摘要
以鸡痘病毒(FPV)疫苗株为载体,将H 5亚型A IV血凝素基因(HA)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-L acZ复合启动子(AT I-P 7.5×20)和单一启动子(P 7.5)下游,构建了携带A IV HA基因和鸡IL-18基因的重组鸡痘病毒转移载体质粒pUTAL-H 5HA-IL 18。将H 5亚型A IV HA基因插入到鸡痘病毒表达载体pUTA 2复合启动子(AT I-P 7.5×20)下游构建了携带H 5亚型A IV HA基因的重组鸡痘病毒转移载体质粒pUTA 2-H 5HA。应用脂质体转染法,将重组鸡痘病毒转移载体质粒与282E 4株鸡痘病毒共转染鸡胚成纤维细胞(CEF),在B rdU药物加压下进行3次蚀斑筛选。以不同代次细胞的mRNA为模板,利用H 5亚型A IV HA基因和鸡IL-18基因特异引物进行RT-PCR和蛋白印迹检测,筛选出能共表达H 5亚型A IV HA基因与鸡IL-18基因和单独表达H 5亚型A IVHA基因的重组鸡痘病毒rFPV-H 5HA-IL 18和rFPV-H 5HA,这些重组鸡痘病毒的构建为A IV活载体疫苗的研制奠定了基础。
Recombinant fowlpox virus（rFPV） vaccine strain coexpressing HA from subtype HSN1 of AIV plus chicken interleukin-18 gene were constructed by inserting cDNA copy of AIV （HSN 1 ） HA gene and cDNA copy of chicken IL-18 gene into the FPV expression plasmid pUTA-16-LacZ and cDNAs respectively regulated by ATI-P7.5 × 20 promoter and P7.5 promoter. This recombinant expression plasmid was named as pUTAL-HSHA-IL18, cDNA copy of subtype HSN1 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-HSHA. CEF cells were co-transferred by these two recombinant expression plasmid vectors and 282E4 FPV with DOTAP Liposomal. The recombinant FPV（rFPV）,rFPV-HSHA-IL18 and rFPV-HSHA strains,which could correctly express AIV HA gene and chicken IL-18 gene ,were created by three cycles drug BrdU screening and verified by RT-PCR and Western blotting assay. This study paved the way for further development of a new AIV recombinant vaccine.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2006年第4期390-393,共4页
Chinese Journal of Veterinary Science
基金
吉林省应用基础资助项目(20010544)
