鲤春病毒血症病毒糖蛋白在杆状病毒表达系统中的表达与鉴定

Expression and Identification of Spring Viremia of Carp Virus Glycoprotein in Baculovirus Expression System

本实验利用杆状病毒表达系统表达鲤春病毒血症病毒(SVCV)糖蛋白。将SVCV糖蛋白基因(G)插入到供体质粒pFastBacHTA中,再用构建的质粒pFastBac-G转化E.coli DH10αBac感受态细胞,经蓝白斑筛选,获得含G基因的重组穿梭载体Bacmid-G。通过脂质体介导将Bacmid-G转染对数生长期的Sf9细胞,获得重组杆状病毒AcNPV-G。AcNPV-G感染Sf9细胞,超声破碎后离心取上清进行SDS-PAGE及Western Blot试验,结果可见大小约为57 kDa的蛋白条带,并与羊抗SVCV血清发生特异性反应。间接免疫荧光试验在感染AcNPV-G的Sf9细胞中观察到荧光信号。试验结果表明,AcNPV-G在Sf9细胞中成功的表达了SVCV糖蛋白,且具有良好的免疫学活性。

In this study,baculovirus expression system was used to express glycoprotein of spring viremia of carp virus（SVCV）. The glycoprotein gene of SVCV was cloned into the pFastBacHTA vector,followed by transformation into competent cells DH10αBac for blue-white selection. The positive recombinant bacmid-G was transfected to Sf9 cell in logarithmic growth phase with Cellfectin Reagent induction to obtain recombinant Baculovirus AcNPV-G. A specific 57 kDa protein band and hybridizing reaction between expressed protein and positive serum of SVCV were observed by SDS-PAGE and Western blot. For indirect immunofluorescence,specific fluorescence signals were observed in Sf9 cells infected by AcNPV-G. The results indicated that SVCV glycoprotein had been expressed in Sf9 cells with immunological activity.