本文選題：真核表達 + 畢赤酵母��； 參考：《山東農業(yè)大學》2015年碩士論文

【摘要】：奇異變形桿菌(Proteus mirabilis,P.mirabilis)是一種急性條件致病菌,廣泛分布于自然界中,各種日齡的雞群均易感染奇異變形桿菌,該菌主要引起雞肢腿癱瘓、腹瀉、腦軟化、敗血癥等癥狀。本病既可水平傳播,又能垂直傳播,很容易引起爆發(fā)性流行疾病。近些年奇異變形桿菌在雞群中的感染率逐漸升高,對養(yǎng)禽業(yè)帶來了巨大的負面影響。目前無有效的疫苗預防,研發(fā)新型疫苗成為當務之急。Omp A是大多數(shù)革蘭陰性菌外膜蛋白的主要成分,Korn等人證明奇異變形桿菌Omp A能夠刺激小鼠B淋巴細胞大量增殖,具有良好的免疫原性。而巴斯德畢赤酵母(Pichia pastoris)表達系統(tǒng)具有表達量高、遺傳穩(wěn)定性強、重組蛋白分泌表達以及對外源蛋白翻譯后修飾等特點。此外,酵母表達的蛋白無內毒素。因此,本研究對奇異變形桿菌Omp A進行巴斯德畢赤酵母真核表達,利用表達產物與槐花多糖佐劑構建成基因工程重組亞單位疫苗。本研究的目的是探索槐花多糖對雞奇異變形桿菌Omp A基因工程亞單位疫苗的免疫增強效果。1.奇異變形桿菌OmpA畢赤酵母表達根據(jù)Gen Bank中發(fā)表的雞奇異變形桿菌Omp A基因序列(參考序列號為NC_010554.1)設計特異性引物,以奇異變形桿菌Q1株為模板PCR擴增Omp A基因,大小為1107bp,構建p MD18T-Omp A克隆載體進行測序。重組質粒p MD18T-Omp A經Eco R I和Not I雙酶切后回收Omp A基因,連接到p PIC9質粒構建表達載體p PIC9-Omp A,經Sac I線性化后Licl轉化法轉入畢赤酵母GS115中,RDB平板篩選陽性子轉化子,菌落PCR鑒定目的基因成功整合到畢赤酵母基因組中。陽性轉化子經1.0%甲醇誘導96 h后Omp A濃度達到0.8μg/m L,SDS-PAGE顯示重組蛋白大小約為52 k Da。Western blot鑒定發(fā)現(xiàn)重組蛋白能與兔多抗原核表達的Omp A血清發(fā)生反應。表明雞奇異變形桿菌Omp A成功表達。2.槐花多糖對雞奇異變形桿菌重組亞單位疫苗的免疫增強效果為研究槐花多糖佐劑對奇異變形桿菌Omp A基因工程亞單位疫苗免疫反應的影響,將提取的槐花多糖與酵母表達的Omp A等體積配比制成重組亞單位疫苗。將360只1日齡SPF雛雞隨機分為6組,分別注射3個不同多糖劑量(100mg/m L、200 mg/m L、300 mg/m L)的Omp A疫苗,弗氏佐劑-Omp A,純化Omp A以及生理鹽水。分別檢測外周血抗體效價、外周血清IL-2濃度、外周血T淋巴細胞增殖率、外周血CD4+、CD8+細胞百分比以及疫苗免疫保護效果。結果表明,200 mg/m L槐花多糖組抗體滴度、IL-2濃度、T淋巴細胞增殖率以及CD4+、CD8+細胞百分比顯著高于其他組,本組疫苗保護率最高88.8%,這表明在真核表達的奇異變形桿菌Omp A可以作為開發(fā)基因工程亞單位疫苗有效地免疫原,槐花多糖可以顯著增強奇異變形桿菌Omp A基因工程重組亞單位疫苗的免疫效果。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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