[Cloning, expression and purification of dust mite allergen Der f 3 and identification of its allergic activity].
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.


CONCLUSIONS
Der f3 gene has been successfully cloned and its prokaryotic expression vector is constructed.
Cai
Cheng-Yu
CY
Allergy and Immunology Institute, Shenzhen University, Shenzhen 518060, China.
Bai
Yu
Y
Liu
Zhi-Gang
ZG
Ji
Kun-Mei
KM
D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D040001:Dermatophagoides farinae / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006967:Hypersensitivity / Q000097:blood; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000485:Allergens; D039741:Antigens, Dermatophagoides; D018076:DNA, Complementary; D007073:Immunoglobulin E