Clonging And Expression Of Human Ameloblastin And A Pilot Study On Its Biological Effects

Enamel is the hardest tissue in mammalian. This specialized structure develops through a series of reciprocal epithelial-mesenchymal interaction. The tissue-specific extracellular matrix synthesized by the ameloblasts facilitates the initiation and orientation of inorganic hydroxyapatite crystallites. Ameloblastin (also known as amelin or sheathlin), an extracellular protein that is synthesized and secreted by ameloblasts, is expressed at high levels in secretory and post-secretory ameloblasts. Immunohistochemistry using antibodies raised against porcine sheathlin extracted from newly formed enamel revealed that they concentrate in the prism sheath space. So it is proposed that ameloblastin regulates the speed of crystal elongation, and stabilizes the growing crystal. It may also decide the prism structure. A precise role for ameloblastin during enamel organic matrix formation is not yet known. Now the ameloblastin cDNA were cloned in mouse, rat, procine and human. But the biological functions of ameloblastin are still poorly characterized. In this study, we cloned the human ameloblastin cDNA by RT-PCR, expressed ameloblastin in E.coli and mammalian cells, purified the recombinant protein, prepared anti-ameloblastin antibodies and labeled a ameloblastin cDNA probe. Then Western blot , immunohistochemistry and in situDepartment of Operative Dentistry & Endodontics-7-hybridization studies were proceeded. Finally, a variety of biological effects of ameloblastin on human dental pulp cells and periodontal ligament cells were observed. This paper includes following 4 parts:1 Cloning and sequencing of human ameloblastin geneIn this study, total RNA was extracted from the tooth germ of 5 months human embryo by acid guandinium thiocyanata-phenol-chloroform method, the desired cDNA products were obtained from the total RNA by RT-PCR with the primers including Olido(dt) and two gene specific primers respectively. The segment(about 1.3kb) was inserted into pGEM-T Easy vector and the recombinant plasmid was transformed into E. coli DH5 a . The positive clones were analyzed with restriction endonuclease mapping and DNA sequencing. The results showed that the nucleotide sequence we cloned were consistent with the GenBank record.2 Expression of human ameloblastin in E. coli and mammalian cellsThe open reading frame of ameloblastin cDNA, the fragment encoding the terminal N peptide of ameloblastin and the fragment encoding the terminal C peptide of ameloblastin were inserted into prokaryotic expression vector pLCM182, pGEX-4T-l and pRSET-A respectively. The recombinant plasmids were transformed into E. coli DH5 a or E. coli BL21(DE3) and induced by raising temperature to 42 癈 or by IPTG according to the expression vector used. After induction, new protein bands appeared on SDS-PAGE gel near 65kDa, 46kDa and 52kDa respectively. The expressed terminal C peptide of ameloblastin was soluble and the cells were induced in large scale. The expressed product was purified and used to immune New Zealand White rabbits with Freund's complete adjuvant to produce antibody. Western blot using this antibody revealed that ameloblastin was specifically expressed in tooth germ but not in brain, heart, liver, spleen, lung, kidney, pancreas, thymus or muscle in human.Department of Operative Dentistry