Keating K M, Segal D B, Craig S J, Nault A K, Semensi V, Wasserman A S, Counsell C M, Bond J F
ImmuLogic Pharmaceutical Corporation, Waltham, MA 02154, USA.
Mol Immunol. 1995 Mar;32(4):287-93. doi: 10.1016/0161-5890(94)00140-v.
In this study we have addressed the question of whether reassociating the two recombinant protein chains that comprise the major cat dander allergen, Fel d I, would change the overall IgE and allergic patient T cell immunoreactivity compared to the native molecule. To accomplish this, the chains were combined under reducing and denaturing conditions, then allowed to reassociate by dilution and extensive dialysis against a physiological buffer. An initial examination of the reaction products using quantitative capture ELISA demonstrated comparable reactivity to Fel d I. Further analysis, using a pool of cat allergic patient plasma, showed that the products of the reassociation reaction (rFel d I) also possessed an enhanced IgE binding capacity. Depletion ELISA results gave only a 5% difference in reactivity between rFel d I and the native protein versus a 20% difference with the mixture of the two chains. Comparative secondary T cell stimulation assays were subsequently performed using cat allergic patient peripheral blood lymphocytes. Here the results demonstrated no loss of reactivity with the reassociated chains as compared to Fel d I or the two mixed recombinant chains. To biochemically characterize the products of the reassociation reaction we have performed reverse phase HPLC and then analysed the isolated fractions by mass spectrometry. It was clear from these results that like the native Fel d I, the products of the reassociation reaction favored heterodimer formation, with no homodimer being detected. This implies that the reassociated protein chains had preferentially adopted a native-like conformation.
在本研究中，我们探讨了一个问题：与天然分子相比，重新组合构成主要猫皮屑过敏原Fel d I的两条重组蛋白链，是否会改变整体IgE和过敏患者T细胞的免疫反应性。为实现这一点，在还原和变性条件下将两条链进行组合，然后通过稀释并与生理缓冲液进行广泛透析使其重新缔合。使用定量捕获ELISA对反应产物进行的初步检测表明，其与Fel d I具有相当的反应性。进一步分析显示，使用一组猫过敏患者血浆，重新缔合反应的产物（rFel d I）也具有增强的IgE结合能力。竞争ELISA结果显示，rFel d I与天然蛋白之间的反应性差异仅为5%，而与两条链的混合物相比差异为20%。随后使用猫过敏患者外周血淋巴细胞进行了比较性二次T细胞刺激试验。结果表明，与Fel d I或两条混合的重组链相比，重新缔合的链没有失去反应性。为了从生化角度表征重新缔合反应的产物，我们进行了反相高效液相色谱，然后通过质谱分析分离的馏分。从这些结果可以清楚地看出，与天然Fel d I一样，重新缔合反应的产物有利于异二聚体的形成，未检测到同二聚体。这意味着重新缔合的蛋白链优先采用了类似天然的构象。
