抗鹅细小病毒卵黄IgG的制备及其间接ELISA检测方法的建立  

Preparation of anti-GPV yolk IgG and development of an indirect-ELISA for detection of goose parvovirus infection in goose

作　　者：刘菲[1,2] 唐卫杰 程安春[1,2] 汪铭书[1,2] 赵婷婷[1] 韩清林[1,3] 高丽芹[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014 [3]四川省大英县畜牧局,四川大英629300

出　　处：《中国兽医科学》2011年第6期601-606,共6页Chinese Veterinary Science

基　　金：四川省教育厅自然科学基金项目(2006A012);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);现代农业产业技术体系建设专项(nycytx-45-12)

摘　　要：采用水稀释-辛酸-硫酸铵法粗提与DEAE50纤维素层析相结合的方法从免疫鹅细小病毒(GPV)的鹅卵内提取鹅卵黄IgG,通过核酸蛋白检测仪和SDS-PAGE测定IgG的浓度和纯度后,制备兔抗鹅IgG酶标抗体;建立了针对GPV的间接ELISA抗体检测方法,确定其最佳工作条件,并对该ELISA的特异性及重复性进行检测;应用该间接ELISA检测了GPV VP3基因疫苗的免疫效果。结果显示,获得的纯化鹅卵黄IgG浓度为10mg/mL,抗体纯度达到94.5%,兔抗鹅IgG的血清琼脂扩散效价约为1∶32;经筛选确定该ELISA的最佳工作条件为:最适抗原包被浓度为20μg/mL,最适血清稀释度为1∶100,酶标抗体的最佳工作浓度为1∶3 000。用建立的间接ELISA检测禽流感病毒、新城疫病毒、传染性支气管炎病毒、传染性法氏囊炎病毒、鸭肝炎病毒、鸭瘟病毒的阳性血清均为阴性,表明该方法特异性强、重复性好。经临床初步应用,能检出GPV VP3基因疫苗产生的抗体,在肌肉注射100μg与200μg基因疫苗的鹅血清中,IgG均从第14天开始上升,并分别在第35、21天达到最大值。表明本试验制备的兔抗鹅IgG酶标抗体具有较好的应用价值。IgG was extracted from egg yolk of goose infected with goose parvovirus（GPV） by the methods of water-dilution-octanoic acid-ammonia sulfate initial extraction and DEAE chromatography.The concentration and purity of the extracted IgG were detected by protein nucleic acid detector and SDS-PAGE.Rabbit anti-goose IgG HRP was prepared.An indirect ELISA assay was developed for detecting the antibody against gosling plague.The optimal working conditions,specificity and reproducibility of the indirect ELISA assay were determined.The efficacy of GPV VP3 gene vaccine was tested by the indirect ELISA.In result,the purified goose egg yolk IgG had high concentration（10 mg/mL） and purity（94.5%）.The titer of the antiserum was 1∶32.In the developed indirect ELISA,the optimal concentration of antigen was 20 μg/mL,the optimal dilution of serum sample was 1∶100,and the optimal dilution of the labeledantibody was 1∶3000.The indirect ELISA was specific,sensitive and good reproducibility for detection of the antibody against GPV,but the antibodies against avian influenza virus,infectious bronchitis virus,infectious bursal disease virus,duck hepatitis virus,or duck plaque virus were not detected by the developed indirect ELISA. In the clinical application test,DNA vaccine-produced antibodies could be detected by the indirect ELISA. The levels of IgG in sera of the geese injected intramuscularly with 100μg or 200μg DNA vaccine were increased on day 14 post-injection,and reached peaks on day 35 and 21 post-injection,respectively,indicating that rabbit anti-goose IgG HRP had a good value.

关 键 词：抗鹅细小病毒卵黄IgG 纯化 间接ELISA 应用 

分 类 号：S852.659.2[农业科学—基础兽医学]