腐食酪螨Tyrp13克隆表达、纯化及免疫鉴定  

Cloning, expression, purification and immunoassay of Tyr p 13 from Tyrophagus putrescentiae(Schrank)

作　　者：钟永浩 袁如意 陈扬 季树宇 杨礼腾 刘晓宇[1] ZHONG Yonghao;YUAN Ruyi;CHEN Yang;JI Shuyu;YANG Litang;LIU Xiaoyu(Institute of Allergy and Immunology,Shenzhen University,Shenzhen,Guangdong 518060,China;Department of Allergy,the Third Affiliated Hospital of Shenzhen University,Shenzhen,Guangdong 518060,China)

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

出　　处：《中国热带医学》2020年第11期1031-1035,共5页China Tropical Medicine

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：目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基因序列(GeneBank登录号AY710432.1)设计引物,PCR大量扩增目的基因。构建原核表达载体pET-32a(+)-Tyr p 13,转化感受态细胞E. coli Rosetta(DE3),用IPTG(异丙基-β-D-硫代半乳糖苷)诱导目的蛋白表达;层析纯化表达产物,免疫印迹(Western Blot)法检测纯化后的表达产物免疫学活性;通过生物信息学软件推测其抗原表位、构建进化树。结果克隆得到的序列经基因测序可知其长度约400 bp,其表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分子量约为14 kD,呈可溶性表达。免疫印迹结果显示,Tyr p 13能够与过敏患者血清IgE特异性结合,表明其具有过敏原性;表位分析进一步证实该过敏原具有免疫原性。结论经克隆表达、纯化后得到纯度较高的腐食酪螨Tyr p 13,为进一步开展临床特异性诊断和治疗提供参考。Objective In this study,Tyrp 13 gene was cloned,the recombinant protein was expressed and purified,the immunological characteristics were identified,and the information of allergen epitope was analyzed by bioinformatics software.Methods The total RNA of the mite was extracted,RT-PCR was used to amplify the cDNA.The primers were designed according to the known Tyr p 13 gene sequence(GenBank Accession No.AY710432.1).A large number of target genes was amplified by PCR.Prokaryotic expression vector pet-32 a(+)-Tyr p 13 was constructed,then transformed into E.coli RosettaTM(DE3) Competent Cells,and the target protein expression was inducted by isopropyl β-d-1-thiogalactopyranoside(IPTG);purification of the expression product by chromatography,Western blot method was used to detect the immunological activity of the purified expression product;its antigen epitope was estimated by bioinformatics software,and its evolution tree was constructed.Results The length of the cloned DNA sequence was about 400 bp,and the expressed product was soluble in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) with molecular weight of about 14 kD.Western blotting showed that Tyr p 13 could specifically bind to IgE in sera of allergic patients,which indicated that Tyr p 13 had allergenicity;epitope analysis further confirmed that the allergen had immunogenicity.Conclusion High purity Tyr p 13 can be obtained by clone expression and purification,which lays a theoretical foundation for further clinical specific diagnosis and treatment.

关 键 词：腐食酪螨 Tyr p 13基因克隆表达 蛋白纯化 抗原表位与过敏原性 

分 类 号：R384.4[医药卫生—医学寄生虫学]