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IMMUNOLOGY / BASIC RESEARCH
Molecular cloning, expression, purification and in silico epitope prediction of cobalamin-independent methionine synthase (Mor a 2), as a novel allergen from Morus alba pollen
Nazlı Arda
2,4
1
Department of Molecular Biology and Genetics, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul, Turkey
2
Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul, Turkey
3
Division of Immunology and Allergic Diseases, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey
4
Center for Research and Practice in Biotechnology and Genetic Engineering (BIYOGEM), Istanbul University, Istanbul, Turkey
Submission date: 2020-10-10
Final revision date: 2021-04-26
Acceptance date: 2021-05-01
Online publication date: 2021-05-12
Corresponding author
Yunus Aksüt
Department of Molecular Biology and Genetics, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul, Turkey
Department of Molecular Biology and Genetics, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul, Turkey
KEYWORDS
Morus albapollen allergyrecombinant allergenMor a 2cobalamin-independent methionine synthase (MetE)Schizosaccharomyces pombeB- and T-cell epitopes
TOPICS
ABSTRACT
Introduction:
Morus alba (white mulberry) pollen is an aero-allergen source that can trigger allergic diseases. Cobalamin-independent methionine synthase (MetE) in M. alba pollen has been proved to be one of the major allergens for some patients living in Istanbul (Turkey). The aim of the present study was to carry out recombinant production and identification of MetE (Mor a 2), a novel allergen from M. alba pollen. The IgE binding reactivity of rMor a 2 produced for the first time was evaluated and some structural features were investigated by in silico methods to better understand its immunogenicity.
Material and methods:
The gene encoding Mor a 2 was cloned in fission yeast, Schizosaccharomyces pombe ura4-D18h- strain, using the pSLF1073 vector. This is the first report of the production of recombinant pollen allergen in S. pombe. After the purification, immunoreactivity of rMor a 2 was confirmed by immunoblotting using sera of a patient allergic to M. alba pollen. Moreover, B-cell epitopes of rMor a 2 were predicted using various bioinformatic tools, namely Bioinformatics Predicted Antigenic Peptides, BepiPred 2.0 and Immune Epitope Database, whereas T-cell epitopes were estimated using NetMHCIIpan-3.2 and NetMHCII 2.3 servers.
Results:
The immunoblotting analysis yielded 11 of 11 positive reactions to rMor a 2. In silico predictions exerted seven B-cell epitopes (22-33, 384-394, 407-423, 547-553, 571-577, 671-678, 736-741) and seven T-cell epitopes (54-62, 161-170, 197-205, 347-358, 622-630, 657-665, 756-764).
Conclusions:
These findings may support the use of rMor a 2 in the diagnosis and treatment of allergic diseases associated with M. alba and/or MetE.
Morus alba (white mulberry) pollen is an aero-allergen source that can trigger allergic diseases. Cobalamin-independent methionine synthase (MetE) in M. alba pollen has been proved to be one of the major allergens for some patients living in Istanbul (Turkey). The aim of the present study was to carry out recombinant production and identification of MetE (Mor a 2), a novel allergen from M. alba pollen. The IgE binding reactivity of rMor a 2 produced for the first time was evaluated and some structural features were investigated by in silico methods to better understand its immunogenicity.
Material and methods:
The gene encoding Mor a 2 was cloned in fission yeast, Schizosaccharomyces pombe ura4-D18h- strain, using the pSLF1073 vector. This is the first report of the production of recombinant pollen allergen in S. pombe. After the purification, immunoreactivity of rMor a 2 was confirmed by immunoblotting using sera of a patient allergic to M. alba pollen. Moreover, B-cell epitopes of rMor a 2 were predicted using various bioinformatic tools, namely Bioinformatics Predicted Antigenic Peptides, BepiPred 2.0 and Immune Epitope Database, whereas T-cell epitopes were estimated using NetMHCIIpan-3.2 and NetMHCII 2.3 servers.
Results:
The immunoblotting analysis yielded 11 of 11 positive reactions to rMor a 2. In silico predictions exerted seven B-cell epitopes (22-33, 384-394, 407-423, 547-553, 571-577, 671-678, 736-741) and seven T-cell epitopes (54-62, 161-170, 197-205, 347-358, 622-630, 657-665, 756-764).
Conclusions:
These findings may support the use of rMor a 2 in the diagnosis and treatment of allergic diseases associated with M. alba and/or MetE.
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| eISSN: | 1896-9151 |
| ISSN: | 1734-1922 |
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