以GPT和GFP为双重筛选标记的禽痘病毒转移载体的构建及鉴定  

Construction and identification of a fowlpox virus transfer vector

作　　者：王艳丽[1,2] 李俊辉[1,2] 姜永萍[1] 夏俊[2] 裴磊[1] 冉多良[2] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室/兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]新疆农业大学动物医学院,新疆乌鲁木齐830052

出　　处：《中国预防兽医学报》2009年第7期501-504,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金："十一五"国家科技支撑计划项目(2006BAD06A05)

摘　　要：为研制和开发以禽痘病毒为载体的重组病毒疫苗,本研究构建了禽痘病毒通用转移载体pSY681-gfp-gpt,该载体含有gfp和gpt2种报告基因及背对的痘苗病毒晚期启动子P11和早期启动子P7.5,P11启动gfp和gpt两个基因,P7.5用于启动外源基因,在早期启动子P7.5下游引入NotⅡ和AflⅡ两个酶切位点,用于外源基因的插入。为检测禽痘病毒转移载体pSY681-gfp-gpt的有效性,将H9亚型禽流感病毒A/Chicken/Shanghai/10/01(H9N2)的HA基因插入到该载体中构建转移载体pSY681-gfp-gpt-HA9,将转移载体转染已感染禽痘病毒S-FPV-017的鸡胚成纤维细胞,利用gfp和gpt同时进行双重筛选、数轮蚀斑纯化后获得了重组病毒rFPV-gpt-gfp-HA9,通过PCR、westernblot鉴定,结果证明,获得了能稳定表达H9亚型禽流感病毒HA蛋白的重组禽痘病毒rFPV-gpt-gfp-HA9,为今后重组禽痘病毒活载体疫苗的研制奠定了基础。Fowlpox virus has been used as a safe vector to construct vector-based vaccine for the control of avian virus diseases. In this study, fowlpox virus transfer vector pSY681-gfp-gpt was constructed by inserting two reporter genes （gfp and gpt） under the late promoter P11 and the exogenous gene under early promoter P7.5. The vector was tested by insertion of the HA gene of H9 subtype avian influenza virus A/Chicken/Shanghai/10/01 （H9N2） and transfected into CEF cells infected with S-FPV-017. Recombinant viruses were selected by gfp and gpt followed by several cycles plaque purification. Western blot and PCR indicated that recombinant FPV could efficiently express HA gene of AIV in CEF.

关 键 词：重组禽痘病毒 绿色荧光蛋白 黄嘌呤鸟嘌呤磷酸转移酶 HA基因 

分 类 号：Q782[生物学—分子生物学]