大鼠p75NTR荧光真核表达载体的构建及其在HEK293细胞中的表达

Construction of Eukaryotic Fluorescence Expression Vector of Rat p75NTR and Its Expression in HEK293 cells

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 eukaryotic fluorescence expression vector of human p75 neurotrophin receptor （p75NTR） cDNA and investigate its expression in human embryo kidney 293 （HEK293） cells. Methods： The pEGFP-N1-RP75 eukaryotic fluorescence expression vector was constructed by cloning a 828bp insertion of rat p75NTR cDNA from pDC316-RP75 plasmid containing the full length open reading frame to pEGFP-N1 vector. After analysis of enzyme hydrolyze and DNA sequence, the recombinant eukaryotic expression vector was transfected into HEK293 cells by lipofection and the expression of rat p75NTR in HEK293 was detected by confocal microscopy and immunohistochemistry staining. Results： The analysis of enzyme hydrolyze and DNA sequence showed that the recombinant eukaryotic expression vector contained rat p75NTR cDNA and the detection of confocal microscopy and immunohistochemistry staining showed that the HEK293 cells transfected with pEGFP-NI-RP75 could express rat p75NTR. Conclusions： The results showed that the recombinant eukaryotic fluorescence expression vector was constructed successfully and the transfected HEK293 cells could express rat p75NTR, which paves the way for firther studies on the mechanism ofp75NTR function.