【作者】 甄宇红；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 奶牛乳腺炎是一种给奶牛养殖业造成巨大经济损失的疾病,主要采用抗生素治疗。抗生素疗法会引起动物体内甚至牛奶中药物残留和细菌耐药性的增强,间接危害人类健康。因此,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。特异性卵黄抗体(Egg yolk immunoglobulin,IgY)可治疗由细菌或病毒感染引起的疾病,具有安全无残留、不产生抗药性、价廉易得等优点,是一种具有广阔应用前景的抗生素替代品。本研究制备了抗奶牛乳腺炎主要致病菌——金黄色葡萄球菌和大肠杆菌特异性卵黄抗体,考察了抗体的体外抑菌活性、对吞噬细胞的吞噬调理作用及对奶牛乳腺炎的治疗作用。分别以灭活的金黄色葡萄球菌和大肠杆菌免疫蛋鸡制备卵黄抗体。ELISA检测表明,抗体最高效价可达25600,抗体高效价(≥6400)可持续100天;抗原浓度、蛋鸡日龄和佐剂等影响免疫效果。特异性IgY含量随时间变化趋势与抗体效价变化趋势基本一致,每毫升卵黄液中金黄色葡萄球菌和大肠杆菌特异性IgY的最高含量分别可达1.86mg和1.88 mg,总IgY的平均含量分别为9.82 mg和9.95 mg。采用水稀释—盐析—超滤—凝胶过滤技术从卵黄中分离纯化IgY,经超滤、凝胶过滤后抗体纯度分别可达87.09%和94.52%,回收率分别为100.6 mg/egg和27.68 mg/egg。水稀释—盐析—超滤法所得抗体纯度高、产量大,适于大规模分离纯化。体外抑菌实验表明,两种特异性IgY均以剂量依赖方式抑制抗原菌在液体培养基中的生长,大肠杆菌特异性IgY浓度为20 mg/mL,金黄色葡萄球菌特异性IgY浓度为10mg/mL时能完全抑制细菌在液体培养基中的生长。两种特异性IgY对牛奶中分离的5株大肠杆菌和5株金黄色葡萄球菌分别具有生长抑制活性。免疫荧光和免疫电镜结果表明特异性IgY与细菌结合,使细菌表面结构发生改变。细菌经荧光素标记,采用流式细胞技术检测特异性IgY对奶牛体内吞噬细胞噬菌作用的影响。特异性IgY浓度为1 mg/mL时对吞噬细胞的吞噬促进作用最强。该浓度IgY使外周血PMN吞噬金黄色葡萄球菌的吞噬率由60.2%升高到90.5%,乳中PMN吞噬金黄色葡萄球菌的吞噬率由52.95%升高到82.45%;乳中PMN和Mφ吞噬金黄色葡萄球菌的吞噬率分别提高29.5%和27.66%,吞噬大肠杆菌的吞噬率分别提高26.5%和26.36%,IgY对两种细胞的吞噬促进作用差异不明显(p＞0.05)。乳池内分别注射抗金黄色葡萄球菌特异性IgY(200 mg)和青霉素(1000 mg)治疗金黄色葡萄球菌引起的实验性乳腺炎和临床型乳腺炎,6天治疗期内牛奶颜色和奶中凝块得到明显改善;体细胞数和细菌计数显著降低。IgY和青霉素对实验性乳腺炎的治愈率分别为83.3%和66.7%,对临床型乳腺炎的治愈率分别为50%和33.3%。综上所述,本研究成功制备出抗奶牛乳腺炎致病菌特异性IgY,考察了其体内外活性,并证明了其对奶牛乳腺炎的疗效优于青霉素。为特异性IgY替代抗生素治疗奶牛乳腺炎提供了依据。更多还原

【Abstract】 Dairy cow mastitis is one of the main causes of economic losses in dairy cow industry. Antibiotics are used widely in prevention and treatment of mastitis.With the present awareness of the problems associated with antibiotics-resistant and residue of antibiotics in milk,it is urgent to find an alternative to antibiotics for mastitis treatment.Chicken egg yolk immunoglobulin(IgY) provides an inexpensive and effective source of antibodies for the passive immunization of animals.It is a promising alternative for the treatment and prevention of bacteria infections,and has shown to be effective against a number of pathogens.In this study,specific IgY against the primary mastitis-causing pathgoens, Staphylococcus aureus and Escherichia coli,were prepared.The in vitro and in vivo activity of these specific IgY and their opsonization to phagocytes were estimated.Specific IgY were produced by hens immunized with formaldehyde killed S.aureus or E. coli O111 in long-standing immunization response(titer≥6400 for 100 days) with the highest titer of 25600.Several factors affect IgY titer during imunization including concentrations of antigens,days of hens and quaility of adjuvants.The contents of specific IgY in yolk varied at the same trend to that of IgY titer,and the highest contents of specific IgY against S.aureus and E.coli O111 were 1.86 mg/mL and 1.88 mg/mL,the average contents of total IgY were 9.82 mg/mL and 9.95 mg/mL,respectively.IgY was isolated from yolks with a purity of 94.52%and a yield of 27.68 mg/egg by water dilution,salt precipitations,ultrafiltration and gel filtration.The purity decreased to 87.09%,but the yield increased to 100.6 mg/egg without gel filtration.Specific IgY inhibited the growth of bacteria in liquid culture medium in a dose-dependent manner.The growth of S.aureus and E.coli were completely inhibited by 10 mg/mL of S.aureus-IgY and 20 mg/mL of E.coli-IgY,respectively.These specific IgY could also inhibit the growth of five strains of S.aureus and five strains of E.coli isolated from mastitic cows.Immunofluorescence and immunoelectron indicated specific IgY bound with bacteria resulting in structural alteration on cell surface.The opsonization of IgY to phagocytes in cows was detected by flow cytometry.Specific IgY enhanced the phagocytosis of polymorphonuclear neutrophil leukocytes(PMN) isolated from cow blood and milk with an optimal concentration of 1 mg/mL.The phagocytic percents of PMN in blood to S.aureus increased from 60.2%to 90.5%with IgY opsonizing,that of PMN in milk increased from 52.95%to 82.45%.The phagocytosis of PMN and macrophages(Mφ) in milk to S.aureus and E.coli were both enhanced by specific IgY.The increment of phagocytic percents for PMN and Mφto S.aureus were 29.5%and 2.7.66%, those to E.coli were 26.5%and 26.36%,respectively.The opsonization of specific IgY to PMN and Mφwas similar(p＞0.05 ).Treatments for clinical mastitis and experimental mastitis were performed by a 6-day course of intramammary infusion with IgY(200 mg) and penicillin(1000 mg).The milk color and clot recovered to normal form during the therapy course,the milk somatic cell counts and bacterial counts decreased.The cure rates for experimental mastitis by IgY and penicillin were 83.3%and 66.7%,and those for clinical mastitis were 50%and 33.3%,respectively.In conclusion,this study indicated specific IgY has potential to use as an alternative of antibiotics for mastitis therapy.更多还原