小鼠PD-1Ig真核表达质粒的构建与表达

Objective: To construct the PD-1Ig encoding plasmid, and prepare it in large scale. It will facilitate the study of the immunotherapy of malignant melanoma by blocking the PD-1 coinhibitory pathway. Methods: First, the ectodomain of murine programmed cell death 1（PD-1）was predicted by TMpred software and UniProt protein knowledgebase, and its encoding cDNA was cloned by RT-PCR from the total RNA of lymph nodes of a C57BL/6 mouse. Then, the eukaryotic expression vector for PD-1Ig, i.e. the ectodomain of murine PD-1 fused to murine Ig Fc-containing domains, was constructed, and verified by sequence analysis. Its expression in vitro and in vivo was detected by ELISA and RT-PCR, respectively. Finally, the pPD-1Ig plasmid was prepared in large scale with QIAGEN EndoFree Plasmid Giga Kit. Results: The prediction of murine PD-1 revealed that the potential signal peptide was from amino acid residues (aa) 1 to 20, and the most possbile starting point of transmembrane domain was aa 169-170. Therefore, the cDNA encoding the ectodomain of murine PD-1 (aa 21-168) was amplified by RT-PCR, and subcloned to pIg-tail plasmid. The concentration of PD-1Ig and Ig-tail in the culture supernatant of corresponding plasmid-transfected B16F10 melanoma cell line were 17.53 ng/mL and 21.81 ng/mL, respectively. The expected size of RT-PCR product for PD-1Ig or Ig-tail was observed in the muscle samples from the injection sites at 1 d and 3 d post-plasmid injection, respectively. Conclusion: The eukaryotic expression vector for PD-1Ig was constructed and verified, and it can be expressed in vitro and in vivo. The pPD-1Ig and pIg-tail plasmid prepared in large scale facilitated the further immunotheapy of maliganant melanoma.?????