目的 表达环形泰勒虫的重组TA04380蛋白（rTA04380）并建立酶联免疫吸附试验（ELISA）检测方法,初步评价该方法的应用价值。 方法 用TMHMM和SignalP对TA04380蛋白进行生物信息学分析,根据GenBank上公布的环形泰勒虫TA04380核苷酸序列设计引物,PCR扩增TA04380基因的截短片段（1~636 bp）,构建pET30a(+)-TA04380表达质粒,转化至Rosetta（DE3）感受态细胞,诱导表达并纯化rTA04380蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定蛋白表达情况;蛋白质免疫印迹（Western blotting）分析rTA04380蛋白的反应原性及其与其他牛梨形虫的交叉反应性。以rTA04380蛋白为靶抗原建立检测抗环形泰勒虫抗体的ELISA方法,筛选确定最佳的抗原包被量、血清和二抗的稀释度、孵育时间以及最优的封闭剂;用所建立的ELISA方法检测56份标准环形泰勒虫阴性牛血清和76份标准环形泰勒虫阳性牛血清以确定该检测方法的阈值;分析rTA04380蛋白包被后第1、2、3周ELISA方法的重复性和稳定性;用建立的ELISA检测125份牛血清,并与已报道的基于子孢子表面抗原（SPAG）蛋白的ELISA方法作比较,计算符合率;用建立的ELISA方法检测571份来自9个省（自治区）的牛血清,评价该方法的应用价值。 结果 生物信息学分析结果显示,环形泰勒虫TA04380蛋白无信号肽序列,在212~413 aa存在4次跨膜区,与东方泰勒虫同源蛋白的氨基酸序列一致性为55%;PCR扩增片段长636 bp。SDS-PAGE结果显示,rTA04380蛋白主要以包涵体的形式存在,纯化后的rTA04380蛋白相对分子质量（M_r）为31 940;Western blotting分析结果显示,rTA04380蛋白能与抗His的单克隆抗体和环形泰勒虫阳性牛血清反应,与东方泰勒虫、中华泰勒虫、双芽巴贝斯虫、牛巴贝斯虫阳性牛血清和健康牛血清无交叉反应。以rTA04380蛋白为抗原建立的ELISA方法,最佳反应条件为：抗原包被浓度为10 μg/ml,1%牛血清白蛋白封闭1 h,1 ∶ 100稀释的血清孵育1 h,1 ∶ 6 000稀释的兔抗牛HRP-IgG孵育1 h;检测阈值为16.9%,敏感性和特异性分别为93.4%和96.4%,假阳性率为3.6%。用所建立的ELISA方法与基于SPAG蛋白的ELISA方法检测牛血清样品的阳性率分别是54.4%（68/125）和49.6%（62/125）,符合率为85.6%。用所建立的ELISA方法检测来自9个省（自治区）的571份牛血清样品,总阳性率为43.1%（246/571）,其中阳性率由高到低依次为吉林（85.7%,24/28）、宁夏（54.2%,13/24）、贵州（50.0%,13/26）、河南（8/16）、甘肃（43.8%,57/130）、内蒙古（42.2%,35/83）、辽宁（41.1%,30/73）、云南（38.2%,42/110）和青海（29.6%,24/81）。 结论 成功表达了rTA04380蛋白,建立的ELISA方法敏感性、特异性高,稳定性和符合性良好,具有大规模现场应用的潜在价值。

Objective To express recombinant TA04380 protein (rTA04380) of Theileria annulata, establish an enzyme-linked immunosorbent assay (ELISA)-based detection method and evaluate its value of application. Methods Bioinformatics analysis of TA04380 protein was performed with TMHMM and SignalP, primers were designed according to the nucleotide sequence of T. annulata TA04380 published in GenBank, and the truncated fragment (1-636 bp) of TA04380 gene was amplified by PCR to construct pET30a(+)-TA04380 plasmid and was transformed into Rosetta (DE3) competent cells, from which the rTA04380 protein was expressed by induction and then purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify the expression of the recombinant protein. The reactivity of the rTA04380 protein and its cross-reaction with other bovine piroplasma were analyzed by Western blotting. The rTA04380 protein was used as the target antigen to establish an ELISA method for the detection of serum antibodies against T. annulata; the optimal antigen coating concentration, serum and secondary antibody concentrations and the time for optimal blocking were determined by screening tests. The established ELISA method was used to detect 56 standard T. annulata negative bovine sera and 76 standard T. annulata positive bovine sera to determine the threshold value. The reproducibility and robustness of the ELISA method in the first, second and third weeks after the rTA04380 protein was coated. A total of 125 bovine serum samples were detected with the established ELISA, and compared with the reported ELISA method based on sporozoite surface antigen (SPAG) protein, the coincidence rate was calculated, and 571 bovine serum samples from 9 provinces were detected by the established ELISA method to evaluate its application value. Results The bioinformatics analysis showed that the T. annulata TA04380 protein has no signal peptide sequence, there are four transmembrane regions in the 212-413 aa region, and the amino acid sequence identity with T. orientalis homologous protein is 55%. The PCR amplification fragment length was 636 bp. The SDS-PAGE showed that the rTA04380 protein mainly existed in the inclusion bodies, and the purified TA04380 protein band was located at a relative molecular weight (M_r) of 31 940; Western blotting analysis showed that the rTA04380 protein could react both with the anti-His monoclonal antibody and T. annulata positive serum, but no cross-reaction was observed with T. orientalis, T. sinensis, B. bovis, B. bigemina reactive sera and healthy bovine serum. The optimal conditions of the ELISA method based on rTA04380 protein were: antigen is coated at a concentration of 10 μg/ml, and blocked with 1% bovine serum albumin for 1 h followed by addition of 1 ∶ 100 diluted serum and incubate for 1 h. The rabbit anti-bovine horseradish peroxidase (HRP)-IgG was added as a secondary antibody at a concentration of 1 ∶ 6 000 and incubated for 1 h. The detection threshold was 16.9%, the sensitivity and specificity were 93.4% and 96.4%, respectively, and the false positive rate was 3.6%. The positive rates of the established ELISA method and the ELISA method based on SPAG protein were 54.4% (68/125) and 49.6% (62/125), respectively, and the coincidence rate between the two methods was 85.6%. a The positive rate of 571 bovine serum samples from 9 provinces was 43.1% (246/571) using the established ELISA method. The positive rate from high to low is Jilin (85.7%, 24/28), Ningxia (54.2%, 13/24), Guizhou (50.0%, 13/26), Henan (8/16), Gansu (43.8%, 57/130), Inner Mongolia (42.2%, 35/83), Liaoning (41.1%, 30/73), Yunnan (38.2%, 42/110) and Qinghai (29.6%, 24/81). Conclusion The rTA04380 protein was successfully expressed, and the established ELISA based method presents high sensitivity and specificity, good reproducibility and coincidence, impliying potential value for wide application in the field.