摘要
从含有禽流感病毒A/Goose/Guangdong/1/96 (H5N1)核蛋白 (NP)基因的质粒pUCNP中切下NP基因片段 ,将其亚克隆到pSY5 38质粒 ,再将带有痘苗病毒启动子P11的LacZ基因也平端克隆到pSY5 38质粒 ,然后切下同时含有NP及LacZ基因的片段 ,再亚克隆到禽痘病毒载体pSY6 81,从而构建出表达核蛋白基因的重组禽痘病毒转移载体pSY(NP +LacZ)。应用脂质体介导的方法将转移载体转染已感染禽痘病毒S FPV 0 17的鸡胚成纤维细胞 ,在X gal存在的条件下 ,利用蓝白斑筛选、数轮蚀斑纯化以及PCR、Western blot鉴定 ,结果证明 ,获得了能高效表达AIVNP蛋白的重组禽痘病毒rFPV NP。SPF鸡体内的免疫保护试验结果表明 ,它能够诱导机体产生较高水平的NP特异性抗体 ,并对H5N1和H7N1这 2种亚型高致病力禽流感病毒的攻击提供一定的保护。
To construct transfer vector, NP gene of A/Goose/Guangdong/1/96(H5N1), which derived from recombinant plasmid pUCNP, was subcloned into EcoRI site of pSY538, and Lac Z gene promoted by p11 was cloned into SmaI site of this plasmid. Both NP and Lac Z gene were cloned into NotI site of FPV vector pSY681. Then it was transferred onto CEF cells infected with S FPV 017. Fowlpox virus recombinants were selected by blue plaque screening with X gal following several cycles purification. The results of identification by PCR and Western blot indicated that recombinant FPV could efficiently express NP gene of AIV in vitro. The results of immunization in SPF chickens indicated that this recombinant virus could induce high titer NP specific antibodies and protect chickens against the lethal challenge with H5N1 and H7N1 HPAIV.
出处
《中国农业科学》
CAS
CSCD
北大核心
2003年第6期704-708,共5页
Scientia Agricultura Sinica
