产气荚膜梭菌α-β2-ε融合蛋白原核表达及其间接ELISA方法的建立

Prokaryotic expression of the fusion protein α-β2-ε of Clostridium perfringens and establishment of an indirect ELISA method

摘要:

【背景】产气荚膜梭菌(Clostridium perfringens)是引起牛魏氏梭菌病(猝死症)的主要病原，严重危害养牛业。酶联免疫吸附法(ELISA)是诊断疫病及抗体检测的重要手段。【目的】表达重组α-β2-ε融合蛋白，并以此为靶抗原建立间接ELISA抗体检测方法，为临床检测牦牛C. perfringens及流行病学调查提供技术支持。【方法】利用生物信息学软件(EditSeq、Protean)和Jameson-Wolf方法分析牛源C. perfringens的α、β2和ε蛋白抗原指数，截取抗原指数较高肽段并用柔性Linker连接，经密码子优化后克隆至载体pET-32a(+)，构建重组表达质粒pET-32α-β2-ε，转化至大肠杆菌(Escherichia coli) BL21(DE3)感受态细胞，经培养、诱导表达、纯化后采用SDS-PAGE和Western blotting鉴定。以重组α-β2-ε融合蛋白作为包被抗原，经棋盘法和控制单一变量法优化反应条件，建立间接ELISA抗体检测方法，通过受试者工作特征(receiver operating characteristic, ROC)曲线确定临界值并对其敏感性、特异性、重复性和临床效果进行评价。【结果】α的241‒403肽段、β2的161‒355肽段、ε的154‒331肽段抗原指数较高，将它们连接并融合表达，经SDS-PAGE和Western blotting鉴定融合蛋白表达正确。ELISA抗体检测方法最佳工作条件为：抗原包被浓度5 μg/mL 4 ℃过夜或37 ℃孵育60 min；5%脱脂乳于37 °C封闭1 h；血清稀释度为1:150于37 °C孵育30 min；酶标二抗1:2 000于37 °C孵育30 min；避光显色10 min。检测临界值为0.470 2；敏感性高，可检测阳性血清最大稀释度为1:3 200；特异性强，与溶血性曼氏杆菌、沙门氏菌、大肠杆菌、金黄色葡萄球菌等阳性血清均无交叉反应；重复性好，批内和批间变异系数均小于10%。四川省甘孜藏族自治州部分地区共277份牦牛血清样品检测结果显示，C. perfringens抗体阳性率为89.89%，与商品化产气荚膜梭菌α毒素ELISA检测试剂盒检测结果基本一致。【结论】成功制备重组α-β2-ε融合蛋白并建立检测C. perfringens的间接ELISA抗体检测方法。

Abstract:

[Background] Clostridium perfringens is the main pathogen causing sudden death syndrome in cattle, seriously endangering the cattle industry. Enzyme-linked immunosorbent assay (ELISA) is an important tool for diagnosis of this disease and antibody detection. [Objective] We expressed the recombinant α-β2-ε fusion protein and used this protein as the target antigen to establish an indirect ELISA method for antibody detection, aiming to provide technical support for the clinical detection of C. perfringens in yaks and epidemiological investigation. [Methods] The bioinformatic tools EditSeq and Protean and the Jameson-Wolf method were employed to analyze the antigenic indices of α, β2, and ε proteins of C. perfringens from cattle. The peptides with higher antigenic indices were captured, ligated, optimized for codons, and then cloned into the pET-32a(+) vector to construct the recombinant expression plasmid pET-32α-β2-ε, which was transformed into BL21(DE3) competent cells. The cells were cultured and induced for protein expression, and the expressed protein was purified and identified by SDS-PAGE and Western blotting. With the recombinant α-β2-ε fusion protein as the coating antigen, we optimized the reaction conditions by the checkerboard assay and single factor tests to establish an indirect ELISA method. The cut-off was determined by the receiver operating characteristic (ROC) curve, and the specificity, sensitivity, reproducibility, and clinical efficacy of the established method were evaluated. [Results] The peptides with high antigenic indices of α (aa 241–403), β2 (aa 161–355), and ε (aa 154–331) toxins were selected for fusion expression, and the fusion protein was determined as correctly expressed by SDS-PAGE and Western blotting. The optimal ELISA conditions for antibody detection were as follows: incubation with the coating antigen (5 μg/mL) at 4 ℃ overnight or 37 ℃ for 60 min; incubation with 5% skimmed milk at 37 ℃ for 1 h; incubation with the serum (dilution at 1:150) at 37 ℃ for 30 min; incubation with secondary antibody (1:2 000) at 37 ℃ for 30 min; color development for 10 min with protection from light. The cut-off of detection was 0.470 2. The established method demonstrated high specificity because of no cross-reactivity with positive sera of Mannheimia haemolytica, Salmonella, Escherichia coli, and Staphylococcus aureus. It showcased high sensitivity, with the maximum dilution of detectable serum being 1:3 200. The coefficients of variation within and between batches were less than 10%, which indicated high reproducibility of the established method. The detection for 277 yak serum samples from Ganzi Tibetan Autonomous Prefecture, Sichuan Province with the established method showed that the antibody positivity rate of C. perfringens was 89.89%, which was basically in agreement with the results obtained by the commercial ELISA kit for the detection of C. perfringens α-toxin. [Conclusion] We successfully constructed the recombinant α-β2-ε fusion protein and established an indirect ELISA method for the detection of C. perfringens.