Preparation And Properties Of Recombinant Collagen Protein And Its Biomaterials

Collagen is a biomacromolecule in the extracellular matrix of animals and is important for maintaining cell structural integrity and other biological functions.In humans,collagen accounts for about one third of the total protein of the body,and is an important structural protein in many extracellular matrices of connective tissue.Over the past few decades,collagen has been widely used in a variety of formats as a safe biomedical material and has been universally recognized by clinicians and patients.However,natural collagen is mainly derived from animal tissue,and has the risk of pathogens or prion contamination,thus,the further development of biomedical materials based on natural collagen is limited.In recent years,researchers have found a new type of collagen with a characteristic triple helix structure of collagen in bacteria,which can form a stable triple helix structure in the absence of hydroxyproline.More importantly,bacterial collagen-like protein can be produced from the E.coli expression system in a high yield by recombinant technology,and the recombinant collagen has many excellent properties,such as non-immunogenic,non-cytotoxic,and so on.And the scientific data provided so far show that recombinant bacterial collagen-like proteins have significant potential to become clinically effective biomedical materials.Therefore,in the study of this paper,we have prepared recombinant collagen and its related biological materials,then its physical and chemical properties were studied.The main contents of the paper include the following three parts:1.The recombinant collagen protein VCL was expressed in E.coli BL 21 strain,then purified using a NI-NTA affinity chromatography column.The recombinant collagen CL with only a triple helix domain was obtained by excising the non-collagenous domain(V domain)in VCL with trypsin.Then CL was characterized by sodium dodecylsulfonate polyacrylamide gel electrophoresis(SDS-PAGE),Fourier transform infrared spectroscopy(FTIR)and circular dichroism(CD).As a result,the purified recombinant collagen protein has high purity,and can form a complete triple helix structure.The melting temperature of the three-helix structure at pH 3.0(31.4?)was lower than pH7.0(35.7?).2.Polyethylene glycol(PEG),modified chondroitin sulfate(ChS-ADH),and CL based hydrogels PEG-ChS-CL were prepared by chemical crosslinking,and hydrogels PEG-ChS without CL were also prepared.Scanning electron microscopy(SEM)was used to observe the morphology of the gel.The rat 5 mm skull defect model was constructed and then implanted with two different types of gels for investigating the effect of CL on the repair of bone defects.We used the computer tomography(CT)and magnetic resonance imaging(MRI)to observe the osteogenesis effect of the model under different time periods.And the results showed that PEG-ChS-CL gel had a significant effect on bone defect repair compared with PEG-ChS gel.The results of HE staining futher demonstrated the promoting effect on bone repair of PEG-ChS-CL hydrogel.This study provides a new idea for recombinant collagen protein CL as a biomedical material for the repair and treatment of bone defects.3.We synthesized fluorescein isothiocyanate(FITC)doped fluorescent silica nanoparticles(FNPs),and then modified it with recombinant collagen protein CL,CLCL and polypeptide POG of variable lengths.The effects of recombinant collagen with different length on the morphology,particle size and maximum emission wavelength of FNPs were studied by different characterization methods.And the results of MTT assay showed that the recombinant collagen modified FNPs had low cytotoxicity and showed good biocompatibility.Thus,they were expected to be used in biomimetic imaging system.