PD-1抗体表达载体的构建以及其功能的探究 |
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引用本文: | 陶雷,;黎江,;朱海莉,;钱其军.PD-1抗体表达载体的构建以及其功能的探究[J].浙江工程学院学报,2014(6):697-701. |
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作者姓名: | 陶雷 ;黎江 ;朱海莉 ;钱其军 |
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作者单位: | [1]浙江理工大学生命科学学院新元医学与生物技术研究所,杭州310018; [2]第二军医大学东方肝胆外科医院病毒基因治疗实验室,上海200438 |
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基金项目: | 国家自然科学基金(81071850) |
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摘 要: | 通过构建含PD-1抗体基因的重组腺病毒表达载体Ad35-anti-PD-1,表达的PD-1抗体以探究其对细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)抗肿瘤功能的影响。用ELISA实验检测Ad35-anti-PD-1在293细胞中PD-1抗体的表达量;利用Protein G亲和层析法纯化PD-1抗体;通过体外杀伤实验研究PD-1抗体阻断PD-1信号通路的CTL杀伤肝癌细胞能力的变化。显示成功构建重组腺病毒载体Ad35-anti-PD-1,滴度为1×1010 pfu/mL;ELISA实验检测在72h时293细胞上清中PD-1抗体的表达量约在600ng/mL;Westernblotting实验证明PD-1抗体包含正确的重链(50kD)和轻链(25kD);ELISA实验检测从100mL细胞上清中得到1mL浓度在40μg/mL的PD-1抗体;体外杀伤实验证明CTL在添加PD-1抗体的环境中杀伤肝癌细胞的能力显著增强。
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关 键 词: | CTL PD-1抗体 抗肿瘤 免疫细胞治疗 免疫抑制 |
Research on Construction of Expression Vector of PD-1 Antibody and Its Function |
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Affiliation: | TAO Lei,LI Jiang,ZHU Hai-li,QIAN Qi-jun(1. Xinyuan Institute of Medicine and Biotechnology, School of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China; 2. Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Second Military Medical University, Shanghai 200438, China) |
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Abstract: | This research explores the influence of PD-1antibody on antitumor function of cytotoxic T lymphocyte(CTL)by establishing recombinant adenovirus expression vector Ad35-anti-PD-1containing PD-1antibody gene;tests the expression quantity of PD-1antibody expressed from Ad35-anti-PD-1in 293 cell through ELISA experiment,purifies PD-1antibody with Protein G affinity chromatography,studies the change of ability of PD-1antibody blocking CTL in PD-1signal path from killing hepatoma carcinoma cell through killing experiment in vitro and successfully constructs recombinant adenovirus carrier Ad35-anti-PD-1with a valence of 1×1010 pfu/mL.ELISA experiment tests that the expression quantity of PD-1antibody in 293 cell supernatant is about 600ng/mL at 72 h.Westernblotting experiment proves that PD-1antibody contains correct heavy chain(50kD)and light chain(25kD).ELISA experiment tests that1 mL PD-1antibody with a concentration of 40μg/mL can be obtained from 100 mL cell supernatant.Killing experiment in vitro proves that the ability of CTL in killing hepatoma carcinoma cell in an environment with PD-1antibody is greatly improved. |
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Keywords: | CTL PD-1 antibody antitumor immune cell therapy immunosuppression |
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