Cloning and sequencing

By the mid-1980s a large number of allergens had been purified using biochemical techniques, bur in general the yields were poor and, apart from physicochemical properties, the molecular structures of these proteins were poorly defined. With the advent of molecular cloning techniques, the primary structures of many allergens were rapidly established and, by screening cDNA expression libraries with IgE antibodies, new allergens were identified and sequenced. More than 100 allergen sequences are now listed in protein databanks (GENBANK) and a partial listing of major allergens is shown in Table 3.

The aim of cloning and sequencing is to obtain the primary structure of the allergen and to express the allergen in recombinant form. For many major allergens, N-terminal or internal amino acid sequences, and monospecific antibodies, were available to confirm the identity of cDNA clones. In some cases, e.g. cat allergen, Fel d 1 or Aspergillus allergen. Asp f 1, the nucleotide sequences were obtained by polymerase chain reaction (PCR) using primers derived from amino acid sequence. However, the usual approach has been to isolate mRNA from allergen source material (e.g. pollen grains, mite or cockroach bodies, fungal spores) and to use reverse transcriptase to generate double-stranded cDNA, which is then packaged into A.gtll or similar expression vectors. The cDNA libraries are screened with pooled IgE antibodies from 6-8 allergic patients, or with high titer monospecific polyclonal antibodies, to identify cDNA clones expressing allergen. Increasingly, allergens are being identified solely based on screening cDNA libraries and this approach has been very successful for allergens which were previously ill defined, e.g. fungi (Alternaria, Aspergillus, Trichophyton,); foods (peanut, shrimp); and insects (cockroaches, fire ants, hornets).

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