马泰勒虫新疆株EMA-1基因的原核表达及间接ELISA检测方法的建立

发布时间：2018-07-03 19:24

  本文选题：马泰勒虫 + 新疆株EMA-　； 参考：《畜牧兽医学报》2017年09期

【摘要】：马泰勒虫裂殖子表面抗原1(EMA-1)是用于马梨形虫病诊断的重要靶抗原之一。为建立实用有效的间接酶联免疫吸附试验方法,笔者根据马泰勒虫EMA-1基因序列设计并合成引物,将新疆株EMA-1全基因序列克隆于原核表达载体pGEX-4T-1上,构建pGEX-4T-1/EMA1重组质粒,转化至BL21(DE3)中,经IPTG诱导得到EMA-1融合蛋白,切胶纯化后作为包被抗原建立检测马泰勒虫抗体的rELISA方法。结果显示:(1)GST-EMA1蛋白相对分子质量56ku,与其理论值基本一致;(2)通过Western blot分析证明其具有很好的特异性和反应原性;(3)以GST-EMA1蛋白作为包被抗原建立的rELISA可明显区分马泰勒虫、驽巴贝斯虫阳性血清和健康马血清;批内和批间重复试验的最大变异系数分别为14.79%和11.06%;(4)使用建立的间接ELISA与cELISA商品试剂盒分别对新疆伊犁马场收集的96份马血清样品进行检测,检出阳性率分别为27.1%(26/96)和25.0%(24/96),两者总符合率为95.8%。结果表明,基于原核表达的GST-EMA1蛋白所建立的rELISA特异性、重复性好,检出率与马泰勒虫临床分离率接近,可为新疆马泰勒虫病(特别是隐性带虫马)的检测、监控提供有效手段。
[Abstract]:Merozoite surface antigen 1 (EMA-1) is one of the important target antigens for the diagnosis of piridiosis. In order to establish a practical and effective indirect enzyme-linked immunosorbent assay (Elisa) method, primers were designed and synthesized according to the sequence of EMA-1 gene of Myrelella martensii. The EMA-1 gene sequence of Xinjiang strain was cloned into the prokaryotic expression vector pGEX-4T-1, and the recombinant plasmid pGEX-4T-1 / EMA1 was constructed. After transformed into BL21 (DE3), the fusion protein EMA-1 was induced by IPTG. The fusion protein was purified and purified as a coated antigen to establish a rELISA method for the detection of marTaylor's antibody. The results showed that: (1) the relative molecular weight of GST-EMA1 protein was basically consistent with its theoretical value; (2) it was proved by Western blot analysis that GST-EMA1 protein had good specificity and reactivity; The maximum coefficients of variation of intra- and inter-lot repeated tests were 14.79% and 11.06%, respectively. (4) 96 horse serum samples collected from Yili horse farm in Xinjiang were detected by using the established indirect Elisa and CELISA commercial kits. The positive rates were 27.1% (26 / 96) and 25.0% (24 / 96), respectively. The results showed that the rELISA based on prokaryotic expression of GST-EMA1 protein was specific and reproducible, and the detection rate was close to that of the clinical isolation rate, which could provide an effective method for detection and monitoring of MalTaylor's disease in Xinjiang.
【作者单位】： 新疆农业大学动物医学学院;伊犁出入境检验检疫局综合技术服务中心;
【基金】：国家自然科学基金-新疆联合基金重点项目(U1403283)
【分类号】：S858.21
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本文编号：2094741