Basic technical operation of recombinant adenovirus construction, amplification and purification
(AdEasy System of Homologous Recombination in Bacteria)
Cloning of a target gene (taking pshuttle-CMV plasmid as an example)
1. Select the appropriate digestion site, perform digestion ligation, and insert the target fragment into the pshuttl-CMV plasmid multiple cloning site.
2. Identification of recombinant plasmids: enzyme identification or sequencing.
3. Amplify the recombinant plasmid, purify and prepare an appropriate amount of shuttle plasmid containing the gene of interest.
4. Cut the linearized recombinant shuttle plasmid with PmeI single enzyme, and electrophoresis identified that the plasmid was completely cut.
5. Reclaim the linearized plasmids for co-transformation.
Total transformation
1 E. coli BJ5183 electroporation preparation
a. Pick a single BJ5183 bacteria from the fresh agar plate, inoculate in LB medium, shake at 37 ℃ overnight.
b. Inoculate 25ml overnight culture in 500ml LB medium, shake at 37 ° C until OD600 reaches 0.4.
c. Quickly place the culture in an ice bath for 30 min until it is sufficiently cooled.
d. Transfer the bacterial solution to a pre-cooled centrifuge tube, centrifuge at 2500r / min for 15 min at 4 ° C, discard the culture solution, and recover the cells.
e. Wash the pellet with 10ml of pre-chilled 10% glycerol and centrifuge at low temperature twice.
f. Add about 1ml (appropriate amount) of pre-cooled 10% glycerol to resuspend the pellet, dilute the suspension 100 times, and measure the OD600 to a dilution concentration of 2-3 × 1010 cells / ml (1.0 OD600 is about 2.5 × 1010 cells / ml). Separately pack and store at -80 ℃ or liquid nitrogen until use.
2 The viral skeleton plasmid is transformed into E. coli, amplified and purified.
3 Add 1-5µl (approximately 1µg) linearized shuttle plasmid and 1µl (approximately 100ng / µl) viral backbone plasmid (such as pAdEasy-1) into an EP tube containing approximately 40µl BJ5183 electrocompetent cells, mix well, and cool on ice .
4 Add the mixture to the electric rotating cup and shock (1250-1500V / mm, 5ms).
5 At the end of the electric shock, remove the sample, add 1ml of SOC or LB culture solution, and shake at low speed at 37 ℃ for 40min.
6 Spread an appropriate volume of electroporated transformed cell fluid on several kanamycin-resistant plates (25-50 mg / ml) and incubate at 37 ° C for 16-20 hr.
On the 7th day, pick the colonies growing on the plate (select the smallest colonies), inoculate 3ml of LB medium containing 25-50mg / ml, and incubate at 37 ℃ for 10-15hr.
8. The plasmid was extracted by alkaline cracking method and screened by 0.8% agarose gel electrophoresis. The large plasmid was a possible positive clone, which was further identified by enzyme digestion. Single digestion with PacI, 0.8% agarose gel electrophoresis shows a large fragment (about 30kb), and a small fragment (about 3.0 or 4.5kb) (also can be identified by other enzyme digestion), then basically determined as positive clone.
9 Take 1-5µl of positive plasmids and transform them into DH5α E. coli cells (BJ5183 is recA +, the plasmid DNA is prone to mutation, DH5α or JM109, XL1-blue strains are recombinant defective strains, which can stably amplify the identified recombinant plasmids), expand Enrich bacteria and purify plasmids.
Three recombinant viral plasmids transduced 293 cells
1 293 cell culture: 24 hours before the transduction, taking a square flask as an example, inoculate 2 × 106 293 cells in a 25cm2 square flask to make the growth density about 50-70%. (You can also use 6-well or 96-well plates)
2 Cut the recombinant virus plasmid with PacI single digestion (about 4µg DNA is required for transduction of 25cm2 square flask). After completely linearized, precipitate with ethanol and dissolve with 20µl ddH2O.
3 Lipofectamine-coated plasmid (Lipofectamine as an example): Each 4µg PacI needs about 20µl Lipofectamine encapsulation. Plasmid and liposome are diluted in 500µl serum-free medium and mixed, and placed at room temperature for 15-30min.
4 Gently wash the culture flask with serum-free medium, add 2.5ml of serum-free medium, and leave at 37 ℃ for 10min.
5 Add the Lipofectamine-DNA mixture to the culture flask and place it in the 37 ° C incubator for 4 hr.
6 After 4 hr, discard the Lipofectamine-DNA mixture and add another 6 ml of DMEM complete medium (containing 10% FCS). If a large number of cells float, do not discard Lipofectamine-DNA solution, add 6ml DMEM complete medium, incubate at 37 ° C overnight, then change the solution.
7 Observe the cell growth during the cultivation process. After about 2 weeks, the appearance of cytopathy (CPE) can be observed (for example, with the pAdTrack-CMV plasmid, green fluorescence can be observed due to the inclusion of GFP).
Four recombinant virus identification
1 After 10-14 days of transduction, collect the cell pellet, add 2ml of sterilized PBS to suspend, freeze and thaw the cells, collect the supernatant after centrifugation and store at -80 ℃.
2 Take 30-50% of the supernatant from Step 1 and infect 293 cells in a 25cm2 square flask with 50-70% saturation. After 2-3 days, obvious cell lesions appeared.
3 3-5 days after infection, collect virus when 1 / 3-1 / 2 cells float. Collect cells according to step 1 and prepare virus supernatant. Identification of recombinant adenovirus production by Western blot and / or PCR.
4 PCR to identify recombinant viruses. Take 5µl virus supernatant and add 10µl proteinase K, incubate at 55 ℃ for 1hr, then boil for 5min. After centrifugation, take 1-2µl for PCR.
5. Recombinant virus amplification and purification
1 Inoculate 293 cells in a 75cm2 square flask to a density of 90%, and add an appropriate amount of virus supernatant to infect the cells. After 3-4 days, the cells almost became round and half of the cells floated, then all cells were collected. Centrifuge at about 500g and discard the supernatant.
2 Resuspend the pellet in sterilized PBS, and freeze and thaw repeatedly 4 times. Centrifuge at 7000g for 5min at 4 ° C. Virus purification requires at least 30 bottles of 75cm2 square bottles.
3 CsCl continuous gradient centrifugal purification: Weigh 4.4g CsCl in a 50ml centrifuge tube, add 8ml of virus to lyse the supernatant, mix well, and the volume is about 10ml. Transfer to a 12ml ultracentrifuge tube (for SW41 rotor) and cover about 2ml of mineral oil. After equilibration, centrifuge at 32000 rpm for 18-24 hr at 10 ° C, and aspirate the centrifuged virus band with a syringe.
(Also CsCl discontinuous gradient centrifugation: slowly add 8 ml CsCl 1.4 (53 g + 87 mL 10 mM Tris-HCl, pH = 7.9) to a 20 ml ultracentrifuge tube, and carefully add 6 mL CsCl 1.2 (26.8 g + 92 mL 10) mM Tris-HCl, pH = 7.9), then carefully add the virus supernatant to a volume of 20ml. After equilibration, centrifuge at 23000rpm for 90min at 4 ° C (SW28 rotor), and draw the lower blue-white virus band with a syringe)
4 Virus dialysis desalination: configure dialysate (10 mM Tris pH 8.0, 2 mM MgCl2, 5% sucrose) and sterilize. Dialysis at 4 ℃, replacement of dialysate 3 times, can basically remove CsCl, the virus is stored at -80 ℃.
Six virus titer determination (TCID50)
1 Cell preparation: Inoculate 100µl of 293 cells in a 96-well plate with approximately 104 cells per well and culture in 2% DMEM
2 Preparation of diluted virus solution: Dilute the virus solution into 8 higher concentrations (such as 10-3-10-10) with 2% DMEM, repeat 10 for each concentration, and add 100 µl of virus dilution solution to each well. The remaining two rows without virus were used as negative controls. Incubate at 37 ° C for 10 days.
3 Observe the cells after 10 days, count the number of holes with CPE in each row, and calculate the cytopathic rate. (If all the cells in each well of a certain concentration are diseased, the ratio is 1, if there is no cell disease, the ratio is 0).
4 Calculate T = 10 × 101 + d (S-0.5) / ml
d = Log 10 dilution (if a 10-fold dilution ℃, d = 1)
S = sum of cytopathic ratios at each concentrated ℃
Laboratory recombinant adenovirus commonly used plasmids: viral backbone plasmids: pAdEasy-1, pAdEasy-2
Shuttle plasmids: p-Shuttle, p-Shuttle-CMV, pAdTrack, pAdTrack-CMV
Here list the not-so-usual dices, or anything else that is close to the function of dice.
A traditional die is a rounded cube, with each of its six faces showing a different number of dotsfrom 1 to 6. When thrown or rolled, the die comes to rest showing on its upper surface a random integer from one to six, each value being equally likely. A variety of similar devices are also described as dice; such specialized dice may have polyhedral or irregular shapes and may have faces marked with symbols instead of numbers. They may be used to produce results other than one through six. Loaded and crooked dice are designed to favor some results over others for purposes of cheating or amusement.
Other Style Dices
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