摘要
以鸡痘病毒（FPV）疫苗株为载体,将H5和H7亚型禽流感病毒血凝素（AIV HA）基因串联后（拥有同一个阅读框）和鸡白细胞介素-18（IL-18）基因分别插入到鸡痘病毒表达载体pUTA-16-LacZ复合启动子（ATI-P7.5×20）和单一启动子1（P7.5）下游,构建了携带AIV HA基因和鸡白细胞介素-18基因的重组鸡痘病毒转移载体质粒pUTAL-H5-H7-IL18;用相同的方法构建重组鸡痘病毒转移载体质粒pUTAL-H5-IL18;将H5亚型AIV HA基因插入到鸡痘病毒表达载体pUTA2复合启动子（ATI-P7.5×20）下游,构建了携带H5亚型AIV HA基因的重组鸡痘病毒转移载体质粒pUTA2-H5.应用脂质体转染法,将重组鸡痘病毒转移载体质粒与282E4株鸡痘病毒共转染鸡胚成纤维细胞（CEF）,经BrdU进行三次加压蚀斑筛选后,以不同代次的细胞mRNA为模板,利用H5亚型AIV HA基因、H7亚型AIV HA基因和鸡IL-18基因特异引物进行RT-PCR和蛋白印迹检测,筛选出H5HA-H7HA融合蛋白基因和鸡IL-18基因共表达的重组鸡痘病毒rFPV-H5HA-H7HA-IL18,H5亚型AIV HA基因和鸡IL-18基因共表达的重组鸡痘病毒rFPV-H5HA-IL18以及单独表达H5亚型AIV HA基因的重组鸡痘病毒rFPV-H5HA.这些重组鸡痘病毒的构建为AIV活载体疫苗的研制奠定了基础.
The cDNAs of subtype H5N1 and H7N1 of AIV HA were connected and named as H5HA-H7HA cDNA,which possess common read frame. The cDNA of H5HA-H7HA and chicken Interleukin-18 were inserted into expression plasmid pUTA-16-LacZ, and respectively regulated by ATI-P7.5 × 20 promoter and P7.5 promoter. This recombinant expression plasmid vectors was named as pUTAL-H5-H7-IL18, and the pUTAL-H5- IL18 was constructed by the same above way. The cDNA copy of subtype H5N1 AIV HA was inserted into the FPV expression plasmid pUTA2, and regulated by ATI- P7.5 × 20 promoter. This recombinant expression plasmid was named as pUTA2-H5. CEF cells were co-transfered by these three recombinant expression plasmid vectors and 282FA FPV with DOTAP Liposomal. The recombinant FPV（rFPV）, rFPV-H5-H7-IL18, rFPV-H5-IL18 and rFPV-H5HA strains, which could correctly express AIV HA gene and chicken IL-18 gene, were created by three cycles drug BrdU screening and being verified by RT-PCR and Western blotting assay. This study paved the way for farther developing new AIV recombinant vaccines.
出处
《高技术通讯》
CAS
CSCD
北大核心
2006年第4期408-413,共6页
Chinese High Technology Letters
基金
国家十五重点攻关项目（2004BA519A12）资助.
关键词
禽流感病毒
血凝素基因
重组禽痘病毒
鸡白细胞介素-18
avian influenza virus （AIV）, hemagglutinin gene （HA）, recombinant fowlpox virus （rFPV）, interleukin- 18 gene （ IL- 18）
