重组腺相关病毒中游离与错误包装宿主细胞DNA检测的两种前处理方法的比较
Comparison of two pretreatment methods for detection of free and mispackaged host cell DNA in recombinant adeno-associated virus
王光裕 1胡倩 2史新昌 1闫书美 2周勇 1刘宾3
作者信息
- 1. 中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京 100050
- 2. 上海泰昶生物技术有限公司,上海 201413
- 3. 上海天泽云泰生物医药有限公司,上海 201203
- 折叠
摘要
目的 采用DNA酶(deoxyribonuclease,DNase)处理与填料亲和两种前处理方法检测重组腺相关病毒(recombi-nant adeno-associated virus,rAAV)游离与错误包装宿主细胞DNA(host cell DNA,HCDNA)残留量,并进行比较.方法 分别采用DNase处理法和填料亲和法分离游离与错误包装HCDNA,再进行核酸提取及qPCR检测.比较两种前处理方法检测HCDNA残留量的准确度和重复性.结果 采用DNase处理的方式,DNase未处理组核酸定量检测结果为HCDNA总残留量,DNase处理组为错误包装HCDNA量,二者差值为游离HCDNA残留量.DNase未处理和处理组回收率均为75％以上,重复性RSD小于30％.采用亲和提取方式,填料偶联亲和配基,结合rAAV,检测错误包装HCDNA回收率为75％以上,检测游离HCDNA回收率仅为36％.结论 DNase处理法可有效检出游离与错误包装HCDNA,可为后续研究奠定基础.
Abstract
Objective To compare the application of two pretreatment methods,deoxyribonuclease(DNase)treatment and ligand affinity,in detecting the residual amount of free and mispackaged host cell DNA(HCDNA)in recombinant adeno-associated virus(rAAV).Methods Free and mispackaged HCDNA were isolated by DNase treatment and ligand affinity respectively,and then the nucleic acid was extracted and detected by qPCR.The accuracy and reproducibility of two pretreatment methods for detecting HCDNA residues were compared.Results Using DNase treatment,the result of nucleic acid quantitative detection in non-DNase-treated group was the total residual amount of HCDNA,that in DNase-treated group was the amount of mispackaged HCDNA,and the difference between them was the residual amount of free HCDNA.The recovery rates of both the untreated and treated groups were more than 75％,and the RSD of reproducibility was less than 30％.Using affinity extraction method,with the affinity ligand combined with rAAV,the result of recovery rate of mispackaged HCDNA was over 75％,and that of free HCDNA was only 36％.Conclusion DNase treatment method can effectively detect free and mispackaged HCDNA,laying a foundation for further research.
关键词
腺相关病毒/DNA酶/亲和填料/PCR/宿主细胞DNA/游离/错误包装Key words
Adeno-associated virus(AAV)/Deoxyribonuclease(DNase)/Affinity ligand/PCR/Host cell DNA(HCDNA)/Free/Mispackaged引用本文复制引用
基金项目
国家重点研发计划(2023YFC3403305)
国家药品质量标准提高课题(2022S07)
中央级公益性科研院所基本科研业务费专项中国医科院项目(2023-PT350-01)
出版年
2024
NETL
NSTL
万方数据