發(fā)布時(shí)間：2018-06-17 20:25

  本文選題：鴨坦布蘇病毒 + NS蛋白��； 參考：《畜牧獸醫(yī)學(xué)報(bào)》2016年05期

【摘要】：為了建立坦布蘇病毒(TMUV)感染鴨群的抗體檢測(cè)方法,以TMUV NS1的原核表達(dá)蛋白質(zhì)作為包被抗原,建立了間接ELISA方法,并對(duì)該方法進(jìn)行了檢測(cè)條件的優(yōu)化。將TMUV NS1全基因序列克隆至pET-28a(+),利用BL21(DE3)表達(dá)NS1蛋白,純化后其質(zhì)量濃度為4.16μg·μL~(-1)。通過(guò)方陣試驗(yàn),確定了NS1蛋白的最佳包被質(zhì)量濃度是100ng·孔-1,檢測(cè)血清的最佳稀釋倍數(shù)為40倍。隨后對(duì)檢測(cè)條件進(jìn)行了優(yōu)化,優(yōu)化后的結(jié)果:抗原的最佳包被條件是4℃作用過(guò)夜;酶標(biāo)二抗的最佳工作條件是稀釋5 000倍,37℃作用1h;陰陽(yáng)血清的臨界值為0.278。一系列試驗(yàn)驗(yàn)證了該檢測(cè)方法具有很強(qiáng)的特異性、敏感性、重復(fù)性。對(duì)采集自山東各地的80份血清樣品進(jìn)行檢測(cè),其檢測(cè)結(jié)果顯示,該方法與經(jīng)典的雞胚中和試驗(yàn)檢測(cè)結(jié)果的符合率為93.5%以上,且比傳統(tǒng)的中和試驗(yàn)更敏感。對(duì)TMUV感染鴨群的血清進(jìn)行檢測(cè),描述了TMUV感染后鴨群抗體水平的消長(zhǎng)規(guī)律,為該病的防治提供重要的理論依據(jù)。以NS1蛋白為包被抗原的間接ELISA方法的建立為臨床上TMUV的感染提供了一種新的抗體檢測(cè)方法,尤其是在TMUV早期感染的檢測(cè)方面有著更為廣泛的應(yīng)用前景。
[Abstract]:In order to establish an antibody detection method for tambusuvirus (TMUV) infected ducks, an indirect Elisa method was established using the prokaryotic expression protein of TMUV NS1 as the coated antigen, and the detection conditions were optimized. The whole gene sequence of TMUV NS1 was cloned into pET-28a (pET-28a, BL21DE3) to express NS1 protein. After purification, the mass concentration of NS1 was 4.16 渭 g / L. The best coating concentration of NS1 protein was determined to be 100ng pore -1 by square array test, and the best dilution multiple of serum was 40 times. Then the detection conditions were optimized. The results showed that the best coating condition of antigen was 4 鈩，

本文編號(hào)：2032339