人α-乳清蛋白基因的克隆及其在转基因小鼠中高效表达

The Cloning of Human α-Lactalbumin Gene and High-level Expression in Milk of Transgenic Mice

从粘粒文库中筛选出人α 乳清蛋白基因 ,构建 9 5kb的转基因表达载体 .利用显微注射的方法获得 6 8只F0 代小鼠 ,经PCR检测和DNA印迹分析证实有 8只小鼠 (4 ♂ ,4♀ )为整合人α 乳清蛋白基因的转基因阳性小鼠 ,整合率为 11 7% ,整合拷贝数在 1至 8之间 .利用SDS 聚丙烯酰胺凝胶电泳检测和蛋白质印迹分析 ,4只雌性F0 代转基因阳性小鼠全部表达了人α 乳清蛋白 .放射免疫测定法测定 ,含量分别为 0 6 2g/L、 0 4 8g/L、0 5 6 g/L、 3 2 1g/L ;同时测定F0 代 5 0号转基因公鼠的后代阳性母鼠 (5 0 2号 )乳样中人α 乳清蛋白含量也达到 1 0 3g/L ,证明由原代转基因公鼠遗传给后代的人α 乳清蛋白基因亦获得了稳定的表达 .所构建的人α 乳清蛋白转基因载体具有结构较小 ,表达量高 ,可以稳定遗传等优点 .为利用人α 乳清蛋白基因改善牛乳成分和品质奠定了基础 .

The transgenic expression vector was constructed by using 9.5kb α-lactalbumin genomic fragment cloned from human cosmid library. Eight transgenic mice (4♂,4♀)were detected by PCR and Southern blot analysis from 68 F 0 mice produced through microinjection. The copy number ranges from 1 to 8, and the integration rate is 11.7%. Human α-lactalbumin have been detected in milk samples of the four founder female transgenic mice by SDS-PAGE gel electrophoresis and Western blotting, and the concentrations of the human α-lactalbumin are quantified to be 0.62g/L, 0.48g/L, 0.56g/L, 3.21g/L respectively by radioimmunoassay (RIA) method. Meanwhile, one offspring of the No.50 male transgenic mice has also expressed the human α-lactalbumin, and the concentration is 1.03 g/L in the milk. The transgenic construct herewith has the characterization of a high-level expression and stable inheritance. A foundation has been provided for improving the quality of cow milk thorough transgenesis of human α-lactalbumin.