云南农业大学学报2011，Vol.26Issue(3)：328-333,6.DOI:10.3969/j.issn.1004-390X(n).2011.03.008
血管内皮生长因子(VEGF)实时荧光定量PCR质粒标准品的构建
Construction of Standard Plasmid for Detecting the Vascular Endothelial Growth Factor Gene (VEGF) by Using Real-time Fluorescence Quantitative PCR
摘要
Abstract
The purpose of this study to build a standard plasmid of the VEGF gene whose function was closely associated with many clinic diseases.This plasmid can be used to develop a method of fast, sensitive and reliable detection system.In this study, Banna mini-inbred pigs (Sus scrofa) , age of 4 to 6 months, were used to construct an animal model.The total RNA from skin wounds was extracted and amplified through RT-PCR and specific primers designated in this study.The purified product ofPCR was legated with a pMD18-T vector and transferred into the bacterium DH5α for replication.The recombinant plasmid picked out from positive clones was amplified by PCR, digested with EcoR I and Hind III, and then sequenced.This process was used to calculate the standard concentration of recombinant plasmids from real-time quantitative PCR.The sensitivity of this method for creating VEGF by expressing mRNA gene through PCR was good.Specifically, the fewest number of copies was with a range of 1.0 x 103 ～ 1.0 x 108 copies.A clear linear relationship was found between the threshold cycle number (Ct) and the PCR system (r2 =0.982) , and amplification efficiency was determined to be 96.92％.The plasmid standard from mini inbred pigs obtained in the present study can serve as a molecular foundation for the study of the VEGF and its role in many clinical diseases.关键词
实时荧光定量/VEGF基因/质粒标准品Key words
real-time fluorescence quantitative/ VEGF/ plasmid standard分类
畜牧业引用本文复制引用
刘丽仙,霍金龙,李颖,赵跃,霍海龙,曾养志..血管内皮生长因子(VEGF)实时荧光定量PCR质粒标准品的构建[J].云南农业大学学报,2011,26(3):328-333,6.基金项目
云南省应用基础研究计划面上项目(2010ZC241) （2010ZC241）
云南农业职业技术学院科学研究与技术开发项目 （）
昆明医学院研究生创新基金项目(2009S03). （2009S03）
