A.LB medium (g/L)
| Reagent | Dosage (g) |
| Tryptone | 10 |
| Yeast Extract | 5 |
| NaCl | 10 |
Add ddH2O to 1 L, sterilization at 121℃ for 20 min.
Solid plus 3 g agar per 100 mL.
B. Kanamycin (kan+) (50 mg/mL)
| Reagent | Dosage |
| kan | 500 mg |
| ddH2O | 10 mL |
Filter and remove bacteria.
Store in 1 mL package.
C. Inoculation: Take 5 μL of cryopreserved glycerol bacteria pET28a and add to 5 mL liquid LB medium in test tube, and then add the final working concentration of kanamycin is 50 μg/mL, culture at 37℃, 220 rpm in constant temperature shaker for 12~18h.
D. After the configured LB solid plate is lowered to room temperature, add the final concentration of 50 μg/mL kan+ and mix well, and then invert on the super clean table.)
E. PCR(μL)
| Reagent | Dosage (μL) |
| 2*mix | 25 |
| NS3-HeL/ZIKA-F | 2.5 |
| NS3-HeL/ZIKA-R | 2.5 |
| DNA | 2 |
| ddH2O | 18 |
| Total | 50 |
PCR procedure (NS3-HeL-1344bp / NS3-ZIKA-1854bp)
| Step | Temperature (℃) | Time |
| 1 | 95 | 5 min |
| 2 | 95 | 30 sec |
| 3 | 55 | 30 sec |
| 4 | 72 | 2 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |
PS: Steps 2 to 4 cycle 30 times.
F. Agarose gel
| Reagent | Dosage |
| 1*TAE | 50 mL |
| Agarose | 0.5 g |
High heat for 2 min to melt. Add 5 μL Nucleic acid dye.
Install the gel plate and insert the electrophoresis comb. Pour the prepared agarose gel into the gel platen at a height of 3~4 mm (avoid bubbles), and keep at room temperature to solidification. When fully set, pull out the electrophoresis comb and transfer the solid gel to the electrophoresis tank, and then we add 1*TAE buffer and completely immerse the gel. Put them into the spot hole of the agarose gel, final add 10 μL DNA Maker. Cover the electrophoresis tank, Loading hole Located at the electric field negative, power on, 150 V, 20 min. Turn off the power, remove the gel, and observe under UV light illuminator.
G. Gel extraction
Cut the gel which have destination fragment. Then weigh, add a certain proportion of gel melting agent to melt agarose gel, mix well and heat in water bath at about 50℃ until the gel is completely melted. Add binding fluid to allow the DNA to selectively bind to the DNA preparation membrane. Add DNA washing solution for cleaning, and then get purified DNA by elution.
H. Plasmid extraction
Take bacterial solution centrifuge at 12000 rpm for 1 min and discard the supernatant, collect bacteria. Add 250 μL buffer S1 to suspend the bacteria. Add 250 μL Buffer S2, gently and fully turn up and down 4 to 6 times and mix well to fully crack the bacteria until the clear solution is formed. Add 350 μL Buffer S3, gently and fully turn up and down 6 to 8 times, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Absorb the supernatant and transfer it to the preparation tube, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add 500 μL Buffer W1, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add 700 μL Buffer W2, centrifuge at 12000 rpm for 1 min, discard filtrate, and then use the same way again. Place the preparation tube back into the 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min. Place the preparation tube back into the 1.5 mL centrifuge tube, let the ethanol in W2 evaporate for 1 to 2 min, eluent or ddH2O preheated at 60-65℃ is added with 50 μL eluent in the center of the preparation membrane, and then water bath at 60-65℃ for 1 to 2 min, centrifuge at 12000 rpm for 1 min. Get plasmid DNA.
I. Digestion plasmid pET28a
| Reagent | Dosage (μL) |
| pET28a Plasmid (<=1ug)< /td> | 10 |
| BamHI | 1 |
| XhoI | 1 |
| Cutsmart | 5 |
| ddH2O | 13 |
| Total | 50 |
PCR Recovered product
| Reagent | Dosage (μL) |
| NS3-Hel/ZIKA | 10 |
| BamHI | 1 |
| XhoI | 1 |
| Cutsmart | 2 |
| ddH2O | 6 |
| Total | 20 |
Reaction at 37℃ for 30 min.
Agarose gel electrophoresis (Same method as above)
Gel extraction and plasmid extraction (Same method as above)
J. Ligation
| Reagent | Dosage |
| pET28a after digestion | 7 |
| NS3-HeL/ZIKA after digestion | 10 |
| T4 DNA Ligase | 1 |
| 10*T4 Ligase DNA buffer | 2 |
| Total | 20 |
Reaction at 16℃ for 30 min.
K. Heat shock transformation
Place centrifuge tube containing DH5α on ice and add 10 μL of the recombinant product, treated with ice bath for 30 minutes, treated with 42℃ hot water bath for 45 s, and then immediately treated with ice bath for 2 minutes. Add 900 μL LB liquid medium, gently blow and mix, and place the centrifuge tube in a shaking table at 37℃ and culture at 200 rpm for 1 hour. And then centrifuge at 5000 rpm for 5 min, leave a little supernatant to reinsert the cells, and coat them on LB plate medium containing kan+, and culture at 37℃ overnight.
L. Verified the transformant DH5α and inoculated DH5α
PCR
| Reagent | Dosage (μL) |
| 2*mix | 10 |
| Primer F | 1 |
| Primer R | 1 |
| ddH2O | 8 |
| Total | 20 |
PS: DNA plate: DH5α
Transferred the constructed recombinant plasmid to BL21(DE3) and plate coated for screening. (Same method as above)
Expanded the culture seed solution and performed IPTG induced expression BL21(DE3). Expanded the culture seed solution: 1% Inoculated volume is transferred to 100 mL LB Liquid medium, and OD600 is grown at 37°C to 0.4 to 0.6 (about 3 to 4 h).The final concentration of 0.4 mM IPTG was added to induce expression, and overnight culture was performed at 14°C.
A.Configuration buffer
a.His buffer A (0.5 L)
| Reagent | Dosage (g) |
| 20 mM Na2HPO4·2H2O | 1.78 |
| 500 mM NaCl | 14.6 |
| 20 mM imidazole | 1.02 |
PS: Adjust pH to 7.4 with HCl (6 M) and set the volume to 0.5 L.
b.His buffer B (0.25 L)
| Reagent | Dosage (g) |
| 20 mM Na2HPO4·2H2O | 0.89 |
| 500 mM NaCl | 7.3 |
| 20 mM imidazole | 8.5 |
PS: Adjust pH to 7.4 with HCl(6M), set the volume to 0.25 L and 0.45 mm filter membrane filtration sterilization.
c.10 mM MOPS buffer (50 mL): 0.1 g powder MOPS, 50 mL ddH2O, pH6.5
d.10 mM Tris-HCl buffer (50 mL): 0.02 g powder Tris-HCl, 50 mL ddH2O, pH6.5
e.2 mM MgCl2 buffer (50 mL): 0.02 g powder MgCl2, 50 mL ddH2O
B.Ultrasonic cell fragmentation
a.Centrifuge to collect bacteria, add 15 mL His buffer A, fully suspended, place it in a 50 mL centrifuge tube, ultrasonic crushing on ice, power 500 w, open 3 s off 3 s, total duration 10 to 20 min.
b.Centrifuge the collected supernatant, 4℃ , 12000 rpm, 30 min, collect the supernatant.
C.Nickel column affinity purified protein
Remove the pre-loaded nickel column and wash 10 column volumes with ddH2O, then wash 10 column volumes with His buffer A. The filtered cel supernatant is Loaded into the nickel column at a flow rate of 1 mL/min. Wash the sampled nickel column with His buffer A. The recombinant protein is eluted with His buffer B. Wash the column with His buffer B, His buffer A and water in turn. Wash the column with 5 mL 20% ethanol.
D.D.Configured SDS-PAGE protein glue
Assemble the glue mold, configure a 1.00 mm thick layer of glue. Configure a 1.00 mm thick layer of glue. Stand for 10 to 15 minutes, wait for the upper layer of glue to solidify, carefully pull out the comb, use the gun head or syringe to absorb the electrophoresis buffer and rinse the sample hole to perform SDS-PAGE electrophoresis.
E.SDS-PAGE protein electrophoresis
Add 40 μL crude protein solution into 6 μL loading buffer, boil at 100℃ for 10 min, centrifuge at 4℃, take 10 μL treated supernatant sample and add into sample hole. Cover the top, connect the electrophoresis device, and control the voltage of the sample before gluing at 100-200 V, about 15-20 min.When the bromophenol blue indicator reaches the separation glue, the voltage rises to 200 V, and the electrophoresis process keeps the voltage stable. Electrophoresis can be stopped when the bromophenol blue indicator reaches 1-2 cm away from the front, about 0.5-1 h.
F.Configure dyeing solution and decolorizing solution
Formula of dyeing solution (1 L) : 400 mL ethanol, 500 mL ddH2O, 100 mL acetic acid, 0.1% R250, filter with filter paper after dissolution.
Decolorizing solution formula: 40% ethanol, 10% acetic acid , 50% ddH2O.
G.Dyeing and decolorization of protein glue
Dyeing: After electrophoresis, turn off the power, take out the glass plate, separate the rubber surface from the glass plate, and then Light R250 for dyeing, about 0.5-1 h. Decolorization: Discard the dyeing solution, wash the rubber surface 2-3 times with ddH2O, and then add the decolorization solution for diffusion decolorization, and change the decolorization solution every 30 min until the protein bands are clear.
Configuration of SDS-PAGE protein glue : Separation glue/Concentrate glue
6% PAGE jelly: 70~300 KD
8% PAGE jelly: 30~200 KD
10% PAGE jelly: 20~80 KD
12% PAGE jelly: 15~60 KD
15% PAGE jelly: 10~45 KD
A.Synthetic DNA substrate: Helicase substrate is synthesized by combining a 1:1 molar ratio with a single strand with a final concentration of 20 μM in 10 mM Tris hydrochloric acid pH 8.5, 95°C 5 min, 75°C 5 min, 55°C 5 min, 35°C 5 min, at room temperature 25°C 1 h.
B. NS3-HEL/NS3-ZIKA: 10 mM MOPS (pH 6.5), 2 mM MgCl2, 2.5 nM-labeled double-stranded substrate, and 9.75 nM NS3 enzyme are mixed, ATP is added until the final concentration is 3 mM, and the test is performed 15 min Later. The reaction is performed at 100 μL and repeated three times. At the same time, blank (distilled water), negative (only NS3 enzyme is added) and positive are set up, and different concentration gradients are set. After reaction for 15 min, fluorescence detection is carried out with enzyme labeling instrument, with excitation wavelength of 620 nm and emission wavelength of 685 nm.
Overall system
| Reagent | Dosage (μL) |
| 10 mM MOPS | 10 |
| 2 mM MgCl2 | 10 |
| 2.5 nM Double stranded substrate | 1 |
| 9.75 nM NS3 protease | 4 |
| 3 mM ATP | 3 |
PS: Supplement with ddH2O to 100 μL