Cloning And Expression Of The Gly Gene Encoding The Glycine Hydroxy Methyl Transferase Of Veillonella Dispar

The dental plaque of oral is a special microbial environment.The bacteriagrow, multiply and metabolism in this status. Due to the restriction of the livingenvironment and the nutritive material, there may occur the symbiosis andantagonism among the bacteria. When they are in a kind of balance the dentalplaque will format which is the basic condition of the dental caries.If thisbalance was destroyed maybe suppress the happen of dental caries.Veillonella is a kind of Gram-negative coccus, it mainly parasitize in oral,intestinal canal and respiratory tract of animals and human. Veillonella hasability of acid resistance to some extant and it can grow in acid circumstances.Some researches supply that after the settable of Veillonella in the oral theStreptococcus mutans and other bacteria could follow,then the dental plaquewould form. The Glycine hydroxy methyl transferase(Gly) is the key enzymeof the metabolism of the Veillonella which catalyze the transfer of serine andglycine. The intermediate product-tetrahydrofolic acid is the important enzymeof DNA in the cell which plays the crucial part in the growth of Veillonella.This experiment investigate the effect of this enzyme in the angle of molecularbiology, it may provide a new way to avoid and cure the caries which may leadto thesuccess of biological anticariesThis experiment is operated in two processes. Firstly:through PolymeraseChain Reaction(PCR) amplification Glycine hydroxy methyl transferase (gly)gene of veillonella dispar then connect it with plasmid PMD18-T.We constructclone vector of gly gene of veillonella dispar successfully. Secondly:amplification the gly gene which contains activity section and conservativesequence by using cloning vector contrasted in the last process as a template. Design primers by the assistant of Primer5. Through connect the gene withplasmid Pet28a the theprokaryotic expression vector of gly gene of veillonelladispar is contrusted. Then transfer the reconstructed recombinant plasmid intothe Ecoil-BL21in the condition of37℃using a final concentration of1.0mmol/L of IPTG to induce expression. Then analyze the result oflectrophoresis of the product by SDS-PAGE electrophoretic analysis comparingwith no IPTG-induced recombinant plasmid,BL21bacteria with norecombinant plasmid and plasmid pET28a.The result shows that: in the first process of the experiment the gene weobtain is gly gene.Through GenBank BLAST the homology of which withVeil-lonella parvula is99%.This gene has been submitted to NCBIGenBank,The submission number is JQ929767.Then the second one showsthere is anticipant band in protein expression to contract we can’t find thecorresponding band of the control group. So we can easily come to theconclusion that expression vector of gly gene can translate and express theprotein. This experiment establish the base for the next step of researching theactivity and function of the protein.