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 ,能够调控外源基因在乳腺表达

Cloning of 5' and 3' Flanking Sequence of Ovine BLG and Regulating the Expression of human proinsulin in Mammary Cell Line and fransgenic mice

and 3' flanking of Sheep BLG and whole sequence of human proinsulin were cloned from sheep and human genomic DNA respectively. The recombinant structure used to direct exogenous gene to express especially in mammary gland was then constructed by joining 4.2kb of 5'flanking with 2.1kb of 3'flanking of BLG. As reporter gene, GFP was fused with pBLG construct and expressed in TD47 mammary cell line by transfection. The results of observation under UV-microscope and the data collected by fluorometer demonstrated that the GFP was expressed validly in TD47. Following the same protocol, human proinsulin gene was digested from pGEM-INS and consequentially inserted into pBLG constructs. By microinjection, 5 transgenic mice were generated and two of the females expressed human insulin in milk. The yield of expression is 37.44 and 39.99mg/L respectively in average. These outcomes showed that the construct of pBLG can direct the expression of exogenous gene in animal mammary gland.