本文選題：牛病毒性腹瀉病毒 + 分子鑒定��； 參考：《河南農(nóng)業(yè)大學》2015年碩士論文

【摘要】：牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV),又稱牛病毒性腹瀉-粘膜病病毒(Bovine viral diarrhea-mucosal disease virus,BVD/MDV),是一種世界范圍內(nèi)廣泛分布的危害動物健康的重要病原體。它能感染牛、羊、豬等多種動物,造成機體的呼吸系統(tǒng)或生殖系統(tǒng)等發(fā)生疾病,感染牛免疫力降低,易繼發(fā)其他病原感染,導致疾病的發(fā)病率和死亡率增加。特別是形成持續(xù)性感染的牛,沒有任何臨床癥狀但可以排毒形成傳播源,給疫病的防控造成極大困難。為了解河南肉牛BVDV分離株的培養(yǎng)和遺傳特性,制備免疫診斷和防治試劑,本研究比較了致細胞病變型(cytopathic,CP)BVDV標準毒和BVDV分離株的細胞培養(yǎng)特性和分子遺傳特性,并通過制備免疫原免疫產(chǎn)蛋雞,獲得了高效的抗BVDV IgY,為BVD免疫學檢測和臨床治療該病提供物質(zhì)和技術(shù)參考。首先,利用細胞培養(yǎng)技術(shù)對RT-PCR檢測為陽性的血清樣品進行病毒分離培養(yǎng),獲得一個穩(wěn)定的分離株,命名為BVDV-MQ03。經(jīng)光學顯微鏡觀察,該分離株為致細胞病變型病毒,細胞培養(yǎng)48h左右即開始出現(xiàn)部分單層細胞呈拉網(wǎng)狀、細胞死亡、脫落等病變。根據(jù)分離株MQ03與瘟病毒屬參考株進行5'-UTR序列分析構(gòu)建進化樹顯示,其屬于BVDV-I型,其與NY-1和Osloss兩個BVDV-Ib亞型毒株處于同一進化分支上,進化關(guān)系較為密切。通過遺傳進化分析和序列相似性分析,MQ03與Osloss毒株的同源性最高,為97.1%,與NY-1的同源性達到96.2%,而與其它毒株的同源性均低于92.0%,證實MQ03屬于BVDV-Ib亞型。通過MDBK細胞增殖MQ01、MQ03和Oregon C24V共三個毒株,收集病毒細胞培養(yǎng)物。利用PEG、蔗糖密度梯度離心純化以上三個毒株。提純病毒經(jīng)磷鎢酸負染,電鏡觀察到略呈圓形,直徑30~80 nm的有囊膜病毒顆粒。將純化的標準毒作為抗原,建立了間接ELISA方法。其中抗原包被濃度為1μg/mL,被檢樣品的稀釋倍數(shù)為1:100,包被液為0.05mol/L,PH 9.6的碳酸鹽緩沖液,酶標抗體工作濃度為1:8000稀釋。最佳封閉條件、血清和酶標抗體的作用最佳條件均為37℃,1 h。上述條件確定后,特異性和重復性檢驗發(fā)現(xiàn),除BVDV陽性卵黃抗體外,該純化抗原不與抗雞新城疫病毒、雞減蛋綜合征病毒、H5亞型禽流感病毒、H9亞型禽流感病毒的陽性血清反應(yīng),隨機抽取的五個卵黃樣品檢測的批內(nèi)變異系數(shù)和批間變異系數(shù)在3.54%和7.28%之間,均小于10%。以濃縮的標準毒和分離毒制作抗原免疫健康產(chǎn)蛋雞,收集雞蛋,用水稀釋硫酸銨鹽析法提取卵黃抗體。間接ELISA法測定IgY效價,發(fā)現(xiàn)蛋雞首次免疫后第14 d,在卵黃中可以檢測到特異性抗體,經(jīng)過3次免疫后,抗MQ01、MQ03和Oregon C24V IgY的效價分別達到1:25600、1:51200和1:51200。獲得的高免卵黃抗體可進一步開發(fā)為抗體制劑并用于臨床該病的預(yù)防和治療。
[Abstract]:Bovine viral diarrhea virus (Bovine viral diarrhea virus), also known as bovine virus viral diarrhea-mucosal disease virus (BVD / MDV), is an important pathogen which is widely distributed in the world and endangers animal health. It can infect many animals, such as cattle, sheep, pigs and so on, and cause diseases such as respiratory system or reproductive system. In particular, cattle with persistent infection, without any clinical symptoms, but can detoxify to form a source of transmission, causing great difficulties in the prevention and control of epidemic disease. In order to understand the culture and genetic characteristics of BVDV isolates from Henan beef cattle, and to prepare immunodiagnostic and preventive agents, the cell culture characteristics and molecular genetic characteristics of cytopathicus CPVDV standard virus and BVDV isolates were compared. The immunogen immunized laying hens were prepared and the highly effective anti-BVDV IgYs were obtained, which provided material and technical reference for the detection of BVD immunology and the clinical treatment of the disease. Firstly, a stable virus isolate was obtained from the serum samples detected by RT-PCR and named BVDV-MQ03. It was observed by optical microscope that the isolated strain was cytopathic virus. After 48 hours of cell culture, some monolayer cells began to appear in the form of pulling net, cell death, exfoliation and so on. The phylogenetic tree analysis of MQ03 and reference strain showed that MQ03 belongs to BVDV-I type, and it is closely related to NY-1 and Osloss BVDV-Ib subtype in the same evolutionary branch. The genetic evolution analysis and sequence similarity analysis showed that MQ03 had the highest homology with Osloss strain (97.1), 96.2% homology with NY-1, and lower homology with other strains than 92.0%. It was confirmed that MQ03 belonged to BVDV-Ib subtype. The virus cell cultures were collected by proliferation of MDBK cell lines MQ01mq03 and Oregon C24V. The above three strains were purified by PEG and sucrose density gradient centrifugation. The purified virus was negatively stained with phosphotungstic acid and was observed to be a circular virus particle with a diameter of 3080 nm by electron microscope. Using the purified standard virus as antigen, an indirect Elisa method was established. The concentration of antigen coating is 1 渭 g / mL, the dilution multiple of the tested sample is 1: 100, the coating solution is 0.05 mol / L PH9.6 carbonate buffer, and the working concentration of enzyme labeled antibody is 1: 8000 dilution. The best conditions for blocking the serum and the enzyme labeled antibody were 37 鈩，

本文編號：2002124