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王巍, 张立, 张朋. 大鼠DAF蛋白真核表达载体的构建及生物活性鉴定[J]. 四川大学学报（医学版）, 2013, 44(3): 333-336.
引用本文: 王巍, 张立, 张朋. 大鼠DAF蛋白真核表达载体的构建及生物活性鉴定[J]. 四川大学学报（医学版）, 2013, 44(3): 333-336.
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WANG Wei, ZHANG Li, ZHANG Peng. Eukaryotic Expression and Identification of the Recombination Protein of Rat Decay-accelerating Factor with Pichia pastoris Expression System[J]. Journal of Sichuan University (Medical Sciences), 2013, 44(3): 333-336.
Citation: WANG Wei, ZHANG Li, ZHANG Peng. Eukaryotic Expression and Identification of the Recombination Protein of Rat Decay-accelerating Factor with Pichia pastoris Expression System[J]. Journal of Sichuan University (Medical Sciences), 2013, 44(3): 333-336.
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大鼠DAF蛋白真核表达载体的构建及生物活性鉴定

Eukaryotic Expression and Identification of the Recombination Protein of Rat Decay-accelerating Factor with Pichia pastoris Expression System

    摘要
  • 摘要: 目的 构建大鼠促衰变因子(DAF)的酵母真核表达载体,诱导其表达并对生物学特性进行鉴定。 方法 采用RT-PCR技术从大鼠肝脏组织中扩增出DAF基因并克隆到毕赤酵母表达载体pPIC9K质粒中。经酶切鉴定后,将pPIC9K-DAF转化至毕赤酵母菌GS115,通过G418-YPD平板筛选高拷贝转化子。转化子经甲醇诱导表达后,对表达的DAF蛋白进行SDS-PAGE及Western blot鉴定并检测重组蛋白的生物活性。 结果 成功克隆大鼠DAF基因,并筛选出pPIC9K载体中的DAF阳性克隆,证实所获得相对分子质量约为70×103大小的重组蛋白具有大鼠DAF蛋白的抗原性和生物学活性。 结论 采用分子克隆的方法成功构建大鼠DAF蛋白的毕赤酵母真核表达载体,能为补体及免疫研究提供高效的大鼠DAF蛋白来源。

     

    Abstract: Objecitve To construct yeast eukaryotic expression vector carrying the gene of rat decay-accelerating factor (DAF), and to induce the expression of recombinant protein. Methods The cDNA of rat DAF was amplified by RT-PCR from the fresh hepatic tissue of rat. The PCR product was inserted into the Pichia pastoris vector pPIC9K. Then the recombinant plasmid pPIC9K-DAF was transformed into yeast GS115 through electroporation. The positive high copy number transformants were rapidly selected by using G418-YPD and were induced by methanol. The induced product was analyzed by DNA sequencing, SDS-PAGE and Western blot. Finally, the bio-activity of recombinant rat DAF was examined. Results The rat DAF cDNA was successfully cloned in pPIC9K. The positive clone in pPIC9K expression vector (pPIC9K-DAF) was sieved, and the pPIC9K-DAF showed the same seqencing result as reported in GenBank. And the recombination protein of rat DAF (relative molecular mass is 70×103) has natural rat immunogenicity and bio-activity. Conclusion Successfully cloning and high-level expression of rat DAF recombination protein in Pichia pastoris laid a foundation for further research on immunity and complement system.

     

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