【摘要】 克隆人干燥综合征A抗原基因(SSA-60kD),为SSA抗原的表达和使用重组抗原建立ELISA法用于自身抗体的临床检测。根据GenBank中检索到的人SSA-60kD cDNA序列,在5’和3’非编码区设计特异性引物,提取人源HeLa细胞总RNA作为模板,经RT-PCR扩增SSA-60kD抗原cDNA。PCR产物纯化后连接至载体PET-30a,导入大肠杆菌DH5α,构建重组质粒PET-30a-SSA-60kD。对后者进行酶切鉴定、测序和分析。在大肠杆菌中表达,产生重组融合蛋白。RT-PCR扩增产物为1632bp。PET-30a-SSA-60kD经EcoR I和V双酶切证实含目的基因片段。序列分析提示与GenBank中一致。利用金属螯合亲和层析法和纯化,得到了高纯度的SSA-60kD重组蛋白。成功克隆人SSA-60kD基因,并构建PET-30a-SSA-60kD,建立了ELISA方法,检测了风湿病患者血清中抗SSA-60kD自身抗体。更多还原

【Abstract】 To clone human Sjogren’s syndrome antigen A(SSA) for expression of antigen SSA-60kD and establish a new clinical detecting method,primers of human SSA-60kD cDNA were designed and synthesized based on the human SSA-60kD cDNA sequence reported in GenBank.Human SSA-60kD cDNA was amplified from RNA of cultured HeLa cell by reverse transcriptase polymerase chain reaction(RT-PCR).The production of amplification was ligated to PET-30a vector and then transformed into the competent bacteria DH5α to construct the recombinant plasmid PET-30a-SSA-60kD.The recombinant plasmid was digested with EcoR Ⅰ and EcoR Ⅴ,and positive clones were sequenced.The recombinant protein was obtained after construction of recombinant expression vector and expression in E.coli system.The Human SSA-60kD cDNA fragment containing 1632bp was amplified by RT-PCR.Restriction endonuclease mapping using EcoR Ⅰ and EcoR Ⅴ showed that the target gene was inserted into the recombinant plasmid.The complete coding sequence of Human SSA-60kD was consistent with that of GenBank through DNA sequencing.Metal chelate resin chromatography column was used for purification of recombinant protein which has SSA-60kD antigenity and high purity.The full length of human SSA-60kD cDNA was successfully cloned and the recombinant plasmid PET-30a-SSA-60kD was constructed.The ELISA method was established by using the recombinant protein as the antigen for the detection of serum SSA-60kD autoantibody in rheumatic patients.更多还原