Preparation Of Recombinant Nerve Growth Factor NGF-DOPA And The Research Of Its Adhesion Property To Conduit Materials

Peripheral nerve injury is still a difficult problem in clinical,and it affects the function of limbs and the quality of life of patients seriously.The development of neural tissue engineering provides a new solution for peripheral nerve repair.Nerve conduit is a common scaffold in neural tissue engineering and biodegradable synthetic polymer material has been a good choice for nerve conduit materials in recent years.However,the bioactivity of these materials is limited and it can not promote nerve regeneration and repair.The nerve growth factor,which has the function of nourishment,is added to the nerve conduit to promoting the growth of nerve cell and the regeneration of myelin sheath.Nerve growth factor(NGF)is a very important nutritional factor in the nervous system that can be applied to a variety of nervous system diseases and nerve injury.NGF is widely used in the repair of central and peripheral nerves because of its nutritional effects to the injured peripheral nerve and the function of promoting regeneration and repair.NGF has a short half-life and it is easily diluted by the surrounding body fluid.Therefore,how the immobilization of NGF on the surface of materials to improve its function is a research hotspot in recent years.Inspired by the strong adhesion of marine mussels,studies have found that the main adhesion components are 3,4-L-dihydroxyphenylalanine(DOPA).We immobilized the nerve growth factor NGF on the nerve conduit with DOPA,which is great significance for the repair of peripheral nerve.In this project,the tyrosine repeats sequences(Tyrosine-Lysine-Tyrosine-Lysine-Tyrosine,YKYKY)are integrated into the end of the NGF by genetic engineering,and then the gene expressed by the E.coli expression system.We purified the target protein NGF-YKYKY by affinity chromatography and transformed tyrosine into DOPA structure by tyrosine hydroxylation.The physicochemical and biological activities of the target protein were detected.Many groups of nerve conduits were prepared by PPC and PLGA material and analyzed by various methods to select a group of composite materials with excellent properties.Finally,the prepared NGF-DOPA was adhered to the PPC-PLGA composite material.A series of tests on the immobilized nerve repair materials were carried out and vitro cell test was made to detect the role of the scaffold in peripheral nerve repair.The experimental results show that the NGF-YKYKY can be expressed in E.coli and the protein was identified as the target protein by specific identification after purification and renaturation.It was found that the addition of YKYKY and DOPA did not affect the bioactive functional zone of NGF.The PPC-PLGA composite membrane was prepared by solution replacement with uniform distribution,good flexibility and high porosity.The results showed that the PPC-75 PLGA composite membrane,which has the best mechanical properties and cell compatibility,is an excellent choice for nerve conduit scaffolds preparing.The adhering NGF-DOPA was fixed on the PPC-75 PLGA composite membrane and its’ properties were detected by ELISA method,the hydrophilic material changes and XPS method.Compared with ordinary NGF and NGF-YKYKY,NGF-DOPA has stronger adhesion.The cell experiment results show that NGF-DOPA adhered on the surface of the material has good biological functions,which can promote the adhesion of Schwann cells and promote the expression of related neurotrophic factors.The composite scaffold containing NGF provides a good repair environment for nerve regeneration that can promotes axonal regeneration and reconstruction.