Application of an Indirect ELISA Assay Based on Em-AGO2 Recombinant Protein in the Early Infection of Alveolar Echinococcosis
ZHANG Jun-Mei1,2, GUO Xiao-La2, CAO Shan-Ling2, TIAN Zheng2, ZHOU Yu2, WU Yi-Xuan2, ZHANG Zheng-Zhe2, LIU Guang-Liang1,2,*, LUO Xue-Nong2,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
Abstract：Alveolar echinococcosis (AE) is a severe zoonotic parasitic disease caused by the larval stage (alveolar hydatid) of Echinococcus multilocularis, which parasitizes the liver of humans and animals. This study aimed to prepare the E. multilocularis Em-AGO2 (Argonaute 2) recombinant protein using prokaryotic expression technology, establish an indirect enzyme-linked immunosorbent assay (ELISA), and preliminarily evaluate its potential for early diagnosis of AE. The N-terminal 1~738 bp fragment of the Em-AGO2 gene was selected to construct the truncated recombinant expression vector pET-28a-Em-AGO2 (1~738 bp). This vector was transformed into an Escherichia coli expression system, and high-level expression was achieved through induction. The Em-AGO2 truncated recombinant protein was then purified by affinity chromatography to obtain a high-purity product. Western blot analysis confirmed that serum samples from E. multilocularis-infected mice specifically recognized the truncated Em-AGO2 recombinant protein, indicating its strong antigenicity. The purified truncated Em-AGO2 recombinant protein was used as the coating antigen. Through stepwise parameter adjustment reaction conditions were optimized: 1 μg/mL of truncated Em-AGO2 recombinant protein was coated onto microplates using 0.05 mol/L (pH 9.6) carbonate coating buffer (CBS) at 4 ℃ overnight; Plates were blocked with 5% skimmed milk at 37 ℃ for 1 h; Test sera (1∶200 diluted in blocking buffer) were added (100 μL/well) and incubated at 37 ℃ for 1 h; Horseradish peroxidase (HRP)-conjugated secondary antibody (1∶10000 diluted in blocking buffer) was added (100 μL/well) and incubated at 37 ℃ for 1 h; after chromogenic substrate development in the dark for 10 min, 50 μL of stop solution was added, and OD450 were measured. ROC-curve analysis revealed that the indirect ELISA based on truncated recombinant Em-AGO2 exhibited 100% diagnostic sensitivity and 100% specificity (AUC=1.00) for Echinococcus multilocularis infection in mice. With no cross-reactivity observed against positive serum samples from sheep infected with Echinococcus granulosus, Taenia multiceps, Fasciola hepatica, Toxoplasma gondii, Haemonchus contortus, or rabbits infected with Echinococcus granulosus cysticercus. When applied to detect early-stage E. multilocularis-infected mouse serum samples, the method showed that the following performance: At 7 d post-infection (dpi), the positive detection rates using crude parasite antigen, Em18 recombinant protein, and truncated Em-AGO2 recombinant protein were 18.75%, 75%, and 71.88%, respectively; at 10 dpi, these rates increased to 34.38%, 90.63% and 84.38%; at 15 dpi, they reached 81.25%,100%, and 100%; At 20 dpi, all 3 assays maintained 100% positivity. In summary, this study successfully developed an indirect ELISA detection method for AE based on the truncated Em-AGO2 recombinant protein. This method demonstrates significant potential in the early diagnosis of animal alveolar echinococcosis and holds important application prospects.
张军梅, 郭小腊, 曹珊菱, 田峥, 周昱, 吴易璇, 张政哲, 刘光亮, 骆学农. 基于Em-AGO2重组蛋白间接ELISA检测方法在多房棘球蚴病早期感染中的应用[J]. 农业生物技术学报, 2025, 33(11): 2526-2535.
ZHANG Jun-Mei, GUO Xiao-La, CAO Shan-Ling, TIAN Zheng, ZHOU Yu, WU Yi-Xuan, ZHANG Zheng-Zhe, LIU Guang-Liang, LUO Xue-Nong. Application of an Indirect ELISA Assay Based on Em-AGO2 Recombinant Protein in the Early Infection of Alveolar Echinococcosis. 农业生物技术学报, 2025, 33(11): 2526-2535.
[1] 顾贤波, 王志鑫, 樊海宁, 等 . 2019. 超声引导下微波消融与手术切除治疗早期泡型肝包虫病疗效对比[J]. 介入放射学杂志, 28(12): 1151-1155. (Gu X B, Wang Z X, Fan H N, et al.2019. Ultrasound-guided microwave ablation versus surgical resection for the treatment of early alveo- lar hepatic echinococcosis: Comparison of curative effect[J]. Journal of Interventional Radiology, 28(12): 1151-1155.) [2] 郭刚, 任远, 焦红杰, 等. 2023. 腹腔感染棘球蚴微囊对小鼠肝脏多房棘球蚴感染及致病的影响[J]. 中国寄生虫学与寄生虫病杂志, 41(02): 156-162. (Guo G, Ren Y, Jiao H J, et al.2023. Effect of intraperitoneal inoculation with Echinococcus microcysts on the infection and pathogenicity of E. multilocularis in mouse liver[J]. Chinese Journal of Parasitology and Parasitic Diseases, 41(02): 156-162.) [3] 赵玉泽, 刘学军, 杜毓锋. 2015. Ago2蛋白在肿瘤中的研究进展[J]. 医学综述, 21(11): 1986-1989. (Zhao Y Z, Liu X J, Du Y F.2015. Research progress of Ago2 protein in tumors[J]. Medical Recapitulate, 21(11): 1986-1989.) [4] Arroyo J D, Chevillet J R, Kroh E M, et al.2011. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma[J]. Proceedings of the National Academy of Sciences of the USA, 108(12): 5003-5008. [5] Bi X J, Shao Y M, Li L, et al.2018. Evaluation of the diagnostic value of the immunoblotting and ELISA tests using recombinant Em18 antigen in human alveolar echinococcosis from Xingjiang China[J]. Experimental & Therapeutic Medicine, 16(4): 3155-3160. [6] Bresson-Hadni S, Spahr L, Chappuis F.2021. Hepatic alveolar echinococcosis[J].Seminars in Liver Disease, 41(3): 393-408. [7] Brunetti E, Kern P, Vuitton D A.2010. Writing panel for the WHO-IWGE. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in hu- mans[J]. Acta tropica, 114(1): 1-16. [8] Carmena D, Benito A, Eraso E.2007. The immunodiagnosis of Echinococcus multilocularis infection[J]. Clinical Microbiology and Infection, 13(5): 460-475. [9] Casulli A, Barth T F E, Tamarozzi F.2019. Echinococcus multilocularis[J]. Trends in Parasitology, 35(9): 738-739. [10] Cheng N, Li Y, Han Z G.2013. Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma[J]. Hepatology, 57(5): 1906-1918. [11] Cubillos-Ruiz J R, Baird J R, Tesone A J, et al.2012. Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer[J]. Cancer Research, 72(7): 1683-1693. [12] Deplazes P, Rinaldi L, Alvarez Rojas C A, et al.2017. Global distribution of alveolar and cystic echinococcosis[J]. Advances in Parasitology, 95: 315-493. [13] Diederichs S, Jung S, Rothenberg S M, et al.2008. Coexpres- sion of Argonaute 2 enhances RNA interference toward perfect match binding sites[J]. Proceedings of the National Academy of Sciences of the USA, 105(27): 9284-9289. [14] Förster S, Koziol U, Schäfer T, et al.2019. The role of fibroblast growth factor signalling in Echinococcus multilocularis development and host-parasite interaction[J]. PLOS Neglected Tropical Diseases, 13(3): e0006959. [15] Gottstein B, Stojkovic M, Vuitton D A, et al.2015. Threat of alveolar echinococcosis to public health-a challenge for Europe[J].Trends in Parasitology, 31(9): 407-412. [16] Kern P, Menezes da Silva A, Akhan O, et al.2017. The Echinococcoses: Diagnosis, clinical management and burden of disease[J]. Advances in Parasitology, 96: 259-369. [17] Kronenberg P A, Deibel A, Gottstein B, et al.2022. Serological assays for alveolar and cystic echinococcosis a comparative multi-test study in Switzerland and Kyrgyzstan[J]. Pathogens, 11(5): 518. [18] Ma T, Wang Q, Hao M, et al.2023. Epidemiological characteristics and risk factors for cystic and alveolar echinococcosis in China: An analysis of a national population based field survey[J]. Parasites & Vectors, 16(1): 181. [19] Shen J, Xia W, Khotskaya Y B, et al.2013. EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2[J]. Nature, 497(7449): 383-387. [20] Tappe D, Zidowitz S, Demmer P, et al.2010. Three-dimensional reconstruction of Echinococcus multilocularis larval growth in human hepatic tissue reveals complex growth patterns[J]. American Journal of Tropical Medicine & Hygiene, 82(1): 126-127. [21] Torgerson P R, Keller K, Magnotta M, et al.2010. The global burden of alveolar echinococcosis[J]. PLOS Neglected Tropical Diseases, 4(6): e722. [22] Vaksman O, Hetland T E, Trope' C G, et al.2012. Argonaute, Dicer, and Drosha are up-regulated along tumor progression in serous ovarian carcinoma[J]. Human Pathology, 43(11): 2062-2069. [23] Wang X, Kui Y, Xue C Z, et al.2025. Past, present and future epidemiology of echinococcosis in China based on nationwide surveillance data 2004-2022[J]. Journal of Infectious Diseases, 90(3): 106445. [24] Wen H, Vuitton L, Tuxun T, et al.2019. Echinococcosis: Advances in the 21st Century[J]. Clinical Microbiology Reviews, 32(2): e00075-18. [25] Wu J, Yang J, Cho W C, et al.2020. Argonaute proteins: Structural features, functions and emerging roles[J]. Journal of Advanced Research, 24: 317-324. [26] Zhang J, Fan X S, Wang C X, et al.2013. Up-regulation of Ago2 expression in gastric carcinoma[J]. Medical Oncology, 30(3): 628. [27] Zhang X, Graves P R, Zeng Y.2009. Stable Argonaute 2 overexpression differentially regulates microRNA production[J]. Biochimica et Biophysica Acta, 1789(2): 153-159. [28] Zheng Y.2013. Phylogenetic analysis of the Argonaute protein family in platyhelminths[J]. Molecular Phylogenetics and Evolution, 66(3): 1050-1054.
