抗沙门氏菌IgY抗体的制备及其特性分析

发布时间：2018-01-05 05:22

本文关键词：抗沙门氏菌IgY抗体的制备及其特性分析　出处：《第四军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的沙门氏菌是一大群寄生于人类和动物肠道中的革兰氏阴性菌,属于肠道菌群,是引起人类腹泻的主要病原体。卵黄免疫球蛋白IgY (Immunoglobulin yolk),也称为卵黄抗体。在结构和免疫特性上不同于哺乳动物的免疫球蛋白IgG,与IgG相比具有物理化学稳定等多种优点。因此,IgY作为替代传统哺乳动物抗体的潜力受到广泛的关注。本文用减毒沙门氏菌作为抗原免疫母鸡制备成抗沙门氏菌的IgY (AS-IgY),并对其理化性质和生物学活性进行分析,这为沙门氏菌的治疗和预防性抗体的研制奠定基础。方法 1.免疫原为减毒沙门氏菌菌株Ty21a型,为沙门氏菌K抗原类。 2.6月龄雌性健康产蛋京白鸡,体重1300±100g,每周产蛋5-6枚,正常饲养1周后免疫。 3.用减毒沙门氏菌菌株Ty21a作为抗原,按照109个细胞/只鸡免疫进行接种;初次免疫时加入等量的福氏完全佐剂,分别于双翅膀皮下、左右背部皮下进行点注射。以后每2周免疫1次,共3次,但不加佐剂,剂量、部位同初次免疫。末次免疫后,每2个月强化免疫1次,使抗体维持在一个较高的水平。免疫1周后,收集鸡蛋并做好标记,4℃保存备用。 4.收集免疫前及免疫后第15、20、25、30、35、40、45天的鸡蛋为研究对象。AS-IgY抗体的纯化采用去除蛋清,收集卵黄液经过蒸馏水粗提,50%饱和硫酸铵盐析,透析、以及聚乙二醇浓缩等步骤得到纯化的IgY抗体。 5.通过蛋白质定量后,再经过SDS-PAGE、Western blot和ELISA等方法对所纯化抗体的特性进行鉴定。 6.通过体外抑菌实验验证其生物学活性。结果免疫后10天左右,即可从卵黄中检测出特异性IgY抗体,30天左右抗体量达到高峰,每个卵黄可提取100～200mg抗体。该抗体具有较强的热稳定性和耐酸性,效价可持续2～6个月。还原性SDS-PAGE显示该抗体呈现两条带,分子量约20KD和70KD。ELISA结果显示,IgY与链球菌、白色念珠菌、大肠杆菌、金黄色葡萄球菌等其他抗原之间无交叉反应,并可在体外抑制沙门氏菌的生长。结论 1.已成功制备出高纯度的抗沙门氏菌IgY抗体(AS-IgY)。 2.通过SDS-PAGE、Western blot以及ELISA等对其理化性质的分析结果表明,此抗体是一种耐酸、耐热、产量高并且制备简便的新型抗体。 3.AS-IgY抗体在体外能够抑制沙门氏菌的生长,与无关抗原无交叉反应。 4.AS-IgY抗体有可能为沙门氏菌感染的治疗和预防提供新的研究手段。
[Abstract]:objective
Salmonella is a group of gram negative bacteria that colonize the intestine of human and animal, belonging to gut bacteria is the major pathogen causing human diarrhea. Yolk immunoglobulin IgY (Immunoglobulin yolk), also known as egg yolk antibody. The structures and immunological characteristics different from mammalian immunoglobulin IgG, compared with IgG it has several advantages of physical and chemical stability. Therefore, IgY as an alternative to traditional mammalian antibody potential has been widely concerned. In this paper, using attenuated Salmonella as immune antigen hen prepared against Salmonella IgY (AS-IgY), and the physicochemical properties and biological activity were analyzed, which laid the foundation for treatment and the prevention of Salmonella antibody development.
Method
1. immunogen was attenuated Salmonella strain Ty21 a, K for Salmonella antigens.
2.6 month old female healthy laying Jingbai chicken, weight 1300 + 100g, weekly egg 5-6, normal feeding 1 weeks after immunization.
3. with the attenuated Salmonella typhimurium strain Ty21a as antigen in 109 cells / chicken immune vaccination; the first immunization when adding the same amount of complete Freund's adjuvant, respectively in the wings subcutaneous, subcutaneous injection of about 2 weeks after each immunization for 1 times, a total of 3 times, but not kasano agent. Parts of the same dose, the first immunization. After the last immunization, immunization for 1 times every 2 months, the antibody is maintained at a high level. 1 weeks after immunization, egg collection and mark, 4 degrees preservation reserve.
4., we collected the eggs before and after 15,20,25,30,35,40,45 days of immunization. The purification of.AS-IgY antibodies was achieved by removing egg white, collecting yolk solution, distilling water, crude 50%, ammonium sulfate salting out, dialysis and polyethylene glycol concentration, and then purified IgY antibody.
5. after quantitation of protein, the characteristics of the purified antibody were identified by SDS-PAGE, Western blot and ELISA.
6. the biological activity was verified by the bacteriostasis experiment in vitro.
Result
About 10 days after immunization, which can detect the specific IgY antibody from egg yolk, 30 days the amount of antibody reached the peak, each egg yolk.contains 100 ~ 200mg antibody. It has high thermal stability and acid resistance of the antibody titer continued for 2 to 6 months. Reduction of SDS-PAGE showed that the antibody showed two bands the molecular weight of about 20KD, and the results of 70KD.ELISA showed that IgY and Candida albicans, Streptococcus, Escherichia coli, no cross reaction between Staphylococcus aureus and other antigens, and can inhibit the growth of Salmonella in vitro.
conclusion
1. the high purity of Salmonella resistant IgY antibody (AS-IgY) has been successfully prepared.
2. through SDS-PAGE, Western blot and ELISA analysis of its physical and chemical properties, it is shown that the antibody is an acid, heat resistant, high yield and simple preparation of new antibodies.
3.AS-IgY antibodies can inhibit the growth of Salmonella in vitro, and have no cross reaction with unrelated antigens.
4.AS-IgY antibodies may provide new research methods for the treatment and prevention of Salmonella infection.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】