)gene of iridovirus by PCR showed that 740 bp and 1367 bp specific genes were obtained.Sequence MCP gene amplified fragment,and phylogenetic analysis revealed that the isolated virus belonged to the genus Megalocytivirus in Family Iridovirade.The purification of the virus by sucrose density gradient centrifugation and transmission electron microscopy observation found that the virus particles were regular hexagon in shape with diameter of 120-150 nm.Polyclonal antibody against large yellow croaker iridovirus was obtained by immunizing mice with purified virus as antigen,with the titer of 1∶7000.Three immune proteins were preliminarily identified by SDS-PAGE and Western blotting.The finding provided a method for the purification of large yellow croaker iridovirus,identification of the immune protein,and theoretical basis for molecular biology research,protein research and vaccine preparation.