Polypeptide inhibitor capable of effectively resisting respiratory tract RNA virus and application thereof

Abstract

The invention discloses a polypeptide inhibitor capable of effectively resisting respiratory tract RNA (Ribonucleic Acid) virus and application thereof. The TP series polypeptide provided by the invention has efficient and broad-spectrum antiviral activity. Therefore, a new strategy is provided for prevention and control of human respiratory viruses, and meanwhile, a new theoretical basis is provided for accelerating research and development of polypeptide small molecule drugs for resisting the respiratory viruses. The TP series polypeptide has a clear antiviral mechanism, can ensure the safety of application and the definition of an optimization approach, is convenient for later further development, improves the stability of a polypeptide drug, improves the administration mode of the polypeptide drug, and is expected to further expand the application range and effect of a polypeptide inhibitor in resisting respiratory tract RNA viruses.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a polypeptide inhibitor capable of effectively resisting respiratory RNA virus and application thereof. BACKGROUND

[0002] Respiratory virus refers to a virus that invades the respiratory tract as a portal, proliferates in respiratory mucosa epithelial cells, and causes local infection of the respiratory tract or lesions of tissues and organs other than the respiratory tract. Common respiratory viruses include influenza virus of Orthomyxoviridae, parainfluenza virus, respiratory syncytial virus, measles virus, mumps virus of Paramyxoviridae, and other viruses such as rhinovirus and coronavirus. Co-infection caused by multiple respiratory viruses is a common clinical phenomenon. Due to the high difficulty and long cycle of vaccine development, it is crucial to develop safe and effective antiviral drugs. In general, the targets of antiviral drugs can be divided into two categories, namely virus targets (Direct-Acting Antivirals, DAA) and host protein targets (Host-Targeting Antivirals, HTA). Drugs targeting viruses aim to inhibit the activity of key viral proteins such as polymerase and protease that regulate viral replication, or directly inactivate viral structural proteins. Drugs targeting hosts mainly disrupt the function of key host proteins that the virus relies on during its entire life cycle, thereby indirectly inhibiting viral replication. However, due to insufficient drug efficacy, increasing drug resistance, and severe side effects, most viral infections currently lack effective treatment drugs. In addition, due to the emergence of drug-resistant viruses as a result of the large-scale use of existing DAA drugs, there is an urgent need to develop HTA broad-spectrum antiviral drugs targeting host proteins required for viral replication, which can be used in combination with existing DAA drugs to improve treatment efficacy.

[0003] As strict intracellular parasites, viruses rely on the vesicle transport system of host cells to successfully complete each link of their life cycle. RAB11, which regulates the recycling endosome pathway of host cells, is very important for the intracellular transport of many negative-strand RNA virus proteins and the assembly and release of virions, and the regulatory mechanisms in the infection processes of different viruses are diverse. Studies have shown that the active transport of the ribonucleoprotein (vRNP) of many negative-strand respiratory viruses, including influenza a virus (IAV), respiratory syncytial virus (RSV), and mumps virus (MuV), from the cytoplasm to the cell membrane depends on the recycling endosome (RE) pathway regulated by RAB11. In addition, the related research of human parainfluenza virus 3 (HPIV3) also confirms the importance of RAB11. In summary, the key regulator of the host vesicle transport pathway, RAB11, has the potential to become a broad-spectrum anti-respiratory virus target.

[0004] Polypeptide-based therapeutic methods are an emerging field that has been proven to be effective in treating diseases caused by pathogenic microorganisms such as viruses and bacteria. Due to their high efficiency and low toxicity, polypeptides may become promising antiviral drugs. Because polypeptide inhibitors have a small molecular weight, they can be administered through the nasal cavity to directly inhibit the replication of viruses in the respiratory tract and reduce the risk of being destroyed by gastrointestinal juices and proteases in the body. Polypeptide drugs have the outstanding advantages of small dosage, good efficacy, and low toxicity in preventing and treating diseases, and therefore have great research and development value. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a polypeptide inhibitor that can effectively resist respiratory RNA viruses and the application thereof.

[0006] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0007] In the first aspect, the present application provides a polypeptide inhibitor that can effectively resist respiratory RNA viruses. The polypeptide inhibitor can bind to RAB11 protein, interfere with the formation process of RAB11-FIPs complex, and thereby inhibit virus infection that depends on the RAB11 transport pathway. The polypeptide inhibitor is a polypeptide TP designed and synthesized in vitro from the RBD sequence of FIP2, and the carboxy terminus of the sequence contains a 9-amino acid length transmembrane peptide (ArgLysLysArgArgGlnArgArgArg) to help TP penetrate the cell membrane and enter the cytoplasm to function.

[0008] Further, the present application makes a series of modifications to the TP polypeptide, including:

[0009] (1) Polypeptide YT (SEQ NO. 4) containing the protected core sequence (SEQ NO. 1) RBD region 9-21 amino acids: it contains 9-21 amino acids of the sequence (SEQ NO. 2) that can inhibit the formation of RAB11-FIP2 complex, and this region is more conserved in the RBD of FIP family members; Specifically:

[0010] TP, SEQ NO. 2:

[0011] Thr His Ile Arg Glu Leu Glu Asp Tyr Ile Asp Asn Leu Leu Val Arg Val Met Glu Glu Thr Pro Ser Ile Leu Arg Val Pro Arg Lys Lys Arg Arg Gin Arg Arg Arg

[0012] YT, SEQ NO. 4:

[0013] Tyr Ile Asp Asn Leu Leu Val Arg Val Met Glu Glu Thr Arg Lys Lys Arg Arg Gin Arg Arg Arg

[0014] (2) Polypeptide YT-DRI containing the protected sequence (SEQ NO. 4): it contains the sequence (SEQ NO. 5) that can inhibit the formation of RAB11-FIP2 complex, which is a new polypeptide formed by replacing the L-type amino acid sequence of YT with D-type amino acid and adjusting the amino acid sequence in reverse order. Specifically:

[0015] YT-DRI, SEQ NO. 5:

[0016] Arg Arg Arg Gin Arg Arg Lys Lys Arg Thr Glu Glu Met Val Arg Val Leu Leu Asn Asp Ile Tyr

[0017] The long-acting respiratory virus inhibiting polymer of the present application can inhibit the transport pathway regulated by the RAB11-FIPs complex. The carboxy terminal of the FIP family member has a very conservative RAB11 binding domain (RAB11 Binding Domain, RBD). According to the RBD sequence of FIP2, a polypeptide TP is designed and synthesized in vitro, which can competitively bind to RAB11 with FIP2, thereby preventing the formation of the RAB11-FIP2 complex, and playing a role in inhibiting the transport pathway in the host cell which is synergistically regulated by RAB11-FIP2, and ultimately achieving the purpose of resisting viruses dependent on the pathway.

[0018] In the above-mentioned amino acid sequence:

[0019] SEQ N0.1 is the full-length sequence of FIP2 protein, which has 266 amino acids;

[0020] SEQ NO.2 is TP, which is a 9-amino acid length of a cell-penetrating peptide added to the carboxy terminal of the RBD sequence (total of 28 amino acids) of FIP2, and the total length is 37 amino acids;

[0021] SEQ NO.3 is the aforementioned 9-amino acid length of a cell-penetrating peptide Tat;

[0022] SEQ N0.4 is YT, which takes the 9th-21st amino acid of TP and adds a cell-penetrating peptide to the carboxy terminal, and the total length is 22 amino acids;

[0023] SEQ NO.5 is YT-DRI, which is a D-type amino acid sequence instead of an L-type amino acid sequence of YT, and the amino acid sequence is adjusted in reverse order to form.

[0024] In the present application, since the premise for the polypeptide to play an antiviral role is to penetrate the cell membrane and enter the cell, it is necessary to detect whether the polypeptide enters the cell. The detection method is immunofluorescence. A small molecule fluorescent substance FITC is labeled at the N terminal of the representative polypeptide TP, so that the polypeptide can spontaneously fluoresce, and the cell nucleus is stained with DAPI, and the positional relationship of green fluorescence and blue fluorescence can be observed. At the same time, the cells infected with viruses are displayed by red fluorescence, and by observing the co-localization of green fluorescence and red fluorescence, it can be judged whether the polypeptide can enter the infected cells. For example, Figure 1 The polypeptide has good cell-penetrating effect, and its cell-penetrating ability is not affected by viral infection.

[0025] In the present application, the cytotoxicity of the polypeptide is detected by CCK8 method, as shown in Figure 2 The representative polypeptide TP has no cytotoxicity within 75 μM.

[0026] In the present application, the method of TCID50 is used to detect the effect of TP on anti-HPIV3 infection in HeLa cells, as shown in the following table. Figure 3 As shown in the following table, at the polypeptide concentration of 3.25 μM (IC50), 50% of virus infection can be inhibited, which proves that the polypeptide TP has good anti-virus effect.

[0027] Further, the method of TCID50 is used to detect the anti-virus effect of TP in MK2 cells and MDCK cells. As shown in the following table, Figure 4 As shown in the following table, TP shows good anti-virus effect in these cells.

[0028] In the second aspect, the present application provides a drug for treating respiratory virus infection, which comprises any one of the polypeptides described above.

[0029] In the third aspect, the present application provides the use of the drug described above as a broad-spectrum inhibitor for inhibiting various respiratory virus infections. The polypeptide as the only active ingredient can effectively inhibit various respiratory virus infections. The virus is any one of HPIV3, RSV, and IAV.

[0030] The technical principles, advantages and beneficial effects of the present application are as follows:

[0031] The important innovation of the present application is that the anti-virus activity of the polypeptide is achieved by destroying the RAB11 transport pathway of the host cell, so theoretically it has a broad-spectrum inhibitory effect on all viruses that depend on this pathway. The interaction between FIPs and RAB11 through the carboxy-terminal RAB11 binding region forms a RAB11-FIPs complex, which is essential for RAB11 to correctly perform its function. Based on the phenomenon that the RAB11-FIPs complex can regulate the transport, assembly and release of various virus components in the cell, and according to the RBD structure composition and sequence characteristics of FIP2, a group of polypeptides that can inhibit the infection of respiratory viruses dependent on the RAB11 transport pathway are designed, which will have great significance for the prevention and treatment of respiratory viruses.

[0032] The present application uses the method of TCID50 to detect the anti-virus activity of these polypeptides in HeLa cells. As shown in the following table, Figure 5 As shown in the following table, the inhibitory effect of the two modified polypeptides on viruses is further improved compared with TP. The IC50 of YT polypeptide is 1.99 μM, and the IC50 of YT-DRI is 0.94 μM. The above series of polypeptides prove that the TP polypeptide designed based on the mechanism of preventing the formation of RAB11-FIP2 complex, and after being truncated or designed and modified with non-natural amino acids, all have the activity of inhibiting HPIV3.

[0033] The amino acids at positions 9-21 of the protected sequence (SEQ NO. 2) are more conserved in the RBD of FIP family members, so it has the potential to inhibit the binding of other FIPs to RAB11 in addition to inhibiting the formation of the RAB11-FIP2 complex, and thus can more comprehensively interfere with the function of RAB11.

[0034] In addition to HPIV3, the infection of two other important respiratory viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV), also depends on the regulation of RAB11, so the application next uses the TCID50 method to detect the broad-spectrum antiviral effect of the optimized polypeptide YT-DRI. As shown in Figure 6 The IC50 of the YT-DRI polypeptide for inhibiting RSV and IAV infection is 0.37 μM and 0.42 μM, respectively.

[0035] In summary, the TP series of polypeptides have high and broad-spectrum antiviral activity. This provides a new strategy for the prevention and control of human respiratory viruses, and also provides a new theoretical basis for accelerating the research and development of polypeptide small molecule drugs against respiratory viruses. The clear antiviral mechanism of the TP series of polypeptides can ensure the safety of its application and the clarity of the optimization pathway, facilitating further development and having high clinical application and promotion value. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 : Detection of the ability of the polypeptide TP to pass through the cell membrane into the cytoplasm by fluorescence labeling.

[0037] Figure 2 : CCK-8 assay of the cytotoxicity of the polypeptide TP. The results show that the polypeptide has no toxicity to cells at a concentration of ≤75 μM.

[0038] Figure 3 : Efficacy of TP in HeLa cells. Tat is a 9-amino-acid transmembrane peptide used as a negative control. The TCID50 method was used to detect the titer of viral particles in the supernatant, and the IC50 value of TP was measured to be 3.25 μM.

[0039] Figure 4 : In addition to HeLa cells, the polypeptide TP has an inhibitory effect on viruses in a variety of cells, including MK2 cells and MDCK cells.

[0040] Figure 5 : The polypeptide TP was modified, and the antiviral effect of the modified polypeptides YT and YT-DRI was further improved.

[0041] Figure 6 : The modified polypeptide YT-DRI has an inhibitory effect on the infection of RSV and IAV.

[0042] Figure 7 : The modified polypeptide YT-DRI can inhibit the co-infection of HPIV3, RSV and IAV.

[0043] Figure 8 : The modified polypeptide YT-DRI can inhibit the infection of HPIV3, RSV and IAV in a mouse model.

[0044] Wherein: a, the animal experiment process for detecting the effect of the polypeptide YT-DRI on the infection of HPIV3; b, the body weight change curve of the HPIV3 experimental group mice; c, the lung tissue virus titer of the HPIV3 experimental group mice; d, the lung tissue pathological section of the HPIV3 experimental group mice; e, the animal experiment process for detecting the effect of the polypeptide YT-DRI on the infection of RSV; f, the body weight change curve of the RSV experimental group mice; g, the lung tissue virus titer of the RSV experimental group mice; h, the lung tissue pathological section of the RSV experimental group mice; i, the animal experiment process for detecting the effect of the polypeptide YT-DRI on the infection of IAV; j, the body weight change curve of the IAV experimental group mice; k, the survival rate of the IAV experimental group mice; and l, the lung tissue pathological section of the IAV experimental group mice. DETAILED DESCRIPTION

[0045] The technical solutions of the present application are further described in detail below in combination with specific embodiments and the accompanying drawings.

[0046] Example 1: Detection of the cell-penetrating efficiency of the polypeptide

[0047] 1. Materials: DMEM medium (gibico), serum (gibico), immunofluorescence dish (NEST), PBS, DAPI, paraformaldehyde, and polypeptide synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd.

[0048] 2. Experimental process

[0049] This experiment is divided into two groups. In order to avoid the influence of the addition of HPIV3 virus on the entry of the polypeptide into cells, one group of experiments adds the polypeptide after the addition of HPIV3 virus, and the other group does not add the virus but only adds the polypeptide, and a negative control is prepared for each group. The steps are as follows:

[0050] The immunofluorescence steps are as follows:

[0051] (1) 1ml of HeLa cells are plated in an immunofluorescence special dish, and the sample is collected when the cells grow to 50% confluence.

[0052] (2) The culture medium is aspirated, and the residual culture medium is washed away with 1ml of 0.01mol / L, pH 7.4 PBS, and washed three times, each time for 5min.

[0053] (3) Prepare 4% paraformaldehyde solution, dissolve 4g paraformaldehyde in 100ml PBS. Add 1ml of prepared 4% paraformaldehyde to each dish, and react for 5min, so as to fix the cells.

[0054] (4) Aspirate the 4% paraformaldehyde, and add 1ml of 0.01mol / L, pH7.4 PBS to wash away the residual paraformaldehyde, and wash for 3 times, each time for 5min.

[0055] (5) Dilute 1mg / ml DAPI solution to 100ng / ml with PBS, and add to the dish, and react for 15min.

[0056] (6) Aspirate the reaction solution, and add 1ml of 0.01mol / L, pH7.4 PBS to wash away the residual reaction solution, and wash for 3 times, each time for 5min.

[0057] (7) Place the dish under a fluorescence microscope to observe.

[0058] As shown in Figure 1, the polypeptide can enter the cells in both the cells without virus and the cells with virus, proving that the polypeptide TP has good membrane penetration ability. Figure 1

[0059] Example 2: Cell toxicity test of polypeptide

[0060] 1. Experimental materials: cck-8 reagent (MCE)

[0061] 2. Experimental process

[0062] The polypeptide not only needs to be able to inhibit the virus in the process of antiviral, but also needs to ensure that it has no toxicity to the cells. Therefore, the cell toxicity test is used to detect this index. The steps are as follows:

[0063] (1) Use HeLa cells to pave 96-well cell plates, 100ul per well.

[0064] (2) When the cells grow to 30%-40% confluence, replace the DMEM medium containing 10% serum with DMEM medium containing 4% serum, and then add TP to the wells according to the polypeptide concentration gradient, and the polypeptide concentration gradient is 0-300um.

[0065] (3) After 24h of culture, sample collection, add 10ul of live cell detection agent cck-8 to each well, and mix well.

[0066] (4) Place at 37 degrees for 4h.

[0067] (5) Use a microplate reader to detect the OD450 absorbance value.

[0068] The results are shown in Figure 2. Figure 2 ​The amount of living cells was 100% in untreated cells. After adding 75 μM polypeptide, the amount of living cells was basically the same as that of the control (untreated cells), which proved that the polypeptide TP had no toxicity to the cells within 75 μM.

[0069] Example 3 Determination of the antiviral efficiency of polypeptide

[0070] 1. Experimental materials

[0071] 2. Experimental process

[0072] 2.1 Amplification of virus

[0073] (1) Use MK2 cells to pave 5 100 mm dishes.

[0074] (2) When the cells grow to 50%-60% confluence, replace the DMEM medium containing 10% serum with serum-free DMEM medium, and add 20 μl of 10^6 PFU / ml HPIV3 virus.

[0075] (3) After 2 days, observe whether CPE occurs in the cells. When the phenomenon is obvious, collect the sample.

[0076] (4) Absorb the supernatant in the 100 mm dish into a 15 ml centrifuge tube, and centrifuge at 1000 g for 5 min.

[0077] (5) Take the supernatant into a new 15 ml centrifuge tube, and filter it with a 50 ml syringe and a 0.45 μm filter membrane to remove bacteria.

[0078] 2.2 Determination of the antiviral efficiency of polypeptide TP

[0079] (1) Use HeLa cells to pave a 24-well plate.

[0080] (2) When the cells grow to 40%-50% confluence, replace the DMEM medium containing 10% serum with serum-free DMEM medium, and add 1 μl of 10^6 PFU / mL HPIV3 virus to each well.

[0081] (3) After 2 h, add polypeptide TP with concentrations of 0 μM, 0.16 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, and 10 μM, respectively.

[0082] (4) Collect the supernatant after 48 h of virus infection, and determine the titer of the virus by the TC1D50 method.

[0083] The results are as follows Figure 3As shown in the figure, the virus particle titer in the supernatant of the HPIV3 virus alone is 100%. After adding the polypeptide TP, the titer decreases with the increase of the polypeptide concentration. When the polypeptide concentration is 10 μM, the titer decreases to 10%, which proves that the polypeptide TP has a good antiviral effect. In combination with Figure 2 , the polypeptide TP has no cytotoxicity within 75 μM, which proves that the polypeptide has a good antiviral effect under the premise of drug safety.

[0084] Example 4 Determination of the antiviral efficiency of the polypeptide TP in various cells

[0085] 1. Materials

[0086] MK2 cells and MDCK cells are self-owned in the laboratory

[0087] 2. Determination of the antiviral efficiency of the polypeptide TP in various cells

[0088] (1) MK2 cells and MDCK cells were respectively used to pave 24-well plates.

[0089] (2) When they grow to 40%-50% confluence, the DMEM medium containing 10% serum was replaced with DMEM medium without serum, and 1 μl of 10^6 PFU / mL HPIV3 virus was added to each well.

[0090] (3) After 2 h, polypeptide TP with concentrations of 0 μM, 0.16 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM and 10 μM was respectively added.

[0091] (4) The supernatant was collected after 48 h of viral infection, and the titer of the virus was determined by the TC1D50 method.

[0092] In Figure 4 , it can be seen that the polypeptide TP can have a significant antiviral effect in HeLa cells, MK2 cells and MDCK cells.

[0093] Example 5 Influence of the modified polypeptide of the polypeptide TP on the antiviral effect

[0094] 1. Materials

[0095] Polypeptides YT and YT-DRI

[0096] 2. Influence of the modification of the polypeptide TP on the antiviral effect

[0097] (1) HeLa cells were used to pave 24-well plates.

[0098] (2) When they grow to 40%-50% confluence, replace the DMEM medium containing 10% serum with DMEM medium without serum, and add 1 μl of 10^6 PFU / mL HPIV3 virus to each well.

[0099] (3) After 2h, add polypeptides TP, YT and YT-DRI at concentrations of 0 μM, 0.16 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM and 10 μM, respectively.

[0100] (4) Collect the supernatant after 48h of viral infection, and determine the viral titer by the TC1D50 method.

[0101] In the Figure 5 application, the polypeptide TP is modified: YT is a sequence of 9-21 amino acids in TP, and YT-DRI is obtained by replacing the normal L-type amino acids in the peptide chain with D-type amino acids and reversing the direction of the main skeleton of the peptide chain. Experiments show that the modified polypeptides can significantly improve the viral inhibition effect. The IC50 of YT is 1.99 μM, and the IC50 of YT-DRI is 0.94 μM.

[0102] Example 6 Analysis of the broad-spectrum antiviral effect of polypeptide YT-DRI

[0103] 1. Materials

[0104] HeLa cells and MDCK cells are self-owned in the laboratory

[0105] 2. Determination of the antiviral efficiency of polypeptide YT-DRI in various cells

[0106] (1) Use HeLa cells and MDCK cells to plate 24-well plates.

[0107] (2) When they grow to 40%-50% confluence, replace the DMEM medium containing 10% serum with DMEM medium without serum, and add 1 μl of 10^5 PFU / mL RSV virus to HeLa cells and 0.1 μl of 10^7 PFU / mL IAV virus to MDCK cells.

[0108] (3) After 2h, add polypeptide YT-DRI at concentrations of 0 μM, 0.16 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM and 10 μM, respectively.

[0109] (4) Collect the supernatant after 48h of viral infection, and determine the viral titer by the TC1D50 method.

[0110] 3. Determination of the efficiency of peptide YT-DRI in inhibiting HPIV3, RSV and IAV co-infection in MDCK cells

[0111] (1) MDCK cells were plated in 12-well plates.

[0112] (2) When the cells grow to 40%-50% confluency, replace the DMEM medium containing 10% serum with DMEM medium without serum, and add a mixture of HPIV3, RSV and IAV to each well.

[0113] (3) After 2 h, add peptide YT-DRI at a concentration of 5 μM.

[0114] (4) 36 hours after virus infection, samples were collected and total RNA of cells was extracted using the Trizol method.

[0115] (5) Discard the supernatant, add 1000 μl of Trizol to the wells, and let stand at room temperature for 5 minutes.

[0116] (6) Add 200 μl of chloroform to the wells, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

[0117] (7) Centrifuge at 12,000 g for 15 min at 4°C.

[0118] (8) Remove the upper aqueous phase into a new 1.5 ml centrifuge tube, add 500 μl of isopropanol, and let it stand at room temperature for 10 minutes.

[0119] (9) Centrifuge at 12,000 g for 10 min at 4°C.

[0120] (10) Discard the supernatant and add 1 ml of 75% ethanol.

[0121] (11) Centrifuge at 12,000 g for 5 min at 4°C.

[0122] (12) Air separation at 12000g for 2 minutes.

[0123] (13) Add 40 μl of DEPC water to dissolve RNA.

[0124] (14) Take 1 μl RNA sample and perform fluorescence quantitative experiment using qRT-PCR.

[0125] exist Figure 6 It can be seen that the peptide YT-DRI can play a significant antiviral effect in both RSV-infected HeLa cells and IAV-infected MDCK cells. Figure 7 It can be seen that in MDCK cells co-infected with HPIV3, RSV and IAV, the polypeptide YT-DRI has a significant inhibitory effect on all three viruses.

[0126] Example 7 Analysis of the broad-spectrum antiviral effect of polypeptide YT-DRI in vivo

[0127] 1. Analysis of the protective effect of the HPIV3 mouse model

[0128] 1.1 Materials

[0129] Polypeptide YT-DRI, HPIV3, 2-week-old Balb / c mice

[0130] 1.2 Determination of the antiviral efficiency of polypeptide YT-DRI

[0131] (1) Day 0: Balb / c mice were divided into 3 groups, 5 mice in each group. After inhalation anesthesia with isoflurane, the normal group was given 20ul PBS by nasal instillation, and the infection group and the YT-DRI group were both given 20uL of virus solution containing about 10^6 pfu of HPIV3 by nasal instillation.

[0132] (2) One hour after infection, the normal group and the infection group were injected intraperitoneally with PBS, and the YT-DRI group was injected intraperitoneally with 2mg / kg YT-DRI.

[0133] (3) Day 0-2: Drug administration was performed once a day, and body weight was recorded.

[0134] (4) Day 3: The mice were sacrificed by cervical dislocation, and lung tissue was taken.

[0135] (5) The right lung was used for the preparation of tissue sections and observation analysis: section preparation was completed by Wuhan Pino Fly Biotechnology Co., Ltd.

[0136] (6) The left lung was used for virus titer determination. Weigh 100mg into a grinding tube, add 1000ul PBS to it, grind the tissue, centrifuge at 13000rpm for 5 minutes, and take the supernatant to detect the virus titer of mouse lung tissue according to the method of Example 3.

[0137] 2. Analysis of the protective effect of the RSV mouse model

[0138] 2.1 Materials

[0139] Polypeptide YT-DRI, RSV, 5-8-week-old female Balb / c mice

[0140] 2.2 Determination of the antiviral efficiency of polypeptide YT-DRI

[0141] (1) Day 0: Balb / c mice were divided into 3 groups, 5 mice in each group. After inhalation anesthesia with isoflurane, the normal group was given 20ul PBS by nasal instillation, and the infection group and the YT-DRI group were both given 50uL of virus solution containing about 10^6 pfu of RSV by nasal instillation.

[0142] (2) After 1 hour of challenge, the normal group and the infection group were injected with PBS intraperitoneally, and the YT-DRI group was injected with 2 mg / kg YT-DRI intraperitoneally.

[0143] (3) Day 0-3: once a day, and record the body weight.

[0144] (4) Day 4: the mice were sacrificed by cervical dislocation, and the lung tissues were taken.

[0145] (5) The right lung was used for the preparation of tissue sections and observation analysis: the section preparation was completed by Wuhan Pinofly Biotechnology Co., Ltd.

[0146] (6) The left lung was used for virus titer determination. 100 mg was weighed into a grinding tube, 1000 ul of PBS was added, the tissue was ground and broken, centrifuged at 13000 rpm for 5 minutes, and the supernatant was detected according to the method of Example 6 to detect the virus titer of the mouse lung tissue.

[0147] 3, Protective analysis of IAV infected mouse model

[0148] 3.1 Materials

[0149] Polypeptide YT-DRI, IAV / WSN, 5-8 week old female Balb / c mice

[0150] 3.2 Determination of antiviral efficiency of polypeptide YT-DRI

[0151] (1) Day 0: the Balb / c mice were divided into 3 groups, 5 in each group. After being anesthetized by inhaling isoflurane, 50 ul of virus solution containing about 4,000 pfu was dropped into the nose.

[0152] (2) After 1 hour of challenge, the infection group was injected with PBS intraperitoneally, the oseltamivir group was injected with 20 mg / kg oseltamivir intraperitoneally, and the YT-DRI group was injected with 2 mg / kg YT-DRI intraperitoneally.

[0153] (3) Day 0-4: once a day, and record the body weight. When the body weight decreased by more than 25%, euthanasia was performed.

[0154] (4) Day 14: the mice were sacrificed by cervical dislocation, and the lung tissues were taken.

[0155] (5) The right lung was used for the preparation of tissue sections and observation analysis: the section preparation was completed by Wuhan Pinofly Biotechnology Co., Ltd.

[0156] (6) The left lung was used for virus titer determination. 100 mg was weighed into a grinding tube, 1000 ul of PBS was added, the tissue was ground and broken, centrifuged at 13000 rpm for 5 minutes, and the supernatant was detected according to the method of Example 6 to detect the virus titer of the mouse lung tissue.

[0157] In Figure 8 the present application, the anti-viral effect of the modified polypeptide YT-DRI was detected in a mouse model. The experimental results in the HPIV3 and RSV animal models showed that the degree of weight loss (b and f), lung tissue viral titer (c and g), and lung tissue inflammatory lesions (d and h) of the mice in the YT-DRI group were reduced to some extent compared with the infection group, the alveolar infiltration area was significantly reduced, the thickening of the bronchiole and lung blood vessel wall was reduced, indicating that YT-DRI had a certain inhibitory effect on viral pneumonia caused by HPIV3 and RSV. In addition, the results of the IAV lethal animal model with the oseltamivir group as the positive control showed that the mice in the YT-DRI group had a survival rate of 80% (k) and a certain degree of reduction in weight loss (j) and lung tissue pathological inflammatory changes (l) compared with the infection group. It is worth noting that the dosage of YT-DRI is only one-tenth of that of oseltamivir.

Claims

1. A polypeptide-based inhibitor effective against a respiratory RNA virus, characterized in that: The polypeptide inhibitor can bind to RAB11 protein, interfere with the formation process of RAB11-FIPs complex, and thus inhibit virus infection dependent on RAB11 transport pathway; the polypeptide inhibitor is a polypeptide TP designed and synthesized according to the RBD sequence of FIP2, the amino acid sequence of FIP2 is shown as SEQ NO. 1, and the amino acid sequence of the polypeptide TP is shown as SEQ NO.

2.

2. The polypeptide-based inhibitor of claim 1, wherein: The polypeptide inhibitor is modified by the polypeptide TP to obtain a polypeptide YT, and the amino acid sequence of the polypeptide YT is shown as SEQ NO.

4.

3. The polypeptide inhibitor according to claim 2, wherein: The polypeptide inhibitor is modified by the polypeptide YT to obtain a polypeptide YT-DRI, and the amino acid sequence of the polypeptide YT-DRI is shown as SEQ NO.

5.

4. The polypeptide inhibitor according to claim 3, characterized in that: The polypeptide YT-DRI sequence is to replace the L-type amino acid sequence of YT with a D-type amino acid, and to adjust the amino acid sequence in reverse order.

5. A medicament for treating a respiratory viral infection, characterized in that: The drug comprises the polypeptide according to any one of claims 1 to 4.

6. The drug according to claim 5 as a broad-spectrum inhibitor for inhibiting various respiratory virus infections.

7. Use according to claim 6, wherein: The polypeptide can effectively inhibit various respiratory virus infections as the only active ingredient.

8. Use according to claim 7, wherein: The virus is any one of HPIV3, RSV, and IAV.

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