CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application

A technology of deoxynucleotide and vaccine composition, which is applied in the field of single-stranded deoxynucleotide, and can solve the problems that have not been reported or disclosed in the literature

Active Publication Date: 2011-05-18
CHANGCHUN HUAPU BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Yet, up to the present invention, do not see relevant bibliographical report or disclosure

Method used

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  • CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application
  • CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application
  • CpG-containing single-stranded deoxynucleotide, its vaccine composition and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0245] Example 1 Design of CpG single-stranded deoxynucleotide

[0246] The design sequence is as follows:

[0247] (1)(G) n (L) n X 1 X 2 CGY 1 Y 2 (M) n (G) n

[0248] X 1 =A, T, G; X 2 =A, T; Y 1 =A, T; Y 2 =A, T, C; L, M = A, T, C, G; n is 0-6

[0249] 5'-ggggTCgTTCgTCgTTgggggg-3' (SEQ ID NO: 2)

[0250] 5'-ggggATAACgTTgCgggggg-3' (SEQ ID NO: 3)

[0251] 5'-ggggTgCAACgTTCAgggggg-3' (SEQ ID NO: 4)

[0252] 5'-ggggTCCTACgTAggAgggggg-3' (SEQ ID NO: 8)

[0253] 5'-ggggTCCATgACgTTCCTgAAgggggg-3' (SEQ ID NO: 23)

[0254] 5'-gggggACgTCgCCggggggg-3' (SEQ ID NO: 37)

[0255] 5'-ggATCCgTACgCATgggggg-3' (SEQ ID NO: 38)

[0256] 5'-gggggAATCgATTCgggggg-3' (SEQ ID NO: 101)

[0257] 5'-gggATgCATCgATgCATCgggggg-3' (SEQ ID NO: 100)

[0258] 5'-ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 102)

[0259] 5'-gggACgTACgTCgggggg-3' (SEQ ID NO: 105)

[0260] 5'-gggggATCgACgTCgATCgggggg-3' (SEQ ID NO: 108)

[0261] 5'-ggCgATCgATCgATCggggggg-3' (SEQ ID NO: 111)

[0262] 5'-ggggTCgATCgATCgAgggggg-3' (SEQ ID NO: 112)

[0263] 5'-g...

Embodiment 2

[0441] Example 2 Synthesis of CpG single-stranded deoxynucleotide

[0442] DNA synthesis adopts the solid-phase phosphoramidite triester method to synthesize DNA fragments. This method has the advantages of high efficiency and rapid coupling, and has been widely used in DNA chemical synthesis.

[0443] DNA chemical synthesis is different from enzymatic DNA synthesis from the 5'→3' direction, but starts from the 3'end. The specific reaction steps are as follows:

[0444] 1. Deprotection group

[0445] Use trichloroacetic acid to remove the protective group DMT (dimethoxytrityl) of the nucleotide linked to CpG (Controlled Pore Glass) to obtain free 5'-hydroxyl end for the next condensation reaction .

[0446] 2. Activation

[0447] The phosphoramidite-protected nucleotide monomer is mixed with the tetrazolium activator and enters the synthesis column to form the phosphoramidite tetrazole active intermediate (its 3'-end has been activated, but the 5'-end is still affected by DMT Protecti...

Embodiment 3

[0457] Example 3 Enhancement effect of CpG-containing single-stranded deoxynucleotide on the immunostimulatory effect of heat shock protein hepatitis C virus antigen peptide fusion protein

[0458] 1. Hepatitis C virus specific cytotoxic T lymphocyte killing test

[0459] 1. Material:

[0460] Mycobacterium tuberculosis heat shock protein 65 (HSP65) and multi-epitope HCV core antigen fusion protein (HSP-HCV), CpG-containing deoxy oligonucleotide (CpG ODN), transfected with B16 of HCV core antigen multi-epitope antigen peptide gene Cell (HCV + B16 cells). CpG ODN1 (SEQ ID NO: 7), CpG ODN2 (SEQ ID NO: 106), CpG ODN3 (SEQ ID NO: 103), CpG ODN4 (SEQ ID NO: 18), CpG ODN5 (SEQ ID NO: 86), CpG ODN6 (SEQ ID NO: 79), CpG ODN7 (SEQ ID NO: 95), CpG ODN8 (SEQ ID NO: 123), CpG ODN9 (SEQ ID NO: 124), CpG ODN10 (SEQ ID NO: 159).

[0461] 2. Experimental animals and groups:

[0462] Using 40 male C57 / BL6 mice 6-8 weeks old, 10 mice in each group, divided into normal saline group (injection of saline...

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Abstract

The present invention relates to one kind of CpG-containing single-stranded deoxynucleotide capable of strengthening the immunostimulation of antigen or antigen composite, the vaccine composition of the single-stranded deoxynucleotide and antigen or antigen composite and its preparation process. The CpG-containing single-stranded deoxynucleotide can strengthen the specific antitumor, antiviral and chlamydia infection resisting activity of heat shock protein-antigen peptide fusion protein induced antigen peptide. In addition, the present invention also relates to the application of the CpG-containing single-stranded deoxynucleotide in preparing vaccine composition and the method of strengthening the immunostimulation. The vaccine composition may be used as treating medicine for viral infection, bacterial infectio, parasite infection, allergic reaction, cancer and relevant diseases.

Description

Technical field [0001] The invention relates to a CpGODN-containing single-stranded deoxynucleotide capable of enhancing the immunostimulatory effect of an antigen or an antigen composition, and a vaccine composition composed of the single-stranded deoxynucleotide and the antigen or the antigen composition. In addition, the present invention also relates to the application of CpGODN-containing single-stranded deoxynucleotides in preparing vaccine compositions, and the application of the vaccine compositions as medicines. Background technique [0002] In 1984, Tokunaga T et al. found that Bacille Calmette-Guerin (BCG) DNA can inhibit tumor growth, activate human peripheral blood mononuclear cells and natural killer (NK) cells, and induce interferon (IFN). These functions depend on the CpG structure in DNA molecules. (Tokunaga T, Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity,...

Claims

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Application Information

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IPC IPC(8): C12N15/117C07H21/00A61K39/39A61P31/00A61P35/00
CPCC12N2310/17C12N15/117A61K2039/55561A61K39/39A61P31/00A61P33/00A61P35/00A61P37/00
Inventor 王丽颖包木胜于永利
Owner CHANGCHUN HUAPU BIOTECHNOLOGY CO LTD
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