Abstract：
Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3-2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.
摘要：
贝类是全球过敏的主要原因，影响了大约十分之一的普通人口。肌浆钙结合蛋白，也被称为过敏原笔M4，是虾过敏的重要因素。我们的目标是评估生产重组Penm4蛋白作为诊断虾过敏的潜在工具的最有效技术。在这项研究中,第一次,我们在真核系统中产生了功能性重组Penm4蛋白，巴斯德毕赤酵母,并在酶联免疫吸附和反相蛋白质微阵列分析中对大肠杆菌产生的等效物进行了分析。基于麦芽糖结合蛋白的双标签系统已成功用于将细菌和酵母中的Penm4的产量提高1.3至2.3倍，分别。免疫学表征表明，N-糖基化对于Penm4的折叠和特异性IgE的识别都不是至关重要的。然而,Ca2耗竭测定表明依赖于血液样品中钙离子的存在。结果表明，比较分析如何阐明基本的过敏原制造点。总之,大肠杆菌产生的与麦芽糖结合蛋白融合的Penm4蛋白应该是对虾变态反应诊断中进一步研究的首选选择。
