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目的:测定携带突变型Rad50基因的重组腺病毒载体Ad-Rad50-GFP在人鼻咽癌细胞株CNE1中的表达及其对CNE1的最适感染倍率(MOI)。方法:终点稀释法测定Ad-Rad50-GFP生物滴度,细胞生长曲线观察重组腺病毒载体对CNE1生长的影响,荧光显微镜下计算重组腺病毒载体的转染效率;免疫印迹实验检测以最适感染量感染后CNE1细胞内突变型Rad50蛋白的表达。结果:Ad-Rad50-GFP生物滴度为1.26×10~(11)pfu/ml,重组腺病毒载体以不高于50的感染倍率转染时对CNE1生长无明显影响,且MOI=50时转染24h后GFP表达水平最高,可检出高表达的突变型Rad50蛋白,在转染72h后仍维持约70%的转染效率。结论:重组腺病毒载体Ad-Rad50-GFP能有效转染CNE1细胞并使其表达突变型Rad50蛋白,MOI=50为Ad-Rad50-GFP对CNE1的最适感染倍率。
OBJECTIVE: To determine the expression of Ad-Rad50-GFP in human nasopharyngeal carcinoma cell line CNE1 and its optimal infection rate against CNE1 (MOI). Methods: The titer of Ad-Rad50-GFP was determined by endpoint dilution method. The cell growth curve was used to observe the effect of recombinant adenoviral vector on the growth of CNE1. The transfection efficiency of recombinant adenoviral vector was calculated by fluorescence microscopy. Expression of mutant Rad50 protein in CNE1 cells after infection. Results: The titer of Ad-Rad50-GFP was 1.26 × 10 ~ (11) pfu / ml. The recombinant adenovirus had no significant effect on the growth of CNE1 when transfected at a multiplicity of infection of no higher than 50, and at MOI = 50 GFP was the highest expression level after 24 h of staining, and the mutant Rad50 protein with high expression level was detected. The transfection efficiency was maintained at about 70% after 72 h of transfection. Conclusion: Recombinant adenovirus vector Ad-Rad50-GFP can effectively transfect CNE1 cells and express mutant Rad50 protein. MOI = 50 is the optimal infection rate of CNE1 by Ad-Rad50-GFP.