慢病毒GFP质粒的构建及脂质体介导鸡胚细胞中瞬时表达动力学初步研究

A Primary Study on the Constructed GFP Lentivirus Vector's Transient Expression Kinetics in the Chicken Embryo Cells by Liposome-mediating

GFP(green fluorescent protein)是一种用于检测细胞的基因表达和蛋白定位的报告基因,pLenti6/V5-D-TOPO是高效的慢病毒类表达载体。实验利用PCR(polymerase chain reaction)从质粒pEGFP-N1中扩增了EGFP基因片段,再利用高效、快速、定向的TOPO克隆法将扩增片段克隆到pLenti6/V5-D-TOPO载体中,构建了既能高效转染细胞又能方便观察蛋白表达情况的pLEGFP重组载体。通过PCR法鉴定及瞬时转染,结果显示,GFP基因正确地插入载体中,并能够在Hela细胞及鸡胚X期细胞中瞬时表达,但重组和对照质粒瞬时转染效果存在明显差异。通过该研究证实分子量大小、质粒和脂质体比例是影响转染的关键因素,也为转基因的研究提供了一种方法。

GFP is a reporter gene by which gene expressing and location of protein can be detected. The pLenti6/VS- D-TOPO is a lentiviral expressing vector with high efficiency. In this expriment, the GFP gene was amplified by PCR from the plasmid pEGFP-N1, then it was ligated into the pLenti6/V5-D-TOPO by the TOPO cloning efficiently, quickly and directly, and constructed the recombined pLEGFP vector that could transfect the cells efficently and display protein expressing expediently. The results of PCR and transient transfecting of the pLEGFP testified that the GFP gene was ligated correctly into the pLenti6/VS-D-TOPO and that the pLEGFP could express transiently in the Hela cells and the chicken embryo ceils at stage X. But transient transfection effect has an obvious difference between recombinant plasmid and control plasmid. The results of research show that the molecular size and the ratio of plasmid and liposome have the key effect on transfection. This work also provides a tool for transgenic chicken research.