Modified oligodeoxynucleotide molecule containing CpG sequence unit and application thereof

A deoxynucleotide and oligomerization technology, used in biochemical equipment and methods, anti-tumor drugs, allergic diseases, etc., can solve the problem of low bioavailability and achieve good oral bioavailability and good immune regulation. , the effect of improving the immune response

Active Publication Date: 2016-09-07
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the full or partial sulfur modification of the backbone of CpG oligodeoxynucleotides can partially solve the problem of CpG oligodeoxynucleotides being hydrolyzed by nucleases, but there is still the problem of low bioavailability. Invention of small molecule modifications to CpG oligodeoxynucleotides to address this issue, particularly the bioavailability of oral administration

Method used

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  • Modified oligodeoxynucleotide molecule containing CpG sequence unit and application thereof
  • Modified oligodeoxynucleotide molecule containing CpG sequence unit and application thereof
  • Modified oligodeoxynucleotide molecule containing CpG sequence unit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the influence of azide and alkynyl concentration, catalyst, reaction time on CuAAC reaction

[0035] A dipeptide AF is selected to optimize the reaction conditions, and the optimized method can be extended to the click reaction between other small molecules and CpG ODN. The premise of all changes in reaction conditions is that the reaction is carried out under shaking at room temperature.

[0036] Table 1 Experimental design of different concentrations of azide and alkynyl, different proportions of catalysts and different reaction times

[0037]

[0038] figure 1 It is the PAGE diagram of the click reaction under different reaction conditions. In (a) and (b), M represents a 20bp DNA marker, and lane 1 is an unmodified CpG ODN; perform PAGE electrophoresis according to the design in Table 1; figure 1 The reaction conditions in (a) refer to the conditions of the classic small molecule click reaction [Rostovtsev, V.V., et al., A stepwise huisgency lo...

Embodiment 2

[0046] Example 2, the ratio of azide small molecule to CpG ODN and the influence of reaction buffer on CuAAC reaction

[0047] After determining the optimal reaction time, clarifying the role of TBTA, optimizing the concentration of CpG ODN and AF and the ratio of the catalyst, the effect of different ratios of AF and CpG ODN and the reaction buffer on the click reaction was investigated, and the coupling product was determined purification method.

[0048] Table 2 Different buffers, the experimental design of different ratios of AF and CpG ODN

[0049]

[0050] figure 2 In (a) and (b), M represents 20bp DNA marker, lane 1 is unmodified CpG ODN; (a) according to Table 2 Designed for electrophoresis; (b) PAGE verification of the purification method, lane 3 is purified CpG-AF;

[0051] figure 2 In (a), 2M pH 7.0 triethylamine acetate buffer was added to the reaction system of lane 2 and lane 3, and there was no buffer in the reaction system of lane 4 and lane 5; the r...

Embodiment 3

[0053] Example 3, the above CuAAC reaction conditions are applied to other small molecules

[0054] Take the fatty acid lauric acid as an example, image 3 Schematic diagram of the CuAAC reaction of azidolauric acid and alkyne-modified CpG ODN;

[0055] Reactant stock solution configuration;

[0056] ①0.5mM Alkyne-modified CpG ODN stock solution:

[0057] Centrifuge the centrifuge tube containing the alkyne-modified CpG ODN powder first. Each tube contains 25 nmol of CpG ODN, add 50 μl of ultrapure water, shake to mix evenly, and store at -20°C.

[0058] ②0.02M azidolauric acid stock solution: 2.3mg azidolauric acid, add 353μl DMF, shake to dissolve, seal and store at 4°C.

[0059] ③ 4mM TBTA stock solution: Weigh 4.2mg TBTA powder, dissolve in 2ml DMF, shake to mix evenly, and store at -20°C.

[0060] ④0.5mM CuSO 4 Stock solution: weigh 12.5mg CuSO 4 ·5H 2 O, dissolved in 1ml ultra-pure water, ready to use now.

[0061] ⑤ 0.2M Sodium Ascorbate stock solution: Weigh 1...

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Abstract

The invention relates to a modified oligodeoxynucleotide molecule containing a CpG sequence unit and application thereof in the technical field of immunotherapy, relates to the application of the modified oligodeoxynucleotide molecule containing the CpG sequence unit to serve as immunopotentiator, and further relates to the application of the modified oligodeoxynucleotide molecule containing the CpG sequence unit to serve as oral or suction preparations for treating tumors. The structure modified CpG oligodeoxynucleotide (CpG ODN) is prepared from peptide fragments with specific combinations, fatty acid with specific chain length and lipoid structure modified CpG ODN with an immune modulating function. The structure modified CpG ODN has better oral bioavailability and immune modulating function, can be used as oral immunopotentiation and immunotherapy medicine, can improve immune response of an organism to antigen and can be applied to anaphylactic diseases, infectious diseases, immunodeficiency diseases, cancers and the like.

Description

[0001] This application is a divisional application of the original application. The filing date of the original application is: 2013.11.08; the application number is: 201310554389.X; its use. technical field [0002] The invention belongs to the technical field of immunotherapy, and specifically relates to a modified oligodeoxynucleotide molecule containing a CpG sequence unit and its application. Background technique [0003] As early as the 1890s, Coley, an American oncologist, found that 40% of the tumors in 900 long-term tumor patients with crude bacterial extracts could regress by themselves. At that time, it was believed that the lipopolysaccharide in the crude bacterial extracts played an active role. , has not attracted enough attention. Later, an in vivo experiment using Bacillus Calmette-Guerin (BCG) in mice found that BCG treated with DNase did not have an anti-tumor effect, indicating that bacterial DNA is the substance that mediates this anti-tumor effect [Tok...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/117A61P35/00A61P37/04
CPCC12N15/117C12N2310/17
Inventor 徐宇虹吴彩兴张冲向小飞
Owner SHANGHAI JIAOTONG UNIV
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