Background

Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity.

Methods and Results

The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients.

Conclusions

This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

中文翻译：

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青蟹青蟹重组原肌球蛋白的分子和致敏性表征

背景

原肌球蛋白是甲壳类动物（包括泥蟹物种）的主要过敏原，但其在橄榄青蟹中的分子和过敏原特性尚不为人所知。因此，本研究旨在从S. olivacea生产重组原肌球蛋白并随后研究其 IgE 反应性。

方法和结果

将原肌球蛋白基因克隆并在大肠杆菌系统中表达，然后进行SDS-PAGE和免疫印迹试验以鉴定重组蛋白的致敏潜力。发现产生的 855 个碱基对的原肌球蛋白基因与锯缘青蟹有 99.18% 的同源性。它的 284 个氨基酸与甲壳类动物、蛛形纲动物、昆虫和肺炎克雷伯菌的原肌球蛋白相匹配，范围从 79.03 到 95.77%。原肌球蛋白含有 89.44% 的 α-螺旋折叠，具有包含同型二聚体七聚体链的双链 α-螺旋卷曲螺旋结构的三级结构。IPTG 诱导的组氨酸标记的重组原肌球蛋白被纯化为 42 kDa 的大小，并使用抗原肌球蛋白单克隆抗体确认为原肌球蛋白。90.9% (20/22) 的螃蟹过敏患者血清中，重组原肌球蛋白的 IgE 结合具有反应性。

结论

本研究成功地从橄榄链球菌中生产出一种致敏性重组原肌球蛋白。这种重组的原肌球蛋白可用作过敏诊断的特异性过敏原。