ABSTRACT
A cDNA clone coding for the major house dust mite allergen Der p 1 was isolated from a lambda gt 11 library. Its sequence correlates with known amino acid sequences of Der p 1 and it produces a fusion protein which reacts with rabbit anti-Der p 1 antiserum.
Subject(s)
Allergens/isolation & purification , Mites/genetics , Allergens/genetics , Amino Acid Sequence , Animals , Antigens, Dermatophagoides , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Mites/immunology , Molecular Sequence DataABSTRACT
A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact residues making up the active site. Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene. These data will now facilitate epitope mapping studies.
Subject(s)
Allergens/genetics , Cysteine Endopeptidases/genetics , Mites/genetics , Amino Acid Sequence , Animals , Antigens, Dermatophagoides , Base Sequence , DNA/genetics , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
A technique is described for obtaining 'prints' of the antibody output of individual antibody-secreting cells (ASC), by repeated exposure of an immobilized ASC monolayer to coverslips coated with antigen. Zones of bound antibody on the coverslips (each being the 'print' of an individual ASC) are subsequently visualized by ELISA technology.
Subject(s)
Antibody-Producing Cells/immunology , Immunoglobulin Isotypes/analysis , Cell Count , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Hemolytic Plaque TechniqueABSTRACT
IgE production was examined in a human myeloma cell line (U266) in cultures in exponential growth and in cultures where cell growth was synchronized by thymidine blockade. Antibody secretion is shown to be restricted to late G1/early S phases of the cell cycle in synchronized cultures, as judged by the slope of the curve relating supernatant IgE levels to time. Employing the ELISA-plaque assay to enumerate cells in active IgE secretion, the frequency of the latter was also shown to peak in S phase. These data collectively suggest that IgE gene transcription occurs only during limited periods of the cell cycle. These results are discussed in terms of using U266 to study the mechanism of action of regulatory T-cell factors which affect IgE synthesis and/or secretion.
