基于VP0蛋白的用以检测1型鸭甲肝病毒抗体的间接ELISA的建立和应用  

Development and application of an indirect ELISA based on VP0 for detection of antibodies against duck hepatitis A virus type 1

作　　者：齐晓燕[1,2,3] 程安春[1,2,3] 汪铭书[1,2,3] 陈舜[1,2,3] 贾仁勇[1,2,3] 朱德康[1,3] 刘马峰[1,2,3] 刘菲[3] 杨乔[1,2,3] 孙昆峰[1,2,3] 陈孝跃[1,3] 

机构地区：[1]四川农业大学动物医学院禽病防治研究中心,四川成都611130 [2]四川农业大学预防兽医研究所,四川成都611130 [3]动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《中国兽医科学》2015年第12期1218-1224,共7页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2015BAD12B05);国家现代农业(水禽)产业技术体系专项(CARS-43-8);四川省创新团队项目(12TD005/2013TD0015)

摘　　要：为检测1型鸭甲肝病毒(DHAV1)抗体,在大肠杆菌中表达获得了DHAV1的重组VP0蛋白,以纯化复性的重组VP0蛋白为抗原建立检测DHAV1抗体的间接ELISA(VP0-ELISA)。其最佳反应条件为:以1.67μg/mL重组VP0蛋白37℃孵育1h后4℃包被过夜;50g/L明胶封闭0.5h;被检血清1∶160稀释,37℃孵育1h;HRP标记的山羊抗鸭IgG进行1∶400稀释,37℃孵育1h;TMB避光显色10min,测定D450nm/D630nm值,阳性阈值为0.375。该方法具有较强的灵敏性、特异性和重复性。用建立的间接VP0-ELISA对60份鸭血清样本进行检测,与以DHAV1作为包被抗原的ELISA相比,阳性检出率为92.3%,阳性符合率为90.0%。上述结果表明,基于VP0蛋白的间接ELISA可用于临床DHAV1抗体的检测和流行病学调查。To detect antibodies against duck hepatitis A virus type 1（DHAV1）,the recombinant protein VP0 of DHAV1was expressed in Escherichia coli.An indirect ELISA based on the purified and renatured protein VP0 was established,the optimal conditions were determined as follows：the optimal concentration of VP0 protein was 1.67μg/mL and the protein was incubated at 37℃for 1h,and then at 4℃for overnight;the blocking buffer and incubating time was 50g/L gelatin and 0.5h,respectively;the test serum was used at 1∶160dilution and incubated for 1h;the optimal dilution of HRP-goat-anti-duck IgG was 1∶400,and the incubating time was 1h;D450nm/D630 nm was detected after coloration for 10 min by TMB.The positive threshold was 0.375,which was determined by testing 56 negative serum samples.The results demonstrated that the ELISA method was sensitive,specific and repeatable.Compared with an indirect ELISA method based on DHAV1,the positive rate was 92.3%,and the coincidence between VP0-ELISA and DHAV1-ELISA was 90.0% by detecting 60 serum samples.This indirect VP0-ELISA method would be applied to detect antibodies against DHAV1 in clinic and the epidemiological investigation.

关 键 词：1型鸭甲肝病毒 VP0蛋白 抗原性 间接VP0-ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]