Anti-coronavirus protein medicine and application thereof

Abstract

The invention relates to a protein for treating new coronavirus and application thereof. The protein is LSR and CRD1 oligopeptide protein derived from the LSR. The protein disclosed by the invention has a relatively strong inhibition effect on original strains and various variant strains of the new coronavirus, and can be used as a medicine for treating diseases caused by the new coronavirus. Through verification, the protein drug provided by the invention can have a strong and broad-spectrum antiviral effect on new crown original strains and mutant strains thereof (Omicro B521 and XBB) from the cellular level, the organoid level and the tissue level, the pathological injury of intestinal tracts and lungs is improved, and the level of proinflammatory cytokines is reduced. In addition, the LSR and the oligopeptide medicine CRD1 derived from the LSR can effectively inhibit the combination of Spike and ACE2, so that the infection rate of SARS-CoV-2 is reduced. The PEG-LSR protein and the CRD1 oligopeptide derived from the LSR are expected to have the potential of preventing and / or treating new variant strains appearing in the future and other various coronaviruses taking ACE2 as receptors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a drug for treating the intestines and lungs infected by the novel coronavirus using LSR protein and CRD1 protein, and its application. Background Art

[0002] Since its outbreak, the novel coronavirus (SARS-CoV-2) has been characterized by its high infectiousness and prolonged treatment. Patients infected with the virus often experience symptoms such as cough, fever, and shortness of breath. In addition to the lungs, the intestines are also a key target organ for SARS-CoV-2 infection. In particular, several studies have reported that COVID-19 involving the gastrointestinal tract is more severe, and gastrointestinal symptoms may even precede respiratory symptoms. As the disease progresses, systemic inflammation and even multi-organ failure can develop. Therefore, the development of effective treatments for COVID-19 has become a hot topic among researchers both domestically and internationally. SARS-CoV-2, the pathogen of COVID-19, is a beta-coronavirus, an RNA virus whose genome encodes four structural proteins: the matrix protein (M protein), the viral envelope (E protein), the spike protein (S protein), and the nucleocapsid (N protein). The E protein is involved in viral assembly and release, primarily altering the permeability of host cell membranes and serving as a key protein for viral entry into the human body. The N protein is primarily involved in the replication and packaging of SARS-CoV-2 and is the most abundant protein in the virus. It can bind to the viral genetic material, RNA, to form a nucleocapsid. Its sequence is conserved and highly immunogenic. The main function of the M protein is to participate in the assembly of the virus and to antagonize interferon. The first step for the novel coronavirus to invade the human body is for the S protein to bind to the host cell receptor, allowing the genetic material to invade the host cell. However, the S protein of the new coronavirus can also induce the body to produce neutralizing antibodies, thereby reducing the virus's ability to infect and invade. Therefore, the S protein is a potential target for clinical diagnosis, therapeutic antibodies, and vaccine design and development, and is also the main antigenic site currently detected for SARS-CoV-2.

[0003] The S proteins of both SARS-CoV and SARS-CoV-2 utilize the angiotensin-converting enzyme 2 (ACE2) receptor for entry into human target cells. Given that S protein binding to the host receptor ACE2 is a critical step in SARS-CoV-2 viral entry and the development of later clinical pathology, interfering with this binding may provide a universal strategy for preventing and treating SARS-CoV-2 infection, applicable to all SARS-CoV-2 variants and other novel coronaviruses that utilize ACE2 as their cellular receptor. Consequently, most vaccines and neutralizing antibodies are designed to abrogate this interaction. Neutralizing antibodies primarily target the trimeric spike glycoprotein, but are hampered by issues related to solubility, unsuitability for oral or inhaled administration, immunogenicity, and mutational escape. Therefore, treating viral infection by modulating the interaction of the viral S protein with the host cellular protein ACE2 may be effective against all current and future SARS-CoV-2 variants, given the relatively conserved interaction between the S protein and its receptor, ACE2. Furthermore, ACE2 is a common receptor for multiple coronaviruses, including SARS-CoV and HCoV-NL63. Therefore, confirming the effectiveness of this strategy could provide a rapidly deployable intervention in future coronavirus outbreaks.

[0004] Current treatments for SARS-CoV-2 mainly include: Drugs used for the prevention and treatment of COVID-19 patients mainly include chemical preparations, biological products and Chinese herbal medicines.

[0005] Chemical agents can generally include drugs that inhibit the entry of viruses into host cells, such as chloroquine; drugs that block the viral replication process, such as ribavirin and favipiravir; and drugs that inhibit the production of viral proteins, such as lopinavir / ritonavir. Although these drugs can achieve certain effects, the actual application of chemical drugs is also accompanied by certain side effects and potential dangers. For example, the side effects of ribavirin include anemia, fatigue, rarely fatigue, headache, insomnia, loss of appetite, nausea, vomiting, mild diarrhea, constipation, and even hemolytic anemia, which can lead to worsening of heart disease and myocardial infarction.

[0006] Chinese herbal medicine includes "three medicines (Jinhua Qinggan Granules, Lianhua Qingwen Capsules, and Xuebijing Injection) and three prescriptions (Qingfei Paidu Decoction, Huashi Baidu Decoction, and Xuanfei Baidu Decoction)". Although they play a role in the treatment of new coronavirus patients, their effect is slow and the mechanism of action is still unclear.

[0007] Biological products primarily include vaccines, recombinant proteins, and peptide drugs. They hinder viral replication, including viral entry, mRNA synthesis, and protein synthesis, thereby reducing viral load and preventing viral spread. However, current recombinant protein and peptide drugs are mostly exogenous proteins or peptides with potential antigenicity, potentially causing other potentially damaging effects on the body.

[0008] The lipolysis-stimulated lipoprotein receptor (LSR) is a transmembrane protein expressed in a variety of tissues, including the liver, lungs, heart, blood vessels, kidneys, and intestines. To date, the functions of LSR that have been discovered include: (1) as a lipoprotein receptor, it helps the liver absorb triglyceride-rich lipoproteins; (2) it maintains the integrity of the epithelial and endothelial barriers and is a key component of the tricellular tight junction; (3) it is a host receptor for the binary toxin Clostridium difficile translocase (CDT); (4) as an upstream negative regulator of the YAP signaling pathway, it can regulate intestinal progenitor cell proliferation and Paneth cell differentiation; (5) it participates in cell migration, infiltration, and proliferation and is a tumor promoter or inhibitor; (6) it is a component of the blood-brain barrier. However, the role of LSR and its functional domain CRD1 in the infection process of coronaviruses, including SARS-CoV-2, has not been reported, and whether it is involved in coronavirus infection is also unclear.

[0009] Through the above analysis, the problems and defects of the existing technology are as follows:

[0010] The role of LSR and its functional domain CRD1 in the infection of coronaviruses including SARS-CoV-2 has not been reported, and whether they are involved in coronavirus infection is also unclear. Summary of the Invention

[0011] In response to the problems existing in the prior art, the present invention provides a drug and its application in preventing and / or treating novel coronavirus infection using LSR protein and its derived CRD1 protein.

[0012] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0013] By designing and constructing LSR gene knockout cell lines, organoids, and mouse models, as well as LSR overexpression cell lines, organoids, and mouse models, the relationship between LSR and novel coronavirus infection was studied. The experimental results showed that:

[0014] (1) Knockout of the Lsr gene: It can significantly increase the viral infection rate in cells and organoids, significantly increase the level of inflammatory factors in the mouse infection model, and increase the damage to the lung and intestinal tissue structure in the mouse infection model, and the viral load is significantly increased.

[0015] (2) Adding LSR protein: It can significantly reverse the above phenomenon. Specifically, it can significantly reduce the viral infection rate in cells and organoids, significantly weaken the level of inflammatory factors in the mouse infection model, and improve the damage to the lung and intestinal tissue structure in the mouse infection model. The viral load is significantly reduced. It can be seen that LSR itself has a protective and ameliorative effect on the infection of the new coronavirus. LSR has a new use in preventing and treating new coronavirus infection.

[0016] By treating cell lines, organoids and mouse models with LSR-derived oligopeptide CRD1, the relationship between LSR-derived oligopeptide CRD1 and new coronavirus infection was studied, and its toxicity was tested. The experimental results showed that with the increase of CRD1 concentration, the viral infectivity decreased in a dose-dependent manner. CRD1 pretreatment significantly attenuated the level of inflammatory factors in the mouse infection model, while improving the damage to the lung and intestinal tissue structure in the mouse infection model, and the viral load was significantly reduced. CRD1 treatment had almost no effect on cell viability.

[0017] It can be seen that the LSR-derived CRD1 oligopeptide protein has a protective and ameliorative effect on the infection of the new coronavirus, and its cytotoxicity is low and can be ignored. The LSR-derived CRD1 oligopeptide protein also has a new use in preventing and treating new coronavirus infection.

[0018] The benefits of the present invention lie in that: through research, it was found that LSR protein and its derived CRD1 protein can significantly reduce the viral infection rate in cells, organoids and mouse models, improve the pathological characteristics of the mouse infection model, have significant therapeutic effects, and have no side effects or complications, and can fundamentally treat and prevent the infection of the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the relative expression of LSR genes in epithelial cells in groups infected with different SARS-CoV-2 strains and the control group;

[0020] Figure 2 This is a graph showing the viral load of WT epithelial cells and LSR KO epithelial cells infected with different SARS-CoV-2 strains;

[0021] Figure 3 This is a graph showing the viral load of epithelial cells infected with different SARS-CoV-2 strains by LSR protein;

[0022] Figure 4 This is a graph showing the viral load of WT organoids and LSR KO organoids infected with different SARS-CoV-2 strains;

[0023] Figure 5 This is a graph showing the viral load of organoids infected with different SARS-CoV-2 strains using LSR protein;

[0024] Figure 6 Figure 2 is the HE staining results of intestinal tissues of the two groups of mice after virus infection, as well as the viral load and the contents of IL2, IL6, IL12, Cxcl10, Tnfα, Ifnα, and Ifnγ;

[0025] Figure 7 Figure 2 is the HE staining results of lung tissues of the two groups of mice after virus infection, as well as the viral load and the levels of IL2, IL6, IL12, Cxcl10, Tnfα, Ifnα, and Ifnγ;

[0026] Figure 8 This is a diagram of the interaction between LSR and ACE2;

[0027] Figure 9 This is a diagram of the interaction between ACE2 and Spike in LSR cells with different expression levels;

[0028] Figure 10 This is a diagram of the co-localization of CRD1 and ACE2 in hACE2-293 cells;

[0029] Figure 11 This is a graph showing the viral load of different SARS-CoV-2 strains after treatment with different concentrations of CRD1;

[0030] Figure 12 This is a graph showing the viral load in the intestinal and lung tissues of the two groups of mice;

[0031] Figure 13 These are the HE staining results of the intestinal and lung tissues of the two groups of mice;

[0032] Figure 14 is a graph showing the levels of IL2, IL6, IL12, Cxcl10, Tnfα, Ifnα, and Ifnγ in the intestinal and lung tissues of the two groups of mice;

[0033] Figure 15 This is a graph showing the cell viability of three epithelial cells after treatment with different concentrations of CRD1. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Example 1 Effect of novel coronavirus infection on LSR protein expression

[0036] 1. Experimental Animals

[0037] hACE2 mice (NO.T037659) were purchased from Nanjing Jicui Yaokang Experimental Animal Technology Co., Ltd.

[0038] 2. Novel Coronavirus Strain

[0039] By passaged SARS-CoV-2 virus (including the WT original strain and various variant strains Omicron BA.5.2 and XBB) in Vero E6 cells, high-titer original virus and variant virus stock solutions were obtained; all work involving infectious viruses (including virus proliferation and titer determination tests) was carried out in the biosafety level 3 (BSL3) facility of the Shandong Provincial Center for Disease Control and Prevention (CDC).

[0040] 3. Preparation of cells overexpressing ACE2

[0041] The coding sequence of the full-length human ACE2 gene (NM_001371415) was cloned into the retroviral vector pQCXIP (Clontech); HEK293 cells, A549 cells, and HIEC-6 cells were infected with human ACE2-expressing retrovirus to generate hACE2-293 cells, hACE2-A549 cells, and hACE2-HIEC-6 cells. Subsequently, the cells were selected with 5 μg / ml puromycin for 14 days before subsequent experiments.

[0042] 4. Establishment of cell infection model

[0043] Caco2, hACE2-A549 and hACE2-HIEC-6 cells were infected with 1MOI SARS-CoV-2 virus (including the WT original strain and various mutant strains Omicron BA.5.2 and XBB) for 24 hours, and cellular RNA was extracted for subsequent experiments.

[0044] 5. RNA Extraction and Real-time Quantitative PCR

[0045] The harvested cell samples were homogenized in Trizol (Ambion, USA) to obtain total RNA. RNA was reverse transcribed using a reverse transcription system kit according to the manufacturer's instructions (Takara, Japan). Fluorescence quantitative PCR was then performed using a Thermo Fisher QS3 PCR instrument according to the instructions in the SYBR Green PCR Master Mix (ABI, USA) kit. The internal reference gene was β-actin, and the expression of the ΔCt=Ct was used. gene -Ct β-actin Calculate the relative expression level of each target gene mRNA and use 2 -ΔCt Value representation.

[0046] 6. Data Analysis

[0047] The experimental data were statistically analyzed using Prism 8 software. Unpaired t-test was used to compare two groups, and one-way ANOVA was used to compare multiple groups. The results were expressed as mean ± standard error (SEM). P < 0.05 was considered statistically significant.

[0048] 7. Experimental results

[0049] The results of the relative expression of LSR genes in the novel coronavirus infected cells, including the original strain WT infected cells, Omicron BA.5.2 infected cells, XBB infected cells and control group cells are as follows: Figure 1 As shown by Figure 1 a It can be seen that in Caco2 cells, the relative expression level of LSR gene in the original strain WT infection group, Omicron BA.5.2 infection group and XBB infection group cells was significantly reduced.

[0050] Depend on Figure 1 b It can be seen that in hACE2-A549 cells, the relative expression level of LSR gene in the original strain WT infection group, Omicron BA.5.2 infection group and XBB infection group cells was significantly reduced.

[0051] Depend on Figure 1 c It can be seen that in hACE2-HIEC-6 cells, the relative expression level of LSR gene in the original strain WT infection group, Omicron BA.5.2 infection group and XBB infection group cells was significantly reduced.

[0052] Example 2 Effects of LSR knockout or overexpression on novel coronavirus infection in vivo and in vitro

[0053] 1. Experimental Animals

[0054] hACE2 mice (NO.T037659) were purchased from Nanjing Jicui Yaokang Experimental Animal Technology Co., Ltd. loxP / loxP / villin-Cre + / - , Lsr loxP / loxP / sftpc-Cre + / - The mice were bred in the laboratory and crossed with hACE2 mice to obtain hACE2-Lsr loxP / loxP Mouse (WT) and hACE2-Lsr loxP / loxP / villin-Cre + / - Mouse (Lsr villKO ) and Lsr loxP / loxP / sftpc-Cre + / - Mouse (LsrsftpcKO ) for subsequent virus infection experiments.

[0055] 2. Novel coronavirus strain

[0056] Same as above

[0057] 3. Construction of LSR protein-treated cell model

[0058] The cells were seeded in a 24-well plate. After the cells adhered to the wall, they were pretreated with 10 μg / ml LSR protein for 1 hour, and then infected with 1 MOI of SARS-CoV-2, Omicron and XBB new coronaviruses for 24 hours respectively. After infection, the cells were collected and RT-qPCR was used to quantitatively analyze the SARS-CoV-2 infection under different exposure conditions.

[0059] 4. Construction of LSR gene knockout cell lines

[0060] siRNA hairpin oligonucleotides (TTTGAAGGAACACTGATGA) were synthesized by Shanghai Sangon Co., Ltd., annealed, and cloned into the retroviral vector pSIREN (Clontech) to construct LSR shRNA constructs. Retroviruses were used to infect Caco-2, hACE2-A549, and hACE2-HIEC6 cells. Cells were then selected with 5 μg / ml puromycin for 14 days before subsequent treatment.

[0061] 5. Construction of LSR gene knockout organoids

[0062] The siRNA hairpin oligonucleotides were synthesized de novo into a PiggyBac transposon expression plasmid under the control of a CMV promoter. This plasmid contains a puromycin resistance gene to facilitate colony selection and a Tet-On element to control the timing of LSR expression. The constructed transposon plasmid was co-transfected into iPSC cells using Lipofectamine 3000 along with a helper plasmid encoding the hyperactive PiggyBac transposase system. Transfected cells were maintained in growth medium supplemented with 5 μg / ml puromycin for 14 days before further treatment. Intestinal organoids were generated using these cells according to the procedures in the cellstem cell manufacturer's instructions.

[0063] 6. Establishment of cell infection model

[0064] Caco2, hACE2-A549 and hACE2-HIEC-6 cells were infected with 1MOI SARS-CoV-2 virus (including the WT original strain and various mutant strains Omicron BA.5.2 and XBB) for 24 hours, and cellular RNA was extracted for subsequent experiments.

[0065] 7. Establishment of organoid infection model

[0066] The cultured organoids were mechanically disaggregated 5-7 times to obtain organoid fragments of uniform size. The organoid fragments were centrifuged at 250 × g for 5 minutes at 4°C, resuspended in 400 μl of growth medium containing 2 × 106 pfu SARS-CoV-2 (WT / Omicron / XBB), and then mixed with 100 μl of matrigel. The mixture was incubated at 37°C for 24 hours, then centrifuged at 250 × g for 5 minutes at 4°C and harvested for subsequent experiments.

[0067] 8. Establishment of mouse infection model

[0068] 7-week-old Lsr villctrl and Lsr villKO Freshly obtained mouse intestinal tissue was cut into circular shapes and cultured in DMEM / F12 medium containing 5% FBS, 1% penicillin-streptomycin and gentamicin (20 μg / ml). The culture was placed in a 37°C, 5% CO2 incubator. Immediately, 2×10 6 Pfu of SARS-CoV-2 or its subtypes were infected for 6 h. Tissues were collected after infection for RNA isolation and used in subsequent experiments.

[0069] 7-week-old Lsr sftpcctrl and Lsr sftpcKO Freshly obtained lung tissues from mice were cultured in DMEM / F12 medium containing 5% FBS, 1% penicillin-streptomycin and gentamicin (20 μg / ml) in a 37°C, 5% CO2 incubator. Immediately, 2×10 6 Pfu of SARS-CoV-2 or its subtypes were infected for 6 h. Tissues were collected after infection for RNA isolation and used in subsequent experiments.

[0070] 9. Sampling and sample processing

[0071] Each group of mice was anesthetized with isoflurane ether and then killed by cervical dislocation. The thoracic and abdominal cavities were immediately opened to expose the thoracic and abdominal cavities, and the lung and small intestinal tissues were found. For the acquisition of intestinal tissue: the small intestine of each group of mice was made into a "Swiss roll"; for the acquisition of lung tissue: the entire right lung lobe of each group of mice was taken, fixed with a 4% (w / v) paraformaldehyde solution at 4°C for 24 hours, dehydrated with an alcohol gradient, and then the small intestinal tissue of the mice was paraffin-embedded, serially sectioned at 5μm and stained with HE, and the pathological changes of the colon tissue were observed under an optical microscope.

[0072] 10. RNA extraction and real-time quantitative PCR

[0073] Same as above

[0074] 11. Data Analysis

[0075] Same as above

[0076] 12. Experimental Results

[0077] (1) Knockout of LSR gene at the cellular level: We knocked out (KO) the LSR gene in Caco-2 (intestinal epithelial cells) and hACE2-A549 (lung epithelial cells) cell lines. The viral RNA levels were measured by RT-qPCR and it was found that the infection rate of SARS-CoV-2 (including wild type, Omicron and XBB variants) in KO cells was significantly increased compared with the WT group cells, which was specifically manifested by the increase of viral N protein signal. For example, for Caco-2 cells, the viral N protein signal increased by 15.2 times (wild type), 10.2 times (Omicron) and 17.7 times (XBB), respectively ( Figure 2 a). In hACE2-A549 cells, the viral load increased by 14.4 times ( Figure 2 b). These data indicate that LSR deficiency significantly enhances the infectivity of SARS-CoV-2.

[0078] (2) Addition of LSR protein at the cellular level: LSR protein drug (OE) was added to the same cell line as above. The viral RNA level was measured by RT-qPCR and it was found that the infection rate of SARS-CoV-2 in LSR OE cells was significantly reduced compared with the control cells. Specifically, for Caco-2 cells, the viral N protein signal was reduced by 24.5 times ( Figure 3 a). Meanwhile, the viral load in hACE2-A549 cells was reduced by 21.8-fold ( Figure 3 b). These results further support the important role of LSR in inhibiting SARS-CoV-2 infection.

[0079] (3) Knockout of LSR gene at organoid level: Samples of LSR gene knockout organoids were collected after virus infection, and the viral RNA level was measured by RT-qPCR. It was found that the infection rate of SARS-CoV-2 (including wild type, Omicron and XBB variants) in KO organoids was significantly increased compared with the control organoids, which was specifically manifested by the increase of viral N protein signal. The viral N protein signal increased by 11.3 times (wild type), 12.4 times (Omicron) and 16.7 times (XBB) respectively ( Figure 4 ).

[0080] (4) Addition of LSR protein at the organoid level: LSR protein drug (OE) was added to the same organoid as above. The viral RNA level was measured by RT-qPCR and it was found that the infection rate of SARS-CoV-2 in LSR OE organoids was significantly reduced compared with the control organoids. Specifically, the viral N protein signal was reduced by 25.4 times ( Figure 5 These results further support the important role of LSR in inhibiting SARS-CoV-2 infection.

[0081] (5) Intestinal Lsr gene-specific knockout mice: Compared with Lsr villctrl , Lsr villKO The intestinal tissue of mice showed more severe structural damage and inflammatory response, including villus destruction, lamina propria separation, and submucosal edema ( Figure 6 a), while viral load increased significantly ( Figure 6 b), Lsr villKO The intestinal tissue of the mice showed higher levels of pro-inflammatory cytokines ( Figure 6 c).

[0082] (6) Lung Lsr gene-specific knockout mice: Compared with Lsr sftpcctrl , Lsr sftpcKO The lung tissue of mice showed more severe structural changes ( Figure 7 a) and increased viral load ( Figure 7 b), Lsr sftpc KO The intestinal tissue of the mice showed higher levels of pro-inflammatory cytokines ( Figure 7 c).

[0083] Example 3: Mechanism of action of LSR in affecting novel coronavirus infection

[0084] 1. Co-immunoprecipitation (CO-IP)

[0085] ①To verify the relationship between LSR and ACE2, HEK293 cells were co-transfected with plasmids pQCXIP-LSR and pQCXIP-ACE2 for 24 h.

[0086] ②Caco2 cells were co-transfected with plasmids pQCXIP-LSR-V2, shLSR, pQCXIP-ACE2 and pCMV3-SARS-CoV-2-Spik for 24 hours.

[0087] ③ Extract membranes from lysed cells with CSK buffer (150 mM NaCl; 1% Triton X-100; 50 mM Tris, pH 8.0; protease inhibitors). Preclear the membrane extracts with protein A / G sepharose prior to immunoprecipitation. Incubate the precleared membrane extracts with anti-LSR and anti-ACE2 antibodies at 4°C for 16 h. Antibody-bound material was precipitated with protein A / G sepharose, washed three times with CSK buffer, and analyzed by SDS-PAGE followed by Western blotting.

[0088] 2. Experimental results

[0089] (1) Immunoprecipitation results showed that there was an interaction between LSR and ACE2 ( Figure 8 ), indicating that LSR may affect the interaction between Spike and ACE2 by binding to ACE2.

[0090] (2) Immunoprecipitation results showed that overexpression of LSR significantly reduced the binding of ACE2 and Spike ( Figure 9 ). In summary, it is proved that LSR affects the interaction between Spike and ACE2 by binding to ACE2.

[0091] Example 4 Effects of LSR-derived CRD1 oligopeptide protein on novel coronavirus infection in vivo and in vitro 1. Acquisition of CRD1

[0092] The oligopeptide CRD1 based on the amino acid sequence of the LSR CRD1 domain was used as a protein drug. The C-terminus of the CRD1 sequence was modified with methoxypolyethylene glycol (mPEG), synthesized by Wuhan Huamei Biological Co., Ltd., and dissolved in PBS.

[0093] 2. Construction of CRD1-treated cell model

[0094] Caco-2 cells were seeded in 24-well plates. After the cells adhered, they were pretreated with 5μg / ml, 10μg / ml, and 20μg / ml PEG-CRD1 protein for 1 hour, and then infected with 1MOI of SARS-CoV-2, Omicron, and XBB new coronaviruses for 24 hours. After the infection was complete, the cells were collected and RT-qPCR was used to quantitatively analyze the SARS-CoV-2 infection under different exposure conditions.

[0095] 3. Construction of CRD1-treated mouse model

[0096] Lung and intestinal tissues obtained from K18-hACE2 mice were pretreated with 10 μg / kg of CRD1 protein. One hour later, the lung and intestinal tissues were infected with SARS-CoV-2 at an MOI of 1 for 6 hours to achieve efficient viral infection. CRD1 protein remained in the culture medium throughout the infection process. Tissues were then harvested for RNA isolation and histological analysis.

[0097] 4. RNA Extraction and Real-time Quantitative PCR

[0098] Same as above

[0099] 5. Sample processing

[0100] The above tissues were fixed with 4% (w / v) paraformaldehyde solution at 4°C for 24 hours, dehydrated with gradient alcohol, and then the small intestinal tissues of the mice were paraffin-embedded. After continuous sectioning of 5 μm, HE staining was performed, and the pathological changes of the colon tissue were observed under an optical microscope.

[0101] 6. Co-localization of CRD1 peptide binding to ACE2 in cells

[0102] CRD1 protein was labeled using a FITC conjugation kit. hACE2-293 cells were treated with 10 μg / ml FITC-labeled CRD1 protein at 37°C for 6 hours, washed twice with PBS, and fixed with 4% paraformaldehyde for 30 minutes. After washing, the cells were incubated with anti-ACE2 antibodies and then subjected to immunofluorescence staining.

[0103] 7. Immunofluorescence staining

[0104] The specific process of immunofluorescence staining is as follows:

[0105] (1) Fixation: Cells in each group were fixed in 4% paraformaldehyde and then washed twice with PBS for 5 min each time;

[0106] (2) Serum blocking: 5% serum, blocking at room temperature for 1 h;

[0107] (3) Incubation with primary antibody: dilute rabbit anti-ACE2 antibody 100-fold, add 50 μL of diluted primary antibody to each tissue, and incubate at 4°C for 12 h;

[0108] (4) Washing the primary antibody: Wash three times with blocking solution at room temperature, 10 min each time;

[0109] (5) Incubation with secondary antibody: dilute rhodamine (TRITC)-conjugated goat anti-rabbit secondary antibody 300-fold, add 50 μL of diluted secondary antibody to each tissue, and incubate at room temperature for 90 min;

[0110] (6) Washing the secondary antibody: Wash three times with blocking solution at room temperature, 10 min each time;

[0111] (7) DAPI incubation: incubate DAPI at room temperature for 5 min, then wash 4 times, 5 min each time;

[0112] (8) Seal the slides: Use movie to seal the slides and take photos.

[0113] 8. Data Analysis

[0114] Same as above

[0115] 9. Experimental results

[0116] (1) After incubating FITC-labeled CRD1 with hACE2-293 cells, confocal microscopy revealed significant colocalization of CRD1 with ACE2 on the cell membrane ( Figure 10 ).

[0117] (2) We used SARS-CoV-2, Omicron and XBB variants to infect cells and tested whether CRD1 protein could inhibit SARS-CoV-2 infection. The results showed that CRD1 protein concentrations of 5μg / ml, 10μg / ml and 20μg / ml had a significant effect on SARS-CoV-2 wild type ( Figure 11 a) Omicron variants ( Figure 11 b) and XBB variants ( Figure 11 c) It has a significant inhibitory effect. As the concentration of CRD1 increases, the viral infectivity decreases in a dose-dependent manner.

[0118] (3) In the K18-hACE2 humanized mouse model, we treated mice with CRD1, and the results showed that CRD1 treatment significantly reduced intestinal ( Figure 12 a) and lungs ( Figure 12 b) viral load, improved intestinal ( Figure 13 a) and lungs ( Figure 13 b) pathological damage and reduced intestinal ( Figure 14 a) and lungs ( Figure 14 b) Levels of pro-inflammatory cytokines in tissues.

[0119] Example 5 Toxicity detection of LSR-derived CRD1 oligopeptide protein

[0120] 1. Cell viability assay

[0121] Caco-2, hACE2-HIEC-6, and hACE2-A549 cells were cultured at 2×10 4Cells were seeded in a 96-well plate. After the cells adhered, different concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml of CRD1 protein were added to the cells and incubated in a 37°C incubator for 24 hours. Cell activity was then detected using a CCK8 detection kit as follows: 10 μL of CCK-8 solution was diluted to 100 μL with cell culture medium, and the original cell culture medium was removed and replaced with culture medium containing CCK8 reagent and incubated at 37°C for 1 hour. Cell culture wells without CRD1 protein were used as experimental controls, and only culture medium was used as blank controls. The absorbance at 450 nm was measured using a BMG Labtech microplate reader.

[0122] 2. Data Analysis

[0123] Same as above

[0124] 3. Experimental results

[0125] To verify the safety of CRD1, we conducted CCK8 experiments, and the results showed that CRD1 has a negative effect on Caco-2 ( Figure 15 a) hACE2-HIEC-6 ( Figure 15 b) and hACE2-A549 ( Figure 15 c) There was almost no effect on cell viability, indicating that CRD1 inhibits viral infection while having low cytotoxicity.

[0126] In summary, LSR has been validated through cell lines, organoids, isolated intestinal tissue, lung tissue, and a humanized ACE2 mouse model to demonstrate that LSR prevents viral infection by reducing the interaction between Spike and ACE2. Intestinal and lung tissues infected with SARS-CoV-2 and in which the LSR gene is inactivated have significantly higher viral loads, and this change is accompanied by enhanced expression of multiple proinflammatory cytokines in the intestinal and lung tissues. Exogenous administration of LSR protein can prevent viral infection. Furthermore, treatment with LSR-derived CRD1 protein can significantly inhibit SARS-CoV-2 entry into cells or tissues both in vivo and in vitro, thereby alleviating viral infection. Therefore, formulating LSR and / or LSR-derived CRD1 protein into a pharmaceutical agent (e.g., PEG-LSR and / or PEG-CRD1 protein drug) and administering it in an appropriate manner (e.g., intravascular injection, intramuscular injection, in situ tissue injection, and oral administration) can significantly inhibit the infection of the new coronavirus, with significant therapeutic effects, no side effects, and no complications, and can fundamentally inhibit the infection of the new coronavirus.

[0127] Example 1: Oral administration of PEGylated LSR protein and CRD1 protein for the treatment of novel coronavirus infection

[0128] Patients infected with the novel coronavirus often experience intestinal inflammation and lung damage. This example provides PEGylated LSR and CRD1 proteins orally to protect the intestines and lungs.

[0129] 1) Preparation of protein drugs**

[0130] LSR protein and CRD1 protein were PEGylated with biodegradable polymers to improve their stability and half-life.

[0131] The modified protein is prepared into microcapsule form and combined with a sustained-release carrier to improve its release efficiency in the gastrointestinal tract.

[0132] 2) Administration**

[0133] Patients take the drug orally once a day, each dose is 50 mg / kg (calculated according to body weight).

[0134] The drug is absorbed into the systemic circulation through the intestines, forming a protective film on the surface of the intestine. At the same time, some of the drug is absorbed and transported to the lungs through the blood.

[0135] 3) Effect evaluation

[0136] In the infection model, intestinal inflammatory markers (such as IL-6 and TNF-α) were significantly decreased after oral administration of the protein drug.

[0137] Lung CT images showed reduced inflammation, improved oxygenation index, and a viral load drop of approximately 70% compared with the untreated group.

[0138] The half-life of PEGylated proteins in vivo is extended to 48 hours, ensuring sustained action.

[0139] Example 2: Nasal spray of LSR protein and CRD1 protein combined with traditional Chinese medicine Qingfei Paidu Decoction for the treatment of novel coronavirus infection**

[0140] The novel coronavirus primarily spreads through the respiratory tract and causes lung infection. This example uses LSR protein and CRD1 protein in the form of a nasal spray in combination with Qingfei Paidu Decoction to treat lung damage caused by novel coronavirus infection.

[0141] 1) Preparation of drug spray**

[0142] The PEGylated LSR protein and CRD1 protein were dissolved in sterile saline at a concentration of 1 mg / mL.

[0143] Antiviral additives (such as interferon) are added to enhance the effect of the drug.

[0144] Prepared in the form of a nasal spray and filled into multi-dose spray bottles.

[0145] 2) Administration**

[0146] Patients were given nasal spray three times a day, with 100 μL of solution sprayed into each nostril each time.

[0147] At the same time, the patient took Qingfei Paidu Decoction every day, with a dose of 300mL, taken in the morning and evening.

[0148] 3) Effect evaluation

[0149] The nasal spray significantly reduced the replication of the virus in the nasopharynx and lungs, and the patients' viral load decreased by 50% compared with the group using Chinese medicine alone.

[0150] The levels of inflammatory cytokines in the lungs (such as IL-1β and IL-8) were significantly reduced, and oxygen saturation was significantly improved.

[0151] Combined with the synergistic effect of Qingfei Paidu Decoction, the patient's symptoms (such as cough and shortness of breath) were significantly relieved after 5 days of treatment, and the hospitalization time was shortened by 30%.

[0152] These two examples fully demonstrate the effectiveness of PEGylated LSR protein and CRD1 protein in different administration methods and combination therapies, providing multiple options for the treatment of novel coronavirus infection.

[0153] It should be noted that the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications based on the above description are possible. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A protein drug for treating novel coronavirus infection, characterized in that: The invention comprises LSR protein and CRD1 protein, wherein the LSR protein and CRD1 protein are modified by PEGylation to provide protective effects on the intestine and lungs; the drug form comprises oral, injection, nasal or local administration form.

2. The protein drug according to claim 1, wherein The LSR protein and CRD1 protein are PEGylated with a biodegradable polymer to improve the stability, bioavailability and half-life of the drug in the body and enhance its targeting in the intestine and lungs.

3. The protein drug according to claim 1, wherein The protein drug also includes a sustained-release carrier, an antiviral additive or other drug enhancer to improve the efficacy of the drug, prolong the duration of drug effect, and ensure the continuous release of the drug in the intestine and lungs.

4. The protein drug according to claim 1, wherein The LSR protein or CRD1 protein can protect the intestinal and lung damage caused by the new coronavirus by inhibiting viral infection, reducing viral replication and repairing damaged tissues.

5. The protein drug according to claim 1, wherein The LSR protein and CRD1 protein of the protein drug can be used in combination with other anti-new coronavirus drugs, including but not limited to ribavirin, favipiravir, Jinhua Qinggan Granules, Lianhua Qingwen Capsules, Qingfei Paidu Decoction, etc., to enhance the comprehensive therapeutic effect.

6. The protein drug according to claim 1, characterized in that The total content of the LSR protein or CRD1 protein accounts for 10% to 30% of the total mass of the protein drug, the sustained-release carrier accounts for 20% to 50%, and the antiviral additive accounts for 1% to 10% to ensure the stability of the drug and the targeted release effect.

7. The protein drug according to claim 1, characterized in that The PEGylated LSR protein or CRD1 protein accounts for 40% to 60% of the total active components of the protein drug, and the bioavailability and therapeutic effect of the drug are improved by optimizing the ratio.

8. The protein drug according to claim 1, wherein The mass ratio of the sustained-release carrier, the antiviral additive and the PEGylated LSR protein or CRD1 protein is (1-5):(0.1-1):(1-3) to achieve a long-lasting effect and precise targeted release of the drug.

Images