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

doi:10.11843/j.issn.0366-6964.2017.09.020

摘要/Abstract

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：①GST-EMA1蛋白相对分子质量56 ku，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:

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. The aim of the present study was to establish an indirect ELISA for practical use. According to the specific sequence of EMA-1 genes of Theileria equi, a pair of primers was designed and synthesized. The EMA-1 gene of Xinjiang strain was cloned, and then was inserted into the prokaryotic vector pGEX-4T-1. The recombinant plasmid (pGEX-4T-1/EMA1) were transformed into Escherichia coli BL12(DE3), and the GST-EMA1 protein was obtained by induction of IPTG.The fusion protein was purified by extracting the inclusion bodies with gel slices,and the purified protein was used as coated antigen for the establishment of indirect ELISA method to detect the antibody to Theileria equi.The results indicated that the expressed EMA-1 had an apparent molecular mass of 56 kDa which was largely consistent with its theoretical value, and expression of protein was identified by Western blot, which confirmed that the protein had highly specificity and reactionogenicity; the purified recombinant GST-EMA1 protein was tested in an ELISA for the detection of antibodies anti-T. equi in horses, and the indirect ELISA could clearly differentiate the T. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera; The intra-and inter-assay demonstrated that the coefficient of maximum variation was 14.79% and 11.06% respectively; 96 serum samples collected from horses in the state of Yili, Xinjiang were used to compare the indirect ELISA and cELISA commercial kit, the resules showed that their positive rates of T. equi infection were 27.1% (26/96) and 25.0% (24/96) respectively, and the total coincidence rate was 95.8%. These results suggest that the GST-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for indirect ELISA test and that provided the means of effective detecting and monitoring for T. equi (especially in recessive infection) in Xinjiang.

中图分类号:

S852.72

宋瑞其, 王盼举, 王振宝, 瓦热斯吐尔松, 闻秀秀, 张杨, 巴音查汗. 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[J]. 畜牧兽医学报, 2017, 48(9): 1737-1743.

SONG Rui-qi, WANG Pan-ju, WANG Zhen-bao, WARESI Tuersun, WEN Xiu-xiu, ZHANG Yang, BA Yinchahan. Prokaryotic Expression of the EMA-1 Gene of Xinjiang Strain of Theileria equi and Establishment of Indirect ELISA Detection Method[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1737-1743.

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