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 f3 gene has been successfully cloned and its prokaryotic expression vector is constructed.
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 f3 gene has been successfully cloned and its prokaryotic expression vector is constructed.