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CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof

A technology of gene sequence and high immunity, applied in the direction of anti-infective drugs, organic active ingredients, medical preparations containing active ingredients, etc., can solve the problems of no treatment methods and achieve the effect of high immune activity

Inactive Publication Date: 2005-11-23
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Excessive CpG-S ODN releases a large number of cytokines such as TNF-α, IL-1, IL-6 and IL-12 by activating the mononuclear-phagocyte system, causing SIRS and sepsis, and the fatality rate is as high as 50 -80%, so far there is no effective treatment

Method used

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  • CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0068] This experimental example is to obtain CpG-S ODN and CpG-N ODN gene sequences by using bioinformatics technology.

[0069] 1. The serotype 2, 5 and 12 Adv DNA and ECDNA sequences were obtained from the Nucleotide database on PubMed (ACCESSION: NC_001405, M73260, NC_001460, NC_000913).

[0070] 2. Using bioinformatics technology and using biological software such as Primer Premier 5 and BioEdit version 5.0.6, 256 CpG-containing hexanucleotide sequences in serotype 2, 5 and 12 Adv DNA and EC DNA were analyzed and compared (See Table 1), based on: ① High frequency in Adv DNA of serotype 2 and 5; ② Low frequency in Adv 12 DNA and EC DNA; ③ Adv2: Adv12≥2 or Adv2: EC≥5 In principle, 19 CpG-containing hexanucleotide sequences unique to serotype 2, 5 Adv DNA were identified.

[0071]

serial number

sequence

Adv 2

Adv 5

Adv 12

EC DNA

Adv2:Adv12

Adv2: EC

Frequency

frequency

Frequency

frequen...

experiment example 2

[0081] This experimental example is to study the synthesis method of oligonucleotides.

[0082] The method of the present invention adopts the solid-phase phosphoramidite triester method. Simply put, monomers in the solution form a 3'→5' phosphodiester bond through a condensation reaction, thereby connecting to a solid-phase support.

[0083] 1. Basic material:

[0084] (1) Support: Solid-phase synthesis is to immobilize nucleic acid on a solid-phase carrier to complete the synthesis reaction. The most commonly used solid-phase carrier is controllable microporous glass beads (CPG, controlled pore glass). The pore size of CPG depends on the synthesized The length of the oligonucleotide depends on the length of the oligonucleotide. Generally, when the synthetic chain length is less than 60nt, choose a pore size of 500 angstroms. Conditions for oligonucleotides up to 175nt. CPG is covalently bonded to the hydroxyl group of the initial nucleotide through a linker compound, and t...

experiment example 3

[0105] This example is to identify the activity of 7 non-obviously irritating ODNs inhibiting CpG-S ODN.

[0106] Isolate and culture human peripheral blood mononuclear cells (hPBMC), adjust the concentration of hPBMC cell suspension to 1×10 6 / ml, added to a 24-well plate, 1ml per well. Set at 37°C CO 2 After culturing in the incubator for 2 hours, add 10 μg / ml of each ODN to be screened for 30 minutes, then add 5 μg / ml of CpG-S ODN (TGG CGC GGGGCG), and place at 37°C CO 2 The cell culture supernatant was taken after 4 hours of incubation in the incubator, and the concentration of TNF-α in the cell culture supernatant was determined by double antibody sandwich ELISA method to clarify the ability of the above 7 ODNs to inhibit the release of TNF-α from hPBMC induced by CpG-S ODN. The experimental results showed that the above 7 ODNs all had the ability to inhibit the release of TNF-α from hPBMC induced by CpG-S ODN, so we officially called it CpG-N ODN (see Table 5).

[010...

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Abstract

Disclosed is a CpG-N ODN gene sequence with high immunological activity CpG-S ODN and antagonism CpG-N ODN and use in preparing medicament for treating infectious diseases, anaphylactic diseases, immunodeficiency diseases, tumor, SIRS and pyaemia.

Description

technical field [0001] The present invention relates to an oligonucleotide and its application in medicine, in particular, to an immune stimulating oligonucleotide (CpG-S ODN) and an oligonucleotide (CpG-S ODN) with an antagonistic effect on it. -N ODN). Background technique [0002] Oligonucleotide fragments containing unmethylated CpG are the smallest unit of bacterial DNA, so oligonucleotides containing unmethylated CpG that have an immunostimulatory effect are called immunostimulatory oligonucleotides (Immnostimulatory oligonucleotides, CpG-S ODN). Its structural feature is a hexamer containing a CpG motif, that is, a complementary palindromic structure of 6 bases. The hexamer must contain at least one CpG dinucleotide, and there must be a purine at the 5' end of CpG. The structure is similar to ApA , GpA or GpT, etc., there must be two pyrimidines such as TpT at the 3' end, and hexamers known to have immunostimulatory effects include AACGTT, AGCGCT, etc. [0003] CpG...

Claims

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Application Information

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IPC IPC(8): C07H21/04
Inventor 周红王良喜郑江
Owner ARMY MEDICAL UNIV
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