Deoxyribozyme for inhibiting mouse norovirus infection

Abstract

The invention discloses deoxyribozyme for inhibiting mouse norovirus infection. The deoxyribozyme DP233 provided by the invention can be used for cutting mRNA (messenger ribonucleic acid) of a mouse norovirus VP2 gene and inhibiting norovirus infection. Besides, the deoxyribozyme DP233 disclosed by the invention has relatively good specificity, and can be combined with and cut norovirus VP2mRNA in a targeted manner, so that norovirus infection is effectively inhibited; and the deoxyribozyme DP233 has low toxicity to host cells, and is suitable for being used as a potential tool for antiviral gene therapy. The deoxyribozyme technology provides a brand new means for virus resistance, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to a deoxyribozyme DP233 capable of cutting mRNA of mouse norovirus minor capsid protein VP2 gene, and is applied in the antiviral field. Background Art

[0002] Norovirus is a small, icosahedral, non-enveloped, positive-sense, single-stranded RNA virus that can cause acute gastroenteritis and severe diarrhea in humans and a variety of mammals (Ford-Siltz et al., 2021). Proteases are proven targets for antiviral therapy, and research has been conducted to develop protease inhibitors for norovirus. These rationally designed transition state inhibitors are composed of dipeptides, tripeptides, and macrocyclic compounds (Chang et al., 2019), but their therapeutic effects are limited. Currently, there is no effective therapeutic drug for norovirus.

[0003] Since there is no recognized broadly reactive neutralizing epitope, the variation of norovirus strains increases the difficulty of vaccine development. At present, human norovirus cannot be cultured in large quantities in vitro, so the use of culturable viruses such as mouse norovirus is helpful to study the basic properties of norovirus (Cates et al., 2020). Norovirus infection requires the attachment of the major capsid protein VP1 to the tissue blood group antigen (HBGA), which is the most important infection step (Peters et al., 2022). VP2 is a structural protein of norovirus, located in the viral capsid. It has been reported that VP2 promotes the stability of the VP1 capsid and coordinates the formation of the VP1 capsid as a structural protein. Subsequently, it was found that the co-expression of VP1 and VP2 increased the production of virus-like particles (Liao et al., 2022). Studies have found that VP2 is a highly alkaline RNA-binding protein. VP2 is essential for delivering norovirus RNA into host cells and is essential for norovirus infection (Ishiyama et al., 2024). Currently, there is no viral treatment for norovirus VP2, and a vaccine for norovirus is still under development. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned background technology, the present invention designs and synthesizes a deoxyribozyme DP233 capable of cleaving the mRNA of the mouse norovirus VP2 gene. In view of the importance of VP2 in norovirus infection and the potential of ribozymes to cleave viral gene mRNA, the deoxyribozyme DP233 can be used to inhibit norovirus infection.

[0005] This application is implemented using the following technical solutions:

[0006] A deoxyribozyme for inhibiting mouse norovirus infection. The deoxyribozyme is deoxyribozyme DP233 capable of cleaving mouse norovirus VP2 gene mRNA, and the nucleotide sequence thereof is: 5'-GGCGCTGATGAGGCCGAACCTTTTTAGGGTCATCATATTTCATCATATGAGCCGAAATGGT-3'. The deoxyribozyme comprises a deoxyribozyme activity center (5'-...CTGATGA...CGAAA...-3') and a flanking sequence (5'-GGCG...TGGT-3') targeting norovirus VP2 mRNA.

[0007] After the deoxyribozyme is incubated with Norovirus VP2 mRNA in a reaction solution (50mmol / L Tris, 25mmol / L MgCl2, pH7.5) at room temperature for 6 hours, the Norovirus VP2 mRNA can be cleaved; at the same time, after the deoxyribozyme is transfected into Norovirus-infected mouse RAW264.7 cells, the expression of the Norovirus VP2 gene is inhibited, and the virus infection is inhibited.

[0008] The beneficial effects of the present invention are:

[0009] In view of the problem that there is no specific medicine for the treatment of norovirus at present, the present invention has discovered a deoxyribozyme DP233, which can cut the mRNA of the mouse norovirus VP2 gene and inhibit norovirus infection. The present invention can provide a new theoretical basis and a new perspective for the research of therapeutic drugs for human norovirus. Subsequently, according to the action characteristics of this deoxyribozyme, a deoxyribozyme that can target human norovirus can be developed to reduce the replication of norovirus and achieve a therapeutic effect.

[0010] In addition, the DNAzyme provided by the present invention also has the following significant improvements:

[0011] 1. Specificity: DNAzyme DP233 targets and cleaves Norovirus VP2 mRNA, thereby effectively inhibiting Norovirus infection.

[0012] 2. Safety: DNAzyme DP233 has low toxicity to host cells and is suitable as a potential tool for antiviral gene therapy.

[0013] 3. New antiviral tool: The DNA enzyme technology of the present invention provides a new means for antiviral and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the structural diagram of the DNAzyme DP233.

[0015] Figure 2 Schematic diagram of the binding of DNAzyme to the mRNA of the Norovirus VP2 gene.

[0016] Figure 3 These are the results of cell experiments showing that DNAzyme inhibits VP2 gene expression.

[0017] Figure 4 These are the experimental results of DNAzyme inhibiting Norovirus infection. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0019] Targeting mouse norovirus VP2, artificially synthesized deoxyribozyme DP233 ( Figure 1 ). The nucleotide sequence of the deoxyribozyme DP233 is 5'-GGCGCTGATGAGGCCGAACCTTTTTAGGGTCATCATATTTCATCATATGAGCCGAAATGGT-3'. Among them, red represents the enzyme activity center, black represents the non-critical region, and green represents the complementary pairing part with the substrate.

[0020] DNAzyme DP233 can bind to mouse norovirus VP2 mRNA and cleave VP2 mRNA ( Figure 2 ). Figure 2 In the figure, red indicates VP2 gene mRNA, black indicates ribozyme, and arrows indicate cleavage sites.

[0021] DNAzyme DP233 was transfected into RAW 264.7 cells. After 36 hours of culture, the cells were infected with mouse norovirus. Norovirus cells without DNAzyme DP233 were used as controls. 48 hours after infection, quantitative PCR was performed to detect the expression of VP2 mRNA ( Figure 3 ). Figure 3 The expression of VP2 mRNA detected by quantitative PCR is shown in (**, p<0.01). The expression of VP2 mRNA was inhibited after transfection with DNAzyme DP233.

[0022] DNAzyme DP233 was transfected into RAW 264.7 cells. After 36 hours of culture, the cells were infected with mouse norovirus. Norovirus cells without DNAzyme DP233 were used as controls. 48 hours after infection, quantitative PCR was performed to detect the number of viral copies ( Figure 4 ). Figure 4 The viral copy number was detected by quantitative PCR (**, p<0.01). Viral infection was inhibited after DNAzyme DP233 transfection.

[0023] Combination Figure 3 and Figure 4It can be seen that DNAzyme DP233 can cut norovirus VP2 mRNA, thereby inhibiting norovirus infection.

Claims

1. A deoxyribozyme for inhibiting mouse norovirus infection, characterized in that: The deoxyribozyme is deoxyribozyme DP233 which can cut the mRNA of mouse norovirus VP2 gene.

2. A DNAzyme for inhibiting mouse norovirus infection according to claim 1, characterized in that: The nucleotide sequence of the deoxyribozyme DP233 is shown in SEQ ID NO.1.

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