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。
Adeno-associated virus(AAV)is a promising viral vector and meets most requirements of being a safe biological agent.However,the commercialization of AAV has been hampered due to the limitation of large-scale production,and only a small number of clinical trials have been launched.In recent years,progresses in scalable manufacturing of AAV have dramatically improved AAVbased clinical researches,and have assisted the development of investigational drug products.An AAV1-based investigational product,Glybera,has been formally approved by European Commission for the treatment of lipoprotein lipase deficiency(LPLD).Glybera was the first gene therapy product in the western world,and the production process involves a scalable baculovirus-insect cell system.However,many problems still need to be solved to improve the productivity and quality of AAV.The present review gives critical insights into current state-of-the-art scalable producing methodologies of AAV,such as baculovirus-insect cell system,HSV complementation system,and Ad complementation system,along with a discussion on the problems,solutions,and developmental trends.Novel AAV-producing platforms in Saccharomyces cerevisiae and vaccinia virus complementation system will also be discussed.
