| Dental caries is a bacterial infectious disease involving multiples factors in which streptococcus mutans plays a major role. Glucan binding proteins (Gbps) are considered to be a cariogenic virulent factor due to their bind glucan ability, which contributes to the adherence and accumulation of the organisms in dental plaque. Today anticarious immunization becomes one of the focuses in caries preventative study. Gene vaccine is a novel immunization method for medical prevention that has just begun since 1990s. Compared with live attenuated vaccine, inactivated vaccine and recombinant subunit vaccine, gene vaccine has advantages such as long-term and stable expression of antigen, the endogenously-produced protein with closer conformation to natural protein, and stronger antigenicity. So far protection against dental caries by gbpB DNA vaccine in vivo has not been reported either at home or abroad.In this study, we cloned all the gene encoding fragments of gbpB from S. mutans Ingbritt genomic DNA, and after being identified, it was subcloned into a prokaryotic expression vector, and then expressed in E. coli. Thus mass recombinant proteins were obtained. Meanwhile we produced the antisera to GbpB with the fusion protein, constructed eukaryotic expression plasmid pcDNA3.1(+)-gbpB as a gene vaccine, and observed its immunization effect through gene vaccine immunize Sprague-Dawley (SD) rats through various methods. Gnotobiotic rats were vaccinated with plasmid and fusion protein, specific immuneresponses in vivo and their protection against dental caries were observed, paving the way for further study.The present study consists of three parts:Part I: Cloning and sequencing of the glucan binding protein B gene of Streptococcus mutansS. mutans Ingbritt was routinely cultured and its genomic DNA was isolated as templates; a couple of primers were designed and synthesized in accordance with the sequence reported by Genbank, then all the gene encoding fragments of gbpB were obtained by polymerase chain reaction (PCR); the fragments were inserted into cloning vector pBluescript KS(pBS) and the resulted plasmid was used to transform competent E. coli DH5, positive recombinant clones were selected and identified; then the two-stranded DNA was sequenced. The sequencing result was found consistent with that reported.Part II: Prokaryotic and eukaryotic expression of the glucan binding protein B gene of Streptococcus mutansThe sequenced fragment was subcloned into prokaryotic gene expression vector pPROEX橦Tb in a certain direction and the resulted plasmid pPROEX橦Tb-gbpB was used to transform competent E. coli DH5; after selection and identification, the positive clone was induced by IPTG to express GbpB fusion protein. Then SDS-PAGE was employed to detect the expression products. A new protein band with Mr 50+103 appeared in the SDS-PAGE gel, and its size was approximately the same as expected.The target protein was purified by Ni2+-NTA affinity chromatography. Rabbits were routinely immunized with the purified protein to produce antisera to GbpB. The antisera were identified by ELISA. The results suggested that we acquired successfully the antisera to GbpB, whose effective deepness was 1:10,000.The sequenced fragment was also subcloned into eukaryotic gene expression vector pcDNA3.1(+) in a certain direction, then the plasmid pcDNA3.1(+)-gbpBwas constructed ,and introduced into COS-7 cells by Lipofectamine reagent. The transient expressed protein was detected by immunochemistry technique in the cell plasma of COS-7 . This result indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine.Part III: Animal immunization and anticarious experiment of the glucan binding protein BPlasmid pcDNA3.1(+)-gbpB DNA vaccine was delivered into Sprague-Dawley (SD) rats in two different routes: intranasal (i.n.) and targeted salivary gland immunization (TSG). The dynamic variety of specific antibodies in sera and saliva were checked by ELISA. The level of s...
|
PDF Full Text Request