TaqMan荧光定量PCR法检测CHO细胞中外源基因拷贝数的方法建立

Establishment of TaqMan fluorescence quantitative PCR method to detect the copy numbers of exogenous gene in CHO cells

目的以β2-微球蛋白(B2M)为内参基因,建立一种检测CHO细胞中外源基因H拷贝数的TaqMan荧光定量PCR方法。方法使用含有目的基因H和内参基因B2M的质粒作为标准品,分别建立目的基因H和内参基因B2M的标准曲线。提取重组CHO细胞基因组DNA,并对所提基因组中目的基因H和内参基因B2M的拷贝数同时进行qPCR检测。结果成功建立了目的基因H和内参基因B2M的标准曲线,扩增效率分别为92.09%和94.96%。标准曲线的相关系数均在0.99以上,且具有良好的重复性。随着细胞传代的增加,目的基因的拷贝数相对稳定。结论成功建立了TaqMan荧光定量PCR法检测CHO细胞中外源基因拷贝数的方法,该方法可用于外源基因在CHO细胞中的遗传稳定性研究。

Objective Usingβ2-Microglobulin(B2M)gene as reference gene,to establish a TaqMan fluorescence quantitative PCR method for detecting the copy numbers of exogenous gene H in CHO cells.Methods Plasmids containing target gene H and reference gene B2M were used as standard,the standard curves of target gene H and reference gene B2M were generated,respectively.The genomic DNA of recombinant CHO cells was extracted,the copy numbers of target gene H and reference gene B2M in the extracted genomic DNA were detected by qPCR,simultaneously.Results The standard curves of target gene H and reference gene B2M were successfully established,the amplification efficiencies were 92.09%and 94.96%,respectively.The correlation coefficients of standard curves were above 0.99,and both standard curves showed good reproducibility.The copy numbers of target gene were relatively stable,with the increase of cell passage.Conclusion A TaqMan fluorescence quantitative PCR method to detect the copy numbers of exogenous gene in CHO cells was successfully developed,this method can be used to study the genetic stability of exogenous gene in recombinant CHO cells.