【摘要】：將口瘡病毒(orf virus,ORFV)ORFV B2L蛋白基因克隆至原核表達(dá)載體p ET-28a(+)中進(jìn)行重組B2L蛋白的誘導(dǎo)表達(dá);對(duì)純化復(fù)性后的B2L蛋白進(jìn)行Western-blot鑒定;用復(fù)性后的B2L蛋白作為包被抗原,優(yōu)化反應(yīng)條件,建立檢測(cè)血清ORFV抗體水平的間接ELISA,并進(jìn)行特異性、靈敏性和重復(fù)性試驗(yàn),最后對(duì)臨床樣品進(jìn)行檢測(cè)。結(jié)果,原核表達(dá)出42 ku的重組B2L蛋白。Western-blot結(jié)果表明,重組蛋白具有良好的特異性和抗原反應(yīng)性。最佳優(yōu)化反應(yīng)條件為:抗原包被量每孔600 ng,血清以1∶200倍稀釋作用1 h,酶標(biāo)二抗以1∶5 000倍稀釋作用30 min,TMB顯色時(shí)間為10 min。在該優(yōu)化條件下,D_(450)≥0.342為陽(yáng)性,D_(450)0.342為陰性;特異性試驗(yàn)表明,此間接ELISA對(duì)其他陽(yáng)性血清的檢測(cè)結(jié)果為陰性;對(duì)121份陽(yáng)性血清進(jìn)行檢測(cè),敏感性為99.2%;重復(fù)性試驗(yàn)表明,批內(nèi)和批間D_(450)值的變異系數(shù)分別在1.81%~6.23%和1.70%~7.45%之間。本試驗(yàn)建立的體系對(duì)臨床上676份山羊血清樣品進(jìn)行檢測(cè),陽(yáng)性率為99.4%。上述結(jié)果表明,本研究建立的間接ELISA可用于ORFV抗體的檢測(cè)。
[Abstract]:The ORFV B2L protein gene of aphthous sore virus (orf virus,ORFV) was cloned into prokaryotic expression vector p ET-28a () for induction and expression of recombinant B2L protein, and the purified and renatured B2L protein was identified by Western-blot. Using the renatured B2L protein as coating antigen, the reaction conditions were optimized, the indirect ELISA, was established to detect the level of serum ORFV antibody, and the specificity, sensitivity and reproducibility test were carried out. Finally, the clinical samples were detected. The results showed that 42 ku recombinant B2L protein was expressed in prokaryotes. Western-blot results showed that the recombinant protein had good specificity and antigen responsiveness. The optimum reaction conditions were as follows: the coating amount of antigen was 600 ng, per pore, the serum was diluted with 1 鈮，

本文編號(hào)：2477856