编码鳜传染性脾肾坏死病毒结构蛋白新基因ORF90.5L的鉴定分析

Identification of a new gene ORF90.5L encoding putative virus structural protein of mandarin fish infectious spleen and kidney necrosis virus (ISKNV)

鳜传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus,ISKNV)全基因组序列于2001年完成测定,全基因组为111362bp,含有124个推测的开放阅读框(ORF)。2007年,Eaton等对12株已完成序列测定的虹彩病毒代表株进行分析比较,除了已经推测的ORF外,认为ISKNV基因组中还存在一个的核心基因(Core gene)ORF90.5L。本研究以ISKNV的cDNA为模板,根据Eaton等报道中推测的上下游位点设计引物扩增出一段963bp的PCR产物。该序列编码一个320个氨基酸的蛋白质,推测分子量为35kD,含有预测的跨膜区,与虹彩病毒属(Iridovirus)其他成员编码的肉豆蔻基膜蛋白有较高的同源性。将该片段克隆到原核表达载体pET32a(+)中,纯化表达的重组蛋白pET90.5L免疫昆明鼠获得高效价多克隆抗体。利用抗pET90.5L抗体与纯化病毒进行免疫印迹,结果表明,抗pET90.5L的多抗可以特异性识别病毒粒子中大小约为35kD的蛋白条带,ORF90.5L编码的蛋白应为病毒的结构蛋白。本研究旨在为ISKNV基因工程疫苗的研发和病源侵染机制研究提供基础参考。

In 2001,the genome of mandarin fish infectious spleen and kidney necrosis virus（ ISKNV） was completely determined which consist of 111 362 bp nucleic acids with 124 putative open reading frames（ ORFs）. In 2007,Eaton et al re-annotated and defined ORFs of 12 iridovirus strains with clear genome information,and ORF90.5L was defined as a new core gene within the genome of ISKNV. Using the cDNA of ISKNV and the primers designed according to Eaton’s paper,a specific PCR product with 963 bp was amplified. The presumed ORF90.5L encodes a 320 amino acid protein with putative molecular weight of 35kDa and a transmembrane helix. The putative protein encoded by ORF90.5L shares high identity with similar ORFs which encoding putative myristylated membrane protein. The PCR product of ORF90.5L was cloned into expression vector pET32a（＋）,and the recombinant protein was expressed and purified followed by anti-pET90.5L mouse antibody was prepared. The Western blot analysis showed that specific viral protein could be detected by anti-pET90.5L antibody which suggested the protein encoded by ORF90.5L is possible to be structural protein of ISKNV.