Cloning, sequence analysis and expression in E. coli of the group 3 allergen of Dermatophagoides farinae.
BACKGROUND
The dust mites, which are mostly represented by Dermatophagoides spp. (Acari: Pyroglyphidae), are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites.


METHODS
Total RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a (+), expressed in E. coli BL21 at the aid of the inducer isopropyl-D-thiogalactopyranoside (IPTG). The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software.


RESULTS
The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f 3 cDNA clones in comparison with the reference (GenBank Accession No. AY283291), which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites (53-68 AA, 108-122 AA and 205-217 AA), one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites.


CONCLUSIONS
Der f 3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f 3 gene but this needs to be further confirmed in the future.
Cui
Yu-bao
YB
Department of Pathogenic Biology, Yancheng Health Vocational & Technical College, Yancheng, Jiangsu 224006, China.
Cai
Hong-xing
HX
Li
Li
L
Zhou
Ying
Y
Gao
Cui-xiang
CX
Shi
Wei-hong
WH
Yu
Ming
M
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D019295:Computational Biology; D040001:Dermatophagoides farinae / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D000485:Allergens; D039741:Antigens, Dermatophagoides