Tume C B, Ngu J L, McKerrow J L, Seigel J, Sun E, Barr P J, Bathurst I, Morgan G, Nkenfou C, Asonganyi T, Lando G
Faculty of Medicine and Biomedical Sciences, University of Yaounde, Cameroon.
Am J Trop Med Hyg. 1997 Nov;57(5):626-33.
A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.
一种酵母（酿酒酵母）表达系统已被改造用于生产试剂级数量的盘尾丝虫主要抗原Ov33。利用针对第三期幼虫产生的一组单克隆抗体，筛选了从成年旋盘尾丝虫构建的cDNA文库。分离出27个cDNA，其中两个与Ov33具有序列同源性，Ov33是一种假定的天冬氨酸蛋白酶抑制剂，是旋盘尾丝虫的免疫显性抗原。这些cDNA在酿酒酵母的细胞内或通过分泌途径高水平表达。使用针对纯化重组蛋白产生的抗血清进行的定位研究表明，Ov33是一种非常丰富的寄生虫蛋白，存在于雌虫的皮下组织、肌肉和子宫以及胚胎微丝蚴中。酵母分泌的可溶性重组蛋白（C71）对天冬氨酸蛋白酶胃蛋白酶具有抑制活性。针对重组蛋白的抗体介导白细胞对皮肤微丝蚴的黏附和杀伤。使用来自441名患者的血清评估了使用重组Ov33的诊断试验的敏感性。两种重组构建体C27和C71的平均敏感性分别为82.2%和85.4%。使用两种重组蛋白的联合敏感性为94%。
