山羊痘病毒ORF103蛋白的原核表达及间接ELISA抗体检测方法的建立与应用

发布时间：2018-02-03 09:10

  本文关键词： 山羊痘病毒 ORF蛋白 原核表达 间接ELISA　出处：《中国兽医科学》2017年05期 　论文类型：期刊论文

【摘要】：将山羊痘病毒ORF103蛋白基因克隆至原核表达载体pET-28a(+)中进行重组ORF103蛋白的诱导表达,对表达产物进行纯化,并进行Western-blot鉴定;用纯化后的重组ORF103蛋白作为包被抗原,优化反应条件,建立血清间接ELISA检测方法,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,原核表达获得了大小约为35 ku的重组ORF103蛋白;Western-blot结果表明,此重组蛋白具有良好的特异性和抗原反应性。山羊痘病毒间接ELISA的优化反应条件为:抗原包被量每孔500 ng,血清以1∶200稀释后作用1 h,酶标二抗以1∶5 000稀释后作用1 h,TMB显色时间为10 min。在该优化条件下,D_(450)≥0.362为阳性,反之为阴性;特异性试验表明,对小反刍兽疫病毒、口疮病毒、马耳他布氏杆菌和产气荚膜梭菌阳性血清的检测结果均为阴性;对60份山羊痘病毒阳性血清进行检测,敏感性为96.6%;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%~3.20%和1.34%~7.38%之间;用本研究建立的方法对临床上162份血清进行检测,阳性率为70.4%。上述结果表明,本研究建立的ELISA可用于山羊痘病毒抗体水平的检测。
[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，

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