Basic study for development of DNA vaccine against dental caries

变形链球菌具有三种葡萄糖转移酶(GTF)，它们协同合成粘附性葡聚糖，促进变形链球菌细胞在牙齿表面的黏附和堆积。因此，GTFS被认为是龋病的主要致病因素。我们构建了针对GTFS的DNA疫苗，然后试图确定它们的免疫原性。将GTF的催化(CAT)和葡聚糖结合(GBD)两个功能区作为DNA疫苗的靶标，通过聚合酶链式反应(PCR)扩增编码这两个结构域的DNA片段，将其连接到真核表达载体pcDNA3中，构建成DNA疫苗载体pcDNA3/CAT和pcDNA3/GBD。将pcDNA3/CAT和pcDNA3/GBD分别导入小鼠胚胎细胞系NIH3T3，逆转录-聚合酶链式反应(RT-PCR)检测CAT和GBD基因的表达，Western印迹检测重组蛋白的表达。RT-PCR检测到CAT和GBD特异的mRNA在pcDNA3/CAT和pcDNA3/GBD中表达，而Western印迹分析也表明CAT和GBD特异的重组体在各自的细胞中表达。我们的结果表明，这些DNA疫苗在哺乳动物细胞中表达重组蛋白作为抗原，并具有诱导免疫应答的能力。

Streptococcus mutans possesses three glucosyltransferase (GTF) enzymes that coordinate to synthesize adhesive glucan, which facilitates adhesion and accumulation of S. mutans cells to tooth surfaces. Thus, GTFs are considered to be a major virulence factor of dental caries. We constructed DNA vaccines against GTFs and then attempted to determine their immunogenicity. Two functional domains of GTFs, the catalytic (CAT) and glucan-binding (GBD) domains, were used as targets of the DNA vaccines.DNA fragments coding these domains were amplified by PCR and ligated into an eukaryotic expression vector, pcDNA3, which resulted in the DNA vaccine plasmids pcDNA3/CAT and pcDNA3/GBD. A mouse embryo cell line, NIH 3T3, was transfected with either pcDNA3/CAT or pcDNA3/GBD and the expressions of mRNA and recombinant proteins for CAT or GBD in the transfected cells were subjected to reverse transcription-PCR (RT-PCR) and Western blot analyses, respectively. Using RT-PCR, CAT-and GBD- specific mRNA was detected in pcDNA3/CAT and pcDNA3/GBD transfected cells, while Western blot analysis also revealed that the CAT- and GBD- specific recombinants were expressed in the respective cells. Our results indicated that these DNA vaccines expressed recombinant proteins as antigens in mammalian cells and possessed the ability to induce immune responses.