口疮病毒B2L蛋白的原核表达及间接ELISA检测方法的建立
发布时间：2019-05-15 23:44
【摘要】：将口疮病毒(orf virus,ORFV)ORFV B2L蛋白基因克隆至原核表达载体p ET-28a(+)中进行重组B2L蛋白的诱导表达;对纯化复性后的B2L蛋白进行Western-blot鉴定;用复性后的B2L蛋白作为包被抗原,优化反应条件,建立检测血清ORFV抗体水平的间接ELISA,并进行特异性、灵敏性和重复性试验,最后对临床样品进行检测。结果,原核表达出42 ku的重组B2L蛋白。Western-blot结果表明,重组蛋白具有良好的特异性和抗原反应性。最佳优化反应条件为:抗原包被量每孔600 ng,血清以1∶200倍稀释作用1 h,酶标二抗以1∶5 000倍稀释作用30 min,TMB显色时间为10 min。在该优化条件下,D_(450)≥0.342为阳性,D_(450)0.342为阴性;特异性试验表明,此间接ELISA对其他阳性血清的检测结果为阴性;对121份阳性血清进行检测,敏感性为99.2%;重复性试验表明,批内和批间D_(450)值的变异系数分别在1.81%~6.23%和1.70%~7.45%之间。本试验建立的体系对临床上676份山羊血清样品进行检测,阳性率为99.4%。上述结果表明,本研究建立的间接ELISA可用于ORFV抗体的检测。
[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，
本文编号：2477856
[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，
本文编号：2477856
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2477856.html
