猪博卡病毒间接ELISA诊断方法的建立

发布时间：2018-07-14 21:20

【摘要】：猪博卡病毒(PBo V)最早于2009年在瑞典被发现,迄今为止,对于其传播方式,侵染机制,及其基因组功能都不甚明了,但是世界各国学者在进行该病毒的流行病学调查时发现,该病毒在PMWS患猪中阳性率较高。另外还有学者提出猪博卡病毒可能与呼吸道疾病病原共感染机体。目前大多认为其基因组编码3个开放阅读框(ORF)分别对应非结构蛋白NS1、抗原蛋白VP、和非结构蛋白NP1,VP蛋白为猪博卡病毒主要的抗原蛋白,但变异性较高,非结构蛋白NP1,编码一个相对保守且高度磷酸化的非结构性蛋白质。目前世界各国对该病毒的检测也仅仅局限于一些不成熟的PCR检测方法,并无血清学检测方法的问世,因而有效的血清学诊断方法的研究迫在眉睫,本研究成功的在河北地区病猪体内提取出猪博卡病毒基因,分别对NP1基因和VP2基因进行B细胞线性抗原表位的预测分析,并对保守的NP1基因和截短的相对保守的VP2基因进行克隆表达,分别将纯化的NP1蛋白和VP2截短蛋白作为包被抗原,经过优化条件和相对比较建立了快速检测猪博卡病毒病的血清学诊断方法。根据NCBI系统中Gen Bank已发表的猪博卡病毒NP1和VP2基因序列,设计合成特异性引物,采用降落PCR技术扩增NP1基因和截短的VP2基因序列,克隆后进行测序,测序正确后进行克隆载体与加酶切位点的目的片段的连接转化,并分别将质粒命名为PMD-NP1和PMD-VP2。对重组质粒PMD-NP1和PMD-VP2进行双酶切得到带酶切位点的NP1基因和截短的VP2基因并将其与原核表达载体p ET32a连接,构建表达质粒p ET32a-NP1和p ET32a-VP2。经IPTG诱导后SDS-PAGE电泳可分别检测到分子质量约为44ku和35 ku的蛋白,与预期的蛋白分子量大小基本一致,以可溶性形式存在于上清中,同时在包涵体中也大量存在。利用原核表达载体p ET32a上带有的His标签对p ET32a-NP1和p ET32a-VP2重组蛋白进行纯化,纯化蛋白经Western-blotting结果显示,重组蛋白与PBOV阳性血清具有良好的反应原性。表明分子质量约43 ku和37 ku的蛋白即为目的蛋白。以纯化的两种重组蛋白分别作为包被抗原,通过对各自反应条件的优化,建立了检测PBo V抗体的间接ELISA检测方法,并比较两种ELISA检测方法。优化结果显示,以NP1蛋白为包被抗原的抗原最佳包被浓度为4μg/m L;封闭液为2%的明胶工作液;阴阳性血清最佳稀释度为1∶80,二抗的最佳稀释度为1∶1000,底物显色时间为10 min。该方法与CSFV、PRRSV、PPV、PCV2阳性血清不发生交叉反应,批内和批间重复性试验的变异系数(CV)均值都小于10%,而以VP2蛋白为包被抗原的抗原最佳包被浓度为2μg/m L;封闭液为2%的明胶工作液;阴阳性血清最佳稀释度为1∶80,酶标抗体最佳稀释度为1∶1000,底物显色时间为15 min。该方法与CSFV、PRRSV、PPV、PCV2阳性血清不发生交叉反应,批内重复性试验和批间重复性试验的变异系数(CV)均小于10%,说明该方法具有较好的特异性和重复性,同时通过比较发现以NP1蛋白为包被抗原的ELISA方法比以VP2蛋白为包被抗原的ELISA方法检出率高。
[Abstract]:The PBo V virus (PBo V) was first discovered in Sweden in 2009. So far, its transmission mode, infection mechanism, and its genome function are not very clear. However, in the epidemiological investigation of the virus, scholars from all over the world have found that the virus has a high positive rate in PMWS pigs. In addition, some scholars have suggested that the swine Boka virus can be used. The 3 open reading frames (ORF) are considered to correspond to the non structural protein NS1, the antigen protein VP, and the non structural protein NP1, and the VP protein is the main antigen protein of the porcine Boka virus, but the variability is high, and the non structural protein NP1 is encoded by a relatively conservative and highly phosphorylated protein. At present, the detection of the virus in the world is only limited to some immature PCR detection methods, and there is no serological detection method. Therefore, the effective serological diagnosis method is imminent. This study successfully extracted the porcine Boka virus gene in the infected pigs in Hebei area, and respectively to NP1 The gene and VP2 gene were used to predict the linear antigen epitopes of B cells and to clone and express the conservative NP1 gene and the truncated and relatively conservative VP2 gene. The purified NP1 protein and the VP2 truncated protein were used as the envelope antigen respectively. The rapid detection of the serological diagnosis of porcine Boka virus disease was established through the optimization conditions and relative comparison. Methods. Specific primers were designed and synthesized according to the NP1 and VP2 gene sequences of the porcine Boka virus published by Gen Bank in the NCBI system. The NP1 gene and the truncated VP2 gene sequence were amplified by landing PCR technique, and then sequenced and sequenced. The recombinant plasmid PMD-NP1 and PMD-VP2 were named PMD-NP1 and PMD-VP2., and the NP1 gene and the truncated VP2 gene with the enzyme cut site were obtained by double enzyme digestion, and they were connected with the prokaryotic expression vector p ET32a, and the expression plasmid P ET32a-NP1 and P ET32a-VP2. were constructed to detect the molecular mass of the molecular weight approximately and 35. Protein, in accordance with the expected protein molecular weight, exists in the soluble form in the supernatant and also exists in the inclusion bodies. The recombinant protein of P ET32a-NP1 and P ET32a-VP2 is purified by the His tag on the prokaryotic expression vector p ET32a. The purified protein is shown by Western-blotting, and the recombinant protein and PBOV Yang are shown. The sex serum has a good reactivity. It shows that the protein of about 43 Ku and 37 Ku is the target protein. The purified two recombinant proteins are used as the envelope antigen respectively. By optimizing the reaction conditions, the indirect ELISA detection method for detecting PBo V antibodies is established and two ELISA detection methods are compared. The optimization results show that The best envelope of antigen for NP1 protein was 4 mu g/m L and 2% gelatin working liquid in closed liquid; the optimum dilution of the sera was 1: 80, the best dilution of two resistance was 1: 1000, and the chromogenic time of the substrate was 10 min.. The method was not cross reacting with CSFV, PRRSV, PPV, PCV2 positive blood, and repeated test in batch and interbatch. The mean value of variation coefficient (CV) was less than 10%, and the best envelope of antigen with VP2 protein as antigen was 2 g/m L, and the closed liquid was 2% gelatin working liquid; the optimum dilution of the sera was 1: 80, the best dilution of the enzyme antibody was 1: 1000, and the chromogenic time of the substrate was 15 min.. The method did not exchange with the CSFV, PRRSV, PPV, and PCV2 positive serum. The coefficient of variation (CV) of the interbatch and interbatch repeatability test (CV) was less than 10%, indicating that the method had better specificity and repeatability. At the same time, it was found that the detection rate of the ELISA method with the NP1 protein as the envelope antigen was higher than the ELISA method with the VP2 protein as the envelope antigen.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651

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