Mrkic Ivan, Abughren Mohamed, Nikolic Jasna, Andjelkovic Uros, Vassilopoulou Emilia, Sinaniotis Athanassios, Petersen Arnd, Papadopoulos Nikolaos G, Gavrovic-Jankulovic Marija
Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000, Belgrade, Serbia.
Mol Biotechnol. 2014 Jun;56(6):498-506. doi: 10.1007/s12033-013-9719-8.
Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.
对香蕉果实过敏似乎已成为欧洲水果过敏的一个重要原因。在已发现的五种过敏原中，被命名为Mus a 5的β-1,3-葡聚糖酶被确定为香蕉过敏组分解析诊断的候选过敏原。由于香蕉果实中蛋白质水平存在差异，在本研究中，Mus a 5在大肠杆菌中作为与谷胱甘肽-S-转移酶的融合蛋白产生。重组Mus a 5在天然条件下通过亲和、离子交换和反相色谱法组合进行纯化。通过埃德曼降解法确认N端序列，通过质谱指纹图谱鉴定55%的一级结构，同时通过圆二色光谱法评估二级结构。重组蛋白的IgG反应性在二维免疫印迹中与抗Mus a 5抗体显示，而在免疫印迹抑制试验中，重组Mus a 5抑制了IgG和IgE与天然Mus a 5的结合。在一组对香蕉果实敏感的十人中，重组Mus a 5的IgE反应性在ELISA中显示。重组Mus a 5是一种适用于香蕉过敏组分解析诊断的新型试剂。
