重组人ADAM15去整合素区域蛋白与人血清白蛋白融合蛋白的表达及其活性

Expression and activity of human serum albumin-recombinant human disintegrin domain of ADAM15 fusion protein

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 investigate the expression of recombinant human disintegrin domain of ADAM 15 （a disintegrin and metalloproteinase 15） and human serum albumin （HSA） fusion protein （HSA-rhddADAM15-His） in Pichiapastori and deter- mine the activity of expressed product. Methods The rddADAM15-His gene was amplified by PCR using rhddADAM15 gene as a template, and cloned into plasmid pBluescript-HSA. The constructed recombinant plasmid was digested with SnaB I and Not I , and the recovered HSA-AD-His fragment was inserted into vector pPIC9k. The constructed recombi-nant plasmid pPIC9k-HSA-rhddADAM15-His was transformed to P. pastoris GSll5 for expression under induction of methanol. The expressed product was analyzed by SDS-PAGE, purified by Blue Sepharose, nickel ion chelate and DEAE anion exchange chromatography, then identified by SDS-PAGE and Western blot, and determined for activity by wound healing test and SRB assay. Results Restriction analysis and sequencing proved that recombinant plasmid pPIC9k-HSA-rhddADAM15-His was constructed correctly. The expressed fusion protein HSA-rhddADAM15-His, with a relative molecular mass of about 76 000, existed in a soluble form, reached a purity of about 75% after purification, and showed specific reaction with rabbit antibody against rhddADAM15. The fusion protein showed significantly inhibitory effect on the migration, while showed no significantly inhibitory effect on the proliferation, of mouse melanoma B16 cells. Conclusion HSA-rhddADAM15-His fusion protein was successfully expressed in P. pastoris and purified, which laid a foundation of study on its action mechanism.