Tick-derived antiviral active polypeptides and uses thereof

Abstract

The application discloses a tick-derived antiviral active polypeptide and application thereof, and belongs to the technical field of biology.The amino acid sequence of the tick-derived antiviral active polypeptide is shown as SEQ ID NO.1, and the polypeptide can well inhibit the replication of a fever with thrombocytopenia syndrome virus (SFTSV) at a low concentration, and has a good prospect in the prevention or treatment of diseases caused by the SFTSV.The antiviral polypeptide has high SFTSV resistance, and can be used for preparing a medicine for preventing and / or treating SFTSV infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an active polypeptide derived from ticks with antiviral function and its application. Background Technology

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a segmented negative-sense RNA virus belonging to the order Bunyavirales and the genus Phlebovirus. The SFTSV genome is divided into three segments: large (L), medium (M), and small (S). The L segment contains 6368 bases and encodes the RNA-dependent RNA polymerase (RdRp); the M segment contains 3378 bases and encodes the glycoproteins Gn and Gc; the S segment consists of a 1744-nucleotide ambiguous RNA segment encoding the antisense nucleocapsid protein NP and the sense non-structural protein NSs.

[0003] Currently, there is no effective treatment for SFTSV infection, and commonly used clinical methods have inconsistent efficacy and unclear side effects. Ribavirin is a common antiviral drug; some literature reports that in patients with low viral loads (<1×10⁶ copies / mL), ribavirin can reduce the mortality rate from 6.25% to 1.16%, but it does not reduce mortality at higher viral loads. Furthermore, ribavirin has significant side effects, such as anemia and hyperamylasemia. Favipiravir (T-705) is a broad-spectrum antiviral drug that primarily inhibits viral RNA polymerase. Studies have shown that favipiravir can inhibit SFTSV replication in vivo and in vitro, but the side effects caused by favipiravir still require further investigation. The key factor in the pathogenesis of severe SFTSV infection is the cytokine storm. Considering the pathogenesis of SFTS, clinicians use steroids to suppress the immune system in patients with severe SFTS. However, steroid treatment within 5 days of symptom onset or in patients with milder symptoms (APACHE II score <14) increases the risk of complications; therefore, steroids should be used with caution. In addition, intravenous immunoglobulin (IVIG) is also considered helpful in reducing viral load in SFTS patients, controlling the spread of SFTSV, and effectively suppressing the cytokine storm, but further research is needed to confirm the effectiveness of IVIG treatment. Convalescent plasma therapy has also been used in some clinical trials, showing a decrease in serum viral load and improvement in clinical course after treatment. However, due to unclear patient inclusion criteria for plasma exchange and unstable treatment effects, establishing plasma exchange or convalescent plasma therapy as a standard SFTSV treatment strategy remains difficult. Specific vaccines against SFTSV are also under intensive development, but no vaccine has yet been approved for marketing. Therefore, it is urgent to develop new, safe, effective and stable anti-SFTSV drugs.

[0004] Defensins are a class of small cationic polypeptides, typically composed of 29-54 amino acids, including 6-8 cysteine ​​residues. These cysteine ​​residues form disulfide bonds within the molecule, resulting in antiparallel β-sheets or α-helices that provide a stable molecular structure. Defensins are widely distributed in animals, plants, and insects, defending against the invasion of various microorganisms and serving as an important component of the organism's immune system. Previous research on defensins has largely focused on antibacterial infection control. In recent years, host defensin polypeptides have been found to possess antiviral activity and have potential applications in the prevention and treatment of various viral diseases. For example, the human antimicrobial peptide LL-37 exhibits antiviral activity against influenza A virus (IAV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1). The human α-defensin HNP4 can inhibit HIV replication by binding to gp120 and CD4. Lactoferrin can interfere with some receptors involved in the pathogenesis of SARS-CoV-2 and can also prevent the virus from entering host cells via ACE2. These studies suggest that defensins may serve as potential antiviral drugs. Summary of the Invention

[0005] The purpose of this invention is to address the lack of anti-SFTSV drugs in existing technologies and to provide a tick-derived antiviral active polypeptide and its application. The polypeptide is a cationic polypeptide expressed in the salivary glands of the tick *Haemaphysalis longicornis*, named HIDfsin2. This invention optimizes the encoding nucleotide sequence of HIDfsin2, successfully amplifies the DNA fragment encoding HIDfsin2 using overlap extension PCR, constructs it into pET-32a to obtain a recombinant plasmid expressing HIDfsin2, and successfully expresses the structurally correct and active tick salivary gland polypeptide HIDfsin2 via prokaryotic expression. Further research on the antiviral activity of HIDfsin2 revealed that the polypeptide HIDfsin2 can inhibit SFTSV replication; 20 μM of HIDfsin2 can achieve an inhibition rate of 46% against SFTSV.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A tick-derived antiviral polypeptide, HIDfsin2, has the following amino acid sequence: GFGCPLNQGACHNHCRSIGRRGGYCAGIIKQTCTCYRK (SEQ ID NO.1, Figure 1 ).

[0008] The preferred sequence of the nucleotide encoding the tick-derived antiviral polypeptide HIDfsin2 is: GGCTTTGGCTGCCCGCTTAACCAGGGCGCCTGCCACAACCATTGCCGTAGCATTGGGCG GCGCGGAGGATACTGCGCGGGAATCATCAAGCAGACTTGCACGTGCTACCGCAAG (SEQ ID NO. 2, Figure 1 ).

[0009] The tick-derived antiviral active polypeptide HIDfsin2 can be synthesized artificially or prepared by expression in cells (such as Escherichia coli).

[0010] A recombinant expression vector containing nucleotides encoding the aforementioned tick-derived antiviral polypeptide HIDfsin2, wherein the recombinant vector, when transformed into cells, can express the tick-derived antiviral polypeptide HIDfsin2. Further, the recombinant expression vector uses pET-32a as the expression vector.

[0011] A recombinant cell containing the aforementioned recombinant expression vector is capable of expressing the tick-derived antiviral polypeptide HIDfsin2. Further, the recombinant cell is *Escherichia coli* BL21.

[0012] The preparation method of the tick-derived antiviral active polypeptide HIDfsin2 includes the following steps: culturing the above recombinant cells to express the tick-derived antiviral active polypeptide HIDfsin2.

[0013] The application of the tick-derived antiviral active polypeptide HIDfsin2 in the preparation of drugs for the prevention and / or treatment of SFTSV infection.

[0014] A drug for the prevention and / or treatment of SFTSV infection, comprising the tick-derived antiviral active peptide HIDfsin2, and may further comprise a pharmaceutically acceptable carrier or excipient.

[0015] Advantages and beneficial effects of the present invention: The tick-derived antiviral active polypeptide of the present invention has the function of inhibiting viral infection, with good inhibitory effect and no side effects. It is significantly effective in preventing and treating infections caused by SFTSV. HIDfsin2 is easy to express, has low production cost, and can be developed and utilized as an anti-SFTSV drug. Attached Figure Description

[0016] Figure 1 It is the tick-derived antiviral active polypeptide HIDfsin2 nucleotide and its amino acid sequence.

[0017] Figure 2This is a schematic diagram of the HIDfsin2 gene coding sequence.

[0018] Figure 3 Coomassie brilliant blue staining of HIDfsin2, a tick-derived antiviral peptide obtained from prokaryotic expression.

[0019] Figure 4 This is an HPLC purity chromatogram of HIDfsin2, a tick-derived antiviral polypeptide obtained from prokaryotic expression.

[0020] Figure 5 This is a circular dichroism chromatogram of HIDfsin2, an antiviral polypeptide derived from ticks and expressed in prokaryotes.

[0021] Figure 6 This is a mass spectrometry image of HIDfsin2, an antiviral polypeptide derived from ticks and expressed in prokaryotes.

[0022] Figure 7 This study is a quantitative analysis of intracellular viral RNA in SFTSV-infected cells using the tick-derived antiviral peptide HIDfsin2 at different concentrations.

[0023] Figure 8 This study is a quantitative analysis of intracellular viral proteins in SFTSV-infected cells by different concentrations of the tick-derived antiviral peptide HIDfsin2. Detailed Implementation

[0024] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0025] Example 1: Preparation of tick-derived antiviral peptide HIDfsin2

[0026] 1. Preparation of HIDfsin2 nucleotide fragments by overlap extension PCR

[0027] (1) Design of HIDfsin2 gene coding sequence

[0028] At the 5' end of the HIDfsin2 gene coding sequence, a protective base, a restriction endonuclease KpnI cleavage site, and an EK enzyme cleavage site were added sequentially. At the 3' end, a terminator (a double terminator of TAATGA can increase termination efficiency), a restriction endonuclease HindIII cleavage site, and a protective base were added sequentially. The resulting nucleotide sequence is shown below. Figure 2 As shown.

[0029] (2) Design of overlapping extension PCR primers

[0030] The designed primer sequences are shown in Table 1 below.

[0031] Table 1

[0032]

[0033] (3) Gene amplification of HIDfsin2

[0034] Following the reagent ratios in Table 2, PCR amplification was performed in the PCR system using primers F2 and R2. The amplified product was then purified using a DNA product purification kit. The purified sample was used as template DNA, and PCR amplification was performed again in the PCR system using primers F1 and R1. 5 μL of the secondary amplification PCR product was subjected to 2% agarose gel electrophoresis to confirm whether the target gene fragment had been successfully amplified. The remaining product was recovered using a DNA product purification kit and used for subsequent enzyme digestion.

[0035] Table 2

[0036]

[0037] (4) KpnⅠ and HindⅢ were used to digest the PCR amplification product and the vector pET-32a.

[0038] Prepare the PCR amplification products and the double digestion system of the pET-32a vector according to Table 3 below, and incubate at 37℃ for 30 min. Perform 2% agarose gel electrophoresis on the digested products, and then use a DNA gel recovery kit to recover the gel and determine the concentration.

[0039] Table 3

[0040]

[0041] (5) Ligation of PCR amplification products with pET-32a vector

[0042] Based on the ratio of vector to target fragment amount of 1:6, calculate the volume ratio of vector plasmid to target fragment, prepare the T4 ligase ligation system as shown in Table 4, and incubate at 16℃ for 3h.

[0043] Table 4

[0044]

[0045] (6) Transformation of recombinant plasmids and identification of positive clones

[0046] 1) Add 10 μL of the ligation product to 50 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 sec, and then place on ice again for 2 min.

[0047] 2) Add 500 μL of antibiotic-free LB liquid medium and incubate at 37°C and 200 rpm for 40 min in a constant temperature shaker.

[0048] 3) Centrifuge at 3700 rpm for 5 min, discard 400 μL of supernatant, mix the remaining liquid by pipetting, and spread it on LB agar plates containing Amp resistance. Incubate overnight at 37°C. The next day, pick a single colony and incubate in 500 μL of LB liquid medium containing Amp resistance. Incubate at 37°C and 200 rpm for 6-8 h, then sequence. Mix the remaining bacterial culture with 30% glycerol at a 1:1 ratio and store at -80°C. Sequence analysis showed that the pET-32a-HIDfsin2 recombinant plasmid was successfully constructed.

[0049] 2. Prokaryotic expression and purification of HIDfsin2

[0050] (1) Recombinant plasmid transformed into BL21 competent cells of expression bacteria

[0051] 60 μL of correctly sequenced bacterial culture was inoculated into 15 mL of LB liquid medium containing Amp resistance and incubated overnight at 37°C and 200 rpm. Plasmids were extracted using a Takara plasmid extraction kit, and their concentration was determined. 1 μL of recombinant plasmid was added to 50 μL of BL21(DE3) competent cells, incubated on ice for 5 min, heat-shocked at 42°C for 45 s, and then placed on ice again for 2 min. All bacterial culture was spread onto LB agar plates containing Amp and incubated overnight at 37°C. The next day, single colonies were picked and placed in 500 μL of LB liquid medium containing Amp, incubated at 37°C and 200 rpm for 6–8 h, then 30% glycerol was added at a 1:1 ratio, mixed well, and stored at -80°C.

[0052] (2) Amplification of the expression bacteria

[0053] Take 20 mL of LB liquid medium containing Amp, inoculate the expression bacteria at a ratio of 1:500, and incubate overnight on a shaker at 37°C and 200 rpm. The next day, inoculate this bacterial culture at a ratio of 1:50 into 1 L of LB liquid medium containing Amp, and incubate on a shaker at 37°C and 200 rpm.

[0054] (3) IPTG-induced expression of target protein

[0055] Measure the OD value of the bacterial culture. 630 IPTG was added at a final concentration of 0.4 mM when the concentration was between 0.4 and 0.6, and the induction was carried out at 37°C and 200 rpm for 4 h.

[0056] (4) Cell disruption

[0057] Centrifuge the induced bacterial culture at 8000 rpm for 6 min at room temperature. Discard the supernatant and resuspend the bacterial cells in 60 mL of 20 mM imidazole. Then, divide the 60 mL resuspended bacterial culture into two portions and sonicate them twice (keeping the temperature low during the disruption process). The program is set as follows: 2 s interval per cycle, 5 s interval per cycle, 99 repetitions per cycle, for two cycles. At this point, the bacterial culture will turn a clear milky white color. Transfer the disrupted bacterial culture to a thick-walled centrifuge tube and centrifuge at 12000 rpm at 4°C for 15 min, collecting the supernatant.

[0058] (5) Separation of target protein by nickel column affinity chromatography

[0059] 1) Pretreatment of nickel column: Assemble the peristaltic pump, nickel column and protein nucleic acid detector in sequence. Adjust the speed of the peristaltic pump to 18 rpm. Rinse the nickel column with 250 mM imidazole. After the detector reading remains unchanged, rinse the nickel column with ddH2O until the detector reading remains unchanged. Finally, equilibrate the nickel column with 20 mM imidazole until the detector reading remains unchanged.

[0060] 2) Sample loading: Adjust the peristaltic pump speed to 8 rpm and load the sample slowly.

[0061] 3) After loading the sample, rinse the nickel column with 20mM imidazole to remove impurities. After the sample is completely bound to the nickel column (the liquid in the nickel column is clear), increase the speed of the peristaltic pump to 12 rpm. At this time, the reading of the protein nucleic acid detector will continue to decrease until it stabilizes.

[0062] 4) Elution: Elution was performed using 250mM imidazole. When the reading on the protein nucleic acid detector rose rapidly, the eluent was collected. When the reading on the detector reached its peak and then dropped to near stability, the collection was stopped.

[0063] 5) Nickel column cleaning: Continue to pump in 250mM imidazole to rinse the nickel column. After the protein nucleic acid detector reading stabilizes, increase the peristaltic pump speed to 15rpm and switch to ddH2O to rinse the nickel column. After the detector reading stabilizes, rinse the nickel column with 20mM imidazole. After the detector reading stabilizes, turn off the instrument and store the nickel column in a refrigerator at 4℃.

[0064] (6) Dialysis and Ultrafiltration

[0065] 1) Boil the dialysis bag in a 2% NaHCO3 and 1mM EDTA solution (pH=8) for 10 minutes. Rinse the dialysis bag thoroughly with distilled water, and boil it again in a 1mM EDTA solution (pH=8) for 10 minutes. After cooling, immerse the dialysis bag in a solution prepared with 0.05% sodium azide, 1% sodium benzoate, 1% formaldehyde, 50% glycerol, and 30% ethanol, and store it in a refrigerator at 4°C for later use.

[0066] 2) Thoroughly clean the dialysis bag with ddH2O, put the protein sample into the dialysis bag and tie it tightly with a thin thread, immerse it in 1L of fresh dialysis solution, and dialyze it at 4℃ for 5 hours, changing the dialysis solution once during the dialysis.

[0067] 3) Take the dialysis protein sample and place it in a 10KD ultrafiltration centrifuge tube. Centrifuge at 4300g and 4℃ for 20 minutes to ensure that the liquid retained in the tube after centrifugation is less than 10mL.

[0068] (7) EK enzyme digestion

[0069] Add 10 μL of Beverages EK enzyme (1 U / μL) to the protein solution obtained after ultrafiltration, and incubate overnight at 25°C for enzymatic digestion.

[0070] (8) Separation of target protein by reverse high performance liquid chromatography (RP-HPLC)

[0071] 1) After enzyme digestion, the sample precipitated. The sample was filtered using a 0.22μm filter membrane.

[0072] 2) Purify the target protein using RP-HPLC. Mobile phase A is 80% methanol, mobile phase B is ddH₂O containing 0.1% TFA, and mobile phase D is acetonitrile containing 0.1% TFA. Use a linear gradient elution for 40 min at a flow rate of 4 mL / min. The elution gradient is 95% to 5% for B and 5% to 95% for D. UV detection is performed at 220 nm. After startup, wash with 95% B and 5% D. Once the baseline stabilizes, equilibrate to zero. Manually load the sample through the injection valve, with a single loading volume less than 5 mL. Collect the protein peak at approximately 19 min, based on preliminary experimental results. After loading, wash the column with 5% B and 95% D until the baseline is linear. Then fill the column with 100% A. Finally, shut down the software and the instrument.

[0073] (9) Lyophilization and quantification of peptides

[0074] The peptides were lyophilized in a freeze dryer. The lyophilized peptide samples were dissolved with an appropriate amount of ddH2O. Protein quantification was performed using the Novozymes Protein BCA Quantification Kit. The remaining samples were lyophilized again and stored at -80°C.

[0075] (10) Identification of HIDfsin2 prokaryotic expression protein

[0076] Coomassie brilliant blue staining was used to identify samples during the prokaryotic expression of the tick-derived peptide HIDfsin2, and the results are as follows: Figure 3As shown in the figure. After bacterial cell lysis, most of the HIDfsin2 protein was expressed in the supernatant, with a small portion existing as inclusion bodies. After purification by nickel column chromatography, EK enzyme digestion, and RP-HPLC, the target band appeared near 6.5 kDa in the supernatant. The HPLC purity of the tick-derived peptide HIDfsin2 isolated by RP-HPLC is shown in the figure. Figure 4 As shown, the elution time of HIDfsin2 was 17.17 min, and it was a single peak. The isolated tick-derived polypeptide HIDfsin2 was identified by circular dichroism chromatography as... Figure 5 The graph shows a positive absorption peak at 190-200 nm and a negative absorption peak at 200-210 nm, indicating that the HIDfsin2 polypeptide possesses both α-helical and β-sheet structures. The molecular weight identified by mass spectrometry is as follows... Figure 6 As shown, the theoretical molecular weight of HIDfsin2 is 4130.79 Da, and the molecular weight after subtracting the three pairs of disulfide bonds formed is 4124.79 Da. The mass spectrometry result of 4125.26 Da is consistent with the theoretical molecular weight.

[0077] Example 2: Inhibition of Fever with Thrombocytopenia Syndrome Virus by Antiviral Peptide HIDfsin2

[0078] 1. Materials:

[0079] (1) Isolation and culture of primary peritoneal macrophages (MPMs) from C57BL / 6J mice

[0080] Six- to eight-week-old C57BL / 6J mice were intraperitoneally injected with 4% thioglycolate for three consecutive days. After euthanasia, the mice were immersed in 75% ethanol for 1-2 minutes, followed by cutting open the peritoneum and rinsing the abdomen with 5 mL of pre-cooled PBS to collect peritoneal macrophages. Cells were centrifuged at 1000 rpm for 6 minutes and resuspended in RPMI 1640 medium (10% FBS + 1% penicillin / streptomycin). After 2-4 hours, unadsorbed cells were washed away, leaving a monolayer of primary mouse peritoneal macrophages. The isolated cells were cultured at 37°C in a 5% CO2 incubator.

[0081] (2) Virus strain: SFTSV virus (GenBank: MW526369.1) was kindly provided by Professor Yu Xuejie of the School of Public Health, Wuhan University.

[0082] 2. Experimental methods and results

[0083] (1) Amplification of SFTSV

[0084] Once Vero cells in the T75 cell culture flasks have reached approximately 90% confluence, the culture medium is aspirated, and 2 mL of DMEM medium containing 2% FBS is added. Then, SFTSV virus solution is added, and the mixture is incubated at 37°C for 1-2 hours. Finally, 8 mL of 10% FBS-DMEM medium is added. After approximately 5-7 days, the cells are repeatedly frozen and thawed 3-5 times. The cells and supernatant are collected into centrifuge tubes, centrifuged at 3000-4000 rpm for 10 minutes at 4°C, cell debris is removed, and the supernatant is aliquoted and stored at -80°C for later use.

[0085] (2) Dose-response relationship detection of HIDfsin2 peptide on SFTSV infection inhibition

[0086] MPMs at approximately 2 × 10 6 Cells were seeded at a rate of 10 cells / well in 24-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. HIDfsin2 peptide was diluted with PBS and added to MPMs at final concentrations of 0, 5, 10, and 20 μM. After 1 h of pretreatment, SFTSV virus solution with an MOI of 1 was added for infection. After 1-2 h, unadsorbed virus was washed away, and the corresponding final concentration of HIDfsin2 peptide was added again. Cell samples were collected after 72 h. Total RNA was extracted from the cells, and the RNA was converted to cDNA using a reverse transcription kit from Novizan. SFTSV vRNA was then quantified using real-time quantitative PCR. The real-time quantitative PCR primers were: SFTSV NP-upstream primer: ATGTCAGAGTGGTCCAGGA; SFTSV NP-downstream primer: TCTCCACCTGTCTCCTTCAG. Analysis of the effect of different concentrations of tick-derived antiviral peptide HIDfsin2 on SFTSV vRNA in SFTSV-infected cells is as follows. Figure 7 As shown, the horizontal axis represents the concentration of HIDfsin2 (μM), and the vertical axis represents the relative viral replication number after treatment with the corresponding concentration of HIDfsin2 peptide. HIDfsin2 can inhibit SFTSV replication in a concentration-dependent manner.

[0087] Following the same treatment method described above, cellular protein samples were collected after 72 hours. The prepared protein samples were then analyzed for SFTSV NP protein using Western blotting. The results are as follows: Figure 8 As shown, the expression level of SFTSVNP protein decreased sequentially with increasing HIDfsin2 concentration.

Claims

1. Use of a tick-borne antivirally active polypeptide for the manufacture of a medicament for the prevention and / or treatment of SFTSV infection, characterized in that: The amino acid sequence of the tick-derived antiviral active polypeptide is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that: The sequence of the nucleic acid encoding the tick-derived antiviral active polypeptide is shown as SEQ ID NO.

2.

3. Use according to claim 1, characterized in that: The preparation method of the tick-derived antiviral active polypeptide comprises the following steps: culturing a recombinant cell to express the tick-derived antiviral active polypeptide. The recombinant cell contains a recombinant expression vector, and the recombinant expression vector contains a nucleic acid with a sequence shown as SEQ ID NO.

2.

4. Use according to claim 3, characterized in that: The recombinant expression vector takes pET-32a as an expression vector.

Images