太鼎生物科技/太鼎食安科技

重組慢病毒lentivirus，廣泛應用於細胞之轉導。轉染後的病毒上清液力價，約106 CFU菌落形成單位（菌落形成單位）/ ml。在高效能病毒力價及體內基因傳遞中，要求較高的病毒純度及力價，且於活體試驗中，避免病毒細胞培養上清液，所造成之污染可能性。因此，病毒上清需要進一步濃縮及純化。

 Abmgood提供一套病毒純化套組。

傳統純化病毒，經由超速離心濃縮。雖然該方法效果很好，但需要特定的的超速離心設備，且技術要求高。此外，以濃縮的病毒上清總體積是有限的超速離心管的大小。報告指出，超速離心過程中，會造成一些不利的物理效應，會影響病毒顆粒的生物活性。

Abmgood發展 Ultra-Pure
Lentivirus Purification Kit (Cat. No.
LV998).，針對病毒濃縮技術及純度做改良。提供一個快速且有效，純化及濃縮病毒的產品。其設計原理，為利用離子交換的結合和獨特緩衝液，最大限度減少病毒顆粒的損壞，且提高病毒力價。

Ultra-Pure Lentivirus Purification Kit 套組內容:

(Cat. No. LV998)

• LentiBind Buffer A
(320ml)

• LentiBind Buffer B
(128ml)

• Lenti-Elution buffer
(32ml)

•
LentiBuffer (40ml)

病毒純化步驟

1. On both day 4
and day 5, centrifuge the collected viral supernatant

at 6000g for 10
minutes at 4°C to
pellet the cell debris. Transfer

supernatant to a new
centrifuge container without disturbing the cell

debris.

2. For every
100ml of viral supernatant to be purified, add 20ml of

LentiBind Buffer A and
8.0ml of LentiBind Buffer B. Mix well and shake

the mixture on a
rotating/orbital shaker (commonly used in Western

Blot applications) at
200-300rpm at 4°C
overnight (for day 4 collection).

3. For day 5
viral supernatant collection, allow the binding for 3

hours only and then
combine both collections before proceeding to

step 4.

4. Centrifuge at
7000g at 4°C for 30 mins. to collect the viral particles.

5. Discard
supernatant completely without disturbing the pellet,

and spin briefly again
if necessary to remove any residual liquid.

6. Add 8.0ml
Lenti-Elution buffer to resuspend the viral pellet completely

with a transfer
pipette and transfer the viral suspension to a 10ml

sterile tube that is
resistant to 10,000g during centrifugation in the following step (Sarstedt Cat
No. 62.515.006). Shake the tubes at 4°C for 30min to elute viral particles
from LentiBind Buffer A.

7. After
incubation, spin at 10,000rpm for 20 min to separate viral

particles from the
binding matrix.

8. Transfer the
supernatant (viral particles) to a 10ml syringe and

filter preparation
through a 0.45μM
filter (Millipore, Cat No. SLHV033RB)

to further clarify the
preparation.

9. Perform buffer
exchange for the viral preparation to get rid of

the high salt
concentrations carried over from LentiBind Buffer B using

a 100kDa cut-off
protein concentrating column (Sartorius stedim biotech,

Cat No. VS2042).

10. Spin the
column at 3000rpm at 20°C until the viral supernatant

volume is close to
1-2ml. To speed up the process, use 1ml transfer pipette to re-suspend the
viral preparation every 30 minutes.

11. Add 10ml
LentiBuffer and spin the column again at 3000rpm at

20°C to exchange for final
buffer until the final volume is 200μl-1.0ml

depending on viral
titre desired. To speed up the process, use 1ml

transfer pipette to
re-suspend the supernatant every 30 minutes.

12. Transfer the
final viral preparation from the buffer exchange column

to a 1-3ml syringe and
sterilize it through a 0.45μM filter. Make sure

to pre-wet the filter
with 100μl of
LentiBuffer to prevent virus loss caused

by residual liquid
left in the filter.

13. Final
lentivirus preparations can either be used immediately for

cell transduction or
aliquoted into smaller volumes (10-20μl) for longterm

storage at -75°C.

【供應廠商】太鼎生物科技有限公司

【連絡人】許虹宜 0920-312382 taiding.biotech@msa.hinet.net

【臺北公司】02-86609496 【台中公司】0935-229803

【相關網址】
www.biopioneer.com.tw

【原廠網址】www.abmgood.com