人干燥综合征A抗原克隆表达与重组蛋白及其临床应用研究

Recombinant Expression of Human Sjogren's Syndrome Antigen A and Its Clinical Application

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。

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 eDNA were designed and synthesized based on the human SSA-60kD eDNA sequence reported in GenBank. Human SSA-60kD eDNA 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 DHsato construct the recom- binant 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 SSA60kD 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.