【摘要】：金黃色葡萄球菌是引起奶牛乳房炎(cow mastitis)的主要病原菌之一,而莢膜多糖和黏附素是金黃色葡萄球菌最主要的毒力因子。當金黃色葡萄球菌侵入機體后,由于莢膜多糖的抗吞噬作用,使其難以被吞噬細胞吞噬清除,進而細菌的黏附素識別并特異性的黏附到組織、細胞的特定分子上是感染的關(guān)鍵,細菌成功定植后,進一步產(chǎn)生酶和毒素,使得感染難以控制。因此本文通過化學(xué)方法將莢膜多糖和黏附素蛋白偶聯(lián),制備偶聯(lián)物,并對其進行免疫學(xué)特性的研究。從而為構(gòu)建金黃色葡萄球菌莢膜多糖-黏附素蛋白二價毒力因子疫苗奠定了理論和實驗基礎(chǔ)。 本實驗研究在哥倫比亞固體培養(yǎng)基中添加不同的營養(yǎng)成分,通過觀察菌落生長情況,從而對金黃色葡萄球菌的培養(yǎng)條件進行優(yōu)化。用此條件培養(yǎng)的金黃色葡萄球菌菌株,采用加酶法對血清8型莢膜多糖進行提取,并用酚提蛋白法和過柱層析法對提取的粗糖進行純化,同時用苯酚-硫酸法測定多糖含量。在獲得純化的血清8型莢膜多糖的基礎(chǔ)之上,以己二酰肼為間橋,碳二亞胺為偶聯(lián)劑,純化的ClfA-FnBPB融合蛋白為載體蛋白,使CP和ClfA-FnBPB偶聯(lián)制備偶聯(lián)物。在偶聯(lián)過程中確定莢膜多糖-蛋白質(zhì)偶聯(lián)物的最佳質(zhì)量比。其次,利用紫外光譜掃描法、苯酚-硫酸法等對偶聯(lián)物進行定性和定量分析并計算偶聯(lián)比。最后,用制備的偶聯(lián)物免疫Ba1B/c小鼠,同時用間接ELISA法檢測抗體水平,并對小鼠進行攻毒保護試驗,評價免疫效果。 結(jié)果顯示,產(chǎn)生血清8型莢膜多糖的金黃色葡萄球菌在加有1%乳糖、2%NaCl和10%胎牛血清的哥倫比亞固體培養(yǎng)基上生長最旺盛。用此條件培養(yǎng)的菌株提純的莢膜多糖,其粗糖的純度為34.43%,精制多糖的純度為79.68%。核酸和蛋白的含量為0.1365mg/mL。用化學(xué)方法制備的莢膜多糖-蛋白質(zhì)偶聯(lián)物在其質(zhì)量比為1：2時可證明偶聯(lián)成功,計算得到偶聯(lián)比為1：1.89。采用間接ELISA法檢測抗體水平,二免后7天,偶聯(lián)物組可產(chǎn)生抗體,且抗體效價最高可達1：6400,說明偶聯(lián)物刺激小鼠產(chǎn)生了免疫應(yīng)答。陰性對照組和單獨多糖組均沒有抗體產(chǎn)生,而單獨融合蛋白組在二免后產(chǎn)生了抗體反應(yīng),但抗體效價較低,最高時僅為1：100。本實驗對三免后兩周的小鼠進行攻毒保護,結(jié)果表明,偶聯(lián)物組的攻毒保護率最高,可達80%,而陰性對照組、單獨多糖組以及單獨融合蛋白組的保護率分別為20%、30%和60%,可見莢膜多糖-蛋白質(zhì)偶聯(lián)物有較好的免疫原性,達到了本實驗的預(yù)期目標。
[Abstract]:Staphylococcus aureus is one of the main pathogens of (cow mastitis) in dairy cattle mastitis, and capsule polysaccharide and adhesin are the main virulence factors of Staphylococcus aureus. When Staphylococcus aureus invades the body, because of the anti-phagocytic effect of capsule polysaccharide, it is difficult to be phagocytized and cleared by phagocytic cells. After successful colonization, the bacteria further produce enzymes and toxins, making the infection difficult to control. In this paper, the conjugate of capsule polysaccharide and adhesin protein was prepared by chemical method, and the immunological properties of the conjugate were studied. Thus, the construction of Staphylococcus aureus capsule polysaccharide-adhesin protein bivalent virulence factor vaccine laid a theoretical and experimental foundation. In this experiment, the culture conditions of Staphylococcus aureus were optimized by adding different nutrient components to the Colombia solid medium and observing the growth of the colony. The strain of Staphylococcus aureus cultured in this condition was extracted by adding enzyme method, the crude sugar was purified by phenol extraction protein method and over-column chromatography, and the content of polysaccharide was determined by phenol-sulfuric acid method. On the basis of purified serotype 8 capsule polysaccharides, the conjugate of CP and ClfA-FnBPB was prepared by using Hexanedihydrazide as the bridge, carbodiimide as the coupling agent and purified ClfA-FnBPB fusion protein as the carrier protein. The optimum mass ratio of capsular polysaccharide to protein conjugate was determined during the coupling process. Secondly, the coupling compounds were qualitatively and quantitatively analyzed and the coupling ratios were calculated by UV scanning and phenol-sulfuric acid methods. Finally, Ba1B/c mice were immunized with the conjugate, and the antibody level was detected by indirect ELISA method. The results showed that Staphylococcus aureus, which produced serotype 8 capsule polysaccharides, grew most strongly on Colombia solid medium containing 1% lactose, 2%NaCl and 10% fetal bovine serum. The purity of crude sugar was 34.43 and the purity of refined polysaccharide was 79.68. The content of nucleic acid and protein was 0.1365 mg / mL. The conjugate of capsule polysaccharide and protein prepared by chemical method can be proved to be successful when its mass ratio is 1:2, and the coupling ratio is calculated to be 1: 1.89. The antibody level was detected by indirect ELISA method. After 7 days, the conjugate group could produce antibody, and the titer of antibody was up to 1: 6400, which indicated that the conjugate stimulated mice to produce immune response. No antibody was produced in the negative control group and the polysaccharide group, but the antibody reaction was found in the fusion protein group, but the titer of the antibody was lower, and the highest antibody titer was only 1: 100. The results showed that the protective rate of the conjugate group was the highest (80%), while that of negative control group, single polysaccharide group and fusion protein group was 20%, respectively. 30% and 60% showed that the conjugate of capsule polysaccharide and protein had good immunogenicity and reached the expected goal of this experiment.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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