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Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3)  Horng-Der Shen, PhDa, Chih-Wen Wang, BSca,

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Presentation on theme: "Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3)  Horng-Der Shen, PhDa, Chih-Wen Wang, BSca,"— Presentation transcript:

1 Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3)  Horng-Der Shen, PhDa, Chih-Wen Wang, BSca, Hong Chou, PhDa, Win-Ling Lin, BSca, Ming F. Tam, PhDb, Mei-Hsiang Huang, BSca, Ming-Ling Kuo, PhDc, Soo-Ray Wang, MD, PhDd, Shou-Hwa Han, MD, PhDe  Journal of Allergy and Clinical Immunology  Volume 105, Issue 4, Pages (April 2000) DOI: /mai Copyright © 2000 Mosby, Inc. Terms and Conditions

2 Fig. 1 Nucleotide and inferred amino acid sequences of cDNA clone PCE2. Numbers to the right are nucleotide positions. Putative glycosylation site (Asn-Ala-Ser) and TAA stop codon are underlined. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2000 Mosby, Inc. Terms and Conditions

3 Fig. 2 Comparison of deduced amino acid sequence of PCE2 and peroxisomal membrane protein allergen (Asp f 3) of A fumigatus. Residues identical to PCE2 sequence are indicated by dot. Dashes, Spaces that have been introduced to optimize alignment. Putative N-glycosylation site is underlined. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2000 Mosby, Inc. Terms and Conditions

4 Fig. 3 IgE immunoblot reactivity to components of P citrinum in asthmatic serum samples. A, Coomassie blue–stained protein profile of P citrinum extract on PVDF membrane. Immunoblots generated from 29 asthmatic serum samples are presented in B. Sera 16, 18, 19, and 20 have negative IgE immunoblotting. Serum 29 was a negative control obtained from mite-sensitive asthmatic patient. Molecular mass standards (Pharmacia, Uppsala, Sweden) used were phosphorylase b (94 kd), BSA (67 kd), ovalbumin (43 kd), carbonic anhydrase (30 kd), soybean trypsin inhibitor (20.1 kd), and α-lactalbumin (14.4 kd). Results of commercial Pharmacia CAP test to P notatum of individual serum sample tested are expressed as RAST classes and are shown in B. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2000 Mosby, Inc. Terms and Conditions

5 Fig. 4 Immunoblot reactivity of PCE2-GST and Asp f 3-GST fusion proteins to IgE antibodies in asthmatic serum samples. A, Coomassie blue–stained SDS-PAGE protein profiles of affinity-purified PCE2-GST and Asp f 3–GST fusion proteins. Strips in B show immunoblot reactivity of recombinant proteins to IgE antibodies in sera from 13 asthmatic patients (serum No in Fig 3). Strip No. 14 represents result of cord serum sample. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2000 Mosby, Inc. Terms and Conditions

6 Fig. 5 Inhibition of IgE immunoblot reactivity of a mixture of serum samples (serum No. 1, 3, 5, 7, 8, 11, and 12 in Figs 3 and 4) that has positive reactivity to PCE2 (A) and Asp f 3 (B). Strips 1 in both panels show Coomassie blue–stained protein profiles of recombinant PCE2 and Asp f 3 on PVDF membranes. Strips 2 show IgE immunoblot reactivity of serum mixture against individual recombinant allergen. Strips 3 to 8 represent results of immunoblot obtained by incubating PVDF blots with same serum mixture that has been mixed and preincubated with 1 and 5 μg, respectively, of PCE2 (strips 3 and 4), Asp f 3 (strips 5 and 6), and BSA (strips 7 and 8). Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2000 Mosby, Inc. Terms and Conditions

7 Fig. 6 Immunoblot reactivity of 6 mAbs to recombinant PCE2 (I-B), Asp f 3 (I-D), crude fungal extracts of P citrinum(II-B) and A fumigatus(II-D). I-A, I-C, II-A, and II-C, Coomassie blue–stained SDS-PAGE protein profiles of the PCE2-GST, Asp f 3–GST, P citrinum, and A fumigatus fungal extracts, respectively. Strips 1-6 in B and D summarize immunoblot reactivity of mAbs PCE2-3, PCE2-7, PCE2-11, PCE2-15, PCE2-19, and PCE2-21 to recombinant proteins (I) and crude fungal extracts (II), respectively. Culture supernatant containing mAb WH20 against house dust mite allergen Der p 7 shows negative result (strips 7). Strips 8 and 9 in all panels show immunoblot results with serum No. 8 in Figs 3 and 4 and a cord serum sample, respectively. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2000 Mosby, Inc. Terms and Conditions

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