Xfect介导重组质粒pEGFP-C2/LAT-ORF3高效转染Vero细胞的方法及其条件的优化

Establishment and Optimization of Eukaryotic Expression Plasmid pEGFP-C2/LAT-ORF3 Transfected into Vero Cell by Xfect as Mediation

旨在用Xfect试剂介导重组质粒pEGFP-C2/LAT-ORF3转染Vero细胞,以期获得转染效率较高的方法,并检测目标片段的表达,从而为进一步研究HSV-2 LAT基因及其ORF3片段的生物学功能奠定基础。用Xfect试剂介导重组质粒pEGFP-C2/LAT-ORF3转染Vero细胞,48 h后观察荧光表达情况,并计算不同转染方法分别在有无血清及质粒与Xfect Polymer比例不同时的转染效率。用RT-PCR检测ORF3片段在Vero细胞中的表达。结果显示,采用贴壁转染法,在有血清及质粒与Xfect Polymer比例为5μg/2μL时转染效率较高;RT-PCR可以获得目的条带,证明ORF3片段在Vero细胞中得到表达。本试验获得的优化条件可以显著提高Xfect Polymer对Vero细胞的转染效率;以绿色荧光蛋白作为标签鉴定目的基因在细胞中表达的方法切实可行。

In order to obtain highly transfection efficiency and detect the expression of target fragment,the recombinant plasmid pEGFP-C2/LAT-ORF3 was transfected into Vero cells by Xfect as mediation.The recombinant plasmid pEGFP-C2/LAT-ORF3 was transfected into Vero cells mediated by Xfect.After 48 hours observing expression of the recombinant plasmid in the cells by fluorescence microscope,the transfection efficiency was calculated by using transfection methods with yes-no blood serum and different proportions of plasmid and Xfect Polymer.Then the expression of ORF3 was detected by RT-PCR.Results showed that the highest transfection efficiency was obtained when the system had blood serum and the proportion of plasmid and Xfect Polymer was 5 μg/2 μL by using adherent transfection.The target band was obtained by RT-PCR,which indicated the expression of ORF3 in Vero cells.It proved that the optimal condition obtained by the experiment could obviously increase the transfection efficiency.It was feasible to use green fluorescent protein as the mark to identify the expression of target DNA in cell.