PD-L1蛋白的表达、纯化与鉴定  

Expression, purification and identification of PD-L1 protein

作　　者：毛丽伟[1] 倪兵[1] 姜曼[1] 黎万玲[1] 肖宇[1] 吴玉章[1] 

机构地区：[1]第三军医大学基础医学部全军免疫学研究所,重庆400038

出　　处：《第三军医大学学报》2005年第5期402-405,共4页Journal of Third Military Medical University

摘　　要：目的克隆人PD -L1的基因,纯化Flp -InCHO系统表达PD- L1蛋白。方法用RT- PCR方法制备PD- L1基因,以哺乳细胞高效表达质粒载体pSec/WG为载体构建重组PD- L1/pSec/WG质粒,重组体用载体上的通用引物为测序引物,鉴定克隆的正确性。再将鉴定过的重组质粒用脂质体法转化转染Flp- InCHO细胞收集上清,G蛋白亲和层析法纯化蛋白。用免疫印迹法鉴定转化细胞所分泌上清的PD -L1基因的表达。结果正确构建了PD- L1/pSec/WG重组质粒,并且在转化细胞所分泌的上清中检测出了PD -L1基因的表达。亲和层析法成功纯化出PD- L1蛋白,分子量为40×103。结论成功地构建了PD -L1/pSec/WG重组质粒,纯化出PD -L1蛋白,可进行下一步mAb制备,进一步研究其功能。Objective To clone human programmed death-1 (PD-L1) gene to purify PD-L1 protein which is expressed stably in Flp-inCHO cell lines. Methods Recombinant PD-L1/pSec/WG plasmid was constructed by ligating PD-L1 gene, which was prepared by RT-PCR and the vector pSec/WG that can express in mammals with high efficiency. The recombinant was sequenced on an automatic sequencer with T7 promoter and T3 promoter sequence as the sequencing primers that locate upstream and downstream of the multiple cloning site (MCS) respectively, and the correctness of the recombinant was determined. The identified recombinant plasmids were then transformed into Flp-In CHO cell lines with Lipofectamine 2000 and the culture medium was collected for purification with protein G-Sepharose 4B column. The expression of PD-L1 was detected by Western blotting. Results The recombinant PD-L1/pSec/WG plasmid was constructed correctly and the expression of PD-L1 gene in the transformed Flp-inCHO cell lines was determined. The protein PD-L1 was purified successfully by protein G-Sepharose 4B column. Conclusion We have constructed successfully the recombinant PD-L1/pSec/WG plasmid and purified the protein PD-L1, based on which we can prepare mAb that aims directly at the protein PD-L1.

关 键 词：PD-L1 RT-PCR 克隆 

分 类 号：Q503[生物学—生物化学] Q513.2