Cloning,sequence analysis and expression in E.coli of the group 3 allergen of Dermatophagoides farinae
10.3760/cma.j.issn.0366-6999.2009.21.025
- Author:
Yu-Bao CUI
1
;
Hong-Xing CAI
;
Li LI
;
Ying ZHOU
;
Cui-Xiang GAO
;
Wei-Hong SHI
;
Ming YU
Author Information
1. Department of Pathogenic Biology,Yancheng Health Vocational & Technical College,Yancheng,Jiangsu 224006,China
- Keywords:
Dermatophagoides farinae;
Derf 3;
recombinant allergen;
cloning;
gene expression;
bioinformatics
- From:
Chinese Medical Journal
2009;(21):2657-2661
- CountryChina
- Language:Chinese
-
Abstract:
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 Derrnatophagoides 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 isopropyI-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 hA),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 f3 gene but this needs to be further confirmed in the future.
