The Study On The Function Of Cell Division Protein FtsK Of Streptococcus Sobrinus

Dental caries is one of the three chief non-contagious diseases which threaten the human health. Mutans streptococci (MS) are the primary cariogenic agents, and Streptococcus mutans and Streptococcus sobrinus are believed to have the closest relationship with human caries. At present, the study of anticaries mainly focuses on a series of events involving the adherence, accumulation and proliferation of MS on tooth surfaces. Researchers have done many studies on anticaries preparation, anticaries drug, anticaries plant, and anticaries immunization. The studies on the proliferation of bacteria mainly focused on anticaries agents which could inhibit bacterial growth. But there has not been any report about the study of the control genes in the cell division of S. sobrinus and their functions in cariogenic process. In this study, we cloned the S. sobrinus's ftsK gene by PCR, expressed it in E.coli, purified the recombinant protein, prepared anti-ftsK antibodies. Then we made ftsK overexpression in S. sobrinus and observed the impact to the division of it. Finally we constructed the mutant of S.sobrinus ftsK gene andstudied it divison situation.Our study consists of three parts:1. Cloning and Sequence Analysis of Streptococcus sobrinus ftsK gene, Prokaryotic Expression and preparation of polyclonal antibody.In this study, amplified ftsK DNA by PCR technique was obtained from the Streptococcus sobrinus's genome with the primers including Olido (dt) and two gene specific primers respectively. The fragment (about 2.1kb) was inserted into pGEM-T Easy vector and the recombinant plasmid was transformed into E. coli DH5 a . The positive clones were analyzed with restriction endonuclease mapping and DNA sequencing. The results showed that the nucleotide sequence we cloned were consistent with the GenBank record.The sequenced fragment was subcloned into prokayotic gene expression vector pPROEX? HTc in a certain direction and the resulted plasmid pPROEX^TMHTc/ftsK was used to transform competent E. coli DH5α. The fusion protein was induced by IPTG After induction, new protein bands appeared on SDS-PAGE gel near 77kDa. The target protein was purified by Ni²⁺-NTA affinity chromatography and used to immune New Zealand White rabbits with Freund's complete adjuvant to produce antibody.. Western blot showed the purified fusion protein could across react with ftsK antiserum.2. Overexpression of ftsK in S.sobrinus and imfection to the cell divisionIn this study, we constructed prokayotic gene expression vector in Streptococcus sobrinus. We obtained ftsK fragment from pGEM/ftsK after modified with Nhe I and BamH I. Then the fragment was inserted into the shuttle vector pDL289 which can be replicated in both E. coli and S.sobrinus. The recombinant plasmid pDL289/ftsK was transformed into S.sobrinus.