【摘要】 目的建立检测土拨鼠肝炎病毒(WHV)核心抗体的ELISA方法,应用于WHV感染的中国旱獭动物模型的研究。方法原核表达重组WHcAg,氯化铯密度梯度离心获得非变性的纯化蛋白;用该纯化蛋白免疫Balb/c小鼠制备多克隆抗体;建立竞争抑制ELISA方法用于检测旱獭血清中的抗-WHc。结果纯化后的蛋白浓度达0.86mg/mL,纯度达89.48%;免疫小鼠后获得抗血清,ELISA间接法显示其多克隆抗体效价达1∶640000,Western blot分析显示该抗体能特异识别WHcAg;建立的竞争抑制ELISA方法对旱獭血清中可能存在的抗-WHc进行检测,诊断特异性及敏感性均较好,重复测定的批内变异系数和批间变异系数均小于10%。结论成功地建立检测旱獭血清中抗-WHc的竞争抑制ELISA方法。该方法具有稳定、简便、特异、敏感的特点,适用于大量旱獭血清抗-WHc的筛查工作,为进一步研究中国旱獭这一新型乙型肝炎病毒动物模型奠定了基础。更多还原

【Abstract】 Objective To establish an ELISA method for detection of Woodchuck hepatitis virus core antibody(WHcAb).Methods Recombinant WHcAg was transfected and expressed E.coli,and purify rWHcAg under native condition by precipitation with saturated ammonium sulfate and CsCl gradient centrifugation;Immunize Balb/c mice with purified rWHcAg to prepare antiserum;Establish a competitive inhibition ELISA method to detect WHcAb in serum of Chinese marmots.Results The concentration of purified rWHcAg is 0.86 mg/ml,The purity of purified antigen was 89.48% ;The titer of polyclonal antibody was 1∶640 000,and western blot analysis showed that the polyclonal antibody could recognize WHcAg specifically;The specificity and sensitivity of diagnosis were as high as 100% when we used the competitive inhibition ELISA method to detect confirmed WHcAb(+)and WHcAb(-)serum of Chinese Marmots,and the coefficients of variation in a lot and among lots were both lower than 10%.Conclusion A simple and fast ELISA method for detection of WHcAb was established successfully.This method has a merit of sensitivity、 specificity and stability.It can be used for screening of WHcAb in serum of woodchuck,which sets up the foundation for further research on Chinese Marmot model.更多还原