A Study On Gene Cloning, Expression And The Distribution Of Tuftelin In Mouse

Tuftelin is one kind of enamel matrix proteins. It has 390 amino acids most of which are Glu and Asp, and its Mw is 44.3 kDa. Enamel is the hardest tissue. The enamel matrix proteins are secreted by ameloblast and alternate by mineral after it was degenerated. So the study on mineralization of enamel was focused on enamel matrix protein. Though tuftelin takes a small proportion in the enamel matrix protein, it might play a role at the initial stage of themineralization because of its acid, hydrophilic property, the strong ability to bind calcium ion as well as its early apperence the earlist experience during the amelogenesis. This might suggest that tuftelin could do an important role in the initiation of enamel biomineralization. The 4 parts of the test studied on gene cloning, expression and the distribution of tuftelin in mouse. It layed the fundation of futher research on the function of tuftelin. 1. Cloning of tuftelinIn the study, total RNA was extracted from the tooth germs of mice. The desired DNA product was obtained from the total RNA by RT-PCR with Oligo(dt) and two gene specific primers. The segment (about 1.2kbp) was inserted into pBluescript vector and the interesting plasmid was transformed into E.Coli host strain XL-1-Blue. The analyzing of restriction endonuclease mapping and DNAsequencing showed that a alternative splicing cDNA which lacked exon 2 was cloned, comparing with the sequence reported by Macdongall in 1998.2. Expression of tuftelin and preparation of polyclonal anti-seraMouse tuftelin was cloned into expression vector pPRoEX橦Tc and expressed in E.coli BL21.The results showed that the restriction endonuclease map and sequence of mouse tuftelin encoding mature protein were consistent with those reported in the references. Tuftelin protein was efficiently expressed in prokaryocytes.Polyclonal antibodies of tuftelin was prepared by immunizing New Zealand rabbit with the purified tuftelin obtained by prokaryotic expression. The tilter of the prepared antibody of tuftelin was 1: 100000 at least.3. Expression vector construction of gene of tuftelin and transfection of tuftelin into COS-7Foreign protein expression by prokaryocytes is characterized by high output, low cost and easy preparation, but it can not fold and glycosylize. Therefore such protein does not has the functions that native protein has. Because tuftelin is a glycoprotein, we selected mammalian cell as expression system. Recombined plasmid of pcDNAS- tuftelin was constructed by inserting the cloned tuftelin into pcDNA3. After being identified by enzyme analysis and computer seqencing, pcDN A3-tuftelin was transfected into COS-7 cell. Western-blot indicated that tuftelin was expressed in COS-7. The success of the expression might have paved the way for function study.4. Tissue distribution of tuftelinWestern-blot analysis was use to detect different kind of tissues. The results showed that the expression of tuftelin was detected in tooth germs, itsmolecular weight being about 42kD, and other mouse tissues, such as muscle, heart, liver, kidney, spleen, lung and brain were also reactive. These findings are in conformation with what has been reported in literature and suggest that tuftelinmay has other functionsThe study on the cDNA cloneing, expressing and preparation of polyclonal anti-sera of tuftelin in mouse laid a foundation for the further study of the structure and function of tuftelin, which may help to discover themolecular mechanism of biomineralization of enamel.