人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 Plasmid of Human P75NTR and Its Expression in HEK293 Cells

构建了人p75神经营养素受体(p75 neurotrophin 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.采用PCR方法从含人p75NTR的pDC316-HP75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-HP75,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Western blot法鉴定人p75NTR的表达.结果表明,酶切鉴定和序列分析证实重组质粒含有人p75NTR编码序列,转染实验表明,重组质粒能够在HEK293细胞中表达出具有活性的人p75NTR片段.

1. Objective： To establish a eukaryotic fluorescence expression plasmid of human p75 neurotrophin receptor （p75NTR） eDNA and investigate its expression in human embryo kidney 293 （HEK293） cells. 2. Methods： The pEGFP-N1-HP75 eukaryotie fluorescence expression vector is constructed by cloning a 835bp insertion of human p75NTR eDNA from pDC316-HP75 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 is transfected into HEK293 cells by lipofection and the expression of human p75NTR in HEK293 is detected by confocal microscopy and Westem blot. 3. Results： The analysis of enzyme hydrolyze and DNA sequence shows that the recombinant eukaryotic expression vector contained human p75NTR cDNA and the detection of confocal microscopy and Western blot show that the HEK293 cells transfected with pEGFP-N1-HP75 could express rat p75NTR. 4. Conclusion： The recombinant eukaryotic fluorescence expression vector is constructed successfully and the transfected HEK293 cells could express human p75NTR, which paves the way for further studies on the mechanism of p75NTR function.