人CD40基因的克隆及其胞外段的原核有效表达

目的克隆人CD40基因并原核高效表达其胞外段。方法采用RT-PCR法,从高表达人CD40抗原的XG-2细胞中扩增CD40全长基因,并将其插入pMD18-T载体中进行测序验证。用特异引物扩增CD40抗原分子的胞外段基因（可溶性CD40、sCD40基因片段）,再亚克隆至原核表达载体pGEX-5x-3,将测序正确的重组表达载体转化大肠杆菌BL21（DE3）,用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达,Western blot检测目的蛋白。结果 RT-PCR能从XG-2细胞总RNA中扩增出特异的目的基因,测序结果显示与GenBank中登录的完全一致,构建的原核表达重组载体pGEX-5x-3/hCD40ECD在表达宿主菌BL21（DE3）中经IPTG诱导能获得有效表达,Western blot证实了目的蛋白条带的特异性。结论获得了人CD40分子的基因及其胞外段原核表达产物,为进一步研究CD40分子的功能和sCD40的应用奠定了良好的基础。

Cloning and Expression of the Extracellular Domain of Human CD40 Antigen Gene

Objective To clone and express the 582 bp cDNA fragment of the extracellular domain of human CD40.Methods The cDNA of human CD40 antigen was amplified from total RNA of XG-2 cells by RT-PCR,and it was inserted into pMD18-T.The extracelluar domain of human CD40 was amplified from the recombinant vector pMD18-T/CD40,then it was subcloned into expression vector pGEX-5x-3,E.coli BL21（DE3） was transfected with the recombinant vector and the expression was induced with IPTG.Results DNA sequencing showed that the obtained 582 bp cDNA sequence was identical with the reported CD40 gene.The target fragment was expressed in bacterial cells.Conclusion The expressed product of 582 bp cDNA fragment of human CD40 obtained lays the foundation for studing the functions of human CD40 antigen.