分泌型核心蛋白聚糖真核表达载体的构建及在HepG2细胞中的表达  

Construction of recombinant eukaryotic expressing vector pcDNA3.1-decorin and expression of decorin in hepatoma HepG2 cells

作　　者：王雅丽[1] 张玉成[1] 杜珍武[1] 张桂珍[1] 

机构地区：[1]吉林大学中日联谊医院中心实验室,长春130033

出　　处：《中国体视学与图像分析》2008年第3期180-184,共5页Chinese Journal of Stereology and Image Analysis

摘　　要：目的构建分泌型核心蛋白聚糖(decorin)真核表达载体,并在肝癌细胞HepG2中表达,为研究核心蛋白聚糖的抗肿瘤作用奠定基础。方法利用特异性引物,应用PCR技术扩增核心蛋白聚糖全长基因cDNA片段,与pcDNA3.1载体进行连接,并转化到大肠杆菌JM109中扩增以获得重组载体,应用双酶切、PCR以及测序鉴定此重组载体;脂质体介导重组载体转染HepG2,经G418筛选建立稳定转染细胞株,分别采用RT-PCR、免疫组化法和westernblot检测其表达。结果获得了约1080bp大小的特异性DNA片段;PCR产物与真核表达载体进行连接,经过双酶切、PCR以及测序鉴定证实分泌型核心蛋白聚糖cDNA片段正确插入真核表达载体pcDNA3.1中。RT-PCR方法可见转染组mRNA表达明显增多,免疫组化和westernblot方法可见转染组细胞decorin蛋白表达明显增高。结论成功构建了真核表达载体pcDNA3.1-decorin,建立了稳定转染decorin的HepG2细胞株。Objective To construct a recombinant eukaryotic expressing vector pcDNA3.1-decorin and make it expressing in human hepatoma HepG2 cells, so as to investigate the effect of recombinant secreting decorin on resistant tumor cells. Methods The full length of decorin cDNA was amplified using decorin gene specific primer and polymerase chain reaction(PCR), The recombinant plasmid-decorin was constructed and transfected into the bacteria JM109.The recombinant eukaryotic expressing vector pcDNA3.1-decorin was ident...

关 键 词：核心蛋白聚糖 聚合酶链反应 HEPG2 

分 类 号：Q78[生物学—分子生物学]