鸡柔嫩艾美耳球虫EtMIC2和SO7蛋白的原核表达及SIgA抗体间接ELISA检测方法建立

Prokaryotic Expression of EtMIC2 and SO7 Proteins of Eimeria tenella and Establishment of an Indirect ELISA Method for Detection of SIgA Antibody

【目的】建立检测鸡肠道黏膜柔嫩艾美耳球虫SIgA抗体的间接ELISA检测方法以期对鸡柔嫩艾美耳球虫特异性黏膜免疫水平进行评价。【方法】将鸡柔嫩艾美耳球虫EtMIC 2和SO 7基因克隆至pET-28a(+)原核表达载体,构建EtMIC2-SO7重组质粒。将重组质粒转化大肠杆菌Rosetta(DE3)感受态细胞,经IPTG诱导表达后,通过SDS-PAGE和Western blotting进行鉴定。采用镍柱亲和层析法对EtMIC2-SO7重组蛋白进行纯化,用Bradford法测定蛋白浓度。以纯化的重组蛋白作为包被抗原,通过方阵滴定法,确定ELISA检测抗原、待检黏膜的最佳工作浓度,通过单一变量法确定最佳待检黏膜孵育时间、封闭液种类、酶标二抗、显色液工作条件,参照ELISA抗体检测试剂盒阴阳性判定标准,以S/P≥0.4为阳性,S/P<0.4为阴性作为黏膜样品阴阳性判定标准,对ELISA检测方法的重复性、特异性、敏感性进行验证并与鸡柔嫩艾美耳球虫全蛋白包被ELISA板检测结果进行符合率比较。【结果】SDS-PAGE和Western blotting结果显示,获得大小约70 ku的EtMIC2-SO7重组蛋白,与预期结果相符。ELISA检测方法的最佳抗原包被浓度为2μg/mL,待检黏膜最适稀释度为1∶40,待检黏膜最适孵育时间为2 h,最适封闭液为5%脱脂奶粉,最适酶标二抗稀释度为1∶100000,最适反应时间为1 h,最适显色时间为15 min;批内、批间重复性试验变异系数均<10%;与新城疫病毒、禽流感病毒、传染性法氏囊病病毒特异性SIgA抗体无交叉反应性;随机选择2份阳性肠道黏膜样品进行敏感性试验,稀释浓度为1∶80时2份样品仍判定为阳性,说明该检测方法敏感性较好;将EtMIC2-SO7重组蛋白和鸡柔嫩艾美耳球虫全蛋白分别包被ELISA板,检测18份肠道黏膜样品,结果表明EtMIC2-SO7重组蛋白包被ELISA板与球虫全蛋白包被ELISA板符合率可达88.9%(16/18)。【结论】本研究采用原核表达系统成功表达并纯化EtMIC2-SO7重组蛋白,并将其作为ELISA包被抗原,成功建立重复性好、特异性强、敏感性高的鸡柔嫩艾美耳球虫SIgA抗体的间接ELISA检测方法,为球虫黏膜免疫疫苗评价提供了初步检测方法。

【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 EtMIC 2 and SO 7 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 2μg/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∶100000,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.