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Isolation of Leishmania DNA fragment for invasion to host cell.

分离利什曼原虫 DNA 片段以入侵宿主细胞。

基本信息

批准号：
09670268
负责人：
MIMORI Tatsuyuki
金额：
$ 2.05万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670268/
关键词：
Leishmania parasite Leishmania amazonensis Molecular biotechnology Invasion Genomic DNA library Deletion mutant Non-pathogenic E.coli Kinetoplast DNA 細胞侵入 kinetoplast(k)DNA DNAライブラリー

项目摘要

Leishmaniases are widely distributed in tropical and temperate zones, where approximately 12 million people are affected. However, there is not drugs for appropriate treatment of Leishmania parasites. Leishmania parasitizes to the mammalina cells. Invasion mechnisum of this parasite are not known yet. We searched the Leishmania invasion molecule using genome and cDNA library and non-pathogenic E.coil system. Genome and cDNA were made cultured promasigotes of Leishmania amazonensis. These DNAs were partially digested with Eco RI and Sau 3A1, then were ligated to plasmid vector (pBluescript IIsk+). These plasmid cotaining Leishmania DNA insert were infected into E.coli (DH5alpha). The E.coli were incubated with mammalian cells (VA-13). Invaded bacteria were selected with washing of antibiotic fluid. Five clones of selected 11 clones had the same 614 base pairs sequences. Furthermore, we made deletion mutants of this insert Leishmania DNA.From this experiment, it was suggested that about 130 base pairs sequences N-terminal was more important site for invasion to the host cell. The GST-fusion protein was made using plasmid (pGEX-2TH) and E.coli (NM522) system. Adhesion activity of this protein was about 20 times compared with control one.

利什曼病广泛分布在热带和温带地区，约有1200万人受到影响。然而，目前还没有适当治疗利什曼原虫的药物。利什曼原虫寄生于哺乳动物细胞。这种寄生虫的入侵机制尚不清楚。我们利用基因组和cDNA文库及非致病性E.coil系统对利什曼原虫入侵分子进行了检索。制备了亚马逊利什曼原虫的基因组和c DNA。用Eco RI和Sau 3A1对这些DNA进行部分酶切，然后连接到pBluescrip IIsk+载体上。将含有利什曼原虫DNA插入片段的重组质粒感染大肠杆菌(DH5pha)。将大肠杆菌与哺乳动物细胞(VA-13)孵育。用抗生素液冲洗的方法筛选入侵细菌。筛选出的11个克隆中有5个克隆具有相同的614个碱基对序列。此外，我们还对插入的利什曼原虫DNA进行了缺失突变，结果表明，约130个碱基对的N末端是利什曼原虫入侵宿主细胞的更重要的部位。以pGEX-2TH为表达载体，以NM522为表达载体，构建GST融合蛋白表达载体。该蛋白的黏附活性约为对照蛋白的20倍。