人促红细胞生成素基因和GFP共表达的输卵管特异性表达载体的构建  

Construction of Oviduct Specifical Expression Vector to Co-express Human Erythropoietin Gene and GFP

作　　者：喻小亮 胡雄贵[2] 谢达平[1] P.Trefil 邓缘[2] 燕海峰[2] 

机构地区：[1]湖南农业大学生物科学技术学院,湖南长沙410128 [2]湖南省畜牧兽医研究所,湖南长沙410131 [3]捷克生物医药研究所

出　　处：《现代生物医学进展》2009年第13期2460-2464,共5页Progress in Modern Biomedicine

基　　金：科技部中捷政府间科技合作项目(中方37-10);湖南省科技攻关项目(06WK3001);长沙市留学人员创业资助基金(k068017)

摘　　要：本实验从蛋鸡输卵管基因组DNA中扩增出约1.3kb的卵清蛋白5＇端调控区（OV）,并从质粒扩增出人促红细胞生成素（hEPO）基因组DNA。将hEPO亚克隆入pEGFP-C1载体的多克隆位点,命名为pEGFP-hEPO,然后将OV片段亚克隆入经Ase I和Vsp I双酶切的切口处,替换掉CMV启动子,经测序和酶切鉴定正确,成功构建了鸡卵清蛋白5＇端调控区调控人促红细胞生成素的共表达载体pOV-GFP-hEPO。并利用脂质体转染法转染鸡输卵管上皮细胞,用荧光倒置显微镜观测绿色荧光蛋白的表达,结果显示共表达载体pOV-GFP-hEPO能够在鸡输卵管上皮细胞定位表达。为制备生产人促红细胞生成素的鸡输卵管生物反应器奠定了基础。In this paper, chicken ovalbumin 5＇-flanking regulatory region （OV） was amplified from the genome of chicken oviduct tissue and human erythropoietin （hEPO） was amplified from the plasmid phEBS-HB. The hEPO was inserted into the MCS of pEGFP-CI （named pEGFP-hEPO）. Then the chicken ovalbumin 5＇- flanking regulatory region was inserted into pEGFP-hEPO which was digested by Ase I and Vsp I （named pOV-GFP-hEPO） and replaced the CMV promoter. The plasmid was confirmed by sequenced and restriction endo-nuclease cutting. Then, the recombination construct with GFP was transfected into the primary chicken oviduct epithelial cells by Lipofectin and detected by green fluorescence. It was demonstrated that the pOV-GFP-hEPO vector can express in the special location of chicken oviduct epithelial cells. It can be used for constructing chicken ovalbumin bioreactor.

关 键 词：卵清蛋白 促红细胞生成素 共表达 生物反应器 

分 类 号：R394.1[医药卫生—医学遗传学] R75[医药卫生—基础医学]