猪PDCD5基因的克隆与真核表达

Molecular Cloning and Eukaryotic Expression of Porcine PDCD5 Gene

用RT-PCR方法从猪肝组织中扩增出猪PDCD5(programmed cell death 5)编码序列,TA克隆至pMD-19T载体中,软件分析猪PDCD5基因的核算序列和蛋白序列,进行染色体定位.构建真核表达载体,将PDCD5基因编码区序列插入绿色荧光蛋白报告基因的真核表达载体pEGFP-C1中.通过脂质体转染法将重组载体瞬转入猪脐静脉血管内皮细胞系(SUVECs)进行瞬时表达.结果表明:该序列编码125个氨基酸,猪PDCD5基因定位于猪6号染色体,含有6个外显子,与人PDCD5基因高度同源.双酶切鉴定和测序表明:重组真核表达载体构建成功,荧光检测和Western blot检测显示PDCD5融合蛋白表达.研究结果为探讨猪PDCD5基因在细胞凋亡调控中的功能提供了基础数据.

An experiment was made to provide basic data for the study of the functions of PDCD5 gene in apoptosis regulation.Porcine PDCD5 gene was obtained by RT-PCR from porcine liver tissue,and the PCR product was cloned into T-easy vector pMD-19T.A software was used to analyze its nucleic acids and protein sequences,and the chromosomal location of porcine PDCD5 was determined.Then the PDCD5 gene was subcloned into the eukaryotic expression vector pEGFP-C1 of the green fluorescent protein（GFP） reporter gene encoding green fluorescence protein.Finally,it was transfected into SUVECs using Lipofectamine2000.The results showed that this gene encoded 125 amino acids,was located on porcine chromosome 6 and was highly homologous to that of human PDCD5 gene.Recombinant vector pEGF-PDCD5 showed the correct orientation and sequence.The expression of GFP in SUVECs transfected with pEGFP-PDCD5 was observed by fluorescence microscopy and detected with western blot.