农业生物技术学报2025，Vol.33Issue(11)：2526-2535,10.DOI:10.3969/j.issn.1674-7968.2025.11.016
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
Application of an Indirect ELISA Assay Based on Em-AGO2 Recombinant Protein in the Early Infection of Alveolar Echinococcosis
摘要
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.关键词
多房棘球蚴病/多房棘球蚴/Argonaute 2(AGO2)/间接ELISA/早期诊断Key words
Alveolar echinococcosis/Echinococcus multilocularis/Argonaute 2/Indirect ELISA/Early diagnosis分类
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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):2526-2535,10.基金项目
国家自然科学基金面上项目(32273031) （32273031）
