应用果蝇S2细胞高效表达甲型H1N1流感病毒可溶性HA蛋白及其活性分析

Soluble expression of A/H1N1 influenza virus HA with Drosophila S2 cell line and its bio-activity identification

目的应用果蝇s2细胞表达甲型H1Nl流感病毒可溶性HA蛋白，并评估其免疫活性。方法以甲型H1N1流感A／Shenzhen／7I／09病毒株作为模版，采用RT-PCR技术扩增HA基因，构建持续性表达载体pAC5．1-HA，协同筛选载体pCoblast共转染至s2细胞，通过Blasticindin抗生素筛选获得稳定转染的s2细胞株。利用WesternBlot技术鉴定细胞上清HA蛋白表达，使用Ni亲和柱纯化重组HA蛋白。使用HA免疫BALB／c小鼠3次，利用ELISA检测HA特异性抗体效价。结果成功克隆A／Shenzhen／7I／09HA基因，长度约1．7×10^3bp。将HA基因克隆入pAC5．1-V5／His载体，经过转染、抗生素筛选后获得稳定表达HA的s2细胞株，目的蛋白以分泌形式表达于上清，相对分子质量约75×10^3D。免疫小鼠后可诱导机体产生抗HA特异性抗体，免疫后10d、30d效价分别为1：1280、1：5120。结论获得可溶性表达的HA蛋白，并具有良好的免疫活性，为后期流感免疫诊断试剂的研制、亚单位疫苗的开发、单克隆抗体的制备奠定了基础。

Objective To express soluble HA of A/H1N1 influenza virus in drosophila S2 cell line and identify its bio-activity. Methods HA gene was amplified from A/Shenzhen/71/09 virus strain using RT-PCR,then we constructed pAC5.1-HA expression vector, which was co-trausfected into S2 cell with pCoblast vector. After transfection,stable S2 cell was selected through Blasticindin. HA in the supernatant was identified with Western Blot assay and purified with Ni-column. Recombinant HA was immunized into BALB/c mice 3 times,and the Abs titers were evaluated with ELISA. Results We successfully cloned HA gene with 1.7 x 10^3 bp of A/Shenzhen/71/09 virus strain and got recombinant pAC5.1-HA expression vector. Stable S2 cell line was established after transfection and selection,which continuously expressed HA with molecular weight 75 x 10^3 D. After immunization with HA, the Abs titers were 1 ： 1280 and 1 ： 5120 respectively on 10 d, 30 d. Conclusion We expressed soluble HA with good bio-aetivity,which contributed to research on immune diagnosis,subunit vaccine,and monoclonal Abs for influenza.