Application of snake venom polypeptide Hc-CATH in the preparation of drugs for preventing and treating Zika virus infection

Abstract

The present invention relates to the application of snake venom polypeptide Hc-CATH in the preparation of drugs for preventing and treating Zika virus infection. The present invention takes the cathelicidin family antimicrobial peptide Hc-CATH derived from the venom gland of blue ring sea snake as the research object, and intends to clarify the role and molecular mechanism of Hc-CATH in ZIKV infection. The research of the present invention found that Hc-CATH can significantly inhibit the infection of ZIKV in vitro and in vivo, and has both preventive and therapeutic effects. The results show that Hc-CATH can directly act on ZIKV, destroy the viral envelope, induce the leakage of the viral genome, and thus inactivate viral particles; Hc-CATH can also reduce the protein level of the host cell viral receptor AXL, reducing the susceptibility of host cells to Zika virus. Combined with the small molecular weight and simple structure of the antimicrobial peptide Hc-CATH, it has broad development and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of snake venom polypeptide Hc-CATH in preparing medicine for preventing and treating Zika virus infection. Background Art

[0002] Zika virus (ZIKV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the flavivirus family. ZIKV is mainly transmitted by mosquitoes, mainly through the bites of Aedes mosquitoes. In addition, ZIKV can also be transmitted through sexual contact, vertical transmission from mother to child, and blood transfusion.

[0003] ZIKV infection can cause a variety of serious neurological and reproductive system diseases, including Guillain-Barrein syndrome, microcephaly, fetal miscarriage or death, male infertility and other serious complications. In addition, ZIKV infection can also cause blindness and a variety of eye abnormalities, including retinal spots, lens subluxation and optic neuritis. However, there is currently no safe and effective vaccine or drug to prevent and treat ZIKV infection in clinical practice. Therefore, it is urgent to develop safe and effective drugs for the prevention and treatment of ZIKV infection.

[0004] Antimicrobial peptides (AMPs) are a class of small natural immune polypeptides encoded by specific genes of biological cells and widely expressed in animals and plants. As important effector molecules of vertebrate natural immunity, antimicrobial peptides can directly kill bacteria and regulate the body's immune response. In recent years, more and more studies have shown that in addition to having broad-spectrum and highly effective antibacterial and immunomodulatory effects, antimicrobial peptides can also play an antiviral role by destroying the viral envelope and regulating the host's antiviral immune response. Therefore, antimicrobial peptides have become an important source for screening antiviral peptides.

[0005] In vertebrates, cathelicidin antimicrobial peptides are one of the main antimicrobial peptide families. In recent years, researchers have identified cathelicidin family antimicrobial peptides from Bungarus bungarus, Bungarus cobra, King Cobra and Cobra. In 2015, a cathelicidin antimicrobial peptide was identified from the venom gland of the Hydrophis cyanocinctus, named Hc-CATH, with an amino acid sequence of KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL and a molecular weight of 3628.59 Daltons. Studies have found that cathelicidin antimicrobial peptides from Hydrophis cyanocinctus have significant antibacterial and anti-inflammatory activities, and have low cytotoxicity and hemolytic activity. However, the role and mechanism of cathelicidin family antimicrobial peptides in snakes in viral infection are still unclear and need to be further elaborated.

[0006] ZIKV infection can cause a variety of serious neurological and reproductive system diseases, including Guillain-Barrein syndrome, microcephaly, fetal miscarriage or death, male infertility and other serious complications. In addition, ZIKV infection can also cause blindness and a variety of eye abnormalities, including retinal spots, lens subluxation and optic neuritis. However, there is currently no safe and effective vaccine or drug to prevent and treat ZIKV infection in clinical practice. Therefore, it is urgent to develop safe and effective drugs for the prevention and treatment of ZIKV infection. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention proposes the use of snake venom polypeptide Hc-CATH in the preparation of drugs for preventing and treating Zika virus infection. The snake venom polypeptide Hc-CATH in the present invention has the characteristics of small molecular weight, simple synthesis, and obvious effect of inhibiting Zika virus infection. It has good application prospects in the research and development of drugs for preventing and treating Zika virus infection.

[0008] The purpose of the present invention is to provide the use of snake venom polypeptide Hc-CATH in the preparation of drugs for preventing and treating Zika virus infection.

[0009] In one embodiment of the present invention, the amino acid sequence of the snake venom polypeptide Hc-CATH from H. cyanocephalus is KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL.

[0010] In one embodiment of the present invention, the dosage of the drug is 1.25 μM-5 μM.

[0011] In one embodiment of the present invention, the drug further includes pharmaceutically or pharmacologically acceptable carriers, salts, esters, hydrates, solvates, crystalline forms, enantiomers, stereoisomers, ethers, metabolites and prodrugs.

[0012] In one embodiment of the present invention, the carrier is selected from one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant and a preservative.

[0013] In one embodiment of the present invention, the filler is selected from one or more of starch, sucrose and lactose; the humectant includes glycerol; and the surfactant includes cetyl alcohol.

[0014] In one embodiment of the present invention, the binder is selected from one or more of cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone.

[0015] In one embodiment of the present invention, the disintegrant is selected from one or more of agar, calcium carbonate and sodium bicarbonate.

[0016] In one embodiment of the present invention, the pharmaceutically acceptable salt is selected from one or more of inorganic acid salts, organic acid salts, alkyl sulfonates and aryl sulfonates.

[0017] In one embodiment of the present invention, the pharmaceutical dosage form is selected from tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts or injections.

[0018] The purpose of the present invention is to provide the function of snake venom polypeptide Hc-CATH in inhibiting Zika virus infection, provide the mechanism of action of snake venom polypeptide Hc-CATH in inhibiting Zika virus infection and provide candidate molecules for the research and development of polypeptide drugs for preventing and treating Zika virus infection.

[0019] The technical solution of the present invention has the following advantages:

[0020] (1) The present invention uses the cathelicidin family antimicrobial peptide Hc-CATH from the sea snake as the research object, and intends to clarify the role and molecular mechanism of Hc-CATH in ZIKV infection. The research of the present invention found that Hc-CATH can significantly inhibit ZIKV infection in vitro and in vivo, and has both preventive and therapeutic effects. Hc-CATH can directly act on ZIKV, destroy the viral envelope, induce the leakage of the viral genome, and thus inactivate viral particles; Hc-CATH can also reduce the protein level of the host cell viral receptor AXL, reducing the susceptibility of host cells to Zika virus.

[0021] (2) Currently, there are no effective vaccines and antiviral drugs that can be used to prevent and treat Zika virus infection in clinical practice. However, the snake venom polypeptide Hc-CATH in the present invention has a small molecular weight, simple synthesis, and a significant effect in inhibiting Zika virus infection. It has a good application prospect in the research and development of drugs for preventing and treating Zika virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0023] Figure 1 This is a diagram showing the cytotoxicity of Hc-CATH in Example 1 of the present invention; wherein Figure 1 -A is a Vero cell cytotoxicity assay diagram of Hc-CATH; Figure 1 -B is a graph showing the cytotoxicity assay of Hc-CATH in A549 cells; Figure 1 -C is the U251 cytotoxicity assay of Hc-CATH (ns: no significance, ***p<0.001);

[0024] FIG2 is a diagram showing that Hc-CATH in Example 2 of the present invention can significantly inhibit the cell pathological changes caused by ZIKV infection; Figure 2-A It is the cytopathic effect diagram; Figure 2-B is the cytopathic effect rate (*p<0.05, **p<0.01, ***p<0.001);

[0025] Figure 3 This is a diagram showing the resistance of Hc-CATH to ZIKV infection in vitro in Example 3 of the present invention; wherein Figure 3 -A is a graph showing the reduction of intracellular viral RNA levels by Hc-CATH; Figure 3 -B is a graph showing that Hc-CATH inhibits the expression of viral NS3 protein; Figure 3 -C is a graph showing that Hc-CATH inhibits the expression of viral E protein; Figure 3 -D and Figure 3 -E: Hc-CATH reduces the virus titer in the cell culture supernatant (ns: no significance, *p<0.05, **p<0.01, ***p<0.001.);

[0026] Figure 4 This is a graph showing the prevention of ZIKV infection by Hc-CATH in C57BL / 6 mice in Example 4 of the present invention (*p<0.05, **p<0.01, ***p<0.001);

[0027] Figure 5 This is a graph showing the treatment of ZIKV infection by Hc-CATH in C57BL / 6 mice in Example 5 of the present invention (***p<0.001);

[0028] Figure 6 This is a graph showing that Hc-CATH-virus-pre inhibits ZIKV infection in Example 6 of the present invention; wherein Figure 6 -A is a schematic diagram of the treatment of the Hc-CATH-virus-pre group; Figure 6 -B is the level of Hc-CATH-virus-pre intracellular viral RNA; Figure 6 -C is the expression diagram of Hc-CATH-virus-pre inhibiting viral NS3 protein; Figure 6 -D is the expression diagram of Hc-CATH-virus-pre inhibiting viral E protein; Figure 6 -E and Figure 6 -F is a graph showing the viral titer in the cell culture supernatant reduced by Hc-CATH;

[0029] Figure 7 This is a diagram showing the binding of Hc-CATH to ZIKV particles and induction of ZIKV genomic RNA leakage in Example 6 of the present invention; Figure 7 -A is the binding diagram of Hc-CATH and ZIKV particles detected by ELISA; Figure 7 -B is a graph for detecting RNA levels of core protein (CAP), precursor membrane protein (PRM), and envelope protein (E) in the viral genome;

[0030] Figure 8 This is a diagram showing the inhibition of ZIKV infection by Hc-CATH-cell-pre in Example 7 of the present invention; Figure 8 -A is a schematic diagram of Hc-CATH-cell-pre treated cells; Figure 8 -B is Hc-CATH-cell-pre that reduces the level of intracellular viral RNA; Figure 8 -C is the expression diagram of Hc-CATH-cell-pre inhibiting the intracellular viral NS3 protein; Figure 8 -D is the expression diagram of Hc-CATH-cell-pre inhibiting intracellular viral E protein; Figure 8 -E and Figure 8 -F is Hc-CATH-cell-pre that reduces the viral titer in the cell culture supernatant;

[0031] Fig. 9 This is a graph showing the expression of Hc-CATH on Vero cell surface receptors in Example 7 of the present invention; Fig. 9 -A is a schematic diagram of Hc-CATH-treated cells; Fig. 9 -B is a Western blot analysis of the changes in the protein levels of Vero cell surface receptors TYRO3, TIM-1, and AXL after Hc-CATH treatment. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] Example 1 Cytotoxicity of Hc-CATH

[0034] In order to determine whether Hc-CATH has an effect on cell viability, Vero, A549, and U251 cells in the logarithmic growth phase were evenly plated in 96-well plates, with approximately 1×10 cells per well. 4 , cultured in a 37°C incubator until the cells were completely attached, Hc-CATH and control peptides AC5 and LL-37 were diluted twice from 50 μM in DMEM culture medium containing 2% FBS and added to each well, an equal volume of PBS was added to the negative control well, and gently mixed, six replicate wells were set for each concentration, and cultured at 37°C for 48 hours, the culture medium was discarded, and serum-free DMEM culture medium containing 10% CCK-8 was prepared, 100 μL was added to each well in the form of liquid replacement, and the absorbance value of each well was detected at 450 nm after 10min-20min.

[0035] Cell viability (cell viability%) = OD value of test well / OD value of control well × 100%

[0036] The results of the cytotoxicity assay of Hc-CATH are shown in Figure 1 As shown, Figure 1 -A shows that Hc-CATH has no toxicity to Vero cells when ≤12.5μM, Figure 1 -B shows that Hc-CATH has no toxicity to A549 cells at ≤6.25 μM. Figure 1 -C showed that Hc-CATH had no toxicity to U251 cells at ≤6.25 μM. Therefore, in subsequent in vitro experiments, the concentrations of Hc-CATH were set to 1.25 μM, 2.5 μM, and 5 μM.

[0037] Example 2 Hc-CATH inhibits cytopathic effect caused by Zika virus infection

[0038] Vero cells will produce obvious cytopathic effect (CPE) after ZIKV infection. Therefore, the effect of Hc-CATH on the cytopathic effect caused by ZIKV infection was tested. Vero cells in the logarithmic growth phase were evenly plated in a 24-well plate, with approximately 5×10 cells per well. 4When the cells were attached and the density was about 70%, ZIKV infection (MOI=1) was added, and Hc-CATH (1.25μM, 2.5μM, 5μM), AC5 (2.5μM), and LL-37 (2.5μM) were added to each well, mixed gently, and cultured at 37°C for 2h. The culture medium was discarded, and DMEM with 2% FBS and the same dose of polypeptide were added. The culture was continued at 37°C for 48h. The cell pathological changes were observed under an inverted fluorescence microscope and pictures were collected. Then, serum-free DMEM culture medium containing 10% CCK-8 was added to each well in the form of liquid replacement. After 10min-20min, the absorbance value of each well was detected at 450nm.

[0039] Hc-CATH can significantly inhibit the cytopathic effect caused by ZIKV infection, as shown in Figure 2. Compared with the uninfected Sham group, the PBS group after ZIKV infection had obvious cytopathic effect, and the CPE value was about 75%. Compared with the PBS group, the addition of Hc-CATH treatment can significantly improve the cytopathic effect caused by ZIKV infection, and the CPE values ​​of the 1.25μM, 2.5μM, and 5μM Hc-CATH treatment groups were approximately 65%, 50%, and 25%, respectively. The above results show that Hc-CATH can inhibit the cytopathic effect caused by ZIKV infected cells, and there is a dose-dependent effect.

[0040] Example 3 Hc-CATH significantly inhibits the replication of Zika virus in Vero cells

[0041] Vero cells in the logarithmic growth phase were evenly plated in a 12-well plate, with the number of cells per well being approximately 1×10 5 , cultured in a 37°C incubator until the cells were completely adhered, ZIKV was added for infection (MOI=1), and then 1.25μM, 2.5μM, 5μM Hc-CATH or an equal volume of PBS (peptide solvent) was added to each well, with 2.5μM AC5 and LL-37 as positive controls. The cells were gently mixed and cultured at 37°C for 2h. The culture medium was discarded, and DMEM with 2% FBS and the same dose of Hc-CATH or an equal volume of PBS were added. The cells were cultured for another 48h. The intracellular viral RNA level, the expression levels of NS3 protein and E protein were detected by Q-PCR, Western blot and immunofluorescence, and the virus titer in the cell culture supernatant was detected by plaque assay.

[0042] Hc-CATH can significantly resist ZIKV infection in vitro. Figure 3As shown in the figure, Q-PCR results showed that Hc-CATH could significantly reduce the RNA level of ZIKV in Vero cells in a dose-dependent manner. At 1.25 μM, 2.5 μM, and 5 μM Hc-CATH, the viral RNA was reduced by about 5%, 33%, and 55%, respectively. Figure 3 -A); Figure 3 -B is the expression diagram of Hc-CATH inhibiting viral NS3 protein, gray value statistics were performed using Image J, and quantification was performed using the ratio of NS3 / β-actin; Figure 3 -C is Hc-CATH inhibiting the expression of virus E protein (green: ZIKV E protein, blue: cell nucleus); Western Blot and immunofluorescence results showed that Hc-CATH could significantly inhibit the expression of ZIKV non-structural protein NS3 and E protein in Vero cells, showing a dose-dependent effect at 1.25μM, 2.5μM, and 5μM ( Figure 3 -B and Figure 3 -C); The results of the virus plaque assay showed that, consistent with the changes in viral RNA and protein levels, Hc-CATH could significantly reduce the viral titer in the cell culture supernatant, showing a dose-dependent effect within the concentration range of 1.25μM, 2.5μM, and 5μM ( Figure 3 -D and Figure 3 -E). The results showed that Hc-CATH could significantly inhibit the replication of ZIKV in Vero cells in a dose-dependent manner.

[0043] Example 4 Hc-CATH can prevent ZIKV infection in mice

[0044] Hc-CATH (5 mg / kg) was injected into C57BL / 6 mice via tail vein. 2 h later, 6×10 6 PFU of ZIKV was injected into the tail vein to infect mice. Four days after infection, C57BL / 6 mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney, and brain of the mice were obtained. The viral RNA levels in various tissues of the mice were detected by Q-PCR.

[0045] The protective effect of Hc-CATH against ZIKV infection in C57BL / 6 mice Figure 4 As shown, the results showed that the viral RNA levels in the heart, liver, spleen, lung, kidney and brain of the Hc-CATH-treated mice were significantly reduced compared with those in the PBS-treated mice, indicating that injecting Hc-CATH into C57BL / 6 mice 2 hours in advance can prevent ZIKV infection.

[0046] Example 5 Hc-CATH can treat ZIKV infection in C57BL / 6 mice

[0047] 6×106 PFU of ZIKV was injected into C57BL / 6 mice via tail vein injection. 2 hours later, Hc-CATH (5 mg / kg) was injected into the mice via tail vein. 4 days after infection, C57BL / 6 mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney, and brain of the mice were removed. The viral RNA levels in various tissues of the mice were detected by Q-PCR.

[0048] The therapeutic effect of Hc-CATH on ZIKV infection in C57BL / 6 mice Figure 5 As shown, the results showed that the viral RNA levels in the heart, liver, spleen, lung, kidney and brain of the Hc-CATH-treated mice were significantly reduced compared with those in the PBS-treated mice, indicating that injection of Hc-CATH into C57BL / 6 mice 2 hours after infection has a therapeutic effect on ZIKV infection.

[0049] Example 6 Hc-CATH directly inactivates Zika virus

[0050] In order to detect whether Hc-CATH directly inactivates ZIKV viral particles and thus inhibits ZIKV infection, 2.5 μM Hc-CATH or an equal volume of PBS was incubated with ZIKV (MOI=1) at 37°C for 2 h, ultracentrifuged, and the precipitate was collected and resuspended, and added to Vero cells. After culturing at 37°C for 2 h, the supernatant was discarded, the cells were washed three times with PBS, and DMEM containing 2% FBS was added. After further culturing for 48 h, real-time fluorescence quantitative PCR, Western blot and immunofluorescence were used to detect the intracellular viral RNA level, NS3 protein and E protein expression levels of the Hc-CATH-virus-pre group, and the plaque assay was used to detect the virus titer in the cell culture supernatant of the Hc-CATH-virus-pre group.

[0051] Hc-CATH-virus-pre treatment can significantly inhibit ZIKV infection. Figure 6 As shown, Figure 6 -C is the expression diagram of Hc-CATH-virus-pre inhibiting viral NS3 protein, gray value statistics were performed using Image J, and quantified by the ratio of NS3 / β-actin; Figure 6 -D is the expression diagram of Hc-CATH-virus-pre inhibiting virus E protein (green: ZIKV E protein, blue: cell nucleus); the results showed that compared with the PBS incubation group, Hc-CATH incubated with ZIKV for 2 hours before infecting cells can significantly reduce the level of viral RNA in Vero cells ( Figure 6 -B), and significantly inhibited the viral nonstructural protein NS3 in Vero cells ( Figure 6 -C) and E protein ( Figure 6-D) expression, and also significantly reduced the viral titer in the cell culture supernatant ( Figure 6 -E and Figure 6 The above results indicate that Hc-CATH can directly inactivate ZIKV virus particles, thereby inhibiting ZIKV infection in Vero cells.

[0052] The above results show that Hc-CATH can significantly inhibit ZIKV infection of cells when it is co-incubated with ZIKV virus at 37°C for 2 hours and then added to Vero cells, indicating that Hc-CATH can directly inactivate ZIKV virus particles. To further clarify how Hc-CATH acts on ZIKV virus, the binding of Hc-CATH to virus particles was first evaluated by ELISA. The plates were coated with 2.5 μM Hc-CATH, AC5 and LL-37, and the control wells were coated with 2.5% BSA. 1×10 6 PFU of ZIKV was used to detect the direct binding between the two. Hc-CATH binds to ZIKV particles and induces the leakage of ZIKV genomic RNA. Figure 7 -A, the results showed that compared with the BSA-coated group, the OD of the Hc-CATH-coated group was 450 The light absorption value increased significantly, indicating that Hc-CATH can directly bind to ZIKV particles.

[0053] Therefore, we further investigated whether Hc-CATH directly inactivates viral particles by acting on the viral envelope. 1 μM, 20 μM, 40 μM Hc-CATH or PBS were mixed with 1×10 5 PFU of ZIKV was incubated at 37°C for 2 hours, micrococcal nuclease (RNase) was added, and incubated at 37°C for 4 hours to digest the genomic RNA exposed outside the viral envelope, and then incubated at 70°C for 30 minutes to inactivate RNase. The remaining undigested viral genomic RNA inside the viral envelope was extracted using the Viral RNAMini kit, and the RNA levels of core protein (CAP), membrane precursor protein (PRM), and envelope protein (E) in the viral genome were detected by Q-PCR.

[0054] The results showed that compared with the PBS control group, the RNA levels of CAP, PRM, and E proteins in the viral genome were significantly reduced after the addition of 1 μM, 20 μM, and 40 μM Hc-CATH. After the addition of 40 μM Hc-CATH, the RNA levels of CAP, PRM, and E in the ZIKV genome were reduced to 87%, 72%, and 84.5%, respectively. Figure 7-B). The results showed that the viral envelope of the PBS-treated group was not destroyed, so it was not digested by RNase, and a relatively complete viral genome was retained. The RNA levels of CAP, PRM, and E in the genome were defined as 100%. After incubation with Hc-CATH, the ZIKV genome was exposed and could be digested by RNase, so the RNA levels of CAP, PRM, and E proteins in the genome were significantly reduced. The results show that Hc-CATH can directly inactivate viral particles by inducing leakage of the viral genome by destroying the viral envelope.

[0055] Example 7 Hc-CATH can reduce the expression level of viral receptors, thereby reducing the susceptibility of host cells to Zika virus

[0056] In order to detect whether Hc-CATH can reduce the susceptibility of Vero cells to ZIKV and thereby inhibit ZIKV infection, 2.5 μM Hc-CATH or PBS was incubated with Vero cells at 37°C for 2 h, the culture medium was discarded, the cells were washed three times with PBS, and then fresh DMEM was added, followed by ZIKV (MOI=1). After incubation at 37°C for 2 h, the supernatant was discarded, and DMEM with 2% FBS was added. After continued culture for 48 h, Q-PCR, Western blot and immunofluorescence were used to detect the intracellular viral RNA level, NS3 protein and E protein expression levels of the Hc-CATH-cell-pre group, and the plaque assay was used to detect the viral titer in the cell culture supernatant of the Hc-CATH-cell-pre group.

[0057] Hc-CATH-cell-pre inhibits ZIKV infection Figure 8 As shown, Figure 8 -C: Hc-CATH-cell-pre can inhibit the expression of intracellular viral NS3 protein, and grayscale value statistics were performed using Image J, and quantified by the ratio of NS3 / β-actin; Figure 8 -D is that Hc-CATH-cell-pre can inhibit the expression of intracellular viral E protein (green: ZIKV E protein, blue: cell nucleus); the results showed that compared with the PBS incubation group, after Hc-CATH was incubated with Vero cells for 2 hours and then infected with ZIKV, the viral RNA level in Vero cells was significantly reduced ( Figure 8 -B), viral NS3 protein ( Figure 8 -C) and viral E protein ( Figure 8 -D) expression level was significantly reduced, and the virus titer in the cell culture supernatant ( Figure 8 -E and Figure 8-F) was also significantly reduced in a dose-dependent manner. The results showed that Hc-CATH can reduce the susceptibility of Vero cells and inhibit the establishment of ZIKV infection in Vero cells.

[0058] TIM-1, TYRO3, and AXL receptors on the cell surface play an important role in the establishment of flavivirus infection. In order to further clarify whether Hc-CATH can directly act on Vero cell surface receptors and thus exert antiviral effects. Vero cells in the logarithmic growth phase were evenly plated in 24-well plates, with approximately 5×10 cells per well. 4 , cultured in a 37°C incubator until the cells were completely attached, 1.25μM, 2.5μM, and 5μM Hc-CATH or PBS were added to each well, mixed gently, and cultured at 37°C for 6h, 12h, and 24h. Western blot was used to detect changes in the levels of Vero cell surface receptors TYRO3, TIM-1, and AXL proteins.

[0059] Effects of Hc-CATH on the expression of receptors on the surface of Vero cells Fig. 9 As shown, Fig. 9 -A is a schematic diagram of Hc-CATH-treated cells; Fig. 9 -B is the change of protein levels of Vero cell surface receptors TYRO3, TIM-1, and AXL detected by Western blot after Hc-CATH treatment. The results showed that compared with the PBS treatment group, the protein levels of Vero cell surface receptors TYRO3, TIM-1, and AXL were not affected after 6h and 12h of Hc-CATH treatment, but after 24h of Hc-CATH treatment, the protein level of Vero cell surface receptor AXL was reduced in a dose-dependent manner, while the protein levels of other receptors (TYRO3, TIM-1) were not affected.

[0060] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Application of snake venom polypeptide Hc-CATH in the preparation of drugs for preventing and treating Zika virus infection; The amino acid sequence of the snake venom polypeptide Hc-CATH is shown in SEQ ID NO.1; The concentration of the snake venom polypeptide Hc-CATH is 1.25 μM-5 μM.

2. The use according to claim 1, It is characterized in that The medicament further comprises a pharmaceutically or pharmacologically acceptable carrier and / or salt.

3. The use according to claim 2, It is characterized in that The carrier is selected from one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant and a preservative.

4. The use according to claim 3, It is characterized in that The filler is selected from one or more of starch, sucrose and lactose; the wetting agent includes glycerin; and the surfactant includes cetyl alcohol.

5. The use according to claim 3, It is characterized in that The binder is selected from one or more of cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone.

6. The use according to claim 3, It is characterized in that The disintegrant is selected from agar.

7. The use according to claim 2, It is characterized in that The pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

8. The use according to claim 1, It is characterized in that The pharmaceutical dosage form is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts or injections.

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