發(fā)布時間：2018-07-03 19:24

  本文選題：馬泰勒蟲 + 新疆株EMA-　； 參考：《畜牧獸醫(yī)學(xué)報》2017年09期

【摘要】：馬泰勒蟲裂殖子表面抗原1(EMA-1)是用于馬梨形蟲病診斷的重要靶抗原之一。為建立實用有效的間接酶聯(lián)免疫吸附試驗方法,筆者根據(jù)馬泰勒蟲EMA-1基因序列設(shè)計并合成引物,將新疆株EMA-1全基因序列克隆于原核表達(dá)載體pGEX-4T-1上,構(gòu)建pGEX-4T-1/EMA1重組質(zhì)粒,轉(zhuǎn)化至BL21(DE3)中,經(jīng)IPTG誘導(dǎo)得到EMA-1融合蛋白,切膠純化后作為包被抗原建立檢測馬泰勒蟲抗體的rELISA方法。結(jié)果顯示:(1)GST-EMA1蛋白相對分子質(zhì)量56ku,與其理論值基本一致;(2)通過Western blot分析證明其具有很好的特異性和反應(yīng)原性;(3)以GST-EMA1蛋白作為包被抗原建立的rELISA可明顯區(qū)分馬泰勒蟲、駑巴貝斯蟲陽性血清和健康馬血清;批內(nèi)和批間重復(fù)試驗的最大變異系數(shù)分別為14.79%和11.06%;(4)使用建立的間接ELISA與cELISA商品試劑盒分別對新疆伊犁馬場收集的96份馬血清樣品進(jìn)行檢測,檢出陽性率分別為27.1%(26/96)和25.0%(24/96),兩者總符合率為95.8%。結(jié)果表明,基于原核表達(dá)的GST-EMA1蛋白所建立的rELISA特異性、重復(fù)性好,檢出率與馬泰勒蟲臨床分離率接近,可為新疆馬泰勒蟲病(特別是隱性帶蟲馬)的檢測、監(jiān)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.
【作者單位】： 新疆農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)學(xué)院;伊犁出入境檢驗檢疫局綜合技術(shù)服務(wù)中心;
【基金】：國家自然科學(xué)基金-新疆聯(lián)合基金重點項目(U1403283)
【分類號】：S858.21
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本文編號：2094741