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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。
Objective To clone human CD40 gene and express its extracellular domain in prokaryotic cells. Methods The full-length CD40 gene was amplified by RT-PCR from XG-2 cells highly expressing human CD40 antigen and inserted into pMD18-T vector for sequencing. The extracellular domain of CD40 antigen molecules (soluble CD40 and sCD40 gene) were amplified by specific primers and subcloned into the prokaryotic expression vector pGEX-5x-3. The correct recombinant expression vector was transformed into E. coli BL21 (DE3) The expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the target protein was detected by Western blot. Results RT-PCR was able to amplify specific target gene from total RNA of XG-2 cells. The sequencing results showed that the recombinant plasmid pGEX-5x-3 / hCD40ECD was identical with that of GenBank. (DE3) induced by IPTG to obtain effective expression, Western blot confirmed the specificity of the target protein band. Conclusion The prokaryotic expression product of human CD40 and its extracellular domain were obtained, which laid a good foundation for further study on the function of CD40 and the application of sCD40.