B亚群禽白血病病毒GX14FF03株感染性克隆构建与拯救  

Construction and Rescue of Infectious Clones from Avian Leukosis Virus Subgroup B Isolate GX14FF03

作　　者：赵亚楠 石梦雅 李海娟 林璐璐 王培坤 韦平 ZHAO Ya'nan;SHI Mengya;LI Haijuan;LIN Lulu;WANG Peikun;WEI Ping(Institute of Microbe and Host Health,Linyi University,Linyi,Shandong 276005;Institute for Poultry Science and Health,Guangxi University,Nanning,Guangxi 530004)

机构地区：[1]临沂大学微生物与宿主健康研究所,山东临沂276005 [2]广西大学养禽与禽病学研究所,广西南宁530004

出　　处：《中国家禽》2020年第4期29-33,共5页China Poultry

基　　金：山东省自然科学基金项目(ZR201807070202);大学生创新创业训练计划立项项目(S201910452031);2017广西科技重大专项(AA17204057)。

摘　　要：为探究从灵山土鸡分离的携带J亚群禽白血病病毒(ALV)U3区片段的B亚群ALV(ALV-B)毒株GX14FF03的致病性,试验以GX14FF03毒株核酸为模板,采用PCR方法分2段扩增病毒的cDNA,PCR扩增产物经克隆、酶切后依次连接到pBluescriptⅡKS(+)质粒,最终获得一个含有ALV-B完整前病毒cDNA的重组质粒pBlue-GX14FF03。重组质粒转染DF-1细胞进行病毒拯救,拯救病毒在DF-1细胞盲传3代后收集细胞上清液保存于-80℃,细胞固定后用于间接免疫荧光检测。另取保存细胞上清进行ALV p27抗原检测。间接免疫荧光检测结果及ALV p27抗原检测结果全部为阳性。拯救病毒及亲本病毒接种DF-1细胞后进行体外复制能力测定,结果显示拯救病毒在1、3、5、7、9 d体外复制能力与亲本病毒基本一致,差异不显著(P>0.05)。上述结果表明病毒拯救成功,命名为rGX14FF03。In order to explore the pathogenesis of avian leukosis virus subgroup B(ALV-B),an infections clone of ALV-B(pBlue-GX14 FF03)was constructed by combing of two fragments using PCR method from GX14 FF03(this virus was one natural recombinant ALV-B with a ALV-J-like U3 region)isolated from commercial native chickens in Guangxi Province.The sequence of the provirus cDNA was amplified by PCR from GX14 FF03 and the infectious clone of pBlue-GX14 FF03 was constructed based on the vector of pBluescriptⅡKS(+).The rescued virus of rGX14 FF03 was generated by transfecting pBlue-GX14 FF03 into the DF-1 cells and identified by ALV group-specific antigen(p27)detection and indirect immunofluorescence assay(IFA).DF-1 cells were infected with rGX14 FF03 and GX14 FF03 to measure the proliferative properties of rescue virus and parental virus,and the results of in vitro replication showed that there was no difference between the rescue virus and the parent virus(P>0.05),indicating that the rescue was successful.

关 键 词：B亚群禽白血病病毒 感染性克隆 病毒拯救 体外复制能力 

分 类 号：S855.3[农业科学—临床兽医学]