摘要
【目的】原核表达小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)的N蛋白,并以N蛋白为包被抗原建立血清间接ELISA检测方法。【方法】克隆PPRV N蛋白基因,将其克隆至原核表达载体pET-28a中进行重组N蛋白的诱导表达,对表达产物进行纯化复性,并进行Western-blot鉴定。用复性后的N蛋白作为包被抗原,优化反应条件,建立血清中PPRV抗体水平的间接ELISA检测方法,并对其进行特异性、灵敏性和重复性试验,最后用该方法对临床样品进行检测,检验其应用效果。【结果】原核表达获得大小为58ku的重组N蛋白,Western-blot分析表明重组蛋白具有良好的特异性和反应原性。PPRV间接ELISA法优化反应条件为:抗原包被量每孔400ng,血清以1∶200倍稀释作用2h,酶标二抗以1∶8 000倍稀释作用1h,TMB显色时间20min,在该优化条件下,OD450≥0.309为阳性,反之为阴性。特异性试验表明,该条件下对羊痘、羊口疮、羊布氏杆菌和羊魏氏梭菌血清检测结果均为阴性;对80份阳性血清进行检测,敏感性为98.7%。重复性试验表明,批内和批间OD450值的变异系数分别在2.29%~8.65%和2.13%~5.68%。用本试验建立的ELISA检测体系对临床197份血清进行检测,阳性率为85.79%,与标准试剂盒的符合率为96.4%。【结论】建立的ELISA检测方法可用于PPRV抗体水平的有效检测。
【Objective】Prokaryotic expression of des petits peste ruminants virus（PPRV）N protein was conducted and the indirect ELISA detection method using N protein as antigen was established.【Method】PPRV N protein gene was cloned into E.coli expression vector pET-28 ato induce the expression of recombinant N protein.N protein of PPRV was purified and tested by Western-blot.Using the recombinant N protein as antigen,the reaction conditions were optimized and an indirect ELISA detection method was established to detect PPRV antibody level in serum.Sensitivity and reproducibility tests were also carried out before clinical samples were tested.【Result】The recombinant protein with size of 58 ku was obtained.Western blot showed that the purified N protein had good specificity and antigen reactivity.The optimal reaction conditions were established as follows：coating amount of antigen was 400 ng per hole,the serum dilution was 1∶200,for 2 h,the second antibody dilution was 1∶8 000 for 1 h,and the TMB reacting timewas 20 min.Serum samples with OD450≥0.309 were determined as positive.Specificity tests showed that Orf virus,Capripox virus,Brucella and Clostridium welchi were all negative.The sensitivity of 80 positive sera was 98.7%.The repeatability test showed that the coefficients of variation of OD450 within same batches and between different batches were 2.29%-8.65% and 2.13%-5.68%,respectively.The clinical197 serum samples were tested using established system and the positive rate was 85.79% and the coincidence rate with the standard kit was 96.4%.【Conclusion】The established system in this study can be used for effectively monitoring the antibody level of PPRV.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2017年第11期1-8,17,共9页
Journal of Northwest A&F University(Natural Science Edition)
基金
陕西省农业科技创新与攻关项目(2016NY-092)
陕西省重点产业创新链项目(2016KTZDNY02-06)
