马头山羊羊口疮病毒的分离鉴定及其B2L基因的特征分析  

Isolation and Identification of ORFV in Matou Goats and Analysis of B2L gene

作　　者：向敏 刘辰晖 余婕 周源 胡修忠 夏永春 王定发 夏瑜 程蕾 XIANG Min;LIU Chenhui;YU Jie;ZHOU Yuan;HU Xiuzhong;XIA Yongchun;WANG Dingfa;XIA Yu;CHENG Lei(Insitute of Animal Husbandry and Veterinary,Wuhan Academy of Agricultural Sciences,Wuhan 430208,China;Qingqing Matou Goat Breeding Cooperative of Yunxi County,Shiyan 442624,China)

机构地区：[1]武汉市农业科学院畜牧兽医研究所,湖北武汉430208 [2]郧西县青青马头羊养殖专业合作社,湖北十堰442624

出　　处：《中国兽医杂志》2024年第11期32-39,共8页Chinese Journal of Veterinary Medicine

基　　金：湖北省重点研发技术项目(2021BBA247);动物胚胎工程及分子育种湖北省重点实验室2021年开放课题(KLAEMB-2021-3);武汉市农科院郧西县技术合作项目。

摘　　要：为确定湖北省某马头山羊养殖场发生的疑似羊口疮(Orf)的病原及其分子生物学特征。本试验利用牛睾丸细胞对马头山羊唇部痂皮病料进行病毒分离培养,通过细胞病变观察、病毒滴度测定、PCR扩增和测序等方法对分离毒株进行了鉴定。并采用MegAlign、MEGA 7.0、DNAMAN和Bioedit等软件分析了分离毒株和China Vaccine毒株B2L基因的核苷酸序列相似性,并预测了蛋白二级结构和疏水性。结果显示,接种病料上清液的牛睾丸细胞盲传5代后出现细胞病变,经PCR扩增的分离毒株B2L基因大小为1137 bp;所分离羊口疮病毒(ORFV)病毒滴度可达105.5TCID50/mL;分离毒株与AH1404、FJ-YX和GX-BH毒株B2L基因同源性为99.3%;与China Vaccine毒株同源性为89.5%。分离毒株与China Vaccine毒株B2L基因序列分析显示处在不同分支,两者存在8个位点突变(aa11、aa80、aa101、aa123、aa126、aa294、aa327和aa333);B2L蛋白为稳定的疏水性蛋白;分离毒株和China Vaccine毒株的蛋白二级结构存在差异。结果表明,该养殖场ORFV的B2L基因出现变异,有关部门应当加强对ORFV流行情况的监测。To determine the pathogen responsible for a suspected outbreak of orf in a Maotou goat farm in Hubei Province,as well as its molecular characteristics,this study isolated and cultured the virus from scab samples taken from the lips of Maotou goats using bovine testicular cells.The isolated strain was identified through cell lesion observation,viral titer determination,PCR amplification,and sequencing.MegAlign,MEGA 7.0,DNAMAN,and Bioedit software were used to analyze the nucleotide sequence similarity of the isolated strain's B2L gene,and predict the secondary structure and hydrophobicity of the protein.The results showed that after five blind passages in bovine testicular cells,cytopathic effects appeared.The PCR-amplified B2L gene from the isolated strain was 1137 bp in size.The viral titer of the isolated orf virus(ORFV)reached 105.5 TCID 50/mL.The B 2 L gene of the isolated strain shared 99.3%homology with the AH1404,FJ-YX,and GX-BH strains,and 89.5%homology with the China Vaccine strain.Sequence analysis of the B2L gene between the isolated strain and the China Vaccine strain revealed that they clustered in different branches,with eight mutation sites(aa11,aa80,aa101,aa123,aa126,aa294,aa327,and aa333).The B2L protein was found to be a stable hydrophobic protein,and differences were observed in the secondary structure of the B2L protein between the isolated strain and the China Vaccine strain.These findings indicate that the B2L gene of the ORFV in this goat farm has undergone mutations.Authorities should strengthen monitoring of the ORFV epidemic.

关 键 词：羊口疮病毒(ORFV) 分离鉴定 遗传演化分析 B2L基因 

分 类 号：S851.3[农业科学—预防兽医学]