Immobilization Of Heparin Onto The Surfaces Of Polycaprolactone And The Expression Of Five Heparin Modification Enzymes

Polycaprolactone(PCL),a kind of biodegradable polymer material,is widely used in biomedical fields.In order to enhance the biocompatibility of PCL,it is necessary to immobilize heparin molecules on PCL surfaces.Heparin molecules were immobilized on PCL surfaces by the method of end-point attachment through the Schiff base reaction.Measured by the toluidine blue staining method,heparin immobilization efficiency reached 4.8?g/cm~2.X-ray photoelectron spectroscopy(XPS)and Fourier infrared spectrum(FTIR-ATR)were used to further test material structures.XPS analysis showed that the heparin coating increased nitrogen,sulfur and sodium signals compared with PCL.The FTIR-ATR analysis further showed PCL surface increased C-N and N-H vibration peaks.These experimental results proved that heparin molecules had formed covalent bonding with PCL.In order to study the structure and function of immobilized heparin,gor trxB mutant Escherichia coli was constructed to express four kinds of sulphate transferase and C5-epimerase.Glutathione reductase(Gor)and thioredoxin reductase(TrxB)are two important enzymes in the redox system.Knockouting trxB gene and gor gene can block the redox pathway in Escherichia coli cells,enhance the formation of disulfide bonds and ensure the normal folding of exogenous proteins.We constructed the gor trxB mutant strain by molecular biology techniques.Four kinds of sulphate transferase and C5-epimerase were successfully expressed and purified by the gor trxB mutant strain.These four kinds of sulphate transferase and C5-epimerase can be used to explore the structure and biological activity of PCL-heparin in later period.End-point attachment method can immobilize heparin on PCL surface efficiently and laid the foundation for its further functional and applied research.