Construction of Fibulin-5 eukarvotic expression vector and establishment of its stable transfected bladder cancer cell line

Abstract

Objective To construct eukaryotic expression vector of Fibulin-5 and establish stable human bladder cancer cell line.Methods Fibulin-5 cDNA in hDANCE_CFLAG plasmid was amplified by polymerase chain reaction (PCR).The amplified products were cloned into pMD-19T simple vector.The pMD-19T-Fibulin-5 vector was cut by Xho Ⅰ and EcoR Ⅰ to generate a Xho Ⅰ-Fibulin5-EcoR Ⅰ fragment that was ligated into enhanced green fluorescent protein carrier ( p-EGFP-N1 ) plasmid to synthesize p-EGFP-Fibulin-5 plasmid,which was then cut by Bg Ⅲ.Finally,the p-EGFP-Fibulin-5 plasmid was transfected into bladder cancer cell line 5637 by liposomes.Results The length of PCR products was 1429 bp.The results of DNA sequencing for the fragments by Xho Ⅰ and EcoR Ⅰ suggested the correct insertion of Fibulin5 cDNA.A band with about 450 bp was generated after p-EGFP-Fibulin-5 plasmids were digested by BgⅢ.The successfully transfected cells by pEGFP-Fibulin-5 or pEGFP-N1 emitted green fluorescence.Conclusion Fibulin-5 eukaryotie expression vector was successfully constructed and transfected to human bladder cancer cell line. Key words: Bladder carcinoma; Eukaryotic expression vector; Transfection; Fibulin-5