Study On The Expression,Purification And Guiding Of Biomimetic Mineralization Of Human Ameloblastin C Telopeptide

Experiment 1 Prokaryotic expression and optimization of expression conditions of human ameloblastin C telopeptideOBJECTIVE:AMBN-C prokaryotic expression conditions were optimized to obtain high expression protein.Methods:1.The pET15b-AMBN-C recombinant plasmid was transferred into E.coli BL21?DE3?and cultured.2.When the OD value of the bacterial solution reaches 0.6,0.5mm IPTG is added to it and cultured at different temperatures and times;The expression conditions of pet15b-ambn-c recombinant plasmid were determined by SDS-PAGE and Western Blot detection results.Results:1.The pET15b-AMBN-C recombinant plasmid was successfully transferred into E.coli BL21?DE3?,and the recombinant expression bacteria were cultured.2.Determining the optimal expression conditions:when the OD value of the bacterial solution reaches0.6,0.5mm IPTG was added to it and cultured at 20?for 16hours.AMBN-C recombinant protein is expressed in the form of inclusion bodies.Experiment 2:Mass expression,purification and identification of AMBN-C recombinant proteinObjective:To obtain AMBN-C recombinant protein with high expression and purity.Methods:1.When the OD value of the bacterial solution reaches0.6,0.5mm IPTG was added to it and cultured at 20?for 16 hours.AMBN-C is expressed in the form of inclusion bodies.2.The inclusion bodies was lysed,washed,and dissolved to obtain soluble AMBN-C recombinant protein;3.The AMBN-C recombinant protein was restored to its normal structure and biological activity by dialysis renaturation.4.The AMBN-C recombinant protein was purified by Ni²⁺affinity chromatography and detected by SDS-PAGE.Results:SDS-PAGE showed that AMBN-C recombinant protein was obtained after purification by Ni²⁺affinity chromatography,and its concentration was 0.66 mg/ml.Experiment 3 AMBN-C recombinant protein guides enamel biomimetic remineralizationObjective:To study the ability of AMBN-C recombinant protein to guide enamel biomimetic remineralization.Methods:1.Observing the self-assembly of AMBN-C protein and its crystallization in artificial saliva by transmission electron microscopy?TEM?;2.Using AMBN-C as a template for biomimetic mineralization,mineralized in artificial saliva for 7 days.Scanning electron microscopy?SEM?was used to observe the structure of the new layer,and X-ray diffraction?XRD?to characterize the new layer.Results:1.When the pH of the AMBN-C protein solution changed from 3.5 to 7.6,the protein gradually agglomerated into oligomers from the initial monomers,and then further self-assembled into nano-microspheres.And finally assembled into a beaded network structure;2.When the pH of AMBN-C protein in artificial saliva changes from 3.5 to 7.6,it crystallized with Ca²⁺?P⁺,and the protein-mineral aggregates formed nano-chains,which were further assembled into plate-like mineral aggregates.3.AMBN-C is used as a biomimetic template to guide the remineralization of enamel in artificial saliva.SEM and TEM showed that:AMBN-C guided the formation of hydroxyapatite crystals along the C axis;XRD further confirmed that the new layer was hydroxyapatite crystals.