牛肠道病毒VP2蛋白免疫原性研究及诊断方法的建立

发布时间：2018-06-04 21:51

本文选题：牛肠道病毒(BEV) + VP2　；参考：《东北农业大学》2015年硕士论文

【摘要】：牛肠道病毒(Bovine enterovirus,BEV)属于小RNA病毒科,肠道病毒属成员,该病毒于1959年被Moll和Davis首次报道,已在全世界主要养牛国家和地区导致疾病发生和流行。目前该病在我国内蒙古、山东、吉林、黑龙江、北京均有发现。感染可引起下痢和呼吸道症状,食欲下降,严重的还有便血,产奶量大幅下降,给养牛业造成了严重的经济损失。目前,我国对牛肠道疾病的诊断和防治还很薄弱,完善该病的诊断技术及研发亚单位疫苗对我国奶牛养殖业具有重大意义。牛肠道病毒基因组分为不分节段的的单股正链的RNA,可直接作为mRNA翻译出一个多聚蛋白,经过一系列降解,产生4种结构蛋白(VP1、VP2、VP3和VP4)和7种非结构蛋白(2A、2B、2C、3A、3B、3C和3D)。据研究证实,抗原决定簇位于暴露在病毒颗粒表面的VP1、VP2、VP3上,且抗VP2抗体中和病毒能力最好,表明VP2蛋白是研究BEV的首选特异性抗原。而3D区域较为保守,可作为BEV分子生物学检测的首选序列。基于此,该实验做了以下方面的研究工作。1开展牛肠道病毒VP2蛋白免疫原性研究,为亚单位疫苗研究奠定基础。应用RT-PCR方法扩增BEV VP2基因,构建重组质粒pET32a(+)-VP2。转化大肠杆菌菌株BL21,IPTG诱导其表达VP2融合蛋白,Western-blot检测目的蛋白。将纯化的重组蛋白(pET32a(+)-VP2)免疫新西兰大白兔,制备VP2多克隆抗体,iELISA方法检测抗体效价,同时利用血清中和实验,评估其诱导中和抗体的能力。SDS-PAGE分析显示,融合蛋白的分子量为50KD,与预期值大小相符。检测其抗体效价为1:25600。血清中和实验表明,原核表达的VP2蛋白可诱导产生的中和抗体效价为1:107.4。本研究成功克隆和表达了具有免疫原性的VP2蛋白,其可诱导产生较高效价的多克隆抗体,为BEV血清学诊断方法的建立及亚单位疫苗的研制奠定了基础。2建立牛肠道病毒分子生物学检测方法,为流行病学调查及疫情诊断提供支撑。首先根据GenBank中公布的BEV 3D基因保守序列,设计并合成一对特异性引物,以构建的含有该引物扩增序列的重组质粒(pEAST-T3-3D)作为阳性标准品,优化各种条件,建立了检测BEV的SYBR GreenⅠ实时荧光定量PCR方法,结果表明,该方法线性关系良好,与牛病毒性腹泻等其它几种牛的病毒均无交叉反应;其检出敏感度达7.13×101拷贝/μL,敏感性比常规PCR检测方法高10倍。应用该方法检测了3个规模化奶牛场送检的41份奶牛腹泻样品和3份气溶胶样品,腹泻样品阳性检出率为39.02%(16/41);3份气溶胶样本均为阳性,阳性检出率为100%。初步临床检测的结果表明,该方法灵敏度高、特异性强、重复性好,可同时检测大量样品。本方法的建立为BEV的检测和定量分析提供了一种技术手段,为进一步研究BEV的流行病学调查及疫情诊断提供了支撑。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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