猪丹毒丝菌保护性抗原筛选及鉴别诊断ELISA方法初步建立

发布时间：2018-03-11 08:53

本文选题：丹毒丝菌　切入点：保护性抗原筛选　出处：《湖南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：本研究从猪红斑丹毒丝菌全基因组水平着手,选择可编码金属转运相关蛋白、表面蛋白、膜蛋白、脂蛋白的阅读框进行跨膜区域分析、信号肽分析后设计51对引物。利用PCR基因克隆技术对选取蛋白进行基因克隆表达,经过PCR扩增、酶切、连接、转化后用特异性引物检测,结果显示51个基因片段成功插入pET-28a(+)载体。将构建好的重组质粒转入大肠杆菌BL21 (DE3)中进行诱导表达,经SDS-PAGE电泳鉴定显示有23个蛋白成功得到表达,其中包括14个可溶性表达蛋白及9个包涵体表达蛋白。对23个蛋白进行小鼠免疫保护试验,最终显示Su-1(即surface protective antigen SpaA)与丹毒丝菌商品苗免疫组的保护率一致,均达到100%保护；同时Surface proteinA(Spa)节段表达的Su-2-2蛋白与Su-2-4蛋白以及脂蛋白Lp-2蛋白免疫组小鼠也有17%的存活率,也显示了一定的免疫保护效果。本实验室对SpaA氨基端的免疫保护区域构建重组SpaA-N蛋白并对其进行了小鼠攻毒保护试验,结果显示了Spa-N蛋白对小鼠有100%的保护率。本研究以该重组蛋白制备A1(OH)3佐剂亚单位疫苗,并进行猪的免疫攻毒试验,对免疫猪进行丹毒丝菌攻毒也显示了100%的保护性,结果表明Spa-N蛋白丹毒丝菌亚单位疫苗候选蛋白具有一定的可行性。亚单位疫苗诱导机体产生的抗体为疫苗所含抗原对应的抗体,而野毒感染产生的抗体比较复杂,因此可以建立一种评估方法用来鉴别诊断丹毒丝菌野毒感染抗体与SpaA-N免疫抗体,本研究选取10个可溶性表达且表达量高的蛋白作为候选包被抗原包被ELISA反应板检测丹毒丝菌感染猪血清,结果显示自然感染丹毒丝菌猪两周后采得血清就能与Fe-2蛋白包被ELISA抗原板产生较强的反应,且Fe-2蛋白包被ELISA抗原板与大肠杆菌、葡萄球菌、链球菌、副猪嗜血杆菌、巴氏杆菌、沙门氏菌等细菌制备的高免血清的特异性试验显示其有较好的特异性,最终选定对丹毒丝菌自然感染血清反应值较好的Fe-2蛋白作为鉴别诊断丹毒丝菌野毒感染抗体与SpaA-N免疫抗体的包被抗原,通过实验优化ELISA条件为：抗原包被量100ng/孔,血清稀释倍数1/100,血清作用时间、酶标二抗作用时间均为30min,底物作用时间15min。
[Abstract]:In this study, the reading frame encoding metal-transport associated protein, surface protein, membrane protein and lipoprotein was selected from the whole genome level of erysipelas erythematosus for transmembrane region analysis. After the signal peptide analysis, 51 pairs of primers were designed. The selected protein was cloned and expressed by PCR gene cloning technique. The selected protein was amplified by PCR, digested by enzyme, ligated, transformed and detected by specific primers. The results showed that 51 gene fragments were successfully inserted into pET-28a () vector. The constructed recombinant plasmid was transferred into E. coli BL21 DE3 to induce expression, and 23 proteins were successfully expressed by SDS-PAGE electrophoresis. There were 14 soluble expression proteins and 9 inclusion body expression proteins. The protective rate of Su-1 (surface protective antigen SpaA) was the same as that of commercial vaccine of Rhizoma erysipelis, and the protective rate of Su-1 (surface protective antigen SpaA) was 100%. At the same time, the mice immunized with Su-2-2 protein, Su-2-4 protein and lipoprotein Lp-2 protein also had a survival rate of 17%. The recombinant SpaA-N protein was constructed in the immunological protection region of SpaA amino terminal and the mice were tested for the protection of the recombinant SpaA-N protein. The results showed that Spa-N protein had a protective rate of 100% to mice. In this study, the recombinant protein was used to prepare the A1OHH3 adjuvant subunit vaccine, and the porcine immunization test was carried out. The protective effect of the recombinant protein on immunized pigs was also shown to be 100%. The results showed that the candidate protein of Spa-N protein subunit vaccine was feasible. The antibody produced by subunit vaccine was the antibody corresponding to the antigen contained in the vaccine, but the antibody produced by wild virus infection was more complex. Therefore, an evaluation method can be established to differentiate the antibody against the field virus infection of Rhizoma erysipelas from the immune antibody of SpaA-N. In this study, 10 soluble and high expression proteins were selected as candidate envelope antigen coated ELISA reaction plates for detection of porcine serum infected with Rhizoma erysipelas. The results showed that the serum collected after two weeks of natural infection could react strongly with Fe-2 protein coated with ELISA antigen plate, and Fe-2 protein coated ELISA antigen plate with Escherichia coli, Staphylococcus, Streptococcus, Haemophilus parasuis, Pasteurella spp. The specificity test of high immunity serum prepared by Salmonella and other bacteria showed that it had good specificity. Finally, the Fe-2 protein with good serum response value to natural infection of Rhizoma erysipelas was selected as the coating antigen for differential diagnosis of the antibody against Rhizoma erysipelas and the immune antibody against SpaA-N. The optimal conditions of ELISA were as follows: the amount of antigen envelope was 100ng / well. The dilution times of serum 1 / 100, the time of serum action and the time of action of enzyme labeled second antibody were 30 min and 15 min respectively.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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