禽流感病毒血凝素与核蛋白在重组禽痘病毒中的共表达  

Co-Expression of Avian Influenza Virus Haemagglutinin and Nucleoprotein in Recombinant Fowlpox Virus

作　　者：乔传玲[1] 张建林[1] 贾永清[2] 邓国华[1] 姜永萍[1] 王秀荣[1] 孟庆文[1] 于康震[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 [2]中国农业科学院东北农业大学动物医学院,黑龙江哈尔滨150030

出　　处：《动物医学进展》2003年第1期81-83,共3页Progress In Veterinary Medicine

摘　　要：为克服禽流感病毒(AIV)血凝素(HA)仅诱导机体产生亚型特异性免疫应答的不足,本研究拟将病毒核蛋白(NP)基因与HA基因在重组禽痘病毒中实现共表达。首先构建转移载体,从测序质粒pUCNP中切下目的基因克隆到载体pSY538早晚期启动子LP2EP2的下游,再将含有禽痘病毒早晚期启动子LP2EP2的NP基因片段亚克隆到已有的AIVHA基因重组禽痘病毒转移载体中,即得到同时含有HA和NP两种基因的重组转移载体pSY(HA+NP)。将转移载体脂质体转染已感染禽痘病毒亲本株S-FPV-017的鸡胚成纤维细胞。根据报告基因β-半乳糖苷酶(LacZ)基因的表达,蓝白斑法筛选重组病毒,经数轮蚀斑纯化,PCR鉴定及West-ern-blot分析,结果表明所获得的重组病毒能高效表达AIVHA及NP两种蛋白。此研究为进一步研制更为有效的禽流感基因工程活病毒载体疫苗奠定了基础。To overcome the pitfall that haemagglutinin(HA) of avian influenza virus(AIV) could only induce subtypespecific immune response, the recombinant fowlpox virus coexpressing HA and Nucleoprotein (NP) protein of AIV was constructed. Firstly, NP gene of AIV A/Goose/Guangdong/1/96(H5N1), which derived from recombinant plasmid pUCNP, was subcloned into plasmid pSY538, followed by the fowlpox virus early/late promoter LP2EP2. The fragment of NP promoted by LP2EP2 was then subcloned into pSY(HA+LacZ), which was the recombinant fowlpox virus (rFPV) of H5 HA gene transfer vector had been constructed previously. After transfer vector named as pSY(HA+NP) was constructed, it was transferred on CEF cells infected with parent fowlpox virus SFPV017. Recombinant FPV was selected by blue plaque screening based on the expressing of LacZ gene, purified by 6 cycles cloning of blue plaque. The results of identification by PCR and Westernblot indicated that recombinant FPV could efficiently express HA and NP gene of AIV in vitro. This study would lay the foundation for developing AI genetically engineering viral vectorbased vaccine.

关 键 词：禽流感病毒 血凝素基因 核蛋白基因 重组禽痘病毒 基因共表达 基因工程活病毒载体疫苗 

分 类 号：S855.3[农业科学—临床兽医学] S852.5[农业科学—兽医学]