Studies on Antigenic Molecules of Plasmodium Falcipalum Using Polyclonal Antibody from Infected Human Subjects

1989年，从输入性疟疾病例和居住在不同流行地区的个人收集了样本。用来自不同流行背景的人的血清检测了分离的恶性疟原虫抗原分子的反应性。用免疫印迹技术检测到寄生虫分子能与感染急性期的血清发生特异性反应。因此，希望该分子可用于在社区一级和在个别病例的诊断中用血清学方法确定最近的感染。1990年，从培养的恶性疟原虫中提取RNA，利用EcoRI适配器构建了lambda gt11基因的表达文库。在用与寄生虫分子高度反应的人血清进行免疫筛选后，重组lambda gtll的单个克隆通过生长转基因E.coil Y1090得到了大量扩增。将插入的DNA用BamHI切下，连接到BamHI酶切的pUC118上。将重组DNA注入大肠杆菌JM109中。对转化细胞进行二次培养，获得BamHI消化后的DNA片段。将得到的DNA片段Nick翻译成32P标记的寄生虫DNA产物，用于BamHI片段基因组DNA的Southern杂交。因此，通过凝胶双脱氧基测序分析，获得了编码抗原分子的部分寄生虫DNA序列。该寄生虫DNA与已登记的寄生虫基因不一致，但与克氏锥虫的VSG基因有相似性。

In 1989, sere from imported malaria cases and from individuals living in various endemic areas were collected. The reactivity of the SDS-fractionated Plasmodium falciparum antigen molecule was tested using the human sera taken from subjects with varying endemic background. Aparitcular parasite molecule was found to be specifically reactive with the sera from acute phase of the infection by Western blot technique. Thus it was hoped that the molecule can be useful to define recent past infection by serological method both at the community level and also in the diagnosis of individual cases. In 1990, the RNA from cultured P. falciparum were extracted, and the lambda gt11 cDNA expression library was prepared using EcoRI adapter. Following immunoscreening using a human serum which is highly reactive with the parasite molecule, a single clone of the recombinant lambda gtll was largery amplifie by growing transfected E. coil Y1090. The insert DNA was cut out by BamHI and ligated to BamHI digested pUC118. The recombinant DNA was injected into E. coli JM109. The transformant cells were again grown to obtain BamHI digested DNA fragment was of the parasite. The resulted DNA fragment nick translated to prepare 32P labeled parasite DNA prode which was applied to Southern hybridazation of BamHI fragmented genomic DNA of the parasite. Thus, a part of the sequence of parasite DNA encoding the antigen molecule was read by means of dideoxyl sequencing analysis on gel. The parasite DNA was not conincidental with already registered parasite gene, however, a similarity was noted with VSG gene of Trypanosoma cruzi.