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盘基网柄菌DJ-1基因真核表达载体的构建

Construction of Eukaryotic Expression Vector of DJ-1 Gene in Dictyostelium discoideum

  • 摘要: DJ-1突变或缺失导致帕金森病相关症状,但其具体功能和致病机理尚在研究和探讨阶段。盘基网柄菌Dictyostelium discoideum是研究神经退化性疾病的模式生物之一,为了研究DJ-1基因在帕金森病中的作用机制,本研究设计特异性引物对盘基网柄菌DJ-1基因或其部分片段(75~479 bp)进行PCR扩增,再通过两步克隆法先对目的基因和pUC18质粒进行酶切、连接、大肠杆菌Escherichia coli DH5α转化、菌株蓝白斑筛选和目的片段测序,构建了盘基网柄菌DJ-1基因的原核表达载体pPROF681和pPROF682。经酶切鉴定和序列测定确认后,再利用引物中的第二限制性内切酶(SacⅠ, Bam HⅠ, Eco RⅠ和XhoⅠ)进行酶切,将目的基因插入盘基网柄菌质粒pDNeo2和pPROF267,构建了DJ-1基因的重组真核表达载体pPROF688和pPROF690,为后期培育含重组载体的盘基网柄菌转化株、进行菌株筛选和鉴定、分析DJ-1的表达量与盘基网柄菌表现型之间的相关性,并进一步建立帕金森病相关基因DJ-1的盘基网柄菌模型奠定基础。

     

    Abstract: The mutations or loss of DJ-1 gene cause Parkinson’s disease(PD), but the role of DJ-1 and its relative mechanisms for PD are still under study and investigation. Dictyostelium discoideum is one of the recognized models for study of neurodegenerative disease. To study the role of DJ-1 in PD and its mechanisms, the specific primers were designed to amplify the full-length Dictyostelium discoideum DJ-1 and its fragment from 75~479 bp using two-step cloning strategy. The DNA of interest and pUC18 vector were digested and ligated with T4 ligase. The product of ligation was transformed into Escherichia coli DH5α and followed by screening of strains of interest using Blue/white selection and gene sequencing. The D. discoideum prokaryotic expression construct pPROF681 and pPROF682 were built and verified by endonuclease digestion and gene sequencing. The genes of interest were then cut by SacⅠ, Bam HⅠ, Eco RⅠ and XhoⅠ designed in primers from pPROF681 and pPROF682 and inserted into D. discoideum vectors p DNeo2 and pPROF267 to create D. discoideum eukaryotic expression vectors pPROF688 and p PROF690. This work provides a very important basis for further achievement of D. discoideum strains con-taining pPROF688 and pPROF690, selection and verification of interested transformants, analysis of the relativities between expression level of DJ-1 and the phenotypes of D. discoideum and built of D. discoideum model of DJ-1 for PD.

     

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