Artificially synthesized polypeptide and application thereof in resisting herpes simplex virus type I

Abstract

The invention belongs to the field of biological medicine, and discloses an artificially synthesized polypeptide and application thereof in resisting herpes simplex virus type I. The polypeptide provided by the invention has a very good antiviral effect and slight toxic and side effects, and can resist brain and systemic infection caused by herpes simplex virus type I. The antiviral mechanism of the polypeptide is clear, and favorable guarantee is provided for application and optimization of subsequent polypeptide inhibitors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a synthetic polypeptide and its application in anti-Herpes simplex virus type I. Background Art

[0002] Herpes simplex virus type 1 (HSV-1) is an infectious pathogen that widely infects humans. It is generally transmitted through the air by respiratory secretions such as saliva, droplets, and sputum, and can also be transmitted by direct contact. After HSV-1 infects the body, it achieves latent infection by antagonizing the host antiviral strategy. The main latent site is the peripheral nervous system. When the host immunity declines or is stimulated by stress factors, the virus can be reactivated for recurrent infection. HSV-1 is very common in the global population. According to the World Health Organization's estimate, approximately 3.8 billion people under the age of 50 are infected with this virus. Infections caused by HSV-1 are usually asymptomatic or only cause moderate symptoms, such as oral herpes or cold sores, while recurrent infections during the latent period are usually accompanied by painful symptoms, such as lesions in the facial or genital area. In addition, HSV-1 infection may be associated with many more serious complications, such as herpetic stromal keratitis (HSK), which is the main cause of infectious corneal blindness, and herpes simplex encephalitis (HSE), which may lead to severe neurological impairment and a high mortality rate. In addition, individuals with a weakened immune system or those undergoing immunosuppressive therapy are more likely to be infected with this virus, with a stronger severity and a higher recurrence frequency.

[0003] HSV-1 is a double-stranded DNA enveloped virus belonging to the Alphaherpesvirinae subfamily of the Herpesviridae family. The full-length genome is approximately 152 kilobase pairs (kbp) and contains at least 84 open reading frames (ORFs). The virus particle presents a three-layer structure: the core part is an icosahedral capsid that encloses the viral DNA, and this capsid is composed of proteins such as VP5, VP19C, and VP23; the outer layer is a lipid envelope rich in glycoproteins (such as gB, gC, and gD); between the core and the envelope is the tegument containing various regulatory proteins (such as VP16 and vhs protein). The genome of HSV-1 consists of a long unique region (UL) and a short unique region (US), with inverted repeat sequences (RL and RS) at both ends, and the genome isomerization process is mediated by the "a" sequence, ultimately forming four functionally equivalent isomers.

[0004] After invading the body, HSV-1 undergoes productive (lytic) infection, during which a large number of infectious viruses and gene expression products at various lytic stages are produced. According to the order of these gene expressions, they can be classified into immediate-early genes, early genes, and late genes. Meanwhile, the productive infection of HSV-1 activates the host's innate immune system, and multiple proteins encoded by HSV-1 can reduce the host's innate immune attack on the virus by antagonizing the activation of pathways such as TLR, cGAS-STING, and NF-κB, ensuring the effective replication of the virus itself.

[0005] So far, all approved antiviral drugs against HSV-1 (e.g., acyclovir and related nucleoside analogs) have the same therapeutic target, viral DNA polymerase. In addition, resistance and even cross-resistance to acyclovir and other anti-HSV-1 nucleoside analogs often occur. Therefore, it is very necessary to identify new therapeutic targets and develop new antiviral strategies. Summary of the Invention

[0006] The object of the present invention is to provide a synthetic polypeptide, the polypeptide is WSYYIGYLRLILPELQARIRTYNQHYNNLL, and the polypeptide W30L added with the TAT sequence is: YGRKKRRQRRRWSYYIGYLRLILPELQARIRTYNQHYNNLL.

[0007] Another object of the present invention is to provide a truncated polypeptide based on the above polypeptide, which is LILP; the polypeptide L4P added with the TAT sequence is: YGRKKRRQRRRLILP.

[0008] The object of the present invention is to provide the application of the above polypeptide in anti-herpes simplex virus type I.

[0009] To achieve the above object, the present invention takes the following technical measures:

[0010] The inventive concept of the present invention is as follows:

[0011] The UL38 (also known as VP19C) protein of HSV-1 is not only an important component of the viral capsid but also has crucial DNA-binding and nucleocytoplasmic shuttle activities, which are essential for virus assembly and replication. The applicant found that UL38 effectively blocked the STING-TBK1-IRF3 signaling pathway by interacting with STING, thereby inhibiting cGAS-STING-dependent immune signal transduction. Based on the amino acid sequence of the interaction between UL38 and STING, a series of polypeptides WSYYIGYLRLILPELQARIRTYNQHYNNLL targeting the STING-binding interface in UL38 were designed, and the sequence was truncated at different positions. Subsequently, a cell-penetrating peptide sequence TAT that can penetrate the cell membrane was added to the N-terminus of its amino acid sequence, thereby obtaining polypeptides W30L, I10R, G10L, Q10H, L4P, and their sequences are shown as SEQ ID NO.1 to SEQ ID NO.5. Polypeptides W30L and L4P can effectively disrupt the interaction between STING and UL38, thereby releasing the host's innate immune response. In a mouse model, polypeptide L4P significantly enhanced the immune response against HSV-1 and effectively improved the symptoms of brain and systemic infections caused by HSV-1, showing strong therapeutic potential.

[0012] The polypeptide L4P of the present invention can be obtained by artificial synthesis or common microbial expression methods. The protection scope of the present invention includes:

[0013] A synthetic polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO.6.

[0014] A synthetic polypeptide, the amino acid sequence of the polypeptide is LILP.

[0015] A synthetic polypeptide with a cell-penetrating peptide added to the N-terminus of the above polypeptide.

[0016] For the above-mentioned polypeptides, preferably, the added cell-penetrating peptide is TAT. The amino acid sequence of the polypeptide shown as SEQ ID NO.6 after adding the cell-penetrating peptide is shown as SEQ ID NO.1, and the amino acid sequence of the polypeptide shown as LILP after adding the cell-penetrating peptide is shown as SEQ ID NO.5.

[0017] The protection scope of the present invention also includes:

[0018] A fusion protein obtained by fusing the above-mentioned polypeptide with a cell-penetrating peptide added to the N-terminus with a protein tag.

[0019] A gene encoding the above-mentioned polypeptide with a cell-penetrating peptide added to the N-terminus or the above-mentioned fusion protein.

[0020] An expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell having the above-mentioned coding gene.

[0021] A composition comprising the polypeptide with a transmembrane peptide added to the N-terminus as described above.

[0022] Use of the polypeptide with a transmembrane peptide added to the N-terminus as described above, fusion protein, gene encoding the polypeptide with a transmembrane peptide added to the N-terminus or the fusion protein as described above, expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell having the above-mentioned coding gene, or the above composition in the preparation of an inhibitor of herpes simplex virus type I.

[0023] Use of the polypeptide with a transmembrane peptide added to the N-terminus as described above, fusion protein, gene encoding the polypeptide with a transmembrane peptide added to the N-terminus or the fusion protein as described above, expression cassette, recombinant vector, recombinant microorganism or isolated recombinant cell having the above-mentioned coding gene, or the above composition in the preparation of a medicament for treating or preventing herpes simplex virus type I.

[0024] In the above-mentioned use, preferably, the herpes simplex virus type I is Human Herpesvirus 1.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The present invention introduces an L4P polypeptide with a significant effect against herpes simplex virus type I. This polypeptide can precisely target the UL38-STING interface and does not interfere with the normal physiological functions of STING. In addition, the L4P polypeptide shows unique advantages in avoiding drug resistance. When the HSV-1 virus evolves to produce certain UL38 mutations to escape the action of the L4P polypeptide, these mutants will also lose the ability to interact with STING, thus unable to achieve immune escape, which puts the virus at a disadvantage in evolution. Given that all current anti-HSV-1 drugs, such as acyclovir, target viral DNA polymerase and there have already been drug resistance and cross-resistance to acyclovir and other anti-HSV-1 nucleoside analogs, determining the STING antagonist function of UL38 as a new drug target and developing this drug resistance strategy as an alternative therapy for HSV-1 and related diseases has extremely important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 For the screening of polypeptides against HSV-1.

[0028] Figure 2 For the cytotoxicity of W30L, L4P, G10L polypeptides.

[0029] Figure 3 For the anti-HSV-1 IC50 of the L4P polypeptide in HeLa cells.

[0030] Figure 4 The IC50 of L4P polypeptide against HSV-1 in MLF cells.

[0031] Figure 5 The effect of L4P on the expression levels of HSV-1-induced inflammation-related genes in HeLa cells.

[0032] Figure 6 The effect of L4P on the expression levels of HSV-1-induced inflammation-related genes in MLF cells.

[0033] Figure 7 The inhibition of the interaction between STING and UL38 by L4P and W30L in 293T cells.

[0034] Figure 8 The antiviral ability of L4P in HeLa and HeLa STING- / - cells.

[0035] Figure 9 The detection of the distribution of L4P in animals by a small animal imaging device.

[0036] Figure 10 The detection of the distribution of L4P in animal tissues and organs by a small animal imaging device.

[0037] Figure 11 The statistical analysis of the radiation efficiency of L4P in animal tissues and organs by a small animal imaging device.

[0038] Figure 12 The difference in the levels of viral replication in the lungs of mice with systemic HSV-1 infection treated with L4P at different times.

[0039] Figure 13 The difference in the levels of viral replication in the spleens of mice with systemic HSV-1 infection treated with L4P at different times.

[0040] Figure 14 The difference in the levels of inflammation-related genes in the lungs of mice with systemic HSV-1 infection treated with L4P at different times.

[0041] Figure 15 The difference in the levels of inflammation-related genes in the spleens of mice with systemic HSV-1 infection treated with L4P at different times.

[0042] Figure 16 The difference in the levels of viral replication in the brain tissues of mice with intracranial HSV-1 infection treated with L4P at different times.

[0043] Figure 17 The difference in the levels of inflammation-related genes in the brain tissues of mice with intracranial HSV-1 infection treated with L4P at different times.

[0044] Figure 18 Survival rate of mice with systemic HSV-1 infection treated with L4P for 14 days.

[0045] Figure 19 Survival rate of mice with intracranial HSV-1 infection treated with L4P for 14 days. Detailed implementation manners

[0046] The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified; the reagents or materials are commercially available unless otherwise specified.

[0047] The 24-well plates and 50 ml syringes were purchased from Diyue Innovation Company. The Total RNA extraction reagent (FORGENE) was purchased from Fujie, the 0.22 μm filter membrane (millipore) was purchased from Feiyi Company, and the One step q-PCR kit (takara) was purchased from Youming Company. The DMEM medium (Gibco) and FBS fetal bovine serum (Gibco) were purchased from Invitrogen.

[0048] The HSV-1 used in the present invention is HSV-1F strain (from Microbial Culture and Virus Collection Center of Wuhan Institute of Virology, CAS; CSTR: 16533.IVCAS6.9705).

[0049] Example 1:

[0050] Design and screening of anti-HSV-1 virus polypeptides:

[0051] 1. Materials

[0052] The applicant designed a series of polypeptides against the UL38 protein of the HSV-1 protein according to its interaction interface with STING and its sequence characteristics, and added a cell-penetrating peptide sequence TAT (YGRKKRRQRRR) to the N-terminus of the amino acid sequences of these polypeptides. Finally, the polypeptides to be screened were obtained: W30L, G10L, I10R, Q10H, L4P. These polypeptides were synthesized by Nanjing Genscript Corporation, and the One step q-PCR kit (takara) was purchased from Youming Company.

[0053] The amino acid sequences of the polypeptides to be screened are as follows:

[0054]

[0055] 2. Amplification of virus

[0056] (1) Inoculate Vero E6 cells into a T75 cell flask;

[0057] (2) When the cells reach 80%-90% confluence, replace the DMEM medium containing 10% serum with the DMEM medium containing 2% serum, and add 10 μl of HSV-1 at 10^6 PFU / ml.

[0058] (3) After 2 days, observe whether the CPE phenomenon occurs in the cells, and collect the samples when the lesion phenomenon is obvious.

[0059] (4) Freeze-thaw the T75 flask at -80 °C three times repeatedly, then transfer the solution to a 50 ml centrifuge tube and centrifuge at 5000 g for 30 min.

[0060] (5) Transfer the supernatant after centrifugation to a new 50 ml centrifuge tube, and filter and sterilize it using a 50 ml syringe and a 0.22 μm filter membrane.

[0061] (6) Take 100 μl of the virus for plaque assay to detect the virus titer.

[0062] 3. Determine the antiviral efficiency of different polypeptides in cells:

[0063] (1) Seed HeLa cells in a 24-well plate.

[0064] (2) When the cells reach 80%-90% confluence, discard the DMEM medium containing 10% serum, wash the cells with PBS, and then add the DMEM medium containing 2% serum (0.5 ml per well), and add the corresponding dose of HSV-1 virus to each well to make the infection dose 0.1 MOI.

[0065] (3) After 1 h, add different lengths of polypeptides in Table 1 with a final concentration of 10 μM respectively; use the group infected with HSV-1 virus without adding polypeptides as the control group.

[0066] (4) Collect the samples 24 h after virus infection, and lyse and inactivate the cells with Trizol.

[0067] (5) Discard the supernatant, add 1 mL of Trizol RNA lysis solution to the wells, and lyse for more than 30 min to inactivate the virus.

[0068] (6) Add 200 μl of chloroform to the lysis solution, shake well, let it stand, and centrifuge at 12000 g for 10 min, then collect the upper layer liquid after centrifugation.

[0069] (7) Add an equal volume of isopropanol to the collected liquid, mix well, precipitate at -20 °C for 10 min, and centrifuge at 12000 g for 15 min. (8) Discard the supernatant, retain the precipitate, add 1 ml of 75% ethanol solution, and centrifuge at 12000 g for 5 min.

[0070] (9) Discard the supernatant and retain the precipitate. Dissolve the precipitate with 50 μl of DEPC water.

[0071] (10) Take 3 μl of the RNA sample and perform fluorescence quantitative experiments using a one-step qRT-PCR kit. Each group has 3 replicates.

[0072] The results are as Figure 1 shown in Table 1. After adding 10 μm polypeptides with different lengths (TAT, W30L, G10L, I10R, Q10H, L4P), the three polypeptides W30L, L4P, and G10L have the ability to resist HSV-1 virus.

[0073] The calculation method of the virus RNA percentage (%) is: the relative virus quantity value corresponding to each sample / the average relative virus quantity of the control group * 100.

[0074] Table 1

[0075]

[0076] Example 2:

[0077] Toxicity determination of the three polypeptides W30L, L4P, and G10L on HeLa cells

[0078] 1. Experimental materials

[0079] CCK-8 reagent (MCE) was purchased from Qidong Co., Ltd. 2. Experimental procedure

[0080] This study evaluated the cytotoxicity of the polypeptides W30L, L4P, and G10L, mainly using the CCK-8 reagent to detect this index. Cells without any treatment were used as the control group.

[0081] The steps are as follows:

[0082] (1) Seed HeLa cells in a 96-well cell culture plate, 100 μl per well.

[0083] (2) When the cells grow to 80%-90% confluence, remove the DMEM medium containing 10% serum. After washing the cells with PBS, add DMEM medium containing 2% serum (0.5 ml per well), and add polypeptides W30L, L4P, or G10L at a certain concentration gradient, so that the final polypeptide concentrations in the wells are 0, 2.34375, 4.6875, 9.375, 18.75, 37.5, 75, 150, 300 μM, respectively. The group added with TAT polypeptide was set as the control group, and each group had 6 replicates.

[0084] (3) After adding the polypeptide and incubating for 24 h, samples were collected. 10 μl of the live cell detection reagent CCK-8 was added to each well and mixed evenly.

[0085] (4) Incubate at 37 °C for 4 h.

[0086] (5) Measure the absorbance value at OD450 using an enzyme-linked immunosorbent assay (ELISA) reader.

[0087] The results are as Figure 2 shown in Table 2, Table 3, and Table 4. When 300 μM of the L4P polypeptide was added to the cells, there was no significant difference in cell viability compared to the control group. However, W30L and G10L showed obvious cytotoxicity at a concentration of 37.5 μM. This indicates that the L4P polypeptide screened in this study has no toxic effect on HeLa cells when the concentration does not exceed 300 μM.

[0088] Calculation method of cell survival rate:

[0089] Absorbance value at OD450 of the experimental group / Average absorbance value at OD450 of the control group * 100 (the survival rates in the following examples are all calculated according to this formula).

[0090] Table 2

[0091]

[0092] Table 3

[0093] Table 4 Example 3: Determination of the antiviral efficiency of polypeptide L4P in HeLa cells and MLF cells 1. Materials HeLa cells and MLF cells were used. The polypeptide L4P was synthesized by GenScript (Nanjing). The One Step q-PCR Kit (Takara) was purchased from Youming Company. 2. Determination of the antiviral efficiency of polypeptide L4P in various cells (1) Seed HeLa cells and MLF cells in 24-well plates. (2) When the cell confluence reaches 80%-90%, discard the DMEM medium containing 10% serum. After washing the cells with PBS, add DMEM medium containing 2% serum (0.5 ml per well), and add the corresponding titer of HSV-1 to each well to make the infection dose 0.1 MOI. (3) After 1 h, add polypeptide L4P at a certain concentration gradient to different cells, so that the final concentration of L4P in the wells is 0, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 μM of polypeptide L4P in different cells respectively. The group adding TAT polypeptide was used as the control group. (4) Harvest the samples 24 h after virus infection, and lyse and inactivate the cells with Trizol. (5) Discard the supernatant, add 1 mL of Trizol RNA lysis solution to the wells, and lyse for more than 30 min to inactivate the virus.

[0094] (6) Add 200 μl of chloroform to the lysate, mix well by shaking, let it stand, and centrifuge at 12,000 g for 10 min. Collect the upper layer liquid after centrifugation.

[0095] (7) Add an equal volume of isopropanol to the collected liquid, mix well, precipitate at -20 °C for 10 min, and centrifuge at 12,000 g for 15 min. (8) Discard the supernatant, retain the precipitate, add 1 ml of 75% ethanol solution, and centrifuge at 12,000 g for 5 min.

[0096] (9) Discard the supernatant, retain the precipitate, and dissolve the precipitate with 50 μl of DEPC water.

[0097] (10) Take 3 μl of the RNA sample and perform a fluorescence quantitative experiment with the One Step qRT-PCR Kit.

[0098] The results are as shown in Figure 3 , Figure 4 Table 5 and Table 6. On two HSV-1 susceptible cells, HeLa cells and MLF cells, polypeptide L4P can effectively inhibit virus replication, suggesting that polypeptide L4P has good antiviral effects and shows concentration dependence. The IC50 values of polypeptide L4P in HeLa cells and MLF cells are 1.535 μM and 1.637 μM respectively.

[0099] The calculation method of the percentage of viral RNA is: the relative viral quantity value corresponding to each sample / the average relative viral quantity of the control group * 100.

[0100] Table 5

[0101]

[0102] Table 6

[0103]

[0104] Example 4:

[0105] Evaluation of the effect of polypeptide L4P on the STING innate immune pathway 1. Materials

[0106] HeLa cells, MLF cells. Polypeptide L4P was synthesized by GenScript Corporation (Nanjing). One step q-PCR kit (takara) was purchased from Youming Company.

[0107] 2. Evaluation of the effect of polypeptide L4P on the STING innate immune pathway

[0108] (1) Seed HeLa cells and MLF cells in 24-well plates.

[0109] (2) When the cell confluence reaches 50%-60%, discard the DMEM medium containing 10% serum. After washing the cells with PBS, add DMEM medium containing 2% serum (the addition volume per well is 0.5 ml). Add polypeptide L4P or TAT to different cells so that the final concentration of L4P or TAT in the wells is 10 μM. The group adding TAT polypeptide is used as the control group. (3) Add HSV-1 moi = 1 12 h after adding the polypeptide. Harvest the samples 24 h after adding the polypeptide, and lyse and inactivate the cells with Trizol.

[0110] (4) Discard the supernatant, add 1 mL of Trizol RNA lysis solution to the wells, and lyse for more than 30 min to inactivate the virus.

[0111] (5) Add 200 μl of chloroform to the lysis solution, shake well, let stand, and centrifuge at 12000 g for 10 min. Collect the upper layer liquid after centrifugation.

[0112] (6) Add an equal volume of isopropanol to the collected liquid, mix well, precipitate at -20 °C for 10 min, and centrifuge at 12000 g for 15 min. (7) Discard the supernatant, retain the precipitate, add 1 ml of 75% ethanol solution, and centrifuge at 12000 g for 5 min.

[0113] (8) Discard the supernatant and retain the precipitate, and dissolve the precipitate with 50 μl of DEPC water.

[0114] (9) Take 3 μl of the RNA sample and perform a fluorescence quantitative experiment using a one step qRT-PCR kit to detect IFNB1, ISG15, and RANTES.

[0115] The results are as Figure 5 , Figure 6 , as shown in Table 7 and Table 8, the polypeptide L4P can effectively inhibit the viral replication in the lungs and spleens of C57BL / 6 mice infected with HSV-1, suggesting that the polypeptide L4P has good antiviral effects in mice.

[0116] The calculation method of the relative expression level of gene RNA is: the relative virus quantity value corresponding to each sample / the average relative virus quantity of the control group, and the calculation results of the P values of the differences between data groups are marked in the figure.

[0117] Table 7

[0118]

[0119] Table 8

[0120]

[0121]

[0122] Example 5:

[0123] Effects of polypeptides TAT, W30L, and L4P on the interaction between STING protein and HSV-1 UL38 protein 1. Materials

[0124] 293T cells and polypeptide L4P were synthesized by GenScript Corporation (Nanjing). Protein A+G agarose beads were purchased from Beyotime (Shanghai), and FLAG M2 was purchased from sigma (USA).

[0125] 2. Effects of polypeptides TAT, W30L, and L4P on the interaction between STING protein and HSV-1 UL38 protein in 293T cells

[0126] (1) Seed 293T cells in a 6-cm dish and divide them into 4 groups.

[0127] (2) When the cell fusion reaches 70%-80% confluence, use the transfection reagent lipo2000 to transfect the expression plasmids of FALG-UL38 protein and HA-STING protein into the cells.

[0128] (3) After 24 h, add to the four dishes respectively: solvent, 10 μM TAT, 10 μM W30L, 10 μM L4P. Collect the samples after 24 h. (4) Lyse the cells with RIPA lysis buffer and lyse at 4 °C for 30 min.

[0129] (5) Centrifuge the samples at 4 °C, 12000 g, for 15 min.

[0130] (6) Subsequently, collect the supernatant, add 10 μl of protein A+G agarose beads and 2 μl of FLAG M2 antibody thereto, and incubate on a rotary shaker at 4 °C for 1 h.

[0131] (7) Centrifuge the above samples at 12000 g for 10 s, discard the supernatant, add 1 mL of RIPA, and repeat three times.

[0132] (8) After the last centrifugation, add 1x protein loading buffer to the agarose beads and boil the samples at 95 °C for 15 min.

[0133] (9) Detect the corresponding proteins of the samples by western blot.

[0134] The results are as Figure 7 shown. There is an interaction between STING and UL38 in the Control group and the TAT group, TAT has no effect on their interaction, and W30L and L4P significantly weaken the interaction between STING and UL38. This indicates that W30L and L4P can intervene in the STING-UL38 interaction interface and weaken the binding of UL38 and SITNG..

[0135] Example 6:

[0136] Determination of the antiviral efficiency of polypeptides TAT and L4P on STING-deficient cells

[0137] 1. Materials

[0138] HeLa, HeLa STING - / - cells. The polypeptides TAT and L4P were synthesized by GenScript Corporation (Nanjing). The One step q-PCR kit (takara) was purchased from Youming Company.

[0139] 2. Determination of the antiviral efficiency of polypeptides TAT and L4P on STING-deficient cells

[0140] (1) Seed the HeLa and HeLa STING - / - cells in 24-well plates.

[0141] (2) When the cells reach 80%-90% confluence, discard the DMEM medium containing 10% serum. After washing the cells with PBS, add DMEM medium containing 2% serum (the addition volume per well is 0.5 ml), and add the corresponding titer of HSV-1 virus to each well to make the infection dose 0.1 MOI.

[0142] (3) After 1 h, add a certain concentration of polypeptide L4P to different cells so that the final concentration of L4P in the wells is 10 μM. The group adding 10 μM TAT polypeptide is used as the control group.

[0143] (4) Sample collection is carried out 24 h after virus infection, and RNA is extracted using a total RNA extraction kit.

[0144] (5) Discard the supernatant, add 250 μl of cRL1 lysis solution to the wells, and lyse on a shaker for 30 min.

[0145] (6) First add the lysis solution to the DNA column, centrifuge at 12000 g for 1 min, and collect the centrifuged liquid.

[0146] (7) Add 400 μl of cRL2 solution to the centrifuged liquid and pipette to mix evenly.

[0147] (8) Transfer the solution to the RNA extraction column and centrifuge at 12000 g for 1 min.

[0148] (9) Add RNA washing buffer1 solution to the RNA column and centrifuge at 12000 g for 30 s.

[0149] (10) Add RNA washing buffer2 solution and centrifuge at 12000 g for 1 min.

[0150] (11) Centrifuge the RNA column empty at 12000 g for 2 min to completely remove the residual RNA washing buffer.

[0151] (12) Add 50 μl of DEPC water to the RNA column and centrifuge at 12000 g for 2 min.

[0152] (13) Take 3 μl of RNA sample and perform a fluorescence quantitative experiment using a one step qRT-PCR kit, with 3 replicates for each group.

[0153] The results are as Figure 8 , shown in Table 9 and Table 10. In HeLa STING - / - cells, compared with the group adding TAT polypeptide, the relative content of the virus in the group adding polypeptide L4P has no obvious change.

[0154] The calculation method of the relative expression level of viral RNA is: the relative viral quantity value corresponding to each sample / the average relative viral quantity of the control group, and the calculated results of the P value of the difference between data groups are marked in the figure.

[0155] Table 9

[0156]

[0157] Table 10

[0158]

[0159] Example 7:

[0160] Polypeptide L4P is rapidly distributed in mice

[0161] 1. Materials

[0162] Polypeptide L4P was synthesized by GenScript. C57BL / 6 mice.

[0163] 2. Distribution of polypeptide L4P in mice

[0164] (1) Six 8-week-old C57BL / 6J mice were randomly divided into two groups, and were intraperitoneally injected with 10 mg / kg Cy5-L4P or the control group (n = 3) (for background fluorescence measurement).

[0165] (2) One hour after injection, the distribution of Cy5-L4P in mice was imaged by an IVIS Lumina K Series III in vivo imaging system (Perkin Elmer).

[0166] (3) The mice were sacrificed by injecting sodium pentobarbital, and the corresponding tissues were obtained, and the brain, heart, lung, liver, and kidney of the mice were imaged and fluorescence evaluated.

[0167] (4) Living Image 4.4 software was used to calculate the relevant radiant efficiency (Ps-1cm-2sr-1) (μW-1cm2).

[0168] The results are as Figure 9 , Figure 10 , Figure 11 , as shown in Table 11, polypeptide L4P can be rapidly distributed in various organs (heart, liver, spleen, lung, kidney, brain, intestine) of mice 1 h after intraperitoneal injection. Figure 9 The right group is the Cy5-L4P injection group, Figure 10 The lower group is the dissected organ group of mice injected with Cy5-L4P, and the rest are controls.

[0169] The radiation efficiency is calculated by reading the values of the regions with Cy5 signals in the organs in the figure and calculating the relevant radiation efficiency (Ps-1cm-2sr-1) (μW-1cm2).

[0170] Table 11

[0171]

[0172] Example 8:

[0173] Evaluation of the antiviral effect of polypeptide L4P against systemic HSV-1 infection in mice

[0174] 3. Materials

[0175] Polypeptide L4P was synthesized by GenScript Corporation. C57BL / 6 mice.

[0176] 2. Antiviral activity of polypeptide L4P in mice

[0177] (1) Thirty-six 8-week-old C57BL / 6 mice were divided into 6 groups of 6 mice each. These 36 mice were challenged by intraperitoneal injection, and the infection dose of the HSV-1 strain was 10 7 PFU / mouse.

[0178] (2) One hour after challenge, the 6 groups of mice were treated with drugs by intraperitoneal injection. Three groups were given 10 mg / kg of L4P, and the other three groups were given 10 mg / kg of TAT as a control.

[0179] (3) The drugs and the vehicle were administered intraperitoneally twice a day for 7 consecutive days. (4) At 0, 4, and 7 days after challenge, the mice treated with TAT and L4P were euthanized, and their lung and spleen tissues were collected. First, the tissues were ground and inactivated with Trizol, and then the total RNA in the tissues was extracted. (5) 200 μl of chloroform was added to the RNA lysate, shaken well, allowed to stand, centrifuged at 12,000 g for 10 min, and the upper layer of the centrifuged liquid was collected.

[0180] (6) An equal volume of isopropanol was added to the collected liquid, mixed well, precipitated at -20 °C for 10 min, and centrifuged at 12,000 g for 15 min. (7) The supernatant was discarded, the precipitate was retained, 1 ml of 75% ethanol solution was added, and centrifuged at 12,000 g for 5 min.

[0181] (8) The supernatant was discarded, the precipitate was retained, and the precipitate was dissolved in 50 μl of DEPC water.

[0182] (9) 3 μl of the RNA sample was taken, and fluorescence quantitative experiments were performed using a one step qRT-PCR kit, with 6 replicates in each group.

[0183] The results are as Figure 12 , Figure 13 , Figure 14 , Figure 15 , as shown in Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, Table 18, Table 19, Table 20, and Table 21. The polypeptide L4P can effectively inhibit virus replication in the lungs and spleens of C57BL / 6 mice infected with HSV-1, suggesting that the polypeptide L4P has good antiviral effects in mice. At the same time, the polypeptide can upregulate the expression levels of Ifnb1, Isg15, Rantes, and Tnfα to fight the virus on the 4th day of HSV-1 infection. On the 7th day of treatment, due to the decrease in virus load, the levels of Ifnb1, Isg15, Rantes, and Tnfα in the L4P group are lower than those in the TAT group, indicating that L4P does not induce persistent inflammation and cause damage.

[0184] The calculation method of the relative gene RNA expression level is: the relative virus quantity value corresponding to each sample / the average relative virus quantity of the control group. The calculation results of the P values for differences between data groups are marked in the figure.

[0185] Table 12

[0186]

[0187] Table 13

[0188]

[0189]

[0190] Table 14

[0191]

[0192] Table 15

[0193]

[0194] Table 16

[0195]

[0196] Table 17

[0197]

[0198] Table 18

[0199]

[0200]

[0201] Table 19

[0202]

[0203] Table 20

[0204]

[0205] Table 21

[0206]

[0207]

[0208] Example 9:

[0209] Evaluation of the antiviral effect of polypeptide L4P against HSV-1 infection in the mouse brain

[0210] 1. Materials

[0211] The polypeptide L4P was synthesized by GenScript Corporation. C57BL / 6 mice.

[0212] 2. Antiviral activity of polypeptide L4P in the mouse brain

[0213] (1) Forty-eight 8-week-old C57BL / 6 mice were divided into 8 groups of 6 mice each. These 48 mice were challenged by intracranial injection, and the infection dose of the HSV-1 strain was 10 4 PFU / mouse.

[0214] (2) One hour after challenge, the 8 groups of mice were treated with drugs by intraperitoneal injection. Four groups were given 10 mg / kg of L4P, and the other four groups were given 10 mg / kg of TAT as a control.

[0215] (3) The drugs and the vehicle were administered intraperitoneally twice a day for 5 consecutive days.

[0216] (4) At 0 day, 0.5 day, 3 days, and 5 days after challenge, the mice treated with TAT and L4P were euthanized, and their brain tissues were taken. First, the tissues were ground and inactivated with Trizol, and then the total RNA in the tissues was extracted.

[0217] (5) 200 μl of chloroform was added to the RNA lysate, shaken and mixed well, allowed to stand, and centrifuged at 12000 g for 10 min, and the upper layer liquid after centrifugation was collected.

[0218] (6) An equal volume of isopropanol was added to the collected liquid, mixed well, precipitated at -20 °C for 10 min, and centrifuged at 12000 g for 15 min. (7) The supernatant was discarded, the precipitate was retained, 1 ml of 75% ethanol solution was added, and centrifuged at 12000 g for 5 min.

[0219] (8) Discard the supernatant and retain the precipitate, and dissolve the precipitate with 50 μl of DEPC water.

[0220] (9) Take 3 μl of the RNA sample and perform a fluorescence quantitative experiment using a one step qRT-PCR kit, with 6 replicates in each group.

[0221] The results are as Figure 16 , Figure 17 , Table 22, Table 23, Table 24, Table 25, Table 26, Table 27, Table 28 show that the polypeptide L4P can effectively inhibit viral replication in the brain tissues of C57BL / 6 mice infected with HSV-1, suggesting that the polypeptide L4P has good antiviral effects in mice. At the same time, the polypeptide can upregulate the expression levels of Ifnb1, Isg15, Rantes, and Tnfα to fight the virus at 0.5 days after HSV-1 infection. On the 5th day of treatment, due to the decrease in viral load, the levels of Ifnb1, Isg15, Rantes, and Tnfα in the L4P group are lower than those in the TAT group, indicating that L4P will not induce continuous inflammation and cause damage.

[0222] The calculation method for the relative expression level of gene RNA is: the relative virus quantity value corresponding to each sample / the average relative virus quantity of the control group, and the calculation results of the P values for differences between data groups are marked in the figure.

[0223] Table 22

[0224]

[0225] Table 23

[0226]

[0227] Table 24

[0228]

[0229]

[0230] Table 25

[0231]

[0232] Table 26

[0233]

[0234] Table 27

[0235]

[0236] Table 28

[0237]

[0238] Example 10:

[0239] Salvage rate of polypeptide L4P in systemic HSV-1 infection in mice

[0240] 1. Materials

[0241] Polypeptide L4P was synthesized by GenScript. C57BL / 6 mice.

[0242] 2. Antiviral activity of polypeptide L4P in the brains of mice

[0243] (1) Twenty 8-week-old C57BL / 6 mice were divided into 2 groups, with 10 mice in each group. These 20 mice were challenged by intraperitoneal injection, and the infection dose of the HSV-1 strain was 10 7 PFU / mouse.

[0244] (2) One hour after challenge, the 2 groups of mice were treated with drugs by intraperitoneal injection. One group was given 10 mg / kg of L4P, and the other group was given 10 mg / kg of TAT as a control.

[0245] (3) The drug and the solvent were administered intraperitoneally twice a day for 14 consecutive days. The survival rate of the mice was recorded.

[0246] The results are as Figure 15 shown. Polypeptide L4P can effectively rescue the survival rate of C57BL / 6 mice infected with HSV-1, indicating that polypeptide L4P has good antiviral effects in mice.

[0247] Example 11:

[0248] Salvage rate of polypeptide L4P in HSV-1-infected brains of mice

[0249] 1. Materials

[0250] Polypeptide L4P was synthesized by GenScript. C57BL / 6 mice.

[0251] 2. Antiviral activity of polypeptide L4P in the brains of mice

[0252] (1) Twenty 8-week-old C57BL / 6 mice were divided into 2 groups, with 10 mice in each group. These 20 mice were challenged by intracranial injection, and the infection dose of the HSV-1 strain was 10 4 PFU / mouse.

[0253] (2) One hour after challenge, the 2 groups of mice were treated with drugs by intraperitoneal injection. One group was given 10 mg / kg of L4P, and the other group was given 10 mg / kg of TAT as a control.

[0254] (3) The drug and the solvent were administered intraperitoneally twice a day for 14 consecutive days. The survival rate of the mice was recorded.

[0255] The results were as Figure 16 shown that the polypeptide L4P could effectively rescue the survival rate of the lungs of C57BL / 6 mice infected with HSV-1, suggesting that the polypeptide L4P had good antiviral effects in vivo.

Claims

1. An artificially synthesized polypeptide, the amino acid sequence of the polypeptide is shown in SEQ ID NO.

6.

2. An artificially synthesized polypeptide, the amino acid sequence of which is shown in LILP.

3. A polypeptide having a cell-penetrating peptide added to the N-terminus of the polypeptide according to claim 1 and / or claim 2.

4. The polypeptide according to claim 3, the amino acid sequence of the polypeptide shown in SEQ ID NO.6 after adding the cell-penetrating peptide is shown in SEQ ID NO.1, and the amino acid sequence of the polypeptide shown in LILP after adding the cell-penetrating peptide is shown in SEQ ID NO.

5.

5. A fusion protein obtained by fusing the polypeptide according to claim 3 with a protein tag.

6. A gene encoding the polypeptide according to claim 3 or the fusion protein according to claim 5.

7. An expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the gene according to claim 6.

8. A composition comprising the polypeptide according to claim 3.

9. Use of the polypeptide according to claim 3, the polypeptide fusion protein according to claim 4, the gene according to claim 6, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the gene according to claim 6 or the composition according to claim 8 in the preparation of a herpes simplex virus type I inhibitor.

10. Use of the polypeptide according to claim 3, the polypeptide fusion protein according to claim 4, the gene according to claim 6, the expression cassette, recombinant vector, recombinant microorganism or in vitro recombinant cell having the gene according to claim 6 or the composition according to claim 8 in the preparation of a medicament for treating or preventing herpes simplex virus type I.

Images