【摘要】：免疫磁珠(Immunomagnetic Beads,IMB)是表面包被有抗體(或配體)的磁性微球。能特異性吸附抗原(受體)等物質(zhì),目前已廣泛應用于生物和醫(yī)學等領域。本研究利用化學共沉淀法合成磁珠,再將抗GST標簽蛋白偶聯(lián)到改性后的磁珠表面,免疫試驗動物制備大量雞抗GST多克隆抗體,與此同時驗證其免疫佐劑效應。將GST多抗包被磁珠,制備得到IMB-GSTAb,然后用IMB-GSTAb捕獲GST-VP2(禽法氏囊病毒VP2抗原)、GST-HN(禽新城疫HN抗原)融合蛋白,用于免疫實驗動物雞,探索新基因工程納米疫苗免疫增強作用和免疫保護力。動物實驗證明IMB具有一定的免疫增強作用和良好的生物安全性。本研究為尋找一種操作簡便、安全有效且價格低廉的新型納米佐劑提供了參考,同時也為禽主要疾病的疫苗生產(chǎn)及防控提供了新的思路。 1.雞抗GST多克隆抗體的制備 培養(yǎng)含GST表達載體(pGEX-KG)的大腸桿菌BL21,收獲GST蛋白。過Sepharose4B柱子純化得到的GST蛋白,包被于自制磁珠的表面,免疫2周齡艾維因雞,ELISA檢測血清效價達1.6×104,證明自制磁珠具有免疫增強作用。采血得到雞血清,按辛酸-硫酸銨法提純雞抗GST多克隆抗體。Western blot試驗證明自制GST多抗具有生物學活性和特異性。 2. IMB-GSTAb的制備 采用化學共沉淀法合成Fe304納米粒子,并對其表面進行修飾,使其表面帶有-CHO基團,得到平均粒徑在50-100nm之間并具有超順磁性的磁珠,改性后磁珠的濃度為5.5mg/mL 將磁珠與GST抗體偶聯(lián)制備IMB-GSTAb,用IMB-GSTAb捕獲GST-VP2、 GST-HN融合蛋白。通過控制和改變緩沖介質(zhì)、溶液pH值、反應時間和溫度等參數(shù),優(yōu)化磁珠偶聯(lián)抗體的條件。通過用紫外分光光度計測定反應前后溶液中蛋白濃度,對磁珠偶聯(lián)抗體的效果進行評價,確定偶聯(lián)抗體的最佳條件為0.1MTBS(pH6.0)緩沖液,37℃混勻儀中孵育3h；捕獲極值為45-50μg/mg;封閉IMB的最佳條件為5%脫脂奶粉,37℃混勻儀中封閉2h；IMB-GSTAb捕獲GST-VP2融合蛋白的最佳反應條件為0.01MPBS(pH7.4)緩沖液,37℃混勻儀中孵育1h,捕獲極值為15μg/mg; IMB-GSTAb捕獲GST-HN融合蛋白的最佳反應條件為0.01MPBS(Ph6.8)緩沖液,37℃混勻儀中孵育1h,捕獲極值為12μg/mg。 3.禽用磁珠納米疫苗的應用 用捕獲GST-VP2(B組)和GST-HN (E組)的IMB免疫1周齡雞,同時設對照組,分別為GST-VP2組(A組)、GST-HN組(D組)、法氏囊病毒滅活苗組(C組)、新城疫病毒滅活苗組(F組)和PBS組(G組),每組5只,于四免疫后翼下靜脈采血,ELISA方法測定血清中抗體水平,結果顯示,B組與A組存在極顯著差異,與C組無顯著差異；E組與D組抗體效價水平存在顯著差異,與F組無顯著差異。證明自制的磁珠納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，

本文編號：2357557