Cloning And Prokaryotic Expression Of Two Genes Relative To Adhesion Or Proliferation Of Mutans Streptococci And Preliminary Study Of Their Function

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. Adhesion and proliferation of MS are the basis of cariogensis. 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 cloning, functions and anticaries immunization of the genes relative to adherence and accumulation. These genes contain surface protein antigen I /II (Ag I / II) , glucosytransferase (GTFs) and glucan binding protein (Gbps). Ag I /II, GTFs and GbpB can protect against dental caries. GbpA of S. mutans contains a C-terminal GBD homologous to the GBDs of the GTFs, but the function of anticaries immunization of GbpA has not been reported. DNA vaccines are new methods that were developed in the lastdecade of the 20th century. The animal immunization experiments on DNA vaccines of T cell and B cell epitope in Streptococcus mutans surface protein antigen and GTF have shown that these DNA vaccines can induce protective immune response. 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, the GBD of glucan binding protein of 5. mutans was cloned,found expressed in E. coll and was purifed. The fusion protein was used to immunize rats. At the same time, the rats were infected by S. mutans. The antiserum was collected and the caries score was evaluated. In addition, we used the "genome walking system" to clone the complete sequence of putative cell division protein ftsK of S. sobrinus, then expressed it in E. coli and observed the impact of the overexpression of the putative cell division protein ftsK of S. sobrinus on the growth of E. coli.Our study consists of two parts:Part One: Preparation of GBD polypeptide and DNA vaccine of glucan binding protein of Streptococcus mutans and study on anticarious immunization.The following are the main experiments conducted:1. The GBD of gbpA of S. mutans was cloned by PCR and then was inserted into vector PUC19 to constructed plasmid PUC19-GBD. The identified plasmid was sequenced. The sequenced result was consistent with other reports.2. The sequenced fragment was subcloned into prokayotic gene expression vector pPROEX?HTb in a certain direction and the resulted plasmid pPROEX?HTb-GBD was used to transform competent E. coli DH5a. The fusion protein was induced by IPTG. The target protein was purified by Ni2+-NTA affinity chromatography. Western blot showed the purified fusion protein could cross reactwith gbpA antiserum.3. The plasmid pcDNA3.1-GBD was constructed and was introduced into COS-7 cells by Lipofectamine reagent. The transient expressed protein was detected by immunochemistry technique in cell plasma of COS-7 cells.4. The rats were immunized respectively with GBD fusion protein and pcDNA3.1-GBD DNA vaccines. Immunized animals demonstrated significantly higher serum IgG and salivary IgA antibody levels to GBD than non-immunized animals. Serum IgG could react with GBD fusion protein in Western blot. Immunization with GBD fusion protein and pcDNA3.1-GBD DNA vaccines resulted in significantly reduced dental caries after infection with S. mutans Ingbritt.Part Two: Cloning cell division protein ftsK of Streptococcus sobrinus. and study on its basic functionWe cloned a fragment from S. sobrinus genome DNA. Homology analysis by genebank demonstrated that it is a fragment of a new gene. The new gene has homology with cell division protein ftsK of E. coli. and SpoIIIE gene of B. subtilis. So it has been named as cell division protein ftsK of S. sobrinus. In this study,...