Vallverdú A, Asturias J A, Arilla M C, Gómez-Bayón N, Martínez A, Martínez J, Palacios R
IFIDESA-ARISTEGUI, Research and Development Department, Bilbao, Spain.
J Allergy Clin Immunol. 1998 Mar;101(3):363-70. doi: 10.1016/S0091-6749(98)70249-0.
Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua.
We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy.
Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen.
Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin.
This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.
在大戟科植物一年生山靛中，已鉴定出两种主要过敏原（Mer a 1A和Mer a 1B），初步认定为肌动蛋白结合蛋白。
我们试图从一年生山靛花粉中克隆并鉴定这些主要过敏原，并获得具有免疫活性的可溶性重组过敏原，用于诊断和治疗。
用简并引物通过聚合酶链反应扩增来分离cDNA克隆。利用基于T7表达系统的载体在大肠杆菌BL21（DE3）中进行表达，通过聚（L-脯氨酸）-琼脂糖亲和层析分离重组过敏原。采用电泳方法（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、等电聚焦和二维聚丙烯酰胺凝胶电泳）和免疫化学方法（蛋白质印迹法和酶联免疫吸附测定）对重组过敏原进行鉴定。
克隆并测序了两个编码一年生山靛花粉肌动蛋白结合蛋白（Mer a 1）的cDNA插入片段。全长Mer a 1 cDNA在大肠杆菌中表达为非融合蛋白。经聚（L-脯氨酸）-琼脂糖单次纯化步骤后，每升培养基中重组Mer a 1的最终产量高达5 mg。重组Mer a 1与对一年生山靛、油橄榄和蓖麻花粉过敏患者血清中的IgE抗体的反应性与天然过敏原相当，但乳胶肌动蛋白结合蛋白与一年生山靛肌动蛋白结合蛋白无交叉反应。结果表明重组Mer a 1与向日葵肌动蛋白结合蛋白共有B表位。
该方法适用于生产特定且纯化的重组过敏原，从而能够对这些蛋白质进行更详细的免疫学鉴定，并开发出更准确的诊断方法和特异性抗过敏治疗方法。
