DOI: 10.57760/sciencedb.28371
title: Prokaryotic Expression, Immunoactivity Analysis, and Preliminary Investigation of the Inflammatory Mechanism of a Novel House Dust Mite Allergen RP2-like Protein
title: 屋尘螨RP2样过敏原原核表达、免疫活性鉴定及致炎机制初探

description: Objective: To clone, express, and purify the ribosomal protein P2-like protein (RP2) from Dermatophagoides pteronyssinus, and to evaluate its immunoreactivity as well as preliminarily explore its pro-inflammatory mechanisms. Methods: Total RNA was extracted from D. pteronyssinus, and the Der p RP2 gene was amplified using reverse transcription polymerase chain reaction (RT-PCR). The amplified product was cloned into the pET-28a (+) vector and transformed into E. coli BL21(DE3) competent cells. After induction and purification, the recombinant protein rDer p RP2 was analyzed by 12% SDS-PAGE and Western blot with anti-IgG antibody. Its immunoreactivity was evaluated via IgE-ELISA by detecting specific IgE in sera from mite-allergic patients. BEAS-2B cells were stimulated with rDer p RP2 at concentrations of 0, 10, 20, 30, and 40 µg/mL, and mRNA expression levels of IL-25 and IL-33 were quantified using quantitative real-time PCR (qPCR). Additionally, transcriptome sequencing and qRT-PCR validation were performed on BEAS-2B cells treated with 30 µg/mL rDer p RP2 to assess the expression of inflammation-related genes. Results: A 342-bp band was successfully amplified by RT-PCR. SDS-PAGE and Western blot confirmed that the molecular weight of rDer p RP2 was approximately 16 kDa. IgE-ELISA results showed that 14.29% of allergic patient sera tested positive for rDer p RP2-specific IgE. Stimulation with rDer p RP2 significantly upregulated IL-25 (F = 33.48, P &lt; 0.000 1) and IL-33 (F = 7.57, P &lt; 0.01) mRNA expression in BEAS-2B cells. Transcriptome analysis identified 200 upregulated and 129 downregulated genes following treatment with 30 µg/mL rDer p RP2. Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes (DEGs) were primarily associated with calcium ion response, metal ion response, lipid oxidation metabolism, and immune-related biological processes and molecular functions. KEGG pathway analysis revealed significant enrichment in immune and metabolic pathways, including arachidonic acid metabolism, lipid and atherosclerosis, ABC transporters, antigen processing and presentation, and natural killer cell-mediated cytotoxicity. The qPCR results were consistent with the transcriptomic sequencing data, showing that rDer p RP2 significantly up-regulated the expression of inflammation-related genes SULF1, TNFSF10, HLA-C, and S100A4, while down-regulating the expression of RNH1 (all P &lt; 0.01). Conclusion: The recombinant Der p RP2 protein from D. pteronyssinus was successfully obtained. Serological analyses confirmed its immunoreactivity, while cellular experiments and transcriptomic sequencing further supported its immunogenic potential, suggesting that it may constitute a novel allergen. These results offer a theoretical foundation for component-resolved diagnosis and allergen-specific immunotherapy.
description: Objective: To clone, express, and purify the ribosomal protein P2-like protein (RP2) from Dermatophagoides pteronyssinus, and to evaluate its immunoreactivity as well as preliminarily explore its pro-inflammatory mechanisms. Methods: Total RNA was extracted from D. pteronyssinus, and the Der p RP2 gene was amplified using reverse transcription polymerase chain reaction (RT-PCR). The amplified product was cloned into the pET-28a (+) vector and transformed into E. coli BL21(DE3) competent cells. After induction and purification, the recombinant protein rDer p RP2 was analyzed by 12% SDS-PAGE and Western blot with anti-IgG antibody. Its immunoreactivity was evaluated via IgE-ELISA by detecting specific IgE in sera from mite-allergic patients. BEAS-2B cells were stimulated with rDer p RP2 at concentrations of 0, 10, 20, 30, and 40 µg/mL, and mRNA expression levels of IL-25 and IL-33 were quantified using quantitative real-time PCR (qPCR). Additionally, transcriptome sequencing and qRT-PCR validation were performed on BEAS-2B cells treated with 30 µg/mL rDer p RP2 to assess the expression of inflammation-related genes. Results: A 342-bp band was successfully amplified by RT-PCR. SDS-PAGE and Western blot confirmed that the molecular weight of rDer p RP2 was approximately 16 kDa. IgE-ELISA results showed that 14.29% of allergic patient sera tested positive for rDer p RP2-specific IgE. Stimulation with rDer p RP2 significantly upregulated IL-25 (F = 33.48, P &lt; 0.000 1) and IL-33 (F = 7.57, P &lt; 0.01) mRNA expression in BEAS-2B cells. Transcriptome analysis identified 200 upregulated and 129 downregulated genes following treatment with 30 µg/mL rDer p RP2. Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes (DEGs) were primarily associated with calcium ion response, metal ion response, lipid oxidation metabolism, and immune-related biological processes and molecular functions. KEGG pathway analysis revealed significant enrichment in immune and metabolic pathways, including arachidonic acid metabolism, lipid and atherosclerosis, ABC transporters, antigen processing and presentation, and natural killer cell-mediated cytotoxicity. The qPCR results were consistent with the transcriptomic sequencing data, showing that rDer p RP2 significantly up-regulated the expression of inflammation-related genes SULF1, TNFSF10, HLA-C, and S100A4, while down-regulating the expression of RNH1 (all P &lt; 0.01). Conclusion: The recombinant Der p RP2 protein from D. pteronyssinus was successfully obtained. Serological analyses confirmed its immunoreactivity, while cellular experiments and transcriptomic sequencing further supported its immunogenic potential, suggesting that it may constitute a novel allergen. These results offer a theoretical foundation for component-resolved diagnosis and allergen-specific immunotherapy.