Biotechnology Department, Razi Vaccine and Serum Research Institute, Mashhad, Iran.
Appl Biochem Biotechnol. 2011 Apr;163(7):895-905. doi: 10.1007/s12010-010-9093-y. Epub 2010 Sep 25.
Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b+ vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni-NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.
藜是藜属的一种杂草一年生植物。C.album 花粉是伊朗主要的过敏原来源。主要的 C.album 花粉过敏原已被描述为 Che a 1、Che a 2 和 Che a 3。本工作旨在将 Che a 1 在大肠杆菌中进行克隆，以建立重组 Che a 1(rChe a 1)的过表达系统。为了克隆这种过敏原，花粉被进行 RNA 提取。通过从提取的 RNA 合成的第一链 cDNA 的聚合酶链反应扩增，制备编码 Che a 1 的全长片段。通过将 cDNA 插入 pET21b+载体进行克隆，然后将构建体转化为大肠杆菌 Top10 细胞，并通过 IPTG 诱导蛋白表达。通过重组蛋白中的组氨酸标签，使用 Ni-NTA 亲和层析纯化 rChe a 1。进行 IgE 免疫印迹、ELISA 和抑制 ELISA 以评估纯化蛋白的 IgE 结合。总之，成功地克隆了 C.album 花粉主要过敏原 Che a 1 的 cDNA，并纯化了 rChe a 1。抑制试验表明，过敏患者的血清与 rChe a 1 反应，类似于粗提物中天然 Che a 1 的反应C.album 花粉。本研究首次报道了使用大肠杆菌作为原核系统克隆 Che a 1 和生产 rChe a 1。
