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核苷酸结合寡聚化结构域样受体蛋白6重组腺病毒过表达载体的构建与鉴定

Construction and identification of nucleotide-binding oligomerization domain-like receptor protein 6 recombinant adenoviral overexpression vector

    摘要
  • 摘要:
    目的 构建及鉴定携带小鼠核苷酸结合寡聚化结构域样受体蛋白6(NLRP6)基因(NM_133 946.2)的过表达重组腺病毒载体。
    方法 合成小鼠NLRP6基因的编码序列, 经酶切后插入腺病毒载体(GL2003), 转化大肠杆菌感受态细胞DH-5α进行培养，挑取转化子，进行聚合酶链反应(PCR)鉴定后送测序进行比对。采用蛋白质印迹法(Western blot)验证重组质粒中NLRP6蛋白的表达。利用Admax包装系统获得重组腺病毒载体Ad-NLRP6, 导入HEK293细胞，经扩增纯化后测定病毒滴度。
    结果 双酶切及DNA测序证明NLRP6成功构建入表达质粒中，同时检测其携带的FLAG标签蛋白，表明NLRP6表达水平显著升高。包装并生产携带NLRP6的重组腺病毒，进一步将病毒感染HEK293细胞, Ad-NLRP6细胞表达绿色荧光蛋白提示感染成功，收集病毒，经扩增纯化后测定病毒滴度为4×1010 PFU/mL。
    结论 成功构建携带小鼠NLRP6基因的过表达重组腺病毒载体，这一结果为进一步研究NLRP6分子学功能提供了有效工具。

     

    Abstract:
    Objective To construct and identify an overexpressing recombinant adenovirus vector carrying the mouse nucleotide-binding oligomerization domain-like receptor protein 6 (NLRP6) gene (NM_133 946.2).
    Methods The coding sequence of the mouse NLRP6 gene was synthesized, digested with enzymes, and inserted into the adenovirus vector (GL2003). The transformed cells were then cultured in strain DH-5α, transformants were selected, and after identification by polymerase chain reaction (PCR), samples were sent for sequencing and comparison. The expression of NLRP6 protein in the recombinant plasmid was validated using the Western blot. The recombinant adenovirus vector Ad-NLRP6 was obtained using the Admax packaging system, then transduced into HEK293 cells, amplified, purified, and the viral titer was determined afterwards.
    Results The successful integration of NLRP6 into the expression plasmid was confirmed through double enzyme digestion and DNA sequencing. Concurrent detection of the FLAG-tagged protein indicated a significant increase in the expression level of NLRP6. Recombinant adenovirus carrying NLRP6 were packaged and produced, followed by infecting HEK293 cells with the virus. The cellular expression of green fluorescent protein in Ad-NLRP6 indicated successful infection. The virus was collected, amplified, purified, and the viral titer was determined to be 4×1010 PFU/mL.
    Conclusion The overexpression recombinant adenovirus vector carrying the mouse NLRP6 gene has been successfully constructed, providing an effective tool for further exploring the molecular function of NLRP6.

     

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