| Streptococcus mutans has been implicated as a causative agent of human dental caries. Vaccination was regarded as an effective method to control the incidence of dental caries. The surface protein of S.mutans is one of important pathogenic factor. The A region of the surface protein antigen pac gene (pacA) enriched T-cells and B-cells epitope. Previous the immune responses of pac-A in mice have been investigated, which w/flibetter than other DNA fragment as DNA vaccine. Although DNA anticaries vaccines have more advantages than traditional vaccines, it still has some problems need to be solved, Such as the lower immune response in vivo. In this study, we focus on constructing traceable DNA anticaries vaccine to indicate the expression of target gene in vitro and vivo, obtain a better gene carrier to improve the effects of gene delivery and increase immune response stimulated by DNA anticaries vaccine encapsulated with poly-D L-lactide-polyethylene glycol (PELA).According to the gene sequence, a pair of primers was designed. The portion of the pac gene was amplified by PCR with the plasmid pPC41 (including pac gene) as template DNA. The PCR product was a fragment 1.3kb named as pac-A. The fragment was purified by PCR recovery kit, then digested with Kpn I and Apa I , and subsequently inserted into the pEGFPCl vector digested with thesame enzymes. The recombinant plasmid named as pEGFPCl-pacA wastransformed into E.coli HB101. The positive colonies carrying recombinant plasmid were screened by LB plate containing Km. The pEGFPCl-pacA was extracted from HB101 (pEGFPCl-pacA) using Plamid Extraction Kit. Plasmid pEGFPCl-pacA was identified by restriction endonuclease digestion. The single fragment with 6kb in size was obtained through Kpn I or Apa I digestion; Two fragment with 1.3kb and 4.7kb in size through Kpn I and Apa I digestion. The result of sequencing for the insertional gene of pEGFPC 1 -pacA displayed that it was accordance with the portion of pac gene from base pair 314 to 1630. It suggested that the pac-A gene had been inserted correctly into eukaryotic expression vector pEGFP-Cl. The phase and orientation of the target gene inserted into the vector were also correct and their ORF didn't change.COS 1 cell was transfected with the recombinant plasmid by Lipofectin in order to identify the transcription and expression of recombinant plasmid pEGFPC 1 -pacA in mammalian cells. The expression of GFP was detected by observation of green fluorescent using invert fluorescent microscope, and the efficiency of transfection was measured using flow cytometry. The expression of pacA and gfp was detected by RT-PCR and Western blot. The cells with green fluorescent protein (GFP) were observed. The fluorescent intensity of transfected cells with the recombinant plasmid was higher than that of controlled cells. The mRNA of pac-A was detected by RT-PCR with total RNA as template, and the fragment with 1.3kb in size was confirmed by RT-PCR. Moreover the gene expression products in cell culture supernatant were detected . Two proteins with Mr of 56KD and 89KD corresponded with predicted Mr of PAC (39-477aa) and Mr of PAC (39-477aa) plus GFP. The proteins were able to react with the specific anti-PAC rabbit sera. The results may be related to the expression of fusion gene in eukaryotic cells. The fusion gene could be code fusion protein, but some fusion proteins were divided into two parts during processing. It suggested that the recombinant plasmid correctly transcripted and translated in eukaryotic cells, and pacA gene could be expressed solely or with GFP together, and the activities of two proteins weren't disturbed each other. Moreover, Detection of GFP is simple, safe and effective in living cells.Gene delivery has been regarded as a powerful tool for curing many hereditary diseases and treating acquired diseases, but DNA vaccines and gene therapy wetelimited in practice by the difficulty of gaining efficient expression of DNA in target cells. In this study we reported that a technique of DNA/PELA... |
