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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 L1重组蛋白抑制移植排斥反应等研究提供了条件
OBJECTIVE: To clone the cDNA of human PD L1 gene and construct the prokaryotic expression vector of extracellular domain of PD L1, which was expressed in E. coli. Methods: The PD L1 cDNA was amplified and cloned from total RNA of human lymphocytes activated by RT-PCR. The prokaryotic expression vector of extracellular domain of PD L1 was constructed and expressed in E. coli BL21 (ED3) . Results: The full length sequence of PD L1 cDNA coding region was cloned and confirmed by DNA sequencing to be consistent with the reported sequence. At the same time, we constructed a prokaryotic expression vector of PD L1 extracellular domain with His6 tag at the carboxyl end and expressed it in E. coli. Immunoblot analysis showed that the relative molecular mass (Mr) of the extracellular domain of PD L1 expressed after IPTG induction was 2 50 0 0, which was consistent with the theoretical value. The recombinant protein can specifically react with anti-His6 tag mAb. CONCLUSION: The successful cloning of PD L1 gene and the expression of its extracellular domain protein in E. coli provide the conditions for the study of graft rejection by PD L1 transgene or inhibition of graft rejection with PD L1 recombinant protein