口疮病毒B2L蛋白的原核表达及间接ELISA检测方法的建立

Prokaryotic expression of B2L protein of orf virus and establishment of indirect ELISA for detection of antibodies against orf virus

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。

Using B2 L protein as an antigen,an indirect ELISA was established.ORFV B2 L protein gene was cloned into E.coli expression vector p ET-28a（＋） in this study to induce expression of recombinant B2 L protein.B2 L protein of ORFV was purified and tested by Western-blot.Using recombinant B2 L protein as an antigen,the indirect ELISA was established to test the level of ORFV antibody in serum. Specificity,sensitivity and reproducibility tests were carried out,and the clinical samples were tested.The recombinant B2 L protein with the size of 42 ku was obtained.The results of Western blot showed that the protein had good specificity and antigen reactivity.The optimal reaction conditions were established as follows ：the coating amount of antigen was 600 ng per hole,closed 1.5 h.The serum dilution was 1 ∶ 200,and incubated for 1 h.The second antibody dilution was 1 ∶ 5 000,and incubated fo r 30 min.The TMB reacting time was 10 min.Criteria for determining the negative and positive results of serum samples were D450≥0.342 and D450〈0.342,respectively. Specificity tests showed that the results of other positive sera were negative.121 positive sera were tested and the sensitivity was 99.2%.The repeatability test showed that the coefficient of variation of D450 nm between the same batch and different batch were from1.81% to 6.23% and from 1.70% to 7.45%,respectively.The clinical 676 serum samples from goat were tested using established system and the positive rate was 99.4%.In conclusion,the reaction system established in this study can be used for monitoring effectively the antibody level against orf virus.