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Basic study for development of DNA vaccine against dental caries

龋齿DNA疫苗研制的基础研究

基本信息

批准号：
13470449
负责人：
FUJIWARA Taku
金额：
$ 10.62万
依托单位：
Nagasaki University (2002)Osaka University (2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470449/
关键词：
DNA vaccine Streptococcus mutans Glucosyltransferase Dental caries S.mutans 哺乳類発現ベクター

项目摘要

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.

变形链球菌具有三种葡糖基转移酶（GTF），它们协同合成粘附性葡聚糖，促进变形链球菌的粘附和聚集。牙齿表面的变形细胞。因此，GTF被认为是龋齿的主要毒力因子。我们构建了针对GTF的DNA疫苗，然后试图确定其免疫原性。以GTF的催化（CAT）和葡聚糖结合（GBD）结构域作为DNA疫苗的靶点，通过PCR扩增GTF的CAT和GBD结构域的DNA片段，并将其连接到真核表达载体pcDNA 3中，构建成DNA疫苗质粒pcDNA 3/CAT和pcDNA 3/GBD。将pcDNA 3/CAT和pcDNA 3/GBD分别转染小鼠胚胎细胞系NIH 3 T3，用RT-PCR和Westernblot分析转染细胞中CAT和GBD的mRNA和重组蛋白的表达。用RT-PCR检测到CAT和GBD特异性mRNA在pcDNA 3/CAT和pcDNA 3/GBD转染的细胞中表达，而Western blot分析也显示CAT和GBD特异性重组体在各自的细胞中表达。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。