Composition for inhibiting coronavirus and application thereof

Abstract

The invention discloses an application of a protein substance in preparation of a composition for inhibiting coronavirus. The coronavirus inhibition comprises the following steps: inhibiting adhesion of the coronavirus on host cells; the invasion of coronavirus to host cells is hindered; the after-cell entry process of coronavirus infection is inhibited. In-vitro verification is carried out on the antiviral effect of the coronavirus GXP2V on various nutritional ingredients (whey protein, milk fat globule membrane, lactoferrin and milk-derived osteopontin), and the result shows that the coronavirus resisting activity of the whey protein, the milk fat globule membrane, the lactoferrin and the milk-derived osteopontin (LPN, short for lactopontin). At present, it is rarely reported that protein substances are used for resisting the coronavirus, the coronavirus GXP2V is a novel coronavirus-like virus, a new thought is developed for resisting the novel coronavirus-like virus, and the application value and prospect are very good.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to compositions for inhibiting coronaviruses and their applications. Background Technology

[0002] Researchers isolated multiple coronaviruses from pangolin tissue samples. Among them, the spike protein (S) of coronavirus GX_P2V showed 92.2% homology with the spike protein of SARS-CoV-2. The S protein is a crucial viral structural protein for SARS-CoV-2 recognition and cell entry to initiate infection, and is also a key target for vaccine development. Angiotensin-converting enzyme 2 (ACE2) is the receptor used by SARS-CoV-2 to infect cells and can be recognized and bound by the S protein. Researchers tested whether ACE2 is involved in GX_P2V infection by using siRNA-mediated ACE2 expression knockdown. The results showed that the expression levels of both ACE2 mRNA and viral RNA were significantly reduced in cells infected with GX_P2V, indicating that ACE2 is also a receptor for GX_P2V infection. However, it is noteworthy that GX_P2V's entry into cells via the ACE2 receptor is not related to viral pathogenicity; no human infections associated with GX_P2V were found or suspected, suggesting that the virus is non-pathogenic in humans. Therefore, GX_P2V can be routinely cultured in a biosafety level 2 laboratory. Furthermore, the reported anti-SARS-CoV-2 drugs remdesivir, chloroquine, hydroxychloroquine, nefinavir, and lopinavir also inhibit GX_P2V, further validating that GX_P2V can serve as a good alternative model for screening anti-SARS-CoV-2 drugs (Hu et al., 2021).

[0003] Whey protein (WP) is a mixture of various proteins found in the whey portion of milk, including lactoferrin, α-lactalbumin, β-lactoglobulin, osteopontin, lysozyme, and immunoglobulins.

[0004] Lactoferrin (LF) is found in biological fluids including breast milk, saliva, and semen, and is the most abundant whey protein. It is also present on mucosal surfaces and in polymorphonuclear leukocyte granules. The richest sources of LF are human milk and cow's milk. The concentration of LF in breast milk varies greatly depending on the stage of lactation and species. Human colostrum contains over 5 g / L of LF, while mature breast milk contains 2-3 g / L. Bovine colostrum contains approximately 0.8 g / L of LF, while cow's milk contains only 0.03-0.49 g / L. Studies have found that higher LF levels in colostrum can protect breastfed infants from bacterial infections (Artym & Zimecki, 2005). Lactoferrin can inhibit viral cell adhesion; it can also enhance the production and activity of natural killer cells, interferon α / β, and helper T cells; and it has inhibitory effects on various Gram-positive and Gram-negative bacteria. Because lactoferrin itself has anti-digestive properties, and the digestive capacity of an infant's gastrointestinal tract is relatively low, it has the potential to exert biological activity in the infant's gastrointestinal tract.

[0005] The milk fat globule membrane (MFGM) is a structure derived from the cell membrane that encapsulates the fat in milk. The MFGM contains approximately 20% protein and 50%–70% phospholipids, with the proteins embedded within the lipids. It also contains some acidic sugars. The MFGM serves as a carrier for various phospholipids, active proteins, and some acidic sugars in breast milk. MFGM proteins account for 1%–2% of the total protein content in bovine milk, and currently, 50–120 proteins with molecular weights ranging from 10 to 300 kDa can be isolated from the MFGM. Among these, mucin 1 and mucin 15 have been reported to have antiviral functions, and lactoglucosin has intestinal antiviral activity.

[0006] Osteopontin (OPN) is a phosphorylated acidic protein with a relatively high concentration (50-180 mg / L) in human milk, compared to only 18 mg / L in cow's milk. OPN was initially discovered in bone and is expressed in many tissues and organs, but its highest concentration was found in human milk; milk-derived osteopontin is abbreviated as lactopontin (LPN). OPN possesses various biological activities, such as promoting cell proliferation and differentiation, enhancing immune function, and promoting bone development. OPN plays a positive role in early infant development, especially in early immune regulation. In early life, insufficient production and low Th1 cytokine response may be the main reasons for low innate cellular immunity and a shift towards a Th2 immune response in newborns. Research indicates that the key to OPN's effectiveness lies in its regulation of the Th1 and Th2 immune balance. Clinical studies have shown that infants fed formula fortified with OPN (oligomeric proton pumped) have good tolerance and excellent growth and development. Compared to regular formula, OPN-fortified formula reduces the incidence of fever and the production of pro-inflammatory immune responses (cytokines) in infants. (e.g., 2016). Furthermore, studies have shown that OPN can stimulate intestinal epithelial cell proliferation, supporting intestinal development and health. Research has found that feeding piglets with OPN-fortified formula can reduce the severity of necrotizing enterocolitis in newborn piglets (Moller et al., 2011). In addition, OPN has been found to promote the synthesis of myelin-related proteins and the formation of myelin, thereby promoting cognitive development. Experiments have shown that mice fed milk rich in OPN exhibited better memory and learning abilities in cognitive tests (Jiang et al., 2020). Therefore, OPN has numerous benefits, including improving infant immune function, promoting intestinal health, and promoting cognitive development.

[0007] The above studies have shown that whey protein, milk fat globule membrane, lactoferrin, and osteopontin can exert a variety of positive effects on the human body, but there are few reports on the application of whey protein, milk fat globule membrane, lactoferrin, and osteopontin to coronaviruses. Summary of the Invention

[0008] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide an application of protein-like substances in the preparation of compositions for inhibiting coronaviruses, thus opening up a new approach for combating SARS-CoV-2.

[0009] A second objective of this invention is to provide a composition containing protein-like substances that can be used to inhibit coronaviruses.

[0010] One of the objectives of this invention is achieved through the following technical solution:

[0011] The application of protein-based substances in the preparation of compositions for inhibiting coronaviruses, including: inhibiting the adhesion of coronaviruses to host cells; hindering the invasion of coronaviruses into host cells; and inhibiting the post-infection process of coronavirus infection.

[0012] In this invention, inhibiting coronavirus can be understood as preventing or treating coronavirus. The composition containing protein substances can interfere with the adsorption of the virus on host cells (including specific adsorption / binding and non-specific adsorption / binding), inhibit the process of viral entry into cells, inhibit the post-infection process of the virus, and inhibit viral replication. The above-mentioned inhibition, prevention, and treatment processes are all within the scope of this invention.

[0013] In a preferred embodiment of the present invention, the coronaviruses include GX_P2V, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV. Among them, coronavirus GX_P2V is a novel coronavirus-like virus, also known as pangolin coronavirus GX_P2V.

[0014] In a preferred embodiment of the present invention, the protein substance includes one or any combination of whey protein (WP), milk fat globule membrane (MFGM), lactoferrin (LF), and osteopontin (OPN). For example, the protein substance may be only whey protein; or only milk fat globule membrane; or only lactoferrin; or only osteopontin; or a combination of lactoferrin and osteopontin; or a combination of whey protein and osteopontin; or a combination of milk fat globule membrane and osteopontin; or a combination of whey protein, milk fat globule membrane, lactoferrin, and osteopontin; or a combination of any three of whey protein, milk fat globule membrane, lactoferrin, and osteopontin.

[0015] In a preferred embodiment of the present invention, the osteopontin is milk-derived osteopontin, which is abbreviated as lactopontin (LPN).

[0016] In a preferred embodiment of the present invention, the composition is one or more of the following: food, food additive, beverage, beverage additive, nutritional supplement, or pharmaceutical. Food includes, but is not limited to, infant formula, adolescent formula, formula for the elderly, modified infant formula, modified adolescent formula, modified formula for the elderly, and protein powder. When the composition is a pharmaceutical, the pharmaceutical may contain pharmaceutically acceptable diluents, binders, excipients, lubricants, sweeteners, flavorings, wetting agents, or absorbents.

[0017] In a preferred embodiment of the present invention, the composition may be in various forms, such as solution, suspension, emulsion or solid.

[0018] In a preferred embodiment of the present invention, the composition is in solution form, and the concentration of the protein substance in the composition is 0.02-10 mg / mL; preferably 0.03-5 mg / mL; more preferably 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.63 mg / mL, 0.31 mg / mL, 0.15 mg / mL, 0.078 mg / mL or 0.039 mg / mL.

[0019] In a preferred embodiment of the invention, the composition further includes oligosaccharides, such as, but not limited to, one or a combination of HMOs, FOS, and GOS, wherein the HMOs are selected from one or any combination of 2'-FL, 3-FL, 3'-SL, 6'-SL, LNT, LNnT, DFL, and LNFP-I. The oligosaccharides have a suitable concentration in the composition; when the composition is in liquid form, the concentration of oligosaccharides is preferably 5 mg / mL.

[0020] In a preferred embodiment of the invention, the composition may further include probiotics, such as Bifidobacterium.

[0021] In a preferred embodiment of the invention, the protein substance is derived from one or a combination of cattle, horses, sheep, and humans. Cow's milk is preferred as it is a widely available source.

[0022] In a preferred embodiment of the invention, the composition provides 3 mg to 375 mg / kg body weight / day of lactoferrin, within which coronaviruses can be well inhibited; in particular, the novel coronavirus GX_P2V can be well inhibited.

[0023] In a preferred embodiment of the invention, the composition provides a milk fat globule membrane at a dosage of 3 mg to 375 mg / kg body weight / day, within which coronaviruses can be well inhibited; in particular, the novel coronavirus GX_P2V can be well inhibited.

[0024] In a preferred embodiment of the invention, the composition provides osteopontin at a dose of 12 mg to 375 mg / kg body weight / day, within which coronaviruses can be well inhibited; in particular, the novel coronavirus GX_P2V can be well inhibited.

[0025] In a preferred embodiment of the invention, the composition provides 6 mg to 375 mg / kg body weight / day of whey protein, within which coronaviruses can be well inhibited; in particular, the novel coronavirus GX_P2V can be well inhibited.

[0026] In a preferred embodiment of the invention, the composition provides one or any combination of lactoferrin (3 mg / kg body weight / day), milk fat globule membrane (3 mg / kg body weight / day), osteopontin (12 mg / kg body weight / day), and whey protein (6 mg / kg body weight / day). For example, it can be a combination of any two, any three, or all four of whey protein, milk fat globule membrane, lactoferrin, and osteopontin.

[0027] In a preferred embodiment of the present invention, the composition is a ready-to-use product, for example, the composition can be taken orally directly, or the composition is in powder form and can be used after being reconstituted with water.

[0028] In a preferred embodiment of the invention, the composition is suitable for oral administration, oral drinking, or oral ingestion.

[0029] The second objective of this invention is achieved by the following technical solution:

[0030] A composition comprising protein substances, the composition including one or any combination of whey protein, milk fat globule membrane, lactoferrin and osteopontin, the composition being used to inhibit coronavirus; preferably, the composition is used to inhibit novel coronavirus-like GX_P2V.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] This invention proposes the application of protein-based substances in the preparation of compositions for inhibiting coronaviruses. The antiviral effects of various nutrients (whey protein, milk fat globule membrane, lactoferrin, and osteopontin) were verified in vitro using a SARS-CoV-2-like virus GX_P2V assay. The results showed that whey protein, milk fat globule membrane, lactoferrin, and osteopontin exhibited anti-SARS-CoV-2 activity. Currently, there are few reports on the use of protein-based substances against SARS-CoV-2. This invention opens up a new avenue for combating SARS-CoV-2 and has significant application value and prospects. Attached Figure Description

[0033] Figure 1 This is a graph illustrating the inhibition rate of four proteins against GX_P2V infection in Example 1 of the present invention;

[0034] Figure 2 This is a graph illustrating the inhibition rate of the lactoferrin and osteopontin composition against GX_P2V infection in Example 1 of the present invention.

[0035] Figure 3 This is a diagram showing the results of the EC50 and CC50 determination experiments of four proteins in Example 2 of the present invention;

[0036] Figure 4 This is a diagram showing the measurement results of infectious particle production after intervention with the antiviral active ingredient in Example 3 of the present invention;

[0037] Figure 5 This is a diagram showing the results of the Western blot assay for the antiviral active ingredient in Example 4 of the present invention.

[0038] Figure 6 This is a diagram showing the results of the preliminary investigation of the antiviral mechanism (cell entry and post-cell entry effects) of four protein substances in Example 5 of the present invention.

[0039] Figure 7 This is a diagram showing the results of the preliminary investigation of the antiviral mechanism (entry and post-entry effects) of lactoferrin in Example 5 of the present invention.

[0040] Figure 8 This is a diagram showing the results of the preliminary investigation into the antiviral mechanism of four protein substances (inhibition of virus adhesion to cells) in Example 6 of the present invention;

[0041] Figure 9 This is a diagram showing the results of RBD docking in two conformations of lactoferrin and SARS-CoV-2 in Example 7 of the present invention. Detailed Implementation

[0042] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] The whey protein, milk fat globule membrane, lactoferrin, and osteopontin used in this invention are all derived from bovine milk. These four proteins exist in powder form and are stored at -20°C in the dark. They are prepared as solutions before use. This invention uses these four proteins to perform in vitro verification against SARS-CoV-2 (pangolin coronavirus GX_P2V). The cells used for in vitro testing are African green monkey kidney cells (Vero E6), the virus is pangolin coronavirus GX_P2V (accession number: MT072864.1), and the culture conditions are 37°C and 5% CO2.

[0045] Example 1

[0046] Inhibition rate against GX_P2V infection

[0047] Methods: Vero E6 cells were pre-seeded in 96-well cell culture plates. Once the cells reached 80-90% of the well area, the culture medium was discarded, and an appropriate concentration of GX_P2V (10⁻⁶ g / L) was rapidly added according to the optimal MOI. 4 The study included the addition of pfu / mL of the active ingredient. The protein component and milk fat globule membrane (MFGM) were selected at a concentration of 5 mg / mL, with each component prepared in triplicate to ensure reproducibility. The negative control group received only the virus. Incubation was performed at 37°C for 2 hours to allow sufficient viral interaction with the cells. The liquid in the wells was aspirated, and the cells were washed once with PBS to remove free virus. Culture medium containing the same final concentration of the active ingredient (5 mg / mL) was quickly added to the wells, while the negative control group received only culture medium. Incubation continued for approximately 46 hours, until significant cytopathic effects were observed under a microscope in the negative control group. Two independent replicate experiments were performed to ensure accuracy.

[0048] Results: Whey protein, milk fat globule membrane, lactoferrin, and osteopontin all exhibited excellent antiviral effects. Figure 1 As shown, the inhibition rate was calculated based on the relative expression level of viral RNA within cells. The inhibition rates of whey protein concentrate, milk fat globule membrane protein, and lactoferrin against GX_P2V infection were 99.97%, 99.98%, and 99.99%, respectively, while the inhibition rate of osteopontin against GX_P2V infection was 52.86%. Figure 2 As shown, a combination of lactoferrin and osteopontin with a mass concentration ratio of 2:1 (lactoferrin 5 mg / mL, osteopontin 2.5 mg / mL) also showed good anti-GX_P2V infection efficacy, with an inhibition rate of 87%.

[0049] Example 2

[0050] EC50 value determination experiment and CC50 value determination experiment

[0051] EC50 value: The EC50 value represents the concentration at which a drug achieves 50% of its effect. Vero E6 cells were pre-seeded in 48-well cell culture plates. Once the cells reached 80-90% of the well area, the culture medium was discarded, and an appropriate concentration of GX_P2V (10⁻⁶ g / L) was rapidly added according to the optimal MOI. 4 The active ingredient (pfu / mL) was added. The active ingredient was diluted 2-fold to obtain eight concentrations (0.039 mg / mL, 0.078 mg / mL, 0.15 mg / mL, 0.31 mg / mL, 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL), with each concentration inoculated into two wells. The negative control group was supplemented with only virus. The steps and conditions of the viral infection process were the same as in Example 1.

[0052] Discard the cell culture medium and wash the cells once with PBS. Add the appropriate reagents (including buffer VL, 70% ethanol, buffer RW1, and buffer RW2) according to the steps of the nucleic acid extraction kit (tissue RNA extraction kit; catNo: RNE11-02) to obtain an RNA sample. Reverse transcribe the RNA to obtain a cDNA sample. Perform real-time quantitative PCR analysis on an appropriate amount of cDNA sample, using specific primers to detect the GX_P2VE gene and the cellular GAPDH gene, obtaining the cycle initiation value (Ct value). Calculate the relative expression level of viral mRNA based on the Ct value, denoted as B. Calculate the EC50 of the components using Graphpadprims 8.0.2.

[0053] CC50 value: Used to evaluate the cytotoxicity / safety of potential active ingredients. Vero E6 cells were pre-seeded in 96-well cell culture plates. Once the cells reached 80-90% of the well area, the culture medium was discarded, and an appropriate concentration of the active ingredient was rapidly added according to the optimal MOI. The active ingredient was diluted 2-fold to eight concentrations, with each concentration seeded in three wells. The negative control group received only culture medium. After 28 hours of culture, 20 μL of rezinazuma dye was added to each well. The values ​​(RFU) were read using a multi-functional plate reader at 0, 30, 60, 90, and 120 minutes after dye addition, under the following conditions: excitation wavelength 554 nm, emission wavelength 593 nm. The cytotoxicity rate was calculated based on the readings and denoted as C. The formula is as follows:

[0054] C active ingredient = 1 - (RFU component - RFU positive control) × 100%

[0055] The CC50 of the components was calculated using Graphpad 8.0.2 software based on the cytotoxicity rate.

[0056] Results: The EC50 value refers to the component concentration when the virus is inhibited by half, while the CC50 value refers to the component concentration when half of the cells die. The former reflects the efficacy of the component, while the latter reflects its toxicity. Based on the relative expression levels of viral mRNA in cells, the half-maximal effective concentrations (EC50) of the four components (whey protein, milk fat globule membrane, lactoferrin, and osteopontin) and the combination of lactoferrin and osteopontin were calculated using the log(inhibitor) vs. response formula in the nonlinear analysis of Graphpad Prism software to be 0.8 mg / mL, 0.442 mg / mL, 0.397 mg / mL, 0.625 mg / mL, and 1.875 mg / mL, respectively. The results are as follows. Figure 3 As shown in the figure, the CC50 values ​​of whey protein, milk fat globule membrane, lactoferrin, and osteopontin were all greater than 5 mg / mL. The results showed that whey protein exhibited antiviral efficacy (32%–99%) within seven concentration ranges: 0.078 mg / mL, 0.15 mg / mL, 0.31 mg / mL, 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL. Milk fat globule membrane showed antiviral efficacy (45%–99%) within eight concentration ranges: 0.039 mg / mL, 0.078 mg / mL, 0.15 mg / mL, 0.31 mg / mL, 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL. Lactoferrin showed antiviral efficacy (30%–99%) across eight concentration ranges: 0.039 mg / mL, 0.078 mg / mL, 0.15 mg / mL, 0.31 mg / mL, 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL. Osteopontin showed antiviral efficacy (30%–80%) across five concentration ranges: 0.31 mg / mL, 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL. The combination of lactoferrin and osteopontin (lactoferrin 5 mg / mL, osteopontin 2.5 mg / mL) showed antiviral efficacy greater than 50% across three concentration ranges: 7.5 mg / mL, 3.75 mg / mL, and 1.875 mg / mL.

[0057] Example 3

[0058] The determination of infectious particle production after intervention with antiviral active ingredients was expressed as TCID50 (PFU / mL).

[0059] Methods: Vero E6 cells were pre-seeded in 96-well cell culture plates and kept ready for use. The cell culture supernatant after viral infection and intervention with the effective active ingredient was collected (the liquid from the culture wells was taken directly after incubation at 37°C for approximately 48 hours) and serially diluted 10-fold. -1 ~10 -7 Each dilution was prepared in 1000 μL volumes. The culture medium in the 96-well cell culture plate was discarded, and 100 μL of virus dilution was added to each well. Ten wells were inoculated for each dilution gradient, for a total of seven dilution gradients. The remaining wells were replenished with virus-free culture medium as a negative control. After 72 hours of incubation, each well was observed under an optical microscope for cytopathic effects. Wells showing cytopathic effects were marked as positive wells, and those without were marked as negative wells. The TCID50 (PFU / mL) value was calculated based on the number of positive and negative wells in each dilution gradient. This value represents the amount of virus required to induce cytopathic effect (CPE) in the wells of the culture plate or test tube, thus characterizing the titer of infectious viral particles.

[0060] Results: TCID50 refers to the viral load required for 50% of cells to be infected, measured in PFU / mL. Based on the initial screening results, the content of infectious viral particles in the supernatant of infected cells was tested after treatment with 5 mg / mL whey protein, milk fat globule membrane, lactoferrin, and osteopontin. Figure 4 It is evident that among these four active ingredients, lactoferrin exhibited the best inhibitory effect on viral production. Treatment with 5 mg / mL lactoferrin reduced the TCID50 value of the virus in the cell supernatant by 1.5 Log10, meaning that the number of infectious viral particles was 4.48% of the untreated group, with an inhibition rate of 95.42%. Figure 4 As can be seen, treatment with the same concentration of whey protein and milk fat globule membrane reduced the Log10 value by 0.89 and 0.88, respectively, with inhibition rates of 87.57% and 87.86%, respectively. All of the above differences were statistically significant.

[0061] Example 4

[0062] Western blot assay to verify antiviral active ingredients

[0063] Methods: After viral infection, the supernatant of the cells was discarded, and 100 μL of RIPA lysis buffer was added, followed by lysis for 10 minutes. Protein quantification was performed using the BCA kit according to the manufacturer's instructions. After all samples were diluted to the same protein concentration, 20 μL of the diluted sample was mixed with 4 μL of loading buffer. The mixture was then treated at 100 °C for 10 minutes to denature the protein. The mixture was then centrifuged at 12,000 rpm for 2 minutes at 4 °C. Samples and protein markers were loaded onto a 12% SDS-PAGE gel and separated by electrophoresis at 80 V for 30 minutes, then 120 V for 60 minutes. The proteins on the gel were transferred to a polyvinylidene fluoride (PEDV) membrane using a transfer apparatus at 15 V for 60 minutes. The PEDV membrane loaded with proteins was blocked with 5% skim milk powder (dissolved in TBST) at room temperature for 2 hours. Because GX_P2V shares over 92.5% amino acid homology with SARS-CoV-2 (NCBI, Protein BLAST), an anti-SARS-CoV-2 nucleocapsid protein antibody was used to detect the GX_P2V nucleocapsid protein. Considering the over 93% homology between the N protein amino acid sequences of GX_P2V and SARS-CoV-2 and the availability of SARS-CoV-2 N protein antibodies, anti-SARS-CoV-2 N protein nucleocapsid protein antibody from Genscript (USA) and anti-GAPDH antibody from Proteintech (USA) were diluted 1:200 and 1:10000, respectively, and incubated at room temperature for 2 hours. The primary antibody was removed by washing three times with TBST, and the secondary antibody (1:20000 diluted with goat anti-mouse IgG (H+L) enzyme-labeled antibody) was used for incubation again for 2 hours. The mixture was then incubated with chemiluminescent buffer for 5 minutes and washed three times with TBST. The luminescence of the PEDV film was then detected using the SuperSignal WestFemto Maximum Sensitivity Chemiluminescent Substrate (Thermo Scientific, USA).

[0064] Result: As Figure 5 As shown, within the range of 0.6 mg / mL to 10 mg / mL, the production of viral N protein in infected cells was dose-dependently inhibited in the lactoferrin-treated groups. Clearly, at a lactoferrin concentration of 10 mg / mL, viral N protein expression was almost undetectable intracellularly; at a lactoferrin concentration of 5 mg / mL, only trace amounts of viral N protein expression were detectable intracellularly. Figure 5 b. As can be seen, the milk fat globule membrane significantly inhibited the production of intracellular viral N protein at a concentration of 10 mg / mL, and trace amounts of viral N protein expression were detected. Figure 5 In this context, NP refers to the GX_P2V nucleocapsid protein; GAPDH refers to the cellular glyceraldehyde-3-phosphate dehydrogenase.

[0065] Example 5

[0066] Preliminary investigation into the antiviral mechanism of active protein substances: cell entry and post-cell entry effects

[0067] Methods: The dosing time method was used to preliminarily determine the stage of the viral life cycle at which the antiviral active ingredient exerts its inhibitory effect. Vero E6 cells were seeded into 48-well plates (1×10⁶ cells / wells). 5 Cells were allowed to adhere and grow in wells (cells / well). GX_P2V at an MOI of 0.01 and an appropriate concentration of antiviral component were uniformly mixed and added to the cells. The mixture was incubated at 37°C for 2 hours to allow viral adsorption and entry into the cells. The virus-containing culture medium was then washed away, and the cells were washed twice with PBS. Cells were replenished with fresh culture medium and cultured for 48 hours post-infection. To investigate whether the active component exerts its effect after viral entry into cells, GX_P2V was used to infect cells for 2 hours at an MOI of 0.01. The virus-containing culture medium was removed, and the cells were washed twice with PBS. The same final concentration of the active component was added to the cells, and the cells were cultured for another 48 hours. Cells were collected for subsequent nucleic acid extraction and RT-qPCR analysis to determine the expression of viral mRNA in the cells to characterize the viral load.

[0068] Result: As Figure 6-7 As shown, after adding whey protein, milk fat globule membrane, lactoferrin, and osteopontin (5 mg / mL) 2 hours after GX_P2V infection, and co-incubating the cells and virus for 46 hours, the relative expression level of viral mRNA in the cells decreased to varying degrees. This indicates that whey protein, milk fat globule membrane, lactoferrin, and osteopontin can inhibit the post-infection process of the virus. Among them, lactoferrin showed the most significant antiviral effect, achieving an inhibition rate of greater than 95% even when added 2 hours after viral infection within the range of 0.625–2.5 mg / mL. For the group that added whey protein and milk fat globule membrane (5 mg / mL) only during the viral infection phase, the relative expression level of viral mRNA in the cells also decreased, indicating that whey protein and milk fat globule membrane can inhibit the viral entry process.

[0069] Example 6

[0070] Preliminary investigation into the antiviral mechanism of active protein substances: their role in inhibiting virus adhesion to cells.

[0071] Methods: Adsorption assays were used to investigate whether the antiviral active ingredient could inhibit the attachment of GX_P2V to cells. Vero E6 cells were seeded into 48-well plates (1×10⁶ cells / wells). 5Incubate overnight in cells / wells. High concentrations of GX_P2V were incubated with certain concentrations of the active ingredients whey protein, milk fat globule membrane, lactoferrin, and osteopontin at 4°C for 2 hours to allow for sufficient interaction between the active ingredients and the virus. Then, the mixture of virus and active ingredients was added to the cells and incubated at 4°C for 2 hours to allow for sufficient interaction between the virus and the cells. The mixture in the culture wells was discarded, and adherent cells were washed three times with PBS. Cells were then collected for subsequent nucleic acid extraction and RT-qPCR analysis to determine the expression of viral mRNA on the cell surface to characterize the viral load.

[0072] Result: As Figure 8 As shown, four active substances—whey protein, milk fat globule membrane, lactoferrin, and osteopontin—were used in an experiment to intervene in virus adsorption on cells. The results were repeated twice to ensure reliability. The results showed that all four components, at concentrations of 10 mg / mL and 5 mg / mL, significantly reduced GX_P2V adhesion to cells. Figure 8 As shown, at an intervention concentration of 10 mg / mL, the inhibition rates of virus attachment to cells were 75.55%, 76.24%, and 94.33%, 75.49%, respectively. Virus attachment assays indicated that whey protein, milk fat globule membrane, lactoferrin, and osteopontin all significantly reduced GX_P2V attachment to cells and inhibited viral infection.

[0073] Example 7

[0074] Computer simulation of the binding of lactoferrin to SARS-CoV-2 spike protein and cellular ACE2 receptor.

[0075] Methods: Protein-protein molecular docking was performed on lactoferrin (PDB: 1LFG) and RBDs of wild-type SARS-CoV-2 in two conformations, as well as human ACE2 (the corresponding structure was extracted from the complex with PDB 7DF4). Water molecules were removed from the structures before docking. The docking platform was ZDOCK 3.0.2 (https: / / zdock.umassmed.edu / ). The docking complex with the highest docking score was selected for analysis using PDBePISA (https: / / www.ebi.ac.uk / msd-srv / prot_int / pistart.html) to obtain the interfacial area (A) of the docking complex. 2 The docking free energy (ΔG) and its P value, the number of hydrogen bonds formed at the docking interface, and the number of salt bridges were determined. The docking complex was visualized using PyMol.

[0076] Results: The docking of lactoferrin and SARS-CoV-2 RBD in two conformations is as follows: Figure 9 As shown in A and 9B, Figure 9A shows lactoferrin and the SARS-CoV-2 receptor binding domain (an open conformation with RBD up); Figure 9 B shows the binding domain of lactoferrin and the SARS-CoV-2 receptor (an open conformation with three RBDs down). RBD (a RBD-up) refers to a conformation in the spike protein where one RBD protrudes upwards, and the spike protein binds to ACE2 on the cell surface in this conformation. RBD (three RBD-down) refers to a conformation in the spike protein where all three RBDs are tightly bound and none protrude upwards. Based on the docking results in Table 1, lactoferrin can spontaneously bind to both conformations of the RBD, exhibiting a free energy of less than 0 kcal / mol. Hydrogen bonds and salt bridges form at the binding interface. Analysis shows that lactoferrin cannot spontaneously bind to ACE2, with a binding free energy of 0.5 kcal / mol. These results suggest that lactoferrin prevents the binding of the RBD to ACE2 by binding to the RBD on the SARS-CoV-2 spike protein.

[0077] Table 1. Record of Docking Results

[0078]

[0079] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. The use of protein-like substances in the preparation of compositions for inhibiting coronaviruses, characterized in that, Inhibiting coronaviruses includes: inhibiting the adhesion of coronaviruses to host cells; blocking the invasion of coronaviruses into host cells; and inhibiting the post-infection process of coronavirus infection; the protein substances include osteopontin.

2. The use of the protein substance of claim 1 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The protein substances also include one or any combination of whey protein, milk fat globule membrane and lactoferrin.

3. The use of the protein substance of claim 1 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The composition is one or more of the following: formula milk powder, modified milk powder, protein powder, nutritional supplements, and drugs.

4. The use of the protein substance of claim 1 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The composition is in solution form, suspension form, emulsion form, or solid form.

5. The use of the protein substance of claim 4 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The composition is in solution form, and the concentration of the protein in the composition is 0.02–10 mg / mL; preferably 0.03–5 mg / mL; more preferably 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.63 mg / mL, 0.31 mg / mL, 0.15 mg / mL, 0.078 mg / mL, or 0.039 mg / mL.

6. The use of the protein substance of claim 1 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The coronaviruses include GX_P2V, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV.

7. The use of the protein substance of claim 1 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The composition also includes oligosaccharides.

8. The use of the protein substance of claim 1 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The protein-like substances are derived from one or a combination of cattle, horses, sheep, and humans.

9. The use of the protein substance according to any one of claims 1-8 in the preparation of a composition for inhibiting coronaviruses, characterized in that, The composition provides 3 mg to 375 mg / kg body weight / day of lactoferrin; Alternatively, the composition provides a milk fat globule membrane of 3 mg to 375 mg / kg body weight / day; Alternatively, the composition provides 12 mg to 375 mg / kg body weight / day of osteopontin; Alternatively, the composition provides 6 mg to 375 mg / kg body weight / day of whey protein; Alternatively, the composition may provide one or any combination of 3 mg to 375 mg / kg body weight / day of lactoferrin, 3 mg to 375 mg / kg body weight / day of milk fat globule membrane, 12 mg to 375 mg / kg body weight / day of osteopontin, and 6 mg to 375 mg / kg body weight / day of whey protein.

10. A composition comprising a protein-like substance, characterized in that, The composition includes osteopontin; it also includes one or any combination of whey protein, milk fat globule membrane and lactoferrin; the composition is used to inhibit coronavirus; preferably the composition is used to inhibit novel coronavirus-like GX_P2V.

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