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

疟疾是一种由蚊子传播的达拉西亚疾病，是世界上大多数热带地区的主要死亡原因，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替代方法来根除这种疾病。这项研究项目的目的是开发一种有效的疟疾疫苗。攻击入侵的子孢子寄生虫。为达到这一目的，我们在1991年(-1991年)完成了以下工作：1)环子孢子表面蛋白(CSP)部分人生长激素与氨基酸重复片段(PNAN)_8(PNVD)_2融合蛋白(FP)在大肠杆菌中的表达和纯化，及其二级结构的CD测定或推测。用木瓜酶部分消化分离Fab片段(Ab-Fab)。3)用酶联免疫吸附试验(EL ISA)测定Fp与抗体(Ab)的亲和力，并测定其解离常数。4)抗原(Fp)、抗体(Ab-Fab)及其与Fp的络合物的结晶实验。在不同温度下使用几种沉淀剂，PHS已经启动，抗体晶体在两到三周内生长到0.8 mm，但对于X射线数据采集来说，它们的尺寸仍然太小。5)1990年开始在巴黎巴斯德研究所按照G.温特程序进行抗体的氨基酸序列测定。我们于1991年完成了抗体Fab重链氨基酸序列的测定，并与其他抗体序列进行了比较，发现了两个一致的半胱氨酸残基。6)我们成功地大量生产了一种新的融合蛋白(FP-L)，该融合蛋白由FP和CSP中发现的一个肽组成并起作用。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.