pIRES2-EGFP-LR真核表达载体的构建及其在293T细胞中的表达

为探讨层黏蛋白受体的生理功能及其在疾病发生、发展过程中的作用,以培养的SD大鼠大脑皮质神经元的总RNA为模板,运用RT-PCR方法扩增了SD大鼠层黏蛋白受体基因.将其克隆到pMD18-T载体中,经酶切纯化后再将其亚克隆到带有加强型绿色荧光蛋白报告基因的pIRES2-EGFP真核表达栽体中,对重组质粒pIRES2-EGFP-LR进行酶切和测序鉴定后,采用阳离子脂质体转染法将重组质粒转染到293T细胞,荧光显微镜和激光共聚焦显微镜观察其表达情况.结果表明,真核表达载体plRES2-EGFP-LR构建成功并可以在293T细胞中表达.该表达载体可以作为研究层黏蛋白受体生理功能的重要工具.

Construction of eukaryotic expression plasmid pIRES2-EGFP-LR and its expression in 293T cells

To explore the physiologic function of laminin receptor (LR) and its role in the development of diseases,a LR gene was amplified by RT-PCR with the total RNA extracted of the cultured cortical neu-rons of SD rats as template. To construct pMD18-T-LR, the amplified gene was cloned into the vector pMD18-T. The LR gene from pMD18-T-LR digested by restriction enzymes Bgl Ⅱ +Sal I was cloned into the eukaryotic expression vector pIRES2-EGFP with the reported gene EGFP (enhanced green fluores-cence protein). The positive recombinant was deisignated with pIRES2-EGFP-LR. After being identified by restriction enzyme digestion and sequencing,the recombinant pIRES2-EGFP-LR was transfected into 293T cells by liposomes. The expression of the recombinant pIRES2-EGFP-LR in the 293T cells was observed by fluorescence microscope and laser scanning confocal microscope. The results showed that the LR of pIRES2-EGFP-LR was successfully expressed in the 293T cells.