双价抗蛇毒鸡卵黄抗体的制备及其特性研究

发布时间：2018-05-24 06:22

本文选题：眼镜蛇 + 蝰蛇　；参考：《广州医学院》2009年硕士论文

【摘要】： 背景:抗蛇毒抗体根据来源可分为抗蛇毒血清(antivenene,AV)及抗蛇毒鸡卵黄抗体(immunoglobulins in yolk, IgY)两种,前者从哺乳动物的血液中分离而得,后者系从禽类的卵黄中提取。自20世纪90年代以来,国外学者已从鸡卵黄中制备出了抗cobra、Russell's viper、Saw-scaled viper,B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca和N. mossambica等数种IgY,并能有效中和相应蛇毒[1,2,3]。在国内,抗眼镜王蛇[4]、抗蝮蛇[5]、抗眼镜蛇[6,7]和抗蝰蛇毒IgY[8]的制备及其特性研究亦有报道。与从其他哺乳动物(如鼠、兔、猪、马、牛等)血液中获得抗体相比,卵黄抗体具有无法比拟的优越性[9,10](Schade et al.1992; Larsson et al.,1991):不与蛋白G或蛋白A结合,不会激活补体系统,不与类风湿因子结合,避免了免疫检测中的假阴性和假阳性问题;鸡蛋来源方便,生产成本低,适于大规模生产;由免疫母鸡所生的鸡蛋黄中只有一种抗体,均一性好,理化性质稳定,耐酸,耐热,易于保存,运输和使用均很方便。另外鸡与哺乳动物种系关系远,少量刺激时即容易产生强免疫反应。研制一种针对华南地区多种剧毒蛇的通用蛇伤防、治特效制剂—多价AV,可在无需明确蛇伤的毒蛇种类情况下立即使用,以赢得有效的最佳治疗时机,对避免严重病变的发生和降低死亡率有着重要作用。在本实验室先前制备有单价抗眼镜蛇及抗蝰蛇毒IgY,本实验拟进一步研制双价抗蛇毒鸡卵黄抗体,为蛇伤救助提供新的途径。目的:通过制备双价抗眼镜蛇、蝰蛇毒鸡卵黄抗体,检测其生物活性,并观察其以口服制剂的形式对眼镜蛇、蝰蛇伤小鼠的保护作用,为蛇伤的预防和治疗提供一种新的方法,为替代马源性抗蛇毒血清奠定基础。方法: 一、双价抗蛇毒IgY的制备与提取 1、双价抗蛇毒IgY的制备制订合适的免疫方案,利用本所专利——蛇毒取毒器制备抗原眼镜蛇、蝰蛇毒,分别与福氏佐剂按1:1(V/V)置自制的乳化器充分乳化,初次免疫采用福氏完全佐剂乳化蛇毒,以后均用福氏不完全佐剂乳化蛇毒。每种抗原在初次免疫后的第三周、第五周各进行1次加强免疫,免疫剂量间歇、逐渐加量。两种单一抗原按顺序依次交替注入单只鸡体内(双翼、胸、腹部和背部皮下及肌肉多位点注射),注入间隔周期为一周。 2、双价抗蛇毒IgY的检测双向免疫扩散和免疫电泳鉴定卵黄中的特异性抗体;SDS-PAGE测定抗体产物的纯度;以间接ELISA法检测特异性IgY抗体效价的动态变化以及IgY在血浆内的效价变化。 3、双价抗蛇毒IgY的提取采用本实验过程中发明的卵黄分离收集器从卵清中分离出卵黄,用水稀释法提取IgY抗体。二、双价抗蛇毒IgY的生物特性研究 1、利用双向免疫扩散法测定抗蛇毒IgY与中国常见10种蛇毒的交叉免疫反应及其反应强度。2、眼镜蛇、蝰蛇毒对卵黄膜均有不同程度的溶膜作用,用自制的卵黄膜溶膜检测仪观察双价抗蛇毒IgY对蛇毒溶膜活性的中和作用。 3、双价抗蛇毒IgY分别与眼镜蛇、蝰蛇毒在体外中和,腹腔注射小鼠,观察双价抗蛇毒IgY对蛇毒致死活性的中和作用。三、双价抗蛇毒IgY的相关动物实验 1、双价抗蛇毒IgY按高、中、低三个浓度灌胃小鼠,在不同时间点杀鼠取血分离血浆,用间接ELISA法检测灌胃小鼠血浆中双价抗蛇毒IgY的效价。 2、双价抗蛇毒IgY按高、中、低三个浓度灌胃小鼠,在不同时间点分别为小鼠腹腔注射眼镜蛇、蝰蛇毒,检测口服制剂双价抗蛇毒IgY对小鼠的保护作用。四、统计采用t检验,SPSS软件等统计分析实验数据。结果: 1、利用蛇毒取毒器制备的蛇毒活性高,含杂质量少。用眼镜蛇、蝰蛇毒粗毒、初次小剂量免疫后,卵黄中第9天即可检测到特异性卵黄抗体。经间歇、逐渐加量加强免疫,抗体效价持续增高,达到较高水平后,能在较长时间内一直维持高效价。 2、在实验过程中制备了卵黄分离收集器(专利号:2006200559807.3),利用此仪器收集试验用卵黄,卵清、卵黄分离度高,卵黄膜光洁、完整,卵清残留量少,有利于进一步的分离纯化。 3、水稀释法去脂效果好,若进一步经硫酸铵盐析后,蛋白回收率约为40%。如能进一步优化条件,经嗜硫色谱、疏水色谱及凝胶层析等方法纯化抗体,有望得到纯度更高的抗体。 4、经双向免疫扩散法测定,所制双价抗蛇毒IgY与舟山眼镜蛇、眼镜王蛇反应最明显,反应沉淀线最清晰,与眼镜蛇亚科和蝰蛇亚科的部分蛇毒有强弱不等的交叉反应;与蝮蛇亚科的蝮蛇、尖吻蝮等部分蛇毒无明显交叉反应。 5、根据卵黄膜溶膜检测仪的时间结果证明,一定量的水提物抗蛇毒IgY完全能对抗眼镜蛇、蝰蛇毒对卵黄膜的溶膜作用。 6、体外中和实验表明,一定浓度的水提物抗蛇毒IgY可以完全保护实验小鼠免受2倍LD50眼镜蛇、蝰蛇毒的攻击。 7、不同浓度剂量的抗蛇毒IgY能对眼镜蛇、蝰蛇伤小鼠有不同程度的保护作用;当IgY剂量足够高时,能够完全保护小鼠免受眼镜蛇、蝰蛇毒的攻击。结论: 1、以眼镜蛇、蝰蛇毒粗毒免疫莱杭母鸡,经初次小剂量、间歇、逐渐加量加强免疫,可持续数周获得含有高效价、特异性抗体的鸡卵黄抗体。 2、自制的卵黄分离收集器可批量分离卵黄,卵黄膜溶膜检测仪可以精确地检测蛇毒或其他毒素的溶膜活性,在工业化生产IgY方面有较好的应用前景。 3、经水稀释法提取分离抗体,纯度可达电泳纯,抗体活性强,回收率高、活性好,方法经济、简单易行。 4、口服制剂的抗蛇毒IgY,若能增加其免疫抗原的种类,抗体纯化程度更高、效价更高,则其保护作用和应用范围将得以极大扩展。
[Abstract]:Background: the anti snake venom antibody can be divided into two kinds, antivenene (AV) and anti venom chicken egg yolk antibody (immunoglobulins in yolk, IgY). The former is isolated from the blood of mammalian and the latter is extracted from the yolk of poultry. Since 1990s, foreign scholars have prepared the anti cobra, Ru from the chicken egg yolk. Ssell's viper, Saw-scaled viper, B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and corresponding venom are effective in the domestic, anti Cobra king snake, anti Agkistrodon snake, the preparation and characteristics of the anti Cobra and viper venom. Compared to the antibodies in the blood (such as rats, rabbits, pigs, horses and cattle), the yolk antibody has an incomparable superiority [9,10] (Schade et al.1992; Larsson et al., 1991): not combining with protein G or protein A, not activating the complement system, not combining with the rheumatoid factor, avoiding the false negative and false positive problems in the immunoassay; the egg source side The production cost is low and suitable for large-scale production; the egg yolk produced by the immune hen has only one antibody, with good homogenization, stable physicochemical properties, acid resistance, heat resistance, easy preservation and convenient transportation and use. In addition, the chicken is far from the mammalian species, and a strong immune response is easily produced when a small amount of spiny is stimulated. A kind of Southern China area is developed. A variety of venomous snakes are common snakes and prevention, treatment preparation, multivalent AV, can be used immediately in the case of venomous snakes without the need for snakebite, in order to win the best time for the best treatment, to avoid the occurrence of serious diseases and to reduce the mortality rate. In this laboratory, we have first prepared the monovalent anti Cobra and the viper IgY, which was first prepared in our laboratory. The aim of the experiment is to further develop the bivalent anti snake egg yolk antibody, so as to provide a new way for rescuing snakebite.
Objective: to prepare bivalent anti Cobra and viper chicken egg yolk antibody and to detect its biological activity, and to observe the protective effect of its oral preparation on Cobra and viper injured mice, and provide a new method for the prevention and treatment of snake wound, and lay a foundation for replacing horse derived serpent sera.
Method:
Preparation and extraction of bivalent anti snake venom IgY
1, the preparation of a bivalent anti snake venom IgY for preparation of the appropriate immunization scheme, using the patent - snake venom device to prepare the antigenic cobra, viper venom, fully emulsified the emulsifier self-made by 1:1 (V/V) and emulsified snake venom with FF's complete adjuvant in the first immunization, and then emulsified snake venom with FF's incomplete adjuvant. At the first third weeks after the first immunization, the immunization was carried out in 1 times at the fifth week, and the immunization dose was intermittent and gradually added. The two single antigens were alternately injected into the single chicken in order (double wing, chest, abdomen and back subcutaneous and muscle multipoint injection), and the interval of injection interval was one week.
2, bivalent anti snake venom IgY was detected by bi-directional immuno diffusion and immunoelectrophoresis to identify specific antibodies in the yolk; SDS-PAGE was used to determine the purity of the antibody products; the dynamic changes in the titer of specific IgY antibodies and the change in the titer of IgY in the plasma were detected by indirect ELISA.
3, the extraction of bivalent anti snake venom IgY was separated from egg white by yolk separator. The IgY antibody was extracted by water dilution.
Study on biological characteristics of two, bivalent anti snake venom IgY
1, the cross immunization of anti snake venom IgY and 10 common snake venom in China and its reaction intensity.2 were measured by bi-directional immuno diffusion method. The cobra and viper venom had different degree of membrane effect on the yolk membrane. The neutralization effect of bivalent anti snake venom IgY on the snake venom was observed by the homemade yolk membrane detector.
3, bivalent anti snake venom IgY was neutralized with cobra, viper venom in vitro, and intraperitoneal injection respectively. The neutralization effect of IgY on the lethal activity of snake venom was observed.
Three, bivalent anti venom IgY related animal experiments
1, bivalent anti snake venom IgY was administered to mice at high, medium and low concentrations at three concentrations. Blood separation plasma was taken at different time points, and the titer of bivalent anti snake venom IgY in the plasma of gavage mice was detected by indirect ELISA.
2, bivalent anti snake venom IgY was intragastric mice at high, middle and low three concentrations. The mice were intraperitoneally injected with cobra snakes and viper venom at different time points, and the protective effect of bivalent anti snake venom IgY on mice was detected.
Four, statistics were analyzed by t test and SPSS software.
Result:
1, snake venom has high activity and less impurity.
After the initial small dose of immunization, the specific yolk antibody can be detected in the yolk for ninth days. After the intermittent, the immunization is gradually added, the titer of the antibody continues to increase, and the high level can be maintained for a long time, and the effective price can be maintained for a long time.
2, the egg yolk separation collector (patent number: 2006200559807.3) was prepared during the experiment. Using this instrument, the egg yolk, egg white, the yolk separation degree was high, the yolk membrane was clean, the yolk membrane was clean, and the residual amount of egg white was less, which was beneficial to further separation and purification.
3, the water dilution method has good effect on degreasing. If further ammonium sulfate is salted out, the recovery rate of protein is about 40%., if the conditions can be further optimized, the antibody is purified by the method of sulfur eosinophilic chromatography, hydrophobic chromatography and gel chromatography, and the higher purity of antibody is expected.
4, by the bi-directional immuno diffusion method, the bivalent anti snake venom IgY has the most obvious reaction with the Zhoushan Cobra and the king cobra. The reaction precipitation line is the clearest, and the cross reaction is not equal to the venom of the cobra subfamily and viper subfamily, and the venom of the Agkistrodon hutpistrodon hkhaghistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackkistrodon ackkistrodon ackistrodon hkhaghagi Agkistrodon acutus.
5, according to the time results of the yolk membrane dissolved film detector, a certain amount of water extract can protect against snake venom IgY.
6, in vitro neutralization experiments showed that a certain concentration of water extracts against snake venom IgY could completely protect the experimental mice from 2 times the attack of LD50 Cobra and viper.
7, the anti snake venom IgY with different concentrations can have different protective effects on the cobra and viper injured mice. When the dose of IgY is high enough, it can completely protect the mice from the attack of the cobra and viper venom.
1, the Brunei hens were immunized with cobra and viper venom. After the first small dose, intermittent, and gradually increased immunity, the chicken yolk antibody with high effective price and specific antibody was obtained for several weeks.
2, the homemade yolk separation collector can separate the yolk in batch, and the yolk membrane detector can accurately detect the membrane activity of the snake venom or other toxin. It has a good application prospect in the industrial production of IgY.
3, the extraction and isolation of antibodies by water dilution method can achieve the purity of electrophoresis, strong antibody activity, high recovery rate and good activity. The method is economical and simple.
4, the protective effect and application range of the antivenom IgY of oral preparation will be greatly expanded if it can increase the type of its immune antigen, the degree of antibody purification is higher and the titer is higher.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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