| 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 |
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| 引用本文： | 喻晓亮,谢达平,胡雄贵,邓缘,燕海嶂. 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[J]. 生物磁学, 2009, 0(13): 2460-2464 |
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| 作者姓名： | 喻晓亮 谢达平 胡雄贵 邓缘 燕海嶂 |
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| 作者单位： | [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 |
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| 基金项目： | 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） |
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| 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。
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| 关 键 词： | 卵清蛋白 促红细胞生成素 共表达 生物反应器 |
Construction of Oviduct Specifical Expression Vector to Co-express Human Erythropoietin Gene and GFP |
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| Affiliation: | YU Xiao-liang, XIE Da-pin, HU Xiong-gu, DENG Yuan, YAN Hai-feng (1 College of Biology Science and Technology, Hunch Agricultural University, Changsha, 410128, China; 2 Hunch Institute of Animal and Veterinary Science, Changsha, 410131, China) |
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| Abstract: | 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. |
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| Keywords: | Ovalbumin Erythropoietin Co-express Bioreactor |
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