Park Sang Myun, Kim Jongsun
Department of Pharmacology and Chronic Inflammatory Disease Research Center, Ajou University School of Medicine, Suwon, Republic of Korea.
J Virol Methods. 2008 Jan;147(1):1-9. doi: 10.1016/j.jviromet.2007.08.001. Epub 2007 Sep 10.
Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne viral zoonosis characterized by fever, hemorrhagic manifestations, and renal disorder. The causative agent of HFRS has been identified as a hantavirus. Hantavirus nucleocapsid proteins have recently been shown to be immunodominant antigens in HFRS, inducing an early and long-lasting immune response, and their amino termini are sensitive tools for the detection of specific antibodies in HFRS patient sera. Previous work has demonstrated that the introduction of the acidic tail of alpha-synuclein (ATS) into heat-labile proteins protects them from heat-induced aggregation. In this study, the ATS peptide was introduced into the N-terminal antigenic portion of the nucleocapsid proteins (amino acid residues 1-70) of the Hantaan virus (HTNV-delta N) and Seoul virus (SEOV-delta N). The recombinant ATS-HTNV-delta N and ATS-SEOV-delta N fusion proteins were heat-resistant, and the proteins purified by heat treatment were immunoreactive to sera from patients with HFRS. Compared with sera from patients with leptospirosis and scrub typhus, sera from patients with HFRS showed much higher reactivity in ATS-HTNV-delta N- or ATS-SEOV-delta N-based IgG ELISAs. Immunoblotting analysis revealed that only sera from patients with HFRS specifically recognized the ATS-HTNV-delta N and ATS-SEOV-delta N, indicating that the ATS-HTNV-delta N and ATS-SEOV-delta N were highly purified species without any other immunoreactive proteins as contaminants. These data demonstrate that the ATS-HTNV-delta N and ATS-SEOV-delta N fusion proteins offer a safe and inexpensive source of pure and specific antigen for large-scale diagnosis and seroepidemiological studies of HFRS, and that ATS-fusion technology can also be utilized to solubilize other antigens that could be used for large-scale diagnosis and seroepidemiological studies of other diseases.
肾综合征出血热（HFRS）是一种由啮齿动物传播的病毒性人畜共患病，其特征为发热、出血表现和肾脏功能紊乱。HFRS的病原体已被确定为汉坦病毒。最近研究表明，汉坦病毒核衣壳蛋白是HFRS中的免疫显性抗原，可诱导早期且持久的免疫反应，其氨基末端是检测HFRS患者血清中特异性抗体的灵敏工具。先前的研究表明，将α-突触核蛋白的酸性尾巴（ATS）引入热不稳定蛋白可保护它们免受热诱导的聚集。在本研究中，将ATS肽引入汉滩病毒（HTNV-δN）和汉城病毒（SEOV-δN）核衣壳蛋白的N端抗原部分（氨基酸残基1-70）。重组ATS-HTNV-δN和ATS-SEOV-δN融合蛋白具有耐热性，经热处理纯化的蛋白对HFRS患者的血清具有免疫反应性。与钩端螺旋体病和恙虫病患者的血清相比，HFRS患者的血清在基于ATS-HTNV-δN或ATS-SEOV-δN的IgG ELISA中显示出更高的反应性。免疫印迹分析表明，只有HFRS患者的血清能特异性识别ATS-HTNV-δN和ATS-SEOV-δN，这表明ATS-HTNV-δN和ATS-SEOV-δN是高度纯化的，没有任何其他免疫反应性蛋白作为污染物。这些数据表明，ATS-HTNV-δN和ATS-SEOV-δN融合蛋白为HFRS的大规模诊断和血清流行病学研究提供了一种安全、廉价的纯特异性抗原来源，并且ATS融合技术还可用于溶解其他抗原，这些抗原可用于其他疾病的大规模诊断和血清流行病学研究。
