荧光定量PCR法检测重组毕赤酵母工程菌的外源基因拷贝数  

Detection of Restructuring of Transgenic Copy Number of Pichia Engineering Bacteria with Fluorescence Quantitative PCR Method

作　　者：郭晋霞[1] 何云凤[1] 范开 

机构地区：[1]重庆理工大学药学与生物工程学院,重庆400054 [2]重庆富进生物医药有限公司,重庆400050

出　　处：《重庆理工大学学报（自然科学）》2016年第2期77-83,共7页Journal of Chongqing University of Technology：Natural Science

摘　　要：提出了重组毕赤酵母工程菌p PICZ-DT30/GS115基因组中外源基因DT30拷贝数的实时荧光定量PCR方法。该重组工程菌用来表达人胰岛素前体(PI-DT30),其表达量的高低、菌种稳定性对工艺研究及整体项目成本等有重大影响。外源基因拷贝数是检测表达量的一个重要指标。该方法中,利用毕赤酵母的看家基因GAP(Glyceraldehyde-3-phosphate dehydrogenase)为内参,分别建立含GAP基因和DT30基因的双标准曲线。将工程菌基因组进行荧光定量PCR,根据标准曲线和Ct值计算目的基因DT30在重组毕赤酵母工程菌中的拷贝数,结果为7个。A method for the reconstruction of the copy number of foreign gene DT30 in genome of the recombinant engineered yeast p PICZ-DT30 / GS115 by real-time fluorescent quantitative PCR was developed. The yeast was used to express human insulin precursor（ PI-DT30）,and its expression andstability have largely impact on both the process research and the overall cost. The copy number of foreign gene is always an important indicator in detecting expression quantity. In this study,GAP gene,the housekeeping gene in Pichia pastoris,was used as a reference,and the double standard curves of GAP gene and DT30 gene were generated respectively. Then the genome of p PICZ-DT30 /GS115 was analyzed by real-time fluorescent quantitative PCR,and the copy number of DT30 was calculated with the standard curves and Ct value. The results have seven.

关 键 词：荧光定量PCR 表达 胰岛素前体 基因拷贝数 

分 类 号：Q39[生物学—遗传学]