Gene cloning of a novel merozoite rhoptry protein from Plasmodium falciparum

通过对免疫亲和纯化的约140 kDa约氏疟原虫杆状蛋白条带的肽序列分析，我们获得了约氏疟原虫的部分肽序列。为了确定编码约氏疟原虫140 kDa棒状蛋白的基因，根据部分氨基酸序列设计了简并的寡核苷酸，从cDNA中进行了PCR扩增。通过基于聚合酶链式反应的基因行走，我们完成了编码由7个内含子组成的1292个氨基酸的开放阅读框的gDNA和cDNAs序列。Deduded氨基酸序列在N端(aa1-24)有SignalP预测的可能的分泌信号序列，没有明显的跨膜结构域。通过对约氏拟青霉基因组数据库的TBLASTN分析，发现该基因存在一个同源基因，因此我们将从多肽序列中鉴定出的该基因命名为金针菇，第二个基因命名为金龟子1b。以果蝇推导的氨基酸序列为查询条件，通过对恶性疟原虫基因组数据库的TBLASTN搜索，发现了一个编码PyRhop H1同源基因的基因家族。通过免疫印迹分析和免疫荧光显微镜观察，确定了该基因的大小和定位。PYRH1ADNA疫苗产生的抗血清在免疫印迹中仅反应140 kDa条带，在免疫荧光显微镜下，顶端细胞器呈现典型的点状图案，与抗PyRHopH1单抗的图案相同。

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.