山羊地方性鼻内肿瘤病毒ENTV-2FJ株的分离纯化和全基因组序列分析  

Isolation, Purification and Genome Sequence Analysis of Enzootic nasal tumor virus of Goats(Capra hircus) Strain ENTV-2FJ

作　　者：江锦秀[1] 林裕胜 张靖鹏 游伟[1] 黄丽丽[2] 毛坤明[3] 胡奇林[1] JIANG Jin-Xiu;LIN Yu-Sheng;ZHANG Jin-Peng;YOU Wei;HUANG Li-Li;MAO Kun-Ming;HU Qi-Lin(Institute of Animal Husbandry&Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013;Fujian Normal University,Fuzhou 350108;Fuqing Technical Service Center of Animal Husbandry and Veterinary Medicine,Fuzhou 350300,China)

机构地区：[1]福建省农业科学院畜牧兽医研究所,福州350013 [2]福建师范大学,福州350108 [3]福清市畜牧兽医技术服务中心,福州350300

出　　处：《农业生物技术学报》2020年第2期313-324,共12页Journal of Agricultural Biotechnology

基　　金：福建省科技计划项目-省属公益类科研院所重点项目(2018R1023-1)

摘　　要：近几年山羊地方性鼻内肿瘤病毒(Enzootic nasal tumour virus of goats, ENTV-2)引起的山羊地方性鼻内肿瘤病(enzootic nasal adenocarcinoma, ENA)在我国多省均有报道。为了丰富山羊(Capra hircus)ENTV-2的分子流行病学资料,本研究采集患病羊的鼻液,用差速离心结合蔗糖密度梯度离心纯化ENTV-2FJ株,进行电镜观察和SDS-PAGE;取纯化的病毒,用反转录(reverse transcription PCR, RT-PCR)方法分段扩增出山羊鼻内肿瘤病毒福建株(ENTV-2FJ)的7条c DNA片段,并对其全基因组序列进行了克隆测序(Gen Bank No. MK559457)分析。ENTV-2FJ株经过纯化后,在电镜下看到病毒粒子呈球形,直径为90~110 nm,有囊膜和纤突;经SDS-PAGE分析,ENTV-2FJ株的蛋白显示9条带,分子量分别为118、79、60、51、28、20、16、13和11 ku。ENTV-2FJ株全基因组序列长7 469 bp,与ENTV四川株(ENTV-2SC)、ENTV陕西株(ENTV-2Shaanxi)、ENTV陕西株2 (ENTV-2Shaanxi2)、ENTV陕西株3 (ENTV-2Shaanxi3)、及ENTV陕西株4 (ENTV-2Shaanxi4)的同源性分别为95.1%、95.4%、95.1%、94.9%和95.4%;经ENTV-2全基因组、群相关抗原(group associated antigen, gag)和囊膜蛋白(envelope protein, env)基因核苷酸序列构建的进化树分析发现,ENTV-2FJ株与ENTV-2SC、ENTV-2Shaanxi、ENTV-2Shaanxi2、ENTV-2Shaanxi3和ENTV-2Shaanxi4处于同一进化分支。上述结果为ENTV-2检测方法的建立和遗传变异研究等提供了理论依据。Enzootic nasal adenocarcinoma(ENA) caused by Enzootic nasal tumour virus of goats(ENTV-2)has been reported in many provinces in China in recent years. In order to clarify the virology and genomewide characteristics of ENTV-2 FJ strain, In this study, the nasal fluid of the diseased sheep was collected, and the virus of ENTV-2 FJ strain was purified by differential centrifugation combined with sucrose density gradient centrifugation, and then subjected to electron microscopic observation and SDS-PAGE;Seven c DNA fragments of ENTV-2 FJ isolate were amplified by reverse transcription PCR(RT-PCR), the amplified products were cloned into p MD19-T plasmid vector and sequenced, the whole genome sequence of the ENTV-2 FJ strain was obtained by splicing with DNAstar 7.1 software and the sequence was analyzed. After purification of the virus of ENTV-2 FJ strain, pure virions can be observed under electron microscope. The virions were spherical, 90~110 nm in diameter, with envelope and spikes;By SDS-PAGE analysis, the structural proteins of the ENTV-2 FJ strain showed 9 bands with molecular weights of 118, 79, 60, 51, 28, 20, 16, 13 and 11 ku, the genome of the ENTV-2 FJ strain was 7 469 bp in length, exhibiting 95.1%, 95.4%, 95.1%, 94.9% and 95.4%homology with ENTV-2 SC, ENTV-2 Shaanxi, ENTV-2 Shaanxi2, ENTV-2 Shaanxi3, ENTV-2 Shaanxi4,respectively. Phylogenetic analysis based on the nucleotide sequence of ENTV-2 whole genomes, gag and env genes revealed that the ENTV-2 FJ strain was in the same evolutionary branch as ENTV-2 SC, ENTV-2 Shaanxi, ENTV-2 Shaanxi2, ENTV-2 Shaanxi3, and ENTV-2 Shaanxi4. These results lay the foundation for the establishment of the ENTV-2 assay and genetic variation analysis.

关 键 词：山羊地方性鼻内肿瘤病毒(ENTV-2) 纯化 电镜观察 SDS-PAGE 全基因组测序 

分 类 号：S855.3[农业科学—临床兽医学]