Expression Of Alcohol Dehydrogenase Gene In Escherichia Coli And Construction Of Suicide Vector For The Replacement Therapy Of Dental Caries

Streptococcus mutans is the predominant pathogen of caries. Lactatedehydrogenase, which is considered an important virulence factor ofStreptococcus mutans, can metabolize pyruvic acid into lactic acid , anddecrease the pH within local plaque, promote the teeth loss of calcium, andfinally result in caries. Previous studies in rodent model have had demonstratedthat Streptococcus mutans which lack the enzyme activity of lactatedehydrogenase (LDH), had significantly less cariogenic. Based on this property,many efforts on preventing caries through prohibiting the activity of LDH havebeen taken, proposing the establishment of an effector strain in the replacementtherapy of dental caries in human. However, following some unsuccessfulattempts investigators demonstrated that entirely inactive ldh gene was alwaysfollowed by cell death. Because LDH deficiency was found to be a lethalmutation in Streptococcus mutans, an auxiliary ADH activity from Zymomonasmobilis was simultaneously introduced to circumvent this problem.The recombinant pasmid pMDLA has been constructed in our previousexperiment by substituting the lactate dehydrogenase gene ofStreptococus mutans for the alcohol dehydrogenase Ⅱ gene of Zymomonasmobilis. In this research the gene of alcohol dehydrogenase Ⅱ was obtainedby digesting the recombinant vector pMDLA and inserted into the expressionvector pET-28a and then expressed in E.coli BL21. Restriction enzymesNcoⅠand XhoⅠ were used to digested DNA fragment from the recombinantplasmid pMDLA. Following agarose gel purification, the pET28a fragmentand the adhB fragment were ligated and transformed into E.coli BL21.Transformants were selected on LB plates containing ampicillin. Restrictionenzyme digestion and sequence analysis were used to confirm the size andproper orientation. IPTG was used to induce the E.coli BL21 for the expressionof adhB. A specific 40KD band appearance on the SDS-PAGE gel and theADH 11 activities were detected by aldehyde indicator plates. It is comfirmedthat the adhB gene can be expressed in E.coli BL21, which makes a base for thefurther study of the construction of LDH–deficient mutant of S.mutans.In the second research, we construct a suicide vector PMDLAT base onpMDLA. Tet gene which is obtained from PBR322 vector by PCR was insertedinto the recombinant plasmid pMDLA. Because the G-vectors can notreplicate in G+ bacteria. Pay attention to include the promoter of tet gene sothat the tet gene can express normally in the Streptococcus mutans of JH1140.The sequencing of PMDLAT show that there was only one mutant , the NO.1134 base got mutant from C to T, but the mutant was already happen inPBR322 vector. Thus, the suicide vector PMDLAT can express tetracyclineresistance normally.The application of replacement therapy to prevent and treat bacterialinfection has certain potential advantages than other conventional therapeuticapproaches. Ideally, a single application of the effector strain to the tissues atrisk should result in its persistent colonization at levels sufficient to protect thehost from infection by a pathogen. Becoming part of the normal flora of thehost, a well-designed effector strain might provide life-long protection against adisease while requiring minimum compliance. This approach also has thepotential advantage of providing herd protection by the natural transmission ofthe effector strain within the population. National? studies on replacementtherapy of dental caries haven't been found yet. We believed that thereplacement therapy of dental caries would be successful ultimately and providea novel approach for the prevention of bacterial disease such as dental caries.