禽流感病毒H5N1亚型HA蛋白在昆虫细胞中的表达、纯化及反应原性分析

Expression,purification and reactivity analysis of HA protein of Avian influenza virus H5N1 subtype in insect cells

为了获得可溶性的禽流感病毒H5N1亚型HA蛋白,并将其用于单克隆抗体的制备和筛选,试验利用Bac-to-Bac杆状病毒表达系统进行HA蛋白的表达和纯化,以含全长HA基因的重组质粒为模板,经PCR扩增得到HA片段并克隆至pFastBac1载体,将鉴定正确的供体质粒转化至DH10Bac感受态细胞,获得Bacmid重组质粒,转染Sf 9细胞后获得重组杆状病毒。通过Western-blot和间接ELISA验证HA蛋白的表达情况和反应原性。结果表明:成功获得含有禽流感病毒H5N1亚型HA基因重组杆状病毒,HA蛋白可以分泌表达,大小约为63 ku,与预期大小一致,且能与H5N1标准阳性血清发生特异性反应;纯化的HA蛋白与免疫小鼠血清反应性良好。说明本研究成功表达并纯化获得了禽流感病毒H5N1亚型HA蛋白,并且具有良好的反应原性。

In order to achieve soluble expression of HA protein of Avian influenza virus H5 N1 subtype,which was used for the preparation and screening of monoclonal antibody,the Bac-to-Bac baculovirus expression system was used to express HA protein of Avian influenza virus H5 N1 subtype in this experiment,and the purification was carried out.The recombinant plasmid containing the full-length HA gene was used as the template,HA fragments were amplified by PCR and were cloned into pFastBac1 vector.The correct donor plasmid was identified and was transformed into DH10 Bac competent cells to obtain recombinant plasmid Bacmid.The Bacmid plasmid was transfected into Sf 9 cells for the preparation of recombinant baculovirus.The HA recombinant protein expression and its reactivity were verified by Western-blot and indirect ELISA.The results showed that recombinant baculovirus containing H5 N1 HA was successfully obtained.The HA protein can be expressed in secreted form,with a size of about 63 ku,which was consistent with the expected size,and can react specifically with the standard positive H5 N1 serum.The purified HA protein reacted well with the serum of immunized mice.The results suggested that H5 N1 HA protein was successfully expressed and purified,and had good reactivity.