传染性法氏囊病病毒抗体间接ELISA检测方法的建立

为了建立传染性法氏囊病病毒(IBDV)抗体间接ELISA检测方法,本研究首先将VP2基因分3段(部分序列重叠),分别为VP2(1~600)、VP2(376~975)和VP2(751~1350),并根据大肠杆菌对密码子的偏爱性进行优化,将优化的3个VP2基因片段分别克隆于原核表达载体pET28a(+)中,转化E.coli Rosetta(DE3)感受态细胞,经IPTG诱导后,进行SDS-PAGE和Western-blot,结果3个重组VP2蛋白均获得了表达。用纯化的3个重组VP2蛋白rVP2(1~600)、rVP2(376~975)和rVP2(751~1350)分别作为包被抗原,建立了IBDV抗体间接ELISA(rVP2-ELISA)。用rVP2-ELISA、IDEXX-ELISA(IBDV)及中和试验对96份临床血清样本进行检测,结果显示,3个重组VP2蛋白建立的IBDV抗体间接ELISA检测方法、IDEXX-ELISA(IBDV)与中和试验比较,相对敏感性分别为97.1%、61.8%、58.8%和100%;相对特异性分别为85.5%、82.3%、83.9%和25.8%;符合率分别为89.6%、75.0%、75.0%和52.08%。本研究为研制IBDV抗体间接ELISA检测试剂盒(以重组VP2蛋白作为包被抗原)奠定了基础。

Development of a ELISA using E.coli-expressed truncated VP2 for specific and sensitive detection of antibodies against infectious bursal disease virus

Infectious bursa disease(IBD)is an infectious disease caused by infectious bursa disease virus(IBDV)that mainly affects the bursa of young poultry.The aim of the present study is to velop an enzyme-linked immunosorbent assay(ELISA)for detecting the specific antibodies to IBDV using the VP2 protein of IBDV as coating antigen.Three truncated VP2 gene(designated VP2(3-600),VP2(376-975)and VP2(751-1350)were synthesized and expressed in E.coli Rosetta(DE3).These recombinant VP2 proteins were purified and used in enzyme-linked immunosorbent assay(ELISA)for detecting IBDV antibodies in chicken.A total of 96 serum samples obtained from chickens vaccinated with commercial activated vaccine(B87)and non-vaccinated chickens against IBDV were tested using r VP2(3-600)-ELISA,r VP2(376-975)-ELISA,r VP2(751-1350)-ELISA,IDEXX-ELISA(IBDV)and neutralization assay,respectively.The relative sensitivitiy between r VP2(3-600)-ELISA,r VP2(376-975)-ELISA,r VP2(751-1350)-ELISA,IDEXX-ELISA(IBDV)and neutralization assay were 97.1%,61.8%,58.8%and 100%,respectively.The relative specificity between r VP2(3-600)-ELISA,r VP2(376-975)-ELISA,r VP2(751-1350)-ELISA,IDEXX-ELISA(IBDV)and neutralization assay were 85.5%,82.3%,83.9%and 25.8%,respectively.The agreement ratio between r VP2(3-600)-ELISA,r VP2(376-975)-ELISA,r VP2(751-1350)-ELISA,IDEXX-ELISA(IBDV)and neutralization assay were 89.6%,75.0%,75.0%and 52.08%respectively.The sensitivity of the r VP2(3-600)-ELISA,r VP2(376-975)-ELISA and r VP2(751-1350)-ELISA were lower than that of the commercial IDEXX-ELISA(IBDV),but the specificity and agreement ratio were better than that of the commercial IDEXX-ELISA(IBDV).In conclusion,the results demonstrate the potential applicability of the r VP2(3-600)-ELISA,r VP2(376-975)-ELISA and r VP2(751-1350)-ELISA for the determination of antibodies to IBDV in chickens.