本文選題：奶牛　切入點(diǎn)：病毒性疾病　出處：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：近年來,我國奶牛養(yǎng)殖業(yè)高速發(fā)展,飼養(yǎng)規(guī)�；⒓s化程度不斷提高,牛傳染性疾病的防控形勢也越來越嚴(yán)峻�？谔阋�(FMD)、牛病毒性腹瀉(BVD)、牛傳染性鼻氣管炎(IBR)、牛輪狀病毒(BRV)、牛冠狀病毒(BCV)等病毒性疾病在我國規(guī)模化牧場廣泛流行并造成重大損失,這些疾病已經(jīng)嚴(yán)重危害到我國奶牛養(yǎng)殖業(yè)的健康發(fā)展。上海作為我國奶牛養(yǎng)殖業(yè)的發(fā)源地,奶牛養(yǎng)殖全部實(shí)現(xiàn)規(guī)�；�,重視并實(shí)現(xiàn)特定疫病的監(jiān)測和防控。針對目前奶牛病毒性疾病的流行情況,我們迫切需要研制出高效經(jīng)濟(jì)的抗病毒和免疫增強(qiáng)性藥物。干擾素(Interferon,IFN)是動物細(xì)胞在受到誘生刺激后產(chǎn)生的一組具有抗病毒、參與免疫調(diào)節(jié)等多種生物活性的細(xì)胞因子,分為三類,即Ⅰ、Ⅱ型和Ⅲ型干擾素。Ⅰ型干擾素具有強(qiáng)大的抗病毒活性,Ⅱ型干擾素可以調(diào)節(jié)機(jī)體免疫增強(qiáng)疾病抵抗力。目前人干擾素和豬干擾素已經(jīng)實(shí)現(xiàn)商品化生產(chǎn)并廣泛運(yùn)用于臨床實(shí)踐,然而由于生產(chǎn)工藝和成本等問題,牛干擾素的研制還停留在實(shí)驗(yàn)室階段,并未在臨床上使用。本試驗(yàn)對上海地區(qū)多個規(guī)�；翀鲞M(jìn)行了奶牛常見病毒性疾病的血清學(xué)調(diào)查,為疫病防控提供數(shù)據(jù)支持;同時采用原核表達(dá)系統(tǒng)表達(dá)獲得具有抗病毒活性的牛αα、y干擾素,為牛干擾素在奶牛臨床上的應(yīng)用奠定基礎(chǔ)。1上海地區(qū)規(guī)�；翀鏊姆N奶牛病毒性疾病血清學(xué)調(diào)查對上海地區(qū)多個規(guī)模化牧場荷斯坦奶牛的牛病毒性腹瀉(BVD)、牛傳染性鼻氣管炎(IBR)、牛冠狀病毒(BCV)和牛輪狀病毒(BRV)的血清學(xué)感染情況進(jìn)行調(diào)查。使用ELISA檢測方法對19個規(guī)�；翀龅腂VDV血清抗原、IBRV血清抗體;9個規(guī)�；翀龅腂CV、BRV血清抗體進(jìn)行檢測。檢測BVDV、IBRV血清樣品920份,檢測BCV、BRV血清樣品200份,并結(jié)合這些牧場的飼養(yǎng)管理、繁殖、疾病等資料數(shù)據(jù)進(jìn)行分析。檢測結(jié)果表明BVDV抗原陽性率平均為3.70%,IBRV、BCV、BRV抗體陽性率分別為46.74%、97.00%、97.50%。2牛α、γ干擾素的原核表達(dá)為在大腸桿菌表達(dá)系統(tǒng)中高效表達(dá)具有抗病毒活性的重組牛干擾素α和γ,將經(jīng)密碼子優(yōu)化后的牛干擾素α、γ成熟蛋白基因合成后克隆至表達(dá)載體pColdⅡ中,然后將重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)中進(jìn)行蛋白誘導(dǎo)表達(dá),獲得可溶性牛干擾素α和γ蛋白。對誘導(dǎo)條件采用單因素比較優(yōu)化以獲得最佳誘導(dǎo)條件,結(jié)果顯示在IPTG濃度為0.64mmol/L,誘導(dǎo)溫度為15℃,誘導(dǎo)時間為9h的條件下,可溶性牛α干擾素的表達(dá)量最高;在IPTG濃度為0.32 mmol/L,誘導(dǎo)溫度為15℃,誘導(dǎo)時間為12h的條件下,可溶性牛γ干擾素的表達(dá)量最高。重組蛋白經(jīng)鎳柱純化后的蛋白純度為99.2%和91.1%,表達(dá)量為36mg和32mg每升菌液。重組蛋白在MDBK細(xì)胞上抗水泡性口炎病毒的活性為1.O×10~6U/mg和1.6×10~5U/mg。
[Abstract]:In recent years, the dairy cattle breeding industry in China has developed at a high speed. The situation of prevention and control of bovine infectious diseases is becoming more and more serious. The virus diseases such as foot-and-mouth disease (FMD), bovine viral diarrhea (BVD), bovine infectious rhinotracheitis (IBR), bovine rotavirus (BRV) and bovine coronavirus (BCVV) are widespread in large scale pastures in China and cause great losses. These diseases have seriously jeopardized the healthy development of the dairy cattle breeding industry in China. As the birthplace of the dairy cattle breeding industry in China, all dairy cattle farming in Shanghai has achieved scale. Pay attention to and realize the surveillance, prevention and control of specific epidemic diseases. In view of the current epidemic situation of viral diseases in dairy cows, We urgently need to develop highly efficient and economical antiviral and immune-enhancing drugs. Interferon Interferon IFNs are a group of cytokines that have anti-virus, participate in immune regulation and other biological activities produced by animal cells stimulated by induction. There are three types of interferon, I. E. interferon 鈪，

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