vMIP-Ⅱ-TfN融合蛋白在毕赤酵母中的表达和纯化

Expression and Purification of vMIP-Ⅱ-TfN in Pichia pastoris

目的:构建pPIC-vMIP-II-TfN酵母表达载体,表达纯化vMIP-II-TfN融合蛋白。方法:利用PCR方法扩增编码人转铁蛋白N端半分子的基因片段,通过酶切、连接、转化等分子克隆方法构建pPIC-vMIP-II-TfN酵母表达载体;电击法转化X33酵母菌;用甲醇诱导重组酵母菌表达融合蛋白,利用硫酸铵沉淀、透析、Ni-NTA层析等技术进行蛋白纯化,SDS-PAGE和Western blot检测蛋白表达和纯化情况,利用趋化实验进行纯化蛋白活性检测。结果:经过两次PCR扩增了一个长约1.1kb的包含Xba I酶切位点的IgG3-TfN基因片段,插入pPIC-vMIP-II的Xba I酶切位点,经菌液PCR鉴定获得重组子,测序结果表明构建载体pPIC-vMIP-II-TfN的表达框正确无误,转化X33酵母菌,用甲醇诱导表达出48kDa的vMIP-II-TfN融合蛋白,经硫酸铵沉淀、透析、Ni-NTA纯化后得到纯度约为95%的vMIP-II-TfN融合蛋白。Western印迹结果表明融合蛋白能与转铁蛋白抗体特异性结合。活性检测表明经过诱导表达的vMIP-II-TfN融合蛋白具有趋化抑制活性。结论:成功构建pPIC-vMIP-II-TfN酵母表达载体,重组酵母工程菌经甲醇诱导成功表达出vMIP-II-TfN融合蛋白,纯化后的vMIP-II-TfN融合蛋白具有趋化抑制活性。

Objective： To construct the pPIC-vMIP-II-TfN yeast expression vector and to express vMIP-II-TfN fusion protein.Method： PCR was employed to amplify the N-terminal half-molecular of human transferrin gene.Molecular cloning methods including digestion,ligation and transformation were used to construct the pPIC-vMIP-II-TfN yeast expression vector.The linearized pPIC-vMIP-II-TfN was transformed into Pichia pastoris X33 strain by electroporation.The transformant was induced by methanol to express target protein and ammonium sulfate precipitation,dialysis,and Ni-NTA affinity chromatography were used to purify the target protein.SDS-PAGE and Western blot was used to detect the expression and purification of vMIP-II-TfN.The activity of vMIP-II-TfN was detected by chemotaxis inhibition activity assay.Results： A 1.1kb IgG3-TfN DNA fragment containing the Xba I site was amplified and it was inserted into Xba I sites of pPIC-vMIP-II.After PCR identification and sequencing,the recombinant plasmid pPIC-vMIP-II-TfN was obtained successfully and the open reading frame was correctly.The linearized pPIC-vMIP-II-TfN by Sac I was transformed into Pichia pastoris X33 strain.After inducing with methanol for 72 h,the transformant was confirmed to express a recombinant protein with molecular mass of 48 kDa by SDS-PAGE and Western blot.After characterization,the vMIP-II-TfN fusion protein was successfully purified from the supernatant of the broth using ammonium sulfate precipitation,dialysis and affinity chromatography.Furthermore,in vitro bioacitivity assay indicated that vMIP-II-TfN has chemotaxis inhibition activity.Conclusion： The pPIC-vMIP-II-TfN yeast expression vector was constructed successfully.The transformant can express the vMIP-II-TfN fusion protein which had chemotaxis inhibition activity.