Open AccessCloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L 1
Author(s) -
TAO Ailin,
HE Shaoheng
Publication year - 2005
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2005.00194.x
Subject(s) - ambrosia artemisiifolia , biology , recombinant dna , ragweed , gene , complementary dna , genbank , molecular cloning , microbiology and biotechnology , cloning (programming) , allergen , genetics , escherichia coli , allergy , computer science , immunology , programming language
Aim: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LüCao in Chinese) and short ragweed ( Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. Methods: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3′cDNA ends were identified by using the 3′‐RACE method. The gene function conferred by the full‐length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET‐44 RosettaBlue cells were subsequently characterized by N‐terminal end sequencing, IgE binding, and cross‐reactivity. Results: Three full‐lengh cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%−90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in nonfusion forms and were confirmed by using the N‐terminal end sequence identity. Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia , but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross‐reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full‐length cDNA demonstrated that alternative nucleotide degeneracy occurred. Conclusion: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little‐studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.