流感病毒HA球状头部结构域在昆虫杆状病毒表达系统的表达及鉴定

Expression and identification of the globular head domain of influenza virus HA in an insect baculovirus expression system

目的通过昆虫杆状病毒表达系统获得具有良好免疫原性的流感病毒蛋白。方法将Hemagglutinin[Influenza A virus A/California/09/2009(H1N1)]进行截短,取57-271球状头部结构域氨基酸序列,在前面加Igκleader信号肽(METDTLLLWVLLLWVPGSTGD),设计H1HAHead基因。将基因序列按照昆虫细胞密码子偏好性进行优化、合成,通过EcoRI和HindIII双酶切克隆至pFastBAC HTA载体上,获得含有目的基因的重组质粒pFastBAC HTA-H1HAHead。利用热激法将重组质粒转化至大肠埃希菌DH10 Bac^(^(TM) ) 感受态细胞,蓝白斑筛选获得重组杆状质粒rBacmid-H1HAHead。在脂质体的介导下,将重组杆状质粒转染至SF9细胞,对病毒进行拯救,转染后120 h,收取上清病毒液,盲传3代,获得SF9细胞,运用间接免疫荧光法(IFA)和Western blot检测目的蛋白的表达。结果经双酶切鉴定成功插入长度为762 bp的目的基因H1HAHead,通过脂质体介导长度为3192 bp的重组杆状质粒rBacmid-H1HAHead构建成功。杆状病毒系统可表达目的蛋白,其分子质量单位28.25 ku,与预期一致。Western blot鉴定该蛋白能与特异性标签His-tag发生反应,IFA鉴定感染重组病毒的SF9细胞外源蛋白表达。结论通过昆虫杆状病毒表达系统成功表达目的蛋白,该蛋白有反应性,为流感抗体检测和新型疫苗的开发奠定了基础。

Objective To obtain an influenza virus protein with good immunogenicity using an insect baculovirus expression system.Methods Hemagglutinin[influenza A virus A/California/09/2009(H1 N1)]was truncated,a sequence of 57-271 amino acids constituting the globular head domain was obtained,and an Igκleader signal peptide(metdtllwvlllwvpgstgd)was added in front of it.The sequence was cloned in a pFastBAC HTA vector using EcoRI and HindIII digestion.The recombinant plasmid was transformed into DH10 Bac^(TM) competent cells via the heat shock method for blue and white spot screening.White colonies were selected and a second round of blue and white spot screening was performed.White colonies were selected and inoculated on triple antibody medium.Recombinant rod-shaped particles were extracted on an adsorption column.The recombinant plasmid was transfected into Sf9 cells using liposomes.Five hours after transfection,the solution was changed.One hundred and twenty h after transfection,the cell supernatant was collected and centrifuged at 3,500 rpm for 5 min and then filtered with a 0.22-μm membrane.The filtrated was inoculated in Sf9 at a 1%volume.Cells were cultured for 72-96 h,the supernatant was collected,and steps were repeated to obtain a thrice-passaged recombinant virus.The thrice-passaged recombinant virus was inoculated into Sf9 and verified using Western blotting and IFA.Results The target gene h1 hahead,762 bp in length,was successfully inserted using double enzyme digestion.The recombinant rod-shaped plasmid rbacmid-h1 hahead,3,192 bp in length,was successfully constructed using liposomes.The target protein was expressed in a baculovirus system,and its molecular weight was 28.25 ku,which was consistent with the expectation.Western blotting indicated that the protein reacted with his tag.IFA indicated that the protein was expressed in Sf9 cells infected with the recombinant virus.Conclusion The target protein was successfully expressed in a baculovirus expression system.The protein was reactive.This finding has laid the foundation for the detection of anti-influenza antibodies and the development of new vaccines.