滅活口蹄疫病毒抗原的離子交換色譜純化
發(fā)布時間：2018-08-28 19:48
【摘要】：口蹄疫(Foot-and-mouth disease,FMD)是由口蹄疫病毒(Foot-and-mouthdisease virus,FMDV)引起的偶蹄動物的烈性傳染性疾病。曾多次在世界范圍內(nèi)爆發(fā),造成嚴(yán)重的經(jīng)濟(jì)損失。接種滅活口蹄疫病毒疫苗是預(yù)防口蹄疫最有效的手段。隨著對疫苗安全性和質(zhì)量要求的不斷提高,對其進(jìn)行有效的分離純化日益受到重視。色譜分離具有分辨率高、易于規(guī)模放大的優(yōu)勢,在動物疫苗分離純化中具有潛在的應(yīng)用前景。但是如何實現(xiàn)結(jié)構(gòu)復(fù)雜、尺寸達(dá)到28 nm的病毒抗原顆粒在有效分離純化的同時,保持顆粒結(jié)構(gòu)的穩(wěn)定,獲得高的抗原收率,仍然是一個挑戰(zhàn)。本文旨在通過研究滅活FMDV在離子交換色譜分離過程中,影響其分離效率和顆粒穩(wěn)定性的關(guān)鍵因素,以建立高效的純化工藝。主要研究內(nèi)容包括如下幾方面:(1)選取DEAE-FF、DEAE-650M、以及DEAE-POROS三種具有相似配基密度和粒徑,但孔徑顯著不同的陰離子交換介質(zhì),考察了介質(zhì)孔徑對滅活FMDV動態(tài)結(jié)合載量(Dynamic binding capacity,DBC)的影響。發(fā)現(xiàn)滅活 FMDV 在 DEAE-POROS(孔徑214 nm)和DEAE-650M(孔徑106 nm)介質(zhì)上的DBC分別達(dá)到11.53和10.03mg/mL,而在小孔徑的瓊脂糖介質(zhì)DEAE-FF(孔徑32nm)介質(zhì)上的DBC僅為前兩者的1/10。激光共聚焦實驗表明滅活FMDV在DEAE-FF介質(zhì)上的吸附僅限于微球的表面薄層區(qū)域,而在大孔DEAE-POROS介質(zhì)上的吸附能擴(kuò)散進(jìn)入微球內(nèi)部。(2)研究了離子交換介質(zhì)孔徑對滅活FMDV純化收率和穩(wěn)定性的影響。經(jīng)過DEAE-FF、DEAE-650M、以及DEAE-POROS離子交換層析后,滅活FMDV的收率分別為54.46%、66.32%和68.42%。通過高效液相凝膠過濾色譜法(HPSEC)分析發(fā)現(xiàn),滅活FMDV與介質(zhì)吸附-解吸作用會導(dǎo)致其裂解成無免疫活性的12S,且裂解程度隨著介質(zhì)孔徑的增大而減小。利用差示掃描量熱儀(Differential Scanning Calorimetry,DSC)分析滅活FDMV吸附在介質(zhì)上發(fā)生解聚的機(jī)理。滅活FMDV在溶液中發(fā)生裂解生成12S的轉(zhuǎn)變溫度Tm1為48.52℃。而吸附在三種介質(zhì)上之后的Tm1值分別降低為41.73℃,44.04℃和45.37℃,表明在層析介質(zhì)上的吸附導(dǎo)致滅活FMDV更易于發(fā)生裂解,而吸附在大孔徑的DEAE-POROS介質(zhì)上的滅活FMDV的穩(wěn)定性相對較高。(3)建立了大孔DEAE-POROS介質(zhì)純化滅活FMDV的工藝。對緩沖液的電導(dǎo)率、進(jìn)樣蛋白濃度、停留時間等參數(shù)進(jìn)行了優(yōu)化。優(yōu)化條件下,滅活FMDV的IEC收率達(dá)94%。經(jīng)過進(jìn)一步超濾濃縮和凝膠過濾色譜(SEC)精制,最終滅活FMDV收率為79%,純化倍數(shù)為173,宿主DNA的去除率達(dá)95%以上。本文研究結(jié)果表明大孔介質(zhì)純化滅活FMDV相比傳統(tǒng)的瓊脂糖介質(zhì)在動態(tài)結(jié)合載量、活性收率和結(jié)構(gòu)穩(wěn)定性上具有明顯的優(yōu)勢,并探討了可能的作用機(jī)理,對滅活FMDV的色譜純化具有一定的指導(dǎo)意義。
[Abstract]:Foot and mouth disease (Foot-and-mouth disease,FMD) is a severe infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (Foot-and-mouthdisease virus,FMDV). There have been many outbreaks around the world, causing serious economic losses. Inoculation with inactivated foot-and-mouth disease virus vaccine is the most effective method to prevent foot-and-mouth disease. With the improvement of the safety and quality of vaccine, more and more attention has been paid to the effective separation and purification of vaccine. Chromatographic separation has the advantages of high resolution and easy scale amplification, and has a potential application prospect in the purification of animal vaccine. However, it is still a challenge to achieve the complex structure of virus antigen particles of 28 nm, while maintaining the stability of the particle structure and obtaining high antigen yield. The purpose of this paper is to establish an efficient purification process by studying the key factors affecting the separation efficiency and particle stability of inactivated FMDV in ion exchange chromatography. The main contents are as follows: (1) three anion exchange media, DEAE-FF,DEAE-650M, and DEAE-POROS, which have similar ligands density and particle size, but have different pore sizes, are selected to investigate the effect of pore size on the dynamic binding capacity of FMDV (Dynamic binding capacity,DBC). It was found that the DBC of inactivated FMDV on DEAE-POROS (aperture 214 nm) and DEAE-650M (aperture 106 nm) media was 11.53 and 10.03 mg / mL, respectively, while the DBC on small aperture agarose medium DEAE-FF (pore size 32nm) was only 1 / 10 of the former. Laser confocal experiments show that the adsorption of inactivated FMDV on DEAE-FF media is confined to the thin layer area of the surface of the microspheres. However, the adsorption energy on the macroporous DEAE-POROS medium diffuses into the microspheres. (2) the effect of the pore size of the ion exchange medium on the purification yield and stability of the inactivated FMDV was studied. After DEAE-FF,DEAE-650M, and DEAE-POROS ion exchange chromatography, the yields of inactivated FMDV were 54.46% and 66.32%, respectively. By (HPSEC) analysis of high performance liquid phase gel filtration chromatography, it was found that the inactivated FMDV and the adsorption-desorption of the medium would lead to the dissociation of 12s with no immune activity, and the degree of decomposition would decrease with the increase of the pore size of the medium. The mechanism of depolymerization of inactivated FDMV adsorbed on medium was analyzed by differential scanning calorimeter (Differential Scanning Calorimetry,DSC). The transition temperature (Tm1) of inactivated FMDV to form 12s in solution is 48.52 鈩，
本文編號：2210431
[Abstract]:Foot and mouth disease (Foot-and-mouth disease,FMD) is a severe infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (Foot-and-mouthdisease virus,FMDV). There have been many outbreaks around the world, causing serious economic losses. Inoculation with inactivated foot-and-mouth disease virus vaccine is the most effective method to prevent foot-and-mouth disease. With the improvement of the safety and quality of vaccine, more and more attention has been paid to the effective separation and purification of vaccine. Chromatographic separation has the advantages of high resolution and easy scale amplification, and has a potential application prospect in the purification of animal vaccine. However, it is still a challenge to achieve the complex structure of virus antigen particles of 28 nm, while maintaining the stability of the particle structure and obtaining high antigen yield. The purpose of this paper is to establish an efficient purification process by studying the key factors affecting the separation efficiency and particle stability of inactivated FMDV in ion exchange chromatography. The main contents are as follows: (1) three anion exchange media, DEAE-FF,DEAE-650M, and DEAE-POROS, which have similar ligands density and particle size, but have different pore sizes, are selected to investigate the effect of pore size on the dynamic binding capacity of FMDV (Dynamic binding capacity,DBC). It was found that the DBC of inactivated FMDV on DEAE-POROS (aperture 214 nm) and DEAE-650M (aperture 106 nm) media was 11.53 and 10.03 mg / mL, respectively, while the DBC on small aperture agarose medium DEAE-FF (pore size 32nm) was only 1 / 10 of the former. Laser confocal experiments show that the adsorption of inactivated FMDV on DEAE-FF media is confined to the thin layer area of the surface of the microspheres. However, the adsorption energy on the macroporous DEAE-POROS medium diffuses into the microspheres. (2) the effect of the pore size of the ion exchange medium on the purification yield and stability of the inactivated FMDV was studied. After DEAE-FF,DEAE-650M, and DEAE-POROS ion exchange chromatography, the yields of inactivated FMDV were 54.46% and 66.32%, respectively. By (HPSEC) analysis of high performance liquid phase gel filtration chromatography, it was found that the inactivated FMDV and the adsorption-desorption of the medium would lead to the dissociation of 12s with no immune activity, and the degree of decomposition would decrease with the increase of the pore size of the medium. The mechanism of depolymerization of inactivated FDMV adsorbed on medium was analyzed by differential scanning calorimeter (Differential Scanning Calorimetry,DSC). The transition temperature (Tm1) of inactivated FMDV to form 12s in solution is 48.52 鈩，
本文編號：2210431
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