CAJ | 학술논문

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pBacSC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转感染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.
이용PCR방법확증일본을형뇌염병독（JEV）E전장기인,연후장기극륭도간상병독전좌재체pBacSC중,사선중조질립 pBacSC-E,재전화입함유천사재체적감수태세포DH10Bac,경항성급람백반사선득도함E기인적중조간상병독DNA,이지질체개도법장차중조간상병독DNA전감염Sf9곤충세포,종이획득중조간상병독.용차중조간상병독감염Sf9곤충세포,경Western-blot검측표명,E기인재곤충세포중획득표체,표체단백적분자량약53 ku,여예기결과대소일치,차능여일본을형뇌염양성혈청발생특이성반응,저위진일보연구JEV아단위역묘화진단항원전정료기출.
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.