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

Abstract

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.

CLC Number:

S852.72

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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Prokaryotic Expression of the EMA-1 Gene of Xinjiang Strain of Theileria equi and Establishment of Indirect ELISA Detection Method

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