本文選題：牛腸道病毒(BEV) + VP2�。� 參考：《東北農(nóng)業(yè)大學》2015年碩士論文

【摘要】：牛腸道病毒(Bovine enterovirus,BEV)屬于小RNA病毒科,腸道病毒屬成員,該病毒于1959年被Moll和Davis首次報道,已在全世界主要養(yǎng)牛國家和地區(qū)導致疾病發(fā)生和流行。目前該病在我國內(nèi)蒙古、山東、吉林、黑龍江、北京均有發(fā)現(xiàn)。感染可引起下痢和呼吸道癥狀,食欲下降,嚴重的還有便血,產(chǎn)奶量大幅下降,給養(yǎng)牛業(yè)造成了嚴重的經(jīng)濟損失。目前,我國對牛腸道疾病的診斷和防治還很薄弱,完善該病的診斷技術(shù)及研發(fā)亞單位疫苗對我國奶牛養(yǎng)殖業(yè)具有重大意義。牛腸道病毒基因組分為不分節(jié)段的的單股正鏈的RNA,可直接作為mRNA翻譯出一個多聚蛋白,經(jīng)過一系列降解,產(chǎn)生4種結(jié)構(gòu)蛋白(VP1、VP2、VP3和VP4)和7種非結(jié)構(gòu)蛋白(2A、2B、2C、3A、3B、3C和3D)。據(jù)研究證實,抗原決定簇位于暴露在病毒顆粒表面的VP1、VP2、VP3上,且抗VP2抗體中和病毒能力最好,表明VP2蛋白是研究BEV的首選特異性抗原。而3D區(qū)域較為保守,可作為BEV分子生物學檢測的首選序列�；诖�,該實驗做了以下方面的研究工作。1開展牛腸道病毒VP2蛋白免疫原性研究,為亞單位疫苗研究奠定基礎(chǔ)。應(yīng)用RT-PCR方法擴增BEV VP2基因,構(gòu)建重組質(zhì)粒pET32a(+)-VP2。轉(zhuǎn)化大腸桿菌菌株BL21,IPTG誘導其表達VP2融合蛋白,Western-blot檢測目的蛋白。將純化的重組蛋白(pET32a(+)-VP2)免疫新西蘭大白兔,制備VP2多克隆抗體,iELISA方法檢測抗體效價,同時利用血清中和實驗,評估其誘導中和抗體的能力。SDS-PAGE分析顯示,融合蛋白的分子量為50KD,與預(yù)期值大小相符。檢測其抗體效價為1:25600。血清中和實驗表明,原核表達的VP2蛋白可誘導產(chǎn)生的中和抗體效價為1:107.4。本研究成功克隆和表達了具有免疫原性的VP2蛋白,其可誘導產(chǎn)生較高效價的多克隆抗體,為BEV血清學診斷方法的建立及亞單位疫苗的研制奠定了基礎(chǔ)。2建立牛腸道病毒分子生物學檢測方法,為流行病學調(diào)查及疫情診斷提供支撐。首先根據(jù)GenBank中公布的BEV 3D基因保守序列,設(shè)計并合成一對特異性引物,以構(gòu)建的含有該引物擴增序列的重組質(zhì)粒(pEAST-T3-3D)作為陽性標準品,優(yōu)化各種條件,建立了檢測BEV的SYBR GreenⅠ實時熒光定量PCR方法,結(jié)果表明,該方法線性關(guān)系良好,與牛病毒性腹瀉等其它幾種牛的病毒均無交叉反應(yīng);其檢出敏感度達7.13×101拷貝/μL,敏感性比常規(guī)PCR檢測方法高10倍。應(yīng)用該方法檢測了3個規(guī)�；膛鏊蜋z的41份奶牛腹瀉樣品和3份氣溶膠樣品,腹瀉樣品陽性檢出率為39.02%(16/41);3份氣溶膠樣本均為陽性,陽性檢出率為100%。初步臨床檢測的結(jié)果表明,該方法靈敏度高、特異性強、重復(fù)性好,可同時檢測大量樣品。本方法的建立為BEV的檢測和定量分析提供了一種技術(shù)手段,為進一步研究BEV的流行病學調(diào)查及疫情診斷提供了支撐。
[Abstract]:Bovine enterovirus (Bovine enterovirus.BEV) is a member of the small RNA virus family. It was first reported by Moll and Davis in 1959 and has led to the occurrence and prevalence of disease in major cattle breeding countries and regions in the world. At present, the disease has been found in Inner Mongolia, Shandong, Jilin, Heilongjiang and Beijing. Infection can lead to dysentery and respiratory symptoms, reduced appetite, severe bloody stool, a sharp decline in milk production, and a serious economic loss to the cattle industry. At present, the diagnosis and prevention of bovine intestinal diseases in China is still very weak. It is of great significance to improve the diagnostic technology and develop subunit vaccines for dairy cattle breeding in China. The genome of bovine enterovirus is divided into unsegmented single-stranded positive RNAs, which can be directly translated as mRNA to produce a polymeric protein. After a series of degradation, four structural proteins (VP1, VP2, VP3 and VP4) and 7 nonstructural proteins (P2A2A2BO2CN3A3B3C and 3DP3B3C) are produced. It has been confirmed that the antigenic determinant is located on the VP1VP2VP3 exposed to the surface of virus particles and has the best neutralization ability against VP2 antibodies, indicating that VP2 protein is the preferred specific antigen for the study of BEV. The 3D region is conserved and can be used as the preferred sequence for BEV molecular biological detection. Based on this, the following research work was done. 1. The immunogenicity of bovine enterovirus VP2 protein was studied, which laid a foundation for the research of subunit vaccine. The BEV VP2 gene was amplified by RT-PCR and the recombinant plasmid pET32a (pET32a) was constructed. The transformed Escherichia coli strain BL21 was induced to express VP2 fusion protein by Western-blot. New Zealand white rabbits were immunized with purified recombinant protein pET32a (pET-VP2), and VP2 polyclonal antibodies were prepared to detect the titer of antibodies by Elisa. At the same time, serum neutralization test was used to evaluate its ability to induce neutralizing antibodies. SDS-PAGE analysis showed that, The molecular weight of the fusion protein is 50 KD, which is in accordance with the expected value. The titer of the antibody was 1: 25600. Serum neutralization test showed that the neutralizing antibody titer induced by prokaryotic expression of VP2 protein was 1: 107.4. In this study, VP2 protein with immunogenicity was cloned and expressed successfully, which could induce the production of polyclonal antibody with high titer. For the establishment of serological diagnosis method of BEV and the development of subunit vaccine, the molecular biological detection method of bovine enterovirus was established, which provided the support for epidemiological investigation and diagnosis of epidemic situation. Firstly, according to the conserved sequence of BEV 3D gene published in GenBank, a pair of specific primers were designed and synthesized. The recombinant plasmid pEAST-T3-3D containing the amplified sequence of the primers was constructed as the positive standard, and various conditions were optimized. A real-time fluorescence quantitative PCR method for the detection of BEV was established. The results showed that the method had a good linear relationship and had no cross reaction with other bovine viruses such as bovine viral diarrhea. Its sensitivity was 7.13 脳 101 copies / 渭 L, which was 10 times higher than that of conventional PCR. This method was used to detect 41 samples of diarrhea and 3 samples of aerosol from 3 large scale dairy farms. The positive rate of diarrhea samples was 39.02% and 16 / 41% respectively, and the positive rate was 100%. The preliminary clinical results showed that the method was sensitive, specific and reproducible, and could be used to detect a large number of samples at the same time. The establishment of this method provides a technical means for the detection and quantitative analysis of BEV, and provides support for the further study of epidemiological investigation and epidemic diagnosis of BEV.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23

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相關(guān)期刊論文 前1條

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