关键词: 粉尘螨, Der f 3, 变应原, 原核表达, 鉴定

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.

Key words: Dermatophagoides farinae, Der f 3, Allergen, Prokaryotic expression, Identification