羊BLG基因5′和3′调控区克隆及其调控GFP基因在乳腺细胞的表达

and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BLG 5′ and 3′ flanking were the same as that of publication completely.The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2kb 5′ flanking with 2.1kb 3′ flanking.In order to assess the efficiency of BLG regulatory elements,green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47).Through observation under UV microscope and detection by fluorometer,it is demonstrated that the GFP has been successfully expressed in TD47 cell line.By virtue of direct observation and quantitative analysis,the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.

人胰岛素原乳腺表达基因构建及其在转基因小鼠乳汁中的表达

从绵羊和人的基因组中用PCR扩增分别克隆了羊乳球蛋白 (BLG) 5’和 3’调控区及人胰岛素原 (hINS)基因组DNA ,并以此为基础构建了人胰岛素原乳腺表达载体 ( pSh -BLG -hINS)。该表达载体包括 4.2kbBLG 5’调控区和 2 .1kbBLG 3’调控区 ,以及 1 .2kb人胰岛素原基因组DNA。将绿色荧光蛋白基因与BLG 5’和 3’调控区融合后转化乳腺细胞系 (TD47) ,通过荧光显微镜观察和紫外吸收检测 ,证明GFP在TD47乳腺细胞系中获得了表达。用BLG -hINS基因分别注射小鼠 ,通过PCR和限制性内切酶检测分析 ,共获得 5只转基因小鼠。经放免 (RIA)检测 ,两只转基因母鼠乳汁中人胰岛素原的表达量分别为 37.44和 39.99mg/L。该结果表明 ,本文所构建的 pBLG乳腺表达结构 。