聂蓓娜,许晨光,万晓春.科研级嵌合抗原受体慢病毒浓缩方法对比研究[J].集成技术,2020,(3):36-43
科研级嵌合抗原受体慢病毒浓缩方法对比研究
Comparisons of Labtoratory-Grade Lentivirus Concentration Methodsfor Chimeric Antigen Receptor T Cell Preparation
投稿时间:2020-02-27  修订日期:2020-03-30
DOI:
中文关键词:  超离;超滤;慢病毒载体;滴度;回收率
英文关键词:ultracentrifugation; ultrafiltration; lentivirus vectors; titer; recovery rate
基金项目:科技部国家重点研发计划项目(2019YFA0906100);广东省重点领域研发计划项目(2019B020201014);深圳市南山区引进海外 高层次创新人才“领航团队”支持计划项目(LHTD20160004);深圳市科技创新委员会基础研究学科布局项目(JCYJ20170818164619194)
作者单位
聂蓓娜 中国科学院深圳先进技术研究院 深圳 518055;中国科学院大学 北京 100049 
许晨光 中国科学院深圳先进技术研究院 深圳 518055;广东省免疫细胞治疗工程技术研究中心 深圳 518055 
万晓春 中国科学院深圳先进技术研究院 深圳 518055;广东省免疫细胞治疗工程技术研究中心 深圳 518055 
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中文摘要:
      慢病毒载体是基因治疗的重要基因转导工具,在嵌合抗原受体(Chimeric Antigen Receptor, CAR)T 细胞治疗血液肿瘤中发挥着极其重要的作用。制备高质量 CAR-T 细胞的先决条件是获得高滴 度的慢病毒,而高滴度的实现离不开浓缩步骤。其中,超速离心(超离)和超滤是两种潜在的可选浓缩 方法。为探究超离和超滤两种浓缩方法在制备科研级 CAR-T 时的效率和对病毒的活力影响,该研究选 择 GFP 和 CD19 CAR(CAR19)-mCherry 两种质粒包装病毒,并对两种方法产生的病毒滴度和回收率进 行比较。结果显示,超滤产生的慢病毒滴度最高达到 1011 TU/mL,显著高于超离法产生的慢病毒滴度 (P<0.05),同时超滤的回收效率也更高。该结果可为科研级 CAR 慢病毒的浓缩方法提供直接的理论 参考依据,有助于 CAR-T 基础研究的顺利开展。
英文摘要:
      Lentiviral vector is an important gene transduction tool for gene therapy and plays an a crucial role in the treatment of hematologic tumors by chimeric antigen receptor (CAR) T cells. The prerequisite for the preparation of high quality CAR-T cells is to obtain lentivirus with high titer by concentration, and ultracentrifugation and ultrafiltration are two main methods for virus enrichment. In order to determine the efficiency of ultracentrifugation and ultrafiltration for virus concentration, and their effect on virus activity in the preparation of research-grade CAR-T, two lentivirus plasmids (GFP and CD19 CAR(CAR19)-mCherry) were selected to package lentivirus, then titer and recovery rate were compared. The results showed that the titer of virus produced by ultrafiltration reached 1011 TU/mL, which was significantly higher than that by ultracentrifugation (P<0.05), and the higher recovery efficiency was also obtained by ultrafiltration. These results can provide direct theoretical reference for the lentivirus concentration method to support the preparation of laboratory-grade CAR-T, and are helpful for the rapid development of CAR-T basic research.
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