H5N1亚型禽流感病毒HA基因在sf9细胞中的表达

Expression and identification of H5N1 highly pathogenic avian influenza virus HA gene in sf9 cells

本研究选用Bac-To-Bac杆状病毒表达系统构建含有H5N1亚型禽流感病毒HA主要抗原基因的重组杆状病毒,并感染sf9昆虫细胞表达HA抗原蛋白。首先,分析禽流感病毒H5N1亚型HA基因编码的氨基酸序列,选取抗原表位集中特异性保守的HA序列,构建重组供体质粒p Fast HA,转化DH10Bac感受态细胞,经蓝白斑筛选和PCR检测,获得重组穿梭载体Bacmind HA。脂质体法转化对数生长期的sf 9昆虫细胞,获得重组杆状病毒r Bac HA。经Western-blotting检测及间接免疫荧光(IFA)分析,结果表明,HA蛋白在sf9昆虫细胞中正确表达,分子质量约为45 ku,并具有良好的反应原性。为进一步研制H5N1亚单位疫苗的研发及研制H5亚型ELISA抗体试剂盒奠定了基础。

Recombinant baculovirus containing HA gene of H5N1 highly pathogenic avian influenza virus（AIV） was constructed using Bac-to-Bac baculovirus expression system,and recombinant HA protein was expressed by infecting sf9 cell with recombinant baculovirus.Firstly,the coding sequences of H5N1 AIV HA gene was analyzed using bioinformatics software,and conservative antigenicity coding sequences were selected.HA gene was inserted into donor plasmid p Fast-HTB to obtain recombinant donor plasmid p Fast HA,and then was transformed into E.coli DH10 Bac,using blue and white spot selection and PCR assay.The recombinant bacmid plasmid Bacmid HA was obtained.The recombinant baculovirus r Bac HA was transfected into sf9 insect cells during a log growth phase using Cellfection reagent to obtain the production of recombinant viral Bacmid HA.The expressed HA protein was analyzed by Western-blot and indirect immunofluorescence assay（IFA）.The results showed that the recombinant HA protein with a 45 ku molecular weight was expressed successfully,providing a new research direction of H5N1 subunit vaccine of HA protein and H5 subtype antibody ELISA detection kit.