The Study Of The Human Endothelial Cell Specific Molecule-1 Expression In MEK Transfected Endothelial Cells

Objective To construct the recombinant vector which can express human endocellular-specific molecule-1 (ESM-1) in E.Coli, express and purity the target protein in vitro and prepare the anti-ESM-1 polyclonal antibody and monoclonal antibody. To transfect the recombinant MEK (mitogen-activated protein kinase/ external-signal regulated kinase kinase) gene of different activity into endothelial cells and examine the expression quantity of ESM-1 in transfected cells. And to study the relation between ESM-1 and MAPK signal transduction and tumor metastasis. Methods PCR was used to amplify ESM-1 DNA, and the product was ligated into the pGEM-T-Vector to get the recombinant plasmid pGEM-T-ESM-1. After proved by restrictive endonuclease mapping, the recombinant plasmid was amplified, digested by the restriction endonuclease( EcoRI and Hind III ). And the target gene, ESM-1, was inserted into pET-28b to construct expression vector. The positive clones were screened by blue-white color screen and antibiotic resistance, and were proved by restrictive endonuclease mapping analysis and DNA sequencing. IPTG was used to induce the ESM-1 expression. We gather the special bands from the gels after SDS-PAGE to immune the New Zealand rabbit with the target protein which was purified by elctroelution to generate the antibody and measure the tilter of the polyclonal antibody by immnodiffusion. At the same time, we immune the Balb/c mouse with purified ESM-1 protein to acquire the anti-ESM-1 monoclonal antibody and measure the tilter of the monoclonal antibody by ELIS A. We transfect the recombinant MEK gene into human umbilic vein endothelial cells ECV304, then examine the change of ESM-1 expression in cells transfected with recombinant MEK gene of differenceactivity .Results By restriction endonucleases mapping analysis and DNA sequencing we demonstrate that the constructed plasmid was the recombinant one. SDS-PAGE showed that the expressed products was about 24kD and the quantity was about 24% of the whole protein of the bacteria. The tilter of the polyclonal antibody is 1:16 (immnodiffusion) . The tilter of the monoclonal antibody is 1:6000(ELISA). After examining the cells by Western blot and immunofluorescent, we find that the cells transfected with the high activity type recombinant MEK gene express ESM-1 and ERK-2 most, the cells transfected with the wild type recombinant MEK gene express ESM-1 and ERK-2 (external-signal regulated kinase-2) more and the untransfected ECV304 cells express ESM-1 and ERK-2 protein least. Conclusion The expressed plasmid was constructed.ESM-1 was expressed in E.Coli, and the polyclonal and monoclonal antibody was prepared successfully. The ESM-1 expression quantity is different in different transfected cells. The cells transfected with the high activity type recombinant MEK gene express ESM-1 most, the cells transfected with the wild type recombinant MEK gene express ESM-1 more and the untransfected ECV304 cells express ESM-1 protein least.These suggest that ESM-1 may be a important compound in MAPK (mitogen-activated protein kinase) signal tranduction pathway.