Expression, Purification and Structure Prediction of Hum s1, a Major Allergen of Humulus Scandens
Abstract
Hum s1, a major allergen of Humulus Scandens, was cloned, expressed and purified. Its protein structure and function was predicted and analyzed, which provided a foundation for further studies into the diagnostic and therapeutic efficacy of the recombinant allergen. Methods The target gene was amplified by PCR and sub-cloned into the expression vector pET32a throughKpn Ⅰ/Xho Ⅰsite. The recombinant plasmid was transformed into clone strainE.coliDH5α. The positive recombinant plasmid identified by PCR was transformed into the expression strainE.coliBL-21. The expressed fusion protein was induced by IPTG, and purified using fast protein liquid chromatography. The structure and function of the protein was predicted and analyzed. Results Prokaryotic expression plasmid pET32a-G2 was constructed successfully. Hum s1 was expressed and purified. The purity of expressed fusion protein exceeded 90%. It has three potential antigen epitopes and two EF-hand structural domains. A three-dimensional model was successfully constructed. The recombinant protein was proved to have immunological activity, with ELESA showing good attachment to sera samples of patients who were allergic to Humulus Scandens. Conclusion Prokaryotic expression vector ofHum s1 was successfully constructed. The recombinant protein was expressed and purified, with its epitope and three-dimensional model being predicted by bioinformatics. The study provided a basis for further development of recombinant vaccines.
Keywords: Humulus, Scandens, Recombinant allergen, Gene clone
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Wallner Ml, Pichler U, Ferreira F, et al. Recombinant allergens for pollen immunotherapy. Immunotherapy, 2013 ; 5 (12):1323-1338.
Klimek L, Schendzielorz P, Pinol R, et al. Specific subcutaneous immunotherapy with recombinant grass pollen allergens: first randomized dose-ranging safety study. Clin Exp Allergy,2012?42(6):936-945.
Yin J . Ye ST, Gu RJ . et al. An important allergenic pollen in china-humulus. J Allergy Clin Immunol. 1995;95( 1); 164-166.
Akdis M. New treatments for allergen immunotherapy. World Allergy Organ J,2014 ;7( 1):23. doi:10. 1186/1939-4551-7-23. Couderc R, Just J. Recombinant allergens for diagnosis and specific immunotherapy value in pediatric patients. Bull Acad Natl Med,2013; 197(3) :645-659.
Jutel M, Akdis С A. Novel immunotherapy 4'accine development. Curr Opin Allergy Clin Immunol, 2014; 14 ( 6): 557-563.
Dewitt AM. Andersson K, Pelt re G. et al. Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phi p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy .2006; 36(1); 77-86.
Mahler V. Vrtala S, Kuss O. et al. Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen. Bet vl. Clin Exp Allergy, 2004; 34(1): 115-122.
Reuter A, Fortunato D. Perono Garoffo L. et al. Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics. Proteomics,2005; 5( 1); 282-289.
Ferreira F. Hawranek T. Gruber P. el al. Allergic cross- reactivity; from gene to the clinic. Allergy, 2004 ; 59 ( 3 ): 243- 267.
Garmatiuk T, Swoboda I, Twardosz-Kropfmiiller A, et al. Characterization of mutants of a highly cross-reactive calcium- binding protein from Brassica pollen for allergen-specific immunotherapy. Immunobiology,2013;218(9) : 1155-1165.
Garcia-Mayoral MF. Trevino MA, Perez-Piriar T. et al. Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5. a member of the SXP/ RAL-2 protein family. PLoS Negl Trop Dis,2014;8(3) ;e2735. doi: 10. 1371 /journal-pntd. 0002735.
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