Part I Construction of plasmids
1.
Escherichia coli culture
|
LB culture medium/1L
|
|
|
Tryptone
|
10g
|
|
Yeast extract
|
5g
|
|
NaCl
|
10g
|
|
Agar
|
15g (1.5%)
|
2.
Plasmid Extraction
Apparatus: Centrifuge
Using
Vazyme
DC201 plasmid test kit:
1. Take 1-5 ml of the bacterial
solution cultured overnight (12-16 h), add it into the centrifuge tube, and centrifuge at 10,000
rpm(11,500 × g) 1 minute. Discard the culture medium, invert on absorbent paper to absorb the residual
liquid.
2. Add 250 μl Buffer P1
into the centrifuge tube with bacterial precipitation and mixed with
pipette
s
or vortex oscillations.
3. Add 250 μl Buffer P2 to
step 2 and gently mix it upside down for 8-10 times to fully crack the bacteria.
4. Add 350 μl Buffer P3 to
step 3 and immediately gently turn the solution upside down 8-10 times to completely neutralize the
BufferP2. A white flocculent precipitate should appear. Centrifuge at 12,000 rpm(13,400 × g) for 10 min.
5. The FastPure DNA Mini Columns
adsorption column was placed in the Collection Tube 2ml. Put Step 4
supernatant
carefully to the adsorption
column with a pipette, taking care not to absorb precipitate, and centrifuge 30-60 sec at 12,000 rpm(13,400 × g)
. Drain the waste liquid from the collection tube and put the adsorption column back into the collection
tube.
6. Add 500
μl
Buffer PW1 and centrifuge 30-60 sec at 12,000 rpm(13,400 ×
g)
.
7. Add 600 μl Buffer
PW2(check whether it has been diluted with anhydrous ethanol) to the adsorption column. 12,000
rpm(13,400 x g) centrifuge 30-60 sec. Discard the waste liquid and put the adsorption column back into
the collection tube.
8. Repeat Step 7.
9. Place the adsorption column
back into the collection tube. Centrifuge at 12,000 rpm(13,400 × g) for 1 min to dry the adsorption
column, the purpose is to adsorb
t
he residual bleach liquid in the
column is completely removed.
10. Place the adsorption column
in a new sterilized 1.5 ml centrifuge tube. Add a 30-100 μl ddH2O to the center of the membrane of
the column adsorption column. Let stand at room temperature for 2 min, centrifuge at 12,000 rpm(13,400 ×
g) for 1 min and wash
r
emove the DNA.
11. Measure the
concentration.
3. Digestion
Gene synthetic plasmid
treatment: centrifuge at 5000rpm for 1min, and then dissolve in 20ul water with a concentration of
200ng/μL.
There are 5 samples in
total (wuhan, Delta, BQ1.1, XBB1.5 and pET-28a)
Reaction condition:
37
℃
/30min
Apparatus:
PCR
amplifier
Reaction system:
1.
20ul system for DNA fragments
|
Nco
I
|
1
ul
|
|
XhoI
|
1ul
|
|
10X buffer
|
2ul
|
|
DNA fragment
|
5ul
|
|
ddH2O
|
11ul
|
2.50ul system for pET-28a
|
NcoI
|
1ul
|
|
XhoI
|
1ul
|
|
10X buffer
|
5ul
|
|
pET-28a
|
5ul
|
|
ddH2O
|
38ul
|
4.
T4 DNA Enzyme ligation
A
pparatus:
PCR
amplifier
1.
Set up the following reaction in
a microcentrifuge tube on ice.
|
COMPONENT
|
20ul REACTION
|
|
T4 DNA Ligase Buffer (10X)
|
2 ul
|
|
V
ector DNA (4 kb)
|
50 ng (0.020 pmol)
|
|
I
nsert DNA (1 kb)
|
37.5 ng (0.060 pmol)
|
|
Nuclease-free water
|
to 20 μl
|
|
T4 DNA Ligase
|
1 μl
|
3.
Gently mix the reaction by
pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends,
incubate at 16°C overnight or room temperature for 10 minutes.
4. Heat inactivate at 65°C for
10 minutes.
4.
Extraction
Apparatus: Centrifuge
1.
PCR reaction solution extraction
Use Vazyme DC301 and recycle
4
samples
1
)
Transient centrifuge PCR
products
.
Measure the body with a pipette
gun
.
Accumulate and transfer to
sterilized 1.5 ml or 2 ml centrifuge tubes
,
refill with sterilized
water to 100 µl.
2
)
Add 5 times the volume of
Buffer GDP and mix upside down or vortex. If less than 100 bp DNA fragments need to be recovered, add
1.5 times the volume (the volume of sample +Buffer GDP) of anhydrous ethanol.
3
)
Enclose the adsorption
column in the collection tube. Transfer solution to the adsorption column. 12,000 rpm (13,800 x g)
centrifuge 30-60 sec.
4)
Discard the filtrate and place
the adsorption column in the collection pipe. Add 700 µl Buffer GW(with anhydrous ethanol added) to
the adsorption column. 12,000 rpm (13,800 × g) centrifuge 30-60 sec.
Repeat Step 5.
5)
Discard the filtrate and put the
adsorption column back into the collection tube. Centrifuge at 12,000 rpm (13,800 × g) for 2 min.
6)
Place the adsorption column in a
1.5ml sterilized centrifuge tube and add 20-30µl Elution Buffer to the center of the adsorption
column.
Leave for 2 minutes. Centrifuge
at 12,000 rpm (13,800 × g) for 1 min. The adsorption column was discarded and the DNA was stored at -20
° C
7)
Measure the
concentration
2.
Gel extraction
Apparatus: Microwave Oven, UV transilluminator, Electro,
Bainmarie
Use
Vazyme
DC301 and recycle
1
samples
1)
Agarose gel
Electrophoresis
2
）
After DNA electrophoresis,
quickly cut the gel containing the target DNA fragment under the ultraviolet lamp.Weigh the gel weight
(remove the empty tube weight), 100 mg gel equal tothe same volume as 100 μl,
as a gel volume.
3
)
Add equal-volume Buffer GDP.
50-55
℃
water bath 7-10 min, according to the size of the gel appropriate
adjustment time, indeed
t
he adhesive block is completely
dissolved. Inversely mix the accelerative sol twice during the water bath.
4
)
Centrifuge briefly to collect
droplets on the tube wall. The FastPure DNA Mini Columns-Gadsorption column was placed in the Collection
Tubes 2 ml collection tube, transfer liquid into the adsorption column, centrifuge 30 -60 secat 12,000
rpm (13,800 × g)
.
5
)
Discard the filtrate and
place the adsorption column in the collection pipe. Add 300 µl Buffer GDP to the adsorption column.
Let stand for 1 minute. 12,000 rpm (13,800 × g) centrifuge 30-60 sec.
6
)
Discard the filtrate and
place the adsorption column in the collection pipe. Add 700 µl Buffer GW(with anhydrous ethanol
added) to the adsorption column. 12,000 rpm (13,800 × g) centrifuge 30-60 sec.
Repeat Step 5.
7
)
Discard the filtrate and
put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm (13,800 × g) for 2
min.
8
)
Place the adsorption column in a
1.5ml sterilized centrifuge tube and add 20-30µl Elution Buffer to the center of the adsorption
column.
Leave for 2 minutes. Centrifuge
at 12,000 rpm (13,800 × g) for 1 min. The adsorption column was discarded and the DNA was stored at -20
° C
9
)
Measure the
concentration
5.
Transformation
A
pparatus: Bainmarie, oscillations incubator
1）
Remove 100ul of Escherichia coli receptor cells
(
DH5α
)
from the -80
℃
refrigerator and thaw them on ice;
2）
Add 1ul plasmid (generally no more than 50ng), gently shake well, and place on ice
for 10min;
3）
42
℃
water bath 90s, quickly transferred to the ice for 2min;
4）
In the
clean bench: add 100ul LB liquid medium
into the EP tube and mix with the bacterial solution;
5）
Apply the bacterial solution to the LB plate containing antibiotics, let the
bacterial solution dry, and then invert
Overnight at 37
℃
(about 16h)
Ps：
A total of 4 samples were transformed: ①wuhan- pET-28a, ② Delta-pET-28a, ③
BQ.1.1-pET-28a, ④XBB.1.5- pET-28a
; Coating on 4 Kana/LB plates (4
plates required)
,
Overnight culture at
3.37
℃
Part 2 Protein expression and purification
1.
IPTG inducible expression
Apparatus: oscillations incubator , Centrifuge
When OD=0.6~0.8 (visual
turbidity), add the final concentration in the medium as 0, 0.1, 0.25, 0.5,
0.75, 1, 2mM
IPTG,
One group was cultured at
37
℃
200 rpm for about 3h
.
Two groups were cultured
overnight at 16
℃
at 200 rpm
.
(
After induction: 10000g/2min centrifuge collection of bacteria
(packed with 50ml centrifuge tube), 10 times the volume (15ml)
The 50 mM Tris-HCl buffer (pH 8.0) is suspended (eventually combined into 7 tubes)
and temporarily stored in a 4
℃
refrigerator.)
2. SDS-PAGE
1) 15%
separation gel
|
D
istilled water
|
2.4mL
|
|
30% Acr-Bis(29:1)
|
5.0mL
|
|
Gel buffer A
|
2.5mL
|
|
10% APS
|
0.1mL
|
|
TEMED
|
0.006mL
|
A layer of isopropyl alcohol is
coated on the lower gel solution to keep the gel surface flat
2) 5% concentrating gel
|
D
istilled water
|
1.0mL
|
|
30% Acr-Bis(29:1)
|
0.5mL
|
|
Gel buffer B
|
1.5mL
|
|
10% APS
|
0.03mL
|
|
TEMED
|
0.003mL
|
3) Electrophoresis
Electrophoretic buffer:
Tris-Glycine-SDS buffer
Loading quantity: small hole
10ul/ large hole 30-40UL
Top glue 80V, 15min
Glue layer 120V,
45min
3.
C
oomassie brilliant blue
1)
Dyeing: Use directly with Koomas
Bright Blue -R250 dyeing solution
2)
D
estainin
g solution
:
|
Ethanol
|
400mL
|
|
Acetic acid
|
100mL
|
|
Distilled water
|
500mL
|
2)
Dyeing: Soak in petri dish for
30min
Decoloration: Wash with water
first, and then use the
destaining
solution to decolorize
overnight.
3.
Extend Culture
Apparatus: : oscillations incubator
10ml of E. coli BL(DE3) cultured overnight were
added to 500 mL of LB with kana resistant liquid medium at
37
℃
at 200 rpm
c
ulture for about 4h.
Added 312.5ul IPTG
4.
Ultrasonic crushing----Collecting protein
Apparatus
：
S
onicator
1)
Collection of inclusion body:
ultrasonic crushing on ice, power 500W (40%), on 3s off 3s, total duration: 10min. Centrifuge at
8000 rpm at 4
℃
for 30 min and remove the precipitate (inclusion body).
2
)
Us
ing
10 times the volume
(15-20ml) of the washing buffer(50 mM Tris-HCl, pH 8.0, 1 M NaCl, 2 M
Urea) wash the inclusion body
twice. Centrifuge at 8000 rpm at 4
℃
for 10min
.
3
)
Dissolved inclusion body:
Dissolved buffer (50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA
,
15 mM DTT, 6 M GuHCl guanidine
hydrochloride)was added to the inclusion body.
A
fter the inclusion body is
completely or mostly dissolve
d
, centrifuge
(5000g/5min/4
℃
) to obtain supernatant.
4. The concentration of
superalbumin was determined by A280, and the concentration of superalbumin was adjusted to 0.5 mg/mL
with dissolved buffer.
6. Dialyze
1
)
Configure the solution
① 2% (W/V) sodium bicarbonate and 1 mmol/L EDTA-2Na (pH=8.0)
:
Weigh
1g sodium bicarbonate is added
water
to the beaker for dissolv
ing
, then
weigh
0.168g EDTA, and finally the
volume was set to 500ml;
② 500ml of 1 mmol/L EDTA-2Na (pH=8.0)
:
W
eigh
0.168g of EDTA, fixed volume to
500 mL.
2
)
Take a dialysis bag of the
required length for the experiment, with 2% (W/V) sodium bicarbonate and 1 mmol/L EDTA-2Na in 500 mL
(pH=8.0)
.
Boil the dialysis bag for 10
min. After thoroughly cleaning with distilled water, place at 500 mL 1 mmol/L EDTA-2Na
(pH=8.0)
boil
for 10 min. After removal, the
dialysis bag was thoroughly cleaned with distilled water.
7. Renaturation
1
)
The inclusion body solution was
transferred to the dialysis bag and dialysis was performed at 4
℃
with 10
X
volume (300ml) of retaliated
buffe
r for
4h.
2) Replace the fresh dialysate
for the second dialysis for 4h, and put the
one last dialysate
in the refrigerator at
4
℃
for overnight dialysis.
8. Ni-NTA Purification
1
)
Use a
pipette
to suck out the
renaturated inclusion body from the dialysis bag
2
)
Balance column: Use 5
times nickel column volume (5ml) balance buffer to wash nickel column
3
)
Pass the column: add the
final concentration of 5mM imidazole (1ml-10UL of 500mM mi) to the inclusion body solution after
refolding
. T
he flow fluid was collected to
detect the ability of protein binding to the nickel column (
collect
sampl
e 1
).
4
)
Washing: Wash the nickel
column using a
B
alance
B
uffer of 10 times the nickel
column volume (10ml) (collect sample 2)
5
)
Wash: Wash the nickel
using a
W
ash
ing
Buffer (
B
alance
B
uffer + 20 mM imidazole) of 3
times the nickel column volume (3 mL) and collect the flow fluid (collect sample 3)
6) Elution: Elution
B
uffer (
B
alance
B
uffer + 200 mM imidazole) with 5
times nickel column volume (5ml) is used to elute
protein. C
ollect eluent into EP tube.
(Collect sample 4)
7
)
A280 was used to determine
protein concentration (BCA quantification), and SDS-PAGE was used to detect protein purity
Part 3 Protein expression and purification
1. RBD coating ELISA high binding plate
The purified RBD protein was diluted with PBS to 0,0.0064, 0.032, 0.16, 0.8, 4, 20,
100, 500 ng/mL, 100µL per well, three multiple
w
ells per sample, incubation for 2 hours at 37
℃
(this step is done in advance to the day before, overnight incubation is used)
2
. ELISA test
1)
Wash: PBST (PBS-0.05% Tween/ 0.5mL), 350µl per well, wash three times (add the
solution, then pour it out, use absorbent paper inverted suction)
2)
Closure: 5%BSA dissolved in PBS, 100 µL per well, sealed at 37℃ for 2 hours (Do not
replace the sealing solution with skim milk powder, skim milk powder contains Biotin that will produce
high background)
3)
Wash: PBST, 350 µl per well, washed three times
4)
Incubate: Biotinylated-ACE2 (1mg/mL)1:2000 Diluted with PBST, 100 µL per well, and
incubated at 37℃ for 2h
5)
Wash: PBST, 350µL per well, wash three times
6)
Incubate: Streptavidin-HRP (1mg/mL) 1:2000 diluted with PBST, 100 µL per well, and
incubated at 37℃ for 1h
7)
Wash: PBST, 350 µl per well, wash five times
8)
Color rendering:TMB substrate solution, 100 µL per well, 10 min at room temperature
(time needs to be observed, and add when the substrate begins to turn yellow when viewed by the naked
eye end with concentrated sulfuric acid)
9)
Termination: 0.5 M H2SO4/termination solution, 100 µ L per well, detected within 15
minutes
3
.
OD detection
OD450 and OD630
Appendix
Apparatus: Electronic balance
1.
IPTG
After dissolving 1g IPTG in 4ml distilled water, it is filled with distilled water to 5ml
and filtered with a disposable needle sterilize, then divide into 1ml small portions and store at -20℃.
(Concentration 0.8mol/l)
2.
Tris-HCl (500mL)
Dilute 1M Tris-HCl solution at pH 8.0 (Take 50ml of 1M Tris-HCl and add 950ml
water)
3.
Wash buffer (200mL)
|
Tris
|
1.211g
|
|
NaCl
|
11.7g
|
|
Urea
|
24g
|
4.
Solution buffer (200mL)
|
Tris
|
1.211g
|
|
EDTA-2Na
|
0.067g
|
|
DTT
|
0.46g
|
|
GuHCl
|
114.636g
|
5.
Retaliating buffer (1L)
|
Glycerol
|
100ml
|
|
PB (0.2M)
|
100ml
|
|
EDTA-2Na
|
0.06g
|
|
l-arginine
|
87.1g
|
|
GSH
|
0.55g
|
|
GSSG
|
0.55g
|
|
Urea
|
120g
|
6.
PB (200mL, 0.2M)
2.4g NaH2PO4 dissolved in 100ml water 5.68g
Na2HPO4 is dissolved in 200ml of water
Take 38ml of NaH2PO4, take 162ml of Na2HPO4 and mix
7.
Balance buffer (500mL)
|
Glycerol
|
50ml
|
|
PB
|
50ml
|
|
Urea
|
30g
|
|
Imidazole
|
0.17g
|
8.
Imidazole mother liquor (50mL, 500mM)
Dissolve 1.7g of imidazole in water and fill to 50ml
9.
Nickel column washing buffer
Add an additional 4ml of 500mM of imidazole mother liquid to the 100ml balance
buffer
10.
Elution buffer
Add an additional 33ml of 500mM of imidazole mother liquid to the 50ml balance
buffer