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
