屋尘螨过敏原Der p 4的克隆、表达、免疫原性鉴定及生物信息学分析  

Cloning,expression,immunogenicity and bioinformatics analysis of Dermatophagoides pteronyssinus allergen Der p 4

作　　者：李启松 杨李 腾飞翔 马桂芳 LI Qisong;YANG Li;TENG Feixiang;MA Guifang(School of Public Health and Management,Jiangsu Vocational College of Medicine,Yancheng 224005,Jiangsu,China)

机构地区：[1]江苏医药职业学院公共卫生与管理学院,盐城224005

出　　处：《中国寄生虫学与寄生虫病杂志》2024年第1期42-47,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：江苏省高校哲学社会科学研究项目(2019SJA2332);江苏高校哲学社会科学研究基金(2018SJA1582)。

摘　　要：目的克隆表达屋尘螨过敏原Derp 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Derp4基因序列,设计PCR引物,提取屋尘螨总RNA,逆转录PCR(RT-PCR)扩增Derp4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/LIPTG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Derp 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1090 bp的条带。重组质粒测序结果显示,插入的片段为全长1090 bp的Derp 4基因序列。SDS-PAGE分析结果显示,Derp 4重组蛋白为包涵体蛋白,相对分子质量(Mr)约60000,经纯化后的重组蛋白纯度>90%。Western blotting分析结果显示,Derp 4重组蛋白可与尘螨过敏患者血清中的IgE特异性结合,在Mr60000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Derp4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(RTPCR)and amplify Der p 4 gene.The sequenced gene fragment was cloned into the pET28a(+)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 sulfatepolyacrylamide gel electrophoresis(SDSPAGE),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,CDsearch,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 1090 bp was amplified by RTPCR.The sequencing results of the recombinant plasmid showed that the inserted fragment was Der p 4 gene with 1090 bp length.The SDSPAGE analysis results showed that the Der p 4 recombinant protein was an inclusion body protein with a relative molecular weight(Mr)of approximately 60000.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 De

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 

分 类 号：R384.42[医药卫生—医学寄生虫学]