转基因克隆奶山羊大量生产重组人的抗凝血酶Ⅲ蛋白质(rhATⅢ)

Large Scale Production of Recombinant Human Antithrombin Ⅲ(rhATⅢ) in Transgenic Cloned Goats

利用转基因克隆奶山羊乳腺生物反应器大量生产重组人的抗凝血酶Ⅲ(rhATⅢ)蛋白质。其中包括:筛选出人的抗凝血酶Ⅲ蛋白基因的cDNA序列;利用山羊的β-酪蛋白基因的启动区,终止信号和Enterokinase蛋白酶酶切DNA序列,构建在乳腺中特异表达rhATⅢ的表达载体。同时在转基因载体的末端连接一个新酶素筛选基因(Neomycin)。再以细胞转染、G418筛选和体细胞核移植(动物克隆)等过程,最后获得含有人的抗凝血酶Ⅲ基因的转基因克隆奶山羊。我们共获得了5个原代转基因公羊。第一只克隆羊在出生后78 d死亡,解剖表明:羊的肺部和肾脏等器官有异常。其它克隆公羊经过与崂山种母羊交配,得到转基因后代,其中两个原代转基因羊的后代母羊已成熟、所获得的奶经蛋白质电泳证明:转基因克隆羊后代奶中含有约60 kD大小的rhATⅢ糖蛋白;经Elisa检测表明:在奶中含有大量活性的rhATⅢ,在来源于两个不同克隆公羊的后代母羊奶中的rhATⅢ含量分别为0.4mg/L和3 g/L。此研究证明:转基因奶山羊可以大量地生产具有很高活性的rhATⅢ。用这种方法生产的rhATⅢ通过蛋白质提纯,制成注射针剂,将可用于人的抗凝血酶Ⅲ缺乏症的治疗、预防血栓和重大手术过程中的止血的作用等。

We report the production of recombinant human antithrombin Ⅲ （rhATⅢ） in transgenic cloned goats. Human ATⅢ mRNA was isolated from human liver tissues, then transcripted into cDNA. The human ATⅢ cDNA （without the first 96 base pair sequence）and Enterokinase recognised peptide-DNA sequence were ligated to a goat beta-casein promoter and poly（A） singling se-quences, neomycin selection gene was linked at the end of the poly（A） singling region. The mammary expression construct was transfected into in vitro cultured goat foetal fibroblast cells and the cells were selected with 500ug/ml G418. Five transgenic cloned goats were derived by nuclear transplantation and their transgenic status were confirmed by Southern and/or PCR analysis. The first cloned goat died at 78 day old with abnormalities in respiratory system and kidney. Two of the male cloned goats were bred to pro- duce female offspring. Milk analysis from the transgenic lines revealed that the size of the rhATⅢ in milk was about 60 kD. One line could produce rhATⅢ at 0.4 mg/L in their milk; another line could produce rhATⅢ at a large scale of 3 g/L in their milk. rhATⅢ was also a glycoprotein, which was similar to human serum ATⅢ.