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Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin).
J Allergy Clin Immunol. 1998 Mar; 101(3):363-70.JA

Abstract

BACKGROUND

Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua.

OBJECTIVES

We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy.

METHODS

Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen.

RESULTS

Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin.

CONCLUSION

This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Authors+Show Affiliations

IFIDESA-ARISTEGUI, Research and Development Department, Bilbao, Spain.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9525453

Citation

Vallverdú, A, et al. "Characterization of Recombinant Mercurialis Annua Major Allergen Mer a 1 (profilin)." The Journal of Allergy and Clinical Immunology, vol. 101, no. 3, 1998, pp. 363-70.
Vallverdú A, Asturias JA, Arilla MC, et al. Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin). J Allergy Clin Immunol. 1998;101(3):363-70.
Vallverdú, A., Asturias, J. A., Arilla, M. C., Gómez-Bayón, N., Martínez, A., Martínez, J., & Palacios, R. (1998). Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin). The Journal of Allergy and Clinical Immunology, 101(3), 363-70.
Vallverdú A, et al. Characterization of Recombinant Mercurialis Annua Major Allergen Mer a 1 (profilin). J Allergy Clin Immunol. 1998;101(3):363-70. PubMed PMID: 9525453.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin). AU - Vallverdú,A, AU - Asturias,J A, AU - Arilla,M C, AU - Gómez-Bayón,N, AU - Martínez,A, AU - Martínez,J, AU - Palacios,R, PY - 1998/4/3/pubmed PY - 1998/4/3/medline PY - 1998/4/3/entrez SP - 363 EP - 70 JF - The Journal of allergy and clinical immunology JO - J Allergy Clin Immunol VL - 101 IS - 3 N2 - BACKGROUND: Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES: We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS: Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION: This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments. SN - 0091-6749 UR - https://www.unboundmedicine.com/medline/citation/9525453/Characterization_of_recombinant_Mercurialis_annua_major_allergen_Mer_a_1__profilin__ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0091-6749(98)70249-0 DB - PRIME DP - Unbound Medicine ER -