Hu Zhen-hua, Chen Yong-jing, Wang Qin, Shi Bi-min, Bai Li-xiong, Zhang Xue-guang
Medical Biotechnology Institute of Soochow University, Suzhou, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 Mar;26(3):207-10.
To express human PD-1Deltaex3(DeltaPD-1) gene in eukaryotic expressing vector and identify the biological activity of the recombinant protein.
The target gene encoding full length human PD-1(PD-1) was cloned by RT-PCR, then two fragments of PD-1Deltaex3 gene were amplified and assembled by TP-PCR, PD-1Deltaex3 gene was obtained. Then the two genes PD-1 and DeltaPD-1 were inserted into the eukaryotic expressing vector pIRES2-EGFP respectively to construct the recombinant vectors pIRES2-EGFP/PD-1 and pIRES2-EGFP/DeltaPD-1. The recombinants were transfected into 293T cells with Lipofect2000 Reagent. The membrane PD-1 protein on the transfected cell surface was detected by flow cytometry. The expression of soluble PD-1 was also analysised by Western blot. The combination of DeltaPD-1 protein to the ligands of PD-1, PD-L1 and PD-L2, were determined by indirect immunofluorescence assay.
The results of enzyme digestion of recombinant vectors and DNA sequencing showed the two genes PD-1 and PD-1Deltaex3 were inserted correctly into plasmid pIRES2-EGFP, the two recombinant vectors were constructed successfully. Flow cytometry and Western blot revealed that 293T cells transfected with vector pIRES2-EGFP/PD-1 could express PD-1 protein on the cell surface but no soluble PD-1 in the supernatant of transfected cells, on the contrary, 293T cells transfected with vector pIRES2-EGFP/DeltaPD-1 could express soluble PD-1 in the culture supernatant but no membrane PD-1. Indirect immunofluorescence assay indicated the DeltaPD-1 protein could bind to the two ligands of PD-1 on the cells surface.
The recombinant eukaryotic expressing vector containing PD-1Deltaex3 was constructed successfully, and the PD-1Deltaex3 gene could encode a soluble form of PD-1 protein. DeltaPD-1 protein remained the biological activity as PD-1. This study provides the initial material for further study of PD-1Deltaex3 in PD-1/PD-L signaling pathway.
在真核表达载体中表达人PD-1Deltaex3（DeltaPD-1）基因并鉴定重组蛋白的生物学活性。
通过RT-PCR克隆编码全长人PD-1（PD-1）的靶基因，然后通过TP-PCR扩增并组装PD-1Deltaex3基因的两个片段，获得PD-1Deltaex3基因。接着将PD-1和DeltaPD-1这两个基因分别插入真核表达载体pIRES2-EGFP中，构建重组载体pIRES2-EGFP/PD-1和pIRES2-EGFP/DeltaPD-1。用Lipofect2000试剂将重组体转染到293T细胞中。通过流式细胞术检测转染细胞表面的膜PD-1蛋白。同时用蛋白质免疫印迹法分析可溶性PD-1的表达。通过间接免疫荧光测定法确定DeltaPD-1蛋白与PD-1配体PD-L1和PD-L2的结合情况。
重组载体的酶切和DNA测序结果表明，PD-1和PD-1Deltaex3这两个基因已正确插入质粒pIRES2-EGFP中，成功构建了两个重组载体。流式细胞术和蛋白质免疫印迹法显示，用载体pIRES2-EGFP/PD-1转染的293T细胞可在细胞表面表达PD-1蛋白，但转染细胞的上清液中无可溶性PD-1；相反，用载体pIRES2-EGFP/DeltaPD-1转染的293T细胞可在培养上清液中表达可溶性PD-1，但无膜PD-1。间接免疫荧光测定表明DeltaPD-1蛋白可与细胞表面的PD-1的两种配体结合。
成功构建了含PD-1Deltaex3的重组真核表达载体，且PD-1Deltaex3基因可编码可溶性形式的PD-1蛋白。DeltaPD-1蛋白保留了与PD-1相同的生物学活性。本研究为进一步研究PD-1Deltaex3在PD-1/PD-L信号通路中的作用提供了初始材料。
