Soto M, Requena J M, Quijada L, Alonso C
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Spain.
J Clin Microbiol. 1998 Jan;36(1):58-63. doi: 10.1128/JCM.36.1.58-63.1998.
In this work, we describe the assembly of a synthetic gene coding for several antigenic determinants found in different Leishmania infantum antigens. Selected epitopes were derived from the ribosomal proteins LiP2a, LiP2b, and LiP0 and from the histone H2A. The resulting gene was overexpressed in Escherichia coli either as a fusion protein (with the vector pMAL-c2) or alone (with the vector pQE). In both cases, high-level bacterial production of the recombinant protein was achieved and the products were found to be stable. Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments confirmed that the corresponding epitopes are present in the engineered protein. Finally, a serological evaluation of this multiple-epitope protein by Falcon assay screening test-ELISA revealed a sensitivity of 79 to 93% and a specificity of 96 to 100% in diagnosis of canine visceral leishmaniasis, indicating that this protein represents a valuable tool for serodiagnosis.
在本研究中，我们描述了一个合成基因的组装，该基因编码在不同婴儿利什曼原虫抗原中发现的几种抗原决定簇。所选表位来源于核糖体蛋白LiP2a、LiP2b和LiP0以及组蛋白H2A。所得基因在大肠杆菌中作为融合蛋白（与载体pMAL-c2）或单独（与载体pQE）过表达。在这两种情况下，均实现了重组蛋白的高水平细菌生产，且发现产物稳定。酶联免疫吸附测定（ELISA）和蛋白质印迹实验证实，相应表位存在于工程蛋白中。最后，通过Falcon检测筛选试验-ELISA对该多表位蛋白进行的血清学评估显示，在犬内脏利什曼病诊断中，其敏感性为79%至93%，特异性为96%至100%，表明该蛋白是血清学诊断的一种有价值工具。
