Cloning Of L Type Calcium Channel α₁D Subunit Specific Gene From Human Odontoblast Cells

Dentin is the principal part of a tooth, unlike bone, under physiologicalcondition, no absorption and reconstruction but mineralization course exists indentin. Dentin formation is a consecutive process in whole life of a tooth andodontoblast cells are responsible for it through secretion and mineralization ofdentin matrix. Thus, taking odontoblast cells as research target would help tobetter understand the mechanisms of dentinogenosis and to some extent providevaluable information in the formation of human mineralized tissues.Odontoblast cells form a monolayer lining the periphery of dental pulp andplay a key role during the formation of dentin calcification. Odontoblasts arederived from embryonic connective cells that are called ectomesenchymal cellsbecause of their well-established origin from the neural crest. Studies haveshowed that many molecules participate in dentin calcification, such as L typecalcium channels, Ca-ATPase, Na+/Ca2+ exchangers and calcium bindingproteins. L type calcium channels play an important role in this process.L type calcium channel is a kind of macromolecule glycoprotein composedof five subunits,α1 ,α2 ,β,γandδ. The pore functional subunit of L type calcium channel, subunitα1, has three isformsα1C,α1D andα1S. Studies have found thattheα1D subunit of L - type calcium channel is the main hypotype in odontoblastcells membrane of mouse. TIt may play a leading role in facilitating the entry ofcalcium into odontoblast cells during the mineralization process of the dentine.TL type calcium channelα1D subunit specific gene fragment from humanodontoblast-like cells had been cloned by our research team.Odontoblast cells, closely neighbor dentin, form a monolayer lining theperiphery of dental pulp, and stretch cell processes into dentinal tubules.Moreover, odontoblast is a terminal differentiated cell, can not furtherdifferentiate and proliferate in vivo, nor in vitro. Thus, it is impossible to obtainlarge number of pure odontoblast cells for research. Our research team hadestablished a method for isolating odontoblasts from human tooth by usingproteolytic enzymes. Odontoblast cell isolated directly from freshly extractedtooth remains original characteristics, and is a better cell model for research.In the present study, cDNA isolated from the human odontoblast cellsusing RT-PCR was cloned into pGM-T．After sequencing, the cDNA wasinserted into prokaryotic expression vector pET32. The recombinant plasmidwas transformed into DH5α．L type calcium channelα1D subunit specific geneof human odontoblast expression was then induced by IPTG．The protein waspurified through Ni-NTA affinity chromatography column and characterized bySDS-PAGE. The engineering of the cDNA encoding CaBvB1.3 of humanodontoblast and its expression will facilitate future studies aimed at determiningits location on the odontoblast cell membrane and tooth slice and studying itsrole that plays in the Ca²⁺ influx mechanisms into odontoblast.