真核表达载体pIRES-EGFP-PDGF-B的构建及在293T细胞中的表达  

Construction of eukaryotic expression vector pIRES-EGFP-PDGF-B and its expression in 293T cells

作　　者：陈松建[1] 徐锡金[1] 霍霞[1] 张宝[1] 梁晓[1] 陈刚建[1] 

机构地区：[1]汕头大学医学院中心实验室,515041

出　　处：《实用医学杂志》2008年第22期3813-3815,共3页The Journal of Practical Medicine

基　　金：广东省科技计划项目(编号:2007B031515015)

摘　　要：目的:构建人基因血小板衍化生长因子B(PDGF-B)真核表达载体pIRES-EGFP-PDGF-B,并在293T细胞中进行瞬间表达。方法:采用PCR方法从购自美国典型培养物保藏中心含有PDGF-B基因全长cDAN序列的质粒中扩增出PDGF-B的cDAN片段,并与真核表达载体pIRES-EGFP连接双酶切鉴定,最后采用Lipofectamine 2000将重组质粒转入293T细胞中,利用荧光显微镜和Westernblot进行表达鉴定。结果:真核表达载体pIRES-EGFP-PDGF-B被成功构建,双酶切鉴定正确,载体能在293T细胞内正确地表达成熟的PDGF-B蛋白,并可分泌到细胞外。结论:成功构建了PDGF-B真核表达载体,为进一步利用PDGF-B对脊髓损伤进行基因治疗奠定基础。Objective To construct the eukaryotic expression vector pIRES-EGFP-PDGF-B and investigate its transient expression in 293T cells. Methods We amplified the full length of PDGF-B cDNA sequences from the plasmid containing PDGF-B whole length cDNA supplied by ATCC with PCR, inserted it into the eukaryotic expression vector pIRES-EGFP, and then identified it with double digestion. Finally we transfected the vector into 293T cells with Lipofectamine 2000 and verified the expression of the vector with fluorescence microscope and Western blot. Results Eukaryotic expression vector pIRES-EGFP-PDGF-B was constructed successfully and verified with double digestion. The constructed vector could express mature PDGF-B protein correctly in 293T cells and the PDGF-B was effectively secreted outside the cells. Conclusion The successful construction of the eukaryotic expression vector PDGF-B lays the foundation for further studying the gene therapy with PDGF-B for acute spinal cord injury.

关 键 词：脊髓损伤 血小板衍化生长因子B 真核表达 

分 类 号：R346[医药卫生—基础医学]