Establishment of an indirect ELISA technique based on a glycoprotein of Dhori virus
In this study,a prokaryotic expression system was utilized to express the glycoprotein(GP)of Dhori virus(DHOV),which was then used as an antigen to establish an indirect ELISA detection method.According to the GP gene of DHOV-GRT169 strain,primers were designed and amplified to obtain gene fragments that removed transmembrane region and signal peptide.The resulting gene fragments were then used to construct the recombinant expression plasmid pET-32a-GP,which was confirmed through se-quencing and transferred into Escherichia coli BL21(DE3)receptor cells for IPTG-induced expression.The recombinant GP protein was purified using nickel column affinity chromatography.Using DHOV-posi-tive goat serum as the primary antibody,the purified protein was detected by Western-blot,and the pu-rified pET-32a-GP was used as the coating antigen.The reaction conditions were optimized by square ma-trix titration,and an indirect ELISA method for detecting DHOV serum antibody was established to eval-uate its repeatability,sensitivity and specificity.The established indirect ELISA method was used to detect DHOV antibody in different animal serum samples collected from various areas in Xinjiang.The results showed successful construction of the prokaryotic expression plasmid pET-32a-GP,with a GP protein size of approximately 66.8 ku and good reactivity.The optimal conditions for ELISA were deter-mined to be a coating concentration of 4 μ g/mL,a primary antibody dilution of 1∶1 000,a 30-minute in-cubation time for the primary antibody,a secondary antibody dilution of 1∶3 000,a 30-minute incuba-tion time for the secondary antibody,and a 4-minute color development time for TMB.Repeated testing showed a coefficient of variation(CV)intra-and inter-assay of less than 10％.Sensitivity testing re-vealed good sensitivity at a positive serum dilution of 1∶1 400.The established indirect ELISA method was negative for detecting GTV,CCHFV and TAMV.A total of 343 animal sera from different regions in Xin-jiang were randomly selected for ELISA detection,with 48 DHOV-positive sera detected,resulting in a positive detection rate of 13.99％.The establishment of this method provides a basis for epidemiologi-cal analysis of DHOV and serves as a theoretical foundation for further research on the biological function of DHOV GP protein,development of detection kits,and creation of vaccines.
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