抗耐甲氧西林金黄色葡萄球菌（MRSA）卵黄免疫球蛋白（IgY）的制备与性质研究
发布时间：2019-05-08 04:50
【摘要】： 免疫球蛋白(immunoglobulin)是具有抗体活性的一种糖蛋白,主要存在于血浆中,也见于其他体液、组织和一些分泌液中。人血浆内的免疫球蛋白可分为五类,即免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)和免疫球蛋白E(IgE),其中IgG是最主要的免疫球蛋白。而母鸡血液中的免疫球蛋白可传递到鸡卵黄中,并且在鸡发育过程中起保护作用,此种免疫球蛋白称为鸡卵黄免疫球蛋白(IgY)。IgG是存在于鸡卵黄中的主要免疫球蛋白,而IgM和IgA则主要存在于血清蛋白中。IgY属多克隆抗体,多克隆抗体在疾病的诊断和防治方面具有许多特定优点,通常一次性大量制备是相当困难的,而多次制备,又导致抗体的均一性差。母鸡一经免疫应答后,可从其卵黄中不断获得特异性的抗体。IgY纯度高、特异性强,不会象其他哺乳动物产生的抗体那样会发生交叉血清反应或过敏反应,因而它是最安全的生物制剂,可替代免疫小鼠、豚鼠或兔等哺乳动物所制备的抗体而大量生产。近年来,IgY的研究和应用是免疫学领域的热点和十分活跃的课题。耐甲氧西林金黄色葡萄球菌是引起院内感染的重要致病菌,其致病性强,对多种抗菌药物耐药,在治疗上十分棘手。本文用耐甲氧西林金黄色葡萄球菌作为抗原免疫母鸡制备了抗耐甲氧西林金黄色葡萄球菌IgY,并对其理化性质和生物学活性进行了研究,旨在为其以后在诊断和防治方面的应用奠定基础。 [目的] 观察鸡卵黄中抗耐甲氧西林金黄色葡萄球菌(MRSA)抗体的含量、纯度、来源及稳定性。 [方法] 用耐甲氧西林金黄色葡萄球菌(MRSA)作为抗原免疫Leghorn鸡;通过改良水稀释法提取卵黄中的IgY;双紫外光波长测定抗体含量,SDS-PAGE电泳检测抗体纯度;Western blot免疫印迹法测定该抗体来源;ELISA检测IgY对温度、酸碱度、胃蛋白酶、胰蛋白酶的稳定性。 [结果] 抗体含量13.3 mg/mL蛋黄液,抗体纯度达96%;免疫印迹证明IgY与鸡血清中的IgG具有相同的分子量和抗原性;IgY具有良好的热稳定性,对酸碱、胃蛋白酶、胰蛋白酶具有一定的耐受力。 [结论] 改良水稀释法能得到高产量、高纯度的特异性IgY,而且其有良好的生物学活性。
[Abstract]:Immunoglobulin (immunoglobulin) is a glycoprotein with antibody activity, mainly in plasma, but also in other body fluids, tissues and some secretions. Human plasma immunoglobulin can be divided into five categories, namely immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE),. Among them, IgG is the most important immunoglobulin. The immunoglobulin in the blood of the hen can be transmitted to the yolk and play a protective role in the development of the chicken. This immunoglobulin is called the egg yolk immunoglobulin (IgY), which is the main immunoglobulin in the chicken yolk. IgM and IgA are mainly found in serum proteins. IGY is a polyclonal antibody. Polyclonal antibody has many specific advantages in the diagnosis, prevention and treatment of diseases, and it is usually very difficult to prepare a large number of antibodies at once, but it is prepared many times, and the polyclonal antibodies have many advantages in the diagnosis and prevention of diseases. It also leads to poor uniformity of antibodies. After immune response, hens can constantly obtain specific antibodies from their yolks. IGY is high in purity and specificity and does not cause cross-serological or allergic reactions like antibodies produced by other mammals. Therefore, it is the safest biological preparation, which can replace the antibody produced by mice, guinea pigs or rabbits and other mammals. In recent years, the research and application of IgY is a hot and very active topic in the field of immunology. Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing nosocomial infection. Its pathogenicity is strong and it is very difficult to treat a variety of antibiotics. Methicillin-resistant Staphylococcus aureus IgY, was prepared from hens immunized with methicillin-resistant Staphylococcus aureus and its physical and chemical properties and biological activity were studied. The aim is to lay a foundation for its future application in diagnosis and prevention. [objective] to observe the content, purity, origin and stability of anti-methicillin-resistant Staphylococcus aureus (MRSA) antibody in chicken yolk. [methods] Methicillin-resistant Staphylococcus aureus (MRSA) was used as antigen to immunize Leghorn chickens, IgY; in yolk was extracted by modified water dilution method, the content of antibody was determined by double ultraviolet light, and the purity of antibody was detected by SDS-PAGE electrophoresis. The source of the antibody was determined by Western blot and the stability of IgY to temperature, pH, pepsin and trypsin was detected by ELISA. [results] the content of antibody in egg yolk liquid was 13.3 mg/mL, the purity of antibody was 96%, the immunoblotting showed that IgY had the same molecular weight and antigenicity as IgG in chicken serum. IgY has good thermal stability and tolerance to acid-base, pepsin and trypsin. [conclusion] the modified water dilution method can obtain high yield, high purity specific IgY, and it has good biological activity.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2471634
[Abstract]:Immunoglobulin (immunoglobulin) is a glycoprotein with antibody activity, mainly in plasma, but also in other body fluids, tissues and some secretions. Human plasma immunoglobulin can be divided into five categories, namely immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE),. Among them, IgG is the most important immunoglobulin. The immunoglobulin in the blood of the hen can be transmitted to the yolk and play a protective role in the development of the chicken. This immunoglobulin is called the egg yolk immunoglobulin (IgY), which is the main immunoglobulin in the chicken yolk. IgM and IgA are mainly found in serum proteins. IGY is a polyclonal antibody. Polyclonal antibody has many specific advantages in the diagnosis, prevention and treatment of diseases, and it is usually very difficult to prepare a large number of antibodies at once, but it is prepared many times, and the polyclonal antibodies have many advantages in the diagnosis and prevention of diseases. It also leads to poor uniformity of antibodies. After immune response, hens can constantly obtain specific antibodies from their yolks. IGY is high in purity and specificity and does not cause cross-serological or allergic reactions like antibodies produced by other mammals. Therefore, it is the safest biological preparation, which can replace the antibody produced by mice, guinea pigs or rabbits and other mammals. In recent years, the research and application of IgY is a hot and very active topic in the field of immunology. Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing nosocomial infection. Its pathogenicity is strong and it is very difficult to treat a variety of antibiotics. Methicillin-resistant Staphylococcus aureus IgY, was prepared from hens immunized with methicillin-resistant Staphylococcus aureus and its physical and chemical properties and biological activity were studied. The aim is to lay a foundation for its future application in diagnosis and prevention. [objective] to observe the content, purity, origin and stability of anti-methicillin-resistant Staphylococcus aureus (MRSA) antibody in chicken yolk. [methods] Methicillin-resistant Staphylococcus aureus (MRSA) was used as antigen to immunize Leghorn chickens, IgY; in yolk was extracted by modified water dilution method, the content of antibody was determined by double ultraviolet light, and the purity of antibody was detected by SDS-PAGE electrophoresis. The source of the antibody was determined by Western blot and the stability of IgY to temperature, pH, pepsin and trypsin was detected by ELISA. [results] the content of antibody in egg yolk liquid was 13.3 mg/mL, the purity of antibody was 96%, the immunoblotting showed that IgY had the same molecular weight and antigenicity as IgG in chicken serum. IgY has good thermal stability and tolerance to acid-base, pepsin and trypsin. [conclusion] the modified water dilution method can obtain high yield, high purity specific IgY, and it has good biological activity.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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