基于TCID_(50)检测AAV9载体制品感染性滴度的方法  

Research on AAV9 Infectious Titer Detection Method Based on TCID

作　　者：王聪[1,2] 李秀 牛苗 戴阳光 董哲岳 董小岩 余双庆 杨怡姝 WANG Cong;LI Xiu;NIU Miao;DAI Yang-guang;DONG Zhe-yue;DONG Xiao-yan;YU Shuang-qing;YANG Yi-shu(Beijing University of Technology,Beijing 100124,China;Beijing FivePlus Gene Technology Co.,Ltd.,Beijing 102629,China)

机构地区：[1]北京工业大学,北京100124 [2]北京五加和基因科技有限公司,北京102629

出　　处：《中国生物工程杂志》2021年第10期28-32,共5页China Biotechnology

摘　　要：目的:建立一种基于半数组织培养感染剂量(median tissue culture infective dose,TCID50)检测9型腺相关病毒(adeno-associated virus type 9,AAV9)载体制品感染性滴度的方法。方法:利用含AAV2 rep和cap基因的1型单纯疱疹病毒(herpes simplex virus type1,HSV1)做为辅助病毒与梯度稀释的AAV9载体制品共同感染HEK-293细胞,培养48 h后用实时荧光定量PCR(quantitative real-time PCR,q PCR)扩增AAV特异性反向末端重复序列(inverted terminal repeats,ITR),根据阳性及阴性感染孔数,利用Krber法计算样品的TCID50。结果:采用携带增强绿色荧光蛋白报告基因的AAV9载体制品确定辅助病毒HSV1-rc最佳感染复数(multiplicity of infection,MOI)为5,AAV9-101的感染性滴度为1.6×109TCID50/m L。结论:对AAV9载体制品进行感染性滴度检测,且具有可重复性。Objective:To establish a method for detecting the infectious titers of adeno-associated virus type9(AAV9)vector products based on the median tissue culture infective dose(TCID50).Methods:HEK-293 cells were co-infected with HSV1 containing rep and cap genes of AAV2 and gradient diluted AAV9 vector products.After 48 hours of culture,the AAV-specific inverted terminal repeats(ITR)were amplified by quantitative real-time PCR(qPCR),and the infectivity titer of the sample was calculated by the Krber method based on the number of positive and negative infected wells.Results:The AAV9 vector product carrying the enhanced green fluorescent protein reporter gene was used to determine the optimal multiplicity of infection(MOI)of the helper virus HSV1-rc as 5.The infectious titer of AAV9-101 is 1.6×109 TCID;/m L.Conclusion:This method can detect the infectious titer of AAV9 vector products with repeatability.

关 键 词：9型腺相关病毒载体 感染性滴度 半数组织培养感染剂量 

分 类 号：Q819[生物学—生物工程]