Studies on purification isolation and immunological characterization of 47kD and 23kD polypeptide from blood stage P.falciparum parasite

恶性疟原虫血期47kD和23kD多肽的纯化分离及免疫学特性研究

• 批准号: 06454197
• 负责人: SUZUKI Mamoru
• 金额: $ 4.74万
• 依托单位: Gunma University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454197/
• 关键词: malaria; vaccine; Western blot; polypeptide; electrophoresis; monoclonal antibody; affinity chromatography; protective immunity; ウェスタンブロ-ト; モノクロナール抗体; アフインティクロマトグラフィ; 熱帯熱マラリア原虫; 抗原蛋白

We have continued this research for several years and have reached the final stage of the study in 1995. The eventual aim of this study is the preparation of 47kD polypeptide which is expressed on the membrane of Plasmodium falciparum parasite at schizont stage of the development. The molecule was focussed in the study on immunoblotting using sera from patients manifesting acute stage clinical symptoms. It looks that the molecule works in the immune response evoked to overcome the acute fulminating stage of the disease. Started from this finding, preparation of the 47kD polypeptide was attempted by means of genetic engineering using cDNA or gDNA libraries. However.successful results were not obtained. In the present study, a preparative SDS-PAGE system was applied to the purification of the target molecule. A large amount of cultured P.falciparum parasites was solubilized and the prepared material was subjected to the purification with the use of Prep-cell econo-auomation system(Bio Rad)by continuous-elution electophoresis. The electrophoresed material was devidedP into 40 fractions each with 8.5ml elutate. A 15mu1 sampled specimen from each fraction was taken and the moleular weight was respectively measured using thin layred polyacril amide gel electrophoresis kit (Excel Gel SDS gradiant 8-18, Pharmacia Biotech). Fractions No.28-32 which contained the polypeptide of target molecular weight were pooled. A new type immuno-affinity chromatography (Affi-Gel Hz lmmunoaffinity) was coupled with anti-47kD monolonal antibody and the pooled material was applied to the prepared column. After running with a large amount of washing buffer, the molecule which was bound to the gel by the monoclonal antibody was eluted by a elution buffer. The resulted material is lyophilized and waits for the study of in vitro immune cell response.

这项研究已进行了数年，并于一九九五年完成最后阶段。本研究的最终目的是制备47kD多肽，该多肽表达于恶性疟原虫发育后期的膜上。该分子集中在使用来自表现出急性期临床症状的患者的血清进行免疫印迹的研究中。看起来这种分子在免疫反应中起作用，以克服疾病的急性暴发阶段。从这一发现开始，尝试通过使用cDNA或gDNA文库的遗传工程来制备47kD多肽。然而，没有取得成功的结果。在本研究中，制备性SDS-PAGE系统应用于纯化的目标分子。溶解大量培养的恶性疟原虫寄生虫，并使用Prep-细胞经济自动化系统（Bio Rad）通过连续洗脱电泳对制备的材料进行纯化。将粗提物分成40个组分，每个组分8.5ml洗脱液。从每个级分中取15 μ l样品，并使用薄层聚丙烯酰胺凝胶电泳试剂盒（Excel Gel SDS gradiant 8 - 18，Pharmacia Biotech）分别测量分子量。合并含有目标分子量多肽的级分28 - 32。本文报道了一种新型的免疫亲和层析（Affi-GelHzImmunoaffinity）与抗47kD单克隆抗体偶联，并将混合物上样于制备好的层析柱上。用大量洗涤缓冲液运行后，用洗脱缓冲液洗脱通过单克隆抗体结合到凝胶上的分子。将所得材料冻干并等待体外免疫细胞应答的研究。

项目成果

Agneza Safranj: "Functional polymeric microspheres synthesized by radiation polymerization" Radiation Physics and Chemistry 46 203〜206, 1995. 46. 203-206 (1995)

Agneza Safranj：“辐射聚合合成的功能性聚合物微球”辐射物理与化学 46 203-206，1995. 46. 203-206 (1995)

Shigeyuki Kano, Hohta Masuda, Mamoru, Suzuki: "Recrudescence of falcparum malaria following treatment with halofantrine" Journal of the Japanese Association for Infectious Diseases. vol.68. 536-538 (1994)

Shigeyuki Kano、Hohta Masuda、Mamoru、Suzuki：“用卤泛群治疗后恶性疟疾复发”日本传染病协会杂志。

Minato Nakazawa, Ryutaro Ohtsuka, Koshio Kawabe, Tetsuo Hongo, Tsuguyoshi Suzuki, Tsukasa Inaoka, Tomoya Akimichi, Shigeyuki Kano, Mamoru Suzuki: "Differntial malaria prevalence among villages of the Gidra in lowland Papua New Guinea" Tropical and Geograp

Minato Nakazawa、Rytaro Ohtsuka、Koshio Kawabe、Tetsuo Hongo、Tsuguyoshi Suzuki、Tsukasa Inaoka、Tomoya Akimichi、Shigeyuki Kano、Mamoru Suzuki：“巴布亚新几内亚低地吉德拉河村庄之间的疟疾患病率差异”热带与地理

Akanmori B. D.: "Immunoglobulin G_<2a> isotype may have a protective role in Plasmodium berghel NK65 infection in Immunised mice" Parasitology Research. 80. 638-641 (1994)

Akanmori B. D.：“免疫球蛋白 G_<2a> 同种型可能对免疫小鼠的伯格尔疟原虫 NK65 感染具有保护作用”寄生虫学研究。

Minato Nakazawa: "Differntial malaria prevalence among villages of the Gidra in lowland Papua New Guinea" Ttopical and Geographical Medicine. 46. 350-354 (1994)

Minato Nakazawa：“巴布亚新几内亚低地 Gidra 村庄中不同疟疾的患病率”Ttopical and Geography Medicine。