Preparation And Characterization Of Recombinant Elastin And Its Hydrogel

Elastin,as a type of structural and functional protein in extracellular matrix,is widely found in connective tissues such as blood vessels,lungs,ligaments and skin of mammals.It is responsible for the tissues in maintaining their morphology,enabling their expansion and deformation,and improving their strength and toughness.Correspondingly,elastin,as an ideal raw material,has great medical potential due to its sound biocompatibility and low immunogenicity.Elastin prepared by gene recombination technology,which has been deemed a hitherto technique of optimum,possesses superiority in controllable sequence,low cost,mass production and low immunogenicity.Therefore,the field of tissue engineering embraces a wide range of hotspot issues coping with efficiently expressing and purifying elastin in prokaryotic recombinant system and preparing elastin adheres to high medical standards in mechanical strength and bioactivity.This paper writing aims to design and develop an efficient elastin sequence expression system,and prepare elastin hydrogel with high mechanical strength and sound biocompatibility,providing new application strategies for the research and development of skin repairing dressings,artificial blood vessels,artificial skin and other medical materials.The main contents are as follows:1.Full-length human elastin being taken as the main reference source for sequence design,key domains was selected to construct human Recombinant Elastin(RE)sequence.Added Sc12 induced folding domain(V-Foldon)into the N-terminus of RE sequence to construct Recombinant Fusion Elastin(RFE)and used the recombinant system of Escherichia coli(BL21)for efficiently expression.After further purified by nickel ion affinity chromatography,RE with high purity was obtained.2.Natural elastin has the character of reversible phase transformation.Reversible phase transformation experiments showed that RE had temperature-sensitive aggregation behavior similar to that of natural elastin.The result of circular dichroism indicated that the secondary structure of natural elastin could be found in RE.On this basis,RE should be crosslinked with Tetrakis Hydroxymethyl Phosphonium Chloride(THPC)to prepare elastin gel materials with stable properties and excellent mechanical properties.Scanning electron microscopy(SEM)showed that the gel material had regular and uniform three-dimensional mesh-shaped structure on the microscopic scale.Cell experiments showed that the material had good biological activity such as supporting cell survival and proliferation on the surface of the material.At the same time,the results of animal experiments showed that the gel had no immune rejection reaction,which consequently reduced the inflammation of the wound surface effectively,promoted the healing and regeneration process of the damaged skin of mice,thus would have high clinical application value in dermatology and other fields.3.RFE had temperature-sensitive aggregation capacity of reversible phase transformation as well as RE.In the meantime,the V-Foldon domain significantly increased the ordering of the secondary structure and regulated the phase transformation aggregation process of elastin,making it appear in the form of collagen "trimer",thus reducing the phase transformation temperature of the recombinant protein,rendering it more similar to natural elastin.In addition,compared with human RE,RFE possessed higher protein expression,more similar biological activity and cross-linking ability.The gel material formed by RFE also had a better skin repairing result on UV damage model of mice skin.