X-ray Structural Studies of Antigen-Antibody Complex Toward Malaria Vaccine Development.

用于疟疾疫苗开发的抗原抗体复合物的 X 射线结构研究。

• 批准号: 01044086
• 负责人: TOMITA Ken-ichi
• 金额: $ 6.46万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01044086/
• 关键词: Malaria vaccine; X-ray structural studies; Repeating amino acid sequence; Circumsporozoite surface protein as antigen; Monoclonal antibodies against circumsporozoite; surface protein; Amino acid Sequence of heavy chain in Fab; 融合蛋白質抗原; モノクロ-ナル抗体 /

Malaria is a Darasitic disease transmitted by mosquitoes and a major cause of death in most tropical areas of the world, because of the appearance of insecticide-resistant mosquitoes and drug-resistant malaria parasites. These have prompted the search for other alternative methods to eradicate it. The objective of this research project was to develop a malaria vaccine which wbold. attack the invaded sporozoite parasite. In order to achieve this purpose, we finished the following subjects in the fiscal years (1989 - 1991).1) Expression and purification of the fusion protein (FP) consisting of a part of human growth hormone and an amino acids-repeating segment, (PNAN)_8(PNVD)_2, of circumsporozoite surface protein (CSP) in E. coli, and the CD measurement or postulation of its secondary structure.2) Purification of the monoclonal antibodies (Ab) against FP produced in BALb/c mice, and isoldrion of Fab segment (Ab-Fab) by partial digestion with papain enzyme.3) ELISA tests for affinity of the antigen (FP) for antibody (Ab) were performed, and the dissociation constants were experimentally determined.4) Crystallization trials of the antigen (FP), the antibody (Ab-Fab) and the complex of AbFab with FP. by using several precipitants under different temperature and pHs have been started, and the antibody crystal grew to 0.8 mm in two to three weeks, but they are still too small in dimension for X-ray data collection.5) The amino acid sequence determination of antibody started in 1990 at the Institut Pasteur in Paris according to G. Winter's procedure. We completely determined the amino acid sequence of the heavy chain in Ab-Fab in 1991, and found two consensus cysteine residues in comparison with the other antibody sequences.6) We succeeded in overproducing a new fusion protein (FP-l) consisting-of FP and a peptide which was found in CSP and acted. as a T-cell epitope and its-crystallization.

疟疾是一种由蚊子传播的Darasitic疾病，是世界上大多数热带地区的主要死因，因为出现了抗药性蚊子和抗药性疟疾寄生虫。这些都促使人们寻找其他的替代方法来消灭它。这个研究项目的目标是开发一种疟疾疫苗，攻击入侵的子孢子寄生虫。为了达到这一目的，我们在1989 ~ 1991年进行了以下研究工作：1）人生长激素部分片段与环子孢子表面蛋白（CSP）氨基酸重复片段（PNAN）_8（PNVD）_2的融合蛋白（FP）在大肠杆菌中的表达和纯化; 2）用木瓜蛋白酶部分消化法纯化BALB/c小鼠产生的抗FP单克隆抗体（Ab）和Fab片段的同源片段（Ab-Fab）; 3）用ELISA法检测抗原（FP）与抗体（Ab）的亲和力，（4）对制备的抗原（FP）、抗体（Ab-Fab）及其与FP的复合物进行结晶试验。在不同的温度和pH值下使用几种沉淀剂，已经开始，并且抗体晶体在两到三周内生长到0.8mm，但是它们的尺寸对于X射线数据收集来说仍然太小。温特的手术1991年，我们对Ab-Fab的重链氨基酸序列进行了全序列测定，并与其它抗体序列进行了比较，发现了两个共有的半胱氨酸残基。6）成功地获得了一种新的融合蛋白（FP-1）。作为T细胞表位及其结晶。

项目成果

M. B. Lascombe et al. (R. J. Poljak): "The three-dimensional structure of Fab R19.9, a monoclonal murine antibody specific for the p-azobenzenearsonate group." Proc. Natl. Acad. Sci. USA.86. 607-611 (1989)

M. B. Lascombe 等人。

G.Boulot et al.(R.J.Poljak): "Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody and of its complex with lysozyme." J.Mol.Biol.213. 617-619 (1990)

G.Boulot 等人 (R.J.Poljak)：“单克隆抗溶菌酶抗体及其与溶菌酶复合物的细菌表达 Fv 的结晶和初步 X 射线衍射研究。”

M. I. Ramos et al. (Y. Omata): "Plasmodium falciparum : Characterization of a 33-kDa soluble antigen." Experimental Parasitology.68. 297-307 (1988)

M.I.拉莫斯等人。

G.E.Armah et al.: "The synthesis,cloning and expression of a repeating segment of the circumsporozoite surface protein of Plasmodium falciparum." Nucleic Acids Res.,Symposium Series.19. 165-169 (1988)

G.E.Armah 等人：“恶性疟原虫环子孢子表面蛋白重复片段的合成、克隆和表达。”

G. E. Armah et al.: "Conformation and immunogenicity of engineered repeating segment of the circumsporozoite surface protein of Plasmodium falciparum." Mol. Biochem. Parasitology.38. 135-140 (1990)

G. E. Armah 等人：“恶性疟原虫环子孢子表面蛋白的工程化重复片段的构象和免疫原性。”