【摘要】：乙型溶血性鏈球菌為鏈球菌感染中的主要致病菌,可根據(jù)其細胞壁中特異性抗原(多糖體)的不同,分為A～H、K～T共18個族；又根據(jù)其表面蛋白質(zhì)M、K、T、S抗原的不同,將各族細菌分為若干個血清型。對人類有致病力者90%為A族(Group Aβ-streptococcus hemolyticus),具有致病性強的特點,常引起人類和動物的多種疾病如皮膚、皮下組織的化膿性炎癥、呼吸道感染、流行性咽炎的爆發(fā)性流行以及新生兒敗血癥、細菌性心內(nèi)膜炎、猩紅熱和風濕熱、腎小球腎炎等變態(tài)反應(yīng)。目前,A族乙型溶血性鏈球菌(Group Aβ-streptococcus hemolyticus)引發(fā)疾病多采用大環(huán)內(nèi)酯類抗生素和β-內(nèi)酰胺類抗生素進行治療,但是在當前耐藥菌不斷增加的情況下,A族乙型溶血性鏈球菌的藥物敏感性越來越得到關(guān)注,使得尋找一種安全、高效的抗生素替代品成為一個亟待解決的問題。 特異性卵黃免疫球蛋白(Egg yolk immunoglobulin, IgY)可治療由細菌或病毒引起的疾病,具有安全無殘留、不產(chǎn)生抗藥性、服用簡便等優(yōu)點,是一種具有廣闊應(yīng)用前景的抗生素替代品。本研究以用超聲波破碎法制備的可溶性的A族乙型溶血性鏈球菌(Group Aβ-streptococcus hemolyticus)為抗原,免疫產(chǎn)卵母雞獲得特異性IgY; IgY經(jīng)分離、純化后,考馬斯亮藍G-250法測定抗體含量,SDS-PAGE電泳檢測抗體純度。間接ELISA測定抗體的效價。并用瓊脂擴散,體外抑菌,免疫熒光等實驗對特異性IgY在體外對A族乙型溶血性鏈球菌的生物活性進行了研究。 結(jié)果顯示提取的抗體含量平均10.8±0.87 mg/蛋,抗體純度達92.5%。ELISA測定抗A族乙型溶血性鏈球菌雞卵黃抗體的效價,于初免后40天左右達到最高值1：5120,并可持續(xù)30天。抗體在溫度低于75℃、pH4-9具有良好的穩(wěn)定性,抗體在pH為2和3時經(jīng)過胃蛋白酶處理短時間內(nèi)迅速失活,-20℃為其適宜貯藏溫度,且反復(fù)凍融不影響其活性。免疫熒光結(jié)果顯示,特異性IgY與A族乙型溶血性鏈球菌有較強的結(jié)合作用。體外抑菌實驗結(jié)果顯示當抗體濃度大于10 mg/mL,特異性IgY能顯著抑制抗A族乙型溶血性鏈球菌的生長,隨著抗體濃度的增加抑菌效果越明顯,最低抑菌濃度為25 mg/mL。研究結(jié)果表明,實驗得到的抗A族乙型溶血性鏈球菌IgY,產(chǎn)量、純度、效價和特異性高,穩(wěn)定性好,在體外能夠特異性地與抗A族乙型溶血性鏈球菌結(jié)合。為特異性IgY在A族乙型溶血性鏈球菌引起的疾病的防治應(yīng)用研究奠定了基礎(chǔ)。
[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 鹵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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