检测猪弓形虫ROP14抗体间接ELISA方法的建立

为丰富和完善弓形虫病的检测方法,本试验基于弓形虫棒状体蛋白14(TgROP14)建立了间接ELISA检测方法。构建原核表达载体pET30α-ROP14,转化至大肠杆菌BL21中,诱导表达并纯化获得TgROP14重组蛋白,通过SDS-PAGE和Western blot试验验证其纯度及免疫原性。利用纯化透析后复性的rTg-ROP14建立猪弓形虫病间接ELISA检测方法,并对反应中各项条件进行了优化。结果表明最佳检测条件为:抗原包被量2μg/孔,血清稀释倍数为1∶20,封闭剂为5%脱脂奶粉,血清作用时间为60 min,二抗稀释倍数为1∶1 000,二抗作用时间为60 min,TMB作用时间为20 min,临界值为0.728。该方法敏感性为1∶80,批内批间重复试验变异系数均小于10%,重复性较好;使用该方法检测猪繁殖与呼吸综合征(美洲型)阳性血清、猪O型口蹄疫阳性血清、猪A型口蹄疫阳性血清和猪瘟阳性血清均未见交叉反应,特异性较好。利用该方法对来自浙江省各个地区的436份猪血清样品进行检测,同时使用商品化的间接血凝试剂盒进行验证,符合率为72.7%(317/436)。综上所述,本试验建立的间接ELISA方法可用于临床样品的检测,为猪弓形虫病的血清流行病学调查提供了依据。

Establishment of an indirect ELISA for detecting antibodies against ROP14 of Toxoplasma gondii in swine

Toxoplasma gondii is a globally distributed apicomplexan,which can parasitize almost all warm-blooded animals and cause toxoplasmosis.In order to enrich and optimize the detection method of toxoplasmosis,an indirect ELISA assay was established in this study,based on T.gondii rhoptry protein TgROP14.The prokaryotic expression vector pET30α-ROP14 was constructed and transformed into E.coli BL21,induced to express the recombinant protein rROP14.The purity and immunogenicity of rROP14 were verified by SDS-PAGE and Western blot assays.An indirect ELISA method for detection of toxoplasmosis in pigs was established by using the renatured soluble protein rTg-ROP14,and the conditions in the reaction were optimized.The results showed that the optimal detection conditions were as follow:antigen coating amount was 2 μg/well,serum dilution was 1∶20, serum incubation time was 60 min,blocking agent was PBS containing 5% skim milk,secondary antibody dilution was 1∶1 000,secondary antibody incubation time was 60 min,TMB incubation time was 20 min,and the cut-off value was 0.728.The sensitivity of the developed method was 1∶80,while the coefficients of variation(CV) in intra-and inter-assays were less than 10%,indicating that this method had a good repeatability.There were no cross reactions with the porcine reproductive and respiratory syndrome(American type)positive serum,swine type O footand-mouth disease positive serum,swine type A foot-and-mouth disease positive serum and swine fever positive serum using the ELISA.436 pig serum samples from various regions of Zhejiang province were tested by this method,verified by commercial indirect hemagglutination kit and the coincidence rate was 72.7%(317/436).In summary,the indirect ELISA method established in this study could be used for detection of clinical samples,providing a new means for the epidemiological investigation of porcine toxoplasmosis.