Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen.
Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV.
Ghosh
B
B
Division of Clinical Immunology, Johns Hopkins Asthma and Allergy, Center, Johns Hopkins University, School of Medicine, Baltimore, MD 21224.
Perry
M P
MP
Marsh
D G
DG
D000485:Allergens / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D000939:Epitopes; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics*; D011058:Pollen; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins
D000485:Allergens; D052179:Antigens, Plant; D000939:Epitopes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C069543:group 5 allergens, Ambrosia