人PD-L1 cDNA的克隆及其胞外区蛋白在大肠杆菌中的表达

Cloning of the cDNA of human PD-L1 gene and the expression of its extracellular domain in Escherichia coli

目的 :克隆人PD L1基因的cDNA并构建PD L1胞外区基因的原核表达载体 ,在大肠杆菌中进行表达。方法 :以RT PCR法从活化的人淋巴细胞总RNA中 ,扩增并克隆PD L1的cDNA ,构建PD L1胞外区基因的原核表达载体 ,在大肠杆菌BL2 1(ED3)中进行表达并鉴定。结果 :克隆到PD L1cDNA编码区的全长序列 ,经DNA测序证明其与已报道的序列一致。同时构建了在羧基端带有His6标签的PD L1胞外区基因的原核表达载体 ,并在大肠杆菌中表达。免疫印迹分析表明 ,在IPTG诱导后表达的PD L1胞外区蛋白 ,相对分子质量 (Mr)为 2 50 0 0 ,与理论值的大小相符。该重组蛋白能与抗His6标签的单抗 (mAb)特异性反应。结论 :成功地克隆PD L1基因 ,其胞外区蛋白在大肠杆菌中获得表达 ,为利用PD L1转基因修饰移植物或以PD

AIM: To clone human PD-L1 cDNA and construct a prokaryotic expression vector for the extracellular domain of PD-L1 gene. METHODS: The human PD-L1 cDNA was cloned by RT-PCR from the total RNA of activated human peripheral lymphocytes. The prokaryotic expression vector for the extracellular domain of PD-L1 gene was constructed and protein expression in E.coli strain BL21(DE3) was determined. RESULTS: The full-length coding sequence of PD-L1 gene was cloned and confirmed by DNA sequencing. The prokaryotic expression vector for the extracellular domain of PD-L1 gene fused with His_6 tag at the C-terminus was then constructed. The recombinant protein was expressed in (E.coli) after IPTG induction and identified by Western blot. The expressed product had a relative molecular mass (M_r) being 22 000, which was consistent with theoretical value. CONCLUSION: The PD-L1 gene was successfully cloned and the extracellular domain of the protein was expressed in E.coli, which lays the foundation for further study of the function of PD-L1.