Expression of erythrocyte binding protein in Plasmodium falciparum merozoites isolated from endemic area

流行区恶性疟原虫裂殖子中红细胞结合蛋白的表达

• 批准号: 13576007
• 负责人: TORII Motomi
• 金额: $ 8.7万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13576007/
• 关键词: infectious disease; malaria; Plasmodium; invasion; protein expression; RT-PCR; parasitology; 赤血球

Mutant erythrocyte that lacked certain proteins on the surface were frequently observed in the malaria endemic areas, and some of them were believed to be generated by the selective advantage against malaria infection. However, Plasmodium falciparum have an ability to invade many types of erythrocyte including these mutant erythrocytes. The molecular base of this ability of P.falciparum could be resulted by the erythrocyte-binding proteins encoded in the multi-gene family involved in the invasion steps. In this study, we explored the possibility if P.falciparum regulated the expression of these multi-gene family in order to overcome the erythrocyte polymorphism by quantitate the transcription and protein expression level of each members of the multi-gene family, PfRhopH1.Firstly, we designed a panel of oligonucleotide primers for real-time PCR method with SYBR green. Complementary DNA was made from the 3D7 clone of P.falciparum and used to make plasmids containing the real-time PCR targets for each member. These plasmids were used to create the standard curve. P.falciparum field samples were collected in the malaria endemic area in Thailand and total RNA were extracted. Simultaneously blood smear were made on the glass slides for the IFA analysis for the protein expression. In order to check the protein expression level, we successfully generated anti-PfRhopHl_2, 3.1, 9 specific sera. Difference in the protein expression was observed among the culture-adapted P. falciparum clones.

在疟疾流行区经常观察到表面缺乏某些蛋白质的突变红细胞，其中一些被认为是由于对疟疾感染的选择性优势而产生的。然而，恶性疟原虫具有侵入包括这些突变红细胞在内的多种类型的红细胞的能力。恶性疟原虫这种能力的分子基础可能是由参与侵袭步骤的多基因家族中编码的红细胞结合蛋白所导致的。本研究通过对多基因家族PfRhopH1各成员的转录水平和蛋白表达水平的定量分析，探讨恶性疟原虫是否通过调控这些多基因家族的表达来克服红细胞的多态性。从恶性疟原虫的3D7克隆制备互补DNA，并用于制备含有每个成员的实时PCR靶标的质粒。这些质粒用于创建标准曲线。在泰国疟疾流行区采集恶性疟原虫现场样本，提取总RNA。同时在玻片上制作血涂片，用于IFA分析蛋白表达。为了检测蛋白表达水平，我们成功地制备了抗PfRhopHl_2、3.1、9特异性血清。在恶性疟原虫的培养适应克隆中观察到蛋白表达的差异。

项目成果

Ling IT, Kaneko O, Narum DL, Tsuboi T, et al.: "Characterization of the rhoph2 gene of Plasmodium falciparum and Plasmodium yoelii"Molecular and Biochemical Parasitology. 127 (1). 47-57 (2003)

Ling IT、Kaneko O、Narum DL、Tsuboi T 等人：“恶性疟原虫和约氏疟原虫的 rhoph2 基因的特征”分子和生化寄生虫学。

Kaneko O, Mu J, Tsuboi T, Su X, Torii M: "Gene structure and expression of a Plasmodium falciparum 220-kilodalton protein homologous to the Plasmodium vivax reticulocyte binding proteins"Molecular and Biochemical Parasitology. (in press).

Kaneko O、Mu J、Tsuboi T、Su X、Torii M：“与间日疟原虫网织红细胞结合蛋白同源的恶性疟原虫 220 千道尔顿蛋白的基因结构和表达”分子和生化寄生虫学。

Kaneko O, Mu J, Tsuboi T, Su X, Torii M.: "Gene structure and expression of a Plasmodium falciparum 220-kDa protein Homologous to the Plasmodium vivax reticulocyte binding proteins"Molecular and Biochemical Parasitology. 121(2). 275-278 (2002)

Kaneko O、Mu J、Tsuboi T、Su X、Torii M.：“与间日疟原虫网织红细胞结合蛋白同源的恶性疟原虫 220-kDa 蛋白的基因结构和表达”分子和生化寄生虫学。

Ling IT, Kaneko O, Narum DL, Tsuboi T, Howwll S, Taylor HM, Scott-Finningan TJ, Torii M, Holder AA: "Characterization of the rhoph2 gene of Plasmodium falciparum and Plasmodium yoelii"Molecular and Biochemical Parasitology. 127(1). 47-57 (2003)

Ling IT、Kaneko O、Narum DL、Tsuboi T、Howwll S、Taylor HM、Scott-Finningan TJ、Torii M、Holder AA：“恶性疟原虫和约氏疟原虫的 rhoph2 基因的表征”分子和生化寄生虫学。

Ling IT, Kaneko O, Narum DL, Tsuboi T, et al.: "Characterization of the rhoph2 gene of Plasmodium falciparum and Plasmodium yoelii"Molecular and Biochemical Parasitology. 127(1). 47-57 (2003)

Ling IT、Kaneko O、Narum DL、Tsuboi T 等人：“恶性疟原虫和约氏疟原虫的 rhoph2 基因的特征”分子和生化寄生虫学。