锥虫早老素蛋白亲水区的克隆和表达纯化

Cloning and Expression of Hydrophilic Regions of Presenilin in Trypanosome

目的体外表达锥虫早老素蛋白亲水区肽段,以制备抗血清用于功能研究。方法根据锥虫早老素蛋白二级结构特性,设计引物分别扩增N端及C端片段的亲水区肽段基因,装入原核表达载体进行表达,并通过变性纯化方法获得足够量表达蛋白,制备兔抗血清。结果成功扩增并克隆锥虫早老素蛋白亲水区片段L2及L7,并分别采用变性磁珠法和变性树脂法进行大量蛋白产物纯化,浓缩纯化产物制备兔抗血清经Western blot杂交验证,出现目的蛋白大小阳性条带。结论成功表达锥虫早老素蛋推测N端及C端亲水肽段,并成功制备抗血清,可用于锥虫早老素蛋白功能分析。

Objective To obtain in vitro expression of the hydrophilic fragments of presenilin of Trypanosome （TbPS） for preparation of antiserum, which could be used for functional analysis. Methods The primers targeting hydrophilie regions of N- and C-terminal fragments were designed according to the secondary structure of TbPS. After amplification, the PCR products were cloned into prokaryotie expression plasmid. The expressed peptides were purified through denature purification methods and concentrated to immunize rabbits for obtaining antiserum of TbPS. Results I2 and L7, the hydrophilie regions of TbPS, were successfully amplified and cloned into pCRT7 vector. 12 protein was purified by denature magnetic bead method and L7 by denature metal affinity resins method. Concentrated proteins were used to immunize rabbits. The obtained antisera were validated by Western blotting with a positive target band. Conclusion Two hydrophilie fragments of TbPS have been successfully expressed in prokaryote. The antiserum from this two fragments are prepared and ready to be used in functional analysis for TbPS.