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Cloning, Recombinant Expression and Activity Studies of a Major Allergen “Enolase” from the Fungus Curvularia Lunata

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Recombinant allergens are required to study allergy at the molecular level and are helpful tools for the improvement of diagnosis and therapy. In the present study, enolase was expressed from Curvularia lunata and analyzed for its immunological reactivity as an allergen. cDNA library was synthesized in λ zap vector and screened with sera obtained from C. lunata allergic patients. A cDNA clone with an ORF of 1.3 kb showed homology to enolases from different fungal sources. It was expressed in E. coli, purified from inclusion bodies yielding 0.5 mg/L and showed enzyme activity of 48 units/mg. It resolved as 48-kDa band on SDS-PAGE and was recognized by all the individual Curvularia positive patient sera in immunoblot and ELISA. r Cur l 2 stimulated patients’ PBMCs and supernatant of these cells showed elevated levels of Th 2 cytokines. Ten B cell epitopes were predicted using computational software and one showed 90% homology to an important IgE epitope of Cla h 6. The various parameters predicted by computational approach can be validated later as a future study to draw conclusive evidence about putative antigenic epitopes. This can further help in generating knowledge about residues important for IgE binding and developing therapeutic modalities.

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Abbreviations

CL:

Curvularia lunata

IEF:

Isoelectric focusing

PEP:

Phosphoenol pyruvate

PGA:

Phosphoglyceric acid

rCur l 2:

recombinant Cur l 2.

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ACKNOWLEDGMENTS

One of the authors (Vidhu Sharma) is recipient of SRF from Council of Scientific and Industrial Research, New Delhi. The present work was supported by Asthma and Allergy Mitigation Task Force, CSIR, Delhi.

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Authors and Affiliations

  1. Institute of Genomics and Integrative Biology, Delhi, India

    VIDHU SHARMA, RATNA GUPTA, ANUPAM JHINGRAN, BHANU PRATAP SINGH, SUSHEELA SRIDHARA & NAVEEN ARORA

  2. Vallabhbhai Patel Chest Institute, Delhi, India

    SHAILENDRA NATH GAUR

  3. Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

    VIDHU SHARMA & ANUPAM JHINGRAN

  4. Allergy and Immunology Section, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road (near Jubilee hall), Delhi, 110007, India

    NAVEEN ARORA

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  1. VIDHU SHARMA
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Correspondence to NAVEEN ARORA.

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SHARMA, V., GUPTA, R., JHINGRAN, A. et al. Cloning, Recombinant Expression and Activity Studies of a Major Allergen “Enolase” from the Fungus Curvularia Lunata . J Clin Immunol 26, 360–369 (2006). https://doi.org/10.1007/s10875-006-9032-4

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  • DOI: https://doi.org/10.1007/s10875-006-9032-4

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