CN115245562A - Composition for inhibiting coronavirus and application thereof - Google Patents
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- CN115245562A CN115245562A CN202111011033.2A CN202111011033A CN115245562A CN 115245562 A CN115245562 A CN 115245562A CN 202111011033 A CN202111011033 A CN 202111011033A CN 115245562 A CN115245562 A CN 115245562A
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Abstract
The invention discloses an application of protein substances in preparing a composition for inhibiting coronavirus, which comprises the following components in part by weight: inhibiting adhesion of coronavirus to a host cell; blocking the invasion of host cells by coronavirus; inhibit the post-cellular processes of coronavirus infection. The invention adopts coronavirus GX _ P2V to carry out in-vitro verification on the antiviral effect of various nutritional ingredients (whey protein, milk fat globule membrane, lactoferrin and milk-derived osteopontin), and the result shows that the coronavirus resistant activity of the whey protein, the milk fat globule membrane, the lactoferrin and the milk-derived osteopontin (LPN, abbreviated as lactobridge protein). The application of protein substances in resisting coronavirus is only reported at present, and the coronavirus GX _ P2V is a new-like coronavirus.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a composition for inhibiting coronavirus and application thereof.
Background
Researchers isolated multiple strains of coronavirus from a tissue sample of pangolin scales. Among them, the spike protein (S) of coronavirus GX _ P2V has 92.2% homology with the spike protein of SARS-CoV-2. The S protein is an important virus structural protein for SARS-CoV-2 to recognize and enter cells to start infection, and is also one of important targets for vaccine development. Angiotensin converting enzyme 2 (ACE 2) is a receptor used by SARS-CoV-2 infected cells and can be recognized and bound by the S protein. Researchers tested whether ACE2 was involved in GX _ P2V infection by using siRNA mediated knock down of ACE2 expression. The results showed that the expression levels of ACE2 mRNA and viral RNA were significantly reduced in GX _ P2V-infected cells, indicating that ACE2 is also a receptor for GX _ P2V-infected cells. However, it is noteworthy that GX _ P2V mediated entry through the receptor ACE2 was not associated with viral pathogenicity, and no human infection associated with GX _ P2V was found or suspected, suggesting that the virus was non-pathogenic in humans. Thus, GX _ P2V can be routinely cultured in secondary biosafety laboratories. In addition, reported anti-SARS-CoV-2 drugs, namely Remdesivir (remdesivir), chloroquine (chloroquine), hydroxychloroquine (hydroxychloroquine), nelfinavir (nelfinavir) and lopinavir (lopinavir), also have an inhibitory effect on GX _ P2V, and further verify that GX _ P2V can be used as a good alternative model for screening anti-SARS-CoV-2 drugs (Hu et al, 2021 years).
Whey Protein (WP) is a mixture of proteins present in the Whey fraction in milk and includes lactoferrin, alpha-lactalbumin, beta-lactoglobulin, osteopontin, lysozyme, immunoglobulins, and the like.
Lactoferrin (LF) is present in biological fluids including milk, saliva and semen and is one of the most abundant proteins in whey proteins. It is also present on mucosal surfaces and in polymorphonuclear leukocyte granules. The most abundant sources of LF are human milk and cow's milk. The concentration of LF in milk varies greatly with lactation stage and species. The LF content in human colostrum is more than 5 g/L, and the LF content in the mature colostrum is 2-3 g/L. The LF content in the bovine colostrum is about 0.8g/L; whereas milk contains only 0.03-0.49 g/l. It was found that higher LF content in colostrum can protect breast-fed infants from bacterial infections (Artym & Zimecki, 2005). Lactoferrin can inhibit cell adhesion of viruses; lactoferrin also enhances the production and activity of natural killer cells, interferon alpha/beta and helper T cells; has inhibitory effect on various gram-positive bacteria and gram-negative bacteria. Since lactoferrin has anti-digestive ability by itself and is relatively low in digestive ability of the gastrointestinal tract of infants, it has the potential to exert biological activity in the gastrointestinal tract of infants.
Milk Fat Globule Membrane (MFGM), in which the fat in Milk is surrounded by a layer of membrane-derived Milk fat globule membrane structure, contains about 20% protein and about 50% -70% phospholipid, and the protein is embedded in the lipid. In addition, the milk fat globule membrane also contains some acidic sugars. The milk fat globule membrane is a carrier for various phospholipids, active proteins and part of acidic sugar of breast milk. The milk fat globule membrane protein accounts for 1-2% of the total protein content of the milk, 50-120 proteins can be separated from the milk fat globule membrane at present, and the molecular weight is 10-300 kDa. Among them, mucin 1 and mucin 15, which are protein components, have been reported to have an antiviral function, and lactadherin has an antiviral function in the intestinal tract.
Osteopontin (OPN) is a phosphorylated acidic protein, and has a high concentration of 50-180mg/L in human milk, whereas milk contains only 18mg/L. Osteopontin OPN was initially found in bone and is expressed in many tissues and organs, and subsequently found in human milk in highest amounts, milk-derived osteopontin, abbreviated as lactobridge protein (L)actiontin, LPN). OPN has a variety of biological activities, such as promoting cell proliferation and differentiation, enhancing immune function and promoting bone development. OPN has a positive effect on early development of infants, in particular on early immunomodulation of infants. Early in life, insufficient production of Th1 cytokines and poor responsiveness may be the primary cause of low innate cellular immunity in newborns, and a shift towards Th2 immune responses. Studies have shown that OPN plays a key role in its regulation of Th1 and Th2 immune balance. Clinical research shows that the infant tolerance is good and the growth and development state is good when the formula powder of the fortified cow milk OPN is eaten; compared with the common formula powder, the formula powder feeding of the enhanced OPN can reduce the generation of fever and proinflammatory immune response (cell factors) of the infants: (
et al.2016). In addition, studies have shown that OPN can stimulate intestinal epithelial cell proliferation, support intestinal development and intestinal health, and that feeding fortified bovine milk OPN formula powder can reduce the severity of necrotizing enterocolitis in newborn piglets (Moller et al, 2011). In addition, OPN has been found to promote myelin-associated protein synthesis and myelin formation, thereby promoting cognitive development, and experimental findings show that mice fed milk-enriched OPN milk exhibit better memory and learning in cognitive testing (Jiang et al, 2020). Thus, OPN has many effects such as improving immune function in infants, promoting intestinal health, and promoting cognitive development.
The above studies indicate that whey protein, milk fat globule membrane, lactoferrin and osteopontin can exert various positive effects on human body, but the application of whey protein, milk fat globule membrane, lactoferrin and osteopontin to coronavirus has been rarely reported.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of a protein substance in preparing a composition for inhibiting coronavirus, and open up a new idea for resisting the novel coronavirus.
It is a further object of the present invention to provide a composition comprising proteinaceous material, which composition is useful for inhibiting coronaviruses.
One of the purposes of the invention is realized by adopting the following technical scheme:
use of proteinaceous material in the manufacture of a composition for inhibiting a coronavirus, the coronavirus inhibiting composition comprising: inhibiting adhesion of coronavirus to a host cell; blocking the invasion of host cells by coronavirus; inhibit the post-cellular processes of coronavirus infection.
In the present invention, inhibiting coronavirus refers to preventing and treating coronavirus, and the composition containing protein substance can interfere with the adsorption (including specific adsorption/binding, non-specific adsorption/binding) of virus on host cell, inhibit the virus cell-entering process, inhibit the virus infection post-cell process, and inhibit virus replication, and the inhibition, prevention and treatment processes are all within the scope of the present invention.
In a preferred embodiment of the 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, also called Pangolin coronavirus GX _ P2V.
In a preferred embodiment of the present invention, the protein material includes one or any combination of Whey Protein (WP), milk Fat Globule Membrane (MFGM), lactoferrin (LF), and Osteopontin (OPN). For example, the proteinaceous material is whey protein only; or the protein matter is only the milk fat globule membrane; or the protein substance is lactoferrin only; or the protein substance is osteopontin only; or the protein substance is the combination of lactoferrin and osteopontin; or the protein substance is the combination of whey protein and osteopontin; or the protein substance is the combination of milk fat globule membrane and osteopontin; or the protein substance is the combination of whey protein, milk fat globule membrane, lactoferrin and osteopontin; or the protein substance is the combination of any three of whey protein, milk fat globule membrane, lactoferrin and osteopontin.
In a preferred embodiment of the present invention, the osteopontin is milk-derived osteopontin, which is abbreviated as Lactobridge Protein (LPN).
In a preferred embodiment of the invention, the composition is one or more of a food, a food additive, a beverage additive, a nutritional supplement, or a medicament. The food includes but is not limited to infant formula milk powder, teenager formula milk powder, middle-aged and old formula milk powder, infant modified milk powder, teenager modified milk powder, middle-aged and old modified milk powder and protein powder. When the composition is a medicament, the medicament may contain pharmaceutically acceptable diluents, binders, excipients, lubricants, sweeteners, flavoring agents, wetting agents, or absorbents.
In a preferred embodiment of the invention, the composition may be in a variety of forms, for example, in the form of a solution, a suspension, an emulsion or a solid.
In a preferred embodiment of the invention, the composition is in the form of a solution, and the concentration of the proteinaceous material in the composition is between 0.02 and 10mg/mL; preferably 0.03 to 5mg/mL; more preferably 5mg/mL,2.5mg/mL, 1.25mg/mL, 0.63mg/mL, 0.31mg/mL, 0.15mg/mL, 0.078mg/mL or 0.039mg/mL.
In a preferred embodiment of the present invention, the composition further comprises oligosaccharides, such as one or a combination including but not limited to HMOs, FOS and GOS, wherein HMOs may be selected from one or any combination of 2' -FL, 3' -SL,6' -SL, LNT, LNnT, DFL and LNFP-I. The oligosaccharide is suitably present in the composition at a concentration, preferably 5mg/mL when the composition is in liquid form.
In a preferred embodiment of the invention, the composition may further comprise a probiotic, such as bifidobacteria.
In a preferred embodiment of the invention, the proteinaceous material is derived from one or a combination of bovine, equine, ovine and human origin. Preferably, the source of cow milk is wide.
In a preferred embodiment of the invention, the composition provides lactoferrin in an amount of 3mg to 375mg per kg body weight per day, within which range coronavirus inhibition is better; in particular, the novel coronavirus GX _ P2V can be well inhibited.
In a preferred embodiment of the invention, the composition provides 3mg to 375mg/kg body weight/day of milk fat globule membrane, in which range the coronavirus is better inhibited; particularly, the novel coronavirus GX _ P2V can be well inhibited.
In a preferred embodiment of the present invention, the composition provides 12mg to 375mg/kg body weight/day of osteopontin, within which range the coronavirus can be preferably inhibited; particularly, the novel coronavirus GX _ P2V can be well inhibited.
In a preferred embodiment of the invention, the composition provides 6mg to 375mg whey protein/kg body weight/day, in which range the coronavirus is preferably inhibited; particularly, the novel coronavirus GX _ P2V can be well inhibited.
In a preferred embodiment of the invention the composition provides one or any combination of lactoferrin in the range of 3mg to 375mg/kg body weight per day, lactoglobulin in the range of 3mg to 375mg/kg body weight per day, osteopontin in the range of 12mg to 375mg/kg body weight per day and whey protein in the range of 6mg to 375mg/kg body weight per day. For example, it may be a combination of any two, any three, or any four of whey protein, milk fat globule membrane, lactoferrin, and osteopontin.
In a preferred embodiment of the invention, the composition is a ready-to-use product, e.g. the composition can be taken orally directly, or the composition is in powder form and is ready to use after reconstitution of the powder composition with water.
In a preferred embodiment of the invention, the composition is suitable for oral administration, oral consumption or oral ingestion.
The second purpose of the invention is realized by adopting the following technical scheme:
a composition containing protein material comprises one or any combination of whey protein, milk fat globule membrane, lactoferrin and osteopontin, and is used for inhibiting coronavirus; preferably the composition is for use in inhibiting neotype coronavirus GX _ P2V.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an application of protein substances in preparing a composition for inhibiting coronavirus, and in-vitro verification is carried out on the antiviral effect of various nutritional ingredients (whey protein, milk fat globule membrane, lactoferrin and osteopontin) by adopting neoid coronavirus GX _ P2V, and the result shows that the neoid coronavirus resisting activity of the whey protein, the milk fat globule membrane, the lactoferrin and the osteopontin is discovered. The application of the protein substances in resisting the new-like coronavirus is only reported at present, and the invention opens up a new idea for resisting the new-like coronavirus and has very good application value and prospect.
Drawings
FIG. 1 is a graph showing the inhibition rate of GX _ P2V infection by four proteins in example 1 of the present invention;
fig. 2 is a graph showing the inhibition rate of the lactoferrin and osteopontin composition against GX _ P2V infection in example 1 of the present invention;
FIG. 3 is a chart showing the results of an assay for the four proteins EC50 and CC50 according to example 2 of the present invention;
FIG. 4 is a graph showing the results of an assay of infectious particle production following intervention with an antiviral active ingredient according to example 3 of the present invention;
FIG. 5 is a graphical representation of the results of the Western blot assay of the antiviral active ingredient of example 4 of the present invention;
FIG. 6 is a graph showing the preliminary results of the antiviral mechanism study (endocytosis, postendocytosis) of four protein substances in example 5 of the present invention;
FIG. 7 is a graph showing the preliminary antiviral mechanism (transcytosis, posttranscytosis) of lactoferrin in example 5 of the present invention;
FIG. 8 is a graph showing the results of investigation of the preliminary antiviral mechanism (effect of inhibiting virus adhesion cells) of four protein substances in example 6 of the present invention;
FIG. 9 is a graph showing the results of RBD docking in both lactoferrin and SARS-CoV-2 conformations according to example 7 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The whey protein, the milk fat globule membrane, the lactoferrin and the osteopontin adopted by the embodiment of the invention are all milk sources, and the four protein substances exist in the form of powder, are stored in the dark at the temperature of-20 ℃, and are prepared into a solution when in use. In the embodiment of the invention, the four protein substances are adopted to carry out in-vitro verification of the anti-neocoronaviruses (pangolin coronavirus GX _ P2V), the cells used in-vitro test are Vero E6, the viruses are pangolin coronavirus GX _ P2V (accession number: MT 072864.1), and the culture conditions are 37 ℃ and 5 percent of CO 2 。
Example 1
Inhibition of GX _ P2V infection
The method comprises the following steps: inoculating vero E6 cells into a 96-well cell culture plate in advance, discarding culture solution when the cells grow to 80-90% of the well area, and rapidly adding GX _ P2V (10) with proper concentration according to the optimal MOI 4 pfu/mL) and active ingredient. Wherein the concentration of the protein component and Milk Fat Globule Membrane (MFGM) is selected to be 5mg/mL, three replicates of each component are made to ensure reproducibility of results. The negative control group was supplemented with virus only. Incubation at 37 ℃ for 2 hours allowed the virus to interact well with the cells. The culture wells were aspirated and the cells were washed 1 time with PBS to remove free virus. Culture medium containing equal final concentration of active ingredient (5 mg/mL) was added to the culture wells quickly, and the culture was continued for about 46 hours until the negative control showed significant cytopathic effect under the microscope. Two independent replicates were performed to ensure the accuracy of the results.
As a result: whey protein, milk fat globule membrane, lactoferrin and osteopontin all showed excellent antiviral effects. As shown in fig. 1, the inhibition rates were calculated based on the relative expression amounts of intracellular viral RNA, and the inhibition rates of the whey protein concentrate, milk fat globule membrane protein, lactoferrin against GX _ P2V infection were 99.97%, 99.98%, and 99.99%, respectively, and the inhibition rate of osteopontin against GX _ P2V infection was 52.86%. As shown in fig. 2, the mass concentration ratio is 2: the composition of lactoferrin and osteopontin (lactoferrin 5mg/mL, osteopontin 2.5 mg/mL) of 1 also exhibited good effect against GX _ P2V infection, with an inhibition rate of 87%.
Example 2
Measurement experiment of EC50 value and measurement experiment of CC50 value
EC50 value: the EC50 value represents the concentration at which the drug acts 50%. Inoculating vero E6 cells into 48-hole cell culture plate in advance, discarding culture solution when the cells grow to 80-90% of the hole area, and rapidly adding GX _ P2V (10) with proper concentration according to optimal MOI 4 pfu/mL) and active ingredient. 2-fold dilution of the active ingredient was carried out to give 8 concentrations (0.039 mg/mL,0.078mg/mL,0.15mg/mL,0.31mg/mL, 0.63mg/mL,1.25mg/mL,2.5mg/mL,5 mg/mL) and 2 wells were inoculated at each concentration. Negative control group only virus was added. The procedure and conditions for the viral infection process were the same as in example 1.
The cell culture medium was discarded and the cells were washed 1 time with PBS. Adding corresponding reagents (including buffervL,70% ethanol, bufferrRW 1 and buffer RW 2) according to the steps of a nucleic acid extraction kit (tissue RNA extraction kit; cat No: RNE 11-02) to obtain an RNA sample. And carrying out reverse transcription on the RNA to obtain a cDNA sample. Taking a proper amount of cDNA samples to perform real-time fluorescence quantitative PCR analysis, and detecting the GX _ P2V E gene and the cell GAPDH gene by using specific primers to obtain a cycle initial value (Ct value). The relative expression level of viral mRNA was calculated from the Ct value and is designated as B. The EC50 of the fractions was calculated using Graphpadstandards 8.0.2.
CC50 value: the potential active ingredients were evaluated for cytotoxicity/safety. Inoculating vero E6 cells into a 96-well cell culture plate in advance, discarding culture solution when the cells grow to 80-90% of the area of the wells, and quickly adding active ingredients with proper concentration according to the optimal MOI. The active ingredient was diluted 2-fold to 8 concentrations, each concentration being inoculated into three wells. Negative control group was supplemented with culture medium only. After 28 hours of incubation, 20 μ L of resazurin dye was added to each well and the values were read with a multifunctional plate Reader (RFU) at 0 min, 30 min, 60 min, 90 min and 120 min after dye addition, respectively, under the conditions: the excitation wavelength is 554nm, and the emission wavelength is 593nm. The cytotoxicity rate was calculated from the readings and was designated as C. The formula is as follows:
c active fraction =1- (RFU fraction-RFU positive control) × 100%
Component CC50 was calculated according to cytotoxicity rate using graphpad8.0.2 software.
As a result: the EC50 value refers to the concentration of the component at which the virus is inhibited by half, and the CC50 value refers to the concentration of the component at which half of the cells die. The former reflects the utility of the ingredient, and the latter reflects the magnitude of toxicity of the ingredient. The half-effective concentrations (EC 50) of the four components (whey protein, milk fat globule membrane, lactoferrin and osteopontin) and the combined components of lactoferrin and osteopontin were 0.8mg/mL, 0.442mg/mL, 0.397mg/mL, 0.625mg/mL and 1.875mg/mL, respectively, as calculated from the log (inhibitor) vs. stress formula in the nonlinear analysis in GraphpadPrism software, based on the relative expression of viral mRNA in cells, as shown in FIG. 3 below. The CC50 values of the whey protein, the milk fat globule membrane, the lactoferrin and the osteopontin are all more than 5mg/mL. The results show that the whey protein has antiviral infection effects (32-99%) in 7 concentration ranges, such as 0.078mg/mL,0.15mg/mL,0.31mg/mL,0.63 mg/mL,1.25mg/mL,2.5mg/mL,5mg/mL, and the like. The milk fat globule membrane has the effect of resisting virus infection (45-99%) in 8 concentration ranges of 0.039mg/mL,0.078mg/mL,0.15mg/mL,0.31mg/mL, 0.63mg/mL,1.25mg/mL,2.5mg/mL,5mg/mL and the like. Lactoferrin has the effect of resisting virus infection (30-99%) in 8 concentration ranges of 0.039mg/mL,0.078mg/mL,0.15mg/mL,0.31mg/mL, 0.63mg/mL,1.25mg/mL,2.5mg/mL,5mg/mL and the like. Osteopontin has antiviral effect (30-80%) in 5 concentration ranges of 0.31mg/mL,0.63mg/mL,1.25mg/mL, 2.5mg/mL,5mg/mL, etc. The antiviral effect of the composition of lactoferrin and osteopontin (5 mg/mL of lactoferrin and 2.5mg/mL of osteopontin) in three concentration ranges of 7.5 mg/mL,3.75mg/mL and 1.875mg/mL is more than 50%.
Example 3
Determination of infectious particle production after intervention with an antiviral active ingredient, the results are expressed as TCID50 (PFU/mL) values.
The method comprises the following steps: vero E6 cells were seeded in 96-well cell culture plates in advance for use. Collecting cell culture supernatant (after culturing at 37 deg.C for about 48 hr, directly collecting liquid in culture well), and performing 10-fold gradient dilution with dilution factor of 10 -1 ～10 -7 The volume of each gradient dilution was 1000. Mu.L. Culture medium from 96 well cell culture plates was discarded, 100 μ L virus dilutions were added to each well, 10 wells were inoculated per dilution gradient for a total of 7 dilution gradients, and the remaining cell wells were supplemented with virus-free culture medium as negative control. After 72 hours of incubation, the appearance of cytopathic effects was observed well by well under an optical microscope. Kong Jiwei positive wells with lesions, whereas negative wells. TCID50 (PFU/mL) values were calculated based on the number of positive and negative wells in each dilution gradient. This value represents the amount of virus required to cause half of the cytopathic effect or death (CPE) in a plate well or in a tube, and thus characterizes the titer of infectious viral particles.
As a result: TCID50 refers to the amount of virus required to infect 50% of the cells in PFU/mL. According to the preliminary screening results, the supernatants of infected cells were tested for their content of infectious virus particles after treatment with 5mg/mL of whey protein, milk fat globule membrane, lactoferrin and osteopontin. As can be seen from fig. 4, among these four active ingredients, lactoferrin showed the best inhibitory effect against virus production, and treatment with 5mg/mL of lactoferrin reduced the TCID50 value of the virus in the cell supernatant by 1.5 Log10, i.e. infectious viral particles were 4.48% of the untreated group, with an inhibition of 95.42%. As can be seen from fig. 4, the treatment of whey protein and milk fat globule membrane at the same concentration reduced the Log10 values by 0.89 and 0.88, respectively, and the inhibition rates were 87.57% and 87.86%, respectively, which are statistically significant.
Example 4
Protein immunoblotting test verification of antiviral active ingredient
The method comprises the following steps: after virus infection, the supernatant was discarded, and 100. Mu.L of RIPA lysate was added thereto and lysed for 10 minutes. Protein quantification was performed using the BCA kit according to the instructions. After all samples were diluted to the same protein content, 20. Mu.L was mixed with 4. Mu.L of loading buffer. The mixture was treated at 100 ℃ for 10 minutes to denature the protein. Then centrifuged at 12000rpm for 2 minutes at 4 ℃. Samples and protein markers were loaded in 12% SDS-PAGE gels, and electrophoretic separation was carried out under the following conditions: 80V, 120V after 30 min, 60 min. Transferring the proteins on the gel to a polyvinylidene fluoride (PEDV) membrane by using a transfer apparatus under the following conditions: 15V,60 minutes. PEDV membranes loaded with protein were blocked with 5% skim milk powder (TBST dissolved) for 2 hours at room temperature. Since GX _ P2V has an amino acid homology of more than 92.5% with SARS-CoV-2 (NCBI, protein BLAST), GX _ P2V nucleocapsid Protein was detected using an anti-SARS-CoV-2 nucleocapsid Protein antibody. Considering that the N protein amino acid sequences of GX _ P2V and SARS-CoV2 have homology of more than 93% with the purchasability of SARS-CoV-2N protein antibody, the nucleic capsid protein antibody against SARS-CoV-2N protein from Genscript, USA and the GAPDH antibody from Proteitech, USA were diluted to 1. The primary antibody was removed by washing 3 times with TBST, and a secondary goat anti-mouse IgG (H + L) enzyme-labeled secondary antibody diluted 1. Incubate with luminophore for 5 min and wash 3 times with TBST. The PEDV film was then detected for luminescence using SuperSignal West Femto Maximum Sensitivity chemistry Substrate (Thermo Scientific, USA).
As a result: as shown in FIG. 5, in the group of lactoferrin preparations in the range of 0.6mg/mL to 10mg/mL, the production of viral N protein in infected cells was dose-dependently inhibited. Obviously, at a lactoferrin concentration of 10mg/mL, the expression of the viral N protein was hardly detectable in the cells; at a lactoferrin concentration of 5mg/mL, only a trace amount of viral N protein expression was detected in the cells. As can be seen from FIG. 5b, the milk fat globule membrane significantly inhibited the production of viral N protein in cells at a concentration of 10mg/mL, with minimal expression of viral N protein detected. In fig. 5, NP refers to GX _ P2V nucleocapsid protein; GAPDH refers to cellular glyceraldehyde phosphate dehydrogenase.
Example 5
Investigation of the primary antiviral mechanism of the active protein substance: action after entry into cells
The method comprises the following steps: the medicine adding time method is adopted to preliminarily determine the stage of the virus life cycle in which the antiviral active component plays the inhibiting effect. Vero E6 cells were seeded in 48-well plates (1X 10) 5 Cells/well) to adhere and grow. GX _ P2V with an MOI of 0.01 and an antiviral component at an appropriate concentration were mixed uniformly, added to the cells, and incubated at 37 ℃ for 2 hours to allow the virus to adsorb and enter the cells. The virus-containing culture medium was then washed away and the cells were washed 2 times with PBS. Cells were supplemented with fresh medium and cultured until 48 hours post infection. To investigate whether the active ingredient functions after the virus entered the cells, GX _ P2V infected the cells for 2 hours at an MOI of 0.01. The virus-containing culture medium was removed, the cells were washed 2 times with PBS, and then the cells were added with the same final concentration of the active ingredient and cultured for another 48 hours. Cells were collected for subsequent nucleic acid extraction and RT-qPCR analysis, and intracellular viral mRNA expression was determined to characterize viral load.
As a result: as shown in FIGS. 6-7, the relative expression level of intracellular viral mRNA was reduced to varying degrees after incubation of the cells with the virus for 46 hours after 2 hours of GX _ P2V infection with the addition of whey protein, milk fat globule membrane, lactoferrin, and osteopontin (5 mg/mL). Whey protein, milk fat globule membrane, lactoferrin and osteopontin are shown to inhibit the post-entry processes of viral infection. The lactoferrin has the most obvious antiviral effect, and can obtain an inhibition rate of more than 95% when being added 2 hours after virus infection within the range of 0.625-2.5 mg/mL. For the group (5 mg/mL) in which whey protein and milk fat globule membrane were added only at the stage of virus-infected cells, the relative expression level of viral mRNA in cells was also reduced, indicating that whey protein and milk fat globule membrane could inhibit the virus entry process.
Example 6
Investigation of the preliminary antiviral mechanism of the active protein substance: inhibiting the adhesion of virus to cells
The method comprises the following steps: adsorption experiments were used to investigate whether antiviral active ingredients could have inhibitory effects on GX _ P2V attachment to cells. Vero E6 cells were seeded in 48-well plates (1X 10) 5 Cells/well) overnight. And (3) incubating GX _ P2V with high concentration and active ingredients of whey protein, milk fat globule membrane, lactoferrin and osteopontin with certain concentration for 2 hours at 4 ℃ to ensure that the active ingredients and viruses fully act. Then, the mixture of the virus and the active ingredient was added to the cells, and incubated at 4 ℃ for 2 hours to allow the virus to sufficiently act on the cells. The mixture in the culture wells was discarded, after 3 adherent cells were washed with PBS, the cells were collected for subsequent nucleic acid extraction and RT-qPCR analysis, and the expression of viral mRNA on the cell surface was determined to characterize the viral load.
As a result: as shown in fig. 8, the intervention virus cell adsorption experiment was performed on the four active substances, whey protein, milk fat globule membrane, lactoferrin and osteopontin, respectively, and the results were repeated twice to ensure the credibility thereof. The results show that the four components can significantly reduce the attachment of GX _ P2V to cells at concentrations of 10mg/mL and 5mg/mL. As shown in fig. 8, the inhibition rates of virus-attached cells under 10mg/mL intervention were 75.55%,76.24% and 94.33%,75.49%, respectively. The virus attachment test shows that the whey protein, the milk fat globule membrane, the lactoferrin and the osteopontin can obviously reduce the attachment of GX _ P2V to cells and inhibit virus infection.
Example 7
Computer simulation of lactoferrin binding to SARS-CoV-2 spike protein and cellular ACE2 receptor
The method comprises the following steps: lactoferrin (PDB: 1 LFG) and SARS-CoV-2 wild type RBD and human cell ACE2 (corresponding structure extracted from PDB 7DF4 complex) under two conformations are subjected to molecular docking on protein-protein level, water molecules in the structure are removed before docking, and ZDOCK 3.0.2 (https:// ZDOCK. Umassmed. Edu /) is adopted as a docking platform. Selecting the docking compound with the highest docking score for analysis, and selecting PDBePISA (https:// www.ebi.ac.uk/msd-srv/prot _ int/prestart. Html) as the platform to obtain the interface area (A) of the docking compound 2 ) The docking free energy (Δ G) and the P value thereof, and the number of hydrogen bonds formed at the docking interfaceAnd the number of salt bridges. The docking complex was visualized with PyMol.
As a result: RBD docking in both lactoferrin and SARS-CoV-2 conformations is shown in FIGS. 9A and 9B, with FIG. 9A showing the lactoferrin and SARS-CoV-2 receptor binding domains (one RBD up open conformation); FIG. 9B shows lactoferrin and SARS-CoV-2 receptor binding domain (three RBD down open conformations). RBD (a RBD-up) refers to a conformation in which there is an upward projection of the RBD in the spike protein, in which conformation the spike protein binds to ACE2 on the cell surface. RBD (three RBD-down) refers to a conformation in which all three RBDs are tightly bound without upward protrusions in the spike protein. According to the docking results analysis of table 1, lactoferrin can spontaneously bind to RBD in both conformations, as shown by a free energy of less than 0kcal/mol. The bonding interface forms hydrogen bonds and salt bridges. The analysis results showed that lactoferrin cannot spontaneously bind to ACE2, with a binding free energy of 0.5kcal/mol. The above results suggest that lactoferrin prevents the binding of RBD to ACE2 by binding to RBD on the spike protein of SARS-CoV-2.
TABLE 1 documentary table of docking results
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. Use of a proteinaceous material in the preparation of a composition for inhibiting a coronavirus, wherein inhibiting a coronavirus comprises: inhibiting adhesion of coronavirus to a host cell; blocking coronavirus invasion of host cells; inhibit the postcellular process of coronavirus infection.
2. Use of proteinaceous material according to claim 1 for the manufacture of a composition for inhibiting coronaviruses, wherein said proteinaceous material comprises one or any combination of whey protein, milk fat globule membrane, lactoferrin and osteopontin.
3. Use of the proteinaceous material of claim 1 for the preparation of a composition for inhibiting coronavirus, wherein the composition is one or more of a formula, a protein powder, a nutritional supplement, and a pharmaceutical.
4. Use of a proteinaceous material according to claim 1 for the preparation of a composition for inhibiting coronavirus, wherein the composition is in the form of a solution, a suspension, an emulsion or a solid.
5. Use of a proteinaceous substance according to claim 4 for the preparation of a composition for inhibiting coronavirus, wherein said composition is in the form of a solution and the concentration of said proteinaceous substance in said composition is 0.02-10 mg/mL; preferably 0.03 to 5mg/mL; more preferably 5mg/mL,2.5mg/mL, 1.25mg/mL, 0.63mg/mL, 0.31mg/mL, 0.15mg/mL, 0.078mg/mL or 0.039mg/mL.
6. Use of a proteinaceous substance according to claim 1 for the preparation of a composition for inhibiting a coronavirus, wherein said coronavirus comprises GX _ P2V, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
7. Use of a proteinaceous material according to claim 1 for the preparation of a composition for inhibiting coronavirus, wherein the composition further comprises an oligosaccharide.
8. Use of a proteinaceous material as claimed in claim 1 for the manufacture of a composition for inhibiting coronavirus, wherein the proteinaceous material is derived from one or a combination of bovine, equine, ovine and human sources.
9. Use of a proteinaceous substance according to any one of claims 1 to 8 for the preparation of a composition for inhibiting coronavirus, wherein said composition provides lactoferrin in an amount of 3mg to 375mg per kg body weight per day;
alternatively, the composition provides milk fat globule membrane between 3mg and 375mg/kg body weight per day;
alternatively, the composition provides 12mg to 375mg/kg body weight/day of osteopontin;
alternatively, the composition provides from 6mg to 375mg/kg body weight/day of whey protein;
still alternatively, the composition provides one or any combination of lactoferrin 3mg to 375mg/kg body weight per day, milk fat globule membrane 3mg to 375mg/kg body weight per day, osteopontin 12mg to 375mg/kg body weight per day, and whey protein 6mg to 375mg/kg body weight per day.
10. A composition comprising proteinaceous matter, wherein the composition comprises one or any combination of whey protein, milk fat globule membrane, lactoferrin, and osteopontin, for use in inhibiting coronaviruses; preferably the composition is for use in inhibiting neotype coronavirus GX _ P2V.
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| CN116602409A (en) * | 2023-02-20 | 2023-08-18 | 合生元（广州）健康产品有限公司 | A composition for inhibiting rotavirus infection |
| CN116602409B (en) * | 2023-02-20 | 2024-04-05 | 合生元（广州）健康产品有限公司 | A composition for inhibiting rotavirus infection |
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