人干燥综合征B抗原重组体的构建及融合表达

Construction and Fusion Expression of Recombinant Human Sjogren's Syndrome Antigen B

目的克隆人干燥综合征B抗原基因(human sjogren’s syndrome antigen B,SSB)并进行原核表达,为使用重组抗原用于自身抗体的临床检测奠定基础。方法根据GenBank中检索到的人SSB cDNA序列,在5′非编码区和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,逆转录RT-PCR扩增人干燥综合征B抗原cDNA。PCR产物纯化后连接至载体PET-30a,导入大肠埃希菌DH5α,构建重组质粒PET-30a-SSB。对重组质粒进行酶切鉴定,选择阳性克隆测序。重组质粒导入大肠埃希菌BL21,阳性克隆经鉴定后在IPTG诱导下表达。结果RT-PCR扩增产物为1245bp。重组质粒PET-30a-SSB经EcoR Ⅰ和HindⅢ双酶切证实含目的基因片段。序列分析提示与GenBank中检索到的一致。SDS-PAGE和Western印迹结果显示融合蛋白相对分子量为73ku,具有天然SSB抗原活性。结论成功克隆SSB基因并表达融合蛋白。

Objective To clone and express human Sjogren＇ s syndrome antigen B （SSB） in E. Coli to establish a new clinical detecting method. Methods According to the human SSB eDNA sequence reported in GenBank, primers of human SSB eDNA were designed and synthesized. Human SSB eDNA was amplified from RNA of cultured Hela cells by reverse transeriptase polymerase chain reaction （RT-PCR）. The product of amplification was ligated to PET-30a vector and then transformed into the competent bacteria DH5α to construct the recombinant plasmid PET-30a-SSB. The recombinant plasmid was digested with EcoR Ⅰ and Hind Ⅲ, and positive clones were sequenced. The recombinant plasmid was transformed into E. Coli BL21. The positive clones were identified by restriction enzymes and induced by IPTG. Results The human SSB eDNA fragment containing 1 245bp was amplified by RT-PCR. Restriction endonuelease mapping using EcoR Ⅰ and Hind Ⅲ showed that the target gene was inserted into the recombinant plasmid. The complete coding sequence of human SSB was consistent with .that of GenBank as confirmed by DNA sequencing. The PET-30a-SSB positive clones produced a fusion protein with molecular weight of 73kD which had natural immunogenicity of SSB as showen by SDS-PAGE and Western blot. Conclusion The full length of human SSB eDNA was successfully cloned and the fusion protein was expressed.