蛋鸡J亚群禽白血病病毒分离株SD1009株感染性克隆的构建与病毒拯救  

Construction and virus rescue of two infectious molecular clones of a subgroup J avian leukosis virus isolate SD1009

作　　者：王琦[1] 王永强[1] 康忠惠[1] 高玉龙[1] 潘伟[1] 秦立廷[1] 李久宽[1] 祁小乐[1] 高宏雷[1] 王笑梅[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2011年第11期1111-1116,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31072146);现代农业肉鸡产业技术体系建设项目(nycytx-42-G3-01);哈尔滨市科技攻关计划项目(2010AA6AN034)

摘　　要：为探究蛋鸡J亚群禽白血病病毒(ALV-J)的致病机理,从山东省某鸡场送检的蛋鸡病料中分离鉴定出1株ALV-J,命名为SD1009株,采用PCR方法分3段扩增出SD1009株的前病毒cDNA,PCR产物经克隆酶切后顺次连接,获得1个含有完整ALV-J前病毒cDNA的重组质粒,命名为pBW-SD1009。全长测序及序列比对分析发现,近年来分离的ALV-J都存在缺失部分rTM和DR区的序列,缺失长度为205bp。通过外源基因嵌合的方式,将205bp嵌合进入,又获得了1个前病毒cDNA的重组质粒,命名为pBW-SD1009A205,将pBW-SD1009和pBW-SD1009A205分别转染DF1细胞,通过间接免疫荧光试验,禽白血病抗原试剂盒检测,反转录酶试剂盒检验,表明拯救出了2株病毒,分别命名为rSD1009和rSD1009A205。本研究成功构建了蛋鸡ALV-J的感染性克隆,并在序列分析的基础上,又构建了第2株补全缺失序列的感染性克隆,结果表明缺失部分rTM和DR区的序列,对病毒的拯救以及病毒的复制动力学无显著影响。In order to investigate the pathogenic mechanism of subgroup J avian leukosis virus（ALV-J）,a full-length infectious clone of ALV-J（pBW-SD1009） was constructed by cloning and combining of three fragments using PCR method from SD1009 isolated from layer chicken in Shandong Province. Compared with other ALV-J classical isolates,a fragment encoding rTM and DR elements with 205bp length was found to be deleted in SD1009. The sequence was then inserted into SD1009 to form another infectious clone and named pBW-SD1009A205. The two viruses pBW-SD1009 and pBW-SD1009A205 were transfected into DF1 cell. The rescued viruses were named rSD1009 and rSD1009A205 respectively and were detected using indirect immunofluorescence assay,avian leukosis virus antigen test kit,and reverse transcriptase assay. All of the results illustrated rSD1009 and rSD1009A205 were resumed,therefore,two rescued viruses of ALV-J isolated from layer chicken were successfully constructed,and it showed that there was no prominent influence on ALV-J virus rescue and virus growth whether the rTM and DR elements deleted or not.

关 键 词：J亚群禽白血病病毒 感染性分子克隆 病毒拯救 生长特性 

分 类 号：S852.659.3[农业科学—基础兽医学]