目的 克隆表达屋尘螨过敏原Der p 4重组蛋白，鉴定其免疫原性并对其生物信息学进行分析。方法 根据已知Der p 4基因序列，设计PCR引物，提取屋尘螨总RNA，逆转录PCR（RT-PCR）扩增Der p 4基因。将测序正确的基因片段克隆至pET-28a(+)载体，提取质粒测序鉴定，将重组质粒转入Rosetta2（DE3）pLysS感受态细胞，1 mmol/L IPTG诱导表达后，采用12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白的表达情况。亲和层析法纯化重组蛋白后，以屋尘螨过敏患者血清为一抗，蛋白质免疫印迹（Western blotting）分析重组蛋白的免疫原性。以纯化后的重组蛋白为包被抗原，间接ELISA法检测30份尘螨过敏患者血清IgE，鉴定其特异性。利用ProtParam tools、SignalP5.0、NetPhos3.1、CD-search、Alphafold、Immune Epitope Database and Analysis Resource（IEDB）等软件和生物信息学预测抗原肽（BPAP）系统等对Der p 4的理化性质、信号肽序列、磷酸化位点、二级和三级结构及B细胞抗原表位进行生物信息学分析。结果 RT-PCR扩增出长度为1 090 bp的条带。重组质粒测序结果显示，插入的片段为全长1 090 bp的Der p 4基因序列。SDS-PAGE分析结果显示，Der p 4重组蛋白为包涵体蛋白，相对分子质量（M_r）约60 000，经纯化后的重组蛋白纯度> 90%。Western blotting分析结果显示，Der p 4重组蛋白可与尘螨过敏患者血清中的IgE特异性结合，在M_r 60 000处有明显条带。间接ELISA检测结果显示，Der p 4重组蛋白与14份（46.67%）尘螨过敏患者血清呈特异性IgE结合。生物信息学分析显示，Der p 4蛋白稳定性较高，无信号肽结构，二级结构和三级结构以α-螺旋和无规则卷曲为主；IEDB和BPAP系统预测到Der p 4蛋白存在15个B细胞抗原表位肽序列。结论 纯化的Der p 4重组蛋白具有免疫原性，与屋尘螨过敏患者血清中的IgE抗体结合较好。

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.