Recombinant polypeptide for broad-spectrum inhibition of novel coronavirus and application thereof

Abstract

The invention discloses a recombinant polypeptide for broad-spectrum inhibition of novel coronavirus and application of the recombinant polypeptide, and relates to the field of synthetic biology and medicine. The recombinant polypeptide is formed by connecting a polypeptide derived from ACE2 and a polypeptide derived from novel coronavirus HR2 through a linker, and six amino acid mutations (AKT / VEW and NHE-DIQ) are introduced into the polypeptide derived from ACE2, so that the interaction between the polypeptide derived from ACE2 and RBD is improved. The recombinant polypeptide provided by the invention has high-efficiency broad-spectrum anti-novel coronavirus activity, and can be used for preparing and developing anti-novel coronavirus drugs. The recombinant polypeptide provided by the invention can efficiently inhibit the novel coronavirus and various mutant strains thereof, can be used for preparing a pharmaceutical composition for preventing or treating the coronavirus, and has the potential to be developed into a broad-spectrum anti-coronavirus drug so as to furthest inhibit the epidemic of the existing coronavirus and the possible new and reappearing coronavirus in the future.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology and medicine, and specifically to a recombinant polypeptide that broadly inhibits the novel coronavirus and its application. Background Art

[0002] The virus particles are spherical, and the spike structures arranged on the surface are composed of four structural proteins: Spike protein (S), Nucleocapsid protein (N), Membrane protein (M) and Envelope protein (E). Among them, the trimeric S glycoprotein directly determines the host range and infection efficiency through conformational dynamic equilibrium and molecular interaction mechanisms.

[0003] The coronavirus S protein plays an important role in the process of virus invasion of target cells. The S protein includes two functional subunits, S1 and S2. The receptor binding domain (RBD) of the S1 subunit is responsible for recognizing and binding to the receptor on the host cell, angiotensin-converting enzyme-2 (ACE2), triggering protease cleavage activation; subsequently, the S2 subunit undergoes a series of conformational changes, prompting the heptapeptide repeat sequence HR1 to form a trimeric coiled coil structure, exposing its hydrophobic pocket area, and then binding to HR2 to form a six-helix bundle (6-HB), ultimately driving the fusion of the virus with the host cell membrane. Therefore, the RBD and HR1 regions are ideal targets for the development of virus entry inhibitors.

[0004] However, during the evolution of coronaviruses, the spike protein RBD and N-terminal domain (NTD) also frequently mutate. These mutations not only enhance the virus's immune escape ability, allowing the virus to evade the attack of the host's immune system, but may also lead to changes in the virus's transmissibility and pathogenicity. Despite this, some hidden sites within the spike protein trimer are relatively conservative, that is, they remain unchanged in different mutant strains. These highly conserved sites provide potential targets for the development of antiviral drugs.

[0005] Therefore, the development of broad-spectrum antiviral drugs that can effectively deal with the virus and its mutants has become the key to the current prevention and treatment of infectious disease outbreaks. In the prior art, for example, the polypeptide disclosed in the patent application with publication number CN115925826A has a strong inhibitory effect on the original strain of the new coronavirus and multiple mutant strains; for example, the polypeptide disclosed in the patent application with publication number CN114437184A has a strong inhibitory effect on the original strain of the new coronavirus and multiple mutant strains. As well as the inventors' previous research (Bi W, Tang K, Chen G, et al. An enhanced broad-spectrum peptide inhibits Omicron variants in vivo. Cell Rep Med. Published online February 3, 2024. doi:10.1016 / j.xcrm.2024.101418), the peptide A1 derived from the ACE2 receptor was fused with the peptide HR2m derived from the highly conserved region HR2 of SARS-CoV-2. The results showed that it can broadly inhibit the infection of the new coronavirus, but the interaction between the peptide A1 and RBD is relatively weak. Summary of the invention

[0006] Based on the deficiencies in the existing basis, the present invention provides a recombinant polypeptide with a broad spectrum of inhibition of the new coronavirus and its application. On the basis of ACE2 polypeptide A1, six amino acid mutations (AKT / VEW and NHE / DIQ) are further introduced to enhance its interaction with RBD, thereby enhancing its antiviral activity.

[0007] The specific technical solutions of the present invention are as follows: The present invention provides a recombinant polypeptide that broadly inhibits the novel coronavirus, wherein the recombinant polypeptide comprises a polypeptide A3M6 derived from ACE2 and a polypeptide HR2 derived from the novel coronavirus HR2; The amino acid sequence of the polypeptide A3M6 is N-terminal-IEEQVEWFLDKFDIQAEDLFYQSSLGGSKGDFR-C-terminal, The amino acid sequence of the polypeptide HR2 is N-terminal-DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ-C-terminal.

[0008] Preferably, the polypeptide A3M6 and the polypeptide HR2 are connected via a linker; the linker is a flexible linker or a rigid linker; The amino acid sequence of the flexible linker is (GGGGS) n , The amino acid sequence of the rigid linker is (MALEK) n , Wherein, n=3-10, and n is an integer.

[0009] Further preferably, the n=7, the recombinant polypeptide is named A3M6L35HR2 (FL), and its inhibitory activity against SARS-CoV-2 is 4.9 nM. The results show that after introducing 6 amino acid mutations into the ACE2-derived polypeptide and fusing it with the HR2 polypeptide through a flexible linker, its inhibitory activity is significantly improved. And the IC of A3M6L35HR2 (FL) for inhibiting SARS-CoV-2 50 A1L35HR2 (IC 50 =157 nM) and HR2 (IC 50 =157 nM) peptide by 7 and 32 times.

[0010] The present invention also provides a nucleic acid molecule encoding the recombinant polypeptide.

[0011] Preferably, the gene sequence of A3M6L35HR2 (FL) is shown in SEQ ID NO.8.

[0012] The present invention also provides a recombinant vector comprising the nucleic acid molecule.

[0013] The present invention also provides a host cell comprising the recombinant vector.

[0014] The present invention also provides a method for preparing the polypeptide, which includes designing and synthesizing the coding gene of the recombinant polypeptide, constructing a recombinant expression vector of the dual-target polypeptide, transforming it into Escherichia coli for expanded culture and induced expression, and separating and purifying it.

[0015] The present invention also provides the use of the recombinant polypeptide, the nucleic acid molecule, the recombinant vector or the host cell in the preparation of a drug for preventing or treating a disease caused by coronavirus infection.

[0016] In the above application, the disease caused by coronavirus infection is a respiratory system infection. The respiratory system infection can be a respiratory tract infection and / or a lung infection.

[0017] In the above application, the coronavirus is the original strain of the new coronavirus and / or a mutant strain of the new coronavirus; the mutant strain of the new coronavirus is D614G, Beta, Delta and / or Omicron series mutant strains.

[0018] In a specific embodiment of the present invention, the Omicron series variants include XBB, BF7, BA.2, EG.5.1, BA.2.86 and KP.2, but are not limited thereto.

[0019] In the embodiments of the present invention, the results show that A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) all have good inhibitory activity against different mutant strains of SARS-CoV-2, and A3M6L35HR2 (FL) has the best inhibitory activity against different mutant strains of SARS-CoV-2, with an IC 50 Between 1.7-16.2 nM.

[0020] The present invention also provides a pharmaceutical composition, which contains the recombinant polypeptide, the nucleic acid molecule, the recombinant vector or the host cell, and a pharmaceutically acceptable carrier.

[0021] Preferably, the pharmaceutical composition is in the form of a nasal spray formulation, an oral formulation, or a parenteral formulation; Further preferably, the oral preparation is selected from tablets, capsules, granules, suspensions and pills; Further preferably, the parenteral preparation is an injectable or bolus preparation; Preferably, the pharmaceutical composition is a vaccine composition.

[0022] In terms of recombinant polypeptide design, simply expressing the polypeptides in series will affect each other in structure and may cover up the target of interaction with the virus. Therefore, simply expressing the polypeptides in series cannot ensure that each polypeptide can form the correct structure or even cannot be expressed. On the one hand, the present invention connects the two polypeptides in series by adding different linkers between them so that the two polypeptides do not interfere with each other in structure. Most importantly, the introduction of six amino acid mutations derived from the ACE2 polypeptide further ensures that the recombinant polypeptide does not affect each other in spatial structure. At the same time, the introduction of six amino acid mutations also further improves the interaction between the polypeptide and the virus, thereby maximizing the efficient and broad-spectrum inhibition of the infection of the new coronavirus.

[0023] The present invention obtains a recombinant polypeptide by a biosynthesis method of microorganism Escherichia coli, and the polypeptide has high-efficiency and broad-spectrum anti-new coronavirus activity, and has the advantages of simple process, low cost, high yield, and easy promotion. However, the chemical solid phase synthesis method of polypeptides is complex and time-consuming, and it is difficult to obtain such polypeptides with a large number of repeated amino acid sequences.

[0024] Beneficial effects of the present invention: The recombinant polypeptides of the present invention include polypeptides derived from ACE2 receptors and polypeptides of novel coronavirus HR2, which are fused and expressed through flexible or rigid linkers. The recombinant polypeptides provided by the present invention can effectively inhibit novel coronavirus and its various mutants, can be used to prepare pharmaceutical compositions for preventing or treating coronavirus, and have the potential to be developed into broad-spectrum anti-coronavirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 For the design of recombinant polypeptides (A) and their identification (B); Figure 2 is the size exclusion chromatography (SEC) analysis of recombinant polypeptides; wherein, AC represents the SEC analysis of three polypeptides, A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2), respectively; Figure 3 is the inhibitory activity of the recombinant polypeptide against SARS-CoV-2; wherein A is the inhibitory activity of A1L35HR2, A3M6L35HR2 (FL), and A3M6L35HR2 (HL2) against SARS-CoV-2 pseudovirus; B is the inhibitory activity of A3M6L35HR2 (FL), A3M6, HR2 polypeptide and A3M6+HR2 mixture against SARS-CoV-2; Figure 4 The effect of the recombinant polypeptide on the activity of target cells; Figure 5 represents the inhibitory activity of the recombinant polypeptide against different mutant strains of SARS-CoV-2; AH represents the inhibitory activity of different mutant strains; Figure 6 The recombinant polypeptide inhibits the cell-cell fusion activity mediated by SARS-CoV-2 D614G S protein; wherein A is a representative diagram of cell-cell fusion; B is a statistical diagram of the cell-cell fusion inhibition activity; Figure 7 for the interaction of the recombinant peptide with SARS-CoV-2 HR1; Figure 8 is the secondary structure of the recombinant polypeptide and its complex; wherein A is the secondary structure of A3M6, HR1 and a mixture of A3M6+HR1; B is the secondary structure of HR2, HR1 and a mixture of HR2+HR1; C is the secondary structure of A3M6L35HR2 (FL), HR1 and a mixture of A3M6L35HR2 (FL)+HR1. DETAILED DESCRIPTION

[0026] Example 1

[0027] 1. Design of recombinant peptides The recombinant polypeptide of the present invention is composed of polypeptides derived from ACE2 and SARS-CoV-2 HR2 through a flexible or rigid linker, and is composed of the following polypeptide structures from the N-terminus to the C-terminus: polypeptide A3M6 derived from ACE2 (sequence as Figure 1 A in FIG), a flexible linker (GGGGS) n or a rigid linker (MALEK) n and a polypeptide HR2 derived from HR2 (sequence as shown in FIG. Figure 1 ), wherein the linker may comprise n tandem repeat sequences, and n may be in the range of 3-10.

[0028] In this embodiment, n=7 is selected as an example, wherein the recombinant polypeptide connected by a flexible linker is named A3M6L35HR2 (FL), and the recombinant polypeptide connected by a rigid linker is named A3M6L35HR2 (HL2). The existing A1L35HR2 polypeptide is used as a comparison. The specific amino acid sequence is as follows Figure 1 As shown in A.

[0029] 2. Construction of recombinant vector pET28a-A3M6L35HR2 (FL) Design the following primers (underlined Nco I and XOt I restriction site): Nco I A3M6 F: ggagatata CCATGG GCATTGAAGAACAAGTGGAATGGTTTCTGGATAAATTTGATATTCAAGCGGAAGATC, 6 HIS XOt I HR2 R: ggtggtggtg CTCGAG CTGTTCATATTTGCCCAG; Using the laboratory pET28a-A1L35HR2 vector as a template, the A3M6L35HR2 (FL) gene fragment was amplified by PCR technology, and the above gene fragment was connected to the NcoI / XOt The pET28a linear vector was obtained by double digestion with I to obtain the pET28a-A3M6L35HR2 (FL) recombinant vector.

[0030] pET28a-A3M6L35HR2 (HL2) was synthesized and recombined by GeneWeizhi Biotechnology Co., Ltd.

[0031] 3. Expression and purification of A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) The plasmids encoding A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) genes (sequences shown in SEQ ID NOs. 7-9) were transformed into Escherichia coli BL21 (DE3), and single clones were selected and cultured in LB medium. The bacterial solution was amplified at 37°C and 220 rpm to a bacterial concentration of OD 600 Reach about 0.5. Add 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) and induce at 37℃ for 5 h. Collect the cells by centrifugation at 1,2000 × g for 3 min, suspend the cells in lysis buffer (10 mM imidazole containing 0.1% Triton), ultrasonically disrupt, and collect the supernatant by centrifugation at 1,2000 × g for 10 min. The supernatant was combined with Ni-NTA at 4℃ for 30 min, and the impurities were washed with 20 mM, 30 mM, 50 mM and 100 mM imidazole, and finally the target protein was eluted with 250 mM imidazole. Finally, the purified recombinant polypeptide was analyzed by SDS-PAGE and immunoblotting.

[0032] like Figure 1 As shown in B, the three polypeptides A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) all migrated to positions close to the expected molecular weight of the band.

[0033] 4. SEC analysis The recombinant peptide obtained by crude purification was further analyzed using a Superdex 75 Increase 10 / 300 GL column. First, the column was connected to the AKTA device, and the column was washed with water for 1 column volume (CV), and then balanced with 1×PBS for 1 CV. The peptide obtained by preliminary purification was filtered through a 0.22 μm filter membrane and then applied to a Superdex 75 Increase 10 / 300 GL column. The detection was 280 nm and the flow rate was 0.5 ml / min.

[0034] like Figure 2 As shown, the peak volumes of the three polypeptides A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) were between 10 and 15 ml, indicating that their molecular weight was around 15 kDa.

[0035] Example 2

[0036] 1. Preparation of SARS-CoV-2 pseudovirus 6.5 × 10 6293T cells were plated in a 10 cm cell culture plate to a cell density of about 80% before transfection. Fresh culture medium was replaced 1 h before transfection, and transfection reagent (Transfection Reagentlipo2000, TR001) and plasmid pNL4-3.Luc.RE expressing Luciferase reporter gene and plasmid pcDNA3.1-SARS-CoV-2-S expressing SARS-CoV-2 S protein and its mutants (Beta, Delta, XBB, BF7, BA.2, EG.5.1, BA.2.86 and KP.2) were diluted with serum-free DMEM. The diluted transfection reagent was added dropwise to the diluted plasmid in a 1:1 system, gently mixed, and placed at room temperature for 20 min. The transfection working solution was added dropwise to the culture medium and placed at 37°C 5% CO 2 Culture in an incubator. After 12 h of transfection, discard the culture medium and replace with fresh cell culture medium. After 48-72 h of transfection, collect the cell supernatant, centrifuge at 3000 × g for 10 min, aliquot, and store at -80℃ for later use.

[0037] 2. Pseudovirus inhibition test Caco-2 cells were plated at 10 4 Cells were plated in a 96-well cell culture plate and cultured overnight to allow the cells to adhere to the wall and grow. A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) peptides were diluted to the required concentration of the experiment (starting concentration 1 μM), and a 4-fold gradient dilution was performed. At the same time, positive (SARS-CoV-2 pseudovirus + Caco-2 cells) and negative (Caco-2 cells) control wells were set. After the SARS-CoV-2 pseudovirus was taken out of the -80℃ refrigerator and dissolved and mixed, it was added to the diluted peptide drug and incubated in a 37℃ incubator for 30 min. The mixture of peptide drug and SARS-CoV-2 pseudovirus was transferred to Caco-2 cells and cultured at 37℃ overnight. The peptide drug and pseudovirus were discarded, and an equal volume of fresh cell culture medium was added at the same time, and the culture was continued for two days. The luciferase enzyme activity (unit is relative light unit, RLU) was detected according to the method of the luciferase reporter gene assay kit, and the IC of the peptide was calculated based on the result. 50 The value was used to further evaluate the inhibitory effect of peptide drugs on viruses.

[0038] The results are as follows Figure 3As shown in A in the figure, the inhibitory activities of A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) against SARS-CoV-2 were 35, 4.9 and 241 nM, respectively. The results showed that after introducing 6 amino acid mutations into the ACE2-derived peptide and fusing it with the HR2 peptide through a flexible linker, its inhibitory activity was significantly improved. The inventors then compared the inhibitory activities of A3M6L35HR2 (FL), A3M6, HR2 peptide and A3M6+HR2 mixture against SARS-CoV-2. The results showed that the A3M6 peptide alone had no obvious inhibitory activity, while A3M6L35HR2 (FL) inhibited SARS-CoV-2 with an IC 50 is 4.8 nM, which is approximately HR2 (IC 50 =157 nM) peptide 32 times ( Figure 3 B in the above example. Figure 5 A to Figure 5 The results showed that A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) all had inhibitory activity against different mutant strains of SARS-CoV-2, and A3M6L35HR2 (FL) had the best inhibitory activity against different mutant strains of SARS-CoV-2, with an IC 50 The above results indicate that A3M6L35HR2 (FL) has a broad spectrum of anti-SARS-CoV-2 activity.

[0039] 3. Safety analysis of A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) peptides A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) peptides were diluted 4-fold in a 96-well plate, 100 μl per well, added to target Caco-2 cells, cultured in a 37°C carbon dioxide incubator for 48 h, then 5 μl CCK-8 was added to each well and cultured for 2 h. The OD450 absorbance was detected and the cell viability was calculated based on the absorbance.

[0040] The results are as follows Figure 4 As shown in the figure, cells treated with different concentrations of A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) did not show obvious cytotoxicity. When A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) were incubated with cells at a high concentration of 20 μM, the viability of Caco-2 cells was above 90%, which was higher than the IC50 of SARS-CoV-2. 50It is about 83 times higher, indicating that it has good safety in inhibiting SARS-CoV-2 at the cellular level.

[0041] 4. Cell-cell fusion inhibition assay Caco-2 target cells were cultured at 10 4 The cells were plated in a 96-well plate and cultured at 37°C overnight. Effector cells (293T / EGFP / D614G cells) expressing EGFP and SARS-CoV-2 D614G S protein were cultured at 2×10 4 After incubation of A1L35HR2, A3M6L35HR2 (FL) and A3M6L35HR2 (HL2) at 37°C for 30 min / well, the effector cells and peptide inhibitor mixture were transferred to the target cells Caco-2. After culturing in a 37°C carbon dioxide incubator for 4-5 h, the cell fusion status was checked under a fluorescence microscope. If the fusion was obvious, the cells were fixed with 4% paraformaldehyde. Finally, the fusion inhibition activity of the peptide was calculated based on the fusion status of each well.

[0042] The results are as follows Figure 6 As shown, the inhibitory activities of A1L35HR2, A3M6L35HR2 (FL), and A3M6L35HR2 (HL2) on S protein-mediated cell-cell fusion were 66, 7, and 180 nM, respectively.

[0043] 5. Biolayer interferometry (BLI) to detect affinity First, the A3M6L35HR2 (FL) peptide was biotinylated at a molar ratio of 1:3 (peptide: biotin) according to the instructions of EZ-Link NHS-PEG12-biotin, and then the unreacted biotin was removed by ultrafiltration through a 3 kDa tube. The biotinylated A3M6L35HR2 (FL) peptide was diluted to 5 μg / ml and the HR1 peptide was diluted to 200 nM using BLI buffer solution (PBS with 0.02% Tween20), and then the detection was performed according to a 2-fold serial dilution. The data were curve fitted using ForteBio software, and k was obtained. on , k off and K D And other related parameters.

[0044] like Figure 7 As shown, A3M6L35HR2 (FL) has a strong interaction with HR1 peptide, and the affinity between the two is 0.03 nM.

[0045] 6. Circular dichroism analysis Circular dichroism can determine the secondary conformation of peptides and the secondary conformational changes after the interaction between peptide samples. The peptide and peptide mixture were diluted to 10 μM using PB buffer solution, and the peptide complex was incubated at 37°C for 30 min. The peptide sample was added to a 0.1 cm quartz sample cup, and the CD value changes in the wavelength range of 180-260 nm were detected at 25°C.

[0046] like Figure 8 As shown in the figure, the single A3M6 and HR1 polypeptides are irregular coils, while the single HR2 and A3M6L35HR2 (FL) form double negative peaks at 208 nm and 222 nm, indicating that both have a certain amount of α-helical conformation. When HR2 and A3M6L35HR2 (FL) are mixed with HR1 polypeptide, the negative peaks of the polypeptide complex at 208 nm and 222 nm are significantly deepened, forming a complex with a higher α-helical content, indicating that HR2 and A3M6L35HR2 (FL) polypeptides can interact with HR1 polypeptide and induce each other to form a large amount of α-helical conformation.

Claims

1. A recombinant polypeptide that broadly inhibits the new coronavirus, characterized in that: The recombinant polypeptides include the polypeptide A3M6 derived from ACE2 and the polypeptide HR2 derived from the novel coronavirus HR2; The amino acid sequence of the polypeptide A3M6 is IEEQVEWFLDKFDIQAEDLFYQSSLGGSKGDFR, The amino acid sequence of the polypeptide HR2 is DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ.

2. The recombinant polypeptide that broadly inhibits the new coronavirus as claimed in claim 1, characterized in that: The polypeptide A3M6 and the polypeptide HR2 are connected via a linker; The linker is a flexible linker or a rigid linker; The amino acid sequence of the flexible linker is (GGGGS) n , The amino acid sequence of the rigid linker is (MALEK) n , Wherein, n=3-10, and n is an integer.

3. The recombinant polypeptide that broadly inhibits the new coronavirus as claimed in claim 2, characterized in that: Said n=7.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the recombinant polypeptide according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that The host cell comprises the recombinant vector according to claim 5.

7. Use of the recombinant polypeptide according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, or the host cell according to claim 6 in the preparation of a medicament for preventing or treating a disease caused by coronavirus infection.

8. The use according to claim 7, characterized in that The disease caused by the coronavirus infection is a respiratory system infection.

9. The use according to claim 7, characterized in that The coronavirus is the original strain of the new coronavirus and / or a variant strain of the new coronavirus; The novel coronavirus variant is at least one of D614G, Beta, Delta and Omicron.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the recombinant polypeptide according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5 or the host cell according to claim 6, and a pharmaceutically acceptable carrier.

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