Supplementary Material for: A Recombinant Sal k 1 Isoform as an Alternative to the Polymorphic Allergen from <b><i>Salsola kali</i></b> Pollen for Allergy Diagnosis

<b><i>Background:</i></b> The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, <i>Salsola kali </i>is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of <i>S. kali </i>pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. <b><i>Methods:</i></b> Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) <i>Escherichia coli </i>cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from <i>S. kali </i>pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. <b><i>Results:</i></b> rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as <i>Platanus acerifolia</i> and Oleaceae members. <b><i>Conclusions:</i></b> rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.<b><i>背景：</i></b> 随着多国荒漠化进程加剧，苋科（Amaranthaceae）花粉过敏的发病率呈上升趋势。在西班牙部分地区，刺沙蓬（<i>Salsola kali</i>）是引发花粉症的主要致敏原之一，其致敏水平几乎与橄榄花粉及禾本科花粉相当。Sal k 1作为刺沙蓬花粉症的致敏标志物，已应用于临床诊断，但目前从花粉中纯化该蛋白的得率较低。本研究旨在制备重组Sal k 1（rSal k 1），使其具备花粉中天然同工型的结构与免疫学特性，并验证其用于临床诊断的潜力。 <b><i>方法：</i></b> 通过聚合酶链式反应（Polymerase Chain Reaction，PCR）扩增特异性互补脱氧核糖核酸（complementary DNA，cDNA），将其克隆至pET41b载体中，随后转化至BL21 (DE3) 大肠杆菌（<i>Escherichia coli</i>）感受态细胞。采用免疫印迹（Immunoblotting）、酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay，ELISA）、嗜碱性粒细胞活化试验（basophil activation test）及皮肤点刺试验（skin-prick test），以从花粉中分离得到的天然Sal k 1为对照，验证该重组蛋白的活性。实验所用样本包括刺沙蓬花粉致敏患者的血清及血细胞，以及特异性单克隆与多克隆抗血清。 <b><i>结果：</i></b> 重组Sal k 1在大肠杆菌中成功表达，每升细胞培养物的得率可达7.5 mg。该蛋白经纯化后达到均一纯度，并通过结构与免疫学实验验证了其与天然蛋白的一致性。相较于二球悬铃木（<i>Platanus acerifolia</i>）花粉及木犀科（Oleaceae）花粉提取物，rSal k 1与花生、杏仁、番茄等植物源性食物提取物的免疫球蛋白E（Immunoglobulin E，IgE）交叉反应活性更高。 <b><i>结论：</i></b> 大肠杆菌表达的重组Sal k 1保留了与花粉源性致敏原一致的完整结构与免疫学特性，能够覆盖花粉中绝大多数天然同工型的免疫学功能，有望替代天然Sal k 1应用于临床诊断。