2 Materials
2.1 DNA Vaccine
Design and Generation
1. Restriction enzymes from New England Biolab (NEB).
2. T4 DNA ligase (NEB).
3. TOP10 competent cells (Life Technologies).
4. 2YT medium (VWR).
5. Innova 40R shaker (Eppendorf).
6. Heraeus Megafuge 16R Refrigerated Benchtop Centrifuge
(Thermo Scientific).
7. Qiagen Endotoxin Free Plasmid Purification kit (Giga
prep size).
8. Nanodrop 2000 (Thermo Fisher).
2.2 Mouse
Vaccination
1. Female C57BL/6 mice of 6–8 week old with ethics approval
from Animal Welfare Committee.
2. Ketamine hydrochloride (100 mg/mL), Medetomidine hydrochloride (1 mg/mL), Atipamezole hydrochloride (5 mg/mL)
and Saline for Injection.
3. 0.5 mL Insulin syringe with 29-G needle (BD).
4. 1 mL Tuberculin syringe and 29-G needle (BD).
5. Goldenrod animal lancet (4 mm) (Medipoint Inc.).
6. Heraeus Fresco17 Refrigerated Microcentrifuge (Thermo
Scientific).
7. CpG2006 Phosphorothioate oligonucleotide (Oligo Factory).
8. Advax™ adjuvant manufactured by Vaxine Pty Ltd (GMP
batch).
9. NEPA21 Electroporator (Nepa Gene).
2.3 JEV Challenge
1. Class II Biosafety cabinet.
2. Individually ventilated mouse cages and a rack.
3. 1 mL Insulin syringe with 29-G needle manufactured by BD.
4. Challenge virus (JEV Nakayama strain).
5. Animal Weighing Scale.
2.4 ELISA
1. Inactivated viruses or recombinant viral proteins of interest.
2. 96-Well ELISA plates (Greiner Bio-One).
3. 24-Well culture plates (Greiner Bio-One).
4. Sterile serological pipettes (Greiner Bio-One).
5. Multichannel pipette (20–200 μL).
6. Variable Adjustable Volume Pipettes.
DNA Vaccine Design and Adjuvant Selection
409
2.1 DNA Vaccine
Design and Generation
1. Restriction enzymes from New England Biolab (NEB).
2. T4 DNA ligase (NEB).
3. TOP10 competent cells (Life Technologies).
4. 2YT medium (VWR).
5. Innova 40R shaker (Eppendorf).
6. Heraeus Megafuge 16R Refrigerated Benchtop Centrifuge
(Thermo Scientific).
7. Qiagen Endotoxin Free Plasmid Purification kit (Giga
prep size).
8. Nanodrop 2000 (Thermo Fisher).
2.2 Mouse
Vaccination
1. Female C57BL/6 mice of 6–8 week old with ethics approval
from Animal Welfare Committee.
2. Ketamine hydrochloride (100 mg/mL), Medetomidine hydrochloride (1 mg/mL), Atipamezole hydrochloride (5 mg/mL)
and Saline for Injection.
3. 0.5 mL Insulin syringe with 29-G needle (BD).
4. 1 mL Tuberculin syringe and 29-G needle (BD).
5. Goldenrod animal lancet (4 mm) (Medipoint Inc.).
6. Heraeus Fresco17 Refrigerated Microcentrifuge (Thermo
Scientific).
7. CpG2006 Phosphorothioate oligonucleotide (Oligo Factory).
8. Advax™ adjuvant manufactured by Vaxine Pty Ltd (GMP
batch).
9. NEPA21 Electroporator (Nepa Gene).
2.3 JEV Challenge
1. Class II Biosafety cabinet.
2. Individually ventilated mouse cages and a rack.
3. 1 mL Insulin syringe with 29-G needle manufactured by BD.
4. Challenge virus (JEV Nakayama strain).
5. Animal Weighing Scale.
2.4 ELISA
1. Inactivated viruses or recombinant viral proteins of interest.
2. 96-Well ELISA plates (Greiner Bio-One).
3. 24-Well culture plates (Greiner Bio-One).
4. Sterile serological pipettes (Greiner Bio-One).
5. Multichannel pipette (20–200 μL).
6. Variable Adjustable Volume Pipettes.
DNA Vaccine Design and Adjuvant Selection
409