PCR-based cloning and immunological characterization of Parietaria judaica pollen profilin.
Profilin has been described as an allergen present in pollen of trees, grasses and weeds. Since Parietaria judaica profilin has a molecular mass similar to other Parietaria allergens (Par j 1 and Par j 2) in the 14-10 kDa range, it is difficult to assess the prevalence of profilin by immunoblotting or to obtain sufficient amounts of purified native profilin for investigation and diagnosis. The aim of this study was to identify P. judaica profilin by PCR-based cDNA cloning and to elucidate its allergenic characteristics. Two cDNA clones encoding P. judaica pollen profilin were isolated by polymerase chain reaction (PCR) amplification using degenerate primers. Sequencing of both clones (Par j 3.0101 and Par j 3.0102) demonstrated a high amino acid sequence homology. Immunodetection of P. judaica pollen after isoelectrofocusing and incubation with rabbit antiserum against profilin indicated the existence of at least 2 isoforms. Expression in Escherichia coli BL21 (DE3) was carried out using a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Cross-reactivity has been found between recombinant P. judaica pollen profilin and profilins from other botanical unrelated plants.
Asturias
J A
JA
Bial-Arístegui, Research and Development Department, Bilbao, Spain. juan.asturias@bial.com
Ibarrola
I
I
Eseverri
J L
JL
Arilla
M Carmen
MC
González-Rioja
R
R
Martínez
A
A
D001483:Base Sequence; D003001:Cloning, Molecular; D003285:Contractile Proteins / Q000276:immunology*; D018076:DNA, Complementary / Q000276:immunology; D006801:Humans; D008840:Microfilament Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D032403:Parietaria / Q000276:immunology*; D028321:Plant Preparations / Q000276:immunology; D011058:Pollen / Q000276:immunology*; D016133:Polymerase Chain Reaction; D051304:Profilins; D012680:Sensitivity and Specificity
D003285:Contractile Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D028321:Plant Preparations; D051304:Profilins