如何制备高滴度的慢病毒？[创新技巧]

如何制备高滴度的慢病毒呢？罗氏公司的研究人员开发出一种新方法。它主要利用了一些升级换代的新产品，能摆脱慢病毒制备过程中的毒性困扰，并实现高达1010TU/ml的产量。更重要的是，重复性好。

慢病毒在基因表达中广受欢迎，因为其能转导大部分的细胞类型，并整合到宿主基因组上，实现长期稳定的表达。不过很多研究人员的困扰是制备出的慢病毒滴度不高，需要纯化和浓缩后才能有效地感染细胞。在此，罗氏公司的研究人员开发出一种制备慢病毒的新方法。它主要利用了一些升级换代的新产品，能摆脱慢病毒制备过程中的毒性困扰，并实现高达10¹⁰TU/ml的产量。更重要的是，重复性好。

实验材料
T175培养瓶：此步骤适合于T175培养瓶。实验中可能需要5-10个培养瓶。
包装细胞系：HEK 293-FT（Invitrogen）
表达载体：第三代的慢病毒表达载体（详见慢病毒表达载体一文）或cFUGW（Cal Tech)
包装质粒：pLP1、pLP2和pLP/VSV-G（Invitrogen）
转染试剂：Fugene HD（罗氏）

操作步骤
Day 1
1. Coat T-175 flask(s) with 10 ml polylysine solution (0.01 mg/ml, or 10 μg/ml) in sterile water/PBS. Leave for at least 1 h.
2. Wash twice with 20 ml sterile water.
3. Split HEK-293FT cells into coated flasks at ~50% confluency around 5:00 PM the day before transfection, with cells growing in 20 ml of 10% FBS Opti-MEM (with GlutaMAX).

Day 2
4. Next morning (9:00 AM), remove media, add 20 ml serum-free (SF) Opti-MEM (with GlutaMAX) supplemented with 25 μM chloroquine.
5. Prepare transfection complex:
a. Add together 10.5 μg pLP1, 7 μg pLP2, 10.5 μg pVSV-G, and 9.3 μg cFUGW. Plasmid preparations should be of extremely high quality and endotoxin free.
b. Dilute DNA into 2 ml of SF Opti-MEM.
c. Add 100 μl FuGENE• HD Transfection Reagent, briefly vortex, and incubate 15 minutes at room temperature.
6. Pipet directly to media in the flask of cells. Agitate flask until contents are distributed.
7. In the early evening of the same day (~5:00 PM), supplement with 10 μM sodium butyrate, directly to the flask medium.

Day 3
8. Next morning (9:00 AM), discard media and replace with 20 ml of SF Opti-MEM (with GlutaMAX and no other supplements).
Note: the discarded media has a small amount of virus and is BSL-2 (bleach disposal, etc.).

Day 4
9. Next morning, transfer the media in each flask (~20 ml) to a Falcon 50 ml centrifuge tube. Replace 20 ml of fresh SF Opti-MEM (with GlutaMAX) media to flask.
10. Spin Falcon tube at 2000× g for 10 min at 4°C and transfer supernatant to new 50 ml tube. Store at 4°C overnight.

Day 5
11. Next morning (9:00 AM), collect ~20 ml of media to a new 50 ml tube.
12. Spin at 2000× g for 10 min at 4ºC and then transfer supernatant to the existing 50 ml tube from the day before, which will now contain approximately 40 ml of viral supernatant.
13. Pass tube contents through a 22 μm vacuum filter (Millipore).
14. Virus can be used as a 1× virus preparation (~1× 107 TU/ml); freeze as 5 ml aliquots at -80° C.

为了确定慢病毒的纯度和浓度是否达到体内应用的要求，将2微升4×10¹⁰ TU的病毒（编码eGFP）注射进入小鼠尾壳核区的前部。一星期后，用免疫组织化学和免疫荧光的方法分析脑区。感染性广，而毒性低，说明这种方法能制备出高质量的慢病毒。

更详细的优化步骤，请登录罗氏公司的网站查看Optimized Production of Lentivirus Using FuGENE HD Transfection Reagent. Biochemica 3/2008: 17-19，或点击索取。