重组人血清白蛋白第3结构域-Nartograstim融合蛋白在毕赤酵母中的表达及初步活性研究

Expression and bioactivity of recombinant 3DHSA-Nartograstim fusion protein in Pichia pastoris

将人血清白蛋白第3结构域(3DHSA)与粒细胞集落刺激因子突变体(Nartograstim)融合基因克隆至载体pPICzαA,置于醇氧化酶启动子(AOX)和α交配因子信号肽作用下构建分泌表达质粒,电击转化入毕赤酵母GS115。SDSPAGE结果显示3DHSA-Nartograstim相对分子质量约为42 kD,Western blot证实其同时具有HSA和G-CSF的抗原性。建立融合蛋白表达的条件为:BMMY培养基pH 6.0、1.5%甲醇、诱导84 h,灰度扫描发现该条件下融合蛋白的纯度为79%。依次采用亲和色谱和疏水色谱纯化得到纯度高于90%的融合蛋白,终产量为86 mg/L。采用MTT法检测融合蛋白对NFS-60细胞株的促增殖能力,结果显示,3DHSA-Nartograstim具有剂量依赖性促进NFS-60细胞增殖的作用。本研究为3DHSA-Nartograstim的进一步研究奠定了基础。

Abstract The gene for coding the fusion protein 3DHSA-Nartograstim was cloned into pPICzαA vector along with the open reading frame of the cx-factor signal under the control of the AOX promoter. The recombinant secret expression plasmid was transformed into Pichia pastoris host strain GS115 by electroporation. The analysis of SDS- PAGE demonstrated that the relative molecular masses of 3DHSA-Nartograstim were about 42 kD, and the results of Western blotting proved that 3DHSA-Nartograstim was recognized specially by HSA and G-CSF antibodies. The established expression condition of 3DHSA-Nartograstim was BMMY pH 6.0, 1.5% methanol for 84 h, and gray scale scanning showed that the purity of 3DHSA-Nartograstim was 79%. Through the combination of affinity chro- matography and hydrophobic chromatography, the purity of 3DHSA-Nartograstim was up to 90% and the final yield was 86 mg/L. The bioactivity of 3DHSA-Nartograstim was measured by NFS-60 proliferation assay. Results demonstrated that 3DHSA-Nartograstim could stimulate NFS-60 proliferation in a dose-dependent manner. The study would be helpful for further research of 3DHSA-Nartograstim.