杆状病毒表达系统（昆虫细胞的培养及杆状病毒滴度测定）.doc

杆状病毒表达系统（昆虫细胞的培养及杆状病毒滴度测定）1昆虫细胞的培养：Insect Cell Lines：Sf9 or Sf21 cells主要用于转染，空斑纯化和滴度测定High Five cells or Mimic Sf9 cells转染效率低，主要用于蛋白表达 Graces Insect Medium with L-glutamine and supplemented with（pH 6.5）： 3330 mg/L lactalbumin hydrolysate 3330 mg/L yeastolate350 mg/L NaHCO310% heat-inactivated fetal bovine serumAtmosphere：air, 100% Temperature：28.0 CGrowth conditions： The recommended media are formulated for use without CO2. Omission ofthe yeastolate or lactalbumin hydrolysate will lead to poor performance by this line.Subculturing：Gently resuspend cells in the spent culture medium by pipetting across the monolayer or scraped by cell scrapers or by hitting the flask against the palm of your hand (175 flask). Preservation：Freeze medium90% serum, 10% DMSO 严格程序降温，过夜后转移液氮保存2转染：细胞70%-80%覆盖率 Baculovirus DNA与脂质体 轻轻 混匀 3扩增病毒：接毒量为MOI=0.05MOI= 0.1 需要的病毒量（ml）=比如：P1代毒滴度为5 106 PFU/ml，细胞量2107 cells/ml，那么需要的病毒量（ml）= =0.4 ml 表达蛋白：接毒量为MOI=1MOI=54杆状病毒滴度测定：两种病毒滴度测定方法的比较：空斑实验法测定滴度依赖于病毒在感染细胞中的复制以及感染周边细胞形成局造型病变；而免疫染色法只需要病毒感染靶细胞并表达病毒所编码的蛋白。因此，免疫染色法（the results of immunostaining expressed as infectious units per ml, or IFU/ml）测定病毒滴度需要的时间比空斑法（the results of the plaque assay expressed as plaque-forming unit/ml, or PFU/ml）更短（Table 1）。Table 1: Comparison of Titration MethodsMeasurementsWhat is required?Virus replication required?Detection of virus by immunostaining (Rapid Titer Kit)Virus must infect cells and express viral protein (PRRSV:N protein; Baculovirus:Gp64)NOPlaque assay or endpoint dilution assay (PFU/ml)Virus must infect cells, replication and then infect surrounding cellsYESBacPAK Rapid Titer Kit:本测定方法，以针对杆状病毒囊膜蛋白gp64的单克隆抗体标记被病毒感染的细胞，然后以HRP-标记的二抗对感染细胞进行染色。通过底物显色，在光学显微镜下计数感染斑点的数量，经过稀释倍数的换算，即可得出病毒的滴度（IFU/ml）。Immunoassay:1. Fix cells2. Detection with an anti-AcMNPV envelope glycoprotein (gp64) MAb3. Stain cells with A secondary HRP-conjugated antibody4. Count foci of infection under light microscopy本病毒滴度测定的关键点：（1） 细胞铺