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Oligopolynucleotide of inhibiting activity of necrosin in human tumor

A tumor necrosis factor, oligonucleotide technology, applied in organic active ingredients, genetic engineering, non-central analgesics, etc., can solve problems such as low affinity and lack of target molecules

Inactive Publication Date: 2004-12-01
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of available target molecules and low affinity, its clinical application is limited.

Method used

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  • Oligopolynucleotide of inhibiting activity of necrosin in human tumor
  • Oligopolynucleotide of inhibiting activity of necrosin in human tumor
  • Oligopolynucleotide of inhibiting activity of necrosin in human tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: In vitro screening of aptamers specifically binding to TNF

[0065] amplified DNA library

[0066] Use primer 1 and primer 2 to amplify the single-stranded DNA library, then take 1 μl for asymmetric PCR, add 200 μl of Binding buffer (2×1M NaCl, 40mM TrisCl, 2mM MgCl2) after the product is recovered, denature at 95°C for 5min, Ice bath for 5min; add 1ul TNF (1ug / μl), incubate at 37°C for 1h, pass through the filter membrane, first wash with PBS, then soak in 300ul elution buffer (10mM EDTA, 7M urea binding buffer), ethanol precipitation and recovery For DNA, 10 μl of the above lysate was used as a template for the next round of screening. In the final rounds of selection, a small amount of TNF was used in order to obtain oligonucleotides expected to have high affinity, and a total of 12 rounds of selection were performed.

[0067] Cloning and sequencing

[0068] After 12 rounds of screening, the final screening product was connected to pGEM T-vector (Pr...

Embodiment 2

[0069] Example 2: Analysis of the secondary structure of the target aptamer

[0070] Using DNASYS v2.5 software to analyze the lowest secondary structure energy of 18 aptamer sequences and simulate their secondary structures, it was found that there may be at least two characteristic secondary structure shapes. One is represented by the following formula I, which is mainly characterized by a double stem-loop structure; the other is represented by the following formula II, which is mainly characterized by a three-stem-loop structure.

[0071]

Embodiment 3

[0072] Embodiment 3: the determination of DNA aptamer and TNF affinity

[0073] Biotin-labeled DNA aptamers were synthesized, and the affinity between the target aptamers and TNF was determined by two methods.

[0074] 1. Conventional ELISA: Coat 96-well ELISA plates with 1ug / ml TNF; block with 1% BSA at 37°C for 2 hours; add 5'-labeled biotin-labeled DNA aptamers to each well after doubling dilution, and incubate at 37°C 1 hour; add 100 μl of horseradish peroxidase-labeled streptavidin diluted 1:400, incubate at 37°C for 1 hour; add chromogenic solution to develop color in the dark, and read OD450 value on a microplate reader. see attached results figure 2 .

[0075] 2. Dot-ELISA method: Mix different concentrations of 5'-labeled biotin nucleic acid with TNF, and bathe in water at 37°C for 1h. The mixture of TNF and labeled nucleic acid was applied to the NC membrane. Wash away unbound nucleic acid, add horseradish peroxidase-labeled streptavidin on the membrane, and inc...

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Abstract

Oligonucleotide sequence i.e. adaptor including DNA sequence and RNA sequence can combine with TNF alpha in specificity so as to restrain damaging effect on L929 cells from TNF alpha. The invented adaptor can be utilized to test TNF alpha and cure disease caused by raised level of TNF alpha. Comparing with current antagon of TNF alpha, the invented adaptor possesses advantages of high specificity, high affinity, fast reaching target tissue and quick plasma clearance etc. The oligonucleotide sequence possesses affinity and specificity of monoclonal antibody as well as permeability and pharmacokinetics characters of small molecule polypeptide. The invention is also related to derivation sequence and modified sequence of oligonucleotide, as well as its usage, preparation and diagnosis in curing disease relevant to TNF alpha.

Description

technical field [0001] The present invention relates to a new set of oligonucleotide sequences. Specifically, the present invention relates to a group of novel oligonucleotides that inhibit the activity of human tumor necrosis factor. The target oligonucleotide is an aptamer (Aptamer) specifically combined with TNFα screened from a random oligonucleotide library using SELEX technology. The invention also relates to derivative sequences of said oligonucleotide sequences, including modified sequences. The present invention further relates to the use of the oligonucleotide sequence in the preparation of medicines for diagnosing or treating diseases related to TNF-α. Background technique [0002] Human tumor necrosis factor α (hTNFα) is a multifunctional cytokine produced by the activation of macrophages / monocytes. It has a wide range of biological activities. It plays an important role and plays a central regulatory role in the human cytokine immune regulatory network. When...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C12N15/115
CPCC12N2310/53C12N15/115C12N2310/322A61K38/00C12N2310/18A61P19/02A61P29/00
Inventor 张智清严馨蕊郭克泰徐春晓
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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