禽用免疫磁珠纳米疫苗的制备及初步应用研究
发布时间：2018-11-25 22:30
【摘要】：免疫磁珠(Immunomagnetic Beads,IMB)是表面包被有抗体(或配体)的磁性微球。能特异性吸附抗原(受体)等物质,目前已广泛应用于生物和医学等领域。本研究利用化学共沉淀法合成磁珠,再将抗GST标签蛋白偶联到改性后的磁珠表面,免疫试验动物制备大量鸡抗GST多克隆抗体,与此同时验证其免疫佐剂效应。将GST多抗包被磁珠,制备得到IMB-GSTAb,然后用IMB-GSTAb捕获GST-VP2(禽法氏囊病毒VP2抗原)、GST-HN(禽新城疫HN抗原)融合蛋白,用于免疫实验动物鸡,探索新基因工程纳米疫苗免疫增强作用和免疫保护力。动物实验证明IMB具有一定的免疫增强作用和良好的生物安全性。本研究为寻找一种操作简便、安全有效且价格低廉的新型纳米佐剂提供了参考,同时也为禽主要疾病的疫苗生产及防控提供了新的思路。 1.鸡抗GST多克隆抗体的制备 培养含GST表达载体(pGEX-KG)的大肠杆菌BL21,收获GST蛋白。过Sepharose4B柱子纯化得到的GST蛋白,包被于自制磁珠的表面,免疫2周龄艾维因鸡,ELISA检测血清效价达1.6×104,证明自制磁珠具有免疫增强作用。采血得到鸡血清,按辛酸-硫酸铵法提纯鸡抗GST多克隆抗体。Western blot试验证明自制GST多抗具有生物学活性和特异性。 2. IMB-GSTAb的制备 采用化学共沉淀法合成Fe304纳米粒子,并对其表面进行修饰,使其表面带有-CHO基团,得到平均粒径在50-100nm之间并具有超顺磁性的磁珠,改性后磁珠的浓度为5.5mg/mL 将磁珠与GST抗体偶联制备IMB-GSTAb,用IMB-GSTAb捕获GST-VP2、 GST-HN融合蛋白。通过控制和改变缓冲介质、溶液pH值、反应时间和温度等参数,优化磁珠偶联抗体的条件。通过用紫外分光光度计测定反应前后溶液中蛋白浓度,对磁珠偶联抗体的效果进行评价,确定偶联抗体的最佳条件为0.1MTBS(pH6.0)缓冲液,37℃混匀仪中孵育3h；捕获极值为45-50μg/mg;封闭IMB的最佳条件为5%脱脂奶粉,37℃混匀仪中封闭2h；IMB-GSTAb捕获GST-VP2融合蛋白的最佳反应条件为0.01MPBS(pH7.4)缓冲液,37℃混匀仪中孵育1h,捕获极值为15μg/mg; IMB-GSTAb捕获GST-HN融合蛋白的最佳反应条件为0.01MPBS(Ph6.8)缓冲液,37℃混匀仪中孵育1h,捕获极值为12μg/mg。 3.禽用磁珠纳米疫苗的应用 用捕获GST-VP2(B组)和GST-HN (E组)的IMB免疫1周龄鸡,同时设对照组,分别为GST-VP2组(A组)、GST-HN组(D组)、法氏囊病毒灭活苗组(C组)、新城疫病毒灭活苗组(F组)和PBS组(G组),每组5只,于四免疫后翼下静脉采血,ELISA方法测定血清中抗体水平,结果显示,B组与A组存在极显著差异,与C组无显著差异；E组与D组抗体效价水平存在显著差异,与F组无显著差异。证明自制的磁珠纳米疫苗具有一定的免疫效果。
[Abstract]:Immunomagnetic beads (Immunomagnetic Beads,IMB) are magnetic microspheres coated with antibodies (or ligands). So far, it has been widely used in biology and medicine. In this study, magnetic beads were synthesized by chemical coprecipitation method, and then conjugated to the surface of modified magnetic beads with anti GST label protein. A large number of chicken anti GST polyclonal antibodies were prepared by immunizing animals, and their immune adjuvant effects were verified. IMB-GSTAb, was prepared by encapsulating GST polyantibodies into magnetic beads, and then IMB-GSTAb was used to capture GST-VP2 (avian bursal virus VP2 antigen) and GST-HN (Newcastle disease HN antigen) fusion protein, which was used to immunize chickens in laboratory animals. To explore the immune enhancement and immune protection of new genetic engineering nano-vaccine. Animal experiments have proved that IMB has a certain immune enhancement and good biological safety. This study provides a reference for finding a new type of nano-adjuvant which is simple, safe, effective and inexpensive, and also provides a new idea for vaccine production and prevention and control of major avian diseases. 1. Preparation of chicken anti-GST polyclonal antibody GST protein obtained from Escherichia coli BL21, containing GST expression vector (pGEX-KG). The GST protein purified by Sepharose4B column was coated on the surface of self-made magnetic beads and immunized with 2-week-old Eviin chicken. The titer of serum detected by ELISA was 1.6 脳 10 4, which proved that the self-made magnetic beads had immune enhancement effect. Chicken serum was collected and purified by octanoic acid-ammonium sulfate method. The. Western blot test of polyclonal antibody against chicken GST proved that the self-made GST polyclonal antibody had biological activity and specificity. 2. The Fe304 nanoparticles were synthesized by chemical coprecipitation method and modified on the surface of IMB-GSTAb to obtain magnetic beads with average diameter between 50-100nm and superparamagnetic particles. The concentration of the modified beads is that the magnetic beads are conjugated with GST antibody by 5.5mg/mL to prepare the GST-VP2, GST-HN fusion protein captured by IMB-GSTAb for IMB-GSTAb,. By controlling and changing the buffer medium, solution pH value, reaction time and temperature, the conditions of magnetic bead coupling antibody were optimized. The concentration of protein in the solution before and after reaction was determined by ultraviolet spectrophotometer, and the effect of magnetic bead coupling antibody was evaluated. The optimum conditions of coupling antibody were determined as 0.1MTBS (pH6.0) buffer, incubated in 37 鈩，
本文编号：2357557
[Abstract]:Immunomagnetic beads (Immunomagnetic Beads,IMB) are magnetic microspheres coated with antibodies (or ligands). So far, it has been widely used in biology and medicine. In this study, magnetic beads were synthesized by chemical coprecipitation method, and then conjugated to the surface of modified magnetic beads with anti GST label protein. A large number of chicken anti GST polyclonal antibodies were prepared by immunizing animals, and their immune adjuvant effects were verified. IMB-GSTAb, was prepared by encapsulating GST polyantibodies into magnetic beads, and then IMB-GSTAb was used to capture GST-VP2 (avian bursal virus VP2 antigen) and GST-HN (Newcastle disease HN antigen) fusion protein, which was used to immunize chickens in laboratory animals. To explore the immune enhancement and immune protection of new genetic engineering nano-vaccine. Animal experiments have proved that IMB has a certain immune enhancement and good biological safety. This study provides a reference for finding a new type of nano-adjuvant which is simple, safe, effective and inexpensive, and also provides a new idea for vaccine production and prevention and control of major avian diseases. 1. Preparation of chicken anti-GST polyclonal antibody GST protein obtained from Escherichia coli BL21, containing GST expression vector (pGEX-KG). The GST protein purified by Sepharose4B column was coated on the surface of self-made magnetic beads and immunized with 2-week-old Eviin chicken. The titer of serum detected by ELISA was 1.6 脳 10 4, which proved that the self-made magnetic beads had immune enhancement effect. Chicken serum was collected and purified by octanoic acid-ammonium sulfate method. The. Western blot test of polyclonal antibody against chicken GST proved that the self-made GST polyclonal antibody had biological activity and specificity. 2. The Fe304 nanoparticles were synthesized by chemical coprecipitation method and modified on the surface of IMB-GSTAb to obtain magnetic beads with average diameter between 50-100nm and superparamagnetic particles. The concentration of the modified beads is that the magnetic beads are conjugated with GST antibody by 5.5mg/mL to prepare the GST-VP2, GST-HN fusion protein captured by IMB-GSTAb for IMB-GSTAb,. By controlling and changing the buffer medium, solution pH value, reaction time and temperature, the conditions of magnetic bead coupling antibody were optimized. The concentration of protein in the solution before and after reaction was determined by ultraviolet spectrophotometer, and the effect of magnetic bead coupling antibody was evaluated. The optimum conditions of coupling antibody were determined as 0.1MTBS (pH6.0) buffer, incubated in 37 鈩，
本文编号：2357557
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