腺相关病毒载体工程毒株精细化纯化  

Refined purification of adeno-associated virus vector-engineered strain

作　　者：高强[1] 宁毅[1] 熊涛 胡珏[1] 聂娟[1] 李玲[1] 肖荣 GAO Qiang;NING Yi;XIONG Tao;HU Jue;NIE Juan;LI Ling;XIAO Rong(Hunan University of Traditional Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]湖南中医药大学,湖南长沙410208

出　　处：《微生物学通报》2021年第11期4468-4476,共9页Microbiology China

基　　金：国家自然科学基金(81803964);湖南中医药大学项目(2019XJJJ026,99821001011);湖南省教育厅项目(20C1431);湖南省卫生计生委项目(C2017027);湖南省“国内一流培育学科”中西医结合基础开放基金(2020ZXYJH03);湖南中医药大学校级重点学科“基础医学”资助项目。

摘　　要：【背景】大量制备高纯度的重组腺相关病毒(Recombinant Adeno-Associated Virus,rAAV),是以腺相关病毒(AAV)为投递载体的基因靶向治疗或基因工程药物研发过程中需要解决的重要问题。【目的】利用层析柱法大量纯化质粒和rAAV细胞培养液,以获得高纯度的rAAV颗粒。【方法】对组装rAAV的3种质粒用HiPrep^(TM)10 Sepharose 6FF和HiTrap PlasmidSelect Xtra凝胶层析柱纯化,目的是得到组装AAV的超螺旋质粒DNA,然后对组装rAAV过程中的AAV-293细胞培养方法进行优化,组装成功病毒后,再对收集到的细胞提取液用HiLoadTM10Q和HiLoadTM10SP阴阳离子交换层析柱纯化,最后用纯化浓缩后的rAAV感染HT1080细胞,通过流式细胞仪检测荧光表达细胞数,计算浓缩后活病毒感染滴度。【结果】HiPrep和HiTrap层析柱纯化后得到大量高纯度的超螺旋质粒DNA,组装病毒后,用HiLoad柱纯化获得大量高纯度的rAAV颗粒,通过测定,rAAV基因组滴度达到(2.46±0.37)×10^(12) TCID50/mL (MOI=1.0×10^(4))。【结论】通过一系列精细化组装纯化制备出高纯度、高滴度rAAV病毒颗粒。[Background] In gene targeted therapy and gene engineering drug development with adeno-associated virus(AAV) as the delivery vector, the challenges of the large-scale preparation of highly purified recombinant AAV(rAAV) should be solved. [Objective] The goal of this study was to obtain highly purified rAAV particles by purification of plasmids and rAAV cell culture medium. [Methods]HiPrep^(TM)10 Sepharose 6 FF and HiTrap PlasmidSelect Xtra gel chromatography columns were used to purify three rAAV plasmids. The study sought to obtain super helical DNA plasmids and optimize the culture of AAV-293 cells in the assembly process. After successful assembly, the virus extract was purified using HiLoadTM10 Q and HiLoadTM10 SP cation exchange columns. HT1080 cells were infected with purified and concentrated rAAV. Flow cytometry was used to enumerate the fluorescent cells to detect the number of live virus and calculate the titer of live virus after concentration. [Results] After HiPrep and HiTrap chromatography columns purification, many copies of highly purified super helical plasmid DNA were obtained. After virus assembly, many highly purified rAAV particles were obtained by cation exchange chromatography. The genome titer of rAAV(TCID50/mL) reached(2.46±0.37)×10^(12)(multiplicity of infection of 1.0×10^(4)). [Conclusion] A high titer of pure rAAV was prepared by a series of assembly refinements and purification.

关 键 词：腺相关病毒 AAV-293细胞 层析柱 纯化 

分 类 号：Q782[生物学—分子生物学]