Construction Of Recombinant Plasmid Through Substituting The Alcohol Dehydrogenase Ⅱ Gene Of Zymomonas Mobilis For The Lactate Dehydrogenase Gene Of Streptococcus Mutans

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,and finally develop caries.Previous studies in rodent model have had demonstrated thatStreptococcus mutans which lack the enzyme activity of lactatedehydrogenase (LDH) , had significantly less cariogenic. Base on thisproperty, many efforts on preventing caries through prohibiting theactivity of LDH were taken, and proposed establishing an effector strainin the replacement therapy of dental caries in humans. However,following some unsuccessful attempt the investigator demonstrate thatentirely inactive ldh gene was always followed by cell death. BecauseLDH deficiency was found to be a lethal mutation in Streptococcusmutans, an auxiliary ADH activity from Zymomonas mobilis wassimultaneously introduced to circumvent this problem.Previously, the ldh gene of Streptococcus mutans and its flankingDNA fragment including 431 bases up-stream and 882 basesdown-stream, was cloned and termed the final recombinant plasmidpMD18-T-ldh. In the present study, the gene encoding alcoholdehydrogenase â…¡was amplified from total DNA of Zymomonasmobilis by PCR with primer A01 and A02. The gene of adhB wasinserted into clone vector pGEM-T and then transformed into E.coliJM109. The entire cloned Streptococcus mutans ldh open readingframe (ORF) in pMD18-T-ldh was deleted by inverse PCR mutagenesiswith primers L01 and L02. Only the first 28 bases, including thetranslation (ATG) start codon and the last 18 bases prior to thetranslation stop codon (TAA), were retained. Restriction enzymesNcoâ… and Xhoâ… were used to digested DNA fragment from inversePCR amplification and recombinant plasmid pGEMA. Following agarosegel purification, the pMDL fragment and the adhB fragment were ligatedand transformed into E.coli JM109. Transformants were selected on LBplates containing ampicillin. Restriction enzyme digestion and sequenceanalysis were used to confirm the size and proper orientation of theinsert in the resulting plasmid, pMDLA, which has the Zymomonasmobilis adhB gene fused in frame to the start and end of the ldh ORF.Sequence analysis of the region showed a high degree of homology tothe known sequence of plasmid pMD18-T-ldh. There was only onemutant , the NO. 880 base to down-side the translation stop codon(TAA). But the mutant was dispeared when conpare it with thesequence of Streptococcus mutans reported in Genebank. Thus, wepresumed that this mutant come from the sequencing error.