Allergen Gene Cloning

Abstract

For gene cloning of known allergens, primers are designed according to individual allergen genes and selective amplification by PCR, however, for cloning of an unknown or unstudied species, the design of the degenerate primers is crucial to its successful amplification by PCR. In order to achieve high expression efficiency of the desired allergen gene in a prokaryotic system or other protein expression system, it is necessary to optimize the allergen gene codon. After codon optimization of the allergen gene sequences, protecting bases and an affinity purification tag are added to the 3′ and 5′ end restriction loci and then sent out for sequence synthesis. The prokaryotic protein expression system is the most commonly used expression system and is also the most affordable. During the prokaryotic protein expression process, high yields of the target protein can be obtained by optimizing the host strain and induction conditions including inducing temperature, inducing agent, inducing time, and IPTG concentration. In the Eschericha coli system, most recombinant proteins are expressed in inclusion bodies. The denaturation and refolding of inclusion bodies are very important to obtain allergens that have biological activities. Finally, recombinant allergens can be purified using chromatography, isoelectric point precipitation, and salt fractionation.