人乳铁蛋白cDNA基因乳腺表达载体的构建与鉴定  

Construction and identification of mammary expressional vector for cDNA of human lactoferrin

作　　者：孟立[1] 张艳丽[1] 许欣[1] 王子玉[1] 闫益波[1] 庞训胜[1] 钟部帅[1] 黄荣[1] 宋洋[1] 王金玉[2] 王锋[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,南京210095 [2]扬州大学动物科技学院,扬州225009

出　　处：《生物工程学报》2011年第2期253-261,共9页Chinese Journal of Biotechnology

基　　金：国家"转基因生物新品种培育"重大专项(No.2008ZX08008-004);江苏省动物繁育与分子设计实验室开放课题(No.YDKT0801)资助~~

摘　　要：为了构建人乳铁蛋白基因（hLF）的乳腺表达载体并验证其在乳腺细胞中的表达情况,本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2作为5′端调控序列,下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9及3′部分基因组片段作为3′端调控序列,长度分别为6.2 kb和7.1 kb,将hLF基因（目的基因）和Neo基因（筛选标记）分别插入到5′端调控序列和3′端调控序列的下游,构建成pBC1-hLF-Neo载体,其全长为25.348 kb。为了检测该载体的生物学功能,用脂质体介导法将其分别导入到山羊乳腺上皮细胞GMC和小鼠乳腺癌细胞株C127中进行表达验证,经G418抗性筛选8~10 d,得到了药物抗性细胞克隆,经催乳素、胰岛素及氢化可的松诱导培养,通过RT-PCR、Western blotting以及重组hLF抑菌圈试验表明,山羊β-casein基因启动子驱动的hLF基因能够在C127和GMC乳腺上皮细胞中转录翻译,且重组hLF具有抑制大肠杆菌生长的生物活性,这为下一步建立稳定整合hLF基因奶山羊胎儿成纤维细胞系奠定了基础。The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin（hLF） gene and then investigate its expression in the mammary gland epithelium cells.The constructed vector contained the 6.2 kb long 5′ flank regulation region including promoter,other elements and the 7.1 kb long 3′ flank regulation region including transcriptional ending signal of a goat＇s β-casein gene.A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing.The results revealed that the structure of the final constructed vector accords with the designed plasmid map.In order to analyze the bioactivity of the vector,we transfected the lined vector DNA into the dairy goat′s mammary gland epithelium cells and C127 cells of a mouse′s mammary epithelium by Lipofectamine.After selection with G418 for 8？10 days,G418-risistant clones were obtained.PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones.After proliferation culture,the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin,insulin and hydrocortisone-a medium capable of inducing recombinant hLF expression.RT-PCR,Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E.coli proliferation inhibition.Paramount to everything,this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.

关 键 词：HLF pBC1-hLF-Neo GMC细胞 C127细胞 生物学功能 

分 类 号：Q78[生物学—分子生物学]