Narum D L, Welling G W, Thomas A W
Department of Chronic and Infectious Diseases, Medical Biological Laboratory, TNO, Rijswijk, Netherlands.
J Chromatogr A. 1993 Dec 31;657(2):357-63. doi: 10.1016/0021-9673(93)80291-F.
A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83,000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-7G8-1. The first step utilizes a new approach to high-performance ion-exchange chromatography (HPIEC) in which elution conditions are not only defined by charge, but also by hydrophobicity. HPIEC fractionation involves successive sodium chloride gradient anion-exchange elutions (A and B), where a change in the non-ionic detergent polyoxyethylenealkylether C10E5 concentration between elutions A and B (from 0.01% to 0.1% (w/v) respectively), results in a fraction that comprises from 2% to 9% PF83-7G8-1. Subsequent column immunoaffinity purification of this fraction on Q-Sepharose CL 4B-28G2dc1 mAb yields a PF83-7G8-1 preparation that is 56% pure. Rat mAb 28G2dc1 recognizes a C-terminal region that is conserved and cross reactive within the AMA-1 family, thus permitting recombinant and native full-length AMA-1 molecules from other species to be purified for molecular analysis. Immunological and molecular characterisation of the vaccine-related characteristics of purified PF83/AMA-1 are now underway.
已开发出一种两步纯化方法，用于纯化恶性疟原虫（PF83/AMA-1）的一种在数量上较少、推测为跨膜的、分子量83,000的顶端膜血期疫苗候选抗原，该抗原已表达为全长杆状病毒重组蛋白PF83-7G8-1。第一步采用了一种新的高效离子交换色谱法（HPIEC），其中洗脱条件不仅由电荷决定，还由疏水性决定。HPIEC分级分离包括连续的氯化钠梯度阴离子交换洗脱（A和B），其中洗脱A和B之间非离子洗涤剂聚氧乙烯烷基醚C10E5浓度的变化（分别从0.01%到0.1%（w/v）），产生一个包含2%至9% PF83-7G8-1的级分。随后在Q-Sepharose CL 4B-28G2dc1单克隆抗体上对该级分进行柱免疫亲和纯化，得到纯度为56%的PF83-7G8-1制剂。大鼠单克隆抗体28G2dc1识别AMA-1家族内保守且具有交叉反应性的C末端区域，从而允许纯化来自其他物种的重组和天然全长AMA-1分子用于分子分析。目前正在对纯化的PF83/AMA-1的疫苗相关特性进行免疫学和分子表征。
