Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis
Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis
复制标题
DOI：
10.1016/j.ijid.2008.03.033
复制
发表时间：
2008-11-01
影响因子：
8.4
通讯作者：
Loukas, Alex
中科院分区：
文献类型：
--
作者：
Laha, Thewarach;Sripa, Jittiyawadee;Loukas, Alex
目的：目的：从肝吸虫（Opisthorchis viverrini）中分离纯化天冬酰胺酰内肽酶（Aspartaminyl endopeptidase，Ov aep 1），并对其表达谱、生物活性和作为免疫诊断抗原的潜力进行研究。viverrini感染O.维韦里尼。研究Ov-AEP-1在寄生虫发育阶段的转录本，并通过系统发育分析、免疫组织化学定位、重组蛋白表达和酶学分析来表征Ov-AEP-1蛋白。结果：Ov-AEP-1是C13半胱氨酸蛋白酶家族的特征性蛋白，其表达产物具有特异性。在成虫和蠕虫、卵和囊蚴中检测到Ov-aep-1转录本。系统进化分析表明Ov-AEP-1与其他吸虫同源蛋白的亲缘关系较近。重组Ov-AEP-1在细菌中以包涵体形式表达并复性为可溶性形式。O. viverrini和重折叠的重组Ov-AEP-1都显示出对诊断三肽底物Ala-Ala-Asn-氨甲基香豆素的催化活性。针对重组Ov-AEP-1产生的兔抗血清识别了成虫体细胞提取物和ES产物中的天然AEP-1蛋白酶。抗Ov-AEP-1 IgG将表达的解剖学位点免疫定位在吸虫的肠道，这意味着在食物消化或其他消化酶激活中发挥生理作用。重组Ov-AEP-1可被后睾吸虫病患者血清抗体识别，但不被其他蠕虫感染者识别，敏感性和特异性分别为85%和100%。阳性预测值为100%，阴性预测值为67%。灵猫，具有肠定位的天冬酰胺酰内肽酶。复性后的重组Ov-AEP-1具有催化活性，有望用于人后睾吸虫病的免疫诊断。(C)2008年国际传染病学会。由爱思唯尔有限公司出版。保留所有权利。
Objectives: To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen.Methods: The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis.Results: Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory-secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala-Ala-Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67%, respectively.Conclusions: The liver fluke, O. viverrini, has a gut-localized asparaginyl endopeptidase. Refolded recombinant Ov-AEP-1 is catalytically active and has potential for immunodiagnosis of human opisthorchiasis. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.