pIRES2-EGFP-hBMP-2真核表达载体的构建及鉴定  

Construction and identification of pIRES2-EGFP-hBMP-2 plasmid

作　　者：何武兵[1] 张旭鸣[1] 许志贤[1] 林世水[1] 许玮[1] 

机构地区：[1]福建医科大学省立临床医学院福建省立医院急诊外科,福州350001

出　　处：《中华实验外科杂志》2013年第1期138-140,共3页Chinese Journal of Experimental Surgery

基　　金：福建省自然科学基金资助项目（2010J01119）;福建省医学创新课题资助项目（2009-CXB-9）;福建省卫生厅青年基金资助项目（2009-1-3）

摘　　要：目的构建带有报告基因增强绿色荧光蛋白（EGFP）的人骨形态发生蛋白-2（hBMP-2）真核表达载体。方法通过逆转录-聚合酶链反应（RT-PCR）法从人外周血淋巴细胞中扩增出骨形态发生蛋白-2（BMP-2）基因，构建BMP-2基因和报告基因EGFP真核表达载体pIRES2-EGFP-hBMP-2，经PCR、酶切、测序等方法进行鉴定，紫外分光光度计测定质粒浓度及纯度。结果真核表达载体pIRES2-EGFP-hBMP-2酶切后显示空载体和hBMP-2基因片段长度分别为5．3kb和366bp，PCR扩增hBMP-2基因片段长度366bp，紫外分光光度计测定空载体和含hBMP-2基因的质粒浓度分别为382mg／L和383mg／L，测序证实pIRES2-EGFP-hBMP-2载体序列正确。结论成功构建真核表达载体pIRES2-EGFP-hBMP-2，可为后续骨修复的基因治疗研究奠定基础。Objective To construct the pIRES2-EGFP-hBMP-2 plasmid carrying human bone morphogenetic protein-2 （BMP-2） gene. Methods BMP-2 gene was amplified using reverse transcription- polymerase chain reaction （RT-PCR） in peripheral blood lymphocytes, then the plRES2-EGFP-hBMP-2 plasmids were constructed. End nuclease cutting, PCR, and sequencing were done to identify the plasmids, and concentration and purity were detected by using ultraviolet spectrophotometer. Results After end nuclease cutting of the pIRES2-EGFP-hBMP-2 plasmids, the fragment length of empty vector and hBMP-2 gene was 5.3 kb and 366 bp respectively. Using PCR amplification, the fragment length of hBMP-2 gene was 366 bp. Using ultraviolet spectrophotometer, the concentration of plasmid carrying empty vector and hBMP-2 gene was 382 and 383 mg/L respectively. The pIRES2-EGFP-hBMP-2 plasmids carrying correct sequence were confirmed by the identification of sequencing. Conclusion We constructed pIRES2-EGFP-hBMP-2 plasmid successfully and established foundation to study its induction of new bone formation using human BMP-2 gene therapy.

关 键 词：骨形态发生蛋白-2基因 真核表达载体 

分 类 号：R[医药卫生]