GST抗体包被免疫磁珠纳米佐剂的初步研究

发布时间：2018-03-11 01:39

  本文选题：免疫磁珠IMB　切入点：GST标签蛋白　出处：《华中农业大学》2011年硕士论文　论文类型：学位论文

【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,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标签蛋白的抗体偶联到改性后的磁珠表面,从而制得表面包被GST抗体的IMB,即IMB-GSTAb,然后用IMB-GSTAb捕获GST-HA1(禽流感病毒HA1抗原)融合蛋白,最后用捕获GST-HA1的IMB免疫实验动物鸡,探索IMB-GSTAb的生物安全性和免疫增强作用。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免疫佐剂提供了参考,同时也为提高禽流感疫苗的免疫效价提供了新的思路。 1.抗GST标签蛋白抗体的制备 培养含GST表达载体(pGEX-KG)的大肠杆菌BL21,收获GST蛋白。将经Sepharose 4B柱子纯化得到的GST蛋白免疫2周龄艾维因鸡,制备鸡抗GST多克隆抗体。ELISA检测血清效价达1.28×104,Western blot结果表明制备的抗体具有生物学活性。采用饱和硫酸铵粗提和葡聚糖凝胶(Sephadex G-200)过柱相结合的方法纯化抗体,得到了纯度较高的抗体IgG,结果显示本研究制备的鸡抗GST多抗与GST-NS1融合蛋白、GST-NP融合蛋白和GST-HA1融合蛋白均具有良好的反应性。 2. IMB-GSTAb的制备 采用化学共沉淀法合成Fe3O4纳米粒子,并对其表面进行修饰,使其表面带有-NH2基团和-CHO基团,得到平均粒径在30-200 nm之间并具有超顺磁性的磁珠,修饰前后磁珠的浓度分别为15.62 mg/mL和5.40 mg/mL。 将磁珠与GST抗体偶联制备IMB-GSTAb,用IMB-GSTAb捕获GST与禽流感病毒HA1的融合蛋白GST-HA1。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。用紫外分光光度计测定、SDS-PAGE分析和IMB-ELISA检测等方法对磁珠偶联抗体的效果进行评价,从而获得抗体最佳偶联条件为0.01M PB (pH7.4)缓冲液,37℃振荡孵育6h;IMB的最佳封闭条件为3%脱脂奶粉,37℃封闭1h; IMB-GSTAb捕获GST-HA1的最佳反应条件为37℃,2 h；-NH2末端IMB最大捕获量为297.2±11.7μg/mL,-CHO末端IMB最大捕获量为299.1±6.9μg/mL 3.IMB佐剂效应检测 用捕获GST-HA1的IMB免疫2周龄鸡(A组),同时设四个对照组,分别为IMB-GSTAb组(B)、GST-HA1抗原组(C)、禽流感灭活苗(Re-5株)组(D)和PBS组(E),每组5只,分别于免疫后7 d、14 d、21 d、28 d、35 d、42 d翅下静脉采血,用血凝(HA)和血凝抑制(HI)试验测定血清抗体效价,结果显示,首免后实验组和D组抗体效价上升较快,到第3周时实验组与B、C和E组均存在显著差异(P0.01),三周后实验组抗体效价降低,表明IMB-GSTAb具有一定的免疫增强作用,能较快的诱导机体产生相应抗体。用不同剂量IMB-GSTAb皮下注射鸡只进行安全性试验,结果显示,当注射磁珠量达108 mg/只时,实验鸡生长未出现异常,剖检观察未发现病理学变化,证明IMB-GSTAb具有良好的生物安全性。
[Abstract]:Immunomagnetic Beadsimb (IMB) is a magnetic microsphere coated with antibodies (or ligands). It can specifically adsorb antigen (receptor) and has been widely used in biological and medical fields. Then the antibody against GST label protein was coupled to the surface of the modified magnetic beads, and the IMB- GSTAb coated with the GST antibody was prepared. Then the fusion protein of GST-HA1 (avian influenza virus HA1 antigen) was captured by IMB-GSTAb. Finally, the GST-HA1 IMB was used to immunize the experimental animal chickens. To explore the biological safety and immune enhancement of IMB-GSTAb. Animal experiments have proved that IMB has a certain immune enhancement and good biological safety. The new immune adjuvant, which is safe, effective and inexpensive, provides a reference for improving the immune titer of avian influenza vaccine. 1. Preparation of antibody against GST label protein. Escherichia coli BL21 containing GST expression vector pGEX-KG was cultured and GST protein was harvested. The GST protein purified by Sepharose 4B column was immunized with 2-week-old Eviin chicken. Preparation of chicken anti-#en0# polyclonal antibody. Elisa assay showed that the antibody had biological activity. The purified antibody was purified by the combination of saturated ammonium sulfate and Sephadex G-200. The antibody IgG with high purity was obtained. The results showed that the fusion protein of chicken anti GST polyclonal antibody and GST-NS1 fusion protein GST-NP and GST-HA1 fusion protein had good reactivity. 2. Preparation of IMB-GSTAb. The surface of Fe3O4 nanoparticles was synthesized by chemical coprecipitation method. The surface was modified with -NH _ 2 group and -CHO group. The magnetic beads with an average particle size of 30-200 nm and superparamagnetic properties were obtained. The concentration of magnetic beads before and after modification were 15.62 mg/mL and 5.40 mg / mL, respectively. IMB-GSTAb was prepared by coupling magnetic beads with GST antibody. The fusion protein GST-HA1 of GST and avian influenza virus HA1 was captured by IMB-GSTAb. The parameters such as buffer medium, pH value of solution, reaction time and temperature were controlled and changed. The conditions of magnetic bead coupling antibody were optimized. The effect of magnetic bead coupling antibody was evaluated by UV spectrophotometer, SDS-PAGE analysis and IMB-ELISA detection. Thus, the optimum coupling conditions for antibody were 0.01M PB (pH7.4) buffer solution at 37 鈩，

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