Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L

• 来源期刊: 中国药理学报（英文版）
• 关键词: pollen allergen profilin gene expression
• 年，卷（期）: 2005,（10）
• 页码范围: 1225-1232
• 页数: 8页
• 语种: 英文

摘要

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 crossreactivity. Results: Three full-length 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.