Cloning and expression of house dust mite allergen Der p 3 and its antigenicity

Abstract

Abstract:

Objective To clone and express Der p 3, an allergen from house dust mite Dermatophagoides pteronyssinus, as a recombinant protein for its antigenicity test. Methods The total RNA was extracted from collected D. pteronyssinus and used for reverse transcription into cDNA. The coding DNA for Der p 3 was amplified from the total cDNA by PCR. The PCR products were sequenced and cloned into E. coli expression vector pET-32a by EcoRⅠand XhoⅠsites. The recombinant Der p 3 protein with His-tag was expressed in E. coli Rosetta under induction of 1 mmol/L IPTG and purified with nickel column. The antigenicity of recombinant Der p 3 was tested by Western blotting with sera from patients of asthma allergic to dust mites. The bioinformatics and structure of Der p 3 was analyzed by using BLAST ProtParam PSIPRED SWISS-MODEL and MEGA toolkit. IEBD and DNA Star was used to predict the epitopes of Der p 3 protein. Results The coding DNA for Der p 3 was amplified from D. pteronyssinus total cDNA by RT-PCR. The open reading frame of Der p 3 was 786 bp, encoding 261 amino acids. The recombinant Der p 3 protein was expressed as insoluble inclusion body in E. coli Rosetta under induction of IPTG, with size of M_r 30 000. The inclusion body was solubilized with urea and purified by nickel column chromatography. Western blotting demonstrated that the recombinant Der p 3 was strongly recognized by IgE in sera from dust mite allergic asthma patients, indicating a potential allergen for asthma. Homology analysis revealed that it shared 80% sequence identity with of Der f 3 from D. farina. Structure of Der p 3 protein was stable containing helix, extension chain and random coil. Six immunogenic epitopes were predicted. Conclusion Der p 3 is cloned from house dust mite D. pteronyssinus and expressed as recombinant protein. The expressed recombinant Der p 3 protein can strongly be recognized by IgE in sera from dust mite allergic asthma patients, indicating its potential as an allergen for asthma.

Key words: Dermatophagoides pteronyssinus, Der p 3, Allergen, Bioinformatics

CLC Number:

R384.4

Xia GU, Chun-yan OUYANG, Ru-yi YUAN, Xie-fang YUAN, Guo-ping LI, Zhi-gang LIU. Cloning and expression of house dust mite allergen Der p 3 and its antigenicity[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(6): 627-631.

Figures/Tables 4

References 22

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