水貂阿留申病毒VP2蛋白Dot-ELISA检测方法的建立  

Establishment of Dot-ELISA detection method of VP2 protein of Aleutian mink disease parvovirus

作　　者：李晶[1] 时坤[2] 曾范利[2] 张妍[1] 孙凡婷 刘杨[1] 杜锐[2,3] 

机构地区：[1]吉林农业大学动物科学与技术学院,吉林长春130118 [2]吉林农业大学中药材学院,吉林长春130118 [3]教育部动物生产及产品质量安全重点实验室吉林省药用动物二级实验室,吉林长春130118

出　　处：《中国预防兽医学报》2015年第8期619-622,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31272565);吉林省经济动物疾病防治创新(20130521023JH)

摘　　要：为建立一种适合于基层快速检测水貂阿留申病毒(ADV)的检测方法,本研究将ADV的VP2基因经重叠延伸PCR法进行扩增后克隆至表达载体p GEX-4T-1中进行原核表达,将纯化的重组蛋白作为包被抗原建立了ADV的Dot-ELISA检测方法,并与对流免疫电泳(CIEP)作对照试验。结果表明,抗原最适包被浓度为20μg/m L,最适血清稀释度为1∶200,酶标二抗最适工作浓度为1∶2 000,血清和酶标抗体最适反应温度为37℃,反应时间为45 min^60 min。阻断试验和交叉试验显示与常见的貂病毒性肠炎和貂犬瘟热阳性血清无交叉反应,表明其具有良好的特异性。采用建立的Dot-ELISA和CIEP对78份临床血清样品进行检测,结果显示Dot-ELISA阳性检出率为84.6%,CIEP的阳性检出率为80.8%,敏感性高于CIEP,两者符合率为96.2%。本研究建立的Dot-ELISA检测方法简便、快速、灵敏、特异、重复性好,适合基层单位用于水貂阿留申病快速诊断和疫病普查。In order to establish a rapid detection method for detection of Aleutian disease virus （ADV）, VP2 gene of ADV was cloned into expression vector pGEX-4T-1 for prokaryotic expression. Subsequently, the purified recombinant protein was used as the coating antigen to establish the Dot-ELISA detection method for ADV detection and the convection immunoelectrophoresis （CIEP） was used as a control. The optimal conditions were coating with 21 μg/mL antigen onto PVDF membrane, diluting the serum sample at 1：200, detecting with the rabbit-anti-mink IgG-HRP at 1：2,000 dilution and incubating the reactionsin 37 ℃ for 45 min to 60 min, respectively. Blocking test and cross test showed the Dot-ELISA was specific for ADV antibody detection and had no cross reaction with the mink viral enteritis and mink distemper positive serum. Repetitive testing resultindicted the assay was repeatability. Tested on 78 clinical serum samples, the positive rates of Dot-ELISA and CIEP were 84.6% and 80.8%, respectively, which showed the sensitivity of Dot-ELISA was higher than that of CIEP. In conclusion, the Dot-ELISA detection method is rapid, sensitive, specific and easy to use, which is suitable for rapid diagnosis and survey of Aleutian disease in mink.

关 键 词：水貂阿留申病毒 VP2基因 原核表达 DOT-ELISA检测 

分 类 号：S852.65[农业科学—基础兽医学]