一种蛙病毒的纯化和酶切分析

Purification and Enzyme Analysis of A Virus from Frog (Rana tigrina rugulosa)

从患病虎纹蛙（Ｒａｎａ ｔｉｇｒｉｎａ ｒｕｇｕｌｏｓａ）蛾料中分离得到一种蛙病毒，感染鱼的 ＦＨＭ、ＣＯ、ＥＰＣ细胞，获得大量的病毒原料，分离纯化得到高纯度的病毒粒子，电镜下可见病毒粒子为二十面体，具囊膜，直径平均为 １２５ ｎｍ．抽提病毒核酸，对其基因组ＤＮＡ进行酶切分析，病毒基因组为双链ＤＮＡ分子，表现脊椎动物虹彩病毒基因组的特点即其胞晒唉 ５’端高度甲基化．以限制性内切酶 Ｘｂａ、ＢａｍＨＩ、ＨｉｎｄⅢ、ＫｐｎＩ和ＰｓｔＩ 酶切病毒基因组 ＤＮＡ，经电泳分离后，分别得到 １３、２５、６、１４、２２条清晰的酶切片段，并根据酶切片段算得基因组的大小超过 １００ ｋｂ．用 ＰＣＲ方法，得到这个病毒的主衣壳蛋白（ＭＣＰ）基因保守序列，与ＦＶ３相比，同源性达９８％．

A virus from frog (Rana tigrina rugulosa) , a member of the Iridoviridae family, is the causative agent of a disease caused high young frog mortalities and severe damage to frog cultures in China. The abundant virus ma- terial was obtained from FHM, CO and EPC cells infected by the virus. Then the virions was isolated and purified. Virions are icosahedron, enveloped, 125 rim in diameter averagely as observed by transmission electron microsopy. The genome of ISKNV is a double-stranded DNA molecule and highly methylated at cytosines in the CpG. As hevi- ral DNA was digested with Xba I , BamH I , Hind Ⅲ, Kpn I and Pst I restriction endonucleases and separat- ed by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the virus DNA was es- timated to he longer than 100 kb. At the nucleotide level of MCP, the virus showed 98% identity to FV3.