Vakili Moghaddam M, Fallahpour M, Mohammadi M, Rasi Varaee F S, Mokhtarian K, Khoshmirsafa M, Jafari R, Shirzad N, Falak R
Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran; Department of Immunology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Department of Allergy and Clinical Immunology, Iran University of Medical Sciences, Tehran, Iran.
Allergol Immunopathol (Madr). 2019 Jul-Aug;47(4):357-364. doi: 10.1016/j.aller.2018.12.006. Epub 2019 Feb 13.
Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera.
Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract.
Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too.
The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.
反枝苋（红根苋）是温带地区致敏花粉的主要来源之一。钙结合蛋白是一种众所周知的泛过敏原，参与不同植物之间的交叉反应。本研究的目的是对钙结合蛋白进行分子克隆和表达，并评估其与反枝苋敏感患者血清的IgE反应性。
从反枝苋花粉中制备致敏提取物，并使用20名反枝苋敏感患者的血清通过ELISA和免疫印迹法测定IgE反应性谱。通过常规PCR方法扩增钙结合蛋白编码序列，并将产物插入pET-21b(+)载体中。重组蛋白在大肠杆菌BL21中表达，并通过金属亲和色谱法纯化。通过ELISA和免疫印迹分析重组蛋白的IgE结合能力，并与粗提物进行比较。
在20名皮肤点刺试验阳性患者中，17名患者的IgE特异性ELISA呈阳性。蛋白质印迹法证实，约53%的ELISA阳性患者与粗提物中的10kDa蛋白发生反应。克隆了编码80个氨基酸残基的反枝苋钙结合蛋白基因，并将其表达为可溶性蛋白，命名为Ama r 3。重组钙结合蛋白在ELISA和蛋白质印迹中显示出与粗提物中10kDa蛋白相当相同的IgE反应性。这些结果也通过抑制方法得到了证实。
反枝苋钙结合蛋白（Ama r 3）的重组形式可以很容易地在大肠杆菌中以可溶性形式产生，并且与其天然对应物显示出相当相似的IgE反应性。
