槐花多糖对鸡奇异变形杆菌OmpA基因工程亚单位疫苗的免疫增强效果

发布时间：2018-06-18 21:34

本文选题：真核表达 + 毕赤酵母　；参考：《山东农业大学》2015年硕士论文

【摘要】：奇异变形杆菌(Proteus mirabilis,P.mirabilis)是一种急性条件致病菌,广泛分布于自然界中,各种日龄的鸡群均易感染奇异变形杆菌,该菌主要引起鸡肢腿瘫痪、腹泻、脑软化、败血症等症状。本病既可水平传播,又能垂直传播,很容易引起爆发性流行疾病。近些年奇异变形杆菌在鸡群中的感染率逐渐升高,对养禽业带来了巨大的负面影响。目前无有效的疫苗预防,研发新型疫苗成为当务之急。Omp A是大多数革兰阴性菌外膜蛋白的主要成分,Korn等人证明奇异变形杆菌Omp A能够刺激小鼠B淋巴细胞大量增殖,具有良好的免疫原性。而巴斯德毕赤酵母(Pichia pastoris)表达系统具有表达量高、遗传稳定性强、重组蛋白分泌表达以及对外源蛋白翻译后修饰等特点。此外,酵母表达的蛋白无内毒素。因此,本研究对奇异变形杆菌Omp A进行巴斯德毕赤酵母真核表达,利用表达产物与槐花多糖佐剂构建成基因工程重组亚单位疫苗。本研究的目的是探索槐花多糖对鸡奇异变形杆菌Omp A基因工程亚单位疫苗的免疫增强效果。1.奇异变形杆菌OmpA毕赤酵母表达根据Gen Bank中发表的鸡奇异变形杆菌Omp A基因序列(参考序列号为NC_010554.1)设计特异性引物,以奇异变形杆菌Q1株为模板PCR扩增Omp A基因,大小为1107bp,构建p MD18T-Omp A克隆载体进行测序。重组质粒p MD18T-Omp A经Eco R I和Not I双酶切后回收Omp A基因,连接到p PIC9质粒构建表达载体p PIC9-Omp A,经Sac I线性化后Licl转化法转入毕赤酵母GS115中,RDB平板筛选阳性子转化子,菌落PCR鉴定目的基因成功整合到毕赤酵母基因组中。阳性转化子经1.0%甲醇诱导96 h后Omp A浓度达到0.8μg/m L,SDS-PAGE显示重组蛋白大小约为52 k Da。Western blot鉴定发现重组蛋白能与兔多抗原核表达的Omp A血清发生反应。表明鸡奇异变形杆菌Omp A成功表达。2.槐花多糖对鸡奇异变形杆菌重组亚单位疫苗的免疫增强效果为研究槐花多糖佐剂对奇异变形杆菌Omp A基因工程亚单位疫苗免疫反应的影响,将提取的槐花多糖与酵母表达的Omp A等体积配比制成重组亚单位疫苗。将360只1日龄SPF雏鸡随机分为6组,分别注射3个不同多糖剂量(100mg/m L、200 mg/m L、300 mg/m L)的Omp A疫苗,弗氏佐剂-Omp A,纯化Omp A以及生理盐水。分别检测外周血抗体效价、外周血清IL-2浓度、外周血T淋巴细胞增殖率、外周血CD4+、CD8+细胞百分比以及疫苗免疫保护效果。结果表明,200 mg/m L槐花多糖组抗体滴度、IL-2浓度、T淋巴细胞增殖率以及CD4+、CD8+细胞百分比显著高于其他组,本组疫苗保护率最高88.8%,这表明在真核表达的奇异变形杆菌Omp A可以作为开发基因工程亚单位疫苗有效地免疫原,槐花多糖可以显著增强奇异变形杆菌Omp A基因工程重组亚单位疫苗的免疫效果。
[Abstract]:Proteus mirabilis (P. mirabilis) is a kind of acute conditioned pathogen, which is widely distributed in nature. The chickens of various days are susceptible to Proteus mirabilis, which mainly causes paralysis of legs, diarrhea, encephalomalacia, septicemia and so on. The disease can be spread both horizontally and vertically, which can easily cause an outbreak of epidemic disease. In recent years, the infection rate of Proteus mirabilis in chickens has gradually increased, which has a huge negative impact on poultry industry. At present, there is no effective vaccine prevention, so it is urgent to develop new vaccine. Omp A is the main component of outer membrane protein of most gram-negative bacteria. Korn et al have demonstrated that Proteus mirabilis Omp A can stimulate the proliferation of B lymphocytes in mice. It has good immunogenicity. The Pichia pastoris expression system of Pichia pastoris was characterized by high expression, strong genetic stability, secretion of recombinant proteins and post-translational modification of foreign proteins. In addition, yeast expressed proteins without endotoxin. Therefore, in this study, Proteus mirabilis Omp A was expressed in E. pastoris and recombinant subunit vaccine was constructed by using the expression product and the adjuvant of Sophora japonica polysaccharides. The aim of this study was to investigate the immune enhancement effect of Sophora japonica polysaccharides against Proteus mirabilis Omp A gene engineering subunit vaccine. Proteus mirabilis OmpA Pichia pastoris expression designed specific primers according to the OmpA gene sequence of Proteus mirabilis published in GenBank (reference sequence number is NCSC010554.1), and amplified Omp A gene by PCR using Proteus mirabilis Q1 strain as template. The cloned vector pMD18T-OmpA was constructed and sequenced. The recombinant plasmid pMD18T-OmpA was digested by Ecor RI and not I, and was ligated to pPIC9 plasmid to construct the expression vector pPIC9-OmpA. The recombinant plasmid pMD18T-OmpA was transformed into Pichia pastoris GS115 with RDB plate to screen positive transformants. The target gene was successfully integrated into the genome of Pichia pastoris by colony PCR. The positive transformant was induced by 1.0% methanol for 96 h, and the concentration of Ompa reached 0.8 渭 g / m L ~ (-1) SDS-PAGE. The recombinant protein was identified to be about 52kDa.Western blot. It was found that the recombinant protein could react with the serum of rabbit polyantigen. The results showed that Proteus mirabilis OmpA was successfully expressed. The immunoenhancement effect of Sophora japonica polysaccharides on the recombinant subunit vaccine of Proteus mirabilis was studied in order to study the effect of polysaccharide adjuvant on the immune response of Proteus mirabilis Omp A gene engineering subunit vaccine. The recombinant subunit vaccine was prepared by matching the extracted polysaccharides from Sophora japonica with the OmpA expressed by yeast. 360 1-day-old SPF chicks were randomly divided into 6 groups, and were injected with three different doses of OmpA vaccine (100mg / mL), Freund's adjuvant -OmpA, and normal saline respectively. The antibody titers of peripheral blood, the concentration of IL-2, the proliferation rate of T lymphocytes in peripheral blood, the percentage of CD4 / CD8 cells in peripheral blood and the protective effect of the vaccine were detected. The results showed that the antibody titer and IL-2 concentration of T lymphocytes and the percentage of CD4 / CD8 cells in 200 mg/m / L Sophora japonica polysaccharide group were significantly higher than those in other groups. The highest protective rate of the vaccine was 88.8%, which indicated that the Eukaryotic expression of Proteus mirabilis Omp A could be used as an effective immunogen for the development of genetic engineering subunit vaccine. Sophora japonica polysaccharides could significantly enhance the immunity of Proteus mirabilis Omp A recombinant subunit vaccine.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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