Research On Expression And Mineralization Of Amelotin

Human tooth enamel is the most hard mineralized tissue in the body and the outermost layer in human denture.Reconstructing enamel-like structures to restore denture has already been an important topic of study in the material sciences and dentistry.Biomimetic mineralization induced by the organic macromolecules as template,to achieve dental restoration,has a very important clinical significance.In this study,AMTN gene was cloned and expressed by gene recombination technique.The purified high purity AMTN protein was used as an organic template for inducing the growth of hydroxyapatite crystals on the dental tissue.The work in this thesis is as follows:1.By extracting the total RNA from the granulosa tissue of enamel development stage and using reverse transcription cDNA as a template,we cloned successfully the AMTN gene.The AMTN gene fragment was cloned into pMD18-T vector,to construct successfully the pMD18-T-AMTN cloning vector.The results of sequencing showed that the cloned AMTN gene was consistent with the AMTN gene published on NCBI.The AMTN gene was then subcloned into pET-28a(+)vector,we also successfully constructed the prokaryotic expression vector of p ET-28a(+)-AMTN.2.The pET-28a(+)-AMTN prokaryotic expression vector was transferred into competent cell E.coli BL21(DE3)to express the recombinant AMTN protein by IPTG induction and optimize expression conditions.SDS-PAGE analysis showed that AMTN protein was almost completely expressed as an inclusion bodies in E.coli BL21(DE3)strain.We optimize the expression conditions,the recombinant prokaryotic expression vector was transferred into host E.coli RossetaTM,to induce the expression of AMTN gene and optimize expression conditions.SDS-PAGE analysis showed that the optimal expression condition was induced by 0.2 mmol/L IPTG for 16 h at 16?.The AMTN protein can be expressed in a partially soluble form in the RossetaTM strain.3.According to the optimized expression conditions,a large number of AMTN protein expression was induced.The AMTN protein in the supernatant of the product was purified by Ni-Agarose affinity chromatography.The purified protein was further purified by Resource Q anion exchange chromatography,and the purity of the purified protein was as high as 90%.Western blotting analysis confirmed that the protein could be specifically recognized by rabbit anti-histidine monoclonal antibody,and the molecular weight is 28 kDa,indicating that the protein is AMTN protein.4.AMTN protein solution(100?g/ m L)was incubated at room temperature for self-assembly.Observed with JEOL 2100 TEM,found that AMTN protein from scattered monomer agglomeration for nanospheres,and then assembled as beaded.Dental slices about 1mm were first etched with 37% phosphoric acid for 60 second,followed by immersion ina 2 mg/m L freshly prepared AMTN protein solution at 4? for 30 min.And then we immersed the dental slices in the mineralization solution formineralization.The precipitates were characterized by SEM and XRD,the results show that the newly precipitates crystals are HA crystals.