重组杆状病毒感染悬浮昆虫细胞及重组蛋白表达策略的优化

Optimization of Recombinant Baculovirus Infection and Its Expression Strategy

为确定嵌合口蹄疫病毒(FMDV)中和表位的猪细小病毒(PPV)衣壳蛋白在昆虫细胞(Sf-9)中的最佳表达条件。通过对比悬浮培养和贴壁培养2种培养条件,探究Sf-9细胞的最佳培养方式;利用血球计数板每隔24 h进行细胞计数,绘制Sf-9细胞生长曲线;将P1代重组病毒传代至P3代,提取重组病毒基因组DNA,进行PCR鉴定,检测插入目的基因的完整性;通过研究感染复数(MOI)和感染时间的关系来摸索重组病毒最佳感染条件,以获得高滴度的重组病毒;利用Western blot检测目的蛋白的表达量并探究重组蛋白的最佳表达条件。结果表明,悬浮培养的Sf-9细胞大小均一、边缘整齐,生长状态良好;PCR鉴定结果表明,插入的FMDV中和表位基因完整,可以进行重组蛋白的表达;Western blot结果显示,重组蛋白同时与FMDV阳性血清和PPV阳性血清产生特异性反应,进一步证明了插入的FMDV中和表位的存在;重组蛋白最佳表达条件是以10个MOI病毒液感染悬浮Sf-9细胞,在72 h时重组蛋白表达量最大。

To screen the optimal expression conditions of chimeric protein,which consisted of porcine parvovirus capsid protein and foot-and-mouth disease virus neutralizing epitopes,in suspended Sf-9 cells,the optimal culturing mode of Sf-9 cells was studied with suspension culturing and adherent culturing.The cell counting was performed every 24 h by using a hemocytometer,and the replication kinetics curve of suspension cells was drawn.After passaging from P1 to P3,the genomic DNA extracted from the baculovirus was identified by PCR for detecting whether the inserted gene was missing.In order to get high titer recombinant baculovirus,the optimal infection conditions were screened by exploring the relationship between the multiplicity of infection(MOI)and infection time,and the expression proteins from different expression conditions were determined by Western blot.The size of the Sf-9 cells in suspension culturing was uniform and the edges were neat,namely more suitable for suspension culturing.The inserted gene fragment was not lost and could express the chimeric protein which could be specific recognized by FMDV positive FMDV sera and PPV positive sera.The optimal expression conditions were 10 MOI and suspension culturing 72 h.