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目的:构建大鼠p75神经营养素受体(p75neurotrophin receptor,p75NTR)cDNA序列的绿色荧光真核表达载体并鉴定其在人胚肾293(human embryo kidney 293,HEK293)细胞中的表达。方法:采用PCR方法从含野生型大鼠p75NTR的pDC316-RP75质粒中扩增目的片段,经EcoRI和SalI双酶切,定向克隆于pEGFP-N1质粒中,构建绿色荧光真核表达载体pEGFP-N1-RP75,经酶切及测序鉴定后,通过脂质体转染HEK293细胞,激光共聚焦及免疫组织化学法鉴定大鼠p75NTR的表达。结果:重组质粒经酶切鉴定和序列分析证实含有大鼠p75NTR的编码序列,转染后经激光共聚焦显微镜及免疫组织化学染色观察表明重组质粒能够在HEK293细胞中表达出具有活性的大鼠p75NTR片段。结论:大鼠p75NTR绿色荧光真核表达载体构建成功并可在HEK293细胞中表达,为进一步研究奠定了基础。
OBJECTIVE: To construct a green fluorescent eukaryotic expression vector of rat p75 neurotrophin receptor (p75NTR) cDNA sequence and identify its expression in human embryo kidney 293 (HEK293) cells. Methods: The pDC316-RP75 plasmid containing the wild-type rat p75NTR was amplified by PCR and cloned into pEGFP-N1 plasmid by EcoRI and SalI digestion. The green fluorescent eukaryotic expression vector pEGFP-N1 -RP75. After identification by restriction enzyme and sequencing, HEK293 cells were transfected by lipofectamine. The expression of p75NTR in rat was identified by laser scanning confocal microscope and immunohistochemistry. Results: The recombinant plasmid was identified by restriction enzyme digestion and sequence analysis confirmed that the coding sequence containing rat p75NTR transfected by confocal laser scanning microscopy and immunohistochemical staining showed that the recombinant plasmid was able to express in HEK293 cells active rat p75NTR Fragment. CONCLUSION: The p75NTR green fluorescence eukaryotic expression vector was successfully constructed and expressed in HEK293 cells, which laid the foundation for further study.