H5亚型AIV HA基因的克隆及其在昆虫细胞中的表达  

Cloning of Structural Protein HA Gene of H5N1 Avian Influenza Virus and its Expression in Insect Cells

作　　者：詹爱军[1] 王新卫[2] 王敬师[1] 周庆丰[1] 马静云[1] 毕英佐[1] 于康震[3] 

机构地区：[1]华南农业大学动物科学学院,广东广州510642 [2]河南农业大学牧医工程学院,河南郑州450002 [3]中国兽药监察所,北京100081

出　　处：《中国家禽》2007年第15期20-23,共4页China Poultry

摘　　要：应用DNAStar软件,参照GenBank中注册的AIV H5亚型毒株HA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HA基因,通过BamHⅠ和XhoⅠ双酶切位点将H5 HA基因插入转移质粒载体pFastBacHTa中,获得重组转移载体pFastBacHTa-H5HA并将其转化DH10Bac细胞,与Bacmid发生位点特异性转座作用,得到重组穿梭载体Bacmid-H5HA,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SDS-PAGE和Western blotting检测,HA基因在High Five细胞中得到了表达,表达产物大小约为67ku,而且具有特异的免疫反应性。A pair of primers was designed by DNAStar software according to registered sequences of HA nucleotide of H5 subtypes AIV on GenBank. Then the HA gene with flanking restriction sites was successfully amplified by RT-PCR. The H5-HA gene was excised with BamHⅠand XhoⅠand inserted into the transfer vehicle pFastBacHTa to obtain the recombinant transfer vectors pFastbacHTa-H5HA,which was used to transform DH10Bac cells,producing specific transposition with Bacmid,Bacmid-H5HA. Bacmid-H5HA was further used to transfect High Five insect cells,when PCR identification proved that the gene was inserted in downstream of the virus polyhedral protein promoter with correct orientation. SDS-PAGE and Western blotting confirmed that the gene had been expressed in High Five cells,and the size of the expression product being approximately 67 ku. The expression product could react to the antisera of AIV specially.

关 键 词：禽流感病毒 血凝素蛋白 昆虫细胞 克隆 

分 类 号：S83[农业科学—畜牧学]