Hernández Pilar, Heimann Michael, Riera Cristina, Solano Marco, Santalla José, Luquetti Alejandro O, Beck Ewald
University of Giessen, Institute of Biochemistry, Friedrichstrasse 24, Giessen, Germany.
Clin Vaccine Immunol. 2010 Oct;17(10):1598-604. doi: 10.1128/CVI.00489-08. Epub 2010 Jul 28.
Many proteins of Trypanosoma cruzi, the causative agent of Chagas' disease, contain characteristic arrays of highly repetitive immunogenic amino acid motifs. Diagnostic tests using these motifs in monomeric or dimeric form have proven to provide markedly improved specificity compared to conventional tests based on crude parasite extracts. However, in many cases the available tests still suffer from limited sensitivity. In this study we produced stable synthetic genes with maximal codon variability for the four diagnostic antigens, B13, CRA, TcD, and TcE, each containing between three and nine identical amino acid repeats. These genes were combined by linker sequences encoding short proline-rich peptides, giving rise to a 24-kDa fusion protein which was used as a novel diagnostic antigen in an enzyme-linked immunosorbent assay setup. Validation of the assay with a large number of well-characterized patient sera from Bolivia and Brazil revealed excellent diagnostic performance. The high sensitivity of the new test may allow future studies to use blood collected by finger prick and dried on filter paper, thus dramatically reducing the costs and effort for the detection of T. cruzi infection.
克氏锥虫是恰加斯病的病原体,其许多蛋白质都含有高度重复的免疫原性氨基酸基序的特征性阵列。与基于粗制寄生虫提取物的传统检测方法相比,使用这些单体或二聚体形式的基序进行的诊断测试已被证明具有显著提高的特异性。然而,在许多情况下,现有的检测方法仍然存在灵敏度有限的问题。在本研究中,我们为四种诊断抗原B13、CRA、TcD和TcE构建了具有最大密码子变异性的稳定合成基因,每种抗原都含有三到九个相同的氨基酸重复序列。这些基因通过编码富含脯氨酸的短肽的接头序列进行组合,产生了一种24 kDa的融合蛋白,该蛋白在酶联免疫吸附测定中用作新型诊断抗原。用来自玻利维亚和巴西的大量特征明确的患者血清对该检测方法进行验证,结果显示其具有出色的诊断性能。新检测方法的高灵敏度可能使未来的研究能够使用手指针刺采集并干燥在滤纸上的血液,从而显著降低检测克氏锥虫感染的成本和工作量。