This work was supported by a grant from State 863 High Technology R&D project of China Research & Development Program “863” (2002AA206111).
从粘粒文库中筛选出人α-乳清蛋白基因,构建9.5 kb的转基因表达载体.利用显微注射的方法获得68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0.62 g/L、0.48 g/L、0.56 g/L、3.21 g/L;同时测定F0代50号转基因公鼠的后代阳性母鼠(50-2号)乳样中人α-乳清蛋白含量也达到1.03 g/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 F0 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.
于舒洋,曹更生,樊宝良,李宁,段颖莉,戴蕴平,王莉莉.人α-乳清蛋白基因的克隆及其在转基因小鼠中高效表达[J].生物化学与生物物理进展,2004,31(3):244-248
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