关键词: 粉尘螨, Der fⅡ, 变应原, 基因多态性, 原核表达

Abstract: Der fⅡ is a major allergen of Dermatophagoides farinae, which can induce the type Ⅰ allergy. In this study, live mites were collected from Shenzhen City, and these identified as D. farinae were picked out and bred in the laboratory. The total RNA was extracted from the bred mites. The Der fⅡ gene fragment was amplified by RT-PCR and sequenced. The Der fⅡ gene was sub-cloned into the expression vector pET-His. The recombinant pET-Der fⅡ plasmid was induced to express Der fⅡ coding protein by IPTG. The recombinant Der fⅡ with 6His-tag was then purified by chelating resin, and its allergic activity was identified by Western blotting. As results, five strains of Der fⅡ gene fragment with 411 bases were determined. Comparison showed that the Der fⅡ gene sequences of the five strains from Shenzhen had an identity from 96.8% to 99.3% at the nucleotide level and 93.8% to 98.6% at the amino acid level with the homologous gene sequence deposited in GenBank (GenBank No.AB195580.1). Then they were sub-cloned into expressing vector pET-His, and five kinds of recombinant allergen Der fⅡ were highly expressed as inclusion bodies, which were then purified by 6His-tag purification system. Using Western-blotting method, the allergic activities of purified recombinant allergens were positively identified as their affinity to IgE antibodies from the mite-allergic sera of patients.  

Key words: Dermatophagoides farinae；Der fⅡ, allergen, gene polymorphism, prokaryotic expression