乙型脑炎病毒E基因在杆状病毒中的表达

Expression of E Gene of Japanese Encephalitis Virus in Baculovirus System

利用PCR方法扩增日本乙型脑炎病毒(JEV)E全长基因,然后将其克隆到杆状病毒转座载体pBac-SC中,筛选重组质粒pBacSC-E,再转化入含有穿梭载体的感受态细胞DH10Bac,经抗性及蓝白斑筛选得到含E基因的重组杆状病毒DNA,以脂质体介导法将此重组杆状病毒DNA转染Sf9昆虫细胞,从而获得重组杆状病毒。用此重组杆状病毒感染Sf9昆虫细胞,经Western-blot检测表明,E基因在昆虫细胞中获得表达,表达蛋白的分子量约53 ku,与预期结果大小一致,且能与日本乙型脑炎阳性血清发生特异性反应,这为进一步研究JEV亚单位疫苗和诊断抗原奠定了基础。

In this study,the full length E gene of Japanese encephalitis virus（JEV） was amplified by PCR and cloned into vector pBacSC to form the recombinant plasmid pBacSC-E.The recombinant pBacSC-E was identified by enzyme digestion.The pBacSC-E was transformed into E.coli strain DH10Bac.Through resistance and blue-white selection,the recombinant baculovirus was obtained from Sf9 insect cells in which recombinant baculovirus DNA transfected through the liposome-mediated.Sf9 insect cells were infected by recombinant baculovirus and Western-blot showed that E protein with a molecular weight of 53 ku was expressed successfully.The recombinant protein can be recognized by JEV-positive serum.This would lay a foundation for developing JEV subunit vaccine and diagnostic antigen.