CpG ODN with immunoregulation function and application of CpG ODN

A technology for immune regulation and application, which can be used in medical preparations containing active ingredients, recombinant DNA technology, DNA/RNA fragments, etc., and can solve problems such as limited

Pending Publication Date: 2021-10-12
CHANGCHUN HUAPU BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current practical application is limited, and more and more in-depth research is needed to design more and more effective sequence structures so that they can exert their potential more widely and be used in clinical practice more safely and efficiently.

Method used

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  • CpG ODN with immunoregulation function and application of CpG ODN
  • CpG ODN with immunoregulation function and application of CpG ODN
  • CpG ODN with immunoregulation function and application of CpG ODN

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The preparation of embodiment 1.CpG ODN

[0088] Using an automatic DNA synthesizer on a certain scale, the solid-phase phosphite amine triester method is used to synthesize the DNA in several steps of deprotection, activation, thiolation, and capping. After synthesis, the oligonucleotides are deprotected using concentrated ammonia, followed by purification and desalting. Purified oligonucleotides were lyophilized as sodium salts and characterized by MS before use. Finally, a CpG ODN sequence with a purity >90% was obtained. The CpG ODNs indicated by ODN1, ODN2, ODN3, ODN4, ODN5, and ODN6 were used in subsequent experiments.

[0089] 5’-DMT dA, dG, dC, dT and other phosphoramidite monomers were purchased from Shanghai Zhaowei Technology Development Co., Ltd. Corresponding vectors were purchased from Chemgenes (Wilmington, MA). 2'-Substituted ribonucleoside phosphoramidites were purchased from Shanghai Zhaowei Technology Development Co., Ltd., Promega (Obispo, CA). ...

Embodiment 2

[0090] Example 2 Effects of CpG ODN on mouse spleen cell T and B cell proliferation

[0091] Isolation and culture of mouse spleen cells:

[0092] Isolate mouse spleen under aseptic conditions, prepare BALB / c mouse spleen cell suspension after grinding and filtering, use RPMI-1640 complete medium to suspend cells, 5××10 5 or 1×10 6 Spread each well in a 96-well U-shaped plate, add different concentrations of CpG (0.03, 0.1, 0.3, 1 and 3uM), and place at 37°C, 5% CO 2 cultured in an incubator.

[0093] After culturing for 16 hours, the cells were collected, washed twice with 1×PBS, and centrifuged at 1500 rpm for 5 minutes. The cells were resuspended in 1×PBS, anti-CD4, anti-CD8 and anti-CD19 antibodies (BD, USA) were added to the cell suspension, and incubated at 4° C. in the dark for 30 min. Wash twice with 1×PBS, centrifuge at 1500rpm for 5min, resuspend cells in 1×PBS, and use BD LSRFortessa TM Flow cytometry (BD, USA) was used for flow cytometric analysis.

[0094] S...

Embodiment 3

[0096] Example 3: Cytokine induction in mouse splenocyte cultures

[0097]Splenocytes from 4-8 week old C57BL / 6 were prepared and cultured in complete RPMI medium. Mouse splenocytes were divided into 5×10 6 Cells / ml were seeded in 24-well culture dishes. CpG ODN dissolved in PBS buffer was added to the cell cultures to final concentrations of 0.03, 0.1, 0.3, 1 and 3 uM, respectively. The cells were then incubated at 37°C for 24 h, and the supernatant was collected for ELISA analysis. The levels of IFN-α, IL-6, TNF-α in the supernatant were determined by sandwich ELISA. The required reagents, including cytokine antibodies and standards, were purchased from BD PharMingen. See the test results figure 2 , 3, 4. in figure 2 It is shown that different CpG ODNs effectively stimulate pDCs to secrete IFN-α levels, and HP3004 is a positive control. image 3 It is shown that different CpG ODNs stimulate B cells to produce IL-6 levels, and HP3004 is a positive control. Figure ...

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Abstract

The invention relates to a CpG ODN with an immunoregulation function and application of CpG ODN. The CpG ODN has good immunostimulation activity, can stimulate B cell proliferation, and meanwhile generates specific cytokines. The CpG ODN can be used as a vaccine adjuvant, can be used alone, can be combined with other adjuvants to play a synergistic role, and can also be applied to the preparation of drugs for preventing or treating tumors, infections and allergies.

Description

technical field [0001] The present invention relates to an immunoregulatory CpG ODN and its composition. The CpG ODN has good immunostimulatory activity, can stimulate B cell proliferation, and produce cytokines, can be used as a vaccine adjuvant alone or in combination with other adjuvants to play a synergistic effect, and can also be used in the preparation of preventive or therapeutic Drugs for tumors, infections, and allergies. Background technique [0002] In 1984, it was found that the nucleic acid components in Bacillus Calmette-Guerin (BCG) had an anti-tumor effect in a mouse tumor-bearing model, and it was successfully applied to the treatment of human tumors (Tokunaga T, Yamamoto H, Shimada S, et al.Antitumor activity of deoxyribonucleic acid fraction from mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J Natl Cancer Inst, 1984, 72(4): 955-962). Degradation analysis with ribonuclease and deoxyribonuclease later pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/117A61K39/39A61P37/04A61P35/00A61P31/00A61P37/08
CPCC12N15/117A61K39/39A61P37/04A61P35/00A61P31/00A61P37/08C12N2310/17A61K2039/55561A61K38/00Y02A50/30A61K39/001156A61K39/001192C12N2310/315C12N2310/321C12N2310/322A61P31/14A61K39/205A61K39/215A61K2039/55505
Inventor 王立公邵彦
Owner CHANGCHUN HUAPU BIOTECHNOLOGY CO LTD
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