H5亚型AIV NS1基因的克隆及其在昆虫细胞中的表达  

Molecular Cloning of Non-Structural Protein NS1 Gene of H5N1 Avian Influenza Virus(AIV)and its Expression in Insect Cells

作　　者：詹爱军[1] 王新卫[2] 王宪文[1] 覃健萍[1] 毕英佐[1] 于康震[3] 曹永长[1] 

机构地区：[1]华南农业大学动物科学学院 [2]河南农业大学牧医工程学院,郑州450002 [3]中国兽药监察所,北京100081

出　　处：《生物技术通报》2008年第2期191-194,198,共5页Biotechnology Bulletin

摘　　要：应用DNAStar软件,参照Genbank中注册的AIV H5亚型毒株NS1基因序列,设计了一对引物,用RT-PCR方法成功地扩增出带双酶切位点的H5亚型AIV的NS1基因,通过BamHⅠ和EcoRⅠ双酶切位点将H5NS1基因插入转移质粒载体pFastbac HTa中,获得重组转移载体pFastbac HTa-H5NS1并将其转化DH10 Bac细胞,与Bacmid发生位点特异性转座作用,得到重组穿梭载体Bacmid-H5NS1,再将其转染昆虫细胞High Five,PCR鉴定证实该基因正确地插入到病毒基因组的多角体蛋白基因启动子下游,经过SDS-PAGE和Western Blot检测,NS1基因在High Five细胞中得到了表达,H5NS1大小约为28kD,而且表达的产物具有特异免疫学反应性。A pair of primers was designed by employing DNAStar software and consulting the NS1 nucleotide sequences of H5 subtypes AIV. Then the NS1 gene with flanking restriction sites was successfully PCR-amplified. The HS-NS1 gene was excised with BamH I and EcoR I and inserted into the transfer vehicle pFastbacHTa to obtain the recombinant transfer vectors pFastbacHTa-H5NS1,which was used to transform DH10 Bac-host cells,producing specific transposition with Bacmid to yield the recombinant shuttle vehicles,Bacmid-H5NS1. And then the Bacmid-H5NS1 was further used to transfect High Five insect cells,when PCR identification proved that the gene was inserted in correct orientation downstream of the virus polyhedral protein promoter;SDS-PAGE and Western blotting detection confirmed that the gene had been expressed in High Five cells,the size of the H5NS1 being approximately 28kD. The expression product could specially react to the sera of AIV.

关 键 词：禽流感病毒 非结构蛋白 杆状病毒表达 克隆 

分 类 号：Q78[生物学—分子生物学]