屋尘螨第十类变应原的克隆、表达、纯化及免疫原性分析  

Cloning,expression,purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus

作　　者：郭楚桐 欧阳春艳 何俊贤 季树宇 杨礼腾 刘晓宇[1] GUO Chu-tong;OUYANG Chun-yan;HE Jun-xian;JI Shu-yu;YANG Li-teng;LIU Xiao-yu(Allergy and Immunology Institute,Shenzhen University,School of Medicine,Shenzhen 518060,China;Allergy Department,The Third Affiliated Hospital of Shenzhen University,Shenzhen 518001,China)

机构地区：[1]深圳大学医学院过敏反应与免疫学研究所,深圳518060 [2]深圳大学第三附属医院变态反应科,深圳518001

出　　处：《中国寄生虫学与寄生虫病杂志》2020年第6期723-729,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.31729002、 No.U1801286、 No.81971514);广州市科学研究计划重点项目(No.201804020043);深圳市孔雀计划团队项目(No.KQTD20170331145453160);深圳市科技计划国际科技合作项目(No.GJHZ2018041819053);南山区“领航团队”支持计划项目(No.LHTD20180007);呼吸疾病国家重点实验室开放课题(No.SKLRD-OP-201909)

摘　　要：目的克隆屋尘螨(Dermatophagoides pteronyssinus)第十类变应原Der p 10基因,表达并纯化Der p 10重组蛋白,分析其免疫原性和生物学信息。方法挑取经纯培养的屋尘螨,提取总RNA,逆转录生成cDNA,RT-PCR扩增Der p 10基因。将pET-24a载体与Der p 10基因连接并转化入Top10克隆菌体中,经BamHⅠ和XhoⅠ双酶切鉴定和测序后转入大肠埃希菌BL21(DE3)中,1 mmol/L IPTG诱导后,采用12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白表达情况。经Ni+离子亲和层析纯化后,以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。使用ProtParam Tools、HNN、SWISS MODEL、DNAStar预测Der p 10的理化性质、原肌球蛋白的二级和三级结构、B细胞抗原表位,Blastp和MEGA工具构建Der p 10系统进化树。结果RT-PCR获得目的基因Der p 10,其开放阅读框为855 bp,可编码284个氨基酸。双酶切结果显示Der p 10已连接至载体上。SDS-PAGE分析结果显示,Der p 10重组蛋白相对分子质量(Mr)为38000,为可溶性蛋白。Western blotting分析结果显示,Der p 10重组蛋白与尘螨过敏患者血清反应呈阳性结果。信息学分析结果显示,Der p 10蛋白不稳定,二级结构和三级结构均以α-螺旋为主。DNAStar预测到Der p 10存在1个B细胞抗原表位肽序列。Blastp和MEGA分析结果显示,与粉尘螨Der f 10亲缘关系较近,序列相似性达94.4%。结论成功构建Der p 10表达菌并纯化出与尘螨过敏患者血清中的IgE抗体结合较高及纯度较好的Der p 10重组蛋白并证实其反应原性。Objective To clone the gene of the tenth-class allergen Der p 10(tropomyosin)of Dermatophagoides pteronyssinus,express and purify recombinant protein Der p 10,and analyze the immunogenicity and biological information of the protein.Methods Cultured mites were picked for total RNA extraction,then cDNA was generated by reverse transcription and underwent RT-PCR to amplify the Der p 10 gene.The pET-24a vector was ligated with the Der p 10 gene and transformed into the Top10 cloned bacteria.After BamH I and Xho I double enzyme digestion and sequencing,the construct was transformed into Escherichia coli BL21(DE3),induced by 1 mmol/L IPTG,and examined in 12%SDS-PAGE to analyze the expression of recombinant protein.After purification by Ni+ion affinity chromatography,the immunogenicity of Der p 10 recombinant protein was analyzed by Western blotting using serum of patient with dust mite allergy as the primary antibody.The ProtParam Tools,HNN,SWISS MODEL,DNAStar were used to predict the physiochemical properties of Der p 10,the secondary and tertiary structure of tropomyosin,and B cell epitope;Blastpand MEGA tool were used to construct Der p 10 phylogenetic tree.Results The target gene Der p 10 was obtained by RT-PCR,with an open reading frame of 855 bp,encoding 284 amino acids.The results of double enzyme digestion and sequencing showed that Der p 10 was linked to the vector.SDS-PAGE analysis showed that the relative molecular mass of Der p 10 recombinant protein was 38000,which was a soluble protein.Western blotting analysis showed that the Der p 10 recombinant protein reacted with the serum of dust mite allergy patient.The bioinformatics analysis indicated that the Der p 10 protein was unstable,and its secondary and tertiary structures were mainly byα-helices.DNAStar predicted the presence of one B cell epitope peptide sequence in Der p 10.Blastp alignment and MEGA analysis showed that it was closely related to Der f 10,with sequence similarity reaching 94.4%.Conclusion The Der p 10 recombinant plasmid was constructe

关 键 词：屋尘螨 Der p 10 表达 纯化 免疫学鉴定 生物信息学 

分 类 号：R384.4[医药卫生—医学寄生虫学]