發(fā)布時(shí)間：2018-03-11 01:39

  本文選題：免疫磁珠IMB　切入點(diǎn)：GST標(biāo)簽蛋白　出處：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【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),已廣泛應(yīng)用于生物和醫(yī)學(xué)等領(lǐng)域。本研究利用化學(xué)共沉淀法合成磁珠,再將抗GST標(biāo)簽蛋白的抗體偶聯(lián)到改性后的磁珠表面,從而制得表面包被GST抗體的IMB,即IMB-GSTAb,然后用IMB-GSTAb捕獲GST-HA1(禽流感病毒HA1抗原)融合蛋白,最后用捕獲GST-HA1的IMB免疫實(shí)驗(yàn)動(dòng)物雞,探索IMB-GSTAb的生物安全性和免疫增強(qiáng)作用。動(dòng)物實(shí)驗(yàn)證明IMB具有一定的免疫增強(qiáng)作用和良好的生物安全性。本研究為尋找一種操作簡(jiǎn)便、安全有效且價(jià)格低廉的新型免疫佐劑提供了參考,同時(shí)也為提高禽流感疫苗的免疫效價(jià)提供了新的思路。 1.抗GST標(biāo)簽蛋白抗體的制備 培養(yǎng)含GST表達(dá)載體(pGEX-KG)的大腸桿菌BL21,收獲GST蛋白。將經(jīng)Sepharose 4B柱子純化得到的GST蛋白免疫2周齡艾維因雞,制備雞抗GST多克隆抗體。ELISA檢測(cè)血清效價(jià)達(dá)1.28×104,Western blot結(jié)果表明制備的抗體具有生物學(xué)活性。采用飽和硫酸銨粗提和葡聚糖凝膠(Sephadex G-200)過(guò)柱相結(jié)合的方法純化抗體,得到了純度較高的抗體IgG,結(jié)果顯示本研究制備的雞抗GST多抗與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應(yīng)性。 2. IMB-GSTAb的制備 采用化學(xué)共沉淀法合成Fe3O4納米粒子,并對(duì)其表面進(jìn)行修飾,使其表面帶有-NH2基團(tuán)和-CHO基團(tuán),得到平均粒徑在30-200 nm之間并具有超順磁性的磁珠,修飾前后磁珠的濃度分別為15.62 mg/mL和5.40 mg/mL。 將磁珠與GST抗體偶聯(lián)制備IMB-GSTAb,用IMB-GSTAb捕獲GST與禽流感病毒HA1的融合蛋白GST-HA1。通過(guò)控制和改變緩沖介質(zhì)、溶液pH值、反應(yīng)時(shí)間和溫度等參數(shù),優(yōu)化磁珠偶聯(lián)抗體的條件。用紫外分光光度計(jì)測(cè)定、SDS-PAGE分析和IMB-ELISA檢測(cè)等方法對(duì)磁珠偶聯(lián)抗體的效果進(jìn)行評(píng)價(jià),從而獲得抗體最佳偶聯(lián)條件為0.01M PB (pH7.4)緩沖液,37℃振蕩孵育6h;IMB的最佳封閉條件為3%脫脂奶粉,37℃封閉1h; IMB-GSTAb捕獲GST-HA1的最佳反應(yīng)條件為37℃,2 h；-NH2末端IMB最大捕獲量為297.2±11.7μg/mL,-CHO末端IMB最大捕獲量為299.1±6.9μg/mL 3.IMB佐劑效應(yīng)檢測(cè) 用捕獲GST-HA1的IMB免疫2周齡雞(A組),同時(shí)設(shè)四個(gè)對(duì)照組,分別為IMB-GSTAb組(B)、GST-HA1抗原組(C)、禽流感滅活苗(Re-5株)組(D)和PBS組(E),每組5只,分別于免疫后7 d、14 d、21 d、28 d、35 d、42 d翅下靜脈采血,用血凝(HA)和血凝抑制(HI)試驗(yàn)測(cè)定血清抗體效價(jià),結(jié)果顯示,首免后實(shí)驗(yàn)組和D組抗體效價(jià)上升較快,到第3周時(shí)實(shí)驗(yàn)組與B、C和E組均存在顯著差異(P0.01),三周后實(shí)驗(yàn)組抗體效價(jià)降低,表明IMB-GSTAb具有一定的免疫增強(qiáng)作用,能較快的誘導(dǎo)機(jī)體產(chǎn)生相應(yīng)抗體。用不同劑量IMB-GSTAb皮下注射雞只進(jìn)行安全性試驗(yàn),結(jié)果顯示,當(dāng)注射磁珠量達(dá)108 mg/只時(shí),實(shí)驗(yàn)雞生長(zhǎng)未出現(xiàn)異常,剖檢觀察未發(fā)現(xiàn)病理學(xué)變化,證明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，

本文編號(hào)：1596050