Cloning,Expression and Purification of Dust Mite Allergen Der f 3 and Identification of its Allergic Activity / 中国寄生虫学与寄生虫病杂志

ABSTRACT

Objective To clone,express and identify Der f 3 gene.Methods Live mites were collected from southern China region,identified as Dermatophagoides farinae,and cultured.The total RNA was extracted.The Der f 3 gene fragment was amplified by RT-PCR and sequenced.The Der f 3 gene fragment encoding a serine protease mature peptide was sub-cloned into the expression vector pET-His.The recombinant pET-Der f 3 plasmid was inserted into E.coli BL21 and induced to express Der f 3 coding protein by IPTG.The recombinant Der f 3 with 6 his-tag was then purified by chelating resin and its allergic activity was identified by Western blotting.Results The Der f 3 gene fragment with 840 bases was determined.Its sequence homology with the published one(GenBank No.D63858) was 99.5% at nucleotide level.It was sub-cloned into expressing vector pET-His and the recombinant allergen rDer f 3 was highly expressed in E.coli BL21(DE3) under induction of IPTG,and purified by 6-His-tag purification system.Using Western blotting method,the allergic activity of the purified recombinant allergen was identified as its affinity to IgE antibodies from the mite-allergic patient sera.Conclusion Der f 3 gene has been successfully cloned and its prokaryotic expression vector is constructed.