人自身抗原52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70,SSB-La的原核表达及纯化鉴定  

Prokaryotic expression and purification identification of human auto-antigen 52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70,SSB-La

作　　者：李欣[1] 吴淡娟[1] 李海侠[1] 褚帅[1] 康霞[1] 裘宇容[1] 

机构地区：[1]南方医科大学南方医院检验科,广东广州510515

出　　处：《中国实验诊断学》2016年第3期364-368,共5页Chinese Journal of Laboratory Diagnosis

基　　金：广东省教育部产学研结合项目(2012B091100461)

摘　　要：目的制备重组人自身抗原52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70以及SSB-La,为建立自制抗核抗体荧光免疫层析快速检测试剂盒奠定基础。方法在NCBI上查询人52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70,SSB-La的碱基序列和氨基酸序列,运用软件预测抗原表位,选择抗原表位并对密码子进行偏嗜性改造,采用人工合成方法合成抗原表位序列并插入至原核表达载体pET30a中。将重组质粒导入大肠杆菌BL21经IPTG诱导表达,表达的重组蛋白用镍离子亲和层析柱纯化,用荧光免疫法测定重组蛋白活性。结果重组人自身抗原SSB-La是可溶性表达,52-kDRo/SSA,rRNP,Jo-1,SmD1以及Scl-70是包涵体形式表达;纯化产物经荧光免疫法测定活性,结果显示:SSB-La,Scl-70以及Jo-1具有较高的抗原活性,52-kDRo/SSA,rRNP以及SmD1抗原活性较弱。结论成功制备了重组人自身抗原52-KDRoSSA,rRNP,Jo-1,SmD1,Scl-70以及SSB-La,为下一步研究开发自制抗核抗体荧光免疫层析快速检测试剂盒奠定了基础。Objective To obtain recombinant human autoantigen 52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70 and SSB-La,for the preparation of antinuclear antibody immunofluorescence chromatography rapid detection kit.Methods Nucleotide and amino acid sequences of 52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70 and SSB-La were found on NCBI.After codon optimization,the epitope sequences selected by using epitope prediction software were linked into prokaryotic expression vector pET-30 ato get recombinant vectors which then were transformed into BL21.The recombinant proteins were got by IPTG induction.After nickel column purification,Recombinant protein activity was measured by fluorescence immunoassay assay.Results SSB-La is a soluble protein,the others are insoluble proteins.Fluorescence immunoassay assay results show：SSB-La,Jo-1and Scl-70 have high activity,but the activity of 52-kDRo/SSA,rRNP,Jo-1and SmD1 is low.Conclusion Recombinant human autoantigen 52-kDRo/SSA,rRNP,Jo-1,SmD1,Scl-70 and SSB-La are successfully obtained in this study,which lays the foundation for ntinuclear antibody immunofluorescence chromatography rapid detection kit.

关 键 词：重组人自身抗原 抗核抗体 抗原表位 荧光免疫法 

分 类 号：R446.6[医药卫生—诊断学]