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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

赵述强, 张瑜, 田浤, 刘超, 高向东

赵述强, 张瑜, 田浤, 刘超, 高向东. 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[J]. 中国药科大学学报, 2013, 44(6): 577-582. DOI: 10.11665/j.issn.1000-5048.20130618
引用本文: 赵述强, 张瑜, 田浤, 刘超, 高向东. 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[J]. 中国药科大学学报, 2013, 44(6): 577-582. DOI: 10.11665/j.issn.1000-5048.20130618
ZHAO Shuqiang, ZHANG Yu, TIAN Hong, LIU Chao, GAO Xiangdong. Expression and bioactivity of recombinant 3DHSA-Nartograstim fusion protein in Pichia pastoris[J]. Journal of China Pharmaceutical University, 2013, 44(6): 577-582. DOI: 10.11665/j.issn.1000-5048.20130618
Citation: ZHAO Shuqiang, ZHANG Yu, TIAN Hong, LIU Chao, GAO Xiangdong. Expression and bioactivity of recombinant 3DHSA-Nartograstim fusion protein in Pichia pastoris[J]. Journal of China Pharmaceutical University, 2013, 44(6): 577-582. DOI: 10.11665/j.issn.1000-5048.20130618

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

基金项目: 江苏省“青蓝工程”资助项目(2010)

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

  • 摘要: 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。SDS-PAGE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 α-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 chromatography 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.
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  • 刊出日期:  2013-12-24

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