关键词: 鸡柔嫩艾美耳球虫; 分泌型免疫球蛋白A(SIgA); ELISA

Abstract: 【Objective】 The aim of this study was to establish an indirect ELISA method for detecting SIgA antibodies against Eimeria tenella in chicken intestinal mucosa in order to evaluate the specific mucosal immune level of Eimeria tenella in chicken.【Method】 The EtMIC2 and SO7 genes of Eimeria tenella were cloned into the prokaryotic expression vector pET-28a(+),and the recombinant plasmid EtMIC2-SO7 was constructed.The recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) competent cells,and the expression of recombinant protein was induced by IPTG and identified by SDS-PAGE and Western blotting.The EtMIC2-SO7 recombinant protein was purified by nickel column affinity chromatography,and the protein concentration was determined by Bradford method. Using purified recombinant protein as the coating antigen,the optimal working concentration for ELISA detection of antigen and mucosa was determined by matrix titration.The optimal incubation time,type of blocking solution,enzyme-linked secondary antibody,and working conditions of the developing solution were determined by single variable method.Referring to the negative and positive criteria of ELISA antibody detection kit,S/P≥0.4 was positive,and S/P＜0.4 was negative as the negative and positive criteria for mucosal samples.The repeatability,specificity and sensitivity of the ELISA detection method were verified,and the coincidence rate was compared with the detection results of Eimeria tenella whole protein coated ELISA plate.【Result】 SDS-PAGE and Western blotting results showed that EtMIC2-SO7 recombinant protein with the size of about 70 ku was obtained,which was consistent with the expected results.The optimal antigen coating concentration of ELISA assay was 2 μg/mL,the optimal dilution of mucosa to be tested was 1∶40,the optimal incubation time of mucosa to be tested was 2 h,the optimal sealing solution was 5% skim milk powder,the optimal dilution of enzyme-conjugate secondary antibody was 1∶100 000,the optimal reaction time was 1 h,and the optimal color development time was 15 min.The coefficients of variation in both intra-batch and inter-batch repeatability tests were less than 10%.No cross-reactivity with specific SIgA antibodies against Newcastle disease virus,Avian influenza virus and Infectious bursal disease virus.Two positive intestinal mucosal samples were randomly selected for sensitivity test.When the dilution concentration was 1∶80,the two samples were still judged to be positive,indicating that the detection method was sensitive.The EtMIC2-SO7 recombinant protein and Eimeria tenella total protein were coated with ELISA plates,and 18 intestinal mucosal samples were detected.The results showed that the coincidence rate of EtMIC2-SO7 recombinant protein coated with ELISA plate and Eimeria tenella total protein coated with ELISA plate was 88.9%(16/18).【Conclusion】 The recombinant EtMIC2-SO7 protein was successfully expressed and purified by the prokaryotic expression system,and was used as the ELISA coated antigen.The indirect ELISA detection method of SIgA antibody against Eimeria tenella was successfully established with good repeatability,strong specificity and high sensitivity,which provided a preliminary detection method for mucosal immune evaluation of coccidium vaccine.

Key words: Eimeria tenella; secretory immunoglobulin A (SIgA); ELISA