t-PA cDNA的克隆及其在毕赤酵母中的表达

Cloning and Expression of t PA cDNA in Pichia pastoris

应用ＰＣＲ方法，在ｔＰＡｃＤＮＡ５′端引入合适的限制酶位点和酵母分泌信号肽，与酵母表达载体ｐＰＩＣ９重组，构建表达质粒ｐＳＴＥＹ，利用ＬｉＣｌ转化法转化酵母菌ＹＳ１０８，在ＭＭ和ＭＤ平板上筛选表型，ＰＣＲ筛选ｔＰＡｃＤＮＡ与酵母染色体整合而形成的阳性克隆，阳性克隆经甲醇诱导表达后，用ＳＤＳＰＡＧＥ证明表达产物的分子量为６ｋＤａ左右，用酪蛋白板溶圈法测定ｔＰＡ的活性。Ｍｕｔｓ表型菌表达产物活性最高为２５００ＩＵ／ｍｌ，Ｗｅｓｔｅｒｎｂｌｏｔ证实表达产物具有天然ｔＰＡ分子的免疫原性。

A suitable restriction enzyme site and yeast secretion peptide sequence was introduced into the 5′ terminal of t PA cDNA by PCR.Recombinant plasmid,pSTE Y,was constructed by inserting t PA cDNA fragment into yeast expression vector pPIC9.The expression plasmid,pSTE Y,was transformed yeast YS108 with lithium chloride method.Phenotypes of transformants were screened in MM and MD plates.The positive transformants were proved with PCR and ensured the integration of t PA cDNA into yeast chromosome DNA.Transformants containing t PA cDNA were induced by methanol for the expression of rt PA.SDS PAGE showd that the molecular weight of the expression product was about 60 kDa.The biological activity of the expression product measured by fibrin plate was more than 2500 IU/ml.The immunogenicity of the rt PA was confirmed by Western blot.