Polypeptide composition for inhibiting novel coronavirus invasion and application thereof

Abstract

The invention relates to a polypeptide composition for inhibiting novel coronavirus invasion and application of the polypeptide composition, S protein derived peptide (SAP1) and membrane fusion inhibition polypeptide (EK1) are adopted, inhibition experiments are carried out on pseudoviruses taking S protein of a novel coronavirus original strain and an Omemitron virus strain as envelope protein and pseudoviruses taking VSV-G as envelope protein, and the results show that the polypeptide composition can be used for inhibiting novel coronavirus invasion. The SAP1 polypeptide, the EK1 polypeptide and the composition of the SAP1 polypeptide and the EK1 polypeptide have no inhibition effect on pseudoviruses taking VSV-G protein as envelope protein, but have inhibition effect on pseudoviruses taking a novel coronal original strain and an Ommi virus strain S protein as envelope protein, and the inhibition effect of the polypeptide composition on viruses is obviously better than that of any polypeptide on viruses. The IC50 of the polypeptide composition to viruses is obviously lower than the IC50 of any one of the polypeptides to viruses.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and specifically relates to a polypeptide composition for inhibiting the invasion of a novel coronavirus and an application thereof. Background Art

[0002] The novel coronavirus mainly expresses four structural antigenic proteins: spike protein (S protein), membrane protein (M protein), envelope protein (E protein) and nucleocapsid protein (N protein). Among them, the process of S protein recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of the host cell is a key step for the virus to invade the human body; the bound S protein undergoes a series of enzyme treatments in the host cell, especially being cut by proteases (such as TMPRSS2) in the host cell, thereby exposing the fusion peptide (FP), which is then further cut by proteases in the host cell, triggering the fusion of the viral membrane with the host cell membrane, allowing the virus to enter the cell. Therefore, the binding of the S protein to the host cell receptor ACE2 and the subsequent membrane fusion process are key steps in virus-infected cells. The patent "Anti-novel coronavirus disease polypeptide composition and its application (CN202310854612.6)" provides an efficient anti-novel coronavirus disease polypeptide composition, in which the three polypeptide inhibitors in the composition can better inhibit various virus variants according to the steps of virus invasion, provide a feasible treatment plan for the treatment or prevention of novel coronavirus, and provide guidance for drug development. Although this anti-new coronavirus disease polypeptide combination has a good inhibitory effect, its reaction mechanism is complex, the cost is high, and there are certain side effects. Moreover, as the virus undergoes various mutations in the process of constantly adapting to the host, it often becomes immune to commonly used inhibitors. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In response to the above problems, a highly effective polypeptide composition for inhibiting the invasion of the new coronavirus is proposed, which only requires a combination of two polypeptides.

[0005] (II) Technical solution

[0006] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0007] On the one hand, the present invention provides a polypeptide composition for inhibiting the invasion of the new coronavirus, comprising: polypeptide 1 and polypeptide 2; the amino acid sequence of polypeptide 1 is TFLDKFNHEAEDLFYQ; the amino acid sequence of polypeptide 2 is SLDQINVTFLDLEYEMKLEEAIKLEESYIDLKEL.

[0008] According to a preferred embodiment of the present invention, in the polypeptide composition for inhibiting the invasion of the new coronavirus, the polypeptide 1 and the polypeptide 2 are mixed in a molar ratio of (0.5-8):(0.5-8).

[0009] Preferably, in the polypeptide composition for inhibiting the invasion of the new coronavirus, the molar ratio of the polypeptide 1 to the polypeptide 2 is 1:1.

[0010] According to a preferred embodiment of the present invention, in the polypeptide composition for inhibiting the invasion of the new coronavirus, the concentration range of the polypeptide 1 is 0.1 to 6000 nM, and the concentration range of the polypeptide 2 is 0.1 to 6000 nM.

[0011] Preferably, in the polypeptide composition for inhibiting the invasion of the new coronavirus, the concentration of polypeptide 1 is 500 nM, and the concentration of polypeptide 2 is 500 nM.

[0012] On the other hand, the present invention also provides an application of a polypeptide composition for inhibiting the invasion of the new coronavirus.

[0013] Furthermore, the polypeptide composition that inhibits the invasion of the new coronavirus can be used to prepare drugs for preventing the new coronavirus disease, treating the new coronavirus disease, and alleviating the new coronavirus disease.

[0014] (III) Beneficial effects

[0015] The present invention provides a polypeptide composition for inhibiting the invasion of the new coronavirus, which is composed of two polypeptides and can effectively inhibit the binding and membrane fusion of the new coronavirus S protein (spike protein) with the host cell receptor ACE2. Experiments have confirmed that the polypeptide composition has a good effect in inhibiting viral invasion, and the inhibitory effect of the polypeptide composition is significantly better than the inhibitory effect of using any one polypeptide alone, and its IC50 (half-maximal inhibitory concentration) is low and much lower than the IC50 of using any one polypeptide alone.

[0016] The polypeptide composition for inhibiting the invasion of the new coronavirus provided by the present invention has a simple reaction mechanism, low cost and can reduce side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1It is the inhibitory effect of two polypeptides and their combination on WT strain and Omicron strain virus. In the figure, Mock represents the luciferase activity without adding any virus and polypeptide to the cell culture fluid, Control represents the luciferase activity of adding only virus without adding any polypeptide to the cell culture fluid, SAP1 and EK1 represent the luciferase activity of adding S protein derived peptide or membrane fusion inhibitory polypeptide to the cell culture fluid at a concentration of 500nM, Mix1:2 represents the luciferase activity of adding a polypeptide combination of SAP1 polypeptide and EK1 polypeptide at a concentration of 500nM and a ratio of 1:2 to the cell culture fluid, Mix2:1 represents the luciferase activity of adding a polypeptide combination of SAP1 polypeptide and EK1 polypeptide at a concentration of 500nM and a ratio of 2:1 to the cell culture fluid, and Mix1:1 represents the luciferase activity of adding a polypeptide combination of SAP1 polypeptide and EK1 polypeptide at a concentration of 500nM and a ratio of 1:1 to the cell culture fluid.

[0019] Figure 2 The invention discloses the inhibitory effects of two polypeptides and their combination on pseudoviruses with VSV-G as envelope protein.

[0020] Figure 3 The dose-effect curves of the two polypeptides and their combination at different concentrations for inhibiting the WT strain and the Omicron strain are shown in FIG. The concentrations used are 0 nM, 0.1 nM, 0.3 nM, 0.9 nM, 2.7 nM, 8.1 nM, 24.3 nM, 72.9 nM, 218.7 nM, 500 nM, 656.1 nM, 1968.3 nM, and 5904.9 nM, respectively.

[0021] Figure 4 The IC50 of the SPK1 polypeptide and the EK1 polypeptide at a concentration of 500 nM and the polypeptide composition in a ratio of 1:1 for inhibiting the WT strain and the Omicron strain. DETAILED DESCRIPTION

[0022] The following is a description of the specific embodiments of the present invention. The polypeptides in this embodiment are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0023] The present invention uses human-derived lung cells Calu-3 to better simulate the targets of COVID-19 infection.

[0024] 1. Cell passaging

[0025] (1) Aspirate the cell culture medium in the culture flask containing Calu-3 (human lung adenocarcinoma cells), rinse once with an appropriate amount of phosphate buffered saline (PBS), and discard the PBS.

[0026] (2) adding digestion solution to the culture bottle, gently shaking the culture bottle so that all the Calu-3 cells at the bottom of the bottle are immersed in the digestion solution, and then placing the culture bottle in a 37° C. incubator to digest the Calu-3 cells; the digestion solution uses 0.25% Trypsin-EDTA;

[0027] (3) After 30 to 60 seconds of digestion, the culture flask was placed under a microscope for observation. It was found that the Calu-3 cells gradually adhered to the wall and tended to be round. When the adhered Calu-3 cells had not yet floated up, the digestion solution was discarded and culture medium was added to terminate the digestion; the culture medium was MEM (containing NEAA) + 10% FBS + 1% double antibody.

[0028] (4) Carefully blow off the adherent cells to form a Calu-3 cell suspension, and then place a portion of the Calu-3 cell suspension in a 37°C incubator for culture at a cell density of 2 to 5 × 10 4 The cells were subcultured every other day at a cell subculture ratio of 1:3, and the cell culture fluid was finally tested for luciferase. Another portion of the Calu-3 cell suspension was divided into plates for transfection to obtain pseudoviruses.

[0029] 2. Preparation and detection of pseudoviruses

[0030] The lentiviral packaging helper plasmid (psPAX2), membrane protein plasmid (pcDNA 3.0-SRAS-CoV-2-S / VSV-G), and plasmid with GFP and Luciferase dual reporter system (Lenti-GFP-Luc) (4.5μg) were transfected into Calu-3 cells, and the supernatant was collected 2 days later. After filtering with a 0.45μM filter, pseudovirus supernatants with S protein of the novel coronavirus original (WT) strain and Omicron strain as envelope protein and pseudovirus supernatants with VSV-G as envelope protein were obtained. The pseudovirus supernatant was added to the cell culture fluid obtained above for luciferase detection.

[0031] Example 1

[0032] The prepared pseudovirus supernatant with the S protein of the WT strain and the Omicron strain as the envelope protein and the SAP1 polypeptide at a concentration of 500 nM were incubated at 37°C for 30 minutes and then added to the Calu-3 cell culture medium. After 2 days, the cells were lysed using the reporter gene lysis buffer (passive lysis buffer) of Promega Company to obtain cell lysate.

[0033] 20 μL of the obtained cell lysate was added to a 96-well plate, followed by 100 μL of luciferase detection reagent (firefly luciferase reporter gene detection kit, Promega), mixed and then detected on a Synergy Mx multi-function microplate reader.

[0034] Example 2

[0035] The 500 nM SAP1 polypeptide added in Example 1 was replaced with 500 nM EK1 polypeptide, and the other conditions were the same as in Example 1.

[0036] Example 3

[0037] On the basis of adding 500 nM SAP1 in Example 1, 500 nM EK1 polypeptide was added, and the molar ratio of SAP1 polypeptide to EK1 polypeptide was 1:1. Other conditions were the same as in Example 1.

[0038] Example 4

[0039] On the basis of Example 3, the contents of the two polypeptides were adjusted so that the molar ratio of SAP1 polypeptide to EK1 polypeptide was 2:1, and the other conditions were the same as those in Example 4.

[0040] Example 5

[0041] On the basis of Example 3, the contents of the two polypeptides were adjusted so that the molar ratio of SAP1 polypeptide to EK1 polypeptide was 1:2, and the other conditions were the same as those in Example 4.

[0042] Luciferase activity can reflect the inhibitory effect on pseudoviruses with WT strain and Omicron strain S protein as envelope protein. The luciferase activity test results of the cell culture fluid without any virus and polypeptide added, the luciferase activity test results of the cell culture fluid with only pseudoviruses with WT strain and Omicron strain S protein as envelope protein without any polypeptide added, and the experimental test results of Examples 1 to 3 were compared. Figure 1 As shown. It can be seen that the luciferase activity of the cell culture fluid without any virus and peptide is very low. After adding the pseudovirus with the S protein of the WT strain and the Omicron strain as the envelope protein, the luciferase activity of the cell culture fluid increased significantly, indicating that the new coronavirus was successfully infected; adding the SAP1 peptide can reduce the luciferase activity by about 23%, indicating that the SAP1 peptide can inhibit the interaction between the S protein and ACE2; adding the EK1 peptide can reduce the luciferase activity by about 62%, indicating that the EK1 peptide can inhibit membrane fusion; adding the combination of SAP1 and EK1 peptides, the luciferase activity is reduced by about 86%, indicating that the combination of SAP1 and EK1 peptides can better inhibit the invasion of the new coronavirus.

[0043] The experimental test results of Examples 4 to 5 show that the peptide composition with a molar ratio of SAP1 polypeptide to EK1 polypeptide of 2:1 can reduce the luciferase activity by about 76%, indicating that increasing the content of SAP1 polypeptide does not help the virus inhibition effect, but reduces the inhibition effect; the peptide composition with a molar ratio of SAP1 polypeptide to EK1 polypeptide of 1:2 can reduce the luciferase activity by about 73%, indicating that increasing the EK1 polypeptide does not help the virus inhibition effect, but reduces the inhibition effect; the experimental test results of Examples 4 to 5 are compared with those of Example 3 as shown in FIG. Figure 1 As shown, it can be seen that the peptide composition with a molar ratio of SAP1 polypeptide to EK1 polypeptide of 1:1 has the best inhibitory effect on the new coronavirus. Because when the new coronavirus invades, the spike glycoprotein (S) embedded in the lipid envelope of the new coronavirus recognizes the receptor ACE2 of the human host cell. After receptor binding occurs, the S protein begins to bend, shortening the distance between the virus and the cell surface. Then, when the distance is close enough, membrane fusion occurs, and then the RNA genome of the virus enters the cell. The SKP1 polypeptide in the polypeptide composition used in the present invention directly inhibits the interaction between the S protein and ACE2, and the EK1 polypeptide is used to inhibit membrane fusion, thereby effectively and synergistically inhibiting the virus in the two steps of virus invasion.

[0044] Comparative Examples 1 to 4

[0045] On the basis of Examples 1 to 4, the pseudovirus supernatants with the S protein of the WT strain and the Omicron strain as the envelope protein were replaced with pseudovirus supernatants with VSV-G as the envelope protein, and the other conditions were the same as in Examples 1 to 4.

[0046] The luciferase activity test results of the cell culture fluid without any virus and polypeptide added, the luciferase activity test results of the cell culture fluid with only pseudovirus containing VSV-G as the envelope protein and without any polypeptide added, and the test results of Comparative Examples 1 to 4 were compared. Figure 2 As shown, the luciferase activity of the cell culture fluid without any virus and polypeptide added is very low. After the pseudovirus with VSV-G as the envelope protein is added, the luciferase activity of the cell culture fluid is significantly increased, indicating that the VSV-G virus has successfully infected; SAP1 polypeptide and EK1 polypeptide and their combination have no inhibitory effect on the invasion of pseudovirus with VSV-G as the envelope protein, indicating the specificity of the polypeptide and its combination for the invasion of the new coronavirus.

[0047] Example 6

[0048] The prepared pseudovirus supernatant with the S protein of the WT strain and the Omicron strain as the envelope protein and a polypeptide composition consisting of SAP1 polypeptide and EK1 polypeptide with a concentration of 0.1 nM and a molar ratio of 1:1 were incubated at 37°C for 30 minutes and then added to the Calu-3 cell culture medium. After 2 days, the cells were lysed using Promega's reporter gene lysis buffer (passive lysis buffer) to obtain a cell lysate.

[0049] 20 μL of the obtained cell lysate was added to each 96-well plate, followed by 100 μL of luciferase detection reagent (firefly luciferase reporter gene detection kit, Promega), mixed and then detected on a Synergy Mx multi-function microplate reader.

[0050] Example 7

[0051] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 0.3 nM, and other conditions were the same as those in Example 6.

[0052] Example 8

[0053] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 0.9 nM, and other conditions were the same as those in Example 6.

[0054] Example 9

[0055] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 2.7 nM, and other conditions were the same as those in Example 6.

[0056] Example 10

[0057] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 8.1 nM, and other conditions were the same as those in Example 6.

[0058] Embodiment 11

[0059] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 24.3 nM, and other conditions were the same as those in Example 6.

[0060] Example 12

[0061] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 72.9 nM, and other conditions were the same as those in Example 6.

[0062] Example 13

[0063] On the basis of Example 8, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 218.7 nM, and other conditions were the same as those in Example 6.

[0064] Embodiment 14

[0065] On the basis of Example 6, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 656.1 nM, and other conditions were the same as those in Example 8.

[0066] Embodiment 15

[0067] On the basis of Example 6, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 1968.3 nM, and other conditions were the same as those in Example 6.

[0068] Example 16

[0069] On the basis of Example 6, the concentrations of SAP1 polypeptide and EK1 polypeptide were adjusted to 5904.9 nM, and other conditions were the same as those in Example 6.

[0070] The experimental test results of Example 3 and Examples 6 to 16 are as follows Figure 3 As shown in the figure, the greater the concentration of the polypeptide composition, the better the inhibitory effect. However, when the concentration of the polypeptide composition is 500nM, the ideal inhibitory effect has been achieved, and the inhibition rate is about 86%. Considering that the higher the concentration, the greater the cost and the more side effects that may be caused, it is believed that the optimal concentration of the polypeptide composition in the present invention is 500nM.

[0071] To summarize the above conclusions, the results show that SAP1 polypeptide and EK1 polypeptide and their combination have a certain inhibitory effect on pseudoviruses with S protein of WT strain and Omicron strain as envelope protein, and the inhibitory effect of the polypeptide combination is significantly stronger than that of any single polypeptide. The concentration of the polypeptide combination is 500nM, and the molar ratio of the three polypeptides is 1:1, which has a better inhibitory effect, and its IC50 is much smaller than the IC50 of any single polypeptide. Figure 4 shown.

[0072] It should be noted that the above embodiments can be freely combined as needed. The above description is only the preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide composition for inhibiting the invasion of the new coronavirus, It is characterized in that It comprises: polypeptide 1 and polypeptide 2; the amino acid sequence of polypeptide 1 is SEQ ID No.1; the amino acid sequence of polypeptide 2 is SEQ ID No.

2.

2. A polypeptide composition for inhibiting the invasion of a novel coronavirus according to claim 1, It is characterized in that The polypeptide 1 and the polypeptide 2 are mixed in a molar ratio of (0.5-8):(0.5-8).

3. A polypeptide composition for inhibiting the invasion of a novel coronavirus according to claim 2, It is characterized in that The molar ratio of the polypeptide 1 to the polypeptide 2 is 1:

1.

4. A polypeptide composition for inhibiting the invasion of a novel coronavirus according to claim 1, It is characterized in that The concentration range of the polypeptide 1 is 0.1 to 6000 nM, and the concentration range of the polypeptide 2 is 0.1 to 6000 nM.

5. A polypeptide composition for inhibiting the invasion of a novel coronavirus according to claim 4, It is characterized in that The concentration of the polypeptide 1 is 500 nM, and the concentration of the polypeptide 2 is 500 nM.

6. Use of a polypeptide composition for inhibiting the invasion of a new coronavirus as described in any one of claims 1 to 5.

7. The use of the polypeptide composition for inhibiting the invasion of the new coronavirus according to claim 6, It is characterized in that The polypeptide composition for inhibiting the invasion of the new coronavirus is used to prepare drugs for preventing, treating and alleviating the new coronavirus disease.

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