摘要
背景:核因子κB在肿瘤生成过程中起重要作用,PDLIM2基因可以介导终止核因子κB的活性,解除核因子κB对肿瘤细胞的凋亡抑制作用。目的:克隆人PDLIM2全长基因,构建pIRES2-EGFP-PDLIM2真核表达载体。方法:采用反转录-聚合酶链反应从新鲜膀胱组织总RNA中克隆PDLIM2全长基因,与经BamHI、XhoⅠ相同双酶切的pIRES2-EGFP质粒载体连接,构建重组质粒pIRES2-EGFP-PDLIM2,经酶切及测序鉴定重组质粒中PDLIM2基因的完整性和忠实性。荧光显微镜下观察重组质粒转染的EJ细胞GFP报告基因表达强度,并对转染细胞PDLIM2的表达进行RT-PCR检测。结果与结论:经酶切和测序证实重组质粒构建正确,并在转染的EJ细胞中获得PDLIM2的高效表达。表明用反转录-聚合酶链反应方法成功从膀胱组织中克隆出PDLIM2全长基因,成功构建pIRES2-EGFP-PDLIM2真核表达载体。
BACKGROUND：Nuclear factor-κB（NF-κB） is a key actor in tumorigenesis,PDLIM2 gene can mediate the termination of NF-κB activation and relieve the apoptosis suppression of NF-κB on tumor cell.OBJECTIVE：To clone PDLIM2 gene and construct a eukaryotic expression vector pIRES2-EGFP-PDLIM2.METHODS：PDLIM2 gene,cloned from total of fresh bladder tissue by reverse transcription polymerase chain reaction（RT-PCR）,and pIRES2-EGFP plasmid were digested by Bam HI and Xho Ⅰ double endonucleases and linked with each other.The integrity and accuracy of PDLIM2 in pIRES2-EGFP-PDLIM2 recombinant plasmid was indentified by enzyme digestion and sequencing.The expression intensity of GFP report gene transfected by EJ cells were observed under a fluorescence microscope and the PDLIM2 expression level was determined by RT-PCR.RESULTS AND CONCLUSION：PDLIM2 gene sequence contained in pIRES2-EGFP-PDLIM2 recombinant plasmid was verified correctly by enzyme digestion as well as sequence analysis.After being transfected into EJ cells,highly efficient expression of PDLIM2 gene contained in pIRES2-EGFP-PDLIM2 recombinant plasmid were detected at mRNA level.PDLIM2 gene can be cloned from bladder tissue by means of RT-PCR.pIRES2-EGFP-PDLIM2 recombinant plasmid has been constructed successfully with high efficient expression in transfected EJ cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2011年第7期1205-1209,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
