目的 采用SYBR Green Ⅰ染料建立实时定量PCR法检测CHO细胞外源抗体轻链(light chain,LC)、重链(heavy chain,HC)基因拷贝数,并进行方法的验证及初步应用.方法 以CHO细胞中稳定表达的B2m(β2-microglobulin)基因作为内参基因,分别设计适宜的LC和HC基因引物及内参基因引物,确定实时定量PCR方法的反应体系和反应程序.对建立的方法进行特异性、线性、精密性及耐用性验证,并采用建立的方法检测重组细胞株工作细胞库(WCB)及不同传代次数细胞中LC和HC基因拷贝数.结果 外源基因与内参基因引物可特异性结合目标片段;B2m、LC、HC基因引物扩增效率分别为106.7％、106.3％和99.1％,线性方程相关系数均大于0.99,具有良好的线性关系;精密性验证的相对标准偏差(relative standard deviation,RSD)均小于1％;短时间内少次冻融对检测结果影响较小.利用建立的方法检测不同代次重组细胞株LC和HC基因拷贝数,未见明显变化.结论 成功建立了 CHO细胞外源基因拷贝数实时定量PCR检测方法,该方法特异性、线性、精密性及耐用性良好,为其他CHO细胞株表达的外源基因拷贝数检测提供了参考.
Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells
Objective To establish a real-time quantitative PCR method using SYBR Green Ⅰ to detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined.The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106.7％,106.3％and 99.1％,respectively,and the correlation coefficients of the linear equations were all greater than 0.99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1％;Few cycles of freeze-thaw in a short period had little effect on the detection results.The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines.
Real-time quantitative PCRCHO cellsAntibodyLight chain(LC)Heavy chain(HC)Gene copy number
万方数据
维普
