【摘要】： 免疫球蛋白(immunoglobulin)是具有抗體活性的一種糖蛋白,主要存在于血漿中,也見于其他體液、組織和一些分泌液中。人血漿內(nèi)的免疫球蛋白可分為五類,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。而母雞血液中的免疫球蛋白可傳遞到雞卵黃中,并且在雞發(fā)育過程中起保護作用,此種免疫球蛋白稱為雞卵黃免疫球蛋白(IgY)。IgG是存在于雞卵黃中的主要免疫球蛋白,而IgM和IgA則主要存在于血清蛋白中。IgY屬多克隆抗體,多克隆抗體在疾病的診斷和防治方面具有許多特定優(yōu)點,通常一次性大量制備是相當(dāng)困難的,而多次制備,又導(dǎo)致抗體的均一性差。母雞一經(jīng)免疫應(yīng)答后,可從其卵黃中不斷獲得特異性的抗體。IgY純度高、特異性強,不會象其他哺乳動物產(chǎn)生的抗體那樣會發(fā)生交叉血清反應(yīng)或過敏反應(yīng),因而它是最安全的生物制劑,可替代免疫小鼠、豚鼠或兔等哺乳動物所制備的抗體而大量生產(chǎn)。近年來,IgY的研究和應(yīng)用是免疫學(xué)領(lǐng)域的熱點和十分活躍的課題。耐甲氧西林金黃色葡萄球菌是引起院內(nèi)感染的重要致病菌,其致病性強,對多種抗菌藥物耐藥,在治療上十分棘手。本文用耐甲氧西林金黃色葡萄球菌作為抗原免疫母雞制備了抗耐甲氧西林金黃色葡萄球菌IgY,并對其理化性質(zhì)和生物學(xué)活性進行了研究,旨在為其以后在診斷和防治方面的應(yīng)用奠定基礎(chǔ)。 [目的] 觀察雞卵黃中抗耐甲氧西林金黃色葡萄球菌(MRSA)抗體的含量、純度、來源及穩(wěn)定性。 [方法] 用耐甲氧西林金黃色葡萄球菌(MRSA)作為抗原免疫Leghorn雞;通過改良水稀釋法提取卵黃中的IgY;雙紫外光波長測定抗體含量,SDS-PAGE電泳檢測抗體純度;Western blot免疫印跡法測定該抗體來源;ELISA檢測IgY對溫度、酸堿度、胃蛋白酶、胰蛋白酶的穩(wěn)定性。 [結(jié)果] 抗體含量13.3 mg/mL蛋黃液,抗體純度達96%;免疫印跡證明IgY與雞血清中的IgG具有相同的分子量和抗原性;IgY具有良好的熱穩(wěn)定性,對酸堿、胃蛋白酶、胰蛋白酶具有一定的耐受力。 [結(jié)論] 改良水稀釋法能得到高產(chǎn)量、高純度的特異性IgY,而且其有良好的生物學(xué)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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