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目的在毕赤酵母中表达重组人ADAM15去整合素区域蛋白(recombinant human disintegrin of ADAM15,rhddADAM15)与人血清白蛋白融合蛋白(HSA-rhddADAM15-His),并测定其活性。方法以rhddADAM15基因为模板,PCR扩增rhddADAM15-His基因,克隆入pBluescript-HSA质粒中,经酶切后与pPIC9k载体连接,构建重组表达质粒pPIC9k-HSA-rhddADAM15-His,将重组表达质粒转化毕赤酵母GS115,甲醇诱导表达。表达产物经Blue-Sepharose、Ni2+螯合层析及DEAE阴离子交换层析纯化,纯化产物经SDS-PAGE和Western blot鉴定后,利用划痕及SRB法检测其活性。结果重组表达质粒经双酶切及测序鉴定证明构建正确;融合蛋白HSA-rhddADAM15-His相对分子质量约76 000,以可溶性分子形式存在于发酵液上清中;纯化的融合蛋白纯度约为75%,可与兔抗rhddADAM15多克隆抗体特异性结合;融合蛋白对小鼠黑色素瘤细胞B16的迁移具有明显的抑制作用,但对其增殖无明显抑制作用。结论成功在毕赤酵母GS115中表达并纯化了HSA-rhddADAM15-His蛋白,为其作用机理的研究奠定了基础。
Objective To express recombinant human disintegrin of ADAM15 (rhddADAM15) and human serum albumin fusion protein (HSA-rhddADAM15-His) in Pichia pastoris and determine its activity. Methods The rhddADAM15-His gene was amplified by PCR using the rhddADAM15 gene as a template and cloned into the pBluescript-HSA plasmid. After digested with pPIC9k vector, the recombinant plasmid pPIC9k-HSA-rhddADAM15-His was constructed and the recombinant plasmid was transformed Pichia pastoris GS115, methanol induced expression The expressed product was purified by Blue-Sepharose, Ni2 + chelate chromatography and DEAE anion exchange chromatography. The purified product was identified by SDS-PAGE and Western blot. Scratches and SRB were used to detect the activity of the product. Results The recombinant plasmid was verified by double enzyme digestion and sequencing. The relative molecular mass of the fusion protein HSA-rhddADAM15-His was about 76 000 and existed in the supernatant of the fermentation broth as a soluble molecule. The purity of the purified fusion protein was about 75% , Which could specifically bind with rabbit anti-rhddADAM15 polyclonal antibody. The fusion protein had obvious inhibitory effect on the migration of mouse melanoma B16 cells, but had no obvious inhibitory effect on the proliferation. Conclusion The HSA-rhddADAM15-His protein was successfully expressed and purified in Pichia pastoris GS115, which laid the foundation for its mechanism of action.