摘要
目的分离纯化人程序死亡蛋白-1(PD-L1)基因,制备重组人PD-L1蛋白,为进一步研究PD-L1在肿瘤免疫逃逸中的作用和机制建立基础。方法通过RT-PCR分离纯化人PD-L1基因,并将其克隆到原核表达质粒pGEX-4T-1中,经酶切,测序鉴定分析后,转化大肠杆菌BL21(DE3),经IPTG诱导表达。产物经GST蛋白纯化系统进行纯化后,用10%SDS-PAGE及Western Blot分析鉴定。结果经RT-PCR分离纯化的PD-L1 cDNA分子由645 bp构成,编码相对分子量约25 KD的蛋白,并与GST形成融合蛋白。GST-PD-L1融合蛋白相对分子量约46 KD。Western Blot结果显示,该蛋白能被兔抗GST和抗PD-L1抗体识别。结论成功构建了PD-L1原核表达载体,并在大肠杆菌BL21中获得高效表达。
OBJECTIVE To further understand the mechanism of PD - L1 in tumor immune evasion, rhPD - L1 cDNA was isolated and expressed in BL21 （ DE3 ）. The protein was purified and indentified for following use. METHODS The extracellular region cDNA of PD - L1 was isolated and purified by RT - PCR. pGEX - PD - L1 plasmid was constructed by enzyme digestion. The plasmid was transformed into E. coli BL - 21 （ DE3 ） following indentified with endonuelease digestion, sequencing and PCR analysis. PD - L1 protein was expressed under IPTG induction and purified by GST protein purification system, then identified by SDS - PAGE and Western blot. RESULTS The fragment of PD - L1 cDNA constructed with 645bp . The relative molecular weight of PD - L1 protein was about 25 KD. The relative molecular weight of GST - PD - L1 fusion protein was 46KD. Western Blot proved that the protein was recognized by rabbit anti - GST polyclonal antibody and anti - PD - L1 antibody. CONCLUSION pGEX -4T - 1/PD - L1 vector was success- fully constructed, and the PD- Llprotein was expressed in E. coli BL21 （DE3） with high efficiency.
出处
《华西药学杂志》
CAS
CSCD
北大核心
2009年第5期487-490,共4页
West China Journal of Pharmaceutical Sciences
关键词
程序死亡蛋白-1
原核表达
纯化和鉴定
大肠杆菌BL-21
Programmed death - 1 ligands （ PD - L1 ）
Prokaryotic expression
Purification and Identification
BL - 21
