人类ZNF434基因的克隆、真核表达和组织表达谱分析

Cloning,Eukaryotic Expression and Expression Profile Analysis of Human ZNF434 Gene

目的:克隆人类ZNF434基因并构建其真核表达质粒,鉴定ZNF434基因的组织表达谱。方法:实时定量PCR检测ZNF434基因在人体10种组织中的表达情况;克隆ZNF434基因的全长开放读码框区并连入真核表达载体pIRES2-EGFP,磷酸钙沉淀法转染HEK293T细胞,荧光显微镜和Western blot鉴定ZNF434蛋白的表达情况。结果:ZNF434基因在检测的人体组织中均有表达,其中骨髓和睾丸表达最高,成功克隆了ZNF434基因并构建了该基因的真核表达质粒ZNF434-pIRES2-EGFP,转染HEK293T细胞可观察到绿色荧光蛋白表达,Western blot可检测出分子量56kDa的Flag-ZNF434融合蛋白表达。结论:明确了人类ZNF434基因的组织表达谱,构建了该基因的真核表达载体,为该基因的进一步研究奠定了基础。

Objective： To construct human ZNF434 gene eukaryotic expression vector and identify its tissue expression profile. Method： Ex- amine the mRNA expression of ZNF434 in 10 human tissues by real - time PCR. The full - length human ZNF434 open reading frame was cloned and constructed into eukaryotic expression plasmid pIRES2 - EGFP. The recombinant plasmid was transfected into HEK293T cells by calcium phosphate method. The expression of EGFP tag was observed by fluorescence microscopy. Western blot were pefl＇ormed to detect the expression of ZNF434. Result：ZNF434 mRNA was expressed in all detected human tissues and highly expressed in bone marrow and testis. The human ZNF434 eukaryotic expression vector ZNF434 - pIRES2 - EGFP was constructed successfully. Green fluorescence was detected in transfected HEK293T cells. Western blot showed the 56kDa Flag- ZNF434 fusion protein was expressed at high lev- el. Conclusion Human ZNF434 tissue expression profile is identified and its eukaryotic expression plasmid is constructed successfully, which provide a foundation for the further research of ZNF434 gene.