pIRES2-EGFP-hTGF-β3真核表达载体的构建及鉴定  

Construction and identification of pIRES2-EGFP-hTGF-β3 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》2014年第10期2278-2280,共3页Chinese Journal of Experimental Surgery

基　　金：国家临床重点专科建设项目,福建省自然科学基金资助项目（2010J01119）

摘　　要：目的 构建带有报告基因增强绿色荧光蛋白（EGFP）的人转化生长因子β3（hTGF-β3）真核表达载体.方法 通过逆转录-聚合酶链反应（RT-PCR）方法从人外周血淋巴细胞中扩增出hTGF-β3基因,构建TGF-β3基因和报告基因EGFP基因真核表达载体pIRES2-EGFP-hTGF-β3,经PCR、酶切、测序等方法进行鉴定,紫外分光光度计法测定质粒浓度及纯度.结果 真核表达载体pIRES2-EGFP-hTGF-β3酶切后显示空载体和hTGF-β3基因片段长度分别为5 300 bp和1 251 bp,PCR扩增hTGF-β3基因片段长度为1 251 bp,紫外分光光度计法测定空载体和含hTGF-β3基因的质粒浓度分别为382 mg/L和411 mg/L,测序证实pIRES2-EGFP-hTGF-β3载体序列正确.结论 成功构建真核表达载体pIRES2-EGFP-hTGF-β3,可为后续的骨修复基因治疗的研究奠定基础.Objective To construct the pIRES2-EGFP-hTGF-β3 plasmid carrying human transforming growth factor-β3 （hTGF-β3） gene.Methods TGF-β3 gene was amplified from peripheral blood lymphocytes using reverse transcription-polymerase chain reaction （RT-PCR）,then the pIRES2-EGFP-hTGF-β3 plasmids were constructed,and identified by end nuclease cutting,PCR,and sequencing.The concentration and purity were detected by ultraviolet spectrophotometer.Results After end nuclease cutting of the pIRES2-EGFP-hTGF-β3 plasmids,the fragment lengths of empty vector and hTGF-β3 gene were 5 300 bp and 1 251 bp respectively.Using PCR amplification,the fragment length of hTGF-β3 gene was 1 251 bp.Using UV spectrophotometer,the concentration of plasmid carrying empty vector and hTGF-β3 gene was 382 mg/L and 411 mg/L respectively.The pIRES2-EGFP-hTGF-β3 plasmids carrying correct sequence were confirmed by the identification of sequencing.Conclusion We constructed pIRES2-EGFP-hTGF-β3 plasmid successfully and established foundation to study its induction of new bone formation using human TGF-β3 gene therapy.

关 键 词：转化生长因子-Β3 真核表达载体 

分 类 号：R346[医药卫生—基础医学]