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

疟疾是一种由蚊子传播的寄生虫病，在世界上大多数热带地区，由于出现了抗杀虫剂的蚊子和耐药的疟疾寄生虫，疟疾是造成死亡的主要原因。这些促使人们寻找其他替代方法来根除它。这一研究项目的目标是研制一种有效的疟疾疫苗。攻击入侵的孢子虫寄生虫。为了实现这一目标，我们在1989 - 1991财政年度完成了以下课题。1)环孢子子表面蛋白（CSP）部分生长激素与氨基酸重复片段（PNAN）_8(PNVD)_2在大肠杆菌中融合蛋白（FP）的表达、纯化及其二级结构的CD测定和推测。2)纯化BALb/c小鼠产生的抗FP单克隆抗体（Ab），用木瓜蛋白酶部分酶切分离Fab片段（Ab-Fab）。3)采用ELISA法测定抗原（FP）与抗体（Ab）的亲和力，并实验测定解离常数。4)抗原（FP）、抗体（Ab-Fab）及AbFab与FP复合物的结晶试验。通过使用几种沉淀剂在不同的温度和ph值下开始，抗体晶体在两到三周内生长到0.8 mm，但它们的尺寸仍然太小，无法进行x射线数据收集。5)抗体的氨基酸序列测定始于1990年巴黎巴斯德研究所根据G. Winter的程序。我们于1991年完全确定了Ab-Fab重链的氨基酸序列，并与其他抗体序列进行比较，发现了两个一致的半胱氨酸残基。6)我们成功地过量生产了一种新的融合蛋白（FP- 1），它由FP和一种在CSP中发现并起作用的肽组成。作为t细胞表位及其结晶。

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.