PD-1抗体表达载体的构建以及其功能的探究

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。

Research on Construction of Expression Vector of PD-1 Antibody and Its Function

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.