耐辐射奇球菌pprM基因回补株的构建与鉴定

Construction and identification of pprM-complemented Deinococcus radiodurans strain

    摘要
  • 摘要:
    目的 构建耐辐射奇球菌pprM基因缺失回补菌株, 为进一步研究PprM蛋白的功能奠定实验基础。
    方法 设计并合成HA-pprM基因(HA为流感病毒血凝素)全长的引物, 以无突变的pGEX-6p-1-pprM载体为模板, PCR扩增出携带NdeⅠ酶切位点的HA-pprM基因全长, 经NdeⅠ酶切后, 与pRADK载体连接并转化大肠杆菌, 经培养、提取质粒及纯化后, 采用酶切鉴定以及基因测序方法确认插入序列的正确性; 采用CaCl2法将构建好的重组质粒pRADK-HA-pprM转化到耐辐射奇球菌pprM基因缺失的菌株中, 通过Western blot验证HA-PprM融合蛋白的表达。
    结果 构建的重组质粒pRADK-HA-pprM经NdeⅠ酶切鉴定结果符合目的条带大小(438 bp), 测序结果显示插入的寡核苷酸序列及方向与预期的一致, Western blot结果显示HA-PprM融合蛋白的相对分子质量约为13000。
    结论 笔者成功构建了重组质粒pRADK-HA-pprM, 为进一步研究与PprM蛋白相互作用的靶DNA的筛选奠定了良好的基础。

     

    Abstract:
    Objective To investigate the function of PprM protein by constructing a pprM-complemented strain of Deinococcus radiodurans.
    Methods HA-pprM gene was amplified by PCR from the plasmid DNA of pGEX-6p-1-pprM(approximately 438 bp) and then inserted into the shuttle expression vector pRADK to construct pRADK-HA-pprM. The correctness of the inserted sequences was confirmed through agarose gel electrophoresis, restriction enzyme digestion, and gene sequencing. The recombinant plasmid was transformed into the pprM mutant strain of Deinococcus radiodurans, and the recombinant protein was analyzed by Western blot.
    Results The recombinant plasmid pRADK-HA-pprM and a complemented strain were obtained. The recombinant protein can be highly expressed in the complemented strain.
    Conclusion This study provided a foundation for further studies on the interaction between the PprM protein and its target DNA.

     

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