Polypeptide and application thereof in preparation of products for resisting respiratory syncytial virus infection

Abstract

The invention provides a polypeptide and application thereof in preparation of a product for resisting respiratory syncytial virus infection, the amino acid sequence of the polypeptide is shown as any one of SEQ ID NO: 1-3, the polypeptide has excellent respiratory syncytial virus resisting activity, and the respiratory syncytial virus is inactivated by destroying the envelope of the respiratory syncytial virus, so that the respiratory syncytial virus infection resistance is improved. The compound can effectively inhibit the activity of the respiratory syncytial virus under the nanomolar concentration, EC50 is 0.309-11.99 [mu] M, the cytotoxicity is low, the safety is good, the action mechanism is different from that of existing drugs for treating diseases related to respiratory syncytial virus infection, and cross drug resistance is not prone to being generated. The molecular weight of each polypeptide is small, chemical synthesis and modification are easy, and a new technical choice is provided for developing a novel preparation for resisting respiratory syncytial virus infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide and its application in the preparation of products for treating respiratory syncytial virus infection. Background Technology

[0002] Respiratory syncytial virus (RSV) is an enveloped RNA virus and a major pathogen causing lower respiratory tract infections in immunocompromised infants and the elderly. Infection can lead to serious illnesses such as bronchiolitis and pneumonia. RSV's high variability and transmissibility, along with its complex immune evasion mechanisms, pose significant challenges to vaccine and drug development, and a wide range of effective treatments remain lacking. Currently, the number of approved anti-RSV drugs is limited, and existing treatments mainly rely on monoclonal antibodies or nucleoside analogs. However, these drugs often suffer from high costs, limited patient populations, limited efficacy, and the risk of drug resistance. Therefore, there is an urgent need to develop drugs with novel anti-RSV infection mechanisms.

[0003] The viral envelope plays a crucial role in RSV infection, serving as an essential structure for RSV adsorption and fusion with host cells, as well as for progeny virus budding. It represents a key breakthrough in the development of anti-RSV drugs. Therefore, developing an anti-RSV drug that can directly target the viral envelope, possesses a novel mechanism of action, has a low risk of drug resistance, and demonstrates high safety is a critical issue that urgently needs to be addressed in current antiviral research. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides a polypeptide and its application in the preparation of products against respiratory syncytial virus (RSV) infection. This polypeptide can directly disrupt the envelope of RSV, inactivating the virus and thus significantly inhibiting RSV infection. The polypeptide provided by this invention exhibits low cytotoxicity and good drug development potential.

[0005] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO:1 to 3.

[0006] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned polypeptide.

[0007] Thirdly, the present invention provides a conjugate comprising the aforementioned polypeptide and a chemical moiety conjugated thereto. In some specific embodiments, the chemical moiety is attached to the polypeptide of the present invention using any number of methods known to those skilled in the art, including covalent and non-covalent attachment methods. The procedure for attaching the chemical moiety to the polypeptide can be tailored to the chemical structure of the chemical moiety.

[0008] Fourthly, the present invention provides a composition comprising the aforementioned polypeptide.

[0009] The composition provided according to the present invention preferably further includes other drugs against respiratory syncytial virus infection.

[0010] Fifthly, a biological material containing the aforementioned nucleic acid molecule, wherein the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism, or cell.

[0011] In a sixth aspect, the present invention provides the use of the polypeptide, the nucleic acid molecule, the conjugate, the composition, or the biomaterial in the preparation of products for treating respiratory syncytial virus infection.

[0012] The use of the polypeptide, nucleic acid molecule, conjugate, composition or biomaterial provided by the present invention in the preparation of a product for resisting respiratory syncytial virus infection, preferably, the resistance to respiratory syncytial virus infection includes disruption of the respiratory syncytial virus envelope.

[0013] The use of the polypeptide, nucleic acid molecule, conjugate, composition, or biomaterial provided by the present invention in the preparation of products for treating respiratory syncytial virus infection, preferably, the products comprise pharmaceuticals or disinfectants. More preferably, the pharmaceuticals further comprise pharmaceutically acceptable carriers.

[0014] In a seventh aspect, the present invention provides the use of the polypeptide, the nucleic acid molecule, the conjugate, the composition, or the biomaterial in the preparation of products for the prevention and / or treatment of respiratory syncytial virus infection-related diseases, preferably, the respiratory syncytial virus infection-related diseases including lower respiratory tract infections.

[0015] The use of the polypeptide, nucleic acid molecule, conjugate, composition, or biomaterial provided by the present invention in the preparation of products for the prevention and / or treatment of respiratory syncytial virus infection-related diseases, preferably, the products comprise pharmaceuticals or disinfectants. More preferably, the pharmaceuticals further comprise pharmaceutically acceptable carriers.

[0016] Eighthly, the present invention provides a method for preparing the polypeptide, which is prepared by solid-phase synthesis according to any of the amino acid sequences shown in SEQ ID NO:1 to 3.

[0017] According to the method for preparing the polypeptide provided by the present invention, preferably, the product obtained by solid-phase synthesis is further purified by high performance liquid chromatography.

[0018] The present invention has the following beneficial effects: The polypeptide provided by this invention exhibits excellent anti-respiratory syncytial virus (RSV) activity. It inactivates RSV by disrupting its envelope, effectively inhibiting RSV activity (ECV) at nanomolar concentrations. 50 The peptides, at concentrations of 0.309 μM, 3.17 μM, and 11.99 μM respectively, exhibit low cytotoxicity, good safety profiles, and mechanisms of action different from existing drugs for treating respiratory syncytial virus (RSV) infection-related diseases, thus reducing the likelihood of cross-resistance. Furthermore, the small molecular weight of each peptide facilitates chemical synthesis and modification, providing a new technological option for developing novel anti-RSV formulations. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a graph showing the statistical results of the inhibition rates of peptides cAVP_4, cAVP_5, and cAVP_6 in the in vitro anti-respiratory syncytial virus infection experiment in Example 2 of the present invention.

[0021] Figure 2 These are transmission electron microscope images of respiratory syncytial virus in the control group and cAVP_4 group in Example 3 of this invention.

[0022] Figure 3 This is a graph showing the detection results of the cytotoxicity of peptides cAVP_4, cAVP_5, and cAVP_6 on 293T cells in Example 4 of this invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0025] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0026] Example 1: Preparation of antiviral peptides Shanghai Qiangyao Biotechnology Co., Ltd. uses a standard solid-phase synthesis method to synthesize corresponding peptides from L-type amino acids. After purification by high-performance liquid chromatography and mass spectrometry, the actual molecular weight of each peptide obtained by purification is consistent with the theoretical value, and the purity is greater than 95%.

[0027] Using this method, the present invention obtained cAVP_4 (amino acid sequence as shown in SEQ ID NO:1), cAVP_5 (amino acid sequence as shown in SEQ ID NO:2), and cAVP_6 (amino acid sequence as shown in SEQ ID NO:3) for subsequent experiments.

[0028] Example 2: Detection of the anti-respiratory syncytial virus activity of the peptide 1. Preservation and dilution of peptides The purified cAVP_4 (amino acid sequence as shown in SEQ ID NO:1), cAVP_5 (amino acid sequence as shown in SEQ ID NO:2), and cAVP_6 (amino acid sequence as shown in SEQ ID NO:3) obtained in Example 1 were stored in DMSO at a concentration of 1 mM to obtain the mother liquor of each peptide.

[0029] Before the experiment, the stock solutions of each peptide were diluted with DMSO to obtain diluted solutions of each peptide with final concentrations of 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM and 0.064 μM, respectively. The solutions were prepared and used immediately.

[0030] 2. In vitro anti-infection experiment with respiratory syncytial virus (1) Experimental methods This experiment referenced existing techniques, specifically “Xu M, Jiao YY, Fu YH, Jiang N, Zheng YB, Yan YF, Zhang M, Zheng YP, Zhu WY, Peng XL, He JS. Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay. Biomed Res Int. 2018Jan 15;2018:8431243. doi: 10.1155 / 2018 / 8431243.”, to prepare recombinant respiratory syncytial virus (rRSV-EGFP) expressing green fluorescent protein (EGFP). rRSV-EGFP with an infection multiplicity (MOI) of 0.5 was mixed with an equal volume of dilution buffer of each peptide at different final concentrations and incubated at 37°C for 2 h. Then, Hep2 cells (laryngeal epidermoid carcinoma cells) seeded in 96-well plates were infected, with 3 replicates for each cell. After incubation for another 2 h, the old culture medium was removed, and the Hep2 cells were washed once with fresh culture medium. Fresh culture medium containing the corresponding concentration of each peptide was then added, and the cells were cultured for another 24 h.

[0031] After culture, the number of virus particles carrying EGFP was counted using the CQ1 high-content cell imaging analysis system (Yokogawa, Tokyo, Japan), and the inhibition rate of RSV virus was analyzed. The half-maximal effective concentration (Cmax) of each peptide was calculated. 50 ).

[0032] (2) Experimental results like Figure 1 As shown, peptides cAVP4, cAVP5, and cAVP6 all significantly reduced the number of respiratory syncytial virus (RSV) in cells, and the EC50 of each peptide was [data missing]. 50 The concentrations were 0.309 μM, 3.17 μM, and 11.99 μM, respectively. This indicates that the three peptides provided by this invention have excellent anti-respiratory syncytial virus (RSV) activity.

[0033] Example 3: Investigation into the mechanism of action of peptides against respiratory syncytial virus The morphological changes of the peptide cAVP_4 after interaction with respiratory syncytial virus particles were observed using transmission electron microscopy. The specific methods are as follows: (1) Purification of respiratory syncytial virus Hep2 cells were used to propagate rRSV-EGFP from Example 2. The cells were lysed by repeated freeze-thaw cycles to release the rRSV-EGFP viral particles. The culture medium after cell lysis was transferred to a centrifuge tube and centrifuged at 7000 rpm for 6 minutes. The supernatant was collected and further filtered through a 0.45 μm filter membrane to obtain purified respiratory syncytial virus.

[0034] (2) Co-incubation The purified respiratory syncytial virus was mixed with the peptide cAVP_4 (final concentration 1.545 μM, obtained by 5×EC) purified in Example 1 of this invention. 50 The calculated values ​​were mixed to obtain the cAVP_4 group. The purified respiratory syncytial virus was mixed with a PBS solution containing 5% v / v DMSO to obtain the control group (i.e., Control). The volume of the PBS solution containing 5% v / v DMSO was the same as that of the purified cAVP_4 peptide obtained in the cAVP_4 group. Both groups were then co-incubated at 37°C for 1 h.

[0035] (3) Transmission electron microscopy characterization After incubation, both groups were fixed with 4% v / v paraformaldehyde solution for 1 h, then negatively stained with tungsten dye to prepare slides, which were observed and photographed under a transmission electron microscope. (4) Experimental results like Figure 2 As shown, transmission electron microscopy can directly observe that respiratory syncytial virus particles treated with peptide cAVP_4 exhibit envelope rupture or shrinkage.

[0036] The peptides cAVP_5 and cAVP_6 from Example 1 were co-incubated with purified respiratory syncytial virus and characterized by transmission electron microscopy according to the above method. The treated respiratory syncytial virus also showed similar envelope rupture or shrinkage phenomena.

[0037] The above results indicate that the various polypeptides provided by this invention exert their antiviral effects by disrupting the envelope of respiratory syncytial virus.

[0038] When the envelope of respiratory syncytial virus (RSV) is destroyed by the polypeptide provided by this invention, it rapidly loses its ability to adsorb onto host cells. At the same time, the internal ribonucleoprotein (RNP) complex is directly exposed to the extracellular environment. The RNP complex is easily degraded by nucleases in the environment. Even if it enters the host cell, it cannot initiate the replication of RSV. Therefore, RSV can no longer infect the host. The polypeptide provided by this invention has a significant anti-RSV infection effect.

[0039] Example 4: Cytotoxicity detection of peptides The cytotoxicity of the peptides purified in Example 1 was determined using the CCK8 Cell Proliferation and Cytotoxicity Assay Kit (Vazyme Biotech Co., Ltd., China). The specific method is as follows: 1. Peptide dilution The stock solutions of each peptide in Example 3 were first diluted 10-fold using PBS solution containing 5% v / v DMSO, and then serially diluted 3-fold to obtain diluted solutions of each peptide with final concentrations of 100 μM, 33.33 μM, 11.11 μM, 3.7 μM, 1.23 μM and 0.412 μM, respectively. The solutions were prepared and used immediately.

[0040] 2. Cell Culture and Processing 293T cells (human embryonic kidney cells) in the exponential growth phase were seeded into 96-well plates and cultured in a cell culture incubator at 37°C with 5% CO2 for 24 h. The supernatant in the wells was replaced with fresh basal medium (containing 2% v / v serum), and different final concentrations of each peptide were added. The cells were then cultured in the cell culture incubator for another 48 h, and three independent technical replicates were performed.

[0041] 3. CCK8 detection CCK8 solution was added to each cell well, and the cells were cultured in a cell incubator for 2 hours. The optical density (OD) of each cell well at 450 nm was then measured. 450 ).

[0042] 4. Data Statistics According to OD 450 The survival rate of 293T cells in each cell well was statistically analyzed, and the half-maximal cytotoxic concentration (CMC) of each peptide was calculated. 50 )value.

[0043] The results are as follows Figure 3 As shown, the cell viability was greater than 80% under different concentrations of each peptide treatment, CC 50 Greater than 100 μM, with high safety.

[0044] In summary, the multiple peptides provided by this invention can effectively inhibit respiratory syncytial virus (RSV) infection, exhibiting low cytotoxicity and good safety. Furthermore, transmission electron microscopy observations show that these peptides can directly disrupt the RSV envelope, leading to viral inactivation. The multiple antiviral peptides provided by this invention possess excellent anti-RSV efficacy and drug development potential.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in any of SEQ ID NO:1 to 3.

2. A nucleic acid molecule, characterized in that, It encodes the polypeptide described in claim 1.

3. A conjugate, characterized in that, It comprises the polypeptide of claim 1, and the chemical moiety conjugated thereto.

4. A composition, characterized in that, It comprises the polypeptide of claim 1; preferably, it also comprises other drugs against respiratory syncytial virus infection.

5. A biomaterial, characterized in that, The biological material contains the nucleic acid molecule of claim 2, wherein the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism or cell.

6. The use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the conjugate of claim 3, the composition of claim 4, or the biomaterial of claim 5 in the preparation of products against respiratory syncytial virus infection.

7. The application according to claim 6, characterized in that, The anti-respiratory syncytial virus (RSV) infection involves disrupting the RSV envelope.

8. The use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the conjugate of claim 3, the composition of claim 4, or the biomaterial of claim 5 in the preparation of products for the prevention and / or treatment of respiratory syncytial virus infection-related diseases.

9. The application according to any one of claims 6 to 8, characterized in that, The products include medicines or disinfectants.

10. The method for preparing the polypeptide according to claim 1, characterized in that, Prepared by solid-phase synthesis according to any of the amino acid sequences shown in SEQ ID NO:1 to 3.

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