本文關(guān)鍵詞： 牛病毒性腹瀉病毒 E2蛋白 真核表達(dá) 間接ELISA 血清流行病學(xué)　出處：《黑龍江八一農(nóng)墾大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：牛病毒性腹瀉/粘膜�。˙ovine viral diarrhea/mucosal disease, BVD/MD）是由牛病毒性腹瀉病毒（Bovine viral diarrhoea virus，BVDV）引起的，表現(xiàn)為腹瀉、繁殖障礙、免疫抑制、持續(xù)性感染和嚴(yán)重的黏膜病等癥狀。該病主要傳染牛，犢牛最為易感，也可感染其他反芻獸和人。與其他瘟病毒屬成員有血清交叉反應(yīng)，給診斷和疫苗免疫帶來了極大困難，對我國養(yǎng)牛業(yè)產(chǎn)生嚴(yán)重的影響。目前，我國BVDV暫無有效疫苗，因此建立一種快速有效的診斷方法尤為重要。 E2蛋白是BVDV的免疫囊膜蛋白，能夠刺激機(jī)體產(chǎn)生中和抗體，可以作為BVD診斷蛋白。本研究利用昆蟲細(xì)胞真核表達(dá)系統(tǒng)成功表達(dá)E2蛋白并純化，經(jīng)間接免疫熒光和Western blot鑒定，測定蛋白濃度為360μg/mL。以純化后的E2蛋白為包被抗原，以BVDV標(biāo)準(zhǔn)陰、陽性血清為陰、陽性對照，建立了BVDV抗體間接ELISA檢測方法。經(jīng)過選擇和優(yōu)化，確定蛋白抗原最佳包被濃度為6μg/mL，最佳血清稀釋倍數(shù)為1：80，最佳包被液選擇碳酸鹽緩沖液，最佳封閉液選擇5%脫脂乳，最佳血清最適作用時間為60min，最佳酶標(biāo)二抗工作濃度為1：10000，最佳底物作用時間為15min。與國外商品化試劑盒比較，總符合率為91.5%。因此，本文建立的BVDV E2抗體間接ELISA診斷方法可以作為BVDV的檢測方法。 為了了解黑龍江地區(qū)奶牛BVD的流行情況，使用建立的BVDV抗體間接ELISA檢測方法，對2012~2014年間黑龍江部分地區(qū)1485份奶牛血清進(jìn)行流行病學(xué)調(diào)查。結(jié)果顯示，這3年的BVDV抗體陽性率分別為59.63%、61.70%、70.33%，且三年間的BVDV抗體總陽性率為64.10%，呈逐年上升趨勢。統(tǒng)計分析發(fā)現(xiàn)，黑龍江地區(qū)奶牛BVD流行情況存在西部地區(qū)高于東部地區(qū)，發(fā)病季節(jié)無明顯差異，規(guī)模化養(yǎng)殖場高于散養(yǎng)戶的特點。
[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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.653

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