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Analysis of antigenic variation mechanism of African trypanosoma applying monoclonal variant-specific antibody

应用单克隆变异特异性抗体分析非洲锥虫抗原变异机制

基本信息

批准号：
60570176
负责人：
FUKUMA Toshihide
金额：
$ 1.09万
依托单位：
Institute of Tropical Medicine, Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

African trypanosomes evade immunological attack in their mammalian host changing their antigen type or serotype. This antigen can be seen as an electron dense structure of 10-15 nm thickness surrounding outer space of plasma membrane of the parasite. The structure is called surface coat and the variant specific antigen is, substantially, uniform glycoprotein. Trypanosome population of different antigen types appear as an undulating parasitemia in loosely ordered manner in an experimentally infected rabbit. In this project, antigenic variation of Trypanosoma brucei gambiense (Tbg) was studied. In the first place, the variation phenomenon was confirmed analysing antigen types which relapsed in temporarily cured mice and number of antibodies which could benoticed in Tbg infected rabbit serum samples according to days after infection. Meanwhile, monoclonal antibodies specific to four different antigen type, 0, 9-1, 9-2 and 11-1. Each of them could agglutinate trypanosomes of homologous ant … More igen type. In the next stage, it was attempted to analyse the mode of antigenic variation in vivo by treating cloned Tbg infected mice with homologous monoclonal antibody and it was realized that even cloned trypanosome population bore trypanosomes of different antigen type(s) in ratio of 10^<-4>-10^<-5>. In the third, the analysis in vitro was planned introducing monoclonal antibody to Tbg culture System. For that purpose, improvement of Tbg culture was investigated. Feeder layer cells from new born mouse brain and muscle gave a stationary growth of Tbg in Eagle's MEM supplemented with fetal bovine serum (5%) and new born bovine serum (5%). Those feeder cells,nevertheless, had ability of supporting Tbg growth in only primary culture. Another problem was difficulty of collecting Tbg because they mainly increased interstitially in foci. To make the system simple and avoid using different population of feeder cell, the culture method without feeder cells found indispensable. After many trials, a better culture method was established as follows. A basal culture medium consisted of Eagle's MEM and Leibovit's L-15 medium. Tbg was cultured in a final culture medium prepared with the basal medium in addition of 5x10^<-4>M EDTA, 10^<-4> M L-cystein,10^<-3> M sodium pyruvate, 3x10^<-6> M 2-mercaptoethanol and fetal bovine serum, 5%. This culture system could be initiated with even cryo-preserved Tbg, and very useful for various experiments. Less

非洲锥虫在其哺乳动物宿主中逃避免疫攻击，改变其抗原类型或血清型。该抗原呈电子致密结构，厚度为10-15 nm，包裹在寄生虫的质膜外层。这种结构被称为表面涂层，变异的特异性抗原基本上是统一的糖蛋白。在实验感染的兔体内，不同抗原类型的锥虫群体表现为松散有序的波状寄生虫病。本课题对布氏冈比亚锥虫的抗原性变异进行了研究。首先，通过对暂愈小鼠复发的抗原类型和感染后天数不同的感染兔血清中可产生抗体的天数的分析，证实了这种变异现象。同时，针对0、9-1、9-2和11-1四种不同抗原类型的单抗。它们都能凝集同源…的锥虫小体更多的伊根型。下一步，用同源单抗处理克隆感染的小鼠，试图分析体内抗原变异的模式，发现即使克隆的锥虫种群也携带不同抗原类型的锥虫(S)，比例为10^&lt；-4&gt；-10^&lt；-5&gt；第三部分，将单抗引入到TBG培养体系中进行体外分析。为此，对TBG的培养条件进行了改进研究。新生小鼠脑和肌肉饲养层细胞在添加胎牛血清(5%)和新生小牛血清(5%)的Eagle‘s MEM中能稳定生长。然而，这些饲养层细胞仅在原代培养中具有支持TBG生长的能力。另一个问题是TBG的收集困难，因为它们主要是在病灶的间质中增加。为了使系统简单，避免使用不同种群的饲养细胞，发现无饲养细胞的培养方法是必不可少的。经过多次试验，确定了较好的培养方法如下。基本培养基组分为Eagle‘s MEM和Leibovit’s L-15。在基础培养液中加入5×10^-4&gt；M EDTA、10^&lt；-4&gt；M L半胱氨酸、10^&lt；-3&gt；M丙酮酸钠、3x10^；-4&gt；-6&gt；M2-巯基乙醇和5%胎牛血清。这种培养体系甚至可以用冷冻保存的TBG来启动，对各种实验都是非常有用的。较少