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Interfering RNA (Ribonucleic Acid) for suppressing hand-foot-and-mouth disease virogene, vector containing the same and application thereof

A hand-foot-and-mouth disease and vector technology, which can be used in DNA/RNA fragments, gene therapy, antiviral agents, etc., can solve the problems of short expression time and low transfection rate of exogenous genes, and achieve good targeting and specificity. Strong, long-lasting effects

Inactive Publication Date: 2013-11-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Minicircular DNA vector overcomes the shortcomings of traditional plasmid vectors such as low transfection rate, easy immune response and short expression time of foreign genes in vivo, and is a safe and efficient new vector

Method used

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  • Interfering RNA (Ribonucleic Acid) for suppressing hand-foot-and-mouth disease virogene, vector containing the same and application thereof
  • Interfering RNA (Ribonucleic Acid) for suppressing hand-foot-and-mouth disease virogene, vector containing the same and application thereof
  • Interfering RNA (Ribonucleic Acid) for suppressing hand-foot-and-mouth disease virogene, vector containing the same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, design of interfering RNA sequence targeting enterovirus type 71 gene, vector construction And cell and animal level pharmacodynamic testing.

[0048] 1. Sequence design of interfering RNA inhibiting enterovirus 71 gene expression and construction of expression vector establish

[0049] 1. Design of interference RNA sequence

[0050] (1), design of interfering RNA sequence targeting enterovirus 71 coding region 3A gene

[0051] Using interference sequence design software ( siRNA design ) to design an interfering RNA sequence targeted at the 3A gene of the enterovirus type 71 coding region, and the designed DNA sequence corresponding to the interfering RNA is as follows figure 1 B, the interference sequence is the conserved region sequence of enterovirus type 71 and Coxsackie virus A16 gene coding region 3A, which can form a small hairpin structure after transcription, so that it can be successfully recognized and cut by dicer enzyme, and res...

Embodiment 2

[0095] Example 2, Design of Interfering Sequence Targeting Coxsackievirus A16 Gene, Vector Construction and Cell and Animal level pharmacodynamic testing.

[0096] 1. Construction of an interfering RNA gene expression vector that inhibits the expression of the Coxsackie virus A16 gene

[0097] 1. Design of interference RNA sequence

[0098] Using interference sequence design software ( siRNA design ) to design an interference RNA sequence targeting the 3A / 3C / 3D gene of the Coxsackie virus A16 coding region, the interference sequence is the conserved region sequence of the 3A / 3C / 3D gene coding region of the Coxsackie virus A16 and enterovirus 71, and the sequence With embodiment 1.

[0099] 2. Construction of minicircle DNA vector

[0100] Construction method is the same as embodiment 1.

[0101] 2. Inhibition of Coxsackie virus A16 by the minicircle DNA interference system at the cellular level

[0102] 1. Inhibition of Coxsackievirus A16 gene expression by...

Embodiment 3

[0117] Example 3: Construction of a bivalent vector targeting Enterovirus 71 and Coxsackievirus A16, fine Cell level and animal level pharmacodynamic testing.

[0118] 1. Construction of bivalent interfering RNA carrier

[0119] The above-mentioned interference fragment expression cassettes (H1+si-3C+U6) and (H1+si-3C+U6) and (H1+si- 3D+U6) were connected by EcoRI and BamHI restriction sites respectively, and connected in series to the corresponding sites of the minicircle DNA vector. Sent for sequencing, pMC-H1+si-3C+si-3D+U6 was successfully constructed. ( Figure 7 )

[0120] 2. Pharmacodynamic detection of interfering RNA

[0121] (1) Pharmacodynamic detection of interfering RNA cell level

[0122] The detection method is the same as above, and the experimental results show that in the RD cell line, the si-3C+si-3D recombinant small circular DNA vector can inhibit the expression of enterovirus type 71 gene by 90%, and the expression of Coxsackie virus A16 ge...

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Abstract

The invention provides a method for suppressing virogene expression causing hand-foot-and-mouth disease, in particular two interfering RNA (Ribonucleic Acid) for two main pathogens of enterovirus 71 and coxsackie virus A16 genomes and a DNA sequence thereof, a carrier containing the interfering RNA and an application of the carrier transcribed interfering RNA in preparation of medicines for preventing and curing hand-foot-and-mouth diseases. It is shown by experiment result that the carrier transcribed interfering RNA can be used for obviously suppressing copying of two viruses and eliminating clinical symptoms of the hand-foot-and-mouth diseases at the same time of reducing pathogen enterovirus 71 and coxsackie virus A16 gene expressions on cell level and experimental animals. The carrier transcribed interfering RNA and the carrier containing the same, provided by the invention, can play am important role in preventing and curing the hand-foot-and-mouth diseases.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the interference RNA for the two main pathogens Enterovirus type 71 and Coxsackie virus A16 genomes that cause hand, foot and mouth disease, the carrier containing the interference RNA, and the RNA transcribed by the carrier Interfering RNA and its application in the preparation of medicines for preventing or treating hand, foot and mouth disease. Background technique [0002] Hand foot mouth disease (HFMD) is a common infectious disease in infants and young children. It is caused by enteroviruses. Clinically, it is characterized by fever, rashes and ulcers on the hands, feet, mouth and other parts. Myocarditis, pulmonary edema, aseptic meningoencephalitis and other fatal complications. Hand, foot and mouth disease is a global infectious disease. There are reports of the prevalence of this disease in most parts of the world, including many provinces in China, which has attra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/63A61K48/00A61P31/14
Inventor 杨倬田波
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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