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

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

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 c DNA克隆的 p GEM- 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 p Fast Bac HTa构建重组转座质粒 p FB- CE2 t,转化 DH10 Bac大肠杆菌 ,获得重组杆状病毒穿梭质粒 Bacmid- CE2 t,以之转染昆虫 Sf9细胞进行外源基因的表达 ,并对其抗原性进行 EL ISA检测。 结果 :SDS- PAGE和 Western blot分析表明Bacmid- CE2 t在 Sf9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

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.