猪流行性腹泻病毒S部分基因的表达、抗体制备及间接ELISA方法的建立
发布时间：2018-11-19 16:34
【摘要】：猪流行性腹泻(PED)是一种急性、感染性高的肠道病毒性疾病,对新生猪仔造成很高的死亡率,猪流行性腹泻病毒(PEDV)为其致病原。PEDV属于尼多病毒目、冠状病毒科,α冠状病毒亚科(或属),基因组为单股正义RNA病毒,基因组大小约28 kb。PEDV变异较大,其临床症状、病理变化以及流行特点都与传染性胃肠炎和轮状病毒病的十分相似。目前,PED在世界范围内广泛流行,但还没有很好的预防和诊断方法。为此本研究选择PEDV S基因的保守序列进行原核表达,制备相应抗体,并初步建立一套检测PEDV抗体的间接ELISA方法。具体研究内容如下:1 PEDV部分S基因的合成及重组质粒的构建通过DNAstar软件对猪流行性腹泻病毒流行毒株S基因进行序列比对与分析,选取位于502-641氨基酸位置处的S1蛋白保守序列,利用大肠杆菌密码子偏嗜性进行密码子优化,Overlap PCR合成,获得417 bp基因片段,连接到克隆载体pJET/1.2-blunt上,并构建重组表达质粒pET32a-S417和pXXGST-S417。2重组质粒的表达与纯化将获得的重组表达质粒转化到BL21感受态细胞进行诱导表达,并通过割胶纯化,获得重组蛋白GST-S417,分子量31 kD左右,浓度0.3 mg/mL左右,纯度达95%以上;通过过柱纯化,获得重组蛋白His-S417,分子量30 kD左右,浓度0.1 mg/mL左右,纯度达90%以上。3多克隆抗体的制备以纯化的GST-S417重组蛋白作为免疫原,免疫新西兰大白兔制备多克隆抗体,免疫量500μg/只。以纯化的His-S417重组蛋白作为包被抗原,检测免疫血清中抗体的效价,ELISA检测结果表明血清效价达1:10000以上,而且制备的多克隆抗体能特异性识别目的蛋白。该研究表明所选的S蛋白区域具有一定的抗原性。4间接ELISA检测方法的初步建立包被纯化的GST-S417蛋白,摸索最佳间接ELISA条件,结果显示:包被量0.32μg/孔,血清最佳稀释比1:800,HRP标记的抗猪抗体稀释比为1:3000时,检测效果最好,阴性临界值为0.340,有良好的重复性和特异性,与市场销售的诊断试剂盒符合率较高,达72.7%,为进一步开发诊断试剂盒提供了理论和技术基础。
[Abstract]:Porcine epidemic diarrhea (PED) is an acute and highly infectious enterovirus disease, which causes a high mortality to newborn piglets. Porcine epidemic diarrhea virus (PEDV) is the causative agent. PEDV belongs to the family Nidoviridae and coronavirus. The genome of 伪 coronavirus subfamily (or genus) is a single-stranded sense RNA virus, and the genome size is about 28 kb.PEDV. The clinical symptoms, pathological changes and epidemic characteristics of 伪 coronavirus are very similar to those of transmissible gastroenteritis and rotavirus disease. At present, PED is popular all over the world, but there is no good method of prevention and diagnosis. In this study, the conserved sequence of PEDV S gene was selected for prokaryotic expression, the corresponding antibody was prepared, and a set of indirect ELISA method for detecting PEDV antibody was established. The main contents are as follows: 1 the synthesis of S gene of PEDV and the construction of recombinant plasmid were used to sequence alignment and analysis of S gene of porcine epidemic diarrhea virus epidemic strain by DNAstar software. The S1 protein conserved sequence located at the 502-641 amino acid position was selected. The codon bias of Escherichia coli codon was used to optimize the, Overlap PCR synthesis. The 417 bp gene fragment was obtained and ligated to the clone vector pJET/1.2-blunt. The expression and purification of the recombinant expression plasmid pET32a-S417 and pXXGST-S417.2 were constructed. The recombinant expression plasmid was transformed into BL21 receptive cells for induction and expression, and the recombinant protein GST-S417, was obtained by gel purification. The molecular weight is about 31 kD, the concentration is about 0.3 mg/mL, and the purity is over 95%. The molecular weight of recombinant protein His-S417, was about 30 kD, the concentration was about 0.1 mg/mL, and the purity of polyclonal antibody was over 90%. The purified recombinant GST-S417 protein was used as immunogen. New Zealand white rabbits were immunized with polyclonal antibodies (500 渭 g / mouse). The purified His-S417 recombinant protein was used as the coating antigen to detect the titer of the antibody in the immune serum. The results of ELISA showed that the titer of the serum was over 1: 10000, and the polyclonal antibody could specifically recognize the target protein. This study shows that the selected S protein region has a certain antigenicity. 4 the preliminary establishment of indirect ELISA detection method for the purification of GST-S417 protein, explore the best indirect ELISA conditions, the results showed that the coating amount of 0.32 渭 g / pore, When the dilution ratio of anti-porcine antibody labeled with 1: 800 or HRP was 1: 3000, the detection effect was the best, the negative critical value was 0.340, which had good repeatability and specificity, and was in good agreement with the diagnostic kit sold on the market. It provides a theoretical and technical basis for the further development of diagnostic kit.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
[Abstract]:Porcine epidemic diarrhea (PED) is an acute and highly infectious enterovirus disease, which causes a high mortality to newborn piglets. Porcine epidemic diarrhea virus (PEDV) is the causative agent. PEDV belongs to the family Nidoviridae and coronavirus. The genome of 伪 coronavirus subfamily (or genus) is a single-stranded sense RNA virus, and the genome size is about 28 kb.PEDV. The clinical symptoms, pathological changes and epidemic characteristics of 伪 coronavirus are very similar to those of transmissible gastroenteritis and rotavirus disease. At present, PED is popular all over the world, but there is no good method of prevention and diagnosis. In this study, the conserved sequence of PEDV S gene was selected for prokaryotic expression, the corresponding antibody was prepared, and a set of indirect ELISA method for detecting PEDV antibody was established. The main contents are as follows: 1 the synthesis of S gene of PEDV and the construction of recombinant plasmid were used to sequence alignment and analysis of S gene of porcine epidemic diarrhea virus epidemic strain by DNAstar software. The S1 protein conserved sequence located at the 502-641 amino acid position was selected. The codon bias of Escherichia coli codon was used to optimize the, Overlap PCR synthesis. The 417 bp gene fragment was obtained and ligated to the clone vector pJET/1.2-blunt. The expression and purification of the recombinant expression plasmid pET32a-S417 and pXXGST-S417.2 were constructed. The recombinant expression plasmid was transformed into BL21 receptive cells for induction and expression, and the recombinant protein GST-S417, was obtained by gel purification. The molecular weight is about 31 kD, the concentration is about 0.3 mg/mL, and the purity is over 95%. The molecular weight of recombinant protein His-S417, was about 30 kD, the concentration was about 0.1 mg/mL, and the purity of polyclonal antibody was over 90%. The purified recombinant GST-S417 protein was used as immunogen. New Zealand white rabbits were immunized with polyclonal antibodies (500 渭 g / mouse). The purified His-S417 recombinant protein was used as the coating antigen to detect the titer of the antibody in the immune serum. The results of ELISA showed that the titer of the serum was over 1: 10000, and the polyclonal antibody could specifically recognize the target protein. This study shows that the selected S protein region has a certain antigenicity. 4 the preliminary establishment of indirect ELISA detection method for the purification of GST-S417 protein, explore the best indirect ELISA conditions, the results showed that the coating amount of 0.32 渭 g / pore, When the dilution ratio of anti-porcine antibody labeled with 1: 800 or HRP was 1: 3000, the detection effect was the best, the negative critical value was 0.340, which had good repeatability and specificity, and was in good agreement with the diagnostic kit sold on the market. It provides a theoretical and technical basis for the further development of diagnostic kit.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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