短串联重复序列图谱分析法鉴定HEK293细胞传代的稳定性

摘要

Objective To identify the passage stability of HEK293 cell which used for vaccine research by short tandem repeat (STR) profiling.Methods HEK293 cell line original from American type culture collection (ATCC)(USA) was passages and set as master seed bank (P40),working seed bank (P43) and manufacturing terminal cell (P65) according the requirements of China pharmacopoeia,and applied STR profiling step by step of cell amplification,DNA extraction,PCR amplification and STR profiling by 20 sites.Results Compared of STR profiling by 20 sites to 9 sites (ATCC) and 16 sites (NICPBP),the STR 20 sites covered all important indicates of 9 sites and 16 sites.Three different passages of HEK 293 P40,P43 and P65 were matched 100％ to ATCC standard cell line marked as 293EcRShh (Adv5),and were in accord with NICPBP test results and also had no exogenous cell pollution.Conclusions The passage stability of the HEK293 cell line is stabilize,has no exogenous cell pollution,and fits to the requirements of vaccine research.%目的 用短串联重复序列(STR)图谱20位点分析法对疫苗研发用HEK293细胞的传代稳定性进行研究.方法 对购自美国菌毒种保管中心(ATCC)的HEK293细胞进行传代,按中国药典三部要求建立主种子库(第40代),工作种子库(第43代)和生产终末细胞(第65代),并进行STR检测；按检测要求进行细胞扩增,DNA提取,PCR扩增及STR20位点分析.结果 STR图谱20位点分析法已全面覆盖美国ATCC 9位点和中检院16位点法,三级传代细胞检测结果与ATCC标准株29EcRShh(Adv5)匹配度100％,与中检院检测结果完全一致,无外源细胞的污染.结论 本实验室传代建库的HEK293细胞遗传稳定,无外源细胞污染,符合疫苗研发用要求.