Gene cloning of a novel merozoite rhoptry protein from Plasmodium falciparum

通过对免疫亲和纯化的约氏疟原虫140 kDa棒状体蛋白条带进行肽段测序，获得PyRhopH 1的部分肽段序列。为了确定编码约氏疟原虫140-kDa棒状体蛋白的基因，使用基于部分氨基酸序列设计的简并寡核苷酸从cDNA进行PCR扩增。最后，通过PCR的基因步移技术，获得了约氏疟原虫RhopH 1的cDNA和gDNA序列，该序列编码由7个内含子组成的1292个氨基酸的开放阅读框架。推导的氨基酸序列在N-末端（aa 1-24）具有SignalP预测的推定的分泌信号序列，并且没有明显的跨膜结构域。对约氏疟原虫基因组数据库的TBLAP 1分析表明，该基因存在一个同源性，因此我们将从肽序列中鉴定的基因命名为pyrhophla，将数据库中发现的第二个基因命名为pyrhoph 1b。利用推导的Pyrhophla的氨基酸序列作为查询条件，在恶性疟原虫基因组数据库中进行TBLAYSTIC搜索，发现了一个编码PyRhopH 1同源物的基因家族。为了确认PyRhopH 1A的大小和定位，进行Western免疫印迹分析和免疫荧光显微镜检查。用pYRH 1A DNA疫苗产生的抗血清在Western免疫印迹上仅反应140-kDa条带，并且通过免疫荧光显微镜显示与抗PyRhopH 1 mAb的模式相同的典型顶端细胞器的点状模式。

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.