Polypeptides, pharmaceutical compositions and uses for treating cysticercosis virus infection

Abstract

This invention belongs to the field of biotechnology, specifically relating to a polypeptide, pharmaceutical composition, and application for treating sacbrood virus (SBV) infection. The polypeptide is derived from the heparin-binding motif of SBV drug-binding pocket protein 2 by replacing A3 with a basic amino acid, N4 with a hydrophobic amino acid, and / or adding a hydrophobic amino acid to the C-terminus. This invention also provides the application of the heparin-binding motif, polypeptide, and antiserum in the preparation of drugs and feed additives for the prevention / treatment of SBV infection, and the application of the heparin-binding motif, polypeptide, and antiserum in the preparation of drugs that block SBV infection in bee larvae. This invention discovers that the heparin-binding motif and its derived peptides have the effect of blocking SBV infection in bee larvae, providing a basis for understanding the SBV-GAG interaction and a reference for developing polypeptides for treating SBV infection based on this interaction.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polypeptide, pharmaceutical composition, and application for treating cysticercosis virus infection. Background Technology

[0002] Sarbood virus (SBV) is a small RNA-like virus in insects that harms the health of bees and is prevalent in many countries around the world. Sarbood primarily affects bee larvae, causing metabolic abnormalities and tissue damage, preventing the larvae from developing into pupae. SBV-rich molting fluid accumulates under the unshed skin, forming sacs.

[0003] Over the past decade, SBV has caused losses to the healthy development of the beekeeping industry and the economic benefits for beekeepers. Although there have been reports of using queen bee replacement, RNAi (RNA interference), and medicinal plant extracts for the prevention and control of SBV infection, these methods still have limitations. Queen bee replacement and "queen imprisonment and brood cutting" are comprehensive management measures for bee disease control, which require a high level of beekeeping skills. While RNAi technology is effective in treating sacbrood, it is prone to off-target effects, which may affect the treatment outcome. Bees are quite sensitive to odors; the pungent odors of medicinal plants and the organic solvents used to extract their active ingredients may cause bee colonies to abscond. In addition, the origin of medicinal herbs has the greatest impact on the efficacy of traditional Chinese medicine; different origins can lead to significant differences in efficacy. Therefore, the industrialization of traditional Chinese medicine preparations is a reliable way to ensure their quality stability.

[0004] The outcome of microbial infections largely depends on the pathogen's ability to utilize and disrupt host components and their activity. Among host components, glycosaminoglycans (GAGs) are a major target for pathogens. The functional conservation of pathogen GAG binding sites has been demonstrated, and pathogens utilize GAGs to promote adhesion and invasion of host cells in almost all infection and pathogenesis processes. GAGs are anionic linear polysaccharides, including heparin and heparan sulfate, composed of repeating disaccharides, and are ubiquitous on the surface of animal cells. The distribution of GAGs within animal tissues varies, and pathogens may utilize a single GAG ​​molecule as an adhesion receptor to achieve infection. Blocking pathogen binding to GAGs may block infection. Developing preventative and therapeutic drugs targeting the binding of pathogens to host cell surface GAGs to block pathogen invasion is a new research area, with antimicrobial peptides and antiviral peptides (AVPs) being among the emerging research areas. In practice, bioactive and safe peptides offer alternative therapeutic measures for bee health and have shown potential as effective drugs against SBV infection.

[0005] SBV is a single-stranded positive-sense RNA virus. The entire SBV genome contains only one open reading frame (ORF). The polyprotein it encodes is cleaved and processed at its amino terminus by the virus's own encoded protease to produce mature structural proteins. In mammalian small RNA viruses, each capsid protein contains a drug-binding pocket domain. These proteins are host receptor binding sites and can bind to GAGs on the host cell surface. SBV capsid proteins contain small RNA virus capsid protein drug-binding pocket domains, with the second domain being the largest.

[0006] Currently, those skilled in the art have limited understanding of the SBV-GAG interaction, and effective methods available for the prevention and control of sacbrood diseases remain limited. Therefore, further exploration of the SBV-GAG interaction is needed to develop effective drugs for the treatment of SBV infection. Summary of the Invention

[0007] This study aimed to identify the heparin-binding motif of drug-binding pocket protein 2 (Dbpp2) and to determine the anti-infective activity of the motif and its derived peptides. This patented study found that the heparin-binding motif and its derived peptides can block SBV infection in wasp larvae, providing support for the development of products based on interactions to treat SBV infection.

[0008] In view of this, one of the objectives of the present invention is to provide a series of peptides based on SBV-GAG interactions, which are derived from the heparin-binding motif of SBV Dbpp2 and have hemolytic and antibacterial activities, thus supporting the development of drugs for the treatment of SBV infection.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The polypeptide based on SBV-GAG interaction is a polypeptide derived from the heparin-binding motif of SBV drug-binding pocket protein 2 by replacing A3 with a basic amino acid, N4 with a hydrophobic amino acid, and / or adding a hydrophobic amino acid to the C-terminus; the amino acid sequence of the heparin-binding motif is shown in SEQ ID NO: 1.

[0011] In this context, A3 represents alanine (A) located at the third position of the motif; N4 represents asparagine (N) located at the fourth position of the motif.

[0012] Furthermore, the basic amino acid is arginine (R) and / or lysine (K).

[0013] Furthermore, the hydrophobic amino acid includes any one or more of alanine, phenylalanine, proline, tryptophan, valine, leucine, isoleucine, and methionine.

[0014] Furthermore, the hydrophobic amino acid is preferably alanine and / or tryptophan.

[0015] This invention has revealed that the R motif in the heparin binding motif plays a crucial role in the binding of heparin to Dbpp2.

[0016] Furthermore, the amino acid sequences of the polypeptide are as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and / or as shown in SEQ ID NO: 6.

[0017] Furthermore, the heparin-binding motif and the polypeptide have hemolytic activity, with the hemolytic activity increasing in the order of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0018] Furthermore, the polypeptides have antibacterial activity, with the antibacterial activity increasing in the order of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0019] Furthermore, replacing A3 in the heparin-binding motif of Dbpp2 with a basic amino acid, replacing N4 with a hydrophobic amino acid, and / or adding a hydrophobic amino acid to the C-terminus can effectively improve its hemolytic and antibacterial activities.

[0020] The in vitro antibacterial activity of the heparin-binding motif and / or the polypeptide is positively correlated with its hemolytic activity.

[0021] A second objective of this invention is to provide an antiserum that can be used to treat cysticercosis virus infection.

[0022] To achieve the above objectives, the present invention adopts the following technical solution:

[0023] The antiserum is obtained by immunizing mice with a drug-binding pocket protein 2 containing the polypeptide described in Target 1 as an antigen; the nucleotide sequence of the drug-binding pocket protein 2 is shown in SEQ ID NO: 7.

[0024] This invention optimizes the coding gene of Dbpp2 and its mutants based on the codon preference of Escherichia coli; adds BamHI and XhoI restriction endonuclease recognition sites to the 5' and 3' ends of the gene, respectively; synthesizes the gene using a chemical synthesis method, clones it into pGEX-4T-1 to construct a recombinant expression vector; induces Escherichia coli BL21 strain containing the recombinant plasmid with IPTG, and purifies the recombinant protein; and prepares antiserum by immunizing mice with the purified recombinant protein Dbpp2.

[0025] A third objective of this invention is to provide the application of SBV drug-binding pocket protein 2 heparin-binding motifs, peptides, and / or antiserum in the preparation of drugs and / or feed additives for the prevention and / or treatment of cysticercosis virus infection.

[0026] To achieve the above objectives, the present invention adopts the following technical solution:

[0027] The application of the heparin-binding motif of SBV drug-binding pocket protein 2, the polypeptide described in objective one, and / or the antiserum described in objective two in the preparation of drugs and / or feed additives for the prevention and / or treatment of cysticercosis virus infection, wherein the amino acid sequence of the heparin-binding motif is shown in SEQ ID NO: 1, and the amino acid sequences of the polypeptide are shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and / or as shown in SEQ ID NO: 6.

[0028] The fourth objective of this invention is to provide the application of SBV drug-binding heparin-binding motif, polypeptide, and / or antiserum of pocket protein 2 in the preparation of a drug that blocks bee larvae infection by cysticercosis virus.

[0029] To achieve the above objectives, the present invention adopts the following technical solution:

[0030] The application of SBV drug binding to the heparin-binding motif of pocket protein 2, the polypeptide described in objective one, and / or the antiserum described in objective two in the preparation of a drug to block bee larvae infected with cysticercosis virus, wherein the amino acid sequence of the heparin-binding motif is shown in SEQ ID NO: 1, and the amino acid sequences of the polypeptide are shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and / or as shown in SEQ ID NO: 6.

[0031] Furthermore, the heparin-binding motif prevents the cysticercosis virus from adhering to or invading host cells by inhibiting the binding of the drug to pocket protein 2 to heparin.

[0032] Furthermore, the heparin binding motif inhibitor binds to pocket protein 2 in a dose-dependent manner.

[0033] Furthermore, the anti-SBV infection efficacy of the polypeptide, the antiserum, and the heparin-binding motif is ranked as follows: antiserum > SEQ ID NO: 4 > SEQ ID NO: 5 > SEQ ID NO: 3 > SEQ ID NO: 6 > SEQ ID NO: 2 > SEQ ID NO: 1.

[0034] Microcalorimetric analysis and fluorescence spectroscopy data on the interaction between membrane-disrupting peptides and model membranes showed that peptides preferentially bind to negatively charged bacterial and cancer cell membranes compared to neutral eukaryotic cell membranes. Peptide binding disrupts the regular membrane bilayer structure. Cationic antimicrobial or antiviral peptides are rich in hydrophobic and basic amino acids; W and R residues are particularly important for their activity, and these two amino acids are also ubiquitous in proteins or peptides with membrane-disrupting activity, allowing these molecules to spontaneously cross membranes. Unlike antibacterial mechanisms, peptides' antiviral effects primarily involve interaction with receptors to prevent viral adhesion or invasion of host cells. Peptide development requires practical application in animals to evaluate their therapeutic efficacy. In bee colony applications, the peptide (KPRARPRR) with the amino acid sequence shown in SEQ ID NO: 4 showed the best therapeutic effect against SBV-infected honeybee larvae, with a therapeutic efficacy of 65.4%. This provides a reference for screening potentially valuable peptides using pathogen-GAG interactions.

[0035] A fifth objective of this invention is to provide a pharmaceutical composition for the prevention and / or treatment of cysticercosis virus infection.

[0036] To achieve the above objectives, the present invention adopts the following technical solution:

[0037] A pharmaceutical composition for the prevention and / or treatment of cysticercosis virus, said pharmaceutical composition comprising the polypeptide of objective one, a heparin-binding motif of SBV drug-binding pocket protein 2 and / or the antiserum of objective two, and a pharmaceutically acceptable carrier.

[0038] The beneficial effects of this invention are as follows:

[0039] 1. This patented study found that SBV and Dbpp2 can bind to heparin.

[0040] 2. This patent study found that R290, R292, and R293 on the C-terminal flexible ring (KPANRPRR) make significant contributions to the binding of Dbpp2 to heparin, indicating that KPANRPRR is a heparin-binding motif of Dbpp2 and can inhibit the binding of Dbpp2 to heparin.

[0041] 3. This patent study found that the heparin-binding motif (KPANRPRR) and its derived peptides both have hemolytic activity. Replacing A3R and N4 with hydrophobic amino acids and adding hydrophobic amino acids to the C-terminus helps to improve hemolytic and antibacterial activities.

[0042] 4. This patent confirms that the heparin-binding motif (KPANRPRR) and its derived peptides have the effect of blocking bee larvae infected with SBV; this patent provides a basis for understanding the SBV-GAG interaction and provides a reference for developing peptides for treating SBV infection based on the interaction. Attached Figure Description

[0043] Figure 1 shows the Dbpp2 GAG binding motif prediction. Specifically... Figure 1A The distribution of R and K in the entire length Dbpp2 is shown. Figure 1B This is a distribution diagram of basic amino acids in the flexible ring (KPANRPRR);

[0044] Figure 2 The image shows the results of heparin inhibiting SBV binding to heparin agarose beads, where M represents the protein marker.

[0045] Figure 3 The graph shows the results of Dbpp2 binding to heparin analysis, where M represents the protein marker.

[0046] Figure 4 The graph shows the results of the heparin binding ability analysis of Dbpp2 and its mutants. In the graph, M represents the protein marker, 1 represents Dbpp23, 2 represents Dbpp22, 3 represents Dbpp21, 4 represents Dbpp2, and 5 represents Dbpp24.

[0047] Figure 5 The graph shows the results of the heparin-binding motif's inhibition of Dbpp2 binding to heparin, where M represents the protein marker.

[0048] Figure 6 The image shows the results of the peptide antibacterial activity assay. In the image, 1 represents KPAARPRR, 2 represents KPRNRPRR, 3 represents KPRARPRR, 4 represents KPANRPRR, 5 represents KPRWRPRRW, 6 represents KPRWRPRR, and 7 represents PBS. Detailed Implementation

[0049] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0050] In this embodiment of the invention, the virus, cells, strains and plasmids: SBV and Escherichia coli K88 strain were isolated and identified by the Veterinary Research Institute of Chongqing Academy of Animal Sciences; expression vector pGEX-4T-1, Escherichia coli DH5α, and BL21(DE3) were provided by Sangon Biotech (Shanghai) Co., Ltd. for a fee.

[0051] In this embodiment of the invention, heparin agarose beads were purchased from Solarbio; heparin and Freund's adjuvant were purchased from Sigma-Alorich; mouse anti-GST monoclonal antibody and alkaline phosphatase-labeled goat anti-mouse IgG antibody were purchased from EarthOx; nitrocellulose membrane was purchased from Merck Millipore; Gluthathione-Sepharose 4B and reduced glutathione were purchased from GE Healthcare; Quick Start Bradford was purchased from Bio-Rad Laboratories (Shanghai) Co., Ltd.; BCIP / NBT was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and the polypeptide composed of L-amino acids was chemically synthesized by Wuxi Maimer Top Biotechnology Co., Ltd., with a purity greater than 95%.

[0052] In this embodiment of the invention, 6-8 week old SPF-grade Kunming mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0053] In Example 2 of this invention, gene synthesis and vector construction were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0054] Example 1. Sequence Analysis

[0055] Using SBV-CQ (GenBank No. KJ716806) as the reference strain, the drug-binding pocket domain and its flanking amino acid sequences (positions 398-702) were selected as the research target and named Dbpp2. Secondary structure prediction (http: / / www.detaibio.com / tools / chou-fasman-forecast.html) and homology modeling (https: / / www.expasy.org / resources / swiss-model) were performed on the sequence using online software to search for flexible loops on the surface of the protein's spatial conformation, as well as basic amino acids and their adjacent hydrophobic amino acids, in order to search for potential heparin-binding motifs.

[0056] Results: Dbpp2 contains 22R and 13K amino acids, with the content of R and K basic amino acids being 11.5% (35 / 305). See details. Figure 1A Based on secondary structure prediction and homology modeling results, the C-terminus of Dbpp2 contains a flexible ring (KPANRPRR) rich in basic amino acids exposed on its surface. Within the ring are hydrophobic amino acids adjacent to the basic amino acids, which may be heparin-binding motifs. (See details...) Figure 1B This motif is also present in other SBV strains.

[0057] The binding of pathogens to GAGs primarily involves the R and K atoms on the flexible ring of pathogen surface proteins. The molecular basis of this binding is the formation of ionic bonds between the positively charged basic amino acid clusters on the flexible ring and the negatively charged GAG. Interactions between the hydrophobic amino acid side chains adjacent to the basic amino acids and the nonpolar groups of the glycans stabilize the protein-glycan binding. Searching for R and K atoms in SBV structural protein sequences revealed that capsid proteins containing small RNA virus drug-binding pocket domains all possess potential GAG adhesion motifs. Dbpp2 (amino acid regions 398-702 of the SBV-CQ partial polyprotein), containing the largest drug-binding pocket domain, was selected as the research focus. The amino acid region 429-739 of the SBV-CQ partial polyprotein was named VP1 and has been confirmed to interact with host cell proteins. Currently, the nomenclature of SBV capsid proteins is based on predictions of the hydrolytic characteristics of small RNA virus 3C proteases. However, the molecular weight predicted based on possible N-terminal cleavage sites on the polyprotein differs from the molecular weight obtained through protein electrophoresis. Based on the characteristic that SBV capsid proteins contain drug-binding pocket domains, this invention names SBV capsid proteins according to the order of these domains from the N-terminus to the C-terminus of the polymer. According to secondary structure prediction and homology modeling results, Dbpp2 has a flexible ring (KPANRPRR) rich in basic amino acids exposed on its C-terminus, with hydrophobic amino acids distributed within the ring adjacent to the basic amino acids. Comparison of the heparin binding affinity between Dbpp2 and its amino acid substitution mutant confirms that R290, R292, and R293 on the C-terminal flexible ring of Dbpp2 significantly contribute to heparin binding, and KPANRPRR is the heparin-binding motif of Dbpp2.

[0058] Example 2. Preparation of recombinant protein and its antiserum

[0059] The Dbpp2 mutant was designed by replacing amino acids in the potential heparin-binding motif; the coding genes of Dbpp2 and its mutants were optimized according to the codon preference of *E. coli*; BamHI and XhoI restriction endonuclease recognition sites were added to the 5' and 3' ends of the gene, respectively; the gene was synthesized chemically and cloned into pGEX-4T-1 to construct a recombinant expression vector; *E. coli* strain BL21(DE3) containing the recombinant plasmid was induced by IPTG, and the recombinant protein was purified; the purified recombinant protein Dbpp2 was used to immunize mice to prepare antiserum; the protein and antiserum preparation methods were performed according to the methods reported in the literature (Shen Kefei, Zhang Yifan, Cao Lan, et al. Expression of structural protein of *Apis sacbrood disease* virus and preparation of its antiserum. *Chinese Journal of Biological Products*); the protein concentration was determined by the Bradford method.

[0060] Example 3. SBV Extraction

[0061] Take the SBV-infected dead larvae sample stored at -80℃, add 5 volumes of Tris-HCl (0.02 mol / L, pH 8.0), and grind thoroughly. Add 1 / 2 volume of chloroform and shake thoroughly. Centrifuge at 12000×g for 10 min at 4℃, and collect the supernatant; repeat the treatment with 1 / 2 volume of chloroform until the supernatant is clear.

[0062] Example 4. SBV Heparin Binding Analysis

[0063] Heparin was dissolved in Tris-HCl solution; different concentrations of heparin (152 μL) were added to 1.5 mL centrifuge tubes along with heparin agarose beads (10 μL) and chloroform-extracted virus (18 μL), respectively, and mixed well; the concentrations of heparin were 0, 10 mg / mL, 30 mg / mL, and 90 mg / mL, respectively; the samples were incubated at 4 °C with gentle shaking for 1 h; centrifuged at 3000 r / min for 10 min at 4 °C, and the precipitate was retained; the sample was added to loading buffer, boiled in a water bath for 10 min, separated by 12% SDS-PAGE, and electrotransferred to a nitrocellulose membrane, blocked with 5% skim milk powder at room temperature for 2 h; washed 3 times with TBST; Dbpp2 antiserum (1:500 dilution) was added and incubated at room temperature for 1 h; washed 3 times with TBST; alkaline phosphatase-labeled goat anti-mouse IgG (1:4000 dilution) was added and incubated at room temperature for 1 h; washed 3 times with TBST; and developed in BCIP / NBT solution.

[0064] Result: As Figure 2 As shown, within the heparin concentration range of 90 mg / mL, the amount of SBV binding to heparin agarose beads decreased with increasing heparin concentration, exhibiting a good dose-dependent relationship.

[0065] Example 5. Dbpp2 Heparin Binding Analysis

[0066] Take 30 μL of heparin agarose bead suspension and wash it three times with pre-cooled Tris-HCl solution. Add 30 μL of 0.3 mg / mL Dbpp2 and heparin solutions of different concentrations to the heparin agarose beads. The concentrations of heparin are: 0, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, and 70 mg / mL. Mix thoroughly and incubate with gentle shaking at 4 °C for 1 h. Centrifuge at 3000 r / min for 5 min at 4 °C. Wash the heparin agarose bead precipitate three times with 200 μL of pre-cooled Tris-HCl solution. Add sample loading buffer and incubate in a boiling water bath for 10 min. Analyze the heparin binding capacity of Dbpp2 using 12% SDS-PAGE.

[0067] Result: As Figure 3 As shown, as the heparin concentration increased from 0 to 70 mg / mL, the amount of Dbpp2 bound to the heparin agarose beads gradually decreased.

[0068] Example 6. Identification of Heparin Binding Motif

[0069] Mix 30 μL of washed heparin agarose beads with 1 mg / mL Dbpp2 or Dbpp2 mutant, and incubate at 4°C with gentle shaking for 1 h; centrifuge at 3000 r / min for 5 min at 4°C, wash the heparin agarose bead precipitate three times with 200 μL of pre-cooled Tris-HCl solution, add loading buffer, and incubate in a boiling water bath for 10 min. Analyze the heparin binding capacity of Dbpp2 and its mutant using 12% SDS-PAGE.

[0070] Results: This invention substituted amino acids in the potential heparin-binding motif (KPANRPRR) to generate Dbpp2 mutants Dbpp21 (R290G), Dbpp22 (R290G+R292G), Dbpp23 (R290G+R292G+R293G), and Dbpp24 (N289R). These mutants altered the heparin binding affinity of Dbpp2 relative to its molecular weight. The amino acid residues affecting the heparin binding affinity of Dbpp2 are R290, R292, and R293; replacing them with G leads to a decrease in the binding affinity of Dbpp2. Replacing N289 with R improves the heparin binding affinity of Dbpp2. This indicates that KPANRPRR is a key motif for heparin binding of Dbpp2, and the R residue in the motif plays a crucial role in the heparin binding affinity of Dbpp2. (See details...) Figure 4 .

[0071] Example 7. Motif Binding Inhibition Analysis

[0072] Dissolve the potential heparin-binding motif using Tris-HCl solution; take 30 μL of heparin agarose bead suspension and wash three times with pre-cooled Tris-HCl solution; add 30 μL of 0.3 mg / mL Dbpp2 and peptide solutions of different concentrations to the heparin agarose beads, with peptide concentrations as follows: 1 × 10⁻⁶ -2 μmol / L, 1×10 -1 μmol / L, 1×10 0 μmol / L, 1×10 1 μmol / L; mix thoroughly, incubate slowly with shaking at 4℃ for 1 h; centrifuge at 3000 r / min at 4℃ for 5 min, wash the heparin agarose bead precipitate 3 times with 200 μL of pre-cooled Tris-HCl solution, add loading buffer, boil in water for 10 min, and analyze the ability of the peptide to inhibit Dbpp2 binding to heparin using 12% SDS-PAGE.

[0073] Result: As Figure 5 As shown, with the KPANRPRR concentration increasing from 1×10⁻⁶, -2 μmol / L increased to 1×10 1 The amount of Dbpp2 bound to heparin agarose beads gradually decreased with μmol / L concentration, indicating that KPANRPRR can inhibit the binding of Dbpp2 to heparin.

[0074] Example 8. Analysis of peptide hemolytic activity

[0075] Heart blood was collected from New Zealand rabbits using a puncture method. After centrifugation at 1000 rpm for 15 min, the supernatant serum and leukocytes were discarded. The lower red blood cell layer was mixed thoroughly with an appropriate amount of PBS (pH 7.4), centrifuged again, and the supernatant was discarded. This process was repeated three times. Finally, a 2% (v / v) rabbit red blood cell solution was prepared with PBS and stored at 4°C for later use. Heparin-binding motifs and their derived peptides were dissolved in PBS to prepare a 1×10⁻⁶ solution. 1 The solution was serially diluted 10-fold with μmol / L solution; 50 μL of peptide solution and 50 μL of rabbit red blood cell suspension were added to the wells of a microagglutination plate, mixed well, and incubated at 37℃ for 30 min to observe hemolytic activity.

[0076] Results: The peptides used for hemolytic activity testing included KPANRPRR and its derivative peptides KPAARPRR, KPRNRPRR, KPRARPRR, KPRWRPRR, and KPRWRPRRW. All of these peptides exhibited hemolytic activity. The order of hemolytic activity from smallest to largest was KPANRPRR, KPAARPRR, KPRNRPRR, KPRARPRR, KPRWRPRR, and KPRWRPRRW.

[0077] Hemolytic activity assays showed that KPANRPRR, similar to antimicrobial and antiviral peptides, exhibited membrane interference activity. Substitution with A3R and N4A improved the hemolytic activity of the peptide; replacing A with the highly hydrophobic W enhanced the hemolytic activity; and adding W to the C-terminus of the peptide further improved its hemolytic activity.

[0078] Example 9. Determination of antibacterial activity of peptides

[0079] Heparin-binding motifs and their derived peptides were dissolved in PBS and formulated into 1×10⁻⁶ ppm solutions. 1 μmol / L solution; seal the plate with sterile agar, place an Oxford cup (8 mm in diameter) on the solidified agar, and then mix the freshly cultured Escherichia coli K88 strain suspension with the nutrient agar medium until the bacterial concentration reaches 1×10⁻⁶. 6 Pour CFU / mL solution onto a plate; after solidification, remove the Oxford cup and add 200 μL of peptide solution to each well; diffuse at 4°C for 1 h, then incubate at 37°C for 16–18 h and observe the diameter of the inhibition zone.

[0080] Result: As Figure 6 As shown, except for KPANRPRR which showed no significant antibacterial activity, all other peptides exhibited antibacterial activity. The order of antibacterial activity from least to most was KPAARPRR, KPRNRPRR, KPRARPRR, KPRWRPRR, and KPRWRPRRW. In KPANRPRR and its derivative peptides, a positive correlation was observed between in vitro antibacterial activity and hemolytic activity.

[0081] Example 10. Analysis of anti-infective effects

[0082] The sugar solution was prepared with a sugar-to-water ratio of 1:1.2, boiled, and heated for 5 minutes. The SBV extracted with chloroform was filtered through a 0.22μm microporous membrane and added to 1000mL of sugar solution to obtain the virus solution. Eighteen days before the challenge, the disease index of the honeybee colonies was measured. Healthy colonies of similar strength were used for the challenge and as controls. The virus solution was sprayed onto the challenge colonies in the evening, spraying both sides of each comb and finally the hive entrance, for three consecutive days. Larval infestation was carried out on the 9th day after the challenge. Mortality and disease index were measured. If a bee colony was found to be diseased, it was used for anti-infection testing. Each polypeptide was randomly assigned to 3 colonies. On the evening of the test day, the bee colonies were sprayed with a sugar solution containing 1 mg / L of polypeptide or a sugar solution containing Dbpp2 antiserum. The healthy control group and the control group without treatment were also sprayed with sugar solution. The drug was administered once every other day for 9 consecutive times, with 50 mL of sugar solution sprayed per full frame of bees each time. On the 9th and 18th day after the first administration, the larval mortality rate was measured, and the control effect of each polypeptide on the bee colony was calculated. Statistical analysis was performed using SPSS 17.0 software, and the chi-square test was used. P < 0.05 was considered statistically significant.

[0083]

[0084] Results: On day 9 post-challenge, the challenged bee colonies showed disease, while the disease index level of healthy colonies remained unchanged. During the 18-day treatment period, the disease index levels of both healthy and untreated colonies remained unchanged. After 18 days of treatment, the larval mortality rate in the peptide-treated groups was significantly lower than that in the untreated groups. The larval mortality rates in the KPRNRPRR, KPRARPRR, and KPRWRPRRW treatment groups were also significantly lower than those in the KPANRPRR and KPAARPRR treatment groups (see Table 1). Among the peptides, KPRARPRR showed the best therapeutic effect on sacbrood disease in Apis sacbrood, with a treatment efficacy of 65.4%. Dbpp2 antiserum showed better therapeutic effects than the peptides on sacbrood disease in Apis sacbrood, with a treatment efficacy of 81.07%.

[0085] Table 1. Results of the analysis of the anti-SBV infection efficacy of peptides.

[0086]

[0087] Note: Different uppercase letters on the shoulder label indicate that the difference between the two groups of data is extremely significant (p<0.01), and different lowercase letters indicate that the difference between the two groups of data is significant (p<0.05).

Claims

1. A polypeptide based on SBV-GAG interaction, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

4.

2. The use of the polypeptide of claim 1 in the preparation of a medicament for treating cysticercosis virus infection.

3. A pharmaceutical composition for treating cysticercosis virus infection, characterized in that, The pharmaceutical composition contains the polypeptide of claim 1, and a pharmaceutically acceptable carrier.

Images