杆状病毒基因治疗应用的生产与纯化
Summary
在该协议中, 杆状病毒是通过瞬态转染杆状病毒质粒进入 Sf9 细胞并在无血清悬浮培养中扩增而产生的。用肝素亲和层析法纯化上清液, 进一步浓缩离心。该协议对用于基因治疗的杆状病毒的制备和纯化具有重要意义。
Abstract
杆状病毒传统上被用于生产重组蛋白和疫苗。然而, 最近, 杆状病毒正逐渐成为基因治疗应用的一个有希望的载体。在这里, 杆状病毒是由 Sf9 细胞黏附培养的杆状病毒质粒 DNA (bacmid) 的瞬态转染而产生的。杆状病毒随后在无血清悬浮培养的 Sf9 细胞中展开, 直到获得所需的体积。然后用肝素亲和层析法从培养上清液中纯化。由于肝素对杆状病毒包络糖蛋白的亲和性, 在上清液中将杆状病毒颗粒结合在一起的肝素柱上上清。该列用缓冲区冲洗, 以去除污染物, 而杆状病毒则是用高盐缓冲柱洗脱的。洗脱液被稀释为一个等渗盐浓度, 杆状病毒粒子进一步集中使用离心。使用这种方法, 杆状病毒可以集中到500倍, 25% 的传染性微粒的恢复。虽然这里描述的协议表明了从培养到1升的杆状病毒的生产和纯化, 该方法可以扩大在封闭系统悬浮培养, 以产生一个临床级的载体, 用于基因治疗的应用。
Introduction
杆状病毒主要用于生产重组蛋白和疫苗的鳞翅目斜纹 fugiperda (Sf) 9 昆虫细胞使用重组杆状使用杆状 multicapsid 核多角体病毒 (AcMNPV)1,2,3,4. 最近, 它正在成为基因治疗应用5的一个有希望的载体。众所周知, 它具有广泛的宿主和组织取向, 感染静止和增殖细胞, 是非致病性的, 并没有集成到主机染色体4,5,6。此外, 杆状病毒可以在无血清悬浮培养, 可伸缩性, 并允许为未来的临床生产关闭系统处理1。
杆状病毒粒子的纯度对于实现有效的转导非常重要, 同时尽量减少细胞毒性7,8,9。杆状病毒可以通过离心或切向流动过滤 (TFF) 来集中, 对其传染性有有限的影响。然而, 这些程序不仅集中了病毒微粒, 而且还有来自 Sf9 文化的细胞碎片和蛋白质, 这可能是有毒的体外(个人观察), 在使用体内时可能诱发炎症或免疫应答。为了避免这种情况, 特别是在使用高度集中的病毒储存时, 感染性杆状病菌需要纯化, 并与污染微粒分离。
报告了几种用于纯化和浓缩杆状病毒载体的方法10,11,12。在现有的方法中, 肝素亲和色谱允许单步高水平的纯化与低浓度的污染蛋白12。该方法是基于硫酸硫酸根的识别作为杆状病毒13,14的受体。在将 Sf9 细胞上移到杆状病毒的柱上并结合后, 该柱可以用生理 (等渗) 缓冲器冲洗, 以去除未绑定或松散的污染微粒。由于对肝素的结合是可逆的, 杆状病毒颗粒可以洗脱高盐缓冲, 这是立即稀释到生理 (等渗) 盐浓度, 以防止通过渗透冲击 12.此外, 杆状病毒的生产, 以及从色谱柱上捕获和洗脱, 可以使用一个封闭系统的过程, 这是符合当前良好的生产实践 (cGMP)。
在这里, 我们提供了一个详细的协议, 以制造, 纯化和浓缩的传染性杆状病毒使用亲和层析和离心。简要地, 我们生产杆状病毒通过转染 Sf9 细胞与杆状病毒质粒 DNA 在黏附培养和进一步扩大传染性杆状病毒在无血清悬浮培养。我们用肝素亲和层析纯化杆状病毒, 并以离心为最后一步, 高度集中载体进行基因治疗应用。
Protocol
有关汇总协议的说明, 请参见图 1 。 1. 杆状病毒质粒 DNA 的纯化 用抗生素在200毫升的 LB 汤中生长含有杆状病毒质粒 DNA (bacmid) 14 的细菌培养;卡那霉素 (50 µg/毫升), 四环素 (10 µg/毫升), 和庆大霉素 (7 µg/毫升), 16 h 在37°c 在一个轨道振动筛设置在 300 rpm。 使用标准碱性裂解协议15从细菌培养中纯化 bacmid DNA,…
Representative Results
所提供的协议在流程图中 (图 1)。步骤包括对 bacmid DNA 的 Sf9 细胞进行瞬态转染, 在板的黏附培养中产生杆状病毒, 随后在无血清悬浮培养中扩增, 核酸酶处理和离心和过滤澄清,用肝素亲和色谱法纯化后, 离心浓度。 解冻后, Sf9 细胞需要至少两个星期的时间来恢复和进入指数阶段的增长。积极生长的 Sf9 细?…
Discussion
本文介绍了在悬浮培养中 Sf9 细胞中杆状病毒的产生和用肝素亲和层析纯化杆状病毒的方法。该协议中使用的参数最大限度地提高了感染杆状病毒的产量, 最大限度地减少了其灭活。此处提供的协议显示, 与其他9实现的恢复相比, 杆状病毒粒子的恢复明显改善。
由于广泛的寄主范围和组织取向, 在中枢神经系统、肝、眼、卵巢、前列腺和睾丸中进行了多项研究…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作的部分原因是由辛辛那提儿童研究基金会 (CCRF) 为 M.N. 和创新核心赠款提供资金, 该基金由国家卫生研究院促进翻译科学中心资助。奖项编号 UL1 TR001425。 内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。
Materials
| Akta Avant 150 | GE Healthcare | 28976337 | Chromatography system |
| POROS Heparin 50 µm Column | ThermoFisher Scientific | 4333414 | Heparin column |
| Ultracentrifuge | Beckman-Coulter | Non-catalog item | Concentrates virus at high-speed |
| Polyallomer Ultracentrifuge tube | Beckman-Coulter | 326823 | Concentrates virus at high-speed |
| MaxQ 8000 orbital shaker incubator | ThermoFisher Scientific | Non-catalog item | Shaker for suspension culture |
| 250 mL Erlenmeyer flasks | ThermoFisher Scientific | 238071 | Flask for suspension culture |
| 1 L Erlenmeyer flasks | ThermoFisher Scientific | 238072 | Flask for suspension culture |
| Microscope (Olympus CKX41) | Olympus | Non-catalog item | Cell monitoring and counting |
| Table top centrifuge | ThermoFisher Scientific | 75253839/433607 | For clarification of Baculovirus supernatant |
| 50 ml Conical tube | ThermoFisher Scientific | 14-959-49A | For collection of Baculovirus supernatant |
| 6-well plate | ThermoFisher Scientific | 07-200-80 | Tissue culture treated plate |
| 10-cm plate | ThermoFisher Scientific | 08-772E | Tissue culture treated plate |
| Stericup-HV, 0.45 µm, PVDF | EMD-Millipore | SCHVU05RE | Filtration unit |
| Kanamycin | ThermoFisher Scientific | 15160-054 | |
| Tetracycline | Sigma-Aldrich | T7660 | |
| Gentamycin | ThermoFisher Scientific | 15750-060 | |
| Bac-to-Bac Vector Kit | ThermoFisher Scientific | 10360-014 | Baculovirus expression system |
| DH10B-T1R Competent cell | ThermoFisher Scientific | 12331-013 | Competent cell for bacmid |
| TE buffer | In-house | Non-catalog item | 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. |
| Plasmid Maxiprep kit | ThermoFisher Scientific | K2100-06 | For bacmid purification |
| Sf9 Cells | ThermoFisher Scientific | 11496-015 | Insect cells |
| Grace’s Insect Cell Culture Medium | ThermoFisher Scientific | 11605-094 | Transfection medium |
| PBS | ThermoFisher Scientific | 20012227 | Washing cells, diluting samples |
| HyClone SFX-Insect cell media | GE Healthcare | SH30278.02 | Serum-free insect cell growth medium |
| Benzonase Nuclease | Sigma-Aldrich | E1014 | Enzyme to degrade DNA and RNA |
| Baculovirus plasmid (bacmid) DNA | In-house | Non-catalog item | Bacmid for Baculovirus Production |
| Cellfectin II | ThermoFisher Scientific | 10362 | Transfection reagent for insect cells |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A4737 | Stabilizes Baculovirus |
| Cryovial | Thomas Scientific | 1222C24 | For storage of Baculovirus |
| HT1080 cell line | ATCC | CCL-121 | Fibroblast cell line |
| DMEM | Sigma-Aldrich | D6429 | Growth media for cell lines |
| Wash buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride |
| Elution buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride |
| Column cleaning buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride |
| Sterile water | In-house | Non-catalog item | For Akta Avant cleaning |
| Sodium hydroxide | Sigma-Aldrich | 1.09137 | For Akta Avant cleaning |
| Ethanol | Sigma-Aldrich | E7073 | For Akta Avant cleaning |
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Cite This Article
Nasimuzzaman, M., van der Loo, J. C., Malik, P. Production and Purification of Baculovirus for Gene Therapy Application. J. Vis. Exp. (134), e57019, doi:10.3791/57019 (2018).