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Structure and application of target programmed cell death protein 5 (PDCD5) anti-influenza virus oligonucleotide

An oligonucleotide, influenza A virus technology, applied in the field of bioengineering drugs, to achieve the effects of less toxic and side effects, important social and economic benefits, and high specificity

Inactive Publication Date: 2012-07-25
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of reliable methods to effectively control the occurrence and prevalence of influenza, and the effective prevention and control of influenza virus methods has become a top priority

Method used

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  • Structure and application of target programmed cell death protein 5 (PDCD5) anti-influenza virus oligonucleotide
  • Structure and application of target programmed cell death protein 5 (PDCD5) anti-influenza virus oligonucleotide
  • Structure and application of target programmed cell death protein 5 (PDCD5) anti-influenza virus oligonucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] This example mainly illustrates the design, synthesis and screening of anti-influenza virus antisense oligonucleotides targeting human PDCD5 gene.

[0035] Materials and Methods

[0036] 1. Design and synthesis of antisense oligonucleotide PDCD5-5

[0037]Retrieve the nucleic acid sequence database in GeneBank, select the PDCD5 mRNA reference sequence NM_004708.2 published by NCBI, and perform computer simulation of the RNA secondary structure, and select 5 unstable stem-loop structures as antisense oligonucleotides target. Through online blast sequence comparison with GeneBank, the selected target sequences have good specificity and will not interfere with the expression of other normal human genes (see Table 1). All oligonucleotides were synthesized with 8909 type automatic DNA synthesizer to synthesize antisense oligonucleotides modified with full sulfur. After the synthesis was completed, the concentrated ammonia solution was cleaved and deprotected at 55°C for 1...

Embodiment 2

[0057] This example mainly illustrates the anti-influenza virus effect of antisense oligonucleotides PDCD5-5 with different lengths.

[0058] 1. Optimization of PDCD5-5 sequence length one

[0059] Referring to the mRNA sequence of the human PDCD5 gene, the PDCD5-5 sequence was increased or decreased by bases at both ends, so that the sequence was 22, 18, 16, and 14 bases. Through online blast sequence comparison with GeneBank, the selected target sequences have good specificity and will not interfere with the expression of other normal human genes. Referring to Example 1, oligonucleotide synthesis, sulfur modification, purification, drying and storage were performed.

[0060] Referring to Example 1, A549 cells were plated on a 96-well cell culture plate, and 60-80% of them were confluent the next day. Dilute PDCD5-5 to 0.25, 0.5, 1.0, 2.0 μM with 1640 containing 1% FBS. The cells were washed once with PBS (pH7.5), and different concentrations of PDCD5-5 with a length of 22...

Embodiment 3

[0072] This example mainly illustrates the research on the inhibition of H1N1 cytopathic activity by rimantadine-modified PDCD5-5 at the level of A549 cells in vitro.

[0073] Materials and Methods

[0074] 1. Coupling of rimantadine to PDCD5-5

[0075] After dehydrochlorination, rimantadine hydrochloride exposes the amino group, and undergoes a nucleophilic substitution reaction with 6-maleimide caproic acid N-succinimide ester to replace the succinimide ester to form a new compound. Re-react with antisense oligonucleotides modified with sulfhydryl groups at 5', and finally form the target rimantadine-antisense oligonucleotide coupling compound. The coupling process is as follows: Figure 10 . Use the optimized synthetic route and the reaction conditions of each step to synthesize, synthesize the rimantadine modified sequence (PJG) of PDCD5-5 coupled with high-purity rimantadine and PDCD5-5, and use various analytical methods to ensure the obtained coupling The compound ha...

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Abstract

The invention relates to a structure and application of target programmed cell death protein 5 (PDCD5) anti-influenza virus oligonucleotide, in particular to a structure and application of the target PDCD5 anti-influenza virus (IV) antisense oligonucleotide, which is found that the PDCD5 can be used as a host target of anti-virus. Found by selection and the verification in the laboratory, viruses infect and interact with the host protein PDCD 5, thus, the antisense oligonucleotide of the target PDCD5 is designed and synthesized. 27 antisense oligonucleotide sequences are actively selected andevaluated on an A549 cell infected by the virus. The invention firstly finds that the oligonucleotide PDCD5-5 complementary to a special functional region of PDCD5 mRNA can effectively inhibit the duplication of IV and the cytopathy caused by H1N1 and H3D5 type IV, and has better specificity and drug property. Therefore, the invention relates to a sequence and a structure of the antisense oligonucleotide infected by the target PDCD 5 mRNA anti IV, and a novel drug for treating the IV virus related diseases and reducing the harmfulness of the IV related diseases.

Description

technical field [0001] The present invention relates to the field of bioengineering drugs, in particular to an antisense oligonucleotide (ASODN, ASODN) targeting programmed cell death factor 5 (PDCD5, Programmed cell death protein 5) for treating influenza virus (IV, influenza virus) infection. Antisense oligodexynucleotide) sequence, structure and therapeutic drugs. Background technique [0002] Influenza virus infection can cause acute respiratory infectious diseases. The disease has a short incubation period, is highly contagious, and spreads rapidly. Influenza A is the most threatening, and it is prone to mutation, which can easily cause outbreaks. From 1918 to 1920, the famous "Spanish flu" caused 20-40 million deaths worldwide. Since then, "Asian flu" caused by influenza A virus (H2N2) in 1957, "Hong Kong flu" caused by influenza A virus (H3N2) in 1968, and "Russian flu" caused by influenza A virus (H1N1) in 1977 Influenza", the highly pathogenic avian influenza vi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/16
Inventor 王升启赵海豹钟芝茵周喆伯晓晨杨静丁晓然林汝仙
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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