Gene cloning of a novel merozoite rhoptry protein from Plasmodium falciparum

恶性疟原虫裂殖子菱形蛋白的基因克隆

基本信息

批准号：
11670242
负责人：
TSUBOI Takafumi
金额：
$ 2.24万
依托单位：
Ehime University
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-11670242/
关键词：
Plasmodium falciparum merozoite rhoptry protein gene cloning ロプトリー

项目摘要

By the peptide sequencing from the bands obtained from the immuno-affinity purified 140 kDa rhoptry protein of Plasmodium yoelii, we obtained partial peptide sequences of PyRhopH1. To determine the gene encoding P.yoelii 140-kDa rhoptry protein, PCR amplifications were done from cDNA using degenerated oligonucleotides designed based on the partial amino acid sequences. Finally, we have identified the Plasmodium RhopH1 in P.yoelii.By the PCR-based gene-walking, we completed gDNA and cDNA sequences that encoded an open reading frame for 1292 amino acids protein separated by 7 introns. Deduded amino acid sequence has putative secretory signal sequence at the N-terminus (aa 1-24) predicted by SignalP and no apparent transmembrane domain. TBLASTN analysis against P.yoelii genome database revealed the existence of a paralogue of this gene, thus we designated the gene identified from peptide sequences as pyrhophla and second gene found in the database as pyrhoph1b. Using deduced amino acid sequence of pyrhophla as a query, TBLASTN search against P.falciparum genome database identified a gene family coding PyRhopH1 orthologues. To confirm the size and localization of PyRhopH1A, Western immunoblot analysis and immunofluorescent microscopy were performed. Antiserum generated with pYRH1A DNA vaccine reacted only 140-kDa band on the Western immunobloting, and showed a punctate pattern typical for the apical organelle as same as the pattern by anti-PyRhopH1 mAb by immunofluorescent microscopy.

通过对约氏疟原虫免疫亲和纯化的140 kDa棒状体蛋白获得的条带进行肽测序，我们获得了PyRhopH1的部分肽序列。为了确定编码 P.yoelii 140-kDa 棒状体蛋白的基因，使用根据部分氨基酸序列设计的简并寡核苷酸对 cDNA 进行 PCR 扩增。最后，我们鉴定了 P.yoelii 中的疟原虫 RhopH1。通过基于 PCR 的基因行走，我们完成了 gDNA 和 cDNA 序列，编码由 7 个内含子分隔的 1292 个氨基酸蛋白质的开放阅读框。推导的氨基酸序列在N端（aa 1-24）具有由SignalP预测的推定分泌信号序列，并且没有明显的跨膜结构域。针对 P.yoelii 基因组数据库的 TBLASTN 分析揭示了该基因的旁系同源物的存在，因此我们将从肽序列中鉴定出的基因指定为pyrhophla，将数据库中发现的第二个基因指定为pyrhoph1b。使用推断的焦叶霉氨基酸序列作为查询，针对恶性疟原虫基因组数据库的 TBLASTN 搜索鉴定出编码 PyRhopH1 直系同源物的基因家族。为了确认 PyRhopH1A 的大小和定位，进行了蛋白质免疫印迹分析和免疫荧光显微镜检查。用 pYRH1A DNA 疫苗产生的抗血清在蛋白质免疫印迹上仅反应 140 kDa 条带，并显示出顶端细胞器典型的点状图案，与免疫荧光显微镜下抗 PyRhopH1 mAb 的图案相同。