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Glycosphingolipid binding protein (Gbp) of Lyme disease Borrelia, Its function and vaccine development.

莱姆病伯氏疏螺旋体的鞘糖脂结合蛋白（Gbp）及其功能和疫苗开发。

基本信息

批准号：
11670267
负责人：
MASUZAWA Toshiyuki
金额：
$ 2.24万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670267/
关键词：
Lyme disease Borrelia Adhesion Galactosylceramide Borrelia burgdorferi glycosphingolipid GAPDH Vaccine

项目摘要

Glyceraldehyde-3-phosphate dehydrogenase (gapdh) homologue gene, open reading flame about 1020 base pair was cloned into pMAL-c2 plasmid vector and transformed into E.coli JM109. The transformant express ca 80kDa GAPDH protein fused with maltose binding protein (MBP). The recombinant fusion purified with amylose resin was subject to SDS-PAGE and transferred onto PVDF membrane. The recombinant protein was specifically bound with galactosylceramide and was named as glycosphingolipid binding protein (Gbp37). The recombinant Gbp37 fusion protein inhibited the binding of Borrelia cells to CHO-K1 cells at dose 200ug/mL.From immuno electron microscpic observation, anti-Gbp37 sera reacted with antigen expressed borrelia cell surface. However, borrelia cells pretreated with surfactant to disrupt the outer membrane did not reacted with the antisera. The observation indicated Gbp37 located borrelia cell surface. Mice immunized with recombinant protein were challenged with live borrelia cells into footpad. The mice were partially protected from borrelia infection, but were not protect completely. The anti-Gbp37 sera cross-reacted with various borrelia strains belonging to variable borrelia spp. The facts indicated that GAPDH homologue (Gbp37) of Borrelia was one of adhesion molecule expressed cell surface, bound with glycosphingolipid and elicited protective immunity.

将甘油醛-3-磷酸脱氢酶（gapdh）同源基因（开放阅读长度约1020 bp）克隆到pMAL-c2质粒载体中，转化大肠杆菌JM 109。表达约80 kDa的GAPDH蛋白与麦芽糖结合蛋白（MBP）融合。用直链淀粉树脂纯化的重组融合蛋白进行SDS-PAGE，并转移到PVDF膜上。重组蛋白与半乳糖神经酰胺特异性结合，命名为鞘糖脂结合蛋白（Gbp 37）。重组Gbp 37融合蛋白在200 μ g/mL浓度下可抑制疏螺旋体细胞与CHO-K1细胞的结合，免疫电镜观察，抗Gbp 37血清可与表达抗原的疏螺旋体细胞表面发生反应。然而，用表面活性剂预处理以破坏外膜的疏螺旋体细胞不与抗血清反应。结果表明Gbp 37定位于疏螺旋体细胞表面。用重组蛋白免疫的小鼠用活的疏螺旋体细胞攻击到足垫中。小鼠部分地免受疏螺旋体感染，但没有完全保护。抗Gbp 37血清与属于可变疏螺旋体属的各种疏螺旋体菌株交叉反应。研究表明，Borrelia的GAPDH同源物（Gbp 37）是一种表达于细胞表面的粘附分子，可与鞘糖脂结合，产生保护性免疫。