Expression and evaluation of IgE‐binding capacity of recombinant Pacific mackerel parvalbumin
Expression and evaluation of IgE‐binding capacity of recombinant Pacific mackerel parvalbumin
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DOI：
10.1111/j.1440-1592.2004.00344.x
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发表时间：
2004
影响因子：
6.8
通讯作者：
Y. Hamada;Hiroyuki Tanaka;Ayako Sato;S. Ishizaki;Y. Nagashima;K. Shiomi
中科院分区：
文献类型：
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作者：
Y. Hamada;Hiroyuki Tanaka;Ayako Sato;S. Ishizaki;Y. Nagashima;K. Shiomi
摘要背景小清蛋白是鱼类中主要的交叉反应性过敏原。鱼类过敏症的诊断和免疫治疗需要足够量的IgE反应性重组鱼小清蛋白。方法人工合成太平洋鲭鱼小清蛋白的DNA片段，将其克隆到原核表达载体pGEX-6p-3中，在大肠杆菌中表达谷胱甘肽S-转移酶（GST）融合小清蛋白。使用RediPack GST纯化模块（阿默舍姆Pharmacia Biotech，白金汉郡，英国）纯化不含GST的重组小清蛋白。采用凝胶过滤和反相高效液相色谱法对日本鳗鲡、竹荚鱼、真鲷、太平洋鲭鱼、鲣鱼、大眼金枪鱼和比目鱼等7种鱼类的小清蛋白进行了纯化。通过ELISA检测IgE结合能力，通过抑制ELISA检测抗原交叉反应性。结果获得了不含GST的重组太平洋鲭鱼小清蛋白。ELISA和抑制ELISA的数据显示，重组小清蛋白含有天然对应物的大部分IgE结合表位。此外，重组小清蛋白抑制IgE反应性的汇集患者血清中纯化的小清蛋白，从六种鱼在几乎相同的幅度作为天然太平洋鲭鱼小清蛋白。结论由于具有天然对应物IgE结合能力的重组太平洋鲭鱼小清蛋白与多种鱼类小清蛋白具有交叉反应性，因此可以成为鱼类过敏诊断和免疫治疗的有用工具。
ABSTRACT Background Parvalbumin is the major and cross-reactive allergen in fish. Sufficient amounts of IgE-reactive recombinant fish parvalbumin are needed for diagnosis and immunotherapy of fish allergy. Methods A DNA fragment corresponding to parvalbumin of the Pacific mackerel Scomber japonicus was synthesized and cloned into the expression vector pGEX-6p-3 to produce glutathione S-transferase (GST)-fusion parvalbumin in Escherichia coli. The GST-free recombinant parvalbumin was purified using the RediPack GST Purification Module (Amersham Pharmacia Biotech, Buckinghamshire, UK). Parvalbumins of seven species of fish (Japanese eel, horse mackerel, red sea bream, Pacific mackerel, skipjack, bigeye tuna and Japanese flounder) were purified by gel filtration and reverse-phase HPLC. The IgE-binding capacity was examined by ELISA and antigenic cross-reactivity by inhibition ELISA. Results The GST-free recombinant Pacific mackerel parvalbumin was obtained in an electrophoretically pure state. Data from ELISA and inhibition ELISA revealed that the recombinant parvalbumin contains most of the IgE-binding epitopes of the natural counterpart. In addition, the recombinant parvalbumin inhibited the IgE reactivities of the pooled patient serum to parvalbumins purified from six species of fish in almost the same magnitude as the natural Pacific mackerel parvalbumin. Conclusions Because the recombinant Pacific mackerel parvalbumin bearing the IgE-binding capacity of the natural counterpart is cross-reactive with various fish parvalbumins, it can be a useful tool for the diagnosis and immunotherapy of fish allergy.