关键词: 屋尘螨, Der p 4, 克隆, 表达, 蛋白纯化, 免疫学鉴定, 生物信息学

Abstract:

Objective To clone the Der p 4 gene of the Dermatophagoides pteronyssinus allergen, express recombinant protein, ascertain the protein immunogenicity and analyze biological information. Methods The PCR primers were designed according to the known Der p 4 gene sequence. The total RNA of D. pteonyssinus was extracted to produce cDNA by reverse transcription PCR (RT-PCR) and amplify Der p 4 gene. The sequenced gene fragment was cloned into the pET-28a (+) vector. The plasmid was extracted for sequence identification, and the recombinant plasmid was transformed into Rosetta2 (DE3) pLysS competent cells to express recombinant protein by induction with 1 mmol/L IPTG. The recombinant protein was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and purified by affinity chromatography. The immunogenicity of the recombinant protein was analyzed by Western blotting with serum from patients with D. pteronyssinus allergy as the primary antibody. The antigen specificity was assessed by indirect ELISA with the purified recombinant protein as the coating antigen for detecting, serum IgE in 30 dust mite allergy patient samples. ProtParam tools, SignalP5.0, NetPhos3.1, CD-search, Alphafold, Immune Epitope Database and Analysis Resource (IEDB) and bioinformatics prediction antigen peptide (BPAP) system were used to analyze the physicochemical properties, signal peptide sequence, phosphorylation sites, the secondary and tertiary structures and antigen epitopes of B cell were analyzed by bioinformatics approaches. Results A target gene band with a length of 1 090 bp was amplified by RT-PCR. The sequencing results of the recombinant plasmid showed that the inserted fragment was Der p 4 gene with 1 090 bp length. The SDS-PAGE analysis results showed that the Der p 4 recombinant protein was an inclusion body protein with a relative molecular weight (M_r) of approximately 60 000. The purity of the purified recombinant protein was > 90%. Western blotting analysis showed that IgE from the serum of patients with dust mite allergy specifically binds to the Der p 4 recombinant protein with a distinct band at M_r 60 000. The results of indirect ELISA showed that the recombinant protein Der p 4 was specifically bound to the serum of 14 (46.67%) dust mite allergy patients. Bioinformatics analysis showed that Der p 4 protein was highly stable, and the secondary and tertiary structures were dominated by α-helix and random coiling. The IEDB and BPAP systems predicted the presence of 15 B-cell epitope peptide sequences in Der p 4. Conclusion Purified Der p 4 recombinant protein shows reactivity and specifie binding to IgE antibodies in the serum of D. pteronyssinus allergy patients.

Key words: Dermatophagoides pteronyssinus, Der p 4, Clone, Expression, Immunological characteristics, Protein purification, Bioinformatics