荧光定量PCR检测重组新蛭素中毕赤酵母基因组DNA的残留量  

Determination of Residual Pichia Yeast Genomic DNA in Recombinant Neorudin by Real-Time PCR

作　　者：刘晶晶[1,2] 郭莹莹[1,2] 李艳琪[2,3] 王文文[2,3] 吴祖泽[2] 靳继德[2] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100022 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]天津大学化工学院,天津300072

出　　处：《生物技术通讯》2014年第3期401-405,共5页Letters in Biotechnology

基　　金：国家新药创制科技重大专项(2012ZX09102301-008)

摘　　要：目的：建立real—timePCR定量检测毕赤酵母基因组DNA残留量的方法，提高重组新蛭素的产品安全性。方法：选择拷贝数高且分布广泛的毕赤酵母5SrRNA基因为靶标基因设计扩增引物，提取酵母基因组DNA，稀释后作为扩增模板。以罗氏荧光定量PCR_Light Cycler 480平台为基础，建立基于SYBR Green Ⅰ荧光染料的real—timePCR的检测方法，并考察用该方法检测重组新蛭素中毕赤酵母基因组残留量的灵敏度、精密度和回收率。结果：该法检测宿主DNA残留量灵敏度高，DNA浓度为0．1～1000pg／μL范围内呈现良好的线性关系，其标准曲线的误差值小于0．2；用该法对5批注射用重组新蛭素（酵母）产品中宿主基因组DNA残留量进行了测定，结果分别为0．03、2.3、0.2、0．6、0．2μg／mg。结论：该方法具有操作简便、灵敏度高等优点，可用于重组产品中酵母基因组残留DNA的定量测定。Objective： To develop a practical method for determination of residual Pichia yeast genomic DNA in recombinant neorudin by real-time PCR. Methods： Specific primers of Pichia yeast 5S rRNA gene with high copy were designed and demonic DNA was extracted. SYBR Green real-time PCR method to detect residual Pichia yeast genomic DNA was established based on Roche PCR_LightCycler480 system. Moreover, sensitivity, precision and recovery of the above method were further determined. Results： The developed real-time PCR holds a high sensitivity and has a good linearity in the DNA concentration range of 0.1-1000 pg/μL since its standard error value was less than 0.2. Application of this method, five batches products of recombinant neorudin for injection were tested and the results showed that the residual DNA of Pichia yeast was 0.03, 2.3, 0.2, 0.6 and 0.2 pg/ mg respectively. Conclusion： This real-time PCR method is convenient and sensitive, and can be used in quantitative detection for residual Pichia yeast genome DNA in recombinant proteins.

关 键 词：荧光定量PCR 毕赤酵母 DNA残留 重组新蛭素 

分 类 号：Q78[生物学—分子生物学]