Human Plasminogen Kringle 5 cDNA Clone with Eukaryotic System and Identification of Its Expression Product

Purpose To construct eukaryotic expression vector coding human plasminogen kringle 5 cDNA and investigate its inhibitory effects on human umbilical vein endothelial cell line(ECV304) as well as human breast adenocarcinoma cell line(MDA?MB?231) by liposome-mediated gene transfer. Methods The K5 cDNA was fused in-frame with human plasminogen signal sequence by PCR and it was inserted into an eukaryotic expression vector pcDNA3,the recombinant plasmid named pcDNA3K5 was assayed by restriction endonucleases and sequenced.Subconfluent human breast carcinoma cells (MDA?MB?231) were transfected with pcDNA3K5 and pcDNA3 by liposome method.Transfected cells were selected at 1 mg/mL G418 in DMEM containing 10%FBS for 2 weeks.Single cell clones were picked and expanded in the pre-sence of G418.The positive clones were verified by PCR.The expression of K5 mRNA and protein was assayed by RT-PCR and Western blot.The 72 h conditioned media of the positive clones were applied to ECV304 testing their antiproliferation effects and assayed by MTT.The proliferation of pcDNA3K5 transfected MDA?MB?231 was also assayed by MTT. Results The recombinant plasmid pcDNA3K5 was verified by restriction endonuclease analysis and sequencing.After it was transfected into MDA?MB?231,three positive clones were verified by PCR.RT?PCR and Western blot detected the expression of K5 mRNA and protein.The conditioned media of the positive clones inhibited the proliferation of ECV304.In contrast,the proliferation of pcDNA3K5 transfected MDA?MB?231 was not affected. Conclusions The biologically active K5 secreted by pcDNA3K5 transfected MDA?MB?231 showed inhibitory effects on the proliferation of ECV304 but had no effects on the proliferation of MDA?MB?231.