TPO模拟肽与人IgG1Fc融合蛋白在毕赤酵母中的表达及活性鉴定

Expression of TMP Fc in Pichia pastoris and Identification of Its Biological Activity

构建TPO模拟肽与人IgG1Fc融合蛋白的酵母表达体系。利用PCR技术 ,从重组质粒pET2 8a TMPFc中 ,扩增TPO模拟肽与人IgG1Fc的DNA片段 ,连入pPICZαA酵母表达载体 ,电激法转化毕赤酵母。用MDH和MMH筛选具有正确表型的重组转化子 ,PCR、蛋白质印迹鉴定融合基因。MTT法鉴定TMPFc对Ba F3 mpl细胞生长的促进作用。构建的重组毕赤酵母实现了TMPFc的分泌表达 ,表达量占外分泌蛋白质的 6 5 %。表达蛋白质的相对分子量约6 4kD ,对Ba F3 mpl生长具有促进作用。TMPFc酵母表达体系表达出可观的二价模拟肽 ,为二价TMP活性的定量研究奠定基础。

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZαA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.