Expression And Purification And Antiangiogenic Function Research Of The The Human Plasminogen Kringle5Mutant

Objective:Exploret the antiangiogenic effect of the K5mutant protein GST-K5m fusion protein at the cellular level.Methods:Use the primer primor5.0software, PCR method get the K5m genes and cloning the purpose gene into the pGEX-4T-3plasmid BamHI and XhoI restrict site by using gene engineering technology. The pGEX-4T-3/K5m was introduced into E. coli strain BL21(DE3) and soluble proteins were released. The proteins were examined by SDS-PAGE and western blot analysis using an antibody specific to GST-tag, and the results shown that the molecular weight of about37kDa get the protein. In order to validate the biological activities of the protein GST-K5m, we use different concentration (25～1280nmol/L) GST-K5m protein respectively handle umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs), liver cells (Be17402) and normal liver cells (NLC). To assess the prolifeation and induction apoptosis effect of GST-K5m fusion protein to HUVECs, Be17402and NLC, We use the MTT methods and the PI single staining and hoechst-33258staining methods. We detects the effects of the GST-K5m recombinant protein inhibites migration to human umbilical vein endothelial cell, we use GST-K5m fusion protein and His6-K5recombinant protein processing HUVECs.Results:GST-K5m recombinant protein can inhibits proliferation of HUVECs in a concentration dependence manner, however Cell survival rate have no significant change in liver cancer cells and normal liver cells; GST-K5m recombinant protein has no obvious apoptosis induction effection in normal liver cells (NLC) and liver cancer cells (Be17402), but found that GST-K5m fusion proteim has the function of induction apoptosis to endothelial cell; GST-K5m recombinant protein and His6-K5recombinant protein were inhibited the migration of HUVECs.Conclusion:1. Construct the pGEX-4T-3/K5m recombinant plasmid successfully, and gain the GST-K5m recombinant protein by the prokaryotic expression system of the E. coli strain BL21(DE3).2. In vitro studies, our reserch confirm that GST-K5m recombinant protein can endothelial specificaly inhibited endothelial cell proliferation and migration, and induced endothelial cell apoptosis, but have no obvious effect for liver cancer cells and normal liver cells.