Lactoferricin and lactoferrampin for the preparation of inhibitors of 3c protease

Abstract

This invention belongs to the field of biomedical technology, specifically relating to lactoferrin peptides and the application of lactoferrin in the preparation of inhibitors of 3C protease. The lactoferrin peptides of this invention contain the amino acid sequence shown in SEQ ID NO.1. The lactoferrin peptides and lactoferrin provided by this invention can specifically bind to and inhibit the activity of 3C protease, directly inhibiting viral replication by blocking its cleavage function of viral polyproteins. The mechanism is clear and has high affinity. Since 3C protease is highly conserved in the Picornaviridae family, it has broad-spectrum inhibitory effects against enteroviruses such as EV-A71 and CV-A9, and has a wide range of applications. Furthermore, it is derived from natural substances, has good biocompatibility and high safety, and can be made into various forms such as pharmaceuticals and antiviral daily necessities, with diverse application scenarios. This invention provides a new approach for developing antiviral drugs targeting 3C protease based on natural proteins, and has significant theoretical and applied value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to lactoferrin peptides and the application of lactoferrin in the preparation of inhibitors of 3C protease. Background Technology

[0002] Picornaviridae viruses (such as enteroviruses, rhinoviruses, and hepatitis A viruses) are a widespread and serious group of pathogens that can cause a variety of diseases in humans and animals. Enteroviruses (such as EV-A71, CV-A6, and CV-A16) are the main pathogens causing hand-foot-mouth disease and herpetic pharyngitis in infants and young children; some strains can also cause serious complications such as meningitis and myocarditis, even leading to death. Rhinoviruses are the primary cause of the common cold. Hepatitis A viruses are transmitted through the fecal-oral route, causing acute hepatitis.

[0003] The replication of these viruses depends on key proteases they encode, among which the 3C protease (3Cpro) is a core functional protein in the viral life cycle. The 3C protease cleaves viral polyproteins to generate active structural and non-structural proteins, providing the necessary conditions for viral RNA replication and particle assembly. Simultaneously, it can interfere with the host cell's immune response, inhibit host protein synthesis, and induce apoptosis, thereby promoting viral proliferation within the host. Due to the irreplaceable role of the 3C protease in viral replication and the highly conserved amino acid sequence among different small RNA viruses, it has become an ideal target for developing broad-spectrum antiviral drugs.

[0004] Currently, clinical treatment options for small RNA viruses remain limited, primarily focusing on symptomatic and supportive care, with a lack of specific antiviral drugs. Existing antiviral drug development largely focuses on 3C protease inhibitors, but existing candidate drugs suffer from poor stability, high toxicity, and insufficient targeting, limiting their clinical application. Furthermore, some viral strains have developed drug resistance, further increasing the difficulty of prevention and control.

[0005] Lactoferrin is a natural iron-binding glycoprotein widely found in bodily fluids such as breast milk and saliva. It possesses various biological activities, including antibacterial, antiviral, and immunomodulatory effects, and exhibits high safety, making it a promising candidate for application in the biomedical field. Recent studies have revealed that lactoferrin and its derived peptides may exert antiviral effects by binding to viral proteins or host cell receptors. However, its specific inhibitory effect on small RNA virus 3C protease and the related mechanisms remain unclear, and research on developing 3C protease inhibitors based on lactoferrin peptides is still in its infancy.

[0006] Therefore, developing an inhibitor based on natural sources, with high safety and the ability to specifically inhibit 3C protease is of great clinical significance and application value for the prevention and treatment of small RNA virus infection. Summary of the Invention

[0007] To address the above shortcomings, this invention provides a short peptide and its application in enterovirus inhibition products.

[0008] Terminology Explanation:

[0009] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] The meanings of the letters for amino acids in this invention are as follows: A represents alanine; C represents cysteine; D represents aspartic acid; E represents glutamic acid; F represents phenylalanine; G represents glycine; H represents histidine; I represents isoleucine; K represents lysine; L represents leucine; M represents methionine; N represents asparagine; P represents proline; Q represents glutamine; R represents arginine; S represents serine; T represents threonine; V represents valine; W represents tryptophan; Y represents tyrosine.

[0012] The technical solution of this invention is as follows: On one hand, the present invention provides the application of lactoferrin peptides or lactoferrin containing lactoferrin peptides in the preparation of 3C protease inhibitors, characterized in that the lactoferrin peptides contain an amino acid sequence as shown in SEQ ID NO.1.

[0013] SEQ ID NO.1: APRKNVRWCTISQPEWLKCHRWQWRMKKLGAPSITCVRRAFVLECIRAITEKKADAVTLDGG.

[0014] Preferably, the amino acid sequence of the lactoferrin peptide is shown in SEQ ID NO.1.

[0015] Preferably, the amino acid sequence of the lactoferrin is shown in SEQ ID NO.4.

[0016] SEQ ID NO.4: MKLFDPALLSLGALGLCLAAPRKNVRWCTISQPEWLKCHRWQWRMKKLGAPSITCVRRAFVLECIRAITEKKADAVTLDGGMVFEAGLDPYKLRPVAAEIYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCHTGLGRSAGWNIPVGILARYLSWTESLEPLQGAVAKFFSASC VPCVDRQAYPNLCQLCKGEGENQCACSPREPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNTRAPVDAFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQEKFGKNKSGSFQLFGSPPGQRDLLFKDSALGFLRIPSKVDSALYLGSRYLTALKNLRE.

[0017] Specifically, the 3C protease inhibitor is used to prepare antiviral infection products.

[0018] Preferably, the virus includes a small RNA virus.

[0019] More preferably, the small RNA virus includes, but is not limited to, any one or more of the following: enterovirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, cardiogenic virus, and oral thrush virus.

[0020] More preferably, the enteroviruses include, but are not limited to, any one or more of the following: EV-A71 virus, CV-A6 virus, CV-A9 virus, CV-A10 virus, CV-A16 virus, CV-A24 virus, CV-B3 virus, CV-B5 virus, ECHO-7 virus, ECHO-11 virus, PV-1 virus, and EV-D68 virus.

[0021] More preferably, the enterovirus is selected from any one or more of EV-A71 and CV-A9 viruses.

[0022] Specifically, the antiviral infection products include antiviral drugs or antiviral daily necessities.

[0023] Preferably, the dosage form of the medicine includes, but is not limited to, any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

[0024] Preferably, the medicine further includes pharmaceutically acceptable excipients.

[0025] More preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solutes, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0026] Preferably, the daily necessities include, but are not limited to, any one or more of the following: antiviral hand sanitizer, antiviral wipes, antiviral disinfectant, antiviral mask, antiviral protective gloves, antiviral laundry detergent, and antiviral cleaning spray.

[0027] The beneficial effects of this invention are as follows: 1. The lactoferrin and lactoferrin peptides of the present invention (including SEQ ID NO.1) can specifically inhibit the function of cleaving viral polyproteins by binding to the catalytic active site of 3C protease, directly blocking the key link in viral replication. Experiments have confirmed that it has a significant inhibitory effect on the 3C protease of enteroviruses such as EV-A71 and CV-A9, without affecting the activity of viral 2A protease. The mechanism of action is clear, with strong targeting and high affinity.

[0028] 2. Since 3C protease is highly conserved in the Picornaviridae family (such as enteroviruses, rhinoviruses, hepatitis A viruses, etc.), the lactoferrin and peptides of the present invention can exert a broad-spectrum antiviral effect by inhibiting the activity of conserved 3C protease.

[0029] 3. Lactoferrin is a glycoprotein naturally found in bodily fluids such as breast milk. Its peptide segment (SEQ ID NO.1) is derived from a functional fragment of a natural protein, exhibiting good biocompatibility and no risk of toxic side effects. Compared to chemically synthesized antiviral drugs, it has higher safety and is suitable for long-term use or for sensitive populations such as infants and young children. Attached Figure Description

[0030] Figure 1 For EV-A71 3C pro A schematic diagram of the in vitro activity testing system.

[0031] Figure 2 For EV-A71 2A pro A schematic diagram of the in vitro activity testing system.

[0032] Figure 3 This refers to the inhibitory effect of lactoferrin on 3C protease.

[0033] Figure 4 IC50 of lactoferrin against 3C protease 50 Measurement.

[0034] Figure 5This refers to the inhibitory effect of lactoferrin on 2A protease.

[0035] Figure 6 This study investigated the inhibitory effects of lactoferrin and lactoferrin peptides on 3C protease.

[0036] Figure 7 It exhibits antiviral activity at the cellular level from lactoferrin peptides.

[0037] Figure 8 Comparison of the conservation of enterovirus 3C protease sequences and Weblogo diagram results; In the diagram, EV-A represents enterovirus group A, EV-B represents enterovirus group B, EV-A71 represents enterovirus A71, CV-A6 represents Coxsackievirus A6, CV-A9 represents Coxsackievirus A9, CV-A10 represents Coxsackievirus A10, CV-A16 represents Coxsackievirus A16, CV-A24 represents Coxsackievirus A24, CV-B3 represents Coxsackievirus B3, CV-B5 represents Coxsackievirus B5, ECHO-7 represents echovirus 7, ECHO-11 represents echovirus 11, PVS-1 represents poliovirus 1, and EV-D68 represents enterovirus D68.

[0038] Figure 9 This study investigated the inhibitory effects of lactoferrin and lactoferrin peptides on CV-A9 3C protease.

[0039] Figure 10 The inhibitory effects of lactoferrin short peptides and mutant short peptides on 3C protease.

[0040] Figure 11 It exhibits antiviral activity at the cellular level in lactoferrin short peptides and mutant short peptides. Detailed Implementation

[0041] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0042] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0043] Basic Example 1 1. EV-A71 3C pro Construction of in vitro activity assay system EV-A71 3Cpro It is involved in the cleavage of almost all proteins in EV-A71 virus replication, and is very important for EV-A71 virus replication. In addition, it also has functions such as inhibiting the host immune system, shutting down the host cell protein synthesis system, and guiding the host cell programmed apoptosis. The specific experimental method is introduced below using the 3C protease inhibition assay based on fluorescence resonance energy transfer as an example.

[0044] EV-A713C was subjected to a series of steps including transformation, single colony selection, 37°C shake culture for expansion, and low-temperature induction. pro Prokaryotic expression; purification of the protease using the nickel column binding principle; synthesis of a polypeptide (Dbcyl-RTATVQGPSLDFE-Edans) with fluorescent groups (Dbcyl quencher and Edans fluorescein) at both ends and containing the EV-A71 polyprotein self-cleavage site 3B-3C (with QG linkage) as a substrate (e.g.) using the FRET (fluorescence resonance energy transfer) principle. Figure 1 (As shown), to build EV-A71 3C pro An in vitro activity testing system.

[0045] 2. CV-A9 3C pro Construction of in vitro activity assay system CV-A9 3C was developed through a series of steps including transformation, single colony selection, 37°C shake culture for expansion, and low-temperature induction. pro The prokaryotic expression was performed; the protease was purified using the nickel column binding principle; and a polypeptide (Dbcyl-KTSAVLQSGFRKME-Edans) containing fluorescent groups (Dbcyl quencher and Edans fluorescein) at both ends and the CV-A9 polyprotein self-cleavage site 3B-3C (with QG linkage) was synthesized using the FRET (fluorescence resonance energy transfer) principle as a substrate to construct CV-A93C. pro An in vitro activity testing system.

[0046] 3. EV-A71 2A pro Construction of in vitro activity assay system EV-A712A was subjected to a series of steps including transformation, single colony selection, 37°C shake culture for expansion, and low-temperature induction. pro Prokaryotic expression; purification of the protease using the nickel column binding principle; synthesis of a polypeptide (Dbcyl-RTSITTLGKFGQQE-Edans) with fluorescent groups (Dbcyl quencher and Edans fluorescein) at both ends and containing the EV-A71 polyprotein self-cleavage site 2A (with LG linkage) as a substrate (e.g.) using the FRET (fluorescence resonance energy transfer) principle. Figure 2 (As shown), to build EV-A71 2Apro An in vitro activity testing system.

[0047] Example 1 1. Inhibitory effect of lactoferrin on 3C protease EV-A71 3C pro An in vitro activity assay system was used, with luteolin as a positive inhibitor, to verify whether lactoferrin or osteopontin had inhibitory activity against this protease. EV-A71 3C pro The in vitro activity testing system is divided into EV-A71 3C. pro Group (3C) pro The groups included: lactoferrin group (BLF group), osteopontin group (OPN group), positive control group (LUT group), and protease-free group (No protein group).

[0048] EV-A71 3C pro Group: EV-A71 3C pro 1×3C protease activity buffer was added to the in vitro activity test system.

[0049] BLF Group: EV-A71 3C pro 10 mg / mL of lactoferrin was added to the in vitro activity assay system; OPN Group: EV-A71 3C pro 10 mg / mL of osteopontin was added to the in vitro activity assay system; Luteolin Group: EV-A71 3C pro 10 mg / mL of luteolin was added to the in vitro activity test system; Protease-free group: only 1×3C protease activity buffer is added.

[0050] After incubating at 4 degrees Celsius for 30 minutes, the 3C protease substrate peptide was added to a final concentration of 30 μM. Fluorescence detection was immediately initiated in a white 96-well plate with an excitation wavelength of 355 nm and an emission wavelength of 538 nm. Detection was performed continuously for 90 minutes, with a detection interval of 50 seconds.

[0051] The measurement results are as follows Figure 3 As shown, the lactoferrin of the present invention can inhibit the activity of 3C protease, while osteopontin has no inhibitory effect on 3C protease.

[0052] 2. IC50 of lactoferrin against 3C protease 50 The inhibitory effect of lactoferrin on 3C protease was tested using different concentrations, and the IC50 of lactoferrin was calculated based on the enzyme activity curve. 50 Value. The specific measurement method is as follows: EV-A71 3C pro In the in vitro activity test system, 0 mg / mL (3C) was added respectively. pro Lactoferrin at concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, 80 mg / mL, and 100 mg / mL was added to 1×3C protease activity buffer as EV-A71 3C. pro Group (control group). After incubation at 4 degrees Celsius for 30 minutes, 3C protease substrate peptide was added to a final concentration of 30 uM. Fluorescence detection was immediately initiated in a white 96-well plate with an excitation wavelength of 355 nm and an emission wavelength of 538 nm. Detection was performed continuously for 90 minutes, with a detection interval of 50 s.

[0053] The inhibitory effects of lactoferrin at different concentrations on 3C protease were tested, and the IC50 of lactoferrin was calculated based on the enzyme activity curve. 50 Values ​​such as Figure 4 The results showed that lactoferrin at concentrations of 2.5-100 mg / mL could inhibit 3C protease, with an IC50 concentration of [missing value]. 50 =4.907 mg / mL.

[0054] 3. Inhibitory effects of lactoferrin and 2A protease The EV-A71 genome encodes the 2A protease (2A... pro ) is a key functional protein in the viral replication cycle, playing an important role in viral polyprotein processing and host cell function interference.

[0055] With EV-A71 2A pro An in vitro activity assay system was used to verify whether lactoferrin has inhibitory activity against this protease. EV-A71 2A pro The in vitro activity testing system is divided into EV-A71 2A pro Group (2A) pro The groups are: lactoferrin group (BLF group) and protease-free group (No protein group).

[0056] EV-A71 2A pro Group: EV-A71 2A pro Add 1×2A of protease activity buffer to the in vitro activity test system.

[0057] BLF Group: EV-A71 2A pro 10 mg / mL of lactoferrin was added to the in vitro activity assay system; Protease-free group: only 1×2A protease activity buffer was added.

[0058] After incubating at 4 degrees Celsius for 30 minutes, the 2A protease substrate peptide with a final concentration of 30 uM was added to the above groups. Fluorescence detection was immediately initiated in a white 96-well plate with an excitation wavelength of 355 nm and an emission wavelength of 538 nm. Detection was performed continuously for 90 minutes, with a detection every 50 seconds.

[0059] Figure 5 The results showed that the lactoferrin of the present invention could not inhibit the activity of 2A protease.

[0060] Example 2 1. A lactoferrin peptide The structure of the lactoferrin-3C protease complex was predicted using AlphaFold3. The results showed that amino acid residues 1-62 at the N-terminus of lactoferrin (SEQ ID NO. 1) can bind to the catalytic triplet of the 3C protease, thereby occupying the catalytically active site of the 3C protease substrate and thus inhibiting enzyme activity. SEQ ID NO. 1: APRKNVRWCTISQPEWLKCHRWQWRMKKLGAPSITCVRRAFVLECIRAITEKKADAVTLDGG.

[0061] 2. Inhibitory effect of lactoferrin peptides on 3C protease SEQ ID NO.1 was expressed and purified in prokaryotes, and its inhibitory effect on 3C protease activity was tested in vitro. EV-A71 3C pro The in vitro activity testing system is divided into EV-A71 3C. pro Group (3C) pro The groups are: Negative group, Lactoferrin group (BLF group), Lactoferrin peptide group (Binder group), and No protein group (Blank group).

[0062] EV-A71 3C pro Group: EV-A71 3C pro Add 1×3C protease activity buffer to the in vitro activity test system; BLF Group: EV-A71 3C pro 25 μM lactoferrin was added to the in vitro activity assay system; Binder Group: EV-A71 3C pro 25 μM lactoferrin peptide (SEQ ID NO. 1) was added to the in vitro activity assay system. Protease-free group: only 1×3C protease activity buffer is added.

[0063] After incubating at 4 degrees Celsius for 30 minutes, the 2A protease substrate peptide with a final concentration of 30 uM was added to the above groups. Fluorescence detection was immediately initiated in a white 96-well plate with an excitation wavelength of 355 nm and an emission wavelength of 538 nm. Detection was performed continuously for 90 minutes, with a detection every 50 seconds.

[0064] The measurement results are as follows Figure 6 As shown, the results indicate that both lactoferrin and lactoferrin peptide (SEQ ID NO.1) can effectively inhibit 3C protease activity extracellularly.

[0065] 3. Inhibitory effect of lactoferrin peptides on EV-A71 virus RD-susceptible cells were seeded in 96-well plates, and EC was performed when the cell density reached 90%. 50 In the experiment, BLF-2 was serially diluted to half-maximal concentrations of 10.00 mg / mL, 5.00 mg / mL, 2.50 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.078 mg / mL, 0.039 mg / mL, and 0.020 mg / mL, respectively, with the corresponding concentrations of lactoferrin peptide (SEQ ID NO.1) and EV-A71 virus at MOI=0.1. The mixture was incubated at 37 °C with 5% CO2 for 24 h. After washing with PBS and adding lysis buffer, viral RNA was extracted using a kit. The inhibitory effect of different concentrations of lactoferrin peptide (SEQ ID NO.1) on the virus was detected by qRT-PCR, and its EC50 was calculated. 50 .

[0066] The measurement results are as follows Figure 7 As shown, lactoferrin peptides have antiviral effects on EC at the cellular level. 50 =5.157 μM.

[0067] Example 3 Sequence alignment of the 3C protease of different enteroviruses was performed. Figure 8 The results show the sequence conservation of enterovirus 3C protease and the Weblogo diagram. The alignment results indicate that the enterovirus 3C protease is highly conserved at the catalytic triplet, therefore lactoferrin and its peptides may broadly inhibit enterovirus 3C enzyme activity.

[0068] The effect of lactoferrin on the reaction kinetics of CV-A9 3C protease was further tested, with CV-A9 3C... pro An in vitro activity assay system was used to verify whether lactoferrin peptides have inhibitory activity against this protease. CV-A9 3C pro The in vitro activity assay system is divided into four groups: CV-A9 3C proThe study included a negative control group, a BLF group (lactoferrin group), a positive drug control group (rutin group), and a protease-free group (No protein group).

[0069] CV-A9 3C pro Group: CV-A9 3C pro Add 1×3C enzyme activity buffer to the in vitro activity test system.

[0070] BLF Group: CV-A9 3C pro 40 mg / mL of lactoferrin was added to the in vitro activity test system; Positive drug control group: CV-A9 3C pro 500 μM rutin was added to the in vitro activity assay system; Protease-free group: only 1×3C enzyme activity buffer was added.

[0071] After incubating at 4 degrees Celsius for 30 minutes, the 2A protease substrate peptide with a final concentration of 30 μM was added to the above groups. Fluorescence detection was immediately initiated in a white 96-well plate with an excitation wavelength of 355 nm and an emission wavelength of 538 nm. Detection was performed continuously for 90 minutes, with one detection every 50 seconds.

[0072] The measurement results are as follows Figure 9 As shown, the lactoferrin and lactoferrin peptide of the present invention (SEQ ID NO.1) can also inhibit the catalytic activity of CV-A9 3C protease.

[0073] Example 4 1. Lactoferrin short peptides and mutant short peptides In this embodiment, a lactoferrin short peptide (SEQ ID NO.2) consisting of 10 amino acids before and after C17 was synthesized, and a mutant short peptide at the A17 site (SEQ ID NO.3) was also synthesized to explore whether the C17 site residue is a key site for exerting antiviral effects.

[0074] SEQ ID NO.2: GALGLCLAAP.

[0075] SEQ ID NO.3: GALGLALAP.

[0076] 2. Inhibitory effect of lactoferrin short peptides and mutant short peptides on 3C protease. SEQ ID NO.2 and SEQ ID NO.3 were expressed and purified in prokaryotes, and their inhibitory effects on 3C protease activity were tested in vitro. EV-A71 3C pro The in vitro activity testing system is divided into EV-A71 3C. pro Group (3C)pro The groups are: Negative group, lactoferrin short peptide group, mutant short peptide group, positive control group (Luteolin group, i.e., Positive group), and No protein group (No protein group, i.e., MOCK group).

[0077] EV-A71 3C pro Group: EV-A71 3C pro Add 1×3C protease activity buffer to the in vitro activity test system; Lactoferrin short peptide group: EV-A71 3C pro 100 μM lactoferrin short peptide was added to the in vitro activity test system; Mutant short peptide group: EV-A71 3C pro 100 μM of the mutant short peptide was added to the in vitro activity assay system; Positive control group: EV-A71 3C pro 10 mg / mL of luteolin was added to the in vitro activity test system; Protease-free group: only 1×3C protease activity buffer is added.

[0078] After incubating at 4 degrees Celsius for 30 minutes, the 2A protease substrate peptide with a final concentration of 30 uM was added to the above groups. Fluorescence detection was immediately initiated in a white 96-well plate with an excitation wavelength of 355 nm and an emission wavelength of 538 nm. Detection was performed continuously for 90 minutes, with a detection every 50 seconds.

[0079] The measurement results are as follows Figure 10 As shown, the results indicate that neither lactoferrin short peptide (SEQ ID NO.2) nor mutant short peptide (SEQ ID NO.3) significantly inhibited the activity of 3C protease.

[0080] 3. Inhibitory effect of lactoferrin short peptide on EV-A71 virus RD-susceptible cells were seeded in 96-well plates, and EC was performed when the cell density reached 90%. 50In the experiment, BLF-2 was serially diluted at half-maximal concentrations of 10.00 mg / mL, 5.00 mg / mL, 2.50 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.078 mg / mL, 0.039 mg / mL, and 0.020 mg / mL, respectively, with corresponding concentrations of lactoferrin short peptide or mutant short peptide and EV-A71 virus at MOI=0.1. The mixture was incubated at 37 °C with 5% CO2 for 24 h, washed once with PBS, and viral RNA was extracted using a kit after adding lysis buffer. The inhibitory effect of different concentrations of lactoferrin short peptide or mutant short peptide on the virus was detected by qRT-PCR, and the EC50 was calculated. 50 .

[0081] Figure 11 The results showed that neither lactoferrin short peptide (SEQ ID NO.2) nor mutant short peptide (SEQ ID NO.3) had any antiviral effect on EV-A71.

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of lactoferrin peptides or lactoferrin containing lactoferrin peptides in the preparation of 3C protease inhibitors, characterized in that, The lactoferrin peptide contains the amino acid sequence shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The 3C protease inhibitor is used to prepare antiviral infection products.

3. The application according to claim 2, characterized in that, The viruses mentioned include small RNA viruses.

4. The application according to claim 3, characterized in that, The small RNA viruses mentioned include any one or more of the following: enteroviruses, rhinoviruses, hepatitis A viruses, foot-and-mouth disease viruses, cardiogenic viruses, and oral thrush viruses.

5. The application according to claim 4, characterized in that, The enteroviruses mentioned include any one or more of the following: EV-A71 virus, CV-A6 virus, CV-A9 virus, CV-A10 virus, CV-A16 virus, CV-A24 virus, CV-B3 virus, CV-B5 virus, ECHO-7 virus, ECHO-11 virus, PVS-1 virus, and EV-D68 virus.

6. The application according to claim 5, characterized in that, The enterovirus is selected from any one or more of EV-A71 and CV-A9 viruses.

7. The application according to claim 1, characterized in that, The aforementioned antiviral infection products include antiviral drugs or antiviral daily necessities.

8. The application according to claim 7, characterized in that, The dosage forms of the medicine include any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

9. The application according to claim 7, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

10. The application according to claim 7, characterized in that, The aforementioned daily necessities include any one or more of the following: antiviral hand sanitizer, antiviral wipes, antiviral disinfectant, antiviral mask, antiviral protective gloves, antiviral laundry detergent, and antiviral cleaning spray.

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