New DNA vaccine adjuvant of the pBOOST3 plasmids expressing the mouse TBK1 gene
20 µg of lyophilized pBOOST3-mTBK1 plasmid expressing the mouse TBK1 gene
• 4 pouches of E. coli Fast-Media Zeo (2 TB and 2 Agar)
Storage and Stability
Products are shipped at room temperature.
Lyophilized DNA is stable for 12 months when stored at -20°C.
Resuspended DNA is stable for 12 months when stored at -20°C. Avoid repeated freeze-thaw cycles.
Store E. coli Fast-Media Zeo at room temperature. Fast-Media pouches are stable 18 months when stored properly.
General Product Use
The pBOOST3 plasmid was developed as a genetic adjuvant for DNA vaccines to potentiate the immune response to a specific antigen. The plasmid contains the mouse TANK-binding kinase 1 (mTBK1) gene. TBK1, a non-canonical IkB kinase, was shown to mediate the adjuvant effect of DNA vaccines. Administration of DNA vaccines induces the production of type I interferons and inflammatory cytokines in a CpG-independent manner but in TBK1-dependent manner.
The method of plasmid DNA vaccine delivery is known to bias the immune response to a specific antigen towards a type 1 (T-cell) response. A DNA vaccine incorporated with genetic adjuvant such as the MyD88 or the TRIF gene has been shown to enhance immune responses. As TBK1 has been shown to play a crucial role in humoral responses, coadministration of a TBK1-expressing plasmid is expected to further boost DNA vaccine-induced immunogenicity.
• mTBK1 TBK1 signaling is thought to be critical for optimal humoral response as well as for helper T (Th)1 cytokine production after DNA vaccination.
• hEF1 / HTLV prom is a composite promoter comprising the Elongation Factor-1α (EF-1α) core promoter4 and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1α promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance stability of RNA.
• SV40 pAn: The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.
• Ori pMB1 is a minimal E. coli origin of replication with the same activity as the longer Ori.
• EM2KC is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.
• Sh ble: The Sh ble gene from Streptoalloteichus hindustanus encodes a small protein that confers resistance to Zeocin by binding to the antibiotic."
Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To obtain a plasmid solution at 1µg/µl, resuspend the DNA in 200 µl of sterile H20. Store resuspended plasmid at -20°C.
Selection of Bacteria with E. coli Fast-Media Zeo
E. coli Fast-Media Zeo is a fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. Fast-Media Zeo is a TB (liquid) or LB (solid) based medium with zeocin. Method
1- Pour the contents of a pouch of Fast-Media into a clean borosilicate glass bottle or flask.
2- Add 200 ml of distilled water to the flask
3- Heat in a microwave on MEDIUM power setting (about 400Watts), until bubbles start appearing (approximately 3 minutes). Do not heat a closed container. Do not autoclave Fast-Media.
4- Swirl gently to mix the preparation. Be careful, the bottle and media are hot, use heatproof pads or gloves and care when handling.
5- Reheat the media for 30 seconds and gently swirl again. Repeat as necessary to completely dissolve the powder into solution. But be careful to avoid overboiling and volume loss.
6- Let agar medium cool to 45°C before pouring plates. Let liquid media cool to 37°C before seeding bacteria.
Note: Do not reheat solidified Fast-Media as the antibiotic will be permanently destroyed by the procedure. Plasmid DNA solution
- Prepare the vaccine plasmid solution by resuspending 10 µg of the vaccine plasmid DNA in 50 µl saline solution.
- Prepare the pBOOST3 solution by mixing 10 µg of pBOOST3-mTBK1 and 90 µg of the mock plasmid pBOOST3-null in 50 µl saline solution for low dose, or 100 µg of pBOOST3-mTBK1 in 50 µl saline solution for high dose.
- Combine both solutions to obtain a total of 110 µg DNA in 100 µl saline solution.
Note: The quantities are per mouse. Intramuscular injections
- Inoculate 6 to 8-week old female BALB/c mice with 100 µl plasmid DNA solution (described above) into the quadriceps at 0 and 4 weeks.
- Collect sera and analyze for antibodies at 8 weeks.