转甲状腺素蛋白基因在酵母中的表达

Expression of Transthyretin Gene in Yeast Pichia pastoris

用限制酶 Ｂａ ｍ Ｈ Ｉ 将含人 Ｔ Ｔ Ｒ 基因的 Ｄ Ｎ Ａ 片段（ 约４９３ｂｐ） 从质粒ｐ Ｓ Ｋ Ｔ Ｔ Ｒ 上切下后并插入到分泌型载体ｐ Ｈ Ｉ Ｌ Ｓ Ｉ 的 Ｂａ ｍ Ｈ Ｉ 位点上，经酶切鉴定重组质粒ｐ Ｈ Ｉ Ｌ Ｓ Ｉ Ｔ Ｔ Ｒ 上的 Ｔ Ｔ Ｒ 基因插入方向正确。将ｐ Ｈ Ｉ Ｌ Ｓ Ｉ Ｔ Ｔ Ｒ Ｄ Ｎ Ａ 用 Ｂｇｌ Ｉ Ｉ 酶切后，转入真核表达系统———毕赤氏酵母。得到的转化子经摇瓶发酵，上清液用１５ ％ Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测，有 Ｔ Ｔ Ｒ Ｆ 的表达。经 Ｄ Ｅ Ａ Ｅ Ｓｅｐｈｅｒｏｓｅ Ｆ． Ｆ 离子交换柱和 Ｓｅｐｈａｃｒｙｌ Ｓ２００ 分子筛层析，得到纯化的 Ｔ Ｔ Ｒ Ｆ。体外实验表明 Ｔ Ｔ Ｒ Ｆ

Plasmid pSK TTR was digested by Bam HI and the DNA fragment containing TTR gene (Transthyretin) was cloned into the Bam HI site of secretion vector pHIL SI TTR.In right orientation and frame.After the recombinant plasmid pHIL SI TTR was digested by Bgl II,the larger fragment of it was transformed into yeast Pichia pastoris. The SDS PAGE analysis showed that the yeast transformants could express and secret TTRF.The pure TTRF could be obtained by DEAE Sepherose F.F.chromatography and SephacrylS 200 chromatography.The vitro test showed inhibition of the growth of hepatoma cells by TTRF obtained from our experiments.