【摘要】 目的构建含截短的HCV核心蛋白基因和绿色荧光蛋白基因的重组杆状病毒表达载体,探讨其在Sf9细胞中的表达和抗原性。方法用PCR法扩增截短的HCV核心蛋白基因片段(Ct)和增强型绿色荧光蛋白(EGFP)基因,将其插入转座子载体pFastBac1,构建成重组表达载体pFastCt-EGFP,转化大肠埃希菌DH10Bac,获得重组杆状病毒穿梭质粒BacmidCt-EGFP,转染昆虫Sf 9细胞。结果SDS-PAGE和western blot鉴定表明成功表达了分子量约40 000的融合蛋白。以ELISA法检测发现该融合蛋白能与28份抗HCV抗体阳性血清中的15份发生反应,阳性率为53.6%。结论该融合蛋白在昆虫Sf 9细胞中成功表达并具有一定的抗原性。更多还原

【Abstract】 Objective To construct recombinant baculovirus expression vector containing hepatitis C virus(HCV) truncated core gene and EGFP gene,and to study the antigenicity of the fusion protein expressed in Sf 9 cells.Methods PCR-amplified HCV truncated core gene and EGFP gene were cloned into the transposed vector pFastBac1 to construct a recombinant plasmid pFastCt-EGFP,by which E.coli DH10Bac was transformed to get the recombinant BacmidCt-EGFP.Insect Sf 9 cells were transfected with BacmidCt-EGFP and the expression of fusion protein Ct-EGFP was screened by SDS-PAGE and Western blotting.Results SDS-PAGE and Western blot analysis showed that fusion protein Ct-EGFP was expressed with the molecular mass of 40 kD.ELISA results showed that the fusion protein reacted with 15 of 28(54%) anti-HCV positive sera.Conclusion The fusion protein Ct-EGFP was expressed in insect Sf9 cells and showed partial antigenicity.更多还原