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.

变形链球菌拥有三种葡萄糖基转移酶 (GTF)，它们协调合成粘附性葡聚糖，促进变形链球菌细胞在牙齿表面的粘附和积累。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，然后尝试确定其免疫原性。 GTF 的两个功能域，即催化 (CAT) 和葡聚糖结合 (GBD) 域，被用作 DNA 疫苗的靶标。编码这些域的 DNA 片段通过 PCR 扩增并连接到真核表达载体 pcDNA3 中，从而产生在 DNA 疫苗质粒 pcDNA3/CAT 和 pcDNA3/GBD 中。用 pcDNA3/CAT 或 pcDNA3/GBD 转染小鼠胚胎细胞系 NIH 3T3，并对转染细胞中 CAT 或 GBD 的 mRNA 和重组蛋白的表达进行逆转录 PCR (RT-PCR) 和 Western 检测。分别进行印迹分析。使用 RT-PCR，在 pcDNA3/CAT 和 pcDNA3/GBD 转染细胞中检测到 CAT 和 GBD 特异性 mRNA，而蛋白质印迹分析也显示 CAT 和 GBD 特异性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，并具有诱导免疫反应的能力。