【求助】腺病毒扩增和纯化...

【求助】腺病毒扩增和纯化

.Plating 293 cells in T-25 flasks



About 293 cells: 293 cells are derived from human embryonic kidney cells (a.k.a., HEK 293 cells) that are stably expressing human adenovirus E1A and E1B genes.As you know, most recombinant adenoviruses are missing the E1A and E1B genes.293 cells provide the E1A and E1B proteins and allow recombinant adenoviruses to replicate and produce more virus particles. Thus, 293 cells are also referred as the packaging line for recombinant adenoviruses.Although there are several other packaging lines available, 293 cells are the most commonly used line for generating and amplifying adenoviruses.In addition, 293 cells are also a popular choice for many transient gene expression studies because of its high transfection efficiency.



Plate 293 cells in T-25 flasks 12-16hrs prior to transfection (approx. 40-60% confluence at transfection). Alternatively, seed 293 cells in T-25 at subconfluency (~60%) 2 ~ 4 hours prior to transfection (i.e., plating cells in early morning if you want to finish transfection within the same day).This approach has been working very well in our lab, and is particularly useful for those who are concerned about potential over-confluency of the overnight plating culture.



Note:A lower confluence could achieve higher transfection efficiency but may yield a lower absolute number of transfected cells.The following procedure can be scaled up or down proportional to the surface area of plate or well with little or no change in the results.





II.Pac I digestion of adenoviral recombinant plasmids (i.e., pAdYFG)



Rationale:The purpose of Pac I digestion is to liberate the two ITRs of adenoviral genome so that viral DNA replication (i.e., adenovirus production) can be initiated effectively by the adenoviral Terminal Binding Protein.



Pac I digestion: you will need to perform 100ul digestion reaction, using 3ul Pac I (NEB).You will need to use ~3ug DNA for the digestion.We usually use Wizard prep DNA.In this case, you may need to use approx. 30ul of DNA for Pac I digestion (as the concentration of a typical Wizard prep is ~100ng/ul). The digestion reaction should only last 30-60 min (>1 hr or overnight digestion is ridiculous and will hurt you!).[Optional: You can check 5-10ul of your digestion reaction mix (NOT your precipitated DNA) on gel to make sure that the Pac I digestion is complete]. Perform ethanol precipitation, and wash the pellet twice with 70% ethanol.Resuspend DNA in 30ul sterile ddH2O.Ready for transfection (see Stage III).



Note: 1) You should always check 1-2ul of your Wizard prep DNA on an agarose gel (and read A260, which is less reliable and optional).2) If the concentration of your Wizard prep DNA is low, and you want to use >30ul Wizard prep for Pac I digestion, you should first precipitate DNA because the Wizard prep is usually dirty, and the impurities could inhibit your Pac I digestion.3)There is no need to perform PC-8 (phenol/chloroform) extraction and/or gel purification of the Pac I digested pAdYFG DNA (If you do, you will get more hurt than helped).





III.Transfection of 293 cells with LipofectAMINE



1.Prepare 1.5 ml microfuge tubes (or use the wells of a 48-well plate), the 30ul of Pac I digested DNA solution, and 200ul of OptiMEM or plain DMEM (i.e., DMEM without FBS) per transfection.

2.Mix 200ul of OptiMEM or plain DMEN, 30ul of Pac I digested DNA, and 15ul of LipofectAMINE (Invitrogen, usually 5ul/ug DNA) per transfection. Let the mix sit in the cell culture hood for 10 min.

3.Meanwhile, remove the complete medium (containing FBS) from T-25 flask, and wash cells gently by adding 5 ml of OptiMEM or plain DMEM (Note: serum free media) to the side of the flask, rocking slowly to allow the medium to cover the cells, and aspirating the medium.

4. [Optional: repeat the wash once. It is NOT desirable to repeat the wash on less adherent cells, such as 293 cells].

5.Add 2.5 ml OptiMEM or plain DMEM to the flask. Return flask to 37°C incubator until DNA/LipofectAMINE mixture is ready.

6.Add the DNA/ LipofectAMINE mix to the other side of the flask. Rock gently and return flasks to the incubator.

7.After 2-6 hours, remove the OptiMEM or plain DMEM and replace with 8-10 ml of complete DMEM medium. [Note: longer transfection time could result in higher transfection efficiency, as long as the cells are healthy].

8.Check GFP at 24 hrs after transfection (if you use pAdTrack, pAdTrack-CMV, or pAdTrack-TO4 based shuttle vector). Check GFP again in one week.Collect the transfected cells at 10-14 days after transfection.





VI. General comments on transfection and adenovirus making in 293 cells:



1) If a significant portion of the transfected cells is floating at the end of your transfection, do not remove Lipo/DNA-containg medium. Instead, just add 6.0ml of complete DMEM medium, and replace it with fresh complete DMEM medium next morning.



2) The transfected cells usually become confluent at 2-3 days after transfection, and will stay confluent until you collect them.The culture medium usually becomes yellowish.Do not panic!There is No Need to change the medium [If you do, you will get hurt as the generated adenoviruses are usually released to the medium, and subsequently infect more 293 cells, leading to higher virus titers].Instead, you can add 1-2ml of the complete DMEM medium every 3-5 days.We usually do not add any fresh medium or only add 2ml fresh complete DMEM at day 10.



3) You should see some intensified GFP foci usually at 5-7 days after transfection.If your transfection efficiency is not very high (i.e., <10%), you may want to leave the transfected cells for up to 20 days in order to obtain higher titers of the initial virus lysate.Waiting for 1-2 more days makes a big difference in terms of enhancing initial virus titers.Patience will pay off as it takes much longer time to amplify low titer viral lysate!

非常感谢！但是这个是转染293的protocal吧，我想问的是转染、扩增之后，纯化的方法，我查到用氯化铯，不知道这个是不是纯化用的呢？具体怎么操作，以及这个东西购买的问题。

有专门的病毒纯化试剂盒的，比如clontech公司的，好像两三千元吧！

我也想知道

腺病毒的包装，纯化和感染
技术背景：Adeasy系统利用了腺病毒具有的高感染能力，可高度浓缩等优点，
并破坏了腺病毒基因组中的早期基因E1，使之成为较为安全、高效的病毒载体。
利用带有 E1 基因的 293 细胞作为包装细胞，通过倍比扩增，富集病毒颗粒，并
通过CsCl 梯度离心，透析进行分离纯化。对绝大多数的细胞株可以达到近乎100
％的感染效率。但需要指出的是 Adeasy 同样具有所有病毒载体或转染试剂都不
可避免的细胞毒性问题。
所需试剂：
? 293细胞；
? 重组好的腺病毒质粒；
? Pac I限制性内切酶；
? 质粒回收相关试剂；
? 细胞培养、转染相关试剂；
? TBS：10mMTris, 0.9% NaCl,pH8.1;
? 40%CsCl：28.45gCsCl 溶于42.7ml 的TBS 中，4度保存；
? 15%CsCl：9.085gCsCl 溶于47.69ml 的TBS 中，4度保存；
? Beckman 离心管：14*89 mm，SW41 转头；
? Polybrene(sigma)，10 mg/ml；
? 透析袋：Spectrum Co. （成卷供应，带少量甘油，硫化物与重金属），
MW=8000~14400；
? 灭菌甘油。
操作流程：
1. 293细胞（E1-transformedhumanembryonickidneycells 在转染前24小时接种
于1或2个60mm 培养盘中，使之在转染时细胞汇合率为50-70%；
2. 在转染前，用Pac I处理目的质粒（一般一个60mm 细胞盘需要6μgDNA）。
处理后质粒用乙醇沉淀并重悬于20μl 无菌水中；
3. 用PEI或其他转染试剂将6μgPac I处理过的质粒转染细胞；
1

4. 8 个小时后移去混合液，加入 4 ml DMEM 完全培养液（10% CBS，1%
Pen/Strep）；
5. 在转染后 7 到 10 天用细胞刮（而不是胰酶）将细胞从瓶中刮下并移入 50ml
锥形管。离心后将细胞重悬于2.0ml PBS 或全培液中。于液氮中冻结细胞，
37℃水浴中溶解，剧烈振荡。此步骤共进行4 次。注意保存时不要反复冻融，
可保存于-20℃；
6. 将293细胞以50-70%的汇合率铺于60mm 培养盘中，以30-50%的体积比加
入含病毒上清液。在感染后2-3天即可看到明显的细胞裂解或CPE现象；
7. 在有 1/3 到 1/2 的细胞脱离漂浮时，通常是感染后 3-5 天收集病毒。可进一
步通过Westernblot 或PCR（5μl 病毒上清加上10μl PCR-gradeProteaseK，
55℃消化1小时，然后煮沸样品5min，离心后取1-2μl 进行PCR反应）证
实重组腺病毒的存在；
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8. 按步骤5的方法收集病毒上清。在这种情况下一般至少可收集到10 infectious
particles/ml 的病毒。每轮的扩增应能提高一个数量级的梯度；
9. 为了进一步扩增，将步骤8获得的病毒上清进一步感染100 mm 培养盘的细
胞，按步骤5的方法收集病毒，并进而将其感染150mm 细胞培养盘中的293
细胞，以获得足够量的病毒；
10. 将15%CsCl 和40%CsCl 加入Beckman 离心管中制备CsCl 梯度溶液；
11. 将最终富集病毒颗粒的病毒上清滴加于CsCl梯度溶液上；
12. 超速离心，30000rpm，4度离心16个小时；
13. 离心后，应有两条带。位置较高，颜色较弱的条带主要为腺病毒空壳，不具
感染能力；位置较低，颜色较亮的条带含有我们需要收集的活病毒颗粒。用
16号针头将这一条带收集；
14. 在TBS 中透析一小时，随后在含有10％甘油的TBS 中透析两次，每次一小
时；
15. 将纯化的腺病毒分装于EP 管中；
16. 在Eppendorf Biophotometer中测量透析液中的总蛋白量，1μg病毒蛋白相当
于4×109病毒颗粒；
17. 长期保存于-70度中，短期可保存于4度，切忌反复冻溶；
2

18. 由于腺病毒对不同细胞株的感染效率存在差异，且考虑到病毒颗粒的细胞毒
副作用，通常通过梯度感染的方法确定所需的病毒用量。以相对细胞数1：1，
10：1，100：1，1000：1的病毒颗粒感染靶细胞；加入相对培养液体积1：
1000的Polybrene。在 37度下平板离心30分钟；
19. 感染 8~12 小时后换液。将含有病毒的培养液小心移入含有消毒液的废液缸
中，加入适量全培液。
参考文献：
1．Proc. Natl. Acad. Sci. USA，Vol. 95, pp. 2509–2514, March 1998，Asimplified
system for generatingrecombinant adenoviruses.
2．Current Protocols inHumanGenetics (2004)12.4.1-12.4.25.
http://life.xmu.edu.cn/hanlab/Protocol/%E8%85%BA%E7%97%85%E6%AF%92%E7%9A%84%E5%8C%85%E8%A3%85%E5%92%8C%E6%84%9F%E6%9F%93.pdf

谢谢。