Construction of human Par-4 eukaryotic expression vector and expression in K562 cells

VernacularTitle:人类前列腺凋亡反应因子4基因真核表达载体的构建及其在K562细胞中的表达
Author: Jie QIN ; Hongwei WANG ; Tao YANG ; Lei ZHU ; Li ZHANG
Publication Type:Journal Article
Keywords: Par-4 gene; Eukaryotic expression vector,Transfection; K562 cell
From: Journal of Leukemia & Lymphoma 2009;18(1):12-14
CountryChina
Language:Chinese
Abstract: Objective To construct an eukaryotic expression vector of human Par-4 gene with green fluorescent protein gene which iS named pIRES2-EGFP/Par-4 and transfect jt into K562 cell line. Methods Using pDNR/Par-4 plasmid as a template.the full length Par-4 cDNA was amplified by PCR and subsequently cloned into T-A vector.Then subcloned into pIRES2-EGFP vector.After identified by digestion of restriefive endonucleases.pIRES2-EGFP/Par-4 was further confirmed by sequencing.Then it was transfected into K562 cells with Superfect reagents.The total proteins were isolated and Par-4 was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/Par-4 vector were confirmed by digestion of restrictive endonucleases and sequencing.After transfection,the expressions of green fluorescent protein were present.The protein expression of Par-4 has been detected in transfected ceils hv Western blotting.Conclusion The vector pIRES2-EGFP/Par-4 has been constructed and could Successfully express Par-4 gene in K562 cells.