空肠弯曲菌PEB1A蛋白的原核表达及间接ELISA方法的建立

发布时间：2018-05-26 11:30

本文选题：空肠弯曲菌 + 间接ELISA　；参考：《中国畜牧兽医》2017年12期

【摘要】：为了建立检测血清中空肠弯曲菌(Campylobacter jejuni)抗体的间接ELISA方法,试验通过PCR扩增并克隆空肠弯曲菌PEB1A基因,构建原核表达载体pET32a-PEB1A,将该表达载体转化大肠杆菌BL21(DE3)感受态细胞,诱导表达获得约47ku的可溶性目的蛋白,通过Western blotting证明所表达的重组蛋白具有良好的生物学活性。以纯化后的重组蛋白作为包被抗原,建立空肠弯曲菌抗体间接ELISA方法,对其特异性、敏感性、重复性进行检测。结果表明,本试验建立的空肠弯曲菌抗体间接ELISA检测方法临界值为0.3424,本方法仅与空肠弯曲菌阳性血清发生特异性反应,与其他抗血清无交叉反应,具有较强的特异性,批内、批间的重复性试验变异系数均5%,具有较好的重复性和稳定性。该方法的建立可应用于血清中空肠弯曲菌抗体的快速检测,为进一步防制空肠弯曲菌腹泻提供依据。
[Abstract]:In order to establish an indirect ELISA method for detecting the antibody of Campylobacter jejuni in serum, the PEB1A gene of Campylobacter jejuni was amplified and cloned by PCR, and the prokaryotic expression vector pET32a-PEB1A was constructed, and the expression vector was transformed into the BL21 (DE3) receptive cell of Escherichia coli and the expression of soluble target protein of about 47ku was obtained. The recombinant protein expressed by Western blotting proved to have good biological activity. The purified recombinant protein was used as the envelope antigen to establish an indirect ELISA method for the antibody of Campylobacter jejuni, and its specificity, sensitivity and repeatability were detected. The results showed that the indirect ELISA detection method for the antibody of Campylobacter jejuni was established in this experiment. The boundary value is 0.3424. This method is only specific to the positive sera of Campylobacter jejuni, and has no cross reaction with other antiserum. It has strong specificity. In batch, the variation coefficient of repeated test in batch is 5%, and has good repeatability and stability. The method can be applied to the rapid detection of the antibody of Campylobacter jejuni in serum. To provide basis for further prevention of Campylobacter jejuni diarrhea.
【作者单位】：吉林农业大学动物科技学院;军事医学科学院军事兽医研究所;吉林省人兽共患病预防与控制重点实验室;
【基金】：国家重点研发项目“宠物疾病诊疗与防控新技术”(2016YFD0501004)
【分类号】：S852.61

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