人乳铁蛋白腺病毒载体构建及细胞表达.docVIP

人乳铁蛋白腺病毒载体的构建及细胞表达 　　(燕山大学环化学院生物技术与工程系，河北 秦皇岛066004) 　　 　　摘要：将人乳铁蛋白基因与腺病毒基因组骨架质粒重组，在293细胞中包装重组腺病毒颗粒，获得真核细胞表达的载体pAd－hLTF#65377;以pcDNA3X为模板，PCR扩增hLTF基因，腺病毒穿梭载体pshuttle－cmv与hLTF重组为pshuttle－cmv－hLTF；再与腺病毒骨架质粒pAdEasy－1同源重组为pAd－hLTF质粒；脂质体法将重组pAd－hLTF质粒转染HEK 293细胞，包装成重组腺病毒颗粒#65377;Western blot和ELISA法测定细胞中hLTF基因的表达#65377;结果表明，pAd－hLTF质粒转染293细胞后，7～10 d后，获得细胞裂解液，将细胞裂解产物再次接种新鲜的293细胞，3～5 d后，80%以上的细胞变圆#65380;膨胀#65380;漂浮#65377;Western blot和ELISA结果显示，上清液中有hLTF基因的表达，为重组人乳铁蛋白的体外表达及其功能分析奠定了基础#65377; 　　关键词：重组人乳铁蛋白；腺病毒载体；重组蛋白质 　　中图分类号：Q513+．2；S852．65+9．1；Q782文献标识码：A文章编号：0439－8114(2008)06－0629－04 　　 　　Construction of Human Lactoferrin Gene Recombinant Adenoviruses and Expression in Eukaryotic Cells 　　 　　HAN Zeng－sheng，LI Jian，GAO Da－wei，LI Qing－wang 　　(Department of Biological Engineering，College of Environment Chemical Engineering，Yanshan University，Qinhuangdao 066004，Hebei，China) 　　 　　Abstract： To construct the recombinant adenoviral vectors containing human lactoferrin gene， the recombinant adenoviruseswas obtained and expressed the recombinant protein in eukaryotic cells． The human lactoferrin gene was amplified from plasmid pcDNA3X； the gene of interest which contained hLTF gene was cloned into the pshuttle－cmv vector． The resulted vector was named pshuttle－cmv－hltf that was linearized by digesting with restriction endonuclease PmeI， and subsequently cotransformed into Escherichia coli BJ5183 cells with an adenoviral backbone vector pAdEasy－1； Homologous recombinants were performed in bacterial cells． Finally， the linearized backbone adenoviral vector was transfected into the packaging cells lines HEK 293 cells by lipofectamine 2000 transfection reagents． The expression of human lactoferrin in cells was investigated by Western blot and ELISA methods． After the transfection， seven to ten days later， the cells lyses were collected and added into the separate 293 cells． The cells become roundness， swell and floats． Western blot and ELISA results showed the high expressed