An Experimental Study On Cloning And Expression Of The Functional Domain Of Glucan Binding Protein B Of Streptococcus Mutans And Anticaries Immunization

Dental caries is one of the most popular bacterial infective diseases. Mutans streptococci (MS) are generally believed to be the primary cariogenic agents, the surface protein antigen I / II (PAc) , glucosytransferase (GTFs) and glucan binding protein (Gbps) are the most important virulence factors.The anticaries immunazition comes to be one of the focuses in caries preventative study. Glucan binding proteins (Gbps) could bind to the glucan synthesized by the GTF on the cell surface of the Mutans streptococci,so it is considered to be the impotant actor for the adhesion and aggregate of the Mutans streptococci. GbpB attracts more and more attention in anticaries studies because of its good immunogenicity.But the studies usally focuse on the complete gene of GbpB not its fuctional domain.In this study, the gene encoding fuctional domain fragment of GbpB from S. mutans Ingbritt genomic DNA was cloned by PCR and subcloned into a expression vector:pET-DsbA .After being identified, it was expressed in E. coli BL21. Thus mass recombinant proteins were obtained, then purified to get the fuctional domain protein. In the following studies,we observed theglucan-binding capability of the fuctional domain protein of GbpB and its immunogenicity by immunizing Sprague-Dawley (SD) rats through TSG injection routes,Gnotobiotic rats were vaccinated with the fuctional domain protein, its protection against dental caries were observed, paving the way for further study.The present study consists of two parts:Part I : Cloning , sequencing, expression and purification of the fuctional domain of glucan binding protein B gene of Streptococcus mutans1. S. 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 reports, then all the gene encoding fragments of fuctional domain of GbpB were obtained by polymerase chain reaction (PCR); the fragments were inserted into cloning vector:pET-DsbA and the resulted plasmid was used to transform competent E. coli; positive recombinant clones were selected and identified. The sequencing result was found consistent with that reported.2. The resulted plasmid was used to transform competent E. coli BL21, the positive clone was induced by IPTG to express the fuctional domain of GbpB fusion protein.Then SDS-PAGE was employed to detect the expression products. A new protein band with Mr 25×103 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 and then cut by enzyme and purified again to get the fuctional domain of GbpB protein.Part II: Animal immunization and anticarious experiment of the fuctional domain of the glucan binding protein B...