Gene Clone And Recombinant Expression For Lactate Dehydrogenase Of Veillonella Dispar

Caries is one of the main diseases that hurt human oral health.A large amount of lactic acid can be produced in the period when streptococcus mutans get energy by using carbohydratein in plaque,which leads to the reduce of the PH.Then,the enamel demineralization cause caries.One of the ways to control caries is reducing the accumulation of lactic acid in the biofilm.The lactate dehydrogenase is a kind of inherent enzyme of bacteria.This kind of enzyme always exists in the cell in the form of high concentration,but it also can be found in the plaque,its function is to catalyze the mutual conversion of pyruvic acid and lactic acid,when bacteria come across highly concentrated sugar,the high level of FDP in the cell can lead to the activation of lactate dehydrogenase,and"lactic acid strobe"is opened,metabo- lism pyruvic acid producting massive lactic acids that cause the dissolution and demineralization of teeth calcium phosphate, leading the decayed tooth to damage at last .So lactic acid is the main factor of lower PH of plaque biofilm that caused by bacterium metabolisming exogenetic carbohydrate. So one way to avoid caries is reducing the accumulation of lactic acid in the plaque biofilm.The catalyzing process between pyruvic acid and lactic acid is bidirectional.The lactate dehydrogenase, lack of endogenous glucose,can produce pyruvic acid by reversible lactic acid oxidation,and pyruvic acid can be transformed into formic acid, acetic acid and ethanol which can provide bacteria with energy in the role of pyruvic acid formic acid lytic enzyme.According to the analysis of hungry plaque organic acids,the main elements are formic acid and propionic acid,PH is neutral.So under certain conditions,The lactate dehydrogenase can catalyze lactic acid to pyruvic acid,which can provide the possibility of reducing the PH and the concentration of lactic acid in plaque.From what we discuss above,we can gain that the function of lactate dehydrogenase is bidirectional.Provided with sugar, it can produce a lot mass of lactic acid and leads to the PH reducing of plaque , and on the other hand,using lactic acid,it can produce pyruvic acid by catalysis.then lactate dehydrogenase can increase the PH of plaque by reducing the lactic acid accumulation in plaque.Therefore,some scholar think that lactate dehydrogenase is some kind of caries reduction factor of the plaque.The veillonella can not make use of carbohydrate and also can not ferment dextrose because having no the key enzyme.By using of other bacteria resulting from the glycolytic lactate,produce propionic acid which is dominant, acetic acid,CO2 and H2. The acidity of propionic acid and acetic acid is no much as lactic acid ,so they can reduce the tooth damage caused by acid .This experiment is operated from two aspects,the first one is that,by PCR amplification lactate dehydrogenase gene of veillonella dispar and its homologous district ,then connected with PMD18-T,we construct prokaryotic expression vector of lactate dehydrogenase gene of veillonella dispar and its homologous district.The second one is increasing the lactate dehydrogenase gene which contains activity section and conservative sequence.By using cloning vector as a template,we design primers according to the principal of primer designing.The size is 798bp,by connecting with Pet28a,we construct the prokaryotic expression vector of lactate dehydrogenase gene of veillonella dispar, and then express the reconstructed recombinant plasmid in the BL21,37℃for using a final concentration of 1.0 mmol/L of IPTG-induced expression.Then analyze the electrop- horesis of the product by using SDS-PAGE electrophoretic analysis,and compare it with no IPTG-induced recombinant plasmid,BL21 bacteria and Pet28a .The result shows that there is positive band at around 30KD in cloning vector of lactate dehydrogenase,and we can't see any band at other place. So,we can easily come to the conclusion that cloning vector of lactate dehydrogenase can translate and express the protein.It establish the basic for the next step of the activity and protein function tests.According to the experiment,we amplificate lactate dehydr- ogenase gene of veillonella dispar and its homologous district.The size of the DNA is 2.3Kb,It contains three pieces of genes which are DNA gyrase A,lactate dehydrogenase,and an unknown one, and lactate dehydrogenase is integrated opening reading frame, by analyzing the compare of gene sequence and amino acid,we can gain that lactate dehydrogenase have typical construction that depend on NAD (nLDH) and its homology with Streptococcus mutans is 99%.This gene has been submited into NCBI GenBank,the submission number is EU518464.Till now,many studies of the veillonella are about physiology,biochemistry characteristic and bacterium isolated culture, researches about the level of gene is hardly to find.the report about lactate dehydrogenase gene of the veillonella dispar can not be found.The propose of this experiment is,by using the veillonella dispar as research object to know the reason why it ferment using lactic acid,to explore new ways of caries prevention.So the research about veillonella lactate dehydrogenase gene is significant,it can provide a new way to avoid and cure the caries.The research about this kind of bacteria may lead to the success of biological anticaries...