Cloning and Expression of Che a 1, the Major Allergen of Chenopodium album in Escherichia coli

• Published in: Applied biochemistry and biotechnology Vol. 163; no. 7; pp. 895 - 905
• Main Authors: Vahedi, Fatemeh, Sankian, Mojtaba, Moghadam, Malihe, Mohaddesfar, Maryam, Ghobadi, Sirous, Varasteh, Abdol Reza
• Format: Journal Article
• Language: English
• Published: New York Humana Press Inc 01.04.2011; Springer; Springer Nature B.V
• Subjects: affinity chromatography; Allergens; Allergens - genetics; Allergens - immunology; Allergies; analysis; annuals; Antigens, Plant; Biochemistry; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; Chenopodium; Chenopodium album; Chenopodium album - chemistry; clones; Cloning; Cloning, Molecular; complementary DNA; diagnosis; E coli; enzyme-linked immunosorbent assay; Escherichia coli; Flowers & plants; Fundamental and applied biological sciences. Psychology; Gene expression; genetics; histidine; Humans; Hypersensitivity; Hypersensitivity - immunology; Immunoblotting; immunoglobulin E; Immunoglobulin E - immunology; immunology; Iran; physiopathology; Plant Proteins; Plant Proteins - genetics; Plant Proteins - immunology; Pollen; Pollen - chemistry; polymerase chain reaction; protein synthesis; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Reverse Transcriptase Polymerase Chain Reaction; Rhinitis, Allergic, Seasonal; Rhinitis, Allergic, Seasonal - diagnosis; Rhinitis, Allergic, Seasonal - immunology; Rhinitis, Allergic, Seasonal - physiopathology; RNA; RNA, Messenger; RNA, Messenger - analysis; Iran; Recombinant allergen; Allergy; Che a 1; Cloning; Escherichia coli; Chenopodiaceae; Chenopodium album; Dicotyledones; Angiospermae; Bacteria; Spermatophyta; Chellopodium album; Enterobacteriaceae; Allergen
• ISSN: 0273-2289; 1559-0291; 1559-0291
• DOI: 10.1007/s12010-010-9093-y

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.