山羊β-酪蛋白基因启动子指导的转基因小鼠乳汁高效表达人凝血因子Ⅸ  

High Expression of Human FIX(hFIX) in Transgenic Mice Directed by Goat β-casein Gene Promoter

作　　者：黄赞[1] 颜景斌[1] 黄缨[1] 孙琼[1] 肖艳萍[1] 黄英[1] 曾溢滔[1] 

机构地区：[1]上海市儿童医院上海医学遗传研究所,上海200040

出　　处：《Acta Genetica Sinica》2002年第3期206-211,T001,共7页

基　　金：国家"8 63"高技术项目 (编号 2 0 0 1AA2 13 0 11);上海市现代生物与新药产业发展基金 (编号 9943 190 4)~~

摘　　要：为探讨应用转基因动物乳腺生物反应器高效表达人凝血因子IX(humanclottingfactorIX ,hFIX)的可行性 ,构建了包括山羊 β 酪蛋白基因启动子和外显子 1、内含子 1、外显子 2共约 6 .7kb片段以及hFIX全长cDNA序列和部分经改造的内含子 1的乳腺表达载体 ,通过转基因技术获得 12个原代转基因小鼠 (9♀ ,3♂ ) ,整合率为 11.2 %。经ELISA和Westernblot鉴定 8只转基因母鼠乳汁中有hFIX的表达并拥有很高的凝血活性 ,其中一只的表达量高达5 2 .9mg/L ,其凝血活性亦高达 2 79.2 %。FISH实验表明不同的转基因小鼠外源基因整合于小鼠的不同染色体上。结果证明所构建的山羊 β 酪蛋白基因启动子乳腺表达载体能有效指导hFIX基因在小鼠乳腺中高效表达 ,并能保持hFIX的生物活性。To probe the feasibility of efficient production of human clotting factor IX(hFIX)with the approach of mammary gland bioreactor of transgenic animals,we constructed hFIX mammary gland expression vector containing promoter,exon 1,intron 1 and exon 2 of the goat β casein gene about 6.7kb fragment as well as full length of hFIX cDNA and its modified intron 1 sequence. By using transgenic products and 12 transgenic founders(9♀,3♂) were produced,and the integration rate thus was 11.2%. ELISA assay and Western blot showed that the milk of 8 female transgenic mice had hFIX expression with high clotting activities. The highest hFIX expression in the milk of one transgenic mouse reached 52.9mg/L,and the highest clotting activity of the transgene milk alwas 279.2%. FISH experiments indicated that hFIX DNA was integrated in different chromosomes in different mice. This result indicated that the hFIX mammary gland expression vector based on the goat β casein promoter can efficiently direct high expression of hFIX gene in the milk of transgenic mice,which maintained high clotting activity.

关 键 词：山羊 β-酪蛋白基因启动子 人凝血因子Ⅸ 转基因小鼠 表达 

分 类 号：Q819[生物学—生物工程]