The Expression And Purification Of Soluble Endocan And Influence Of OPN In Transfected MDCK Cells

Objective: To construct the recombinant vector which can express Endocan in E.Coli, express and purify the target protein in vitro. Eukaryotic expressing plasmid pcDNA3.1-Endocan was constructed, and transfected into MDCK, to observe the expression of Endocan and analyse its influence of OPN.Methods: PCR was used to amplify Endocan cDNA, and the product was ligated into the pET32a. After identified by restrictive endonuclease mapping, the recombinant plasmid was amplified for being prepared and digested by KpnI and XhoI. Endocan was inserted into pET32a to construct the expression vector. The recombinant vector was transformed into DH5α. Positive clones were screened by antibiotic resistance, and were proved by DNA sequencing. After amplification, pET32-Endocan was transformed into Rosetta-gamiTM(DE3). Positive clones were screened by antibiotic resistance, and were proved by PCR. IPTG was used to induce the expression. Supernatant was purified by Ni+ column, which was checked by SDS-PAGE. Endocan full length cDNA was amplified using PCR from pet28-Endocan, subcloned into pcDNA3.1/myc-his A, transient transfected into MDCK cells. Western blot was used to detect the expression of Endocan and OPN.Results: The expressed vector was constructed, Endocan was expressed in Rosetta-gamiTM(DE3) .SDS-PAGE showed the expressed product is about 40kD and the quantity is about 84% of the whole protein of the bacteria.100mM imidazole eluted it from Ni+ column. The construction of pcDNA3.1-Endocan has been successfully established by double enzyme digesting and sequencing. The construction of pcDNA3.1-Endocan has been successfully established by double enzyme digesting and sequencing. The trasfected cells expressed more Endocan and OPN.Conclusion: The recombinant plasmid pET32-Endocan and pcDNA3.1-Endocan was identified by DNA sequencing. Endocan fusion protein was expressed in Rosetta-gamiTM(DE3),and initially purified by Ni+ column. The trasfected cells expressed more Endocan and OPN.