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

doi:10.3969/j.issn.1674-7968.2025.11.016

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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.

Key words： Alveolar echinococcosis Echinococcus multilocularis Argonaute 2 Indirect ELISA Early diagnosis

Received: 23 April 2025

ZTFLH:

S855.9

Corresponding Authors: ^*LiuGuangliang01@caas.cn; luoxuenong@caas.cn

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	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

Cite this article:

ZHANG Jun-Mei,GUO Xiao-La,CAO Shan-Ling, et al. Application of an Indirect ELISA Assay Based on Em-AGO2 Recombinant Protein in the Early Infection of Alveolar Echinococcosis[J]. 农业生物技术学报, 2025, 33(11): 2526-2535.

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https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2025.11.016 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2025/V33/I11/2526

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