小鼠PD-1Ig真核表达质粒的构建与表达
刘毅1，徐丽慧2，何贤辉2，孙建方1
基金项目：高等学校博士学科点专项科研基金（20091106120001） 作者简介：刘毅（1973-），男，副主任医师，主要研究方向：皮肤肿瘤的生物治疗,皮肤病的免疫学发病机制.

（1. 中国医学科学院皮肤病研究所，南京 210042； 2. 暨南大学生命科学学院，广州 510632）
摘要：目 的 构建并大规模制备PD-1Ig编码质粒，为探讨阻断PD-1通路对恶性黑素瘤免疫治疗效应的影响创造条件。 方法 用TMpred软件和UniProt蛋白质数据库预测小鼠程序性死亡受体-1（programmed cell death 1, PD-1）胞外域，RT-PCR克隆其编码序列，构建PD-1Ig编码质粒，并测序验证；然后分别应用ELISA和RT-PCR方法检测其体内外表达情况；并大规模制备pPD-1Ig 和pIg-tail质粒。结 果 预测小鼠PD-1分子第1-20残基为信号肽， 第169-170残基为跨膜结构的起点可能性大。成功扩增小鼠PD-1胞外域（第21-168残基）的编码基因，并克隆至pIg-tail中。pPD-1Ig和pIg-tail质粒分别转染B16F10细胞，ELISA检测培养上清中PD-1Ig和Ig-tail浓度分别为17.53 ng/mL和21.81 ng/mL。注射pPD-1Ig和pIg-tail质粒1 d、3 d后均扩增出与预期大小相符的DNA片段。结 论 成功构建了PD-1Ig真核表达载体，证实其能在体内外表达，并大规模制备了pPD-1Ig和pIg-tail质粒，为后续免疫治疗研究创造了条件。
关键词：程序性死亡受体-1；小鼠；真核表达质粒
中图分类号：R392

Construction and expression of eukaryotic plasmids encoding mouse PD-1Ig
LIU Yi1, XU Lihui2, HE Xianhui2, SUN Jianfang1
(1. Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing 210042; 2. College of Life Science and Technology，Jinan University，Guangzhou 510632)
Abstract: 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.
Key words: kProgrammed cell death 1；Mouse；Eukaryotic plasmids