Molecular cloning and expression of cucumisin (Cuc m 1), a subtilisin-like protease of Cucumis melo in escherichia coli

Sankian, M. and Talebi, F. and Moghadam, M. and Vahedi, F. and Azad, F. J. and Varasteh, A. R. (2011) Molecular cloning and expression of cucumisin (Cuc m 1), a subtilisin-like protease of Cucumis melo in escherichia coli. Allergology International, 60 (1). pp. 61-67.

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Abstract

Background: Oral allergy syndrome resulted from plant-derived foods is frequent among adults. Allergy to melon (cucumis melo) is one of the most frequent fruit allergies in Iran. Three different major allergens have been found in Cucumis melo that Cuc m 1 (cucumisin) has been identified as the major allergen of melon. Cucumisin is an alkaline serine protease that it is found as a 78 kDa protein in precursor form. The aim of this study was production of recombinant Cuc m 1 in Escherichia coli (E. coli) cells and characterization of its allergenicity property. Methods: Production of recombinant Cuc m 1 was carried out by cDNA cloning technique into the pET32b(+) vector using specific primers designed based on cucumisin nucleotide sequence available in Genebank database, cucumisin encoding gene and directional cloning method. Cloned plasmid into E. coli TOP10 was transformed into E. coli BL21 and expression of the protein was induced by IPTG. The recombinant protein was purified via Ni-NTA affinity chromatography using histidine tag in recombinant protein. IgE binding of this protein was assessed by IgE-immunoblotting, ELISA and inhibition ELISA. Results: The directional cloning was resulted in expression of a fusion Cuc m 1. Immunoblotting with sera of patients allergic to melon showed strong reactivity with purified protein band. Inhibition assays demonstrated that purified rCuc m 1 could be the same with natural form of Cuc m 1 in total extract. Conclusions: In the present study, we have provided a functional recombinant cucumisin allergen, rCuc m 1 with 86 kDa, which may be used as a standard allergen for clinical diagnosis and study of allergy to melon. © 2011 Japanese Society of Allergology.

Item Type: Article
Additional Information: Cited By :2 Export Date: 16 February 2020 CODEN: ALINF Correspondence Address: Varasteh, A.-R.; Bu-Ali Research Institute, Immunology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Ferdowsi Square, Bu-Ali Square, Mashhad, 9196773117, Iran; email: varasteha@mums.ac.ir
Uncontrolled Keywords: Allergy Cloning Cucumis melo Cucumisin Subtilisin-like proteases complementary DNA immunoglobulin E nitrilotriacetate nickel nucleotide polyhistidine tag recombinant cucumisin recombinant protein unclassified drug adult affinity chromatography allergenicity article bacterial cell binding affinity blood sampling cantaloupe child clinical article controlled study enzyme linked immunosorbent assay Escherichia coli female food allergy gene expression growth inhibition human immunoblotting immunoreactivity male molecular cloning nonhuman nucleotide sequence plasmid prick test priority journal protein expression protein purification school child
Subjects: QU Biochemistry
QW Microbiology and Immunology
Divisions: Mashhad University of Medical Sciences
Depositing User: mr lib7 lib7
Date Deposited: 30 Apr 2020 07:42
Last Modified: 30 Apr 2020 07:42
URI: http://eprints.mums.ac.ir/id/eprint/17116

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