重组融合蛋白rTMP-GH在巴斯德毕赤酵母中表达的研究

Expression and identification of recombinant fusion protein of thrombopoietin mimetic peptide and growth hormone in Pichia pastoris

目的实现TPO模拟肽(throm bopoietin mimetic peptide,TMP)与人生长激素(human growth hormone,hGH)的重组融合蛋白rTMP-GH在巴氏毕赤酵母中的高效表达。方法以含有rTMP-GH核苷酸序列的原核质粒为模板,PCR方法获得rTMP-GH的编码序列,并亚克隆至载体pPIC9K中,将所构建pPIC9K-rTMP-GH表达质粒转化酵母宿主细胞GS115,MD/MM平板及G418抗性筛选高表达株,然后进行试管补料分批表达,Western blot对表达产物进行鉴定,通过巨核细胞集落形成能力对其生物学活性进行初步检测。结果成功构建pPIC9K-rTMP-GH表达质粒,筛选到高表达rTMP-GH重组融合蛋白的巴氏毕赤酵母细胞株,初步证实所表达产物具有刺激巨核细胞集落形成的能力。结论成功实现了rTMP-GH融合蛋白在巴氏毕赤酵母中的高效表达。

Objective To fulfill high-efficient expression of rTMP-GH, a recombinant fusion protein of thrombopoietin mimetic peptide and human growth hormone, in Pichia pastoris. Methods eDNA fragment coding for rTMP-GH was amplified by PCR, and subsequently cloned into the expression vector pPIC9K. The linearlized plasmid pPIC9K-rTMP-GH was transformed into Pichia pastoris GS115 cells and screened in MD/ MM plates under pressure of G418. The recombinant fusion protein rTMP-GH was identified by Western blot- ting, and its biological activity was analyzed by its ability on colony formation of megakaryoblasts. Results The pPIC9K-rTMP-GH expression plasmid was successfully constructed. GS115 cells with high expression of rTMP-GH was screened out, and the rough purified rTMP-GH showed promotive effect on megakaryoblast colony formation. Conclusion High-efficient expression of rTMP-GH in Pichia pastoris is achieved.