金黄色葡萄球菌血清8型荚膜多糖的提取纯化及其偶联物免疫学特性的研究

发布时间：2018-11-09 12:44

【摘要】：金黄色葡萄球菌是引起奶牛乳房炎(cow mastitis)的主要病原菌之一,而荚膜多糖和黏附素是金黄色葡萄球菌最主要的毒力因子。当金黄色葡萄球菌侵入机体后,由于荚膜多糖的抗吞噬作用,使其难以被吞噬细胞吞噬清除,进而细菌的黏附素识别并特异性的黏附到组织、细胞的特定分子上是感染的关键,细菌成功定植后,进一步产生酶和毒素,使得感染难以控制。因此本文通过化学方法将荚膜多糖和黏附素蛋白偶联,制备偶联物,并对其进行免疫学特性的研究。从而为构建金黄色葡萄球菌荚膜多糖-黏附素蛋白二价毒力因子疫苗奠定了理论和实验基础。本实验研究在哥伦比亚固体培养基中添加不同的营养成分,通过观察菌落生长情况,从而对金黄色葡萄球菌的培养条件进行优化。用此条件培养的金黄色葡萄球菌菌株,采用加酶法对血清8型荚膜多糖进行提取,并用酚提蛋白法和过柱层析法对提取的粗糖进行纯化,同时用苯酚-硫酸法测定多糖含量。在获得纯化的血清8型荚膜多糖的基础之上,以己二酰肼为间桥,碳二亚胺为偶联剂,纯化的ClfA-FnBPB融合蛋白为载体蛋白,使CP和ClfA-FnBPB偶联制备偶联物。在偶联过程中确定荚膜多糖-蛋白质偶联物的最佳质量比。其次,利用紫外光谱扫描法、苯酚-硫酸法等对偶联物进行定性和定量分析并计算偶联比。最后,用制备的偶联物免疫Ba1B/c小鼠,同时用间接ELISA法检测抗体水平,并对小鼠进行攻毒保护试验,评价免疫效果。结果显示,产生血清8型荚膜多糖的金黄色葡萄球菌在加有1%乳糖、2%NaCl和10%胎牛血清的哥伦比亚固体培养基上生长最旺盛。用此条件培养的菌株提纯的荚膜多糖,其粗糖的纯度为34.43%,精制多糖的纯度为79.68%。核酸和蛋白的含量为0.1365mg/mL。用化学方法制备的荚膜多糖-蛋白质偶联物在其质量比为1：2时可证明偶联成功,计算得到偶联比为1：1.89。采用间接ELISA法检测抗体水平,二免后7天,偶联物组可产生抗体,且抗体效价最高可达1：6400,说明偶联物刺激小鼠产生了免疫应答。阴性对照组和单独多糖组均没有抗体产生,而单独融合蛋白组在二免后产生了抗体反应,但抗体效价较低,最高时仅为1：100。本实验对三免后两周的小鼠进行攻毒保护,结果表明,偶联物组的攻毒保护率最高,可达80%,而阴性对照组、单独多糖组以及单独融合蛋白组的保护率分别为20%、30%和60%,可见荚膜多糖-蛋白质偶联物有较好的免疫原性,达到了本实验的预期目标。
[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.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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