PD-L1胞外域密码子优化及原核表达纯化

Codon Optimization and Prokaryotic Expression Purification of PD-L1 Extracellular Segment

目的获取活性的PD-L1胞外域蛋白。方法本研究基于大肠杆菌的原核表达系统,通过稀有密码子优化,将PD-L1胞外域优化后的基因序列转入pET-28a(+)载体中,诱导其以可溶性状态在细菌上清裂解液中表达,再通过SDS-PAGE考马斯亮蓝染色对所得蛋白进行检测分析。结果在密码子优化前,改变温度或者IPTG浓度等诱导条件,目的蛋白始终在包涵体中表达,无法得到保持生物活性、可溶性的PD-L1胞外域蛋白;密码子优化后,通过摸索诱导条件,发现在16℃,0.1mmol/L IPTG的诱导条件下,诱导12h,成功在细菌上清裂解液中得到可溶性的PD-L1胞外域目的蛋白,经镍离子螯合纯化树脂纯化后,获得了保持生物活性及结构的PD-L1胞外域目的蛋白。结论成功获取活性的PD-L1胞外域蛋白为SELEX筛选识别PD-L1胞外域特异性DNA/RNA适体提供实验基础,为通过DNA/RNA适体与PD-L1结合从而阻断PD-1/PD-L1信号通路导致的肿瘤的免疫逃逸提供了新的临床思路。

Objective To obtain the active PD-L1 extracellular domain protein.Methods The optimized gene sequence of PD-L1 extracellular domain was transferred into pET-28a(+)vector and induced its expression in E.coli.PD-L1 extracellular domain protein was analyzed by SDS-PAGE and Coomassie brilliant blue(CBB).Results We found that before codon optimization,the PD-L1 extracellular domain protein was still expressed in bacterial precipitation regardless of change in induction temperature or IPTG concentration.The soluble PD-L1 extracellular domain protein was successfully obtained in the bacterial supernatant lysate under the induction conditions of 16℃and 0.1mmol/L IPTG for 12 h after codon optimization.PD-L1 protein with biological activity and structure was harvested by His-Tag resin.Conclusion Our study found an experimental basis for DNA/RNA aptamers of PD-L1 extracellular domain using SELEX,provided novel insights into the treatment of immune escape of cancer cells.