本文關(guān)鍵詞： 山羊痘病毒 ORF蛋白 原核表達(dá) 間接ELISA　出處：《中國(guó)獸醫(yī)科學(xué)》2017年05期 　論文類型：期刊論文

【摘要】：將山羊痘病毒ORF103蛋白基因克隆至原核表達(dá)載體pET-28a(+)中進(jìn)行重組ORF103蛋白的誘導(dǎo)表達(dá),對(duì)表達(dá)產(chǎn)物進(jìn)行純化,并進(jìn)行Western-blot鑒定;用純化后的重組ORF103蛋白作為包被抗原,優(yōu)化反應(yīng)條件,建立血清間接ELISA檢測(cè)方法,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)獲得了大小約為35 ku的重組ORF103蛋白;Western-blot結(jié)果表明,此重組蛋白具有良好的特異性和抗原反應(yīng)性。山羊痘病毒間接ELISA的優(yōu)化反應(yīng)條件為:抗原包被量每孔500 ng,血清以1∶200稀釋后作用1 h,酶標(biāo)二抗以1∶5 000稀釋后作用1 h,TMB顯色時(shí)間為10 min。在該優(yōu)化條件下,D_(450)≥0.362為陽(yáng)性,反之為陰性;特異性試驗(yàn)表明,對(duì)小反芻獸疫病毒、口瘡病毒、馬耳他布氏桿菌和產(chǎn)氣莢膜梭菌陽(yáng)性血清的檢測(cè)結(jié)果均為陰性;對(duì)60份山羊痘病毒陽(yáng)性血清進(jìn)行檢測(cè),敏感性為96.6%;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%~3.20%和1.34%~7.38%之間;用本研究建立的方法對(duì)臨床上162份血清進(jìn)行檢測(cè),陽(yáng)性率為70.4%。上述結(jié)果表明,本研究建立的ELISA可用于山羊痘病毒抗體水平的檢測(cè)。
[Abstract]:The ORF103 gene of goat pox virus was cloned into the prokaryotic expression vector pET-28a (), and the recombinant ORF103 protein was induced to express, and the expressed product was purified. Western-blot identification was carried out. The purified recombinant ORF103 protein was used as the coating antigen to optimize the reaction conditions and to establish a method for the detection of serum indirect ELISA. The specificity, sensitivity and reproducibility of the method were tested. Finally, the clinical samples were detected. Results the recombinant ORF103 protein of about 35 ku was obtained by prokaryotic expression. Western-blot results showed that. The recombinant protein had good specificity and antigen reactivity. The optimal reaction conditions for indirect ELISA of Goatpox virus were as follows: the amount of antigen coating was 500ng / pore. When the serum was diluted with 1: 200 for 1 hour and the enzyme labeled antibody diluted with 1: 5 000 for 1 h, the reaction time of TMB was 10 min. D450) 鈮，

本文編號(hào)：1486980