A nbp1 single-domain antibody, a tat-nbp1 fusion single-domain antibody and application thereof

Abstract

The application discloses a kind of NbP1 single-domain antibody, TAT-NbP1 fusion single-domain antibody and purposes thereof, belong to biological medicine technical field.The NbP1 single-domain antibody of the application is used to specifically bind new coronavirus PLpro antigen, the NbP1 single-domain antibody is composed of framework region FR and three complementarity determining regions CDR1, CDR2 and CDR3;The amino acid sequence of CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of CDR2 is as shown in SEQ ID NO.2, the amino acid sequence of CDR3 is as shown in SEQ ID NO.3.TAT-NbP1 fusion single-domain antibody is the fusion protein of NbP1 single-domain antibody and HIV-1 virus TAT peptide segment.The NbP1 single-domain antibody and TAT-NbP1 fusion single-domain antibody provided by the application have both antiviral and anti-inflammatory activities, and are safe, which provides a new idea for treating SARS-CoV-2 infection and developing anti-SARS-CoV-2 drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an NbP1 single-domain antibody, a TAT-NbP1 fusion single-domain antibody, and their applications. Background Technology

[0002] Since its emergence in late 2019, the novel coronavirus (SARS-CoV-2) has demonstrated extremely high transmissibility, rapidly transforming into a global pandemic and posing a significant challenge to public health systems worldwide. In recent years, mutant strains of the virus have continued to emerge, partially circumventing the inhibitory effects of existing antiviral drugs and vaccines, a phenomenon that presents a substantial challenge to the development of antiviral drugs.

[0003] Papain-like protein (PLpro) is a key non-structural protein of SARS-CoV-2. It plays a dual role in the viral life cycle: at the viral replication level, PLpro works synergistically with the main protease (Mpro) to process the multiplying proteins pp1a and pp1ab produced during viral replication, thereby generating functional proteins and promoting viral replication; at the immune regulation level, PLpro, through its deubiquitination and deISG-like activities, can specifically cleave ubiquitin-like modifications (such as ISG15) of host proteins, interfering with the host immune response and promoting viral immune escape. Based on these functions, PLpro is considered a valuable drug target in the development of drugs against SARS-CoV-2.

[0004] Antibody therapies with high target specificity are increasingly being used for various human diseases. In recent years, some research has also begun to focus on the development of intracellular targeted antibodies, including intracellular delivery of intracellular antibodies and exogenous antibodies. Among these, a specific fragment (YGRKKRRQRRR) of the trans-activator (TAT) derived from human immunodeficiency virus type 1 (HIV-1) has been shown to deliver biomolecules into cells.

[0005] In the bodies of camels and sharks in nature, there exists a type of antibody that is different from yet similar to human antibodies, called heavy-chain antibody (HcAb), with a molecular weight of approximately 95 kDa. Although this antibody lacks the light chain portion of ordinary monoclonal antibodies, it still exhibits a high-affinity interaction with antigens. In HcAb, the antigen-binding site is formed by the heavy variable domain (VHHs). VHHs typically have a molecular weight of 12-15 kDa and are the smallest antigen-binding fragments found in nature; they are also known as single-domain antibody (sdAb). Due to their small molecular weight, high antigen affinity, low immunogenicity, and good tissue permeability, single-domain antibodies have been applied in various fields of biomedicine, including disease diagnosis and treatment, affinity purification reagents, assistance in protein structure analysis, and biosensors.

[0006] Currently, targeted drug development for PLpro mainly focuses on small molecule compounds. These all suffer from problems such as low selectivity and poor stability to varying degrees. In recent years, antibody therapy using biological macromolecules has been increasingly applied to various human diseases. This is a promising new drug screening direction that can replace small molecule drugs. Therefore, developing an antibody drug that can specifically bind to PLpro and efficiently enter cells using antibody therapy using biological macromolecules is of great significance for overcoming the shortcomings of existing drugs and enriching the treatment options for COVID-19 infection. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide an NbP1 single-domain antibody, a TAT-NbP1 fusion single-domain antibody, and their applications.

[0008] The specific technical solution of this invention is as follows: The first aspect of this invention provides an NbP1 single-domain antibody for specifically binding to the SARS-CoV-2 PLpro antigen. The NbP1 single-domain antibody comprises a backbone region FR and three complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3.

[0009] Furthermore, the amino acid sequence of the NbP1 single-domain antibody is shown in SEQ ID NO. 4.

[0010] A second aspect of the present invention provides a TAT-NbP1 fusion single-domain antibody, wherein the fusion single-domain antibody is a fusion protein of the NbP1 single-domain antibody and the TAT peptide of HIV-1 virus.

[0011] Furthermore, the C-terminus of the NbP1 single-domain antibody is linked to the TAT peptide of the HIV-1 virus; Furthermore, the amino acid sequence of the TAT-NbP1 fusion single-domain antibody is shown in SEQ ID NO. 5.

[0012] A third aspect of the present invention provides a polynucleotide that encodes the NbP1 single-domain antibody or the TAT-NbP1 fusion single-domain antibody.

[0013] Furthermore, the nucleotide sequence encoding the NbP1 single-domain antibody is shown in SEQ ID NO. 6; Furthermore, the nucleotide sequence encoding the TAT-NbP1 fusion single-domain antibody is shown in SEQ ID NO. 7.

[0014] A fourth aspect of the present invention provides a biomaterial expressing the NbP1 single-domain antibody, or expressing the TAT-NbP1 fusion single-domain antibody, or containing the polynucleotide, wherein the biomaterial is at least one of an expression cassette, a vector, a recombinant microorganism, and a cell line.

[0015] The fifth aspect of the present invention provides a method for preparing the TAT-NbP1 fusion single-domain antibody, characterized in that a polynucleotide encoding the TAT-NbP1 fusion single-domain antibody is introduced into a host cell for expression, wherein the host cell is an Escherichia coli prokaryotic expression system.

[0016] The sixth aspect of this invention provides the use of the NbP1 single-domain antibody, or the TAT-NbP1 fusion single-domain antibody, in the preparation of a product, wherein the use in the preparation of the product is any one of the following uses: (1) Preparation of reagents for detecting and / or diagnosing SARS-CoV-2-related diseases; (2) Preparation of reagents for recognizing and / or binding to the SARS-CoV-2 PLpro protein; (3) To prepare drugs for the prevention and / or treatment of SARS-CoV-2 virus infection; Preferably, the SARS-CoV-2-related disease is SARS-CoV-2 PLpro-induced colitis.

[0017] A seventh aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the NbP1 single-domain antibody, or the TAT-NbP1 fusion single-domain antibody, and a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention provides an NbP1 single-domain antibody and demonstrates its applicability for recognizing and binding to the SARS-CoV-2 PLpro antigen. Furthermore, this invention provides a TAT-NbP1 fusion single-domain antibody and demonstrates its ability as a viral replication inhibitor, suppressing the replication of SARS-CoV-2 trVLP, while simultaneously inhibiting the replication of wild-type SARS-CoV-2 and EG.5 variants. It can also act as a PLpro / ISG15 interaction interface blocker to reverse PLpro-induced colonic inflammation in mice, with no toxic side effects on mouse organs after high-dose administration. The development of TAT-NbP1 provides proof-of-concept for novel therapeutic strategies, showing promise in combating SARS-CoV-2 and other emerging coronaviruses by simultaneously inhibiting viral replication and pathological inflammation.

[0019] This invention first evaluated the affinity of the NbP1 single-domain antibody for the SARS-CoV-2 PLpro antigen. Secondly, it selected the SARS-CoV-2 trVLP and live virus system to reveal the inhibitory effect of the TAT-NbP1 fusion single-domain antibody on SARS-CoV-2 replication. Finally, a competitive FP assay verified its ability to block the PLpro / ISG15 interaction interface in vitro, and it was further applied to PLpro-stimulated NCM460 cell line and a mouse colonic inflammation model to evaluate its anti-inflammatory activity.

[0020] The specific results are as follows: 1. In the affinity evaluation of SARS-CoV-2 PLpro, surface ion resonance experiments showed that the dissociation constant of the NbP1 single-domain antibody for recombinant SARS-CoV-2 PLpro protein in vitro was 1.45 × 10⁻⁶. -6 M. This indicates that the NbP1 single-domain antibody specifically binds to SARS-CoV-2 PLpro. In in vitro cell experiments, the TAT-NbP1 fusion single-domain antibody significantly inhibited the replication of SARS-CoV-2 trVLP, wild-type SARS-CoV-2, and EG.5 variants. Furthermore, the TAT-NbP1 fusion single-domain antibody, which can block the PLpro / ISG15 interaction interface in vitro, can reverse PLpro-induced inflammation in the NCM460 cell line.

[0021] 2. In a mouse model of PLpro-induced colitis, the TAT-NbP1 fusion single-domain antibody significantly alleviated PLpro-induced colitis. Compared to treatment with the positive control drug berberine sulfate, the TAT-NbP1 fusion single-domain antibody showed a more significant inhibitory effect on PLpro-induced colitis in mice. Furthermore, pathological sections of mouse organs showed no obvious inflammatory infiltration or tissue damage, indicating that the TAT-NbP1 fusion single-domain antibody has no toxic side effects. Attached Figure Description

[0022] Figure 1 This is an expression diagram of NbP1 single-domain antibody and TAT-NbP1 fusion single-domain antibody.

[0023] Figure 2 A graph showing the surface plasmon resonance detection of NbP1 single-domain antibody against SARS-CoV-2 PLpro.

[0024] Figure 3 Figure showing the results of TAT-NbP1 fusion single-domain antibody inhibiting SARS-CoV2 trVLP replication.

[0025] Figure 4 Figure showing the results of TAT-NbP1 fusion single-domain antibody inhibiting the replication of wild-type SARS-CoV-2 and EG.5 variants.

[0026] Figure 5 The image shows the RT-qPCR results of the effects of the TAT-NbP1 fusion single-domain antibody on the mRNA levels of inflammatory factors and ISG15 in PLpro-induced NCM460 cells. The cells were divided into a model group, a TAT-NbP1 fusion single-domain antibody treatment group, and a positive control group (Coptisine sulfate). In the figure, A shows the expression level of IL-1α after PLpro stimulation and antibody treatment, B shows the expression level of ISG15 after PLpro stimulation and antibody treatment, and C shows the expression level of TNF-α after PLpro stimulation and antibody treatment.

[0027] Figure 6 HE staining images of colon tissue in a mouse colitis model induced by PLpro combined with acetic acid using the TAT-NbP1 fusion single-domain antibody. The mice were divided into a model group, a TAT-NbP1 fusion single-domain antibody administration group, and a positive control group (berberine sulfate).

[0028] Figure 7Figure 1 shows the expression levels of inflammatory factors and ISG15 in the colon tissue of mice after pre-administration of the TAT-NbP1 fusion single-domain antibody and subsequent modeling. Figure A shows the expression level of IL-1α in the colon of mice with the corresponding treatment, Figure B shows the expression level of ISG15 in the colon of mice with the corresponding treatment, and Figure C shows the expression level of TNF-α in the colon of mice with the corresponding treatment.

[0029] Figure 8 HE staining images of pathological sections of five major organs in mice after administration of the TAT-NbP1 fusion single-domain antibody, including staining images of the heart, liver, spleen, lungs and kidneys. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0031] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0032] Unless otherwise specified in the embodiments, all techniques or conditions described in the literature in this field or in the product manual can be followed. The invention will be further described below with reference to specific embodiments, and the advantages and features of the invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the invention.

[0033] Example 1: Preparation of NbP1 single-domain antibody and TAT-NbP1 fusion single-domain antibody I. NbP1 single-domain antibody, TAT-NbP1 fusion single-domain antibody Using SARS-CoV-2 PLpro as the antigen, an NbP1 single-domain antibody was screened. The NbP1 single-domain antibody is used to specifically bind to SARS-CoV-2 PLpro. The NbP1 single-domain antibody consists of a backbone region (FR) and a complementarity-determining region (CDR). The CDRs include CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3. In this embodiment, the amino acid sequence of the NbP1 single-domain antibody is shown in SEQ ID NO. 4. In this embodiment, the nucleotide sequence encoding the NbP1 single-domain antibody is shown in SEQ ID NO. 6.

[0034] The TAT-NbP1 fusion single-domain antibody is a fusion protein of an NbP1 single-domain antibody and a specific fragment (YGRKKRRQRRR) of the trans-activator (TAT) derived from human immunodeficiency virus type 1 (HIV-1). In this embodiment, the amino acid sequence of the TAT-NbP1 fusion single-domain antibody is shown in SEQ ID NO. 5. In this embodiment, the nucleotide sequence encoding the TAT-NbP1 fusion single-domain antibody is shown in SEQ ID NO. 7.

[0035] The amino acid sequence of the NbP1 single-domain antibody complementarity-determining region CDR1 is SEQ ID NO.1: SISRYSY The amino acid sequence of the NbP1 single-domain antibody complementarity-determining region CDR2 is SEQ ID NO.2: LVATITYGGT The amino acid sequence of the NbP1 single-domain antibody complementarity-determining region CDR3 is SEQ ID NO.3: GSYNVEGFG The amino acid sequence of the NbP1 single-domain antibody is SEQ ID NO.4: QVQLQESGGGLVQAGGSLRLSCAASGSISRYSYMGWYRQAPGKERELVATITYGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGSYNVEGFGYWGQGTQVTVSS The amino acid sequence of the TAT-NbP1 fusion single-domain antibody is SEQ ID NO. 5: YGRKKRRQRRRQVQLQESGGGLVQAGGSLRLSCAASGSISRYSYMGWYRQAPGKERELVATITYGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGSYNVEGFGYWGQGTQVTVSS The nucleotide sequence encoding the NbP1 single-domain antibody is SEQ ID NO.6: CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTAGATATTCTTATATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCACTATTACTTATGGTGGTACTACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGTTGGTTCTTATAATGTTGAAGGTTTTGGTTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC Nucleotide sequence encoding the TAT-NbP1 fusion single-domain antibody SEQ ID NO. 7: TATGGCCGCAAAAAACGCCGCCAGCGCCGTCGCCAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTAGATATTCTTATATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCACTATTACTTATGGTGGTACTACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGTTGGTTCTTATAATGTTGAAGGTTTTGGTTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC II. Expression and purification of the NbP1 single-domain antibody and the TAT-NbP1 fusion single-domain antibody In this embodiment, the nucleotide sequence gene of the TAT-NbP1 fusion single-domain antibody was inserted into the pET22b prokaryotic expression vector, and protein expression was performed using the mature *E. coli* BL21(DE3) prokaryotic cell expression system. The plasmid was transformed into BL21(DE3) cells using a heat shock transformation method. The expression conditions for the TAT-NbP1 fusion single-domain antibody were as follows: expression was induced for 12 hours at 16°C and 220 rpm with 0.5 mM IPTG, followed by centrifugation at 4°C and 6700×g for 20 minutes to collect the bacterial cells. The cells were resuspended in 200 mL of pyrogen-free buffer containing 20 mM Tris-HCl (pH 8.0), 1 M NaCl, 5 mM β-ME, and 5 mM imidazole (pH 8.0). The cells were lysed at low temperature under high pressure (800 bar) using a high-pressure cell disruptor, and the supernatant was collected by ultracentrifugation. The TAT-NbP1 fusion single-domain antibody in the supernatant was purified using Ni-NTA affinity chromatography. After purification, protein purity was observed by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Finally, the protein was concentrated using ultrafiltration tubes and transferred to sterile PBS, then stored at -80°C. The expression methods for NbP1 single-domain antibodies and TAT fusion NbP1 single-domain antibodies were the same.

[0036] Figure 1 The images show Coomassie brilliant blue staining of the purified NbP1 single-domain antibody and the TAT-NbP1 fusion single-domain antibody. Due to the small molecular weight of TAT, both the NbP1 single-domain antibody and the TAT-NbP1 fusion single-domain antibody have a molecular weight of approximately 15 kDa.

[0037] Example 2: Affinity evaluation of NbP1 single-domain antibody to PLpro protein This embodiment uses surface ion resonance experiments to analyze the recognition of PLpro by NbP1 single-domain antibodies. Specifically, it includes the following steps: First, a 3D dextran sensor chip coupled with recombinant PLpro was bound to mobile phase NbP1 single-domain antibody at different concentrations. The concentrations were set to 5, 2.5, 1.25, 0.63, and 0.31 μM, respectively. The mobile phase was loaded at a rate of 2 μL / s and flowed through the chip placed on a biomolecular interaction analyzer. The sensor chip then captured the binding interaction between the immobilized PLpro and NbP1.

[0038] Then, the sensor data representing the binding reaction were analyzed using BIEvaluation software. The dissociation constant (K) was calculated by fitting data using this software. D ), K D This represents the affinity index between the NbP1 single-domain antibody and PLpro.

[0039] Experimental results are as follows Figure 2 As shown. Figure 2 Curves showing the affinity of different concentrations of NbP1 single-domain antibody for PLpro, obtained by surface plasmon resonance (SPR). K was calculated after fitting. D 1.45 × 10 -6 M indicates that the NbP1 single-domain antibody can specifically bind to SARS-CoV-2 PLpro.

[0040] Example 3: Evaluation of the inhibitory effect of TAT-NbP1 fusion single-domain antibody on the replication of SARS-CoV-2 trVLP Caco2-N cells were spaced at 2 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates with an MOI of 0.1 using trVLPs and cultured at 37°C and 5% CO2 for 14 hours. The viral load was then discarded, and the cells were washed twice with PBS and replaced with DMEM medium containing gradient concentrations of nanobodies. The initial nanobodies concentration was 1000 nM, and six concentration gradients (15.6–1000 nM) were established through serial dilutions, with three replicates for each concentration. After 48 hours of culture, fluorescence expression in each well was observed under a fluorescence microscope, and the fluorescence intensity was calculated and analyzed using ImageJ software to plot the dose-infection rate curve.

[0041] Experimental results are as follows Figure 3 As shown. Figure 3 Representative immunofluorescence images of Caco2-N cells after 6 h of infection with trVLP followed by 48 h of treatment with serially diluted TAT-fused nanobodies. Figure 3 It can be seen that after 6 hours of trVLP infection and 48 hours of antibody treatment, the antibody can reduce the EGFP fluorescence intensity in the cells in a dose-dependent manner, which indicates that our antibody drug can effectively inhibit trVLP replication.

[0042] Example 4: Evaluation of the inhibitory effect of TAT-NbP1 fusion single-domain antibody on the replication of SARS-CoV-2 virus strain All experiments were conducted in a biosafety level 3 (BSL-3) laboratory. To evaluate the antiviral activity of nanobodies against real SARS-CoV-2, Vero E6 cells were cultured at 2 × 10⁶ cells per well 24 hours prior to the experiment. 4Cells were seeded at a density in 96-well plates. SARS-CoV-2 (wild-type or EG.5 variant) with a multiplicity of infection (MOI) of 0.05 was serially diluted with an equal volume of nanobody (final nanobody concentration range: 0.042–30 μM). After incubation at 37°C for 1 hour, cells were infected with 50 μL of the mixture at 37°C for 1 hour. After removing the seeding medium, 100 μL of serially diluted nanobody was added and incubated at 37°C. After 24 hours of incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Tween-20. Immunofluorescence detection of the viral nucleocapsid (N) protein was performed using a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (catalog number: 40143-T62, Sinopharm, Beijing), followed by labeling with Alexa Fluor488-labeled anti-rabbit secondary antibody (Jackson). All cells were stained with 4,6-diamino-2-phenylindole (DAPI, Sigma, USA) to visualize the nucleus. Fluorescence signals were quantitatively analyzed using an automated imaging cell counter (Nexcelom Celigo) to determine the proportion of infected cells.

[0043] Experimental results are as follows Figure 4 As shown. Figure 4 Representative immunofluorescence images of Vero E6 cells 1 hour after SARS-CoV-2 infection, followed by 48 hours of treatment with serially diluted TAT-fused nanobodies. Figure 4 As can be seen, after infection with real viral strains (wild type, EG.5), treatment with antibody TAT-NbP1 resulted in a dose-dependent reduction in EGFP fluorescence intensity in cells, indicating that our antibody drug can effectively inhibit the replication of SARS-CoV-2 wild type strains and EG.5 mutant strains.

[0044] Example 5: Evaluation of the reversal effect of TAT-NbP1 fusion single-domain antibody on inflammation induced by PLpro stimulation in NCM460 cell lines NCM460 cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). Cells were spaced at 6.0 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight at 37°C and 5% CO2. PLpro protein was diluted to a final concentration of 5 μM in DMEM medium containing 10% FBS and added to the wells to stimulate cells for 24 hours. Cells were then collected for subsequent RNA extraction and RT-qPCR.

[0045] Figure 5To determine the mRNA levels of inflammatory factors and ISG15 in NCM460 cells induced by PLpro after treatment with nanobodies fused with N-terminus TAT ​​at different concentrations in RTqPCR. Figure 5 In the diagram, the horizontal axis represents the NCM460 cell line without any treatment; PLPro represents the group treated with PLPro stimulation only without drug treatment; PLpro+5 μMTAT-CW056 represents the group treated with PLPro stimulation followed by 5 μM of Ebola virus VP30-targeting drug; PLpro+0.19 μMTAT-NbP1 represents the group treated with PLPro stimulation followed by 0.19 μM TAT-NbP1; PLpro+0.56 μMTAT-NbP1 represents the group treated with PLPro stimulation followed by 0.56 μM TAT-NbP1; PLpro+1.67 μMTAT-NbP1 represents the group treated with PLPro stimulation followed by 1.67 μM TAT-NbP1; PLpro+5 μMTAT-NbP1 represents the group treated with PLPro stimulation followed by 5 μM TAT-NbP1; PLpro+5 μM Coptisine sulfate To provide PLpro stimulation, the treatment group was subsequently given 5 μM MCoptisine sulfate. From Figure 5 As can be seen, compared with the PLpro stimulation group alone, both the TAT-NbP1 treatment group and the positive control (Coptisine sulfate) group can significantly reverse the inflammation of NCM460 cell lines caused by PLpro stimulation.

[0046] Example 6: Evaluation of the reversal effect of TAT-NbP1 fusion single-domain antibody on colonic inflammation induced by 1% acetic acid combined with PLpro stimulation in mice. Mice were administered the test drug via intraperitoneal injection for three consecutive days (three doses of each antibody: 2.5, 5, and 10 mg / kg, with berberine sulfate as a positive control). Following treatment with 1% acetic acid combined with PLpro, a colonic inflammation model was established. After model establishment, mice were euthanized, and colonic tissue was collected. Histopathological evaluation was performed using hematoxylin and eosin (H&E) staining, and RNA was extracted from the colonic tissue for RT-qPCR detection of the expression levels of relevant inflammatory factors.

[0047] Figure 6 HE staining images of mouse colon tissue after pre-administration of TAT-NbP1 fusion single-domain antibody and subsequent modeling were obtained, dividing the mice into a model group and a TAT-NbP1 fusion single-domain antibody administration group. Figure 6It can be seen that, compared with the Model group, the TAT-NbP1 group showed a dose-dependent inhibition of colonic inflammation in mice induced by 1% acetic acid combined with PLpro stimulation.

[0048] Figure 7 The expression levels of inflammatory factors and ISG15 in the colonic tissue of mice were investigated after pre-administration of the TAT-NbP1 fusion single-domain antibody and subsequent model establishment. The mice were divided into a model group and a TAT-NbP1 fusion single-domain antibody administration group. Figure 7 In the graph, the horizontal axis represents the group that received no treatment; the horizontal axis represents the group that received model treatment but not prior to drug treatment; the horizontal axis represents the group that received 2.5 mg / kg TAT-NbP1 prior to model treatment; the horizontal axis represents the group that received 5 mg / kg TAT-NbP1 prior to model treatment; the horizontal axis represents the group that received 10 mg / kg TAT-NbP1 prior to model treatment; and the horizontal axis represents the group that received 100 mg / kg Coptisine sulfate prior to model treatment. Figure 7 A represents the expression level of IL-1α in the colon of mice under the corresponding treatment. Figure 7 B represents the expression level of ISG15 in the colon of mice under the corresponding treatment. Figure 7 C represents the expression level of TNF-α in the colon of mice under the corresponding treatment. From Figure 7 It can be seen that, compared with the Model group, the TAT-NbP1 treatment groups, which were administered TAT-NbP1 via intraperitoneal injection from 2.5 mg / kg to 10 mg / kg, showed a dose-dependent reduction in the expression levels of inflammatory factors in the mouse colon induced by 1% acetic acid combined with PLpro stimulation, as well as ISG15.

[0049] Figure 8 HE staining images of mouse organ sections after administration of the TAT-NbP1 fusion single-domain antibody, including the heart, liver, spleen, lung, and kidney. The mice were divided into a model group and a TAT-NbP1 fusion single-domain antibody administration group. After treatment with the TAT-NbP1 fusion single-domain antibody, no inflammatory cell infiltration or tissue damage was observed in the five major organs of the mice. Pathological examination of the heart, liver, spleen, lung, and kidney sections showed no significant abnormalities, indicating that the antibody has good safety at therapeutic doses.

[0050] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any equivalent modifications made by those skilled in the art to the technical solutions of the present invention by reading the present invention specification are covered by the claims of the present invention.

Claims

1. An NbP1 single-domain antibody, characterized in that, The NbP1 single-domain antibody is used to specifically bind to the SARS-CoV-2 PLpro antigen. The NbP1 single-domain antibody consists of a backbone region FR and three complementarity-determining regions CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2 and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The NbP1 single-domain antibody according to claim 1, characterized in that, The amino acid sequence of the NbP1 single-domain antibody is shown in SEQ ID NO.

4.

3. A TAT-NbP1 fusion single-domain antibody, characterized in that, The fusion single-domain antibody is a fusion protein of the NbP1 single-domain antibody of claim 1 or 2 and the TAT peptide of HIV-1 virus.

4. The fusion single-domain antibody according to claim 3, characterized in that, The C-terminus of the NbP1 single-domain antibody is linked to the TAT peptide of the HIV-1 virus. The amino acid sequence of the TAT-NbP1 fusion single-domain antibody is shown in SEQ ID NO.

5.

5. A polynucleotide, characterized in that, The polynucleotide encodes the NbP1 single-domain antibody of claim 1 or 2, or the TAT-NbP1 fusion single-domain antibody of claim 3 or 4.

6. The polynucleotide sequence according to claim 5, characterized in that, The polynucleotide sequence encoding the NbP1 single-domain antibody of claim 1 or 2 is shown in SEQ ID NO. 6; The polynucleotide sequence encoding the TAT-NbP1 fusion single-domain antibody as described in claim 3 or 4 is shown in SEQ ID NO.

7.

7. A biomaterial, characterized in that, The biomaterial expresses the NbP1 single-domain antibody of claim 1 or 2, or the TAT-NbP1 fusion single-domain antibody of claim 3 or 4, or contains the polynucleotide of claim 5 or 6, wherein the biomaterial is at least one of expression cassette, vector, recombinant microorganism and cell line.

8. A method for preparing the TAT-NbP1 fusion single-domain antibody according to claim 3 or 4, characterized in that, The polynucleotide encoding the TAT-NbP1 fusion single-domain antibody of claim 3 or 4 is introduced into a host cell for expression, wherein the host cell is an Escherichia coli prokaryotic expression system.

9. Use of the NbP1 single-domain antibody of claim 1 or 2, or the TAT-NbP1 fusion single-domain antibody of claim 3 or 4, in the preparation of a product, wherein the use in the preparation of the product is any one of the following: (1) Preparation of reagents for detecting and / or diagnosing SARS-CoV-2-related diseases; (2) Preparation of reagents for recognizing and / or binding to the SARS-CoV-2 PLpro protein; (3) To prepare drugs for the prevention and / or treatment of SARS-CoV-2 virus infection; Preferably, the SARS-CoV-2-related disease is PLpro-induced colitis.

10. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition includes the NbP1 single-domain antibody of claim 1 or 2, or the TAT-NbP1 fusion single-domain antibody of claim 3 or 4, and a pharmaceutically acceptable carrier.

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