DOP-PCR法检测单抗残余DNA的探讨  

Discussion of Detecting Residual DNA of Monoclonal Antibody by DOP-PCR

作　　者：李萌[1] 武刚[1] 于传飞[1] 王兰[1] 

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定与标准化重点实验室,北京100050

出　　处：《现代科学仪器》2016年第5期53-58,共6页Modern Scientific Instruments

基　　金：中国食品药品检定研究院中青年基金资助,课题编号：201482;国家“重大新药创制”科技重大专项资助项目（2014ZX09304311-001）

摘　　要：目的：利用简并引物（Degenerate Oligonucleotide-Primed，DOP）PCR法，建立一种物种非特异性的宿主细胞残余DNA检测方法。方法：设计简并引物，优化实时定量PCR的条件，从而建立DOP--PCR法，检测5种重组单抗药物宿主细胞表达系统（CHO细胞、SP2／0细胞和NS0细胞）的残余DNA。结果：简并引物选择51Tag—N6-ATGTGG 3’，定量PCR条件优化为：低严谨性扩增的退火温度由；32℃以3％的斜率升至72℃，严谨性扩增后的荧光检测条件优化为77℃，30s。3个物种的DNA扩增曲线R2均能达到0．99以上，回收率达到80％～132％，3次测定的ESD均小于25％。同时DOP-PCR与CHO细胞特异性的定量PCR进行比较，二者回收率的均值均在80％～130％之间，3次测定的RSD均小于20％。结论：初步建立了物种非特异性的宿主细胞残余DNA的DOP-PCR检测方法，为单抗药物宿主细胞残余DNA检测提供新思路。Objective： A kind of detective method for host cells residual DNA of species non-specificity is established by using DOP-PCR. Methods： DOP-primers and conditions of Q-PCR were optimized, so DOP-PCR was established, in order to detective residual DNA of three kinds of host cells （CHO, SP2/0, NS0）. Result： the optimized DOP-primer was 5＇Tag-N6-ATGTGG 3＇. the optimized Q-PCR condition was that the anneal temperature of low stringency cycle rise from 32 ℃ to 72% by the slope of 3%, and that fluorescence detective conditions of high stringency.cycle was 77 ℃ , 30s. the values of RE of amplification curves of three cells were above 0.99. the recoveries of them were between 80% and 132%, whose RSDs were below 25%. the recoveries of DOP-PCR and general Q-PCR were between 80% and 130%, whose RSDs were below 20%.Conclusion： A kind of detective method for host cells residual DNA of species non-specificity is established by using DOP-PCR, it provides a new idea of detecting host cell residual DNA of monoclonal antibodies.

关 键 词：简并引物 实时定量PCR 残余DNA 单抗药物 

分 类 号：Q789[生物学—分子生物学]