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Recombinant human ameloblastin expression and Study

Author: TianYu
Tutor: XiaoMingZhen
School: Fourth Military Medical University
Course: Clinical Stomatology
Keywords: Ameloblastin Reverse transcription -PCR Recombinant DNA Clone Gene Expression Protein purification Western blotting cDNA probe ALP Pulp Cells Periodontal ligament cells
CLC: R780.2
Type: PhD thesis
Year: 2002
Downloads: 84
Quote: 3
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Abstract

Enamel is the hardest tissue in mammals, the epithelial and mesenchymal interactions product. Enamel-specific extracellular matrix in the hydroxyapatite crystal formation and growth play an important role. Ameloblastin (ameloblastin, also known amelin, sheathlin) by ameloblasts synthesize and secrete extracellular matrix proteins, rat Immunohistochemical studies have shown that it is in the late secretory phase and secretory ameloblasts are highly expressed, ameloblastin mainly located in enamel prism sheaths that ameloblastin role may be controlling the crystal growth rate, maintain the growth of crystals and decided enamel prism structure, but its role in the molecular mechanism is unclear. Currently, cloned rats, mice, cattle, pigs, human ameloblastin gene related ameloblastin function studies are increasingly concerned by scholars. In this study by RT-PCR from human tooth tissue total RNA cloning human ameloblastin genes in prokaryotic and eukaryotic cells was carried out into the glazed expression of prokaryotic expression of ameloblastin C-terminal peptide was purified prepared anti-human ameloblastin antibody, labeled cDNA probes, carried out on human tooth tissue protein blotting (Western Blot), immunohistochemistry and in situ hybridization studies, mRNA and protein levels from the study of human teeth embryos ameloblastin expression features, while using the obtained protein studied outside ameloblastin cultured human dental pulp cells and the biological effects of periodontal ligament cells for further research into the physiological function of enamel proteins laid foundation. The study is divided into the following four parts: First, the human ameloblastin gene analysis using guanidine isothiocyanate-step fetal age from May tooth tissue extracted total RNA, using Oligo (dt) as a primer reverse transcription tooth cDNA, and then using PCR, using a pair of primers amplified from the cDNA of Chinese people ameloblastin gene encoding fragment (about 1.3kb), the amplified gene fragment was inserted into pGEM-T Easy clone vector, transformed into E. coli DH5α, positive clones, plasmid DNA, were identified by restriction enzyme digestion and nucleotide sequencing. The results showed that: a cloned human enamel protein coding region gene fragment sequences in GenBank human ameloblastin Fourth Military Medical University doctoral thesis. 4. CDNA sequence of interest. Second, the human ameloblastin gene in prokaryotic and eukaryotic cells, respectively, cloned human ameloblastin full coding region fragment encoding the N-terminal peptide ameloblastin the CDNA fragment encoding the C-terminal peptide ameloblastin the CDNA fragment was ligated into an expression vector pLCM182, pGEX * T4 and pRSET-A in E. coli and induced expression in 12% SDS-PAGE gels expressed protein bands of relative molecular weight of 65 XIO ', 46 XIO', 52 XIO ', in which the expression of ameloblastin C-terminal peptides in the host bacteria is present in soluble form, its a lot of induction and purified to obtain a high-purity ameloblastin C-terminal peptides. To restructure the purified protein C-terminal peptide into the glaze as antigen mixed with complete / incomplete Freund's adjuvant to immunize New Zealand white rabbits, will be polyclonal antisera by ammonium sulfate preliminary purification, the Western Blot detection of human ameloblastoma proteins in tissue-specific expression. Western Blot Show: ameloblastin in human tooth tissue-specific expression of the total protein in the brain, heart, liver, spleen, lung, kidney, pancreas, thymus, no expression in skeletal muscle and other tissues bands. Respectively, the human ameloblastin non-fusion and fusion gene was cloned into the eukaryotic expression vector pCDNA3 * and PEGFP-C3, the confirmed clone is correct with the carrier liposome-mediated transfection of mammalian cells COS Diao, by immunohistochemistry , Western Blot and other methods to detect ameloblastin expression in mammalian cells. The results, expressed GFP fusion protein fluorescence emitted in the cytoplasm, the nucleus of non-fluorescent, consistent with the results of immunohistochemistry, indicating that human ameloblastin located in the cytoplasm. WesternBlot detection appears in COSJ cells expressed ameloblastin molecular weight of 65X10 \\ secreted into the culture medium ameloblastin a molecular weight of 62X 10 \\ Third, the human ameloblastin tissue expression analysis using anti-human ameloblasts prepared antibody against human tooth tissue sections and dog periodontal tissue sections of immunohistochemical staining was found ameloblastin from the inner enamel epithelium cells differentiate into mature expression began before ameloblasts, secretory ameloblasts highly expressed and continues throughout enamel formation, the newly formed enamel staining; odontoblasts begin before the secretion of dentin matrix protein expressed as enamel, dentin formation in odontoblasts constitutively express ameloblastin, weakly positive, pre-dental essence weakly positive staining: epithelial root sheath cells staining. Dog periodontal tissue staining cementum surface cells were strongly Dopar. ent ofoperatjVE plant enthay \u0026 J blood from me. in = rFWU Fourth Military Medical University doctoral thesis -5 - positive staining is visible in the newly formed cementum positive staining granules reticular distribution. CDNA fragment cloned after double digestion was labeled with digoxigenin labeled probes. Paraffin sections of human tooth germ tissue in situ hybridization showed that ameloblastin in ameloblasts, odontoblasts, some pulp cells, epithelial root sheath cells in the expressed position consistent with the results of immunohistochemistry. Fourth, the expression and purification of human dental pulp cells ameloblastin and periodontal ligament cells to understand the biological role of this protein during tooth development role in the process, the use of four bite salt assay and solid-phase binding assays we observed the training human periodontal ligament cells and recombinant human dental pulp cells in the pack is ameloblastin adhesion and spreading on the surface of the case and found that ameloblastin can promote human periodontal ligament cell attachment, do not promote their stretch of human dental pulp cells The adhesion and spreading without facilitating role. Four Mile salt using colorimetric observed ameloblastin on human periodontal ligament fine?

Recombinant human ameloblastin expression and Study

Abstract

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