CN113827707B - Application of a-lactalbumin in inhibiting coronavirus - Google Patents

Abstract

The invention discloses the application of α-lactalbumin. The application is the application of a-lactalbumin in any of the following: (A1) Preparation of products that inhibit coronavirus, or inhibition of coronavirus; (A2) Preparation of products for the treatment and/or prevention of diseases caused by coronavirus infection, or Treat and/or prevent diseases caused by coronavirus infection; (A3) Prepare products that improve symptoms caused by coronavirus infection, or improve symptoms caused by coronavirus infection; (A4) Prepare products that treat and/or prevent diseases caused by coronavirus infection Products for diseases caused by the replication of coronavirus, or for the treatment and/or prevention of diseases caused by the replication of said coronavirus. The examples of the present invention show that a-lactalbumin can inhibit the adhesion and entry of coronavirus, while also inhibiting the replication of coronavirus after infection.

Description

Application of a-lactalbumin in inhibiting coronavirus

Technical field

The invention belongs to the field of medicine, and specifically relates to the application of α-lactalbumin in inhibiting coronavirus.

Background technique

The 2019 novel coronavirus (2019-nCoV or SARS-CoV-2, which causes COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV are currently Coronaviruses known to infect humans. Among them, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are less pathogenic and generally cause respiratory symptoms, similar to a common cold or pneumonia. The other three coronaviruses are very pathogenic and contagious. The diseases they cause include severe acute respiratory syndrome (SARS, 2002-2004), Middle East respiratory syndrome (MERS, 2012-present) and the new coronavirus. Viral disease (COVID-19). Therefore, how to block the spread of coronavirus, prevent infection and develop new effective drugs are technical issues that need to be solved urgently.

Contents of the invention

One of the objects of the present invention is to provide the application of a-lactalbumin.

The present invention provides the use of α-lactalbumin in any of the following:

(A1) Prepare products that inhibit coronavirus, or inhibit coronavirus;

(A2) Preparation of products for the treatment and/or prevention of diseases caused by coronavirus infection, or the treatment and/or prevention of diseases caused by coronavirus infection;

(A3) Prepare products that improve symptoms caused by coronavirus infection, or improve symptoms caused by coronavirus infection;

(A4) Prepare a product for treating and/or preventing diseases caused by the replication of the coronavirus, or treating and/or preventing diseases caused by the replication of the coronavirus.

Here, a-lactalbumin can be of human origin, animal or plant origin, or can be a recombinant protein, such as the product of Sigma, product number L7269.

Optionally, according to the above application, the coronavirus is selected from at least one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.

Optionally, according to the above application, the disease caused by coronavirus infection is selected from pneumonia, respiratory tract infection, common cold, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and 2019 new coronavirus pneumonia. (COVID-19).

Optionally, according to the above application, the respiratory tract infection is selected from at least one of upper respiratory tract infection, lower respiratory tract infection, tracheitis and acute and chronic stages of bronchitis.

Optionally, according to the above application, the disease caused by the replication of the coronavirus is a disease caused by the coronavirus adsorbing cells, a disease caused by the coronavirus entering a cell, and/or a disease caused by the coronavirus Replication in cells leads to disease.

Optionally, according to the above application, the product is a pharmaceutical.

Optionally, according to the above application, the medicine is a liquid preparation or a solid preparation.

The invention provides a product whose active ingredients include α-lactalbumin; the product has any of the following uses:

(a1) Inhibit coronavirus;

(a2) Treat and/or prevent diseases caused by coronavirus infection;

(a3) Improve symptoms caused by coronavirus infection;

(a4) Treat and/or prevent diseases caused by the replication of the coronavirus.

Optionally, according to the above product, the product is a medicine. The medicine can be formulated into various suitable pharmaceutical preparation forms, for example, it can be in the form of a liquid preparation or a solid preparation. For example, α-lactalbumin can be used alone, or mixed with pharmaceutical excipients (such as excipients, diluents, etc.) to formulate tablets, capsules, granules, syrups, etc. for oral administration or for injection. Powder injection, solution, etc. of medicine.

The embodiments of the present invention show that a-lactalbumin can inhibit the adhesion and entry of coronavirus, and can also inhibit the replication of coronavirus after infection. Taking a-lactalbumin as an example, it was tested with SARS-CoV-2 Luc pseudovirus, and its EC _{50 at the cellular level was 0.1635 mg/ml. At the same time, it was tested with the SARS-CoV-2 infection replication system (trVLP), and its EC 50} at the cellular level was Level _EC50 =0.6809 mg/ml. SARS-CoV-2 enters cells by recognizing the receptor angiotensin-converting enzyme ACE2 and other co-receptors through the spike protein spike. Therefore, spike is a key factor in the virus replication process. As the concentration of α-lactalbumin increases, spike The infection-mediated entry efficiency is reduced, and its relative expression is reduced. Therefore, a-lactalbumin that targets spike and key factors for viral replication can be applied in the treatment of COVID-19.

Description of drawings

Figure 1 shows the experimental results of a-lactalbumin inhibiting the adsorption, entry and post-infection replication of SARS-CoV-2 pseudovirus and trVLP replicon.

Figure 2 shows the inhibition rate of a-lactalbumin in inhibiting SARS-CoV-2 pseudovirus infection.

Figure 3 shows the inhibition rate of a-lactalbumin against SARS-CoV-2 replicon trVLP.

Figure 4 shows the variation of the inhibition rate of a-lactalbumin in inhibiting SARS-CoV-2 pseudovirus and trVLP replicon infection with dose.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

GraphPad 8 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation and tested using asymmetric T test.

The detection principle of the following embodiments:

The new coronavirus replicon model replaces part of the genes of the new coronavirus genome with a green fluorescent protein gene, and stably expresses the replaced viral genes in specific cell lines to experiment with virus replication.

The SARS-CoV-2 replicon virus model (trVLP) was used to avoid the risks and tediousness of P3 laboratory experiments. trVLP can be cultured in ordinary P2 laboratories and can be used as an ideal alternative model against SARS-CoV-2 virus. A large number of basic experiments can be explored on this virus model and then verified with SARS-CoV-2 virus.

Experimental materials and instruments:

Cells: Vero E6, purchased from ATCC.

Huh7.5, a gift from Professor Charles M Rice of Rockefeller University, documented in Luna JM, Scheel TK, Danino T, et al.Hepatitis C virus RNA functionally sequesters miR-122.Cell.2015;160(6):1099-1110.doi :10.1016/j.cell.2015.02.025.

Caco-2-N, a gift from Professor Ding Qiang of Tsinghua University, recorded in Ju X, Zhu Y, Wang Y, et al. A novelcell culture system modeling the SARS-CoV-2 life cycle. PLoS Pathog. 2021; 17(3) :e1009439.doi:10.1371/journal.ppat.1009439.

The above cells were cultured in high-glucose DMEM + 10% fetal calf serum in a 37°C, 5% _CO2 incubator.

Virus: SARS-CoV-2 Luc pseudovirus, a kind gift from researcher Wang Youchun of the China Institute for Food and Drug Control. Documented in Nie J, Li Q, Wu J, et al. Quantification of SARS-CoV-2 neutralizing antibody by apseudotyped virus-based assay. Nat Protoc. 2020; 15(11): 3699-3715. doi: 10.1038/s41596- 020-0394-5.

SARS-CoV-2 GFP pseudovirus was a kind gift from Professor Xia Ningshao of Xiamen University. Described in Xiong HL, Wu YT, Cao JL, et al. Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressingBHK21 cells. Emerg Microbes Infect. 2020; 9( 1):2105-2113.doi:10.1080/22221751.2020.1815589.

trVLP SARS-CoV-2 replicon (hereinafter referred to as trVLP replicon) was donated by Professor Ding Qiang of Tsinghua University and was recorded in Ju X, Zhu Y, Wang Y, et al. A novel cell culture system modeling the SARS-CoV- 2 life cycle.PLoS Pathog.2021;17(3):e1009439.doi:10.1371/journal.ppat.1009439.

Sample: a-lactalbumin, purchased from Sigma, product number: L7269, storage condition -20°C. The alpha-lactalbumin is isolated from human milk.

Detection reagents: as shown in Table 1.

Table 1 Reagent sources

The detection method is detailed as follows.

qRT-PCR (real-time RT-PCR): Follow the manufacturer's instructions and use the RNAprep pure cultured cell/bacterial total RNA extraction kit for RNA extraction. Reverse transcription uses RevertAid First Strand cDNASynthesis Kit. The qPCR amplification system is as follows. Primers:

bjmu-00737-SARS-CoV-2 RNA-F: CGAAAGGTAAGATGGAGAGCC,

bjmu-00738-SARS-CoV-2 RNA-R: TGTTGACGTGCCTCTGATAAG,

bjmu-00061-RPS11-F:GCCGAGACTATCTGCACTAC,

bjmu-00062-RPS11-R:ATGTCCAGCCTCAGAACTTC,

1μl each, 10μM;

POWRUP SYBR MASTER MIX (2X) 5μl (Manufacturer: Thermo Scientific Cat. No.: A25742);

cDNA 3μl;

A total of 10μl amplification system.

Amplification procedure for qPCR Table 2.

Table 2 qPCR amplification procedure

stage repeat temperature Time(min) 1 1 50℃ 2:00 95℃ 0:20 2 40 95℃ 0:03 60℃ 0:35 3(melt) 1 95℃ 0:15 60℃ 0:20 95℃ 0:15 60℃ 0:15

qRT-PCR was performed using QuantStudio 1 Real-Time PCR detection system (Appliedbiosystems, Foster City, CA, USA).

Detection of luciferase expression level: Use Luciferase Cell Culture Lysis Reagent, 5X (diluted to 1X with ddH ₂ O) lysate to lyse cells to obtain cell lysate. Follow the instructions of the Luciferase AssaySystem reagent manufacturer, 20 μl cell lysate Mix with 100 μl of the reaction solution, then add it to the corresponding 96-well chemiluminescence detection plate, and place it in a chemiluminescence detector to read the luminescence value.

Example 1, α-lactalbumin inhibits viral adhesion, entry and replication experiments

In order to study the inhibitory effect of a-lactalbumin on various infection stages of cells infected by SARS-CoV-2 pseudovirus and trVLP replicon, experiments with different treatments were conducted. Vero E6, Huh7.5 and Caco-2-N cells were inoculated into 24-well plates or 96-well plates, and a-lactalbumin was added at different stages of viral infection to explore the effect of a-lactalbumin on the full cycle of viral infection and cell entry. and inhibition of replication after entry into the cell.

1) The experimental method is as follows:

Caco-2-N cells were seeded into 24-well plates, with an inoculation volume of 100,000; Huh7.5 and Vero E6 cells were seeded into 96-well plates, with an inoculation volume of 20,000.

①Adsorption experiment:

Caco-2-N experimental group: The drug α-lactalbumin and viral trVLP replicon were premixed at 4°C for 1 hour to obtain a drug-virus mixture. At the same time, Caco-2-N cells were pre-cooled for 1 hour, and then the drug-virus mixture was added to the cells. , the addition amount is 500 μl per well, in which the trVLP replicon is 0.05MOI, and the concentration of a-lactalbumin is 1 mg/ml after addition. Treat at 4°C for 2 hours, and wash with PBS three times.

Caco-2-N negative control group: Caco-2-N cells were pre-cooled for 1 hour in advance, and then the trVLP replicon was added to the cells in an amount of 0.05 MOI, 500 μl per well, treated at 4°C for 2 hours, and washed three times with PBS.

Caco-2-N positive control group: 1 mg/ml whey protein mixture A17 (described in Fan H, Hong B, LuoY, et al. The effect of whey protein on viral infection and replication of SARS-CoV-2 and pangolin coronavirus in vitro.Signal Transduct TargetTher.2020;5(1):275.Published 2020 Nov 24.doi:10.1038/s41392-020-00408-z) and virus 0.05MOI trVLP replicon were premixed at 4°C for 1h to obtain a mixture, and at the same time Caco-2-N cells were pre-cooled for 1 hour in advance, and then the mixture was added to the cells in an amount of 500 μl per well, in which the trVLP replicon was 0.05 MOI, and the whey protein mixture A17 was added at a concentration of 1 mg/ml, and treated at 4°C for 2 hours. , washed 3 times with PBS.

The Caco-2-N experimental group, Caco-2-N negative control group and Caco-2-N positive control group were used to observe GFP expression under a fluorescence microscope, and use Luciferase Cell Culture Lysis Reagent, 5X (diluted to 1X with ddH ₂ O). The cells (which contain viruses adhering to the cell surface) are collected from the lysate, and RNA is subsequently extracted and qRT-PCR is performed to detect SARS-CoV-2-RNA.

Vero E6 or Huh7.5 experimental group: The drug α-lactalbumin and virus SARS-CoV-2 Luc pseudovirus were premixed at 4°C for 1 hour to obtain the drug virus mixture. At the same time, Vero E6 or Huh7.5 cells were pre-cooled for 1 hour in advance, and then Add the drug-virus mixture to the cells in an amount of 100 μl per well, including SARS-CoV-2 Luc pseudovirus 650 TCID ₅₀ and a-lactalbumin at a concentration of 1 mg/ml. Treat at 4°C for 2 hours and wash 3 times with PBS. Replace with new 10% FBS high-glucose DMEM medium and place it in the incubator for 24 hours.

Vero E6 or Huh7.5 negative control group: Vero E6 or Huh7.5 cells were pre-cooled for 1 hour in advance, and then 650 TCID ₅₀ /well SARS-CoV-2 Luc pseudovirus was added to the cells, the amount added was 100 μl per well, 4 °C for 2 hours, washed three times with PBS, replaced with new 10% FBS high-glucose DMEM medium, and placed in an incubator for 24 hours.

Vero E6 or Huh7.5 positive control group: Whey protein mixture A17 and virus SARS-CoV-2 Luc pseudovirus were premixed at 4°C for 1 hour to obtain the mixture. At the same time, Vero E6 or Huh7.5 cells were pre-cooled for 1 hour in advance, and then the mixture was Add to the cells in an amount of 100 μl per well, including SARS-CoV-2 Luc pseudovirus 650TCID ₅₀ and whey protein mixture A17 at a concentration of 1 mg/ml. Treat at 4°C for 2 hours, wash 3 times with PBS, and replace with new ones. 10% FBS high-glucose DMEM culture medium, placed in an incubator and cultured for 24 hours.

The Vero E6 or Huh7.5 experimental group, Vero E6 or Huh7.5 negative control group, and Vero E6 or Huh7.5 positive control group were detected using the Luciferase Assay System.

Repeat each group 3 times.

② Cell entry experiment:

Caco-2-N experimental group: Add trVLP replicon to Caco-2-N cells at an amount of 0.05 MOI per well, pretreat at 4°C for 2 hours to achieve the same degree of adsorption, wash with PBS 3 times, and add the drug a- Add lactalbumin to the culture medium in which the cells are cultured and mix. The concentration of recombinant protein 1 in the culture medium is 1 mg/ml, and then react at 37°C for 1 hour (the drug blocks the entry into the cells), and wash 3 times with PBS. Then, the cells were cultured in high-glucose DMEM medium containing 10% FBS without viruses and drugs.

Caco-2-N negative control group: Add trVLP replicon to Caco-2-N cells at an amount of 0.05MOI per well, pretreat at 4°C for 2 hours until the adsorption level is consistent, wash with PBS 3 times, and then replace Culture in virus-free high-glucose DMEM medium with 10% FBS.

Caco-2-N positive control group: Add trVLP replicon to Caco-2-N cells at an amount of 0.05MOI per well, pretreat at 4°C for 2 hours until the adsorption level is consistent, wash with PBS 3 times, and emulse the drug Albumin mixture A17 was added to the culture medium in which the cells were cultured and mixed. The concentration of whey protein mixture A17 in the culture medium was 1 mg/ml, and then reacted at 37°C for 1 hour (the drug blocks the entry into the cells), PBS Wash 3 times, and then replace with virus-free and drug-free 10% FBS high-glucose DMEM medium for culture.

The Caco-2-N experimental group, Caco-2-N negative control group and Caco-2-N positive control group were cultured for 48 hours, and the cells were collected using Luciferase Cell Culture Lysis Reagent, 5X (diluted to 1X with ddH ₂ O for use). (containing viruses adhering to the cell surface), RNA was subsequently extracted, qRT-PCR was detected, and the expression of GFP was observed under a fluorescence microscope.

Vero E6 or Huh7.5 experimental group: Add SARS-CoV-2 Luc pseudovirus to Vero E6 or Huh7.5 cells, 650 TCID ₅₀ /well, pretreat at 4°C for 2 hours to achieve the same degree of adsorption, and wash with PBS 3 times , add the drug 1 mg/ml a-lactalbumin to the culture medium of the cells and mix, react at 37°C for 1 hour (the drug blocks the entry into the cells), wash 3 times with PBS, and then replace with virus-free and drug-free Culture in high-glucose DMEM medium with 10% FBS.

Vero E6 or Huh7.5 negative control group: Add SARS-CoV-2 Luc pseudovirus to Vero E6 or Huh7.5 cells, 650 TCID ₅₀ /well, pretreat at 4°C for 2 hours, until the adsorption level is consistent, and wash with PBS for 3 and then replaced with virus-free 10% FBS high-glucose DMEM medium for culture.

Vero E6 or Huh7.5 positive control group: Add SARS-CoV-2 Luc pseudovirus to Vero E6 or Huh7.5 cells, 650 TCID ₅₀ /well, pretreat at 4°C for 2 hours to achieve consistent adsorption, wash with PBS for 3 Once, add 1 mg/ml whey protein mixture A17 to the medium in which the cells were cultured and mix, react at 37°C for 1 hour (drugs are blocked during the cell entry stage), wash 3 times with PBS, and then replace with virus-free Drugs were cultured in high-glucose DMEM medium with 10% FBS.

When the Vero E6 or Huh7.5 experimental group, Vero E6 or Huh7.5 negative control group and Vero E6 or Huh7.5 positive control group were cultured for 24 hours, Luciferase Assay System was used for detection.

Repeat each group 3 times.

③Experiment after entering:

Caco-2-N experimental group: Add the viral trVLP replicon to Caco-2-N cells, 0.05MOI per well, react at 37°C for 1 hour, the virus fully enters the cells, wash 3 times with PBS, and then add the drug a-milk The protein was added to the culture medium in which the cells were cultured and mixed. The concentration of a-lactalbumin in the culture medium was 1 mg/ml. The cells were placed in an incubator and cultured at 37°C.

Caco-2-N negative control group: Add the viral trVLP replicon to Caco-2-N cells, 0.05MOI per well, react at 37°C for 1 hour, the virus fully enters the cells, wash 3 times with PBS, and place in an incubator. Culture at 37°C.

Caco-2-N positive control group: Add viral trVLP replicon to Caco-2-N cells, 0.05MOI per well, react at 37°C for 1 hour, the virus fully enters the cells, wash 3 times with PBS, and then add the drug whey Protein mixture A17 was added to the culture medium in which the cells were cultured and mixed. The concentration of whey protein mixture A17 in the culture medium was 1 mg/ml. The cells were placed in an incubator and cultured at 37°C.

The Caco-2-N experimental group, Caco-2-N negative control group and Caco-2-N positive control group were cultured at three time periods of 48h, 72h and 96h with Luciferase Cell Culture Lysis Reagent, 5X (diluted with ddH ₂ O (Use 1X) lysate to collect cells, then extract RNA and perform qRT-PCR detection. At the same time, DAPI is used to stain the nucleus to collect the trVLP replicon GFP expression results under a fluorescence microscope.

Vero E6 or Huh7.5 experimental group: Add virus SARS-CoV-2 Luc pseudovirus to Vero E6 or Huh7.5 cells, 650 TCID ₅₀ /well, react at 37°C for 1 hour, the virus fully enters the cells, and wash with PBS for 3 Then add the drug a-lactalbumin to the culture medium in which the cells are cultured and mix. The concentration of a-lactalbumin in the culture medium is 1 mg/ml, and then put into an incubator and culture at 37°C.

Vero E6 or Huh7.5 negative control group: Add virus SARS-CoV-2 Luc pseudovirus to Vero E6 or Huh7.5 cells, 650 TCID ₅₀ /well, react at 37°C for 1 hour, the virus fully enters the cells, and wash with PBS 3 times, then placed in the incubator and cultured at 37°C.

Vero E6 or Huh7.5 positive control group: Add virus SARS-CoV-2 Luc pseudovirus to Vero E6 or Huh7.5 cells, 650 TCID ₅₀ /well, react at 37°C for 1 hour, the virus fully enters the cells, and wash with PBS 3 times, then add the drug whey protein mixture A17 to the culture medium for culturing the cells and mix. The concentration of the whey protein mixture A17 in the culture medium is 1 mg/ml, and then put into an incubator and culture at 37°C.

The values of Vero E6 or Huh7.5 experimental group, Vero E6 or Huh7.5 negative control group and Vero E6 or Huh7.5 positive control group were detected using Luciferase Assay System after 24 hours of culture.

Repeat each group 3 times.

The above experimental data processing method is as follows.

SARS-CoV-2 RNA relative expression calculation method:

Caco-2-N experimental group, Caco-2-N negative control group and Caco-2-N positive control group: qRT-PCR was used to detect SARS-CoV-2 and RPS11 (internal reference) RNA expression, and the negative control group was The test results are normalized to 1.

Inhibition rate calculation method:

Vero E6 or Huh7.5 experimental group, Vero E6 or Huh7.5 negative control group and Vero E6 or Huh7.5 positive control group: use the Luciferase Assay System to detect the values, Vero E6 or Huh7.5 experimental group or Vero E6 or Huh7 .5 Divide the value of the positive control group by the value of the Vero E6 or Huh7.5 negative control group and multiply by 100% to obtain the infection rate of the corresponding treatment group. The inhibition rate (%) of the corresponding treatment group = 100% - infection rate (%).

2) Result analysis

The results are shown in Figure 1.

Figure 1A and Figure 1B show the experimental results of the Caco-2-N experimental group, Caco-2-N negative control group and Caco-2-N positive control group, in which a-lactalbumin (1mg/ml) is Caco-2 -N experimental group results, the negative control is the Caco-2-N negative control group results, and the positive control is the Caco-2-N positive control group results. In the adsorption experiment, the specific data are shown in Table 3. The average relative expression amount of RNA in the Caco-2-N positive control group was 0.07; the average relative expression amount of RNA in the Caco-2-N experimental group was 0.05, and the average relative expression amount of RNA in the Caco-2-N experimental group was 0.05. The average relative expression of RNA in the N negative control group was 0.98. In the cell entry experiment, the specific data are shown in Table 4. The average relative expression amount of RNA in the Caco-2-N positive control group was 0.004; the average relative expression amount of RNA in the Caco-2-N experimental group was 0.36. The average relative expression of RNA in the -N negative control group was 0.96. In the post-entry experiment, the specific data are shown in Table 5. The average RNA relative expression in the three time periods of 48h, 72h and 96h in the Caco-2-N positive control group was 0.001, 0.002, 0.0001; Caco-2-N The average RNA relative expression levels in the three time periods of 48h, 72h and 96h in the experimental group were 0.06, 2.68, and 19.23. The average RNA relative expression levels in the Caco-2-N negative control group were 48h, 72h and 96h. They are 0.99, 5.36, and 21.62 respectively.

In the adsorption experiment and cell entry experiment, compared with the negative control group, the relative expression of viral SARS-CoV-2 RNA in the experimental group was significantly reduced. In the post-entry experiment, in the three time periods of 48h, 72h and 96h, the experimental The expression of GFP in each group was less than that of the negative control group, but GFP also gradually increased, indicating that the inhibitory effect after entry was significant but not significant. Especially at 96h, there was no significant difference in the expression of viral RNA. This shows that a-lactalbumin can inhibit the adsorption and entry of trVLP SARS-CoV-2 replicon live virus-infected cells, but the inhibitory effect is not good after entry.

Table 3 Relative expression of RNA in adsorption experiments

repeat Caco-2-N positive control group Caco-2-N experimental group Caco-2-N negative control group 1 0.05 0.12 0.84 2 0.08 0.08 1.17 3 0.09 0.02 0.98

Table 4 Relative expression of RNA in cell entry experiment

repeat Caco-2-N positive control group Caco-2-N experimental group Caco-2-N negative control group 1 0.005 0.30 1.17 2 0.004 0.31 0.94 3 0.005 0.38 0.88

Table 5 Relative expression of experimental RNA after entering cells (Caco-2-N cell results)

Figure 1C shows the experimental results of the Vero E6 or Huh7.5 experimental group, Vero E6 or Huh7.5 negative control group and Vero E6 or Huh7.5 positive control group, in which a-lactalbumin (1 mg/ml) is Vero E6 or Huh7.5 The results of the Huh7.5 experimental group, the negative control (SARS-CoV-2 Luc pseudovirus) is the result of the Vero E6 or Huh7.5 negative control group, the positive control is the result of the Vero E6 or Huh7.5 positive control group, and the post-entry experiment is the entry Experimental results after 24 hours. The specific data of the adsorption experiment are shown in Table 6. The average inhibition rate of the Huh7.5 experimental group was 69.47%, the average inhibition rate of the Vero E6 experimental group was 69.79%, the average inhibition rate of the Huh7.5 negative control group was -3%, and the Vero E6 experimental group had an average inhibition rate of -3%. The average inhibition rate of the E6 negative control group was 0%, the average inhibition rate of the Huh7.5 positive control group was 96%, and the average inhibition rate of the Vero E6 positive control group was 99%. The specific data of the cell entry experiment are shown in Table 7. The average inhibition rate of the Huh7.5 experimental group was 69.52%, the average inhibition rate of the Vero E6 experimental group was 69.63%, the average inhibition rate of the Huh7.5 negative control group was 0%, and the Vero E6 experimental group had an average inhibition rate of 0%. The average inhibition rate of the E6 negative control group was 0%, the average inhibition rate of the Huh7.5 positive control group was 100%, and the average inhibition rate of the Vero E6 positive control group was 100%. The specific data of the experiment after entry are shown in Table 8. The average inhibition rate of the Huh7.5 experimental group was 70.9%, the average inhibition rate of the Vero E6 experimental group was 66.84%, the average inhibition rate of the Huh7.5 negative control group was 0%, and the Vero E6 experimental group had an average inhibition rate of 0%. The average inhibition rate of the E6 negative control group was 0%, the average inhibition rate of the Huh7.5 positive control group was 99%, and the average inhibition rate of the Vero E6 positive control group was 99%. It shows that a-lactalbumin can inhibit the adsorption and entry of SARS-CoV-2 pseudovirus-infected cells.

Table 6 Adsorption experiment inhibition rate (%)

Table 7 Inhibition rate of cell entry experiment (%)

Table 8 Experimental inhibition rate after entry (%)

Example 2, α-lactalbumin inhibits SARS-CoV-2 infection

1) a-lactalbumin inhibits the infection and replication of SARS-CoV-2 pseudovirus

Vero E6 cells were inoculated into a 96-well plate, and the amount of Vero E6 cells in each well was 20,000. The next day, the cells were infected with SARS-CoV-2 Luc pseudovirus, 650 TCID ₅₀ /well (preparation of pseudovirus and TCID ₅₀ determination) , see reference: Nie J, Li Q, Wu J, et al. Quantification of SARS-CoV-2 neutralizing antibody by apseudotyped virus-based assay. Nat Protoc. 2020; 15(11): 3699-3715.

doi: 10.1038/s41596-020-0394-5), and add different concentrations of a-lactalbumin (the concentration of a-lactalbumin in the system is 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ ml, 0.125mg/ml, 0.0625mg/ml), and the negative control was PBS. Incubate in a 37°C, 5% _CO2 incubator for 24 hours. The Luciferase Assay System was used to detect the expression of fluorescent protease in the cell lysate, and the inhibition rate was calculated. Inhibition rate calculation method: There is one group in the experiment that only adds an equal amount of pseudovirus, without a-lactalbumin but an equal amount of PBS. This group is the negative control group; each group uses the Luciferase Assay System to detect the value, and adds a-lactalbumin. Divide the value of the protein group by the value of the negative control group and multiply by 100% to obtain the infection rate of the corresponding group added with a-lactalbumin. The corresponding inhibition rate (%) of the group added with a-lactalbumin = 100% - infection rate (%) . Repeat each group 3 times.

Vero E6 and Huh7.5 cells were inoculated into 96-well plates respectively, and the number of cells in each well was 20,000. The next day, the cells were infected with SARS-CoV-2 GFP pseudovirus, and the amount added to each well was MOI=0.05, and Different concentrations of a-lactalbumin were added respectively (the concentrations of a-lactalbumin in the system were 2 mg/ml, 0.25 mg/ml, and 0ug/ml). Incubate in a 37°C, 5% _CO2 incubator for 48 hours. Observe the expression of GFP using a fluorescence microscope. Repeat each group 3 times.

2) α-lactalbumin inhibits the infection entry of trVLP

Caco-2-N cells were seeded into a 24-well plate, with 100,000 cells in each well. The next day, the cells were infected with trVLP virus at an MOI=0.05 per well, and different concentrations of a- Lactalbumin (the concentration of a-lactalbumin in the system is 2mg/ml, 0.2mg/ml, 0.02mg/ml). Incubate in a 37°C, 5% _CO2 incubator for 96 hours. Observe the expression of GFP using a fluorescence microscope. Repeat each group 3 times.

Caco-2-N cells were seeded into a 6-well plate, with 400,000 cells in each well. The next day, the cells were infected with trVLP virus at MOI=0.05, and different concentrations of α-lactalbumin (α-lactalbumin) were added. The concentration of protein in the system is 2mg/ml, 0.4mg/ml, 0.08mg/ml). Incubate in a 37°C, 5% _CO2 incubator for 96 hours. The negative control was a treatment group in which an equal amount of trVLP virus was added, a-lactalbumin was not added, and an equal amount of PBS was added.

Use western blot to detect the expression of Spike protein. The antibodies used are as follows:

Spike primary antibody: SARS-CoV-2/2019-nCoV Spike Antibody, Rabbit PAb, 40589-T62, Sino Biological;

Spike secondary antibody: Goat anti-Rabbit IgG(H+L)-HRP, BE0101-100, EASYBIO;

Actin primary antibody: Beta Actin Monoclonal antibody, 60008-1-Ig, Proteintech;

Actin secondary antibody: Goat anti-Mouse IgG(H+L)-HRP, BE0102-100, EASYBIO.

qRT-PCR was used to detect viral RNA expression.

Repeat each group 3 times.

3) Results

The results are shown in Figures 2 and 3.

A in Figure 2 is the statistical result of the inhibition rate of a-lactalbumin on SARS-CoV-2 Luc pseudovirus infection. The concentration of a-lactalbumin in the system is 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg /ml, 0.125mg/ml, and 0.0625mg/ml experimental groups, the average inhibition rates were 78.54%, 52.28%, 38.39%, 75.57%, 60.64% and 45.60% respectively. B in Figure 2 shows the GFP expression of SARS-CoV-2 GFP pseudovirus infected by a-lactalbumin. In the experimental group without adding a-lactalbumin, it can be seen that GFP is evenly and obviously expressed at 0.25 mg/ml a-lactalbumin. Under the action of 2mg/ml a-lactalbumin, there is almost no GFP expression, which significantly blocks virus infection, indicating that a-lactalbumin has a negative effect on SARS-CoV-2 Luc pseudovirus and SARS-CoV-2 GFP pseudovirus. The virus has a significant inhibitory effect.

A in Figure 3 shows the expression of GFP in step 2). Under the action of 2mg/ml a-lactalbumin, there is almost no GFP expression. The protein concentration drops to 0.2mg/ml, and the GFP expression increases significantly and is distributed in flakes; protein concentration When it dropped to 0.02 mg/ml, GFP expression further increased and was also distributed in sheets, indicating that cell fusion occurred. B in Figure 3 is step 2) relative expression statistics of viral nucleic acid RNA (relative expression = 2 ^{- ΔCt} (RNA expression in the experimental group) / 2 ^{- ΔCt} (RNA expression in the control group)). When the amount of a-lactalbumin reaches At 2mg/ml, the expression level of viral RNA is very low. As the concentration of a-lactalbumin decreases, the expression level of viral RNA increases. C in Figure 3 is the electrophoresis photo of a-lactalbumin step 2) Spike protein (spike protein) western blot. SARS-CoV-2 enters cells by relying on the spike protein recognition receptor angiotensin-converting enzyme ACE2 and other coreceptors, so it is a key factor in the virus replication process. As the concentration of a-lactalbumin increases, the relative expression of the spike protein decreases, indicating that a-lactalbumin also has a significant inhibitory effect on the live SARS-CoV-2 replicon virus.

Example 3. EC ₅₀ and CC ₅₀ determination of a-lactalbumin inhibiting SARS-CoV-2 Luc pseudovirus and trVLP

1) Vero E6 cells and Caco-2-N cells were seeded into a 96-well plate (ThermoFisher) at a density of 2.0×10 ⁴ cells/well or a 24-well plate at a density of 1.0×10 ⁵ cells/well, and incubated at 37°C. , cultured in a 5% CO ₂ incubator.

2) EC ₅₀ and CC ₅₀ determination of a-lactalbumin inhibiting SARS-CoV-2 Luc pseudovirus.

Vero E6 cells were seeded into a 24-well plate. The next day, the cell culture wells were added with concentrations of 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.0625 mg/ml in the system. ml, 0.03125 mg/ml and 0.015625 mg/ml of a-lactalbumin, followed by the addition of viral SARS-CoV-2 Luc, 650 TCID ₅₀ /well. After 2 hours, discard the supernatant and add PBS buffer to wash three times to remove virus particles that have not entered the cells. Then add again to the cell culture wells the concentrations of 2 mg/ml, 1 mg/ml, and 0.5 mg/ml in the system. ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.03125mg/ml and 0.015625mg/ml a-lactalbumin. The negative control group was Vero E6 cells inoculated into a 24-well plate. The next day, the same amount of PBS as a-lactalbumin was added to the cell culture wells, and then the virus SARS-CoV-2 Luc was added, 650TCID ₅₀ /well. After 2 hours, the supernatant was discarded, and PBS buffer was added and washed three times to remove virus particles that had not entered the cells. 24 hours after virus infection, the intracellular luciferase was quantified using the Luciferase Assay System and the inhibition rate was calculated. Inhibition rate calculation method: Each group uses the Luciferase Assay System to detect the value. The value of the group adding a-lactalbumin is divided by the value of the negative control group and multiplied by 100% to obtain the infection rate of the corresponding group adding a-lactalbumin. The inhibition rate ( %)=100%-infection rate (%). The cytotoxicity of a-lactalbumin against Vero E6 was measured using a CCK8 activity assay.

3) EC ₅₀ and CC ₅₀ determination of a-lactalbumin inhibiting trVLP replicon

Caco-2-N cells were seeded into a 24-well plate. The next day, the cell culture wells were added with concentrations of 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625 mg/ml and 0.03125 mg/ml α-lactalbumin, and then the viral trVLP replicon was added, and the amount added per well was MOI=0.05. After 2 hours, the supernatant was removed, and PBS buffer was added to wash 3 times to remove undesired bacteria. Virus particles that enter the cells are then added to the cell culture wells again at concentrations of 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml and 0.03125mg/ ml of a-lactalbumin. The negative control group was Caco-2-N cells inoculated into a 24-well plate. The next day, the same amount of PBS as a-lactalbumin was added to the cell culture wells, and then the viral trVLP replicon was added. The amount added to each well was MOI=0.05. .After 2 hours, remove the supernatant and add PBS buffer to wash 3 times to remove virus particles that have not entered the cells. 96 hours after viral infection, intracellular viral RNA was quantitatively detected by qRT-PCR. The primers used were

bjmu-00737-SARS-CoV-2 RNA-F: CGAAAGGTAAGATGGAGAGCC;

bjmu-00738-SARS-CoV-2 RNA-R: TGTTGACGTGCCTCTGATAAG;

bjmu-00061-RPS11-F:GCCGAGACTATCTGCACTAC;

bjmu-00062-RPS11-R:ATGTCCAGCCTCAGAACTTC.

Inhibition rate calculation method: (inhibition rate=[1-2 ^-ΔCt (RNA expression amount in experimental group)/2 ^-Δct (RNA expression amount in control group)]x100%). The cytotoxicity of a-lactalbumin to Caco-2-N was measured using a CCK8 activity assay.

4) Result:

The results are shown in Figure 4. Figure 4A shows the EC ₅₀ measurement results of a-lactalbumin inhibiting SARS-CoV-2 Luc pseudovirus. The EC ₅₀ value is 0.1635/mL. The cell activity of a-lactalbumin at the highest experimental concentration of 2 mg/ml is still greater than 90%, so it cannot The CC ₅₀ was effectively estimated, indicating that a-lactalbumin has little toxicity to Vero E6 cells and can be ignored. Figure 4B shows the EC ₅₀ of a-lactalbumin inhibiting the trVLP replicon. The EC ₅₀ value is 0.6809 mg/mL. The cell activity of a-lactalbumin is still greater than 90% at the highest experimental concentration of 2 mg/ml, so the CC ₅₀ cannot be effectively estimated, indicating that The toxicity of a-lactalbumin is very small and can be ignored. The inhibitory effect of a-lactalbumin on SARS-CoV-2Luc pseudovirus and trVLP replicon increases with increasing concentration, indicating that a-lactalbumin inhibits the infection and replication of SARS-CoV-2 pseudovirus and trVLP replicon in a dose-dependent manner.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Claims (5)

1. Application of a-lactalbumin in the preparation of products that inhibit coronavirus infection: The coronavirus is SARS-CoV-2; The product is a medicine. 2. Application of a-lactalbumin in the preparation of products for the treatment and/or prevention of diseases caused by coronavirus infection: The coronavirus is SARS-CoV-2; The product is a medicine. 3. The application according to claim 2, characterized in that: the disease caused by coronavirus infection is 2019 novel coronavirus pneumonia. 4. The application according to claim 1, characterized in that: inhibiting coronavirus infection is inhibiting the adsorption and/or entry of SARS-CoV-2 into cells. 5. The application according to any one of claims 1 to 4, characterized in that the medicine is a liquid preparation or a solid preparation.