经密码子优化的猪Ficolin α在昆虫细胞的表达及其体外抗病毒作用初步分析

摘要

To get native porcine Ficolin α,Bac-to-Bac baculovirus expression system was used.Firstly,rBacmid-Ficolin α was constructed,and then transfected into sf9 insect cells to obtain recombinant baculovirus rBV-Ficolin α.The optimized expression conditions were detected by Western blot and analyzed by gray level using Image J software.Followed by purification,antiviral activity of target protein was assessed in vitro.Western blot results showed that the expression level of Ficolin α in transfected sf 9 insect cells was higher at the infection (MOI) of 7.5 when they were harvested at 96 h,and porcine Ficolin α had the antivirus activity against porcine reproductive and respiratory syndrome virus (PRRSV).In conclusion,porcine Ficolin α was successfully expressed in sf9 insect cells and showed strongly anti-PRRSV activity.This study provided a material basis for the study of antiviral activity and its mechanism of porcine Ficolin α.%利用Bac-to-Bac昆虫杆状病毒表达系统表达具有天然构象的猪Ficolin α.首先构建重组穿梭质粒rBacmid Ficolinα,转染sf9细胞获得重组杆状病毒rBV-Ficolin α;通过Image J软件对优化表达的Western blot图片进行灰度分析确定较佳表达条件;纯化目的蛋白质后,进一步评价其体外抗病毒活性.Western blot结果显示以7.5MOI接种量感染的sf9细胞在96 h时获碍了较高的表达,同时获得的重组猪Ficolin α具有抗猪繁殖与呼吸综合征病毒(PRRSV)活性.猪Ficolin α在sf9昆虫细胞中获得成功表达,且具有良好的抗PRRSV活性.本研究可以为猪Ficolin α的抗病毒活性及作用机制研究提供物质基础.