Preparation Work

Reagents and Materials:

• Tryptone 2×2g
• Yeast extract 2×1g
• NaCl 2×2g
• Agar 4g
• IPTG 0.476g
• Distilled water as needed
• Conical flask × 2
• Weighing paper as needed
• Sealing film × 2
• Digital scale
• Rubber bands as needed
• Test tube × 1
• EP tubes and filters as needed
• Beaker × 1
• Graduated cylinder (or any other equipment for measuring out the amounts of water needed)

Protocol:

To Prepare the Liquid LB:

1. Measure 2g tryptone, 1g yeast extract and 2g NaCl using weighing paper and a digital scale, and add them into a conical flask.
2. Measure 200ml distilled water using a graduated cylinder and add it into the conical flask as well.
3. Cover with sealing film and use a rubber band to secure, then place it in the autoclave.

To Prepare the Solid / Agar LB:

1. Measure 2g tryptone, 1g yeast extract, 2g NaCl and 4g agar using weighing paper and a digital scale, and add them into a conical flask.
2. Measure 200ml distilled water using a graduated cylinder and add it into the conical flask as well.
3. Cover with sealing film and secure with a rubber band, then place it in the autoclave.

To Prepare the Kan Antibiotic Stock Solution:

1. Measure out 1g Kan using weighing paper and a digital scale, and add it to a test tube.
2. Measure 10ml distilled water using a graduated cylinder and add it into the test tube. Mix well.
3. Using a pipette, filter the solution into 2ml EP tubes, and store at -20ºC for later use.

To Prepare the IPTG Antibiotic Stock Solution:

1. Measure out 0.476g of IPTG powder using weighing paper and a digital scale, and add it to a beaker.
2. Measure out 50ml of distilled water using a graduated cylinder, and add it into the beaker.
3. Mix well and store at -20ºC for later use.

Plate Pouring:

1. Prepare autoclaved pipette tips and a new petri dish
2. While the agar LB is still in liquid form, add 1/1000 its volume of Kan solution (e.g. 200mg Kan solution for 200ml agar LB). Shake to mix well.
3. On an ultra-clean bench, light an alcohol lamp and sterilize its top using the flame from the alcohol lamp.
4. Half-open the lid of a petri dish, and pour enough of the agar LB to cover its entire bottom surface.
5. Cover the lid and wait until it solidifies.

Result:

Prepares 500 mL of liquid LB and 300 mL of solid / agar LB, as well as some antibiotic stock solution (Kan solution and IPTG solution), which are used in later experiments. Most importantly, petri dishes of solidified LB medium are prepared, ready for growing E. Coli colonies in the following experiments.

PCR

Reagents and Materials:

From KOD Plus Neo (Toyobo, kod401):

• 10 x PCR Buffer for KOD -Plus- Neo
• KOD -Plus- Neo (1.0 U / µL)
• 25 mM MgSO4
• 2 mM dNTPs

Custom from Shanghai Sangon:

• Primer H3-F
• Primer H3-R

• Distilled water
• 100ng/µL Plasmid solution (previously prepared and extracted)
• 100ng/µL Plasmid backbone (also previously prepared)
• Thermal cycler

Protocol:

PCR (including preparation):

1. Add 25µL KOD Plus Neo Buf., 10µL dNTPs, 1.5µL Primer H3-F, 1.5µL Primer H3-R, 1µL KOD Plus Neo, 2µL MgSO4, 8µL Distilled water, and 1µL Plasmid solution to the PCR strip tube using pipette.
2. Put the PCR solution into thermal cycler and cycle 40 times for the plasmid to amplify. The cycle is as follows:
   1. 94ºC 2 mins
   2. 98ºC 10 secs
   3. 56ºC 30 secs
   4. 68ºC 60 secs
   5. Repeat b-d 40 times
   6. 4ºC indefinitely (for storage)

Result:

Increases the amount of the target DNA for future use

DNA Extraction + Purification

Reagents and Materials:

From Tiangen DP204-02:

• Buffer BL
• Buffer PB
• Buffer PW
• Buffer EB
• Spin column EB2 as needed

• Agarose
• TAE buffer
• Nucleic acid dye
• Marker
• Plasmid backbone
• Liquid LB

Protocol:

1. Add a spin column into a collection tube, then add 50µL BL into it using a pipette
2. Centrifuge, 12000 rpm 1 min to activate the spin column
3. Remove the spin column, remove waste liquid, and put spin column back in
4. Add the mixture containing DNA from the previous section of the experiment (50µL)
5. Add 150µL buffer PB, close the lid and let it set for two minutes
6. Centrifuge, 12000 rpm 1 min, then remove waste liquid from collecting tube
7. Add 600µL buffer PW
8. Centrifuge, 12000 rpm 1 min, remove waste liquid
9. Repeat steps 7 and 8 to assist with removing impurities
10. Centrifuge, 12000 rpm 2 mins, remove waste liquid
11. Open the lid and allow it to dry for 5 mins
12. Transfer the spin column to a new collection tube and discard the old one
13. Add 20µL buffer EB
14. Centrifuge, 12000 rpm 2 mins
15. Pipette out all the liquid from the collection tube and place it back into the spin column (note: do not let the pipette tip touch the filter, and ensure all of the liquid are on the filter, not on the side)
16. Centrifuge, 12000 rpm 2 mins
17. Discard the spin column, and label the collection tube as it now contains the purified DNA.

Result:

Obtains the purified DNA with proteins and other impurities filtered out. Each step uses a different buffer to remove some of the impurities.

Electrophoresis

Reagents and Materials:

• Agarose
• TAE buffer
• Nucleic acid dye
• Marker
• DNA loading buffer

Protocol:

1. Measure 1g agarose into the conical flask.
2. Add 100mL TAE buffer to the conical flask. Heat the solution until boiling, and shake to mix well.
3. Add 10µL nucleic acid dye to the agarose solution. (Note: this should be in the contaminent zone and gloves should be discarded afterwards)
4. Place small-tooth combs in the electrophoresis tank.
5. Pour the solution into the mold. Wait around 20 minutes until the gel solidifies.
6. Transfer 4µL marker to the first hole of the gel.
7. Mix 2µL plasmid solution with 2µL DNA loading buffer then transfer the mixure to the holes in the gel. Be careful not to puncture the gel with the pipette tip
8. Turn on the current for electrophoresis: 10V per centimeter, 300mA for 30 minutes
9. To read the results, use an UV imaging machine linked to a computer

Result:

Tests the samples to verify that PCR and purification were successful.

Preparation 2

Reagents and Materials:

• Liquid LB
• Plasmid backbone solution
• Single-use test tube (>5ml) with lid

Protocol:

1. On an ultra-clean bench, pipette 5ml of liquid LB and 10µL of backbone plasmid template into the single-use test tube, then place the lid on so that it is slightly loose and air can escape.
2. Place it on an orbital shaker at 37ºC, ~200rpm overnight.

Result:

Increases the amount of backbone plasmid template to prepare for purification and use later on.

Plasmid Extraction

Reagents and Materials:

• Plasmid bacterial solution (from previous step)

From Tiangen DP103-02:

• Buffer BL
• Buffer P1
• Buffer P2
• Buffer P3
• Buffer PW
• Buffer EB
• Spin Columns CP3
• Collection tubes

Protocol:

1. Place the CP3 spin column into the collection tube, and add 500µL buffer BL into the CP3 spin column.
2. Centrifuge, 12000 rpm 1 min to activate spin column, then remove waste liquid.
3. Add 750µL (full) plasmid bacterial solution from previous steps into the spin column.
4. Centiruge, 12000 rpm 1 min, then discard clear liquid, keeping the sediments.
5. Pipette 250µL buffer P1 in the spin column and mix using the pipette
6. Pipette 250µL buffer P2 and gently turn the tube 6 times.
7. Pipette 350µL buffer P3 and gently turn the tube 6 times until a white precipitate is formed.
8. Centrifuge, 12000 rpm 10 mins
9. Transfer clear top liquid to the spin column, careful not to collect any precipitate.
10. Centrifuge, 12000 rpm 1 min, discard waste liquid in collection tube.
11. Pipette 600µL buffer PW into the spin column.
12. Centrifuge, 12000 rpm 1 min, discard waste liquid from collection tube
13. Repeat step 11 to 12.
14. Centrifuge, 12000 rpm 2 mins, discard waste liquid from collection tube
15. Let the spin column dry for five minutes.
16. Place the spin column into a clean collection tube then pipette 50µL buffer EB through the spin column.
17. Centrifuge, 12000 rpm 2 mins, transfer solution containing plasmid from the tube back into the spin column.
18. Centrifuge, 12000 rpm 2 mins, discard spin column
19. Close the lid of the collection tube and store it for later use.

Result:

Extracts and purifies the backbone plasmid DNA prepared yesterday for future steps.

Electrophoresis 2

Reagents and Materials:

• Agarose
• TAE buffer
• Nucleic acid dye
• Marker
• DNA loading buffer

Protocol:

1. Measure 1g agarose into the conical flask.
2. Add 100mL TAE buffer and heat until just boiling. Shake to mix well.
3. Prepare the electrophoresis tank with large-tooth combs
4. Add 10µL nucleic acid dye to the agarose solution, then pour the solution into the electrophoresis tank. Wait until the gel solidifies.
5. Mix 10µL plasmid solution with 10µL DNA loading buffer then transfer the mixure to the holes in the gel.
6. Turn on the current for electrophoresis: 10V/cm, 300mA, 30 mins
7. Use an Ultraviolet Spectrophotometer to show results as preparation for the next step.

Result:

Separates the target DNA from any bacterial DNA and helps increase concentration of DNA solution.

Gel Recycling

Reagents and Materials:

• Gel from the previous step
• Sharp utility knife

From Tiangen DP209-02:

• Buffer BL
• Buffer PN
• Buffer PW
• Buffer EB
• Spin column CA2 as needed
• Collection tubes

Protocol:

1. Place the CA2 spin column into the collection tube, and pipette 500µL buffer BL into the spin column.
2. Centrifuge, 12000 rpm 1 min, discard waste liquid from collection tube
3. Cut the DNA strip from the agarose gel using a utility knife and place it in a clean collection tube.
4. Pipette 500µL buffer PN to the agarose gel and place the tube in 50 degree celsius water / dry bath until all the gel has melted.
5. Transfer the gel solution into the spin column and place it under room temperature for two minutes.
6. Centrifuge, 12000 rpm 1 min, discard waste liquid from collection tube
7. Pipette 600µL buffer PW into the spin column.
8. Centrifuge, 12000 rpm 1 min, discard the waste liquid from collection tube
9. Repeat steps 7 to 8
10. 12,000 rpm centrifuge the tube for two minute and discard the waste liquid to make sure no excess buffer PW is left on the spin column.
11. Let the spin column dry for five minutes.
12. Place the spin column into a clean tube then add 30µL buffer EB into the spin column.
13. Centrifuge, 12000 rpm 2 mins, transfer the plasmid solution from the collection tube back through the spin column.
14. Centrifuge, 12,000 rpm 2 mins, discard the spin column.
15. Keep the plasmid solution in the collection tube for later use.

Result:

Recycles the DNA by extracting it from the electrophoresis gel.

Plasmid Construction

Reagents and Materials:

From FlyCut JX201-01:

• 10xFlyCut Buffer
• FlyCut XhoI

From FlyCut JB101-01:

• FlyCut BamHI

• Transgen T4 DNA Ligase FL101-101
• 6x Buffer (Transgen GH101-01)
• dH2O

Protocol:

1. Pipette 10µL plasmid backbone, 5µL 10*Fly cut buffer, 1µL Bam HI, 1µL xhoI, and 33µL distilled water into the test tube.
2. Place the test tube in 37 degree celsius water bath for four hours.
3. Pipette 2µL backbone solution (from previous steps), 4µL target DNA, 0.5µL T4 ligase, 2µL 5* buffer, and 1.5µL distilled water to the test tube.
4. Place the test tube in 25 degree celsius water bath for two hours.

Result:

Cuts the plasmid at two locations, then uses T4 ligase to join the plasmid backbone with the target DNA.

Plasmid Insertion into Bacteria + Cultivation

Reagents and Materials:

From previous experiments:

• Plasmid solution
• Liquid LB
• Solid (agar) AB

• Vazyme DH5α CB502-02

Protocol:

1. Add 5µL plasmid solution and 30µL DH5α to a test tube
2. Place the tube in ice bath for 30 minutes.
3. Transfer the tube to 42ºC water bath for 1 minute 15 seconds. (note: timing should be accurate, use a timer)
4. Place the tube back in the ice bath for 2 minutes.
5. Add 500µL liquid LB to the tube.
6. Place the tube to the 37ºC orbital shacker for 40 minutes.
7. Transfer 100µL bacterial solution to the solid LB petri dish and spread the solution using a spreading rod.
8. Place the petri dish up side down in the incubator at 37ºC overnight.

Result:

Inserts the plasmid into the DH5α E. Coli, then cultivates it to let it grow overnight into bacterial colonies.

PCR 2

Reagents and Materials:

• Vazyme Taq enzyme QL101-01
• dH2O

Custom from Shanghai Sangon:

• H3-R primer
• H3-F primer

From previous experiments:

• Liquid LB
• Petri dishes

Protocol:

1. Add 15µL taq enzyme, 13µL distilled water, 1µL H3-R primer and 1µL H3-F primer to a test tube.
2. Transfer some cultivated bacteria to a new solid LB petri dish and the test tube using the pipette.
   Explanation: to prevent unsuccessfully grown colonies being used, colonies can be first transferred to a separate agar plate and cultivated at 37ºC for a few hours to grow the colonies, and only successfully grown colonies should be used
3. Place the tube into the thermal cycler for 30 cycles. Program:
   1. 98ºC 5 mins
   2. 98ºC 30 secs
   3. 56ºC 10 secs
   4. 72ºC 15 secs
   5. Repeat steps b-d for a total of 30 times
   6. Store at 4ºC
4. Measure 1g agarose into the conical flask.
5. Add 100mL TAE buffer to the conical flask. Heat the solution until well mixed or the TAE is boiling.
6. Setup the elelectrophoresis tank with small-tooth combs.
7. Add 10µL nucleic acid dye to the agarose solution, then pour the solution into the electrophoresis tank. Wait until the gel solidifies (~20 mins)
8. Transfer 20µL marker to the first hole of the gel.
9. Mix 10µL plasmid solution with 10µL DNA loading buffer then transfer the mixure to the holes in the gel.
10. Turn on the current for electrophoresis: 10V/cm, 300mA for 30 minutes.
11. Use a UV imaging machine to visualize results.
12. Add 4mL liquid LB to a test tube.
13. Transfer some newly cultivated bacteria to the test tube.
14. Place the test tube in a 37 degree celcius shaker overnight.

Immunogen Design

Reagents and Materials:

• Computer with internet access
• Access to NCBI Influenza Virus Sequence Database
• MEGA 10 software
• EXCEL (or similar spreadsheet software)
• BioEdit software

Protocol:

1. Open your web browser and go to the NCBI Influenza Virus Sequence Database.
2. Download all available HA2 protein sequences for both Influenza A (H1N1) and Influenza B viruses in FASTA format.
3. Open MEGA 10 software and import all downloaded sequences for sequence alignment.
4. Use the alignment tool to align all sequences, especially focusing on amino acid positions 76–130 in the HA2 protein.
5. Export the aligned sequences and open them in EXCEL or BioEdit.
6. Use the software to score the conservation level for each amino acid position among all aligned sequences.
7. Generate a conservation scoring matrix or heatmap to visualize conservation at each residue.
8. Analyze the matrix to identify regions (epitopes) with the highest conservation scores.
9. Select the most highly conserved epitope sequences for further screening.

Immunogen Screening

Reagents and Materials:

• Internet access to IEDB B-cell epitope prediction
• Kolaskar & Tongaonkar antigenicity analysis tool (on IEDB)
• Computer

Protocol:

1. For each highly conserved sequence identified, open the IEDB B-cell epitope prediction site.
2. Use the Kolaskar & Tongaonkar antigenicity prediction tool to analyze each sequence.
3. Enter the sequence and review the predicted antigenicity scores for each amino acid.
4. Consider factors such as surface accessibility, flexibility, and hydrophobicity, in addition to antigenicity.
5. Identify continuous high-scoring segments as candidate B-cell epitopes.
6. Select the best candidate epitopes for immunogen design, based on all predictive factors.

7. Immunogen Synthesis

   Reagents and Materials:

   • DNA synthesis service (e.g., Tianjin Jiutian Gene Company)
   • Plasmid expression vector: pet28a-sumo
   • Ferritin nanoparticle carrier sequence
   • Design software for DNA constructs
   • Flexible linker sequences

   Protocol:

   1. Design DNA constructs where the 16-amino acid dominant epitopes from Influenza B and H1 are linked together with flexible linker sequences, repeated four times in tandem.
   2. Fuse these tandem repeats to the ferritin nanoparticle carrier sequence to enhance immunogenicity.
   3. Additionally, concatenate H1, B, and homologous H3 epitope fragments and fuse to ferritin for broad-spectrum effect.
   4. Clone the designed immunogen sequences into the pet28a-sumo prokaryotic expression vector.
   5. Submit the recombinant DNA constructs to a synthesis company (such as Tianjin Jiutian Gene Company) for gene optimization and synthesis.
   6. Upon receiving lyophilized plasmid, centrifuge at 12000 rpm for 30 seconds.
   7. Dissolve the pellet in 40 μL ddH₂O, vortex to mix thoroughly.
   8. Measure DNA concentration using a spectrophotometer or fluorometer.
   9. Take 10 μL of the solution to send for sequencing (e.g., Sangon Biotech).
   10. Store the remainder at –20°C for later use.
   11. Plasmid Construction & Nucleic Acid Validation

Transformation for Protein Expression

Reagents and Materials:

• BL21 (DE3) or Transetta (DE3) competent cells (frozen stock)
• LB liquid medium (without antibiotic)
• Solid LB agar plates with antibiotic
• 1.5 mL microcentrifuge tubes
• 42°C water bath

Protocol:

1. Thaw BL21(DE3) or Transetta(DE3) competent cells on ice for 5 minutes.
2. Add 5 μL plasmid to 50 μL competent cells.
3. Follow the same transformation procedure as previously described.
4. Plate on antibiotic LB plates and incubate at 37°C overnight.

Protein Expression, Purification, and Refolding

Reagents and Materials:

• IPTG (for induction)
• LB medium
• Shaking incubator, 28°C and 37°C
• Centrifuge (large volume, cold)
• Inclusion body wash buffer
• Sonicator (with cooling)
• 8M urea
• AKATA pure10 protein purification system
• Nickel affinity purification column
• Imidazole elution buffer (300 mM)
• ddH₂O
• 20% ethanol
• Dialysis tubing (pretreated)
• G+ and G– dialysis buffers
• Ultrafiltration tubes (proper molecular weight cutoff)
• Tabletop centrifuge

Protocol:

1. Inoculate a single colony into LB medium and grow overnight at 37°C.
2. Transfer the culture into fresh LB and grow to OD600 ~0.6–0.8.
3. Add IPTG to a final concentration of 1:1000 (v/v) and induce expression at 28°C, 180 rpm for 10–12 hours.
4. Harvest the induced cells by centrifugation at 4000 rpm for 30 minutes; balance tubes on a scale.
5. Discard supernatant, resuspend pellet in inclusion body wash buffer.
6. Sonicate the resuspended cells at 4°C: 5 s on / 5 s off pulses, total effective sonication time 40 minutes, to lyse cells and remove air bubbles.
7. Centrifuge the lysate at 16000 rpm for 25 minutes at 4°C.
8. Discard the supernatant, resuspend the pellet (inclusion bodies) in 8M urea, and stir at 4°C overnight.
9. Prepare the AKATA pure10 system and nickel affinity column: flush with ddH₂O at 5 mL/min until conductivity is 0, pH 7. Equilibrate further with 2–3 column volumes.
10. Install nickel column, continue flushing with ddH₂O until conductivity stabilizes at 0.
11. Load the inclusion body solution dissolved in 8M urea onto the column using the A pump (100%, 0–3 mL/min).
12. Wash the column with 8M urea to remove unbound or loosely bound protein.
13. Elute the protein with a gradient of imidazole (B pump: 10%, 30%, 50%, 100% of 300 mM imidazole buffer).
14. Collect fractions when UV absorbance increases, stop when the curve stabilizes.
15. Wash the column with 10–20 column volumes of 300 mM imidazole, then with ddH₂O and then with 20% ethanol to store the column.
16. Prepare dialysis tubing (pretreated and stored in laboratory).
17. Dilute the purified protein 1:10 with inclusion body solubilization buffer, load into dialysis tubing, and seal.
18. Dialyze against 5 L G+ buffer, changing buffer 3 times every 6 hours. Then switch to G– buffer, again changing 3 times every 6 hours.
19. After dialysis, concentrate the protein using ultrafiltration tubes by centrifugation at 4000 rpm for 40 minutes.
20. Adjust protein concentration as required for downstream experiments.
21. Aliquot, confirm by native PAGE and store at –80°C.
22. Non-denaturing (Native) PAGE

Reagents and Materials:

• 10% native protein gel
• Bio-Rad PAGE tank and power supply
• 1x Native Running Buffer
• Ice

Protocol:

1. Assemble the 10% native gel in the Bio-Rad PAGE system.
2. Place the electrophoresis tank on ice.
3. Fill tank with 1x Native Running Buffer.
4. Load samples into wells.
5. Run at 80V until samples enter the separating gel, then increase voltage to 120V until completion.

6. TEM and Particle Size Analysis

   Protocol:

   Samples are sent to external service facilities for transmission electron microscopy and particle size analysis.

7. Coomassie Brilliant Blue Staining and Western Blot

   Reagents and Materials:

   • 4x Sample Buffer
   • Heating block (97°C)
   • Bio-Rad PAGE tank and power supply
   • 1x Running Buffer
   • Coomassie Brilliant Blue staining solution
   • Destaining solution
   • Semi-dry transfer system and accessories
   • Nitrocellulose membrane
   • PBST buffer
   • Skim milk powder
   • Primary and secondary antibodies
   • Alkaline phosphatase buffer, NBT, BCIP

   Protocol:

   1. Mix protein sample with 4x Sample Buffer at a 3:1 ratio.
   2. Heat at 97°C for 10 minutes.
   3. Load onto prepared denaturing PAGE gel, fill tank with 1x Running Buffer.
   4. Run at 80V until samples enter separating gel, then increase to 120V until finished.
   5. For staining: Place gel in Coomassie Brilliant Blue solution, oscillate 20 minutes to 2 hours until bands are visible.
   6. Destain on shaker until bands are clear.
   7. For western blot:
      1. Wet nitrocellulose membrane and filter paper with transfer buffer.
      2. Assemble gel/membrane sandwich (hardboard, filter paper, membrane, gel, filter paper, hardboard), avoid air bubbles.
      3. Transfer at 15V for 18 minutes on semi-dry transfer system.
   8. Block membrane with 3% skim milk in blocking buffer for 45 minutes on shaker.
   9. Wash with PBST 3–5 times, 1 minute each.
   10. Incubate with primary antibody (diluted as required), shake for 1.5 hours.
   11. Wash with PBST 3–5 times, 1 minute each.
   12. Incubate with secondary antibody (diluted as required), shake for 1 hour.
   13. Wash with PBST 3–5 times, 1 minute each.
   14. Prepare color development solution: 30 mL alkaline phosphatase buffer, 198 μL NBT, 99 μL BCIP, protect from light.
   15. Add color development solution to membrane, develop color for 5–10 minutes, protect from light.
   16. Stop when bands are clear.