中国兽医科学2017，Vol.47Issue(5)：537-543,7.DOI:10.16656/j.issn.1673-4696.2017.05.001
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
Prokaryotic expression of ORF103 protein of capripox virus and establishment of indirect ELISA for the detection of antibodies against the virus
摘要
Abstract
The capripox virus ORF103 protein gene was cloned into E.coli expression vector pET-28a(+) in this study,and the recombinant ORF103 protein was generated,purified and tested by Western-blot.Using the recombinant ORF103 protein as an antigen,the indirect ELISA was established to detect the levels of capripox virus antibodies in serum.Specificity,sensitivity and reproducibility tests were carried out,and the clinical samples were tested.The recombinant protein with a size of 35 ku was obtained.Western-blot showed that the purified 0RF103 protein had good specificity and antigenic reactivity.The optimal reaction conditions were established as follows:the coating amount of antigen was 500 ng per hole,incubating for 1 h;the serum dilution was 1∶200,reacting for 1 h;the second antibody dilution was 1 ∶ 5 000,reacting for 1 h;the TMBreacting time was 10 min.Criteria for determining the negative and positive results of serum samples were D450≥0.362 and D450＜0.362,respectively.Specificity tests showed that the results of other positive sera were negative.60 positive sera were tested and the sensitivity was 96.6％.The repeatability test showed that the coefficient of variation of D450between the same batch and different batch were from 1.81％ to 3.20％ and from 1.34％ to 7.38％,respectively.The clinical 162 serum samples were tested using the established system and the positive rate was 70.4％.In conclusion,the established system in this test can be used for clinical detection of serum antibodies and epidemiological investigation of GPTV.关键词
山羊痘病毒/ORF103蛋白/原核表达/间接ELISAKey words
capripox virus/ORF103 protein/prokaryotic expression/indirect ELISA分类
农业科技引用本文复制引用
张琪,庞文静,付明哲,史怀平,许信刚..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[J].中国兽医科学,2017,47(5):537-543,7.基金项目
陕西省农业科技创新与攻关项目(2016NY-092,2016NY-095) （2016NY-092,2016NY-095）
陕西省科技统筹创新工程计划项目(2016KTZDNY02-05) （2016KTZDNY02-05）
杨凌示范区农业科技示范推广项目(Ts-2016-13) （Ts-2016-13）
