Preparation method and application of medicine for inhibiting PCV2 from invading host cells

Abstract

The invention provides a preparation method and application of a medicine for inhibiting PCV2 from invading host cells. The invention aims to identify and explore the key host protein participating in PCV2 infection and the action mechanism of the key host protein, and provides a potential target for design of anti-PCV2 drugs or vaccines. According to the invention, a key host protein Annexin A2 (ANXA2) which interacts with Cap is screened out; experiments show that the shRNA (short hairpin ribonucleic acid) targeting ANXA2 or the inhibitor A2ti-1 can obviously reduce PCV2 (porcine circovirus 2) replication; further research finds that the inhibition effect is realized by influencing the adsorption of PCV2 to host cells; therefore, the invention provides a new action target for the prevention and treatment of PCV2, and provides a new vaccine or drug preparation approach for PCV2.

Description

Technical Field

[0001] The present invention relates to the interaction between porcine circovirus type 2 and host cell proteins, and particularly to a method for preparing a drug for inhibiting the invasion of PCV2 into host cells and its application. Background Art

[0002] Porcine circovirus type 2 (PCV2) is a non-enveloped, single-stranded circular DNA virus with an icosahedral structure. The PCV2 genome contains 11 open reading frames (ORFs), among which ORF1 and ORF2 encode two major viral proteins respectively. ORF1 encodes Rep and Rep′, both of which are crucial for virus replication; while ORF2 encodes the capsid protein (Cap), which is the only structural protein of PCV2. As the main component of the PCV2 capsid, Cap has a typical jelly-roll fold structure and plays a key role in the PCV2 life cycle. It mainly mediates the binding of the virus to cells and is endocytosed through host cell receptors, playing a key role in the process of the virus invading host cells. In vitro, 60 PCV2 Cap protein subunits can self-assemble into virus-like particles (VLPs), whose structure is similar to that of wild-type virus and can bind to host cell proteins and invade cells.

[0003] The early attachment and invasion of PCV2 are key steps in virus infection. Glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS-B), are the main receptors of PCV2. In addition to GAGs, Cap can also interact with other cell surface molecules, such as P-selectin and phosphacan, to promote virus infection. Although the above findings highlight the role of host molecules in PCV2 infection, the specific mechanisms mediating PCV2 adsorption and infection have not been fully elucidated, and further exploration of protein targets is needed, and the invasion of PCV2 needs to be targeted and inhibited based on new protein targets.

[0004] In view of this, it is necessary to provide a method for preparing a drug for inhibiting the invasion of PCV2 into host cells and its application to solve or at least alleviate the technical problem of targeting and inhibiting the invasion of PCV2 based on new protein targets. Summary of the Invention

[0005] The main object of the present invention is to provide a method for preparing a drug for inhibiting the invasion of PCV2 into host cells and its application to solve the technical problem of targeting and inhibiting the invasion of PCV2 based on new protein targets.

[0006] To achieve the above object, the present invention provides an application of using ANXA2 protein as a target in the preparation of a drug for inhibiting the invasion of PCV2 into host cells or in the preparation of a PCV2 vaccine.

[0007] The present invention also provides an application of an ANXA2 inhibitor in the preparation of a drug for inhibiting the invasion of PCV2 into host cells.

[0008] Furthermore, the ANXA2 inhibitor is used to inhibit the expression of ANXA2 protein in the host cell and / or the ANXA2 inhibitor is used to inhibit the functional activity of the ANXA2 protein.

[0009] Furthermore, the amino acid sequence of the ANXA2 protein is as shown in SEQ ID NO.1 or as shown in SEQ ID NO.2.

[0010] Furthermore, the ANXA2 inhibitor includes a specific shRNA oligonucleotide sequence targeting porcine ANXA2; the specific shRNA oligonucleotide sequence includes shANXA2-1 or shANXA2-2; the nucleotide sequence of the sense strand in shANXA2-1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the anti-sense strand in shANXA2-1 is as shown in SEQ ID NO.8; the nucleotide sequence of the sense strand in shANXA2-2 is as shown in SEQ ID NO.9, and the nucleotide sequence of the anti-sense strand in shANXA2-2 is as shown in SEQ ID NO.10.

[0011] Furthermore, the ANXA2 inhibitor includes a specific inhibitor that selectively inhibits the ANXA2 / S100A10 heterotetramer.

[0012] Furthermore, the specific inhibitor includes A2ti-1.

[0013] Furthermore, the host cell is a porcine cell; the porcine cell includes porcine kidney epithelial cells.

[0014] The present invention also provides a method for preparing a drug for inhibiting the invasion of PCV2 into host cells, including: using the ANXA2 inhibitor as an active component of the drug for inhibiting the invasion of PCV2 into host cells.

[0015] The present invention also provides an application of an ANXA2 inhibitor in inhibiting the invasion of PCV2 into host cells for non-therapeutic purposes.

[0016] The present invention has at least the following technical effects:

[0017] The present invention aims to identify and explore the key host cell proteins involved in PCV2 infection and their mechanisms of action, providing potential targets for the design of anti-PCV2 drugs or vaccines. The present invention uses virus-like particles (VLPs) assembled by PCV2 Cap to infect cells, and through immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), the key host protein Annexin A2 (ANXA2) that interacts with Cap is screened out; further experiments show that shRNA targeting ANXA2 or inhibitor A2ti-1 significantly reduces the replication of PCV2; ANXA2 located on the surface of host cells promotes the binding of the virus to host cells by interacting with Cap, thereby enhancing the infection efficiency of PCV2. Therefore, the present invention provides new insights into the molecular mechanism of PCV2 infection and potential molecular targets for the development of vaccines and treatment strategies. Brief Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0019] Figure 1 In Example 2 of the present invention, PK-15 cells were infected with different doses of PCV2, and after 48 hours of infection, the mRNA expression level of ANXA2 was detected; in the figure, Mock refers to normal cells without any treatment, MOI (Multiplicity of infection) refers to the multiplicity of infection, 0.5MOI refers to each cell being infected with 0.5 virus particles, 1MOI refers to each cell being infected with 1 virus particle, and 5MOI refers to each cell being infected with 5 virus particles;

[0020] Figure 2 In Example 2 of the present invention, PK-15 cells were infected with different doses of PCV2, and after 48 hours of infection, the protein expression level of ANXA2 was detected; in the figure, in the direction from "-" to "+", the doses of PCV2 are 0MOI, 0.5MOI, 1MOI, and 5MOI respectively;

[0021] Figure 3 In Example 3 of the present invention, the schematic diagram of the design principle of the shRNA sequence targeting ANXA2;

[0022] Figure 4 In Example 3 of the present invention, the expression of ANXA2 was detected by RT-qPCR 24 h after cell transfection; in the figure, shNC refers to the negative control shRNA (non-targeting shRNA), shANXA2-1 refers to the shRNA-1 targeting ANXA2 (corresponding to the label in the text part of the example), and shANXA2-2 refers to the shRNA-2 targeting ANXA2 (corresponding to the label in the text part of the example).

[0023] Figure 5 In Example 3 of the present invention, the expression of ANXA2 was detected by western blot 24 h after cell transfection; in the figure, Mock refers to the empty vector control group, shANXA2-1 refers to the shRNA-1 targeting ANXA2, shANXA2-2 refers to the shRNA-2 targeting ANXA2, and shNC refers to the negative control shRNA (non-targeting shRNA).

[0024] Figure 6 In Example 3 of the present invention, the expression of ANXA2 was detected by western blot 24 h after cell transfection. The western blot results were quantitatively analyzed using Image J software and normalized with actin as the internal reference; in the figure, Mock refers to the empty vector control group, shNC refers to the negative control shRNA (non-targeting shRNA), shANXA2-1 refers to the shRNA-1 targeting ANXA2, and shANXA2-2 refers to the shRNA-2 targeting ANXA2.

[0025] Figure 7 In Example 3 of the present invention, after transfection with shANXA2-1 or shNC, PCV2 was infected and cultured for 36 h, and the expression of Cap was detected; in the figure, Mock refers to the empty vector control group, shNC refers to the negative control shRNA (non-targeting shRNA), shANXA2-1 refers to the shRNA-1 targeting ANXA2, and 4 μg, 3 μg, 2 μg refer to the doses of transfected shANXA2-1 being 4 μg, 3 μg, and 2 μg, respectively.

[0026] Figure 8In Example 3 of the present invention, after transfection with shANXA2-1 or shNC, PCV2 was infected and cultured for 36 h, the expression of Cap was detected, the western blot results were quantitatively analyzed using Image J software, and normalized with actin as an internal reference; in the figure, Mock refers to the empty vector control group, shNC refers to the negative control shRNA (non-targeting shRNA), shANXA2-1 refers to the shRNA-1 targeting ANXA2, and 4 μg, 3 μg, 2 μg refer to the transfection doses of shANXA2-1 being 4 μg, 3 μg, and 2 μg respectively;

[0027] Figure 9 In Example 4 of the present invention, the effect of the inhibitor on cell viability was evaluated by CCK8; in the figure, Control refers to normal cells without any treatment, and 20, 40, 60, 80, 120 refer to the concentrations of inhibitor A2ti-1 (the unit is μM);

[0028] Figure 10 In Example 4 of the present invention, after continued culture for 36 h, the expression level of Cap protein was detected by western blot, the western blot results were quantitatively analyzed using Image J software, and normalized with actin as an internal reference; in the figure, A2ti-1 (μM) refers to the inhibitor concentration, "A2ti-1" is the name of the commercial inhibitor, and μM is the concentration;

[0029] Figure 11 In Example 4 of the present invention, after continued culture for 36 h, qPCR detection of the viral genome copy number was performed; in the figure, A2ti-1 (μM) refers to the inhibitor concentration, "A2ti-1" is the name of the commercial inhibitor, and μM is the concentration;

[0030] Figure 12 In Example 4 of the present invention, after continued culture for 36 h, IFA detection of the expression level of Cap protein was performed; in the figure, Cap refers to the Cap protein of the virus, and the green fluorescence indicates the expression and distribution of Cap protein (using a specific antibody recognizing Cap protein as the primary antibody for immunolabeling); DAPI refers to a DNA fluorescent dye (the full English name is 4',6-diamidino-2-phenylindole, and the full Chinese name is 4',6-diamidino-2-phenylindole), and the blue fluorescence indicates the cell nucleus; Merge refers to the image obtained by superimposing the Cap protein signal (green) and the cell nucleus signal (blue) in the same field of view, A2ti-1 refers to the ANXA2 inhibitor, and 0 μM, 40 μM, 60 μM refer to the inhibitor concentrations;

[0031] Figure 13In Example 5 of the present invention, cells were pretreated with ANXA2 antibody at 4°C and then infected with PCV2, and the copy number of viral genomic DNA adsorbed on the cell surface was detected by qPCR. In the figure, PCV2 refers to the PCV2 infection group only (PCV2 is the name of the virus used in the experiment), Isotype ab+PCV2 refers to the isotype antibody control + virus infection group (cells were first treated with the isotype control antibody, and then infected with PCV2 virus to exclude the influence of non-specific antibody binding on the experimental results), and ANXA2 ab+PCV2 refers to the ANXA2 antibody + virus infection group (cells were first treated with the antibody specifically recognizing ANXA2 (ANXA2 antibody), and then infected with PCV2 virus to study the role of cell surface ANXA2 in the PCV2 adsorption process);

[0032] Figure 14 In Example 6 of the present invention, 293FT cells were co-transfected with Cap-Flag and ANXA2-HA recombinant plasmids. After 48 hours, immunoprecipitation was performed using magnetic beads with HA or Flag-tagged antibodies, and detected by western blot. In the figure, ANXA2-HA refers to the ANXA2 protein with an HA tag (the HA tag is a short peptide with a 9-amino acid sequence, and its amino acid sequence is YPYDVPDYA. The HA tag was fused with the ANXA2 gene for expression, and an anti-HA tag antibody was used to detect the expression of the ANXA2 protein in western blot detection), Cap-Flag refers to the Cap protein with a Flag tag (the Flag tag is a short peptide with an 8-amino acid sequence, and its amino acid sequence is DYKDDDDK. The Flag tag was fused with the cap gene for expression, and an anti-Flag tag antibody was used to detect the expression of the Cap protein in western blot detection), anti-HA refers to the antibody against the HA tag, which is used to detect the HA-tagged protein (i.e., the ANXA2-HA protein), anti-Flag refers to the antibody against the Flag tag, which is used to detect the Flag-tagged protein (i.e., the Cap-Flag protein), and Input refers to the total protein sample in the experiment, which is used for control detection (the total cell protein directly extracted from the cell lysate before immunoprecipitation. The input sample can be used as a positive control in western blot detection to determine that the target protein is normally expressed in the cells and not degraded during the experiment);

[0033] Figure 15In Example 7 of the present invention, the molecular docking results of ANXA2 (pink) and Cap (cyan) predicted by AlphaFold3; the interaction interface is marked with a dashed box, the enlarged view shows the key amino acid residues at the interface, and the non-covalent interactions (including hydrogen bonds and salt bridges) present on the docking surface are represented by yellow dashed lines.

[0034] The realization, functional characteristics, and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the accompanying drawings. Specific Embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0036] Moreover, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0037] Annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein that can be expressed and distributed in the cell membrane, cytoplasm, and nucleus; ANXA2 can participate in the endocytosis and cellular uptake of pathogens by interacting with viral proteins. In the present invention, PK-15 cells were co-incubated with PCV2 VLPs, and immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis were used to find that ANXA2 is a novel host protein that can interact with the capsid protein of PCV2; and the key role of ANXA2 in the early stage of PCV2 infection was further verified. It provides new insights into revealing the molecular mechanism of PCV2 infection and potential targets for the development of PCV2 vaccines and treatment strategies.

[0038] The present invention provides an application of using ANXA2 protein as a target in the preparation of a drug for inhibiting the invasion of PCV2 into host cells or in the preparation of a PCV2 vaccine.

[0039] Specifically, the present invention provides an application of using ANXA2 protein as a target in the preparation of a drug for inhibiting the invasion of PCV2 into host cells, or provides an application of using ANXA2 protein as a target in the preparation of a PCV2 vaccine.

[0040] When used in the preparation of a drug for inhibiting the invasion of host cells by PCV2, ANXA2 protein is used as the inhibitory target, and a drug for inhibiting the invasion of host cells by PCV2 is prepared by inhibiting the expression or functional activity of ANXA2 protein; when used in the preparation of a PCV2 vaccine, the gene sequence of the docking surface peptide segment of the host protein ANXA2 that interacts with the Cap protein is amplified and fused with the cap gene for expression, and the purified fusion protein can self-assemble into chimeric virus-like particles (cVLPs) in vitro for antigen preparation, thereby preparing the PCV2 vaccine.

[0041] Although previous studies have associated ANXA2 with the infection processes of various enveloped viruses such as influenza virus (IV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and the relatively structurally complex non-enveloped virus such as human papillomavirus (HPV), and it was selected for research. However, its role in simple and non-enveloped viruses such as PCV2 has not been reported. Therefore, this study further explored its potential role in PCV2 infection.

[0042] The present invention also provides the use of an ANXA2 inhibitor in the preparation of a drug for inhibiting the invasion of host cells by PCV2.

[0043] In the present invention, the ANXA2 inhibitor is used to inhibit the expression of ANXA2 protein in the host cell and / or the ANXA2 inhibitor is used to inhibit the functional activity of the ANXA2 protein.

[0044] In the present invention, the amino acid sequence of the ANXA2 protein is shown as SEQ ID NO.1, and SEQ ID NO.1 is the sequence of the porcine ANXA2 protein obtained after identification in the present invention; the nucleotide sequence of the porcine ANXA2 gene is referred to GenBank: NM_001005726.1.

[0045] In the examples of the present invention, GenBank: NM_001005726.1 was used as the nucleotide sequence of the porcine ANXA2 gene for experiments; SEQ ID NO.1 was used as the amino acid sequence of the porcine ANXA2 protein for experiments.

[0046] In the present invention, the amino acid sequence of SEQ ID NO.1 is MSTVHEILCKLSLEGDHSTPASAYGSVKAY TNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNEQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDVPKWISIMTERSVCHLQKVFERYKSYSPYDMLESIKKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKGKGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYNYIQQDTKGDYQKALLYLCGGDD.

[0047] The amino acid sequence of the ANXA2 protein in NCBI (GENBANK NO.NM_001005726.1) is shown as SEQ ID NO.2: MSTVHEILCKLSLEGDHSTPASAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTI VNILTNRSNEQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDVPKWISIMTERSVCHLQKVFERYKSYSPYDMLESIKKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKGKGTRDKVLIXIMVSRSEVDMLKIRSEFKRKYGKSLYNYIQQDTKGDYQKALLYLCGGDD. In the present invention, the amino acid sequence of the ANXA2 protein may also be shown as SEQ ID NO.2; it should be noted that, for the purpose of difference explanation, in SEQ ID NO.2, one amino acid is represented by X, and at this position in SEQ ID NO.1, it is specifically represented as R.

[0048] In the present invention, the ANXA2 inhibitor includes a specific shRNA oligonucleotide sequence targeting porcine ANXA2; the specific shRNA oligonucleotide sequence includes shANXA2-1 or shANXA2-2; the nucleotide sequence of the sense strand in shANXA2-1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the anti-sense strand in shANXA2-1 is as shown in SEQ ID NO.8; the nucleotide sequence of the sense strand in shANXA2-2 is as shown in SEQ ID NO.9, and the nucleotide sequence of the anti-sense strand in shANXA2-2 is as shown in SEQ ID NO.10. That is, shANXA2-1 and shANXA2-2 are two specific short hairpin RNAs (shRNAs) targeting porcine ANXA2.

[0049] In the present invention, the ANXA2 inhibitor includes a specific inhibitor that selectively inhibits the ANXA2 / S100A10 heterotetramer (A2t). In the present invention, A2ti-1, a specific inhibitor that selectively inhibits the ANXA2 / S100A10 heterotetramer (A2t), is used; since the A2t complex is crucial for the membrane localization and functional activity of ANXA2 in various cellular processes, inhibiting A2t can evaluate the role of membrane-associated ANXA2 in PCV2 replication.

[0050] In the present invention, the specific inhibitor includes A2ti-1; in the present invention, the inhibitor A2ti-1 is from MedChem Express, USA.

[0051] In the present invention, the host cell is a porcine cell; during the experiment, the porcine cell may include porcine kidney epithelial cells (PK-15); the PCV2 may include the PCV2d strain (Genbank: MH718995); in the examples of the present invention, the PCV2d strain (Genbank: MH718995) is used for infection, and at the same time, this strain is selected to amplify the cap gene. The assembly method of PCV2 VLPs is to put the purified PCV2 Cap protein into a dialysis bag and place it in 1 L of dialysis solution, and dialyze at 4°C for 48 h for in vitro self-assembly.

[0052] In the present invention, the host cell has or is capable of expressing ANXA2 protein.

[0053] The present invention also provides a method for preparing a drug for inhibiting PCV2 from invading host cells, including: using the ANXA2 inhibitor as the active component of the drug for inhibiting PCV2 from invading host cells.

[0054] The present invention also provides an application of an ANXA2 inhibitor in inhibiting the invasion of PCV2 into host cells for non-therapeutic purposes; for example, in scientific research experiments, the ANXA2 inhibitor can be used to explore the dynamic changes during the process of PCV2 invading cells, which helps to analyze the invasion mechanism of PCV2; meanwhile, in high-density farming, the ANXA2 inhibitor can also be used as a coating material for farming equipment to reduce the spread of the virus.

[0055] The following are specific examples of the present invention:

[0056] Example 1

[0057] ANXA2 was identified as a PCV2 Cap binding protein by LC-MS / MS:

[0058] Inoculate PK-15 cells into a cell culture dish with a diameter of 10 cm. When the cells are cultured to form a monolayer with a fusion degree of about 90%, discard the culture medium and wash the cells three times with PBS. Subsequently, change to a serum-free and antibiotic-free medium, add the correctly identified PCV2 VLPs to the medium, mix well and incubate. The cells without VLPs are used as a control; Incubate the commercial magnetic beads and the anti-Cap specific antibody at 4°C for 2 h to obtain an antibody-magnetic bead complex; Discard the cell culture medium, wash three times with PBS, then add 500 μL of cell lysate to the culture dish and let it stand on ice for 30 min. After centrifugation at 10000×g for 1 min, take the supernatant, which is the total cell protein (antigen); Add the total cell protein to the antibody-magnetic bead complex and incubate overnight at 4°C to obtain an antigen-antibody-magnetic bead immune complex; Place the immunocomplex centrifuge tube on a magnetic rack to adsorb the magnetic beads, discard the liquid, wash three times with PBS, resuspend the magnetic beads with 50 μL of elution buffer, let it stand and incubate for 5 min, then separate the magnetic beads with a magnetic rack. The obtained supernatant is the eluted immunoprecipitate; Take 40 μL of the immunoprecipitate, add 10 μL of 5×SDS loading buffer containing a reducing agent, and boil for 5 min; Load the treated sample into the loading well for SDS-PAGE electrophoresis. After the electrophoresis is completed, place the gel in Coomassie Brilliant Blue staining solution and boil for 5 min for staining; Use a gel cutter to cut along the protein bands on the separating gel, and put the cut gel strips into a centrifuge tube. These strips contain the target protein and all its binding proteins; Digest the gel strips with trypsin, extract the peptides, and put them into a liquid chromatography-tandem mass spectrometer (LC-MS / MS) for analysis. Finally, perform mass spectrometry analysis on the gel strips.

[0059] Experimental results:

[0060] As shown in Table 1, multiple host proteins that may interact with PCV2 Cap were found; among them, ANXA2 was selected for further study because of its multiple biological functions in cell membrane repair, cytoskeleton remodeling, and intracellular and extracellular transport.

[0061] Table 1 Host cell proteins potentially interacting with PCV2-VLPs identified by LC-MS / MS in PK-15 cells

[0062]

[0063] PSMs: Peptide spectrum matches.

[0064] Example 2

[0065] PCV2 infection induces ANXA2 expression:

[0066] To investigate the effect of PCV2 infection on ANXA2 expression, PK-15 cells infected with PCV2 were collected and analyzed by RT-qPCR and western blot.

[0067] Virus infection: PK-15 cells were seeded in 6-well cell culture plates and cultured to confluence, then changed to serum-free and antibiotic-free medium. PCV2 was used to infect the cells at doses of MOI = 0.5, 1, and 5 respectively. After 1 h of infection, the cells were changed to fresh medium containing serum and continued to be cultured. Normal cells without virus infection were used as a control (Mock group). 48 h after virus infection, the cells were washed three times with PBS and then subjected to subsequent experiments.

[0068] RT-qPCR detection: 500 μL of Trizol was added to each well to extract total cellular RNA. cDNA of the cells was obtained through a reverse transcription kit. The cDNA samples were subjected to PCR amplification using a commercial ChamQ Universal SYBR qPCR Master Mix enzyme. By normalizing the mRNA level to the internal reference gene TBP and using the 2^-ΔΔCT method to calculate the relative mRNA level. In this experiment, the qPCR primer sequences for porcine ANXA2 were: pANXA2-F (GGAGTGTGTGTCACCTCCAG, denoted as SEQ ID NO.3) and pANXA2-R (AGTTGTACAGGGACTTGCCG, denoted as SEQ ID NO.4), and the qPCR primer sequences for the internal reference gene TBP were: pTBP-F (GATGGACGTTCGGTTTAGG, denoted as SEQ ID NO.5) and pTBP-R (AGCAGCACAGTACGAGCAA, denoted as SEQ ID NO.6).

[0069] Western blot assay: In another cell plate, 200 μL of cell protein lysate was added to each cell well, and the mixture was allowed to stand on ice for 30 min. Subsequently, it was centrifuged at 10,000×g for 5 min, and the supernatant sample was collected. Protein quantification was performed using a BCA kit. 100 μL of the quantified protein sample was taken, and 25 μL of 5×SDS loading buffer containing a reducing agent was added to each sample. After mixing the samples evenly, they were boiled in boiling water for 5 minutes. The protein samples were added to a precast protein gel for electrophoresis separation at 200 V for 30 min. After electrophoresis, the protein gel was placed on a PVDF membrane, and the protein was transferred to the PVDF membrane through a transfer system. Subsequently, the membrane was immersed in 5% non-fat milk powder and incubated at 37 °C for 1 h for blocking treatment. Rabbit anti-ANXA2 antibody (at a ratio of 1:2000), rabbit anti-Cap protein antibody (1:1000), and rabbit anti-actin antibody (1:5000) were diluted according to the ratio and used as primary antibodies and added to the membrane. It was incubated at 37 °C for 1 h, then washed three times with TBST solution, and then incubated with a rabbit-derived HRP-labeled secondary antibody at 37 °C for 1 h. After washing three times with TBST, ECL color development was used, and color development and photography were performed using a chemiluminescence imager. ImageJ software was used to quantify the bands, and the gray-scale ratio of ANXA2 protein to the internal reference protein (actin) was analyzed to evaluate the change in protein expression level.

[0070] Experimental results:

[0071] See Figure 1 and Figure 2 As shown, the mRNA and protein levels of ANXA2 increased in a dose-dependent manner during PCV2 infection; the results indicated that PCV2 infection promoted ANXA2 expression in PK-15 cells.

[0072] Example 3

[0073] Knockdown of ANXA2 inhibits PCV2 infection:

[0074] 1. To explore the effect of ANXA2 on PCV2 proliferation, two specific shRNA sequences targeting ANXA2 were designed to reduce its mRNA level.

[0075] Two specific short hairpin RNAs (shRNAs) targeting ANXA2 were designed using the BLOCK-iT TM RNAi Designer online tool. Meanwhile, a sequence targeting a non-specific region was designed and named shNC. All sequences were synthesized by Beijing Tsing ke Company and cloned into the pLKO.1-EGFP-Puro vector to construct recombinant plasmids.

[0076] PK-15 cells were seeded in cell culture plates. The cells were starved for 1 h before transfection. Two kinds of shRNAs targeting ANXA2 specifically, shNC, and the empty vector pLKO.1-EGFP-Puro (Mock group) were mixed with Opti-MEM at a plasmid DNA amount of 2 μg per well TM to prepare Solution A. 4 μL of Lipofectamine 2000 was mixed with Opti-MEM TM to prepare Solution B. Solution A and Solution B were each allowed to stand at room temperature for 5 min and then mixed together to form a complex. The complex was allowed to stand at room temperature for 20 min and then added to the cell culture medium. The cells were cultured at 37 °C for 6 h, and then replaced with fresh medium containing serum and continued to be cultured. At 24 h after transfection, the RNA of the cells in the shNC group, shANXA2-1 group, and shANXA-2 group was extracted, and the change in the mRNA level of ANXA2 was detected by RT-PCR (the method and detection primers were the same as in Example 2); at 24 h after transfection, the total proteins of the cells in the empty vector control group (Mock), shNC group, shANXA2-1 group, and shANXA-2 group were extracted, and anti-ANXA2 specific antibody and anti-actin antibody were used for western blot to detect the endogenous ANXA2 protein level.

[0077] In this example, referring to Figure 3 the design principle, two specific shRNAs were obtained, namely shANXA2-1 or shANXA2-2.

[0078] The nucleotide sequence of the sense strand in shANXA2-1 is shown in SEQ ID NO.7, and the nucleotide sequence of the anti-sense strand in shANXA2-1 is shown in SEQ ID NO.8.

[0079] The nucleotide sequence of SEQ ID NO.7 is 5’-CCGGGCCTTTGCCTACCAAAGAAGGTCAAGAGCCTTCTTTGGTAGGCAAAGGCTTTTT-3’.

[0080] The nucleotide sequence of SEQ ID NO.8 is 5’-AATTAAAAAGCCTTTGCCTACCAAAGAAGGCTCTTGACCTTCTTTGGTAGGCAAAGGC-3’.

[0081] The nucleotide sequence of the sense strand in shANXA2-2 is shown in SEQ ID NO.9, and the nucleotide sequence of the anti-sense strand in shANXA2-2 is shown in SEQ ID NO.10.

[0082] The nucleotide sequence of SEQ ID NO.9 is 5'-CCGGGCGTGATAAGGTCCTGATTAGTCAAGAGCTAATCAGGACCTTATCACGCTTTTT-3'.

[0083] The nucleotide sequence of SEQ ID NO.10 is 5'-AATTAAAAAGCGTGATAAGGTCCTGATTAGCTCTTGACTAATCAGGACCTTATCACGC-3'.

[0084] Experimental results:

[0085] In PK-15 cells, both shRNAs could reduce the abundance of ANXA2 mRNA. The knockdown efficiency of shANXA2-1 reached 68.1%, significantly higher than 36.4% of shANXA2-2 ( Figure 4 ); correspondingly, the ANXA2 protein levels were reduced by approximately 40.0% and 27.5% respectively ( Figures 5-6 ).

[0086] 2. PK-15 cells were seeded in cell culture plates. The cells were starved for 1 h before transfection. The shANXA2-1 recombinant plasmid was selected. Complexes were prepared with 2 μg, 3 μg, and 4 μg of plasmid DNA and Lipofectamine 2000 respectively. At the same time, the pLKO.1-EGFP-Puro empty vector (Mock group) and shNC were used to prepare complexes with 4 μg of plasmid DNA and Lipofectamine 2000. At 24 h after transfection, the cell culture medium was discarded and replaced with serum-free and antibiotic-free medium. Subsequently, each group of cells was infected with MOI = 1 PCV2. After 1 h of virus infection, the medium was replaced with serum-containing medium and continued to be cultured at 37°C for 36 h. Subsequently, cell proteins were extracted and western blot was performed using anti-Cap protein antibody and anti-actin antibody to detect the change in Cap protein level.

[0087] Experimental results:

[0088] Compared with the untransfected control group, shANXA2-1 reduced the expression level of PCV2 Cap in a dose-dependent manner ( Figures 7-8 ); indicating that ANXA2 is crucial for the efficient replication and proliferation of PCV2.

[0089] Example 4

[0090] A2ti-1 inhibits the in vitro proliferation of PCV2:

[0091] 1. The cytotoxicity of A2ti-1 was evaluated by CCK-8 assay. PK-15 cells were seeded in 96-well plates and cultured until they adhered. The inhibitor A2ti-1 was dissolved in DMSO to prepare a stock solution with a concentration of 50 mM. Subsequently, the inhibitor was diluted with a medium containing 2% FBS to set the final concentration gradients at 20, 40, 60, 80, and 120 μM (as the inhibitor treatment groups). The DMSO group diluted with 2% FBS medium was used as the solvent treatment group. Cells treated with only 2% FBS medium without any other treatment were used as the negative control group, and the group with only 2% FBS medium without cells was used as the blank control group. Each group was set with 5 replicate wells, and the total volume of the medium in each well was 100 μL. After culturing at 37 °C for 48 h, 10 μL of CCK-8 reagent was added to each well of the 96-well plate, and then cultured in the dark at 37 °C for 2 h. The cell plate was placed on an enzyme-linked immunosorbent detector to detect the absorbance at OD 450 nM. Cell viability (%) = (A treatment group - A blank group) / (A negative control - A blank group)]

[0092] × 100%, and the optimal inhibitor concentration was determined by analyzing cell viability.

[0093] Experimental results:

[0094] See Figure 9 As shown, the results showed that the maximum safe concentration of A2ti-1 in PK-15 cells was 60 μM.

[0095] 2. One hour before PCV2 infection, cells were pretreated with A2ti-1 at a concentration of 40 or 60 μM. The inhibitor A2ti-1 was prepared into solutions with final concentrations of 40 μM and 60 μM respectively using serum-free basal medium and added to the cells. At the same time, 0.2% DMSO was used as the negative control group, and cells without any treatment were used as the blank control group. After the cells were incubated for 2 h, they were washed three times with PBS; the MOI = 1 PCV2 virus was added to the inhibitor group and the DMSO control group. After incubation for 1 h, they were washed three times with PBS, and then a serum-containing medium containing the corresponding inhibitor concentration or DMSO was added, and the cells were cultured for another 36 h.

[0096] Western blot detection: Cell proteins were extracted, and anti-Cap antibody or anti-actin antibody was used as the primary antibody for western blot to detect the change in Cap protein level.

[0097] Genomic copy number detection: Cell DNA was extracted, and the plasmid containing the PCV2 genome was used as the standard plasmid. The change in the PCV2 genomic DNA copy number in the cells was detected by fluorescence quantitative PCR.

[0098] Immunofluorescence assay: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, incubated with anti-Cap antibody at 37 °C for 1 h. After washing three times with PBS, Cap protein was labeled with donkey anti-mouse Alexa Fluor 488 fluorescent antibody (green), and the cell nuclei were stained with DAPI (blue). The samples were observed under an inverted fluorescence microscope.

[0099] Experimental results:

[0100] See Figure 10 As shown, Western blot analysis showed that with the increase in the concentration of A2ti-1, the expression level of Cap protein decreased in a dose-dependent manner. At a concentration of 60 μM, the Cap expression level decreased by approximately 60% compared to the untreated group.

[0101] See Figure 11 As shown, the intracellular viral genome content decreased significantly. After treatment with A2ti-1 at concentrations of 40 or 60 μM, it decreased to 48% and 29% of the untreated group, respectively.

[0102] See Figure 12 As shown, immunofluorescence assay showed a similar trend. With the increase in the concentration of A2ti-1, the expression level of Cap protein (green fluorescence) decreased in a dose-dependent manner.

[0103] In summary, the specific inhibitor targeting ANXA2 significantly reduced the expression of PCV2 Cap protein and the proliferation level of its genome in PK-15 cells.

[0104] Example 5

[0105] Surface ANXA2 mediates the binding of PCV2 to host cells:

[0106] Antibody blocking experiment: PK-15 cells were cultured to adhere to a monolayer, washed three times with pre-cooled PBS, changed to serum-free medium for starvation treatment for 1 h. ANXA2-specific antibody or isotype control antibody was diluted to a final concentration of 30 mg / mL with pre-cooled serum-free medium. Cells without any treatment were used as the control group. The cells were incubated at 4 °C for 1 h, and then PCV2 was used to infect the above three groups of cells. After incubation at 4 °C for 1 h, the cells were washed three times with PBS to remove unbound virus, and cell DNA was extracted to detect the PCV2 genome copy number.

[0107] Experimental results:

[0108] See Figure 13 As shown, after treatment with ANXA2-specific antibody, the attached PCV2 particles decreased by approximately 25.2% compared to the untreated group; in contrast, the isotype control group did not show a significant effect.

[0109] Example 6

[0110] Interaction between PCV2 Cap and ANXA2:

[0111] To verify the interaction between Cap and ANXA2, an immunoprecipitation experiment was performed by co-transfecting plasmids expressing Cap (tagged with Flag) and ANXA2 (tagged with HA) into 293FT cells.

[0112] 293FT cells were seeded in 6-well plates. Before transfection, the cells were replaced with serum-free medium and starved for 1 h. Group A: 2 μg of pCI-neo-Cap (tagged with Flag) plasmid or 2 μg of pCDNA3.4-ANXA2 (tagged with HA) plasmid were first separately complexed with 4 μL of Lipofectamine 2000, and then mixed together to form a complex; Group B: 2 μg of pCI-neo-Cap (tagged with Flag) was complexed with Lipofectamine 2000; Group C: 2 μg of pCDNA3.4-ANXA2 (tagged with HA) was complexed with Lipofectamine 2000; After the complexes were left standing at room temperature for 20 min, they were added to the cell culture medium. The cells were cultured at 37 °C for 6 h, and then replaced with fresh medium containing serum and continued to be cultured. After 48 h of transfection, the cells were washed three times with PBS and then added with WB / IP lysis buffer and left standing on ice for 30 min to fully lyse. The cells were centrifuged at 10,000×g for 5 min; The supernatant was incubated with magnetic beads tagged with HA or magnetic beads tagged with Flag by rotation overnight at 4 °C. The samples were eluted with 2×SDS loading buffer. After boiling the mixture for 5 min, electrophoresis and membrane transfer were performed, and western blot detection was carried out using anti-HA or anti-Flag tag antibody as the primary antibody.

[0113] Experimental results:

[0114] See Figure 14 for understanding. The results showed that exogenous ANXA2 interacted with Cap. ANXA2 played a key role in promoting virus adsorption to host cells. Given that Cap plays an important role in the process of virus entry into cells through interaction with host proteins, the direct interaction between Cap and ANXA2 is crucial for this process.

[0115] Example 7

[0116] Molecular docking analysis of Cap and ANXA2:

[0117] To preliminarily reveal the interaction mechanism between ANXA2 and Cap, the binding mode of the Sus scrofa ANXA 2-Cap protein complex was analyzed by AlphaFold3.

[0118] Analysis results:

[0119] See Figure 15 for understanding. The molecular docking results showed that Cap and ANXA2 could form a stable docking interface, and there were multiple hydrogen bonds between the interfaces. These interactions were mainly formed by the loop regions of Cap (loop CD: Asp78; loop EF: Asn128, Thr131, Lys132; loop GH: Gly169, Asp172) and Asp70 on the β-sheet with ANXA 2 domain II (amino acids 122–174). This result further revealed the key role of ANXA2 domain II in the interaction with Cap, and at the same time also demonstrated the interface characteristics of the binding between Cap and ANXA2, providing an important basis for further studying the interaction mechanism between PCV2 virus and host proteins.

[0120] In the above technical solutions of the present invention, the above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made under the technical concept of the present invention by using the content of the specification and drawings of the present invention, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. Use of ANXA2 protein as a target in the preparation of a drug for inhibiting PCV2 from invading host cells or in the preparation of a PCV2 vaccine.

2. Use of an ANXA2 inhibitor in the preparation of a drug for inhibiting PCV2 from invading host cells.

3. The application according to claim 2, wherein The ANXA2 inhibitor is used to inhibit the expression of ANXA2 protein in the host cell and / or the ANXA2 inhibitor is used to inhibit the functional activity of the ANXA2 protein.

4. The application according to claim 3, characterized in that The amino acid sequence of the ANXA2 protein is as shown in SEQ ID NO.1 or as shown in SEQ ID NO.

2.

5. The application according to claim 2, characterized in that The ANXA2 inhibitor includes a specific shRNA oligonucleotide sequence targeting porcine ANXA2; the specific shRNA oligonucleotide sequence includes shANXA2-1 or shANXA2-2; the nucleotide sequence of the sense strand in shANXA2-1 is as shown in SEQ ID NO.7, and the nucleotide sequence of the anti-sense strand in shANXA2-1 is as shown in SEQ ID NO.8; the nucleotide sequence of the sense strand in shANXA2-2 is as shown in SEQ ID NO.9, and the nucleotide sequence of the anti-sense strand in shANXA2-2 is as shown in SEQ ID NO.

10.

6. The application according to claim 2, wherein The ANXA2 inhibitor includes a specific inhibitor that selectively inhibits the ANXA2 / S100A10 heterotetramer.

7. The application according to claim 6, characterized in that, The specific inhibitor includes A2ti-1.

8. The application according to any one of claims 2-7, characterized in that, The host cell is a porcine-derived cell; the porcine-derived cell includes porcine kidney epithelial cells.

9. A method for preparing a drug for inhibiting PCV2 from invading host cells, characterized in that, Comprising: Using the ANXA2 inhibitor as an active ingredient of a drug for inhibiting PCV2 from invading host cells.

10. Use of an ANXA2 inhibitor in inhibiting PCV2 from invading host cells for non-therapeutic purposes.

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