抗A族乙型溶血性链球菌卵黄抗体的制备及生物活性检测

发布时间：2018-07-21 17:10

【摘要】：乙型溶血性链球菌为链球菌感染中的主要致病菌,可根据其细胞壁中特异性抗原(多糖体)的不同,分为A～H、K～T共18个族；又根据其表面蛋白质M、K、T、S抗原的不同,将各族细菌分为若干个血清型。对人类有致病力者90%为A族(Group Aβ-streptococcus hemolyticus),具有致病性强的特点,常引起人类和动物的多种疾病如皮肤、皮下组织的化脓性炎症、呼吸道感染、流行性咽炎的爆发性流行以及新生儿败血症、细菌性心内膜炎、猩红热和风湿热、肾小球肾炎等变态反应。目前,A族乙型溶血性链球菌(Group Aβ-streptococcus hemolyticus)引发疾病多采用大环内酯类抗生素和β-内酰胺类抗生素进行治疗,但是在当前耐药菌不断增加的情况下,A族乙型溶血性链球菌的药物敏感性越来越得到关注,使得寻找一种安全、高效的抗生素替代品成为一个亟待解决的问题。 特异性卵黄免疫球蛋白(Egg yolk immunoglobulin, IgY)可治疗由细菌或病毒引起的疾病,具有安全无残留、不产生抗药性、服用简便等优点,是一种具有广阔应用前景的抗生素替代品。本研究以用超声波破碎法制备的可溶性的A族乙型溶血性链球菌(Group Aβ-streptococcus hemolyticus)为抗原,免疫产卵母鸡获得特异性IgY; IgY经分离、纯化后,考马斯亮蓝G-250法测定抗体含量,SDS-PAGE电泳检测抗体纯度。间接ELISA测定抗体的效价。并用琼脂扩散,体外抑菌,免疫荧光等实验对特异性IgY在体外对A族乙型溶血性链球菌的生物活性进行了研究。 结果显示提取的抗体含量平均10.8±0.87 mg/蛋,抗体纯度达92.5%。ELISA测定抗A族乙型溶血性链球菌鸡卵黄抗体的效价,于初免后40天左右达到最高值1：5120,并可持续30天。抗体在温度低于75℃、pH4-9具有良好的稳定性,抗体在pH为2和3时经过胃蛋白酶处理短时间内迅速失活,-20℃为其适宜贮藏温度,且反复冻融不影响其活性。免疫荧光结果显示,特异性IgY与A族乙型溶血性链球菌有较强的结合作用。体外抑菌实验结果显示当抗体浓度大于10 mg/mL,特异性IgY能显著抑制抗A族乙型溶血性链球菌的生长,随着抗体浓度的增加抑菌效果越明显,最低抑菌浓度为25 mg/mL。研究结果表明,实验得到的抗A族乙型溶血性链球菌IgY,产量、纯度、效价和特异性高,稳定性好,在体外能够特异性地与抗A族乙型溶血性链球菌结合。为特异性IgY在A族乙型溶血性链球菌引起的疾病的防治应用研究奠定了基础。
[Abstract]:Hemolytic streptococcus B is the main pathogenic bacteria in streptococcus infection. According to the specific antigen (polysaccharide body) in its cell wall, it can be divided into 18 groups, and according to the different antigen of its surface protein, The bacteria were divided into several serotypes. 90% of the patients with pathogenicity to human are group A (Group A 尾 -streptococcus hemolyticus), which often cause many diseases in human and animal such as skin, suppurative inflammation of subcutaneous tissue, respiratory tract infection. Outbreak of epidemic pharyngitis and neonatal septicemia, bacterial endocarditis, scarlet fever and rheumatic fever, glomerulonephritis and other allergic reactions. At present, group A group B hemolytic streptococcus hemolyticus disease is mostly treated with macrolide antibiotics and 尾 -lactam antibiotics. However, the drug sensitivity of Group A hemolytic Streptococcus B has been paid more and more attention under the condition of increasing drug resistance, which makes it an urgent problem to find a safe and efficient substitute for antibiotics. Egg yolk immunoglobulin (IGY) is a promising alternative to antibiotics for the treatment of diseases caused by bacteria or viruses. In this study, the soluble group A 尾 -streptococcus hemolyticus (Group A 尾 -streptococcus hemolyticus) prepared by ultrasonic fragmentation method was used as antigen to immunize ovipositing hens with specific IgY; IGY was isolated and purified. The antibody content was determined by Coomassie brilliant blue G-250 method and the purity of antibody was detected by SDS-PAGE electrophoresis. The titer of antibody was determined by indirect Elisa. The bioactivity of specific IGY against group A hemolytic streptococcus B in vitro was studied by Agar diffusion, bacteriostasis in vitro and immunofluorescence. The results showed that the average content of the extracted antibody was 10.8 卤0.87 mg/ eggs, and the purity of the antibody was 92.5. Elisa was used to determine the titer of yolk antibody against group A hemolytic streptococcus hemolyticus. The titer of egg yolk antibody against group A hemolytic streptococcus was 1: 5120 about 40 days after the first immunization, and could last for 30 days. The antibody had good stability at temperature below 75 鈩，

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