人白介素-3乳腺表达载体的构建

人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.

Construction of the Vector for Expressing Human IL-3 Gene Specifically in Bovine Mammary Gland

Interleukin-3(IL-3) is a regulator in haematopoietic system and immune system.It plays important roles in therapy of haematopoietic systemic diseases,tumor,and immune function deficiency.The purpose of present study was to construct a vector for expressing the human IL-3 gene specifically in bovine mammary gland.The plasmid pDsRed2-1 containing the reporter gene of red fluorescent protein and neomycin resistant gene was used as DNA backbone to construct the expression vector.Bovine beta-casein promoter,human interleukin-3 gene and CMV promoter sequences were obtained by PCR amplification,respectively.Then these PCR fragments were inserted into the Multiple Cloning Site of the pDsRed2-1 plasmid.The bovine beta-casein promoter was located at the upstream of the human IL-3 gene for driving the gene expression specifically in bovine mammary gland tissue,while as the CMV promoter was located at the upstream of the red fluorescent protein gene for driving the gene expression in all mammalian cells.The recombinant plasmid was identified by restriction fragment analysis and partial DNA sequencing.The results show that the structure of the final constructed vector accords with the designed plasmid map.