人血小板生成素全长分子在酵母系统中的分泌表达及活性分析

Secretion Expression and Activity Assay of Thrombopoietinin Pichia pastoris

外源基因可通过整合型载体被整合到毕赤酵母（Ｐｉｃｈｉａｐａｓｔｏｒｉｓ）染色体上，获得遗传性稳定分泌表达株．利用酵母信号肽ＭＦα，对该信号肽蛋白酶识别位点的相关序列进行重新设计，使天然人ＴＰＯ成熟肽在毕赤酵母系统中分泌表达成功．表达产物经Ｗｅｓｔｅｒｎ－ｂｌｏｔ进行分析，分子量约为６６ｋＤ处蛋白条带可被ＴＰＯ抗体识别；表达量约为０．１ｇ／Ｌ；Ｎ端氨基酸序列分析结果与设计的一致；表达产物对小鼠骨髓细胞形成巨核细胞集落形成单位（ｍｅｇａｋａｒｙ－ｏｃｙｔｅｃｏｌｏｎｙｆｏｒｍｉｎｇｕｎｉｔ，ＣＦＵ－Ｍｅｇ）具有明显的刺激作用．

Thrombopoietin(TPO)is a cytokine which regulates circulating platelet level,acting on both proliferation and differentiation of megakaryocytes.TPO heterologous gene can be integrated into Pichia pastoris chromosome by integrative expression plasmid generating genetic stale secretion expression strain.By using secretive signal peptide MFα,the related sequence of Kex2 cleavage site was modified and redesigned using for secretion expression in the expression system successfully.Westernblot analysis showed that the positive band with about 66 kD molecular weight was recognized by TPO antibody.Yield of expression was about 01 g/L.Analysis for amino acid sequences showed that the result was consistent with what was designed.Activity assay indicated that expressive product could significantly stimulate formation of colony forming unit of megakaryocyte (CFUMeg) of mice in vitro.