Eukaryotic Expression Of Human Endothelial Cell Specific Molecule-1 And Preliminary Research In Tumor Tissues

Objective To elucidate expression of endothelial cell specific molecule-1 (hESM-1) in tumor tissues and expression and significance of hESM-1 in renal cell carcinoma. To construct the eukaryotic expression vector of pIRES-hESM-1 which can express hESM-1. Methods The monoclonal antibody against hESM-1 was purified with saturated ammonium sulphate and CM-Sepharose FF. The expression of hESM-1 was determined in eight kinds of tumors by Western Blot with the purified monoclonal antibody against hESM-1 as the first antibody. Hybridization in situ and S-P immunohistochemical methods were applied in renal cell carcinoma and normal renal tissue. The target gene was digested by the restrictive endonuclease(EcoR I and Not I) from recombinant plasmid of pET28b-hESM-l and purified by electricelution. Then it was inserted into pIRES to construct eukaryotic expression vector and transformed into E.coli XL1-blue. The positive clones screened by blue-white color screening and antibiotic resistance were proved by restrictive endonuclease mapping analysis and DNA sequencing. The recombinant plasmid pIRES-hESM-1 was transfected into ECV304 by liposomes method. Expression of hESM-1 was detected by in situ hybridization and immunocytochemistry. Results The purity of the monoclonal antibody against hESM-1 was 95% and the concentration was 1.24mg/ml. The hESM-1 protein was expressed in hepatcellular and renal cell carcinoma of the eight kinds of tumors. The hESM-1 mRNA and protein expressed in renal cell carcinoma were located in the vascular endothelial cells. The expression of hESM-1 was higher than that of normal renal tissues. The recombinant plasmid was identificated by restrictive endonucleases mapping analysis and DNA sequencing. The expression of hESM-1 was obviously increased in the level of transcription and protein. Conclusions The monoclonal antibody against hESM-1 is obtained with high purity and concentration. Expression of hESM-1 is exsisted in hepatocarcinoma and renal cell carcinoma, it is related to the occurrence and development of renal cell carcinoma which may be regard as one of markers of its biological behaviors. The eukaryotic expression vector was constructed and transfected into ECV304 successfully. High level of expression of hESM-1 is obtained in ECV304 which lays a good basis for further research.