HCV核心和截短的包膜蛋白E2基因在昆虫细胞中的表达及其抗原性

目的:构建含HCV全长核心基因和截短的包膜蛋白E2基因的重组杆状病毒表达载体,研究其表达和抗原性.方法:以含HCV全长cDNA克隆的pGEM-HCJ4质粒为模板,PCR扩增全长的HCV核心抗原基因和截短的E2基因片段,插入转座载体pFastBacHTa构建重组转座质粒pFB-CE2t,转化DH10Bac大肠杆菌,获得重组杆状病毒穿梭质粒Bacmid-CE2t,以之转染昆虫Sf 9细胞进行外源基因的表达,并对其抗原性进行ELISA检测.结果:SDS-PAGE和Western blot分析表明Bacmid-CE2t在Sf 9细胞表达了HCV C-E2蛋白,并能利用细胞内蛋白酶将其裂解为单独的C蛋白和截短的E2蛋白.纯化后的蛋白能分别与HCV C蛋白、E2蛋白单抗以及慢性丙型肝炎患者血清反应.结论:HCV核心蛋白和截短的E2蛋白在昆虫细胞中成功表达并具有抗原性.

Expression of hepatitis C virus core gene and truncated envelope 2 gene in insect cells and its antigenicity

Objective:To construct recombinant baculovirus expression vector containing hepatitis C virus (HCV) core gene and envelope 2 (E2) gene, and to study its expression and antigenicity in insect cells.Methods:pGEM-HCJ4 plasmid containing full-length cDNA clone of HCV was used as the template to amplify HCV core gene and truncated E2 gene by PCR. The fragments were cloned into the transposed vector pFastBacHTa to construct a recombinant plasmid pFB-CE2t. pFB-CE2t was further transformed into DH10Bac E.coli, the recombinant Bacmid-CE2t was screened and transfected in the Sf 9 cells for the expression of the heterologous gene. Antigenicity of recombinant proteins was detected by ELISA. Results: SDS-PAGE and Western blot analysis demonstrated that recombinant proteins were expressed and cleaved into the core protein and E2 protein in Sf 9 cells. Purified protein reacted strongly with monoclonal antibody (mAb) against HCV core protein, mAb against HCV E2 protein and the serum of patients with chronic HCV respectively. Conclusion:HCV core protein and truncated E2 protein can be expressed in insect cells and have strong antigenicity.