H9N2亚型AIV HA基因的克隆及其在昆虫细胞中的表达

研究目的]利用杆状病毒表达系统表达AIV H9N2亚型HA基因以获得可以利用的HA蛋白;方法]应用DNAStar软件,参照Genbank中注册的AIV H9N2亚型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H9亚型AIV的HA基因,通过BamH Ⅰ和Xho Ⅰ双酶切位点将H9HA基因插入转移质粒载体pFastbacHTa中,获得重组转移载体pFastbacHTa-H9HA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-H9HA,再将其转染昆虫细胞High Five,进行PCR鉴定、SDS-PAGE和Westem Blot检测;结果]HA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,HA基因在High Five细胞中得到了表达,H9HA大小约为67 KD,而且表达的产物具有特异免疫学反应性;结论]HA基因在昆虫细胞获得表达,为进一步生产检测H9N2亚型流感病毒的检测试剂或者构建亚单位疫苗奠定了基础.

Cloning of HA Gene of H9N2 Avian Influenza Virus (AIV) and its Expression in Insect Cells

OBJECTIVE]HA gene of H9N2 AIV was expressed by using Baculovims expression system. METHOD]A pair of primers was designed by employing DNA Star software and consulting the HA nucleotide sequences of H9N2 subtype AIV. Then the HA gene with flanking restriction sites was successfully PCR-amplified. The Hg-HA gene was excised with BamH I and Xho I , and inserted into the transfer vehi- cle pFastbacHTa to obtain the recombinant transfer vectors pFastbacHTa-HgHA, which was used to transform DH10 Bac-host ceils, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-H9HA. And then the Bacmid-H9HA was further used to transfect High Five insect cells. The cells were identified by PCR, SDS-PAGE and Western blotting. RESULTSIHA gene was inserted in correct orientation downstream of the virus polyhedral protein promoter, and had been expressed in High Five cells with the size of the Hg-HA being approximately 67KD. The expression product could specially react to the anti-sera of H9N2 AIV. CONCLUSION]HA gene was successfully expressed in insect cell and this provided a good base for produce diagnostic reagent to detect H9 subtype AIV or construct subunit vaccine against AIV.