Purified CCF protein and GEM particles were prepared and stored according to previous protocols (19 (link), 20 (link)). Briefly, the CCF protein was expressed by Escherichia coli Rosetta (DE3) cells with pET-28a-CCF. The protein was first purified by nickel affinity chromatography (GE Healthcare), followed by anion-exchange chromatography with DEAE Sepharose FF (Amersham Pharmacia Biotech AB, Sweden). The purity of CCF was confirmed by Coomassie blue staining. The GEM particles were prepared by Lactococcus lactis NZ9000 cells using a hot-acid water bath. Vaccine with Alum was prepared with an equal volume of CCF solution and Alum adjuvant. CpG ODN 1826 was obtained from Sangon Biotech Co., Led. (China, Shanghai) and dissolved in CCF solution before intranasal vaccination.
Cpg odn 1826
Manufactured by Sangon
Sourced in China
CpG ODN 1826 is a synthetic oligodeoxynucleotide containing unmethylated CpG motifs. It acts as a toll-like receptor 9 (TLR9) agonist, which can stimulate the immune system.
Automatically generated - may contain errors
Lab products found in correlation
Hpv16 e7 peptide, by GL Biochem (2 mentions)
Nickel affinity chromatography, by GE Healthcare (1 mentions)
Deae sepharose ff, by Cytiva (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Anti cd4 apc, by BD (1 mentions)
Il 12p70, by Thermo Fisher Scientific (1 mentions)
Traut s reagent, by Sangon (1 mentions)
Anti cd80 fitc, by BD (1 mentions)
Ova257 264, by Merck Group (1 mentions)
Ova323 339, by Merck Group (1 mentions)
Lyso tracker red, by Solarbio (1 mentions)
Anti cd11c bb700, by BD (1 mentions)
Anti cd8a percp cy5, by BD (1 mentions)
Anti cd86 pe, by BD (1 mentions)
Anti cd3e fitc, by BD (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
7 protocols using cpg odn 1826
1
Purification and Preparation of CCF Protein and GEM Particles
2
APAP-Induced Liver Injury Mitigation
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NAC (150 mg kg−1, i.p., Sigma‐Aldrich) was administered after 3 h (or 10 h) and DNase I (80 U/dose, Sigma‐Aldrich) was administered by i.v. at 3 and 10 h (or 10 h) after APAP challenge. CpG ODN 1826 (0.5 mg kg−1, i.v., Sangon Biotech) was given at 3 h after APAP. Animals were sacrificed at 12 or 24 h to detect related parameters. In the severe model, the survival rates were monitored for 96 h.
3
Multifunctional Vaccine Delivery System
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Stannous octoate (Sn(Oct)2), polyethyleneimine (PEI), ε-caprolactone, 4-nitrophenyl chloroformate (NPC), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), cysteamine hydrochloride, 2,2′-Dithiodipyridine, N-hydroxysuccinimide (NHS) were obtained from Aladdin (China). CpG ODN1826 and traut's reagent were obtained from Sangon Biotech (Shanghai). HOOC-PEG-NH2 were purchased from Ponsure Biological (Shanghai). Carboxyfluorescein succinimidyl ester (CFSE), OVA, OVA257−264, and OVA323−339 were purchased from Sigma-Aldrich (USA). LysoTracker Red and DAPI were purchased from Beijing Solarbio Science & Technology Co.,Ltd. The anti-SIINFEKL/H-2KbPE, anti-CD11c-BB700, anti-CD86-PE, anti-CD80-FITC, anti-CD3e-FITC, anti-CD4-APC and anti-CD8a-PerCP-Cy5.5 were obtained BD Biosciences (USA). IL-12p70, TNF-α and IFN-γ enzyme-linked immunosorbent assay (ELISA) kits were purchased from Invitrogen (USA). FITC—OVA was prepared according to the previous protocol.
4
HPV16 E7 Peptide and CpG ODN Protocol
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The HPV16 E7 peptide (E7 43–77: GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR) was provided by GL Biochem (Shanghai) Ltd. (Shanghai, China). The purity of the peptide was verified by high-performance liquid chromatography and was found to be routinely >95%. CpG ODN 1826 (5ʹ-TCCATGACGTTCCTGACGTT-3ʹ) was synthesized by Sangon Biotech (Shanghai) Co. Ltd. (Shanghai, China).
5
BrdU Immunohistochemistry Protocol
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Ovalbumin (Sigma), a BrdU kits (Affymetrix eBioscience), rabbit anti-mouse IgG-HRP antibody (Elabscience), and APS (Tianjin Cinorch Pharmaceutical Co., Ltd., Tianjin, China) were purchased. CpG ODN1826 (5′-TCCATGACGTTCCTGACGTT-3′) was synthesized (Sangon Biotech, Shanghai Co., Ltd.) with full chain phosphorothioate modification and PAGE-purified.
6
Synthesis of HPV16 E7 Peptide and CpG-ODN
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The HPV16 E7 peptide (E7 43–77) was synthesized by GL Biochem (Shanghai) Ltd (Shanghai, China). CpG ODN 1826 was synthesized by Sangon Biotech (Shanghai) Co. Ltd (Shanghai, China). The sequences of the peptide and CpG ODN were as follows:
E7 43–77: GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR; CpG-ODN1826: 5′-TCCATGACGTTCCTGACGTT-3′.
E7 43–77: GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR; CpG-ODN1826: 5′-TCCATGACGTTCCTGACGTT-3′.
7
Rv3615c Protein Immunization in Mice
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The Mycobacterium tuberculosis (M.tb) Rv3615c (Esx‐1 substrate protein C, EspC) protein and its peptide array, and CpG ODN 1826 were purchased from Sangon Biotech Co., Ltd (Shanghai, China) with customized synthesis. The peptide array comprises 19 peptides spanning the entire region of the Rv3615c sequence, with each having 15 mers and overlapping by 11 amino acids. The immunization protocol was previously reported.36 Briefly, 10 μg Rv3615c/mouse was mixed with or without 20 μg CpG ODN 1826/mouse. For subcutaneous immunization, the mixture was suspended in PBS and injected twice into the lower quadrant of the abdomen (200 μL/mouse). For intranasal immunization, the mixture was suspended in PBS and injected intranasally through the nasal cavity while the mice were anaesthetized with bromethane (20 μL/mouse). A detail schedule of prime and boost is shown in Figure 1 .
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