Construction And Eukaryotic Expression Of Recombinant Plasmid PEGFP-N1-SrV+ Of Streptococcus Mutans

Dental caries is a form of dental hard tissue diseases, mainly interacted by bacteria as well as many other factors. Streptococcus mutans (S.mutans) is widely recognized as one of the major pathogenic bacteria. Adhesion to teeth surfaces, aggregation, proliferation and acid production of S. mutans is the main process of causing caries. Therefore, the measure of blocking S. mutans's adhesion to teeth surfaces and its aggregation can reduce the incidence of dental caries. At present, the immunological prevention refers to as one of primary measures controlling dental caries. Gene vaccines gradually become a new method of inducing immune responses from the early 1990s, which is another milestone in the history of immunology and honored as the third vaccine revolution. The basic principle of gene vaccines is to deliver exogenous gene into host cells directly; so that host immune system could be induced to produce immune responses against proteins expressed by target genes, and then the purpose of preventing and treating the disease could be achieved.S. mutans surface proteins (surface protein antigen,SpaP) is a main functional area of mediating S. mutans's adhesion and aggregation on teeth surfaces. The variable region of S. mutans (SrV+, V Region) attach two highly conservative terminus called A Region and P Region, and it can take effect of causing dental caries by working together with A or P Region. The SrV+ has the function of adhering and aggregating and helps to promote adhesion and aggregation of S. mutans on teeth surfaces; meanwhile, SrV+ proteins comprise B cell epitope, so the immunogenicity can work alone. In addition, SrV+ proteins can stimulate monocytes to release TNF-a factor through lectin-like substances. Therefore, SrV+ of S. mutans surface proteins is the primary virulence factor involved in the process of causing dental caries, and its coding genes is the srv+.[Object]Based on the reported nucleotide sequence of gene srv+ of the SrV+, we can produce its coding genes chemically firstly, and after the then attach this gene fragment to pEGFP-N1 of the eukaryotic expression vector, so as to construct eukaryotic expression plasmid pEGFP-Nl-SrV+. Then we can instantaneously transect mammalian's Cells 293T, of which the target protein expression could be evaluated as well; therefore, we could be able to lay a foundation for further in animals research of caries-prevention. [Methods] This study falls into two experimentsExperimentâ… :The Construction of Recombinant Plasmid pEGFP-Nl-SrV+Based on the reported nucleotide sequence of Gene srv+ in the SrV+, we have produced the coding genes of srv+ chemically firstly; then, we can create cohesive terminus by means of the process that the vector plasmid pEGFP-N1 and the correct-sequenced fragment of srv+ are double digests separately, so as to conduct the reaction of attachment, and to gain the recombinant plasmid pEGFP-N1-SrV+. It further was done PCR, restriction enzyme digestion and sequencing to be sure right. Experimentâ…¡:The Expressions of the Recombinant Plasmid pEGFP-N1-SrV+in Eukaryotic Cells.By liposome-mediated method, recombinant plasmid pEGFP-N1-SrV+ could be instantaneously transected into mammalian 293T Cells. Then, through immunohistochemistry SABC,and look into the expressions of the target proteins by luminescence microscope, marked by green fluorescent proteins,expressed proteins of the eukaryotic Cells 293T.[Results] Acquisition of the nucleotide sequence of target genes srv+;Sequencing result is in line with the target gene sequence. Successful construction of eukaryotic expression plasmid pEGFP-Nl-SrV+ on the basis of the reaction of attachment after double-restriction. PCR amplification of gene fragments obtained 1.35Kb.KpnI/XhoI double digestion shows 4.7kb and 1.1kb bands.The sequence analysis of recombinant plasmid pEGFP-N1-SrV+ carrying the target gene.The sequence of SrV+ compared with S. mutans UA159 sequence,and homology of more than 96%.on the basis of the confocal laser-scanning and immunohistochemistry, the matter that plasmid pEGFP-N1-SrV+, which can express target proteins inside and outside eukaryotic cells and own immunogenicity, has been proved.[conclusion] The recombinant plasmid pEGFP-N1-SrV+has been successfully constructed; and after the plasmid transformation in mammalian 293T Cells, it can express the target antigen.