BVDV重组E2蛋白间接ELISA检测方法的建立及初步应用

发布时间：2018-02-04 06:04

本文关键词： 牛病毒性腹泻病毒 E2蛋白真核表达间接ELISA 血清流行病学　出处：《黑龙江八一农垦大学》2015年硕士论文　论文类型：学位论文

【摘要】：牛病毒性腹泻/粘膜病（Bovine viral diarrhea/mucosal disease, BVD/MD）是由牛病毒性腹泻病毒（Bovine viral diarrhoea virus，BVDV）引起的，表现为腹泻、繁殖障碍、免疫抑制、持续性感染和严重的黏膜病等症状。该病主要传染牛，犊牛最为易感，也可感染其他反刍兽和人。与其他瘟病毒属成员有血清交叉反应，给诊断和疫苗免疫带来了极大困难，对我国养牛业产生严重的影响。目前，我国BVDV暂无有效疫苗，因此建立一种快速有效的诊断方法尤为重要。 E2蛋白是BVDV的免疫囊膜蛋白，能够刺激机体产生中和抗体，可以作为BVD诊断蛋白。本研究利用昆虫细胞真核表达系统成功表达E2蛋白并纯化，经间接免疫荧光和Western blot鉴定，测定蛋白浓度为360μg/mL。以纯化后的E2蛋白为包被抗原，以BVDV标准阴、阳性血清为阴、阳性对照，建立了BVDV抗体间接ELISA检测方法。经过选择和优化，确定蛋白抗原最佳包被浓度为6μg/mL，最佳血清稀释倍数为1：80，最佳包被液选择碳酸盐缓冲液，最佳封闭液选择5%脱脂乳，最佳血清最适作用时间为60min，最佳酶标二抗工作浓度为1：10000，最佳底物作用时间为15min。与国外商品化试剂盒比较，总符合率为91.5%。因此，本文建立的BVDV E2抗体间接ELISA诊断方法可以作为BVDV的检测方法。为了了解黑龙江地区奶牛BVD的流行情况，使用建立的BVDV抗体间接ELISA检测方法，对2012~2014年间黑龙江部分地区1485份奶牛血清进行流行病学调查。结果显示，这3年的BVDV抗体阳性率分别为59.63%、61.70%、70.33%，且三年间的BVDV抗体总阳性率为64.10%，呈逐年上升趋势。统计分析发现，黑龙江地区奶牛BVD流行情况存在西部地区高于东部地区，发病季节无明显差异，规模化养殖场高于散养户的特点。
[Abstract]:Bovine viral diarrhea/mucosal disease. BVD / MDV is caused by bovine viral diarrhoea virus (BVDVV) and is characterized by diarrhea and reproductive disorders. Immunosuppression, persistent infection and severe mucosal disease. The disease mainly infects cattle, calves are most susceptible to infection, but also can infect other ruminants and humans. At present, there is no effective vaccine for BVDV in China, so it is very important to establish a rapid and effective diagnostic method. E2 protein is the immune envelope protein of BVDV, which can stimulate the production of neutralizing antibody and can be used as a diagnostic protein for BVD. In this study, E2 protein was successfully expressed and purified by insect cell eukaryotic expression system. After indirect immunofluorescence and Western blot identification, the protein concentration was 360 渭 g / mL. The purified E2 protein was used as coating antigen, BVDV standard negative and positive serum as negative. The indirect ELISA detection method of BVDV antibody was established. The optimal coating concentration of protein antigen was 6 渭 g / mL and the optimal dilution multiple of serum was 1: 80 by selection and optimization. The best coating solution was carbonate buffer, the best sealing solution was 5% skimmed milk, the optimal time of serum action was 60 min, and the best working concentration of enzyme labeled second antibody was 1: 10 000. The optimum substrate action time was 15 min. Compared with the commercial kit abroad, the total coincidence rate was 91.5%. The indirect ELISA diagnostic method of BVDV E2 antibody developed in this paper can be used as a detection method for BVDV. In order to understand the prevalence of BVD in dairy cattle in Heilongjiang province, indirect ELISA detection method of BVDV antibody was used. Epidemiological investigation was carried out on 1 485 dairy cattle serums in Heilongjiang province from 2012 to 2014. The results showed that the positive rates of BVDV antibody in the three years were 59.63% respectively. The total positive rate of BVDV antibody in three years was 64.10, which showed an increasing trend year by year. The prevalence of BVD in the west of Heilongjiang province is higher than that in the eastern part of the province, and there is no significant difference in the incidence season, and the characteristics of the large-scale farms are higher than those of the free-range farmers.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.653

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