【摘要】 目的:对用于检测重组技术产品中残余DNA的荧光法及DNA杂交法进行比较性评价。方法:应用地高辛标记的DNA杂交法以及PicoGreen荧光法对3批重组人干扰素α2b中外源DNA残留量进行测定。结果:荧光法检测结果显示3批重组人干扰素α2b制品中DNA残留量分别为每人份剂量(0.695±0.021)ng、(0.664±0.021)ng、(0.916±0.017)ng,而用地高辛标记的DNA杂交实验由于阳性基因组DNA灵敏度偏低以至于结果无法判断。应用琼脂糖凝胶电泳和PicoGreen荧光法测得由生产单位提供的阳性DNA浓度明显偏低,与标示量不符,这说明阳性DNA含量偏低是本次DNA杂交实验失败的主要原因。通过对该阳性DNA重新定量后再进行DNA杂交检测,结果显示3批重组人干扰素α2b制品的DNA残留量均小于每人份剂量1ng,该结果与PicoGreen荧光法相一致。结论:荧光法具有简便、快速,自动化程度高,不受工程菌种类限制,能够精确定量等特点,优于传统的DNA杂交法,更适于重组制品残余外源DNA含量的常规检定。更多还原

【Abstract】 Objective:Evaluation the fluorescence method and DNA hybridization method to determine DNA content for the quality control of residual DNA in recombinant products.Method:To apply Digoxin-labeled DNA hybridization and PicoGreen fluorescence methods to determine residual DNA in three batches of recombinant human interferon α2b.Results:The fluorescence results revealed residual DNA of three batches of recombinant human interferon α2b was(0.695±0.021) ng per dose,(0.664±0.021) ng per dose and(0.916±0.017) ng per dose,respectively.But because of the positive genome DNA,the sensitivity of Digoxin-labeled DNA hybridization was too low to determine the result.DNA electrophoresis and PicoGreen fluorescence method indicated that the concentration of positive genome DNA provided by the manufacturer was obviously low,which was the main reason for the failure of DNA hybridization.When the positive DNA was quantitated,DNA hybridization revealed that the residual DNA of these three batches of recombinant human interferon α2b was less than 1ng per dose.These results were in accord with the PicoGreen fluorescence results.Conclusion:The fluorescence method is convenient,fast,automatic,non-specific and quantitative.It is better than the traditional Digoxin-labeled DNA hybridization and more suitable for routine test for the content of residual extraneous DNA of recombinant biologics.更多还原