CAJ | 학술논문

目的应用果蝇S2细胞表达甲型H1N1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/71/09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Western Blot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/71/09 HA基因,长度约1.7×103 bp.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75×103 D.免疫小鼠后可诱导机体产生抗HA特异性抗体,免疫后10d、30 d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.
목적 응용과승S2세포표체갑형H1N1류감병독가용성HA단백,병평고기면역활성.방법 이갑형H1N1류감A/Shenzhen/71/09병독주작위모판,채용RT-PCR기술확증HA기인,구건지속성표체재체pAC5.1-HA,협동사선재체pCoblast공전염지S2세포,통과Blasticindin항생소사선획득은정전염적S2세포주.이용Western Blot기술감정세포상청HA단백표체,사용Ni친화주순화중조HA단백.사용HA면역BALB/c소서3차,이용ELISA검측HA특이성항체효개.결과 성공극륭A/Shenzhen/71/09 HA기인,장도약1.7×103 bp.장HA기인극륭입pAC5.1-V5/His재체,경과전염、항생소사선후획득은정표체HA적S2세포주,목적단백이분비형식표체우상청,상대분자질량약75×103 D.면역소서후가유도궤체산생항HA특이성항체,면역후10d、30 d효개분별위1∶1280、1∶5120.결론 획득가용성표체적HA단백,병구유량호적면역활성,위후기류감면역진단시제적연제、아단위역묘적개발、단극륭항체적제비전정료기출.
Objective To express soluble HA of A/H1 N1 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-transfected 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 × 103 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 × 103 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-activity,which contributed to research on immune diagnosis,subunit vaccine,and monoclonal Abs for influenza.