Use of icam3 gene / protein in inhibiting replication of novel coronavirus or preventing or treating covid-19

Abstract

This invention relates to the field of molecular biology, specifically to the use of the ICAM3 gene / protein in inhibiting the replication of the novel coronavirus or in preventing or treating COVID-19. This invention provides the use of the ICAM3 gene / protein in inhibiting the replication of the novel coronavirus SARS-CoV-2 or in preventing or treating COVID-19 (Coronavirus Disease 2019). Thus, overexpression of the ICAM3 gene in target cells can effectively inhibit the replication of SARS-CoV-2 in cells, thereby effectively preventing or treating COVID-19.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the use of the ICAM3 gene / protein in inhibiting the replication of the novel coronavirus or in preventing or treating COVID-19. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is a member of the family Coronaviridae, genus Betacoronavirus. It is a membrane-bound, single-stranded, positive-sense RNA virus with a helical structure. Its genome structure, starting from the 5' end, consists of open reading frames (ORFs) 1a and 1b, a spike protein (S), an envelope protein (E), a membrane protein (M), and a nucleocapsid protein (N). ORF1a and ORF1b encode enzymes related to viral transcription and replication. S, E, M, and N primarily encode structural proteins of the virus. Among these, the nucleocapsid protein is mainly responsible for RNA replication and is often used as a diagnostic tool for coronaviruses.

[0003] The novel coronavirus SARS-CoV-2 can cause respiratory infectious diseases in humans, leading to severe pneumonia accompanied by symptoms such as cough, fever, fatigue, sputum production, and / or shortness of breath, seriously threatening people's lives and health. However, after infection with the novel coronavirus, individuals exhibit significantly different disease outcomes. The varying degrees of disease states after infection with the novel coronavirus may be related to underlying factors such as an individual's gender, age, and / or underlying health conditions (e.g., respiratory / cardiovascular diseases and diabetes). For example, studies have shown that male individuals, older individuals, and individuals with poorer underlying health conditions are more likely to develop severe pneumonia after infection with the novel coronavirus.

[0004] However, in some severe cases, despite the absence of the aforementioned potential complications, patients still died from COVID-19 infection, suggesting that genetic factors play a crucial role in the clinical outcome of SARS-CoV-2 infection. Previous studies on Severe Acute Respiratory Syndrome (SARS) have shown that genetic diversity and differences are a key factor determining individual infection status and disease progression. For example, individuals carrying the G-2518A variant of the chemokine ligand 2 (CCL2) gene are able to recruit more monocytes and macrophages, making them more susceptible to SARS-CoV infection. Furthermore, significant differences in susceptibility to viral infection exist among different ethnic groups. For instance, HIV is more likely to infect African populations than European populations due to the polymorphism in the HIV receptor gene CCR5; European populations carry the CCR5-Δ32 variant, which inhibits HIV entry into human T lymphocytes. Therefore, identifying COVID-19 susceptibility genes and elucidating their functional mechanisms is of great significance for the prevention and treatment of COVID-19. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one object of this invention is to provide the use of the ICAM3 gene / protein in inhibiting the replication of the novel coronavirus SARS-CoV-2 or in preventing or treating COVID-19 (Coronavirus Disease 2019). Thus, overexpression of the ICAM3 gene in target cells can effectively inhibit the replication of SARS-CoV-2 in the cells, thereby effectively preventing or treating COVID-19.

[0006] Therefore, the first aspect of the present invention provides the use of the ICAM3 gene or ICAM3 protein in inhibiting SARS-CoV-2 replication.

[0007] The protein encoded by the ICAM3 gene belongs to the immunoglobulin superfamily within the adhesion molecule family. Members of this family all possess an Ig domain; the extracellular domain can interact with various receptors, while the intracellular domain can interact with the cytoskeleton or directly translocate into the nucleus to regulate signal transduction. Previous studies have reported the function of ICAM3 in HIV infection, replication, and the progression to severe illness. For example, the HIV Vpu protein can downregulate the expression of ICAM1 and ICAM3 on the surface of T cells, thereby evading NK cell killing; ICAM3 deficiency can lead to a significant increase in HIV replication in cells. Currently, the biological function and molecular mechanism of ICAM3 in COVID-19 have not been reported. The inventors analyzed data (data source: ERP127339) to suggest a potential link between ICAM3 and COVID-19. The results showed that ICAM3 expression was significantly lower in critical, severe, and moderate cases than in asymptomatic individuals, suggesting that the ICAM3 gene may play a role in inhibiting the development and progression of severe COVID-19. The ICAM3 gene may be a protective gene against COVID-19, suggesting that the ICAM3 gene and protein may have good application value in the clinical diagnosis and treatment of COVID-19.

[0008] Furthermore, through in vitro cell experiments, the inventors demonstrated that overexpression of the ICAM3 gene can significantly inhibit the expression level of the N gene mRNA of SARS-CoV-2 virus; knockdown of the ICAM3 gene can significantly promote the expression level of the N gene mNRA of SARS-CoV-2 virus, suggesting that the ICAM3 gene can inhibit the replication ability of the novel coronavirus.

[0009] A second aspect of the present invention provides a method for inhibiting SARS-CoV-2 replication in target cells. In some embodiments of the present invention, the method includes: overexpressing the ICAM3 gene in the target cells.

[0010] In some embodiments of the present invention, the method further includes:

[0011] The target cells were overexpressed by transfecting them with the ICAM3 gene expression vector.

[0012] A third aspect of this invention provides the use of the ICAM3 gene or ICAM3 protein in the preparation of a medicament. In some embodiments of this invention, the medicament is used to inhibit SARS-CoV-2 replication or to prevent or treat COVID-19.

[0013] A fourth aspect of this invention provides the use of the ICAM3 gene or ICAM3 protein in screening drugs. In some embodiments of this invention, the drug is used to inhibit SARS-CoV-2 replication or to prevent or treat COVID-19.

[0014] In some embodiments of the present invention, the intended use is achieved through the following steps:

[0015] (1) Contact the drug to be screened with mammalian cells infected with SARS-CoV-2;

[0016] (2) Detect the expression level of the ICAM3 gene and / or the content of the ICAM3 protein in the mammalian cells before and after the addition of the drug to be screened. An increase in the expression level of the ICAM3 gene and / or an increase in the content of the ICAM3 protein and a decrease in the viral titer of SARS-CoV-2 after the addition of the drug to be screened are indicators that the drug to be screened is the target drug.

[0017] The fifth aspect of this invention provides the use of the ICAM3 gene or ICAM3 protein in the preparation or screening of COVID-19 diagnostic reagents.

[0018] In some embodiments of the present invention, the use is achieved by at least one of the following methods:

[0019] (1) When used to prepare COVID-19 diagnostic reagents, a reagent that specifically detects the expression level of the ICAM3 gene is used to detect the expression of the ICAM3 gene in the sample to be tested, or a reagent containing the ICAM3 protein level in normal samples is used as a control, and the amount of ICAM3 protein in the sample to be tested is compared with the control.

[0020] (2) When used to screen COVID-19 diagnostic reagents, the ICAM3 gene or ICAM3 protein is used as a target, and the diagnostic reagents are screened based on the ICAM3 gene or ICAM3 protein of the sample to be tested.

[0021] A sixth aspect of this invention provides a COVID-19 diagnostic kit. In some embodiments of this invention, the diagnostic kit includes reagents capable of specifically detecting the ICAM3 gene or the content of ICAM3 protein.

[0022] In some embodiments of the present invention, the reagent capable of specifically detecting the content of the ICAM3 gene or ICAM3 protein is a primer for specifically amplifying the ICAM3 gene or an antibody specifically against the ICAM3 protein.

[0023] A seventh aspect of the present invention provides a pharmaceutical composition. In some embodiments of the present invention, the pharmaceutical composition comprises the ICAM3 protein and / or an ICAM3 gene expression vector.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0026] Figure 1 The relationship between ICAM3 expression levels and different clinical subtypes of COVID-19 patients was shown. The rank-sum test was used for calculation, and a p-value < 0.05 was considered statistically significant.

[0027] Figure 2 This study demonstrated the effect of ICAM3 expression on the expression of the SARS-CoV-2 nucleocapsid gene. Cells were collected at 24, 48, and 72 hours post-infection. (AB) qRT-PCR was used to detect the mRNA expression level of ICAM3 in control cells and A549-ACE2 or Huh7 cells overexpressing ICAM3. (CD) Western blot was used to detect the ICAM3 protein expression level in control cells and A549-ACE2 or Huh7 cells overexpressing ICAM3, assessing the overexpression effect of ICAM3. (EF) qRT-PCR was used to detect the mRNA expression level of the SARS-CoV-2 nucleocapsid gene in control cells and A549-ACE2 or Huh7 cells overexpressing ICAM3. (GH) qRT-PCR was used to detect the mRNA expression level of ICAM3 in control cells and A549-ACE2 or Huh7 cells with knocked-down ICAM3. (IJ) Western blot was used to detect the expression level of the SARS-CoV-2 nucleocapsid gene. Blot experiments were conducted to detect the expression level of ICAM3 protein in control and ICAM3-knockdown A549-ACE2 or Huh7 cells, assessing the knockdown effect of ICAM3 in cells. qRT-PCR was used to detect the mRNA expression level of the SARS-CoV-2 nucleocapsid gene in control and ICAM3-knockdown A549-ACE2 or Huh7 cells. Student's t-test was used for calculation; *** represents P < 0.001, and P < 0.05 was considered statistically significant.

[0028] Figure 3This study demonstrated the effect of ICAM3 expression on the titer of SARS-CoV-2 in cells. Cells were collected at 24, 48, and 72 hours post-infection. (AB) PCR was performed using TCID45. 50 The experiment detected the SARS-CoV-2 titer in control and ICAM3-overexpressing A549-ACE2(A) and Huh7(B) cells to assess viral replication capacity; (CD) via TCID 50 The experiment measured the SARS-CoV-2 titer in control and knockdown ICAM3-mediated A549-ACE2(C) and Huh7(D) cells to assess viral replication capacity. Student's t-test was used for calculation; ** represents P < 0.01, *** represents P < 0.001, and P < 0.05 was considered statistically significant.

[0029] Figure 4This study demonstrated the effect of ICAM3 expression on the expression of interferons and pro-inflammatory factors. Cells used in the experiment were collected 48 hours after SARS-CoV-2 infection. (A) Western blot analysis was used to detect the ICAM3 protein expression level in control and ICAM3-overexpressing A549-ACE2 cells to assess the overexpression effect of ICAM3 in cells. (B) qRT-PCR was used to detect the mRNA expression levels of type I interferon genes (IFNA1 and IFNB1) and type III interferon genes (IFNL1 and IFNL2 / 3) in control and ICAM3-overexpressing A549-ACE2 cells. (C) ELISA was used to detect the expression levels of type I interferon (IFNα and IFNβ) and type III interferon gene (IFNλ) in the supernatant of control and ICAM3-overexpressing A549-ACE2 cells. (D) Western blot analysis was used to detect the expression levels of type I interferon (IFNα and IFNβ) and type III interferon gene (IFNλ) in the supernatant of control and ICAM3-overexpressing A549-ACE2 cells. (E) Using blot experiments, the expression levels of ICAM3 protein in the control group and Huh7 cells overexpressing ICAM3 were detected to evaluate the overexpression effect of ICAM3 in cells; (G) Using qRT-PCR experiments, the mRNA expression levels of type I interferon genes (IFNA1 and IFNB1) and type III interferon genes (IFNL1 and IFNL2 / 3) in the control group and Huh7 cells overexpressing ICAM3 were detected; (F) Using ELISA experiments, the expression levels of type I interferon (IFNα and IFNβ) and type III interferon gene (IFNλ) in the supernatant of the control group and Huh7 cells overexpressing ICAM3 were detected; (G) Using Western blotting... (H) Blot analysis was used to detect the expression level of ICAM3 protein in control and ICAM3-knockdown A549-ACE2 cells to evaluate the overexpression effect of ICAM3 in cells; (H) qRT-PCR was used to detect the mRNA expression levels of type I interferon genes (IFNA1 and IFNB1) and type III interferon genes (IFNL1 and IFNL2 / 3) in control and ICAM3-knockdown A549-ACE2 cells; (I) ELISA was used to detect the expression levels of type I interferon (IFNα and IFNβ) and type III interferon gene (IFNλ) in the supernatant of control and ICAM3-knockdown A549-ACE2 cells; (J) Western blotting was used to detect the expression levels of type I interferon (IFNα and IFNβ) and type III interferon gene (IFNλ) in the supernatant of control and ICAM3-knockdown A549-ACE2 cells; (k) The expression level of ICAM3 protein in the control group and ICAM3 knockdown Huh7 cells was detected by blot experiment to evaluate the overexpression effect of ICAM3 in cells; (k) The mRNA expression level of type I interferon genes (IFNA1 and IFNB1) and type III interferon genes (IFNL1 and IFNL2 / 3) in the control group and ICAM3 knockdown Huh7 cells was detected by qRT-PCR experiment.(L) The expression levels of type I interferon (IFNα and IFNβ) and type III interferon gene (IFNλ) in the supernatant of Huh7 cells with knocked-down ICAM3 were detected by ELISA. Student's t-test was used for calculation; * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. A p-value < 0.05 was considered statistically significant. Detailed Implementation

[0030] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0035] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] This application is based on the inventor's discoveries and understanding of the following facts and problems:

[0037] Genome-wide association studies (GWAS) based on whole-genome SNP microarrays and high-throughput sequencing have identified multiple gene regions significantly associated with severe COVID-19 patients, suggesting that genetic factors play an important role in COVID-19 infection and disease progression. For example, a GWAS study based on populations from countries A and B identified two gene regions (3p21.31 and 9q34.2) significantly associated with severe COVID-19 patients; a study by the Severe Covid-19 GWAS Group showed that SNPs at chromosomal locus 3p21.21, including rs11385942 (the most significant locus in intron 5 of LZTFL1), are associated with respiratory failure in COVID-19 patients. Other GWAS studies have identified more loci associated with severe COVID-19 patients, including rs74956615 (the 3' untranslated exon region of RAVER1 at chromosome 19p13.2), rs2109069 (intron 3 of DPP9 at chromosome 19p13.3), rs6489867 (intron 5 of OAS1 at chromosome 12q24.13), rs10735079 (intron 2 of OAS3 at chromosome 12q24.13), and rs2236757 (intron 6 of IFNAR2 at chromosome 21q22.1). The inventors' research group previously conducted a genome-wide association study of severe COVID-19 patients based on SNP chips. By comparing the genetic differences between mild and severe cases, they identified two susceptibility regions (11q14.2 and 11q23.3) for the first time in the Chinese population. Meanwhile, researchers conducting GWAS studies on the Chinese population found that the 3p21.31 region, which is susceptible to severe COVID-19 in the European population, is also a susceptible region in the Chinese population. However, due to limitations in population size and the fact that the study design used mild cases as controls and severe cases as cases, the statistical power of published GWAS studies on COVID-19 in the Chinese population is relatively insufficient. Furthermore, the functional loci and genes in the identified susceptible regions have not been functionally validated or their mechanisms elucidated. Therefore, existing studies are insufficient to fully elucidate the genetic mechanisms of severe COVID-19 patients in the Chinese population. Based on this, the inventors further conducted a larger-scale genome-wide association study of severe COVID-19 patients in the Chinese population, which included 1,687 COVID-19 patients (908 severe cases and 765 non-severe hospitalized patients) and 50,082 individuals with unknown infection status. Through systematic bioinformatics analysis and population validation, a series of susceptible loci that may be associated with severe COVID-19 patients were finally identified. Among them, the rs2304240 locus located in the 19p13.2 gene region was significantly associated with the occurrence of severe COVID-19 patients.Specifically, using expression profile and genotypic data, the inventors discovered that the protective allele of rs2304240 was significantly associated with high expression of the ICAM3 gene in this region. Through cell biology experiments (overexpression of the ICAM3 gene), the inventors demonstrated that ICAM3 can inhibit the expression of the novel coronavirus N gene in cells. Furthermore, the inventors utilized TCID... 50 The experiments measured viral titers and found that ICAM3 significantly reduced viral titers in cells. Furthermore, the inventors used qRT-PCR to discover that ICAM3 significantly promoted the expression of interferon genes in cells. In summary, these results suggest that the ICAM3 gene plays a role in inhibiting the replication of the novel coronavirus.

[0038] According to a specific embodiment of the present invention, the present invention provides the use of the ICAM3 gene or ICAM3 protein in inhibiting SARS-CoV-2 replication.

[0039] Through in vitro cell experiments, the inventors demonstrated that overexpression of the ICAM3 gene can significantly inhibit the expression level of the N gene mRNA of SARS-CoV-2 virus; knockdown of the ICAM3 gene can significantly promote the expression level of the N gene mNRA of SARS-CoV-2 virus, suggesting that the ICAM3 gene can inhibit the replication ability of the novel coronavirus.

[0040] According to a specific embodiment of the present invention, the present invention provides a method for inhibiting the replication of SARS-CoV-2 in target cells, the method comprising: overexpressing the ICAM3 gene in the target cells.

[0041] Overexpression of the ICAM3 gene or protein in target cells can effectively inhibit the replication of the novel coronavirus in cells, thereby effectively preventing or treating COVID-19.

[0042] According to a specific embodiment of the present invention, the method further includes:

[0043] The target cells were overexpressed by transfecting them with the ICAM3 gene expression vector.

[0044] Gene expression vectors, also known as gene splicing and DNA recombination technologies, are based on molecular genetics. Using molecular biology and microbiology methods, they construct hybrid DNA molecules from genes of different sources in vitro, which are then introduced into living cells to alter the original genetic characteristics of organisms, obtain new varieties, and produce new products. The purpose of their construction is to ensure that the target gene can stably exist in recipient cells and be inherited by the next generation, while simultaneously enabling the target gene to be expressed and function. In this document, "ICAM3 gene expression vector" refers to a vector containing a CDS of the ICAM3 gene that can perform gene transcription, translation, and expression of the ICAM3 protein. It should be noted that there are no particular restrictions on the type of vector or the strength of the promoter on the vector; all expression vectors capable of expressing the ICAM3 gene are covered within the scope of this invention.

[0045] The target cell can be any type of cell; preferably, the target cell is a mammalian cell.

[0046] According to a specific embodiment of the present invention, the present invention provides the use of the ICAM3 gene or ICAM3 protein in the preparation of a drug for inhibiting SARS-CoV-2 replication or for preventing or treating COVID-19.

[0047] By transfecting target cells with the ICAM3 gene expression vector, the target cells can be overexpressed with the ICAM3 gene. In this way, it can be used to inhibit the replication of SARS-CoV-2 in the target cells, and it can also be used to prevent or treat COVID-19.

[0048] ICAM3 gene expression vectors or ICAM3 proteins can be used as drugs to inhibit SARS-CoV-2 replication or to prevent or treat COVID-19.

[0049] According to a specific embodiment of the present invention, the present invention provides the use of the ICAM3 gene or ICAM3 protein in screening drugs for inhibiting SARS-CoV-2 replication or preventing or treating COVID-19.

[0050] According to a specific embodiment of the present invention, drug screening is achieved through the following steps:

[0051] (1) Contact the drug to be screened with mammalian cells infected with SARS-CoV-2;

[0052] (2) Detect the expression level of the ICAM3 gene and / or the content of the ICAM3 protein in the mammalian cells before and after the addition of the drug to be screened. An increase in the expression level of the ICAM3 gene and / or an increase in the content of the ICAM3 protein and a decrease in the viral titer of SARS-CoV-2 after the addition of the drug to be screened are indicators that the drug to be screened is the target drug.

[0053] When mammalian cells are exposed to the drug to be screened, an increase in the expression level of the ICAM3 gene and / or an increase in the content of ICAM3 protein, coupled with a decrease in the viral titer of SARS-CoV-2, indicates that the drug to be screened is effective in inhibiting SARS-CoV-2 replication or in preventing or treating COVID-19.

[0054] According to a specific embodiment of the present invention, the present invention provides the use of the ICAM3 gene or ICAM3 protein in the preparation or screening of COVID-19 diagnostic reagents.

[0055] According to a specific embodiment of the present invention, when used to prepare a COVID-19 diagnostic reagent, a reagent specifically for detecting the expression level of the ICAM3 gene is used to detect the expression of the ICAM3 gene in the sample to be tested, or a reagent containing the ICAM3 protein level in a normal sample is used as a control, and the amount of ICAM3 protein in the sample to be tested is compared with that in the control.

[0056] According to a specific embodiment of the present invention, when used for screening COVID-19 diagnostic reagents, the ICAM3 gene or ICAM3 protein is used as a target, and the diagnostic reagents are screened based on the ICAM3 gene or ICAM3 protein of the sample to be tested.

[0057] According to a specific embodiment of the present invention, the present invention provides a COVID-19 diagnostic kit, the diagnostic kit comprising reagents capable of specifically detecting the content of the ICAM3 gene or ICAM3 protein.

[0058] According to a specific embodiment of the present invention, the reagent capable of specifically detecting the content of the ICAM3 gene or ICAM3 protein is a primer for specifically amplifying the ICAM3 gene or an antibody specifically against the ICAM3 protein.

[0059] It should be noted that the COVID-19 diagnostic kit provided by this invention, in addition to primers for specifically amplifying the ICAM3 gene or antibodies against the ICAM3 protein, may also contain other auxiliary diagnostic reagents. These kits, with or without other diagnostic reagents, are all covered within the scope of protection of this invention. For example, the COVID-19 diagnostic kit may also contain metal ions, ddH2O, staining agents, enzymes, etc. The antibodies against the ICAM3 protein contained in the COVID-19 diagnostic kit can be loaded onto a vector, such as onto a test card, making detection simpler.

[0060] According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition comprising ICAM3 protein and / or an ICAM3 gene expression vector.

[0061] Interferon is a pleiotropic cytokine with antiviral and immunomodulatory properties, and is a key coordinator of the immune response. Interferon specifically binds to cell surface receptors, inducing the synthesis of antiviral proteins to exert its biological activity. Based on interferon structural characteristics, receptor binding, and biological activity, human interferon is classified into three types: type I, type II, and type III interferon. Type I and type III interferon are considered classic antiviral interferons, while type II interferon is the main immunomodulatory interferon. Through in vitro experiments, the inventors discovered that overexpression of the ICAM3 gene significantly promotes the expression of type I interferon (IFNA1 and IFNB1) and type III interferon (IFNL1 and IFNL2 / 3) in cells; while knockdown of the ICAM3 gene significantly inhibits the expression of type I interferon (IFNA1 and IFNB1) and type III interferon (IFNL1 and IFNL2 / 3) in cells. This suggests that the ICAM3 gene may inhibit SARS-CoV-2 replication by promoting interferon production, thereby playing a role in suppressing the development and progression of COVID-19.

[0062] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0063] Example 1: Relationship between ICAM3 expression levels and different clinical subtypes of COVID-19 patients

[0064] To explore the function of the ICAM3 gene in the development and progression of severe COVID-19, we used the public dataset of the COVID-19 cohort (ERP127339) and analyzed and compared the expression levels of ICAM3 in different individuals (64 asymptomatic infected persons, 64 patients with mild symptoms, 34 patients with severe symptoms, and 16 patients with critical symptoms) using the rank-sum test, and studied the relationship between the expression level of ICAM3 and different clinical subtypes of COVID-19 patients.

[0065] Figure 1 This study presents data from the public dataset ERP127339, analyzing the differences in ICAM3 gene expression levels among different clinical subtypes of COVID-19 patients (asymptomatic, mild, severe, and critical). The rank-sum test was used for calculation, and a p-value < 0.05 was considered statistically significant. The results showed that ICAM3 gene expression was significantly lower in critical, severe, and moderate cases than in asymptomatic patients, suggesting that the ICAM3 gene may play a role in inhibiting the development and progression of COVID-19.

[0066] Example 2: ICAM3 significantly inhibited the mRNA expression level of the N gene of the novel coronavirus.

[0067] To investigate the function of ICAM3 in the process of SARS-CoV-2 infection, the inventors constructed cell lines that stably express high levels of ICAM3 and transiently knock down ICAM3 in the A549-ACE2 and Huh7 cell lines.

[0068] First, a cell line stably expressing ICAM3 was constructed. The CDS sequence of ICAM3 was amplified by RT-PCR and constructed into a lentiviral vector with a Flag tag (pLV-Flag). After packaging the virus, it was used to infect human A549-ACE2 and Huh7 cells. After G418 selection, the expression of Flag-ICAM3 was identified by qRT-PCR and Western blot experiments, and a cell line stably expressing ICAM3 was obtained. Figure 2 Tables A and B show the expression levels of ICAM3 in A549-ACE2 and Huh7 cell lines as detected by qRT-PCR. Tables C and D show the ICAM3 protein expression levels in control and ICAM3-overexpressing A549-ACE2 or Huh7 cells detected by Western blot experiments, evaluating the overexpression effect of ICAM3 in cells. The results indicate that A549-ACE2 and Huh7 cells with increased ICAM3 mRNA and protein expression levels were obtained.

[0069] Then, using RNAi technology, the susceptibility gene ICAM3 was knocked down in A549-ACE2 and Huh7 cells. Based on the design principles of small interfering RNA (siRNA) target gene sites, multiple siRNA sequences specifically targeting ICAM3 were designed and synthesized, and transfected into A549-ACE2 and Huh7 cells. The knockdown effect of ICAM3 was identified using qRT-PCR and Western blot experiments. The qRT-PCR and Western blot results showed good ICAM3 overexpression or knockdown effects, ensuring the reliability of subsequent experiments. Figure 2 G and H in the figure show the expression level of ICAM3 in A549-ACE2 and Huh7 cell lines as detected by qRT-PCR. I and J show the expression level of ICAM3 protein in control and knocked-down A549-ACE2 or Huh7 cells by Western blot experiment to evaluate the knockdown effect of ICAM3 in cells. The results show that A549-ACE2 and Huh7 cells with reduced ICAM3 mRNA expression level and protein expression level were obtained.

[0070] In A549-ACE2 or Huh7 cells that overexpressed or knocked down the ICAM3 gene, SARS-CoV-2 was transiently co-transfected (MOI=1). Cells were collected at different time points (24, 48, and 72 hours post-infection), and total cellular RNA was extracted. After reverse transcription, the expression level of the viral N gene was detected using qRT-PCR to indicate intracellular viral replication capacity. Unpaired t-tests were used to analyze differences between groups at each time point to detect the effect of the ICAM3 gene on viral replication. Results are as follows: Figure 2 As shown in the figures, Figures E and F demonstrate that overexpression of the ICAM3 gene significantly inhibits the expression level of the viral N gene mRNA; Figures K and L demonstrate that knockdown of the ICAM3 gene significantly promotes the expression level of the viral N gene mNRA. These results suggest that the ICAM3 gene can inhibit the replication ability of SARS-CoV-2.

[0071] qRT-PCR experimental steps:

[0072] Total RNA was extracted from cells according to the instructions of the RNA extraction kit (CW0599S, Kangwei Century Biotechnology Co., Ltd.). RNA reverse transcription was performed according to the instructions of the reverse transcription kit (RR047A, Baoriyi Biotechnology (Beijing) Co., Ltd.). Real-time quantitative PCR experiments for related genes were conducted according to KAPA. The FAST real-time PCR kit (SFABIKB, Sigma-Aldrich) was used according to the manufacturer's instructions, with all samples in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the mRNA quantification results were normalized using the ΔΔCt method. qRT-PCR primers for the gene were designed using Primer BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer blast / ) on the NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ) website, following the principles of specificity and cross-gene introns. Primers were synthesized by BGI Genomics Co., Ltd., and primer information is shown in Table 1 below.

[0073] Table 1

[0074]

[0075] Western blot experimental procedures:

[0076] Cells from both the control and experimental groups were digested and collected using 0.25% trypsin. The cells were centrifuged at 3000 rpm for 2 min at room temperature, washed once with 1x PBS, and then mixed thoroughly with cell lysis buffer containing protease inhibitors. The cells were then placed on ice for 30 min to lyse. After centrifugation at 12000 rpm for 10 min, the supernatant was transferred to a new centrifuge tube, and an equal volume of 6× protein loading buffer was added. The tube was boiled for 10 min to obtain the total cellular protein sample. An appropriate concentration of SDS-PAGE gel was prepared, followed by loading, electrophoresis, and membrane transfer. After transfer, the cellulose acetate membrane containing the target protein was placed in blocking buffer (prepared with 1×TBST containing 5% skim milk) and blocked at room temperature for 1-2 h. The membrane was washed three times with 1×TBST for 5 min each time, and then incubated with primary antibody overnight at 4°C. The cellulose acetate membrane was then removed, washed three times with 1×TBST for 5 min each time, incubated with secondary antibody at room temperature for 2 h, washed three more times with 1×TBST for 5 min each time, and finally developed.

[0077] Example 3: ICAM3 significantly reduced viral titers in cells.

[0078] Viral titer, or viral virulence, characterizes the amount of virus produced within a certain timeframe after viral infection of cells, reflecting the virus's replication capacity. In Huh-7 or A549-ACE2 cells overexpressing or knocking down the ICAM3 gene, transient co-transfection with SARS-CoV-2 (MOI=1) was performed. At 24, 48, and 72 hours post-infection, the inventors used TCID... 50The viral titer in the cells was detected in the experiment. When the cells reached a density of 80%, they were harvested and seeded into 96-well plates at a density of 15,000 cells per well (6 × 8 wells). Each well contained 100 μl of culture medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture). Virus dilution was then added, with the virus solution serially diluted 10-fold using the culture medium. 100 μl of virus dilution was added to each well for every 10-fold concentration, for a total of 8 gradients. Eight wells served as a negative control group, containing 100 μl of culture medium. At 24, 48, and 72 hours post-infection, the number of wells showing cytopathic effects and the corresponding viral concentrations were observed. The viral titer was calculated using the Reed-Muench method, and unpaired t-tests were used to analyze the differences between groups at each time point.

[0079] The results are as follows Figure 3 As shown, overexpression of the ICAM3 gene significantly reduces viral titer in cells, while knockdown of the ICAM3 gene significantly increases viral titer in cells. This further demonstrates that the ICAM3 gene can inhibit the replication of the SARS-CoV-2 virus.

[0080] Example 4: ICAM3 significantly promotes interferon expression

[0081] To explore the mechanism by which ICAM3 inhibits SARS-CoV-2 replication, the inventors examined the expression of type I and type III interferon genes in virus-infected cells. In Huh-7 or A549-ACE2 cells overexpressing or knocking down the ICAM3 gene, SARS-CoV-2 was transiently co-transfected (MOI=1). 72 hours post-infection, cell supernatants were collected, and total RNA was extracted. For the cell supernatant, ELISA was used to detect interferon expression levels; for the total RNA, qRT-PCR was used to detect interferon gene expression levels. Unpaired t-tests were used to statistically analyze differences between groups.

[0082] The results are as follows Figure 4 The results show that overexpression of the ICAM3 gene significantly promotes the expression of type I interferon (IFNA1 and IFNB1) and type III interferon (IFNL1 and IFNL2 / 3) in cells; while knockdown of the ICAM3 gene significantly inhibits the expression of type I interferon (IFNA1 and IFNB1) and type III interferon (IFNL1 and IFNL2 / 3) in cells. This suggests that the ICAM3 gene may inhibit SARS-CoV-2 replication by promoting interferon production, thereby playing a role in suppressing the development and progression of COVID-19.

[0083] ELISA experimental steps:

[0084] The expression levels of interferons (IFNα, IFNβ, and IFNλ) in cell supernatants were detected using ELISA kits: IFNα (DFNAS0, R&D Systems), IFNβ (DIFNB0, R&D Systems), and IFNλ (DY1598B, R&D Systems) according to the manufacturer's instructions. The procedure is as follows:

[0085] ① Add the cell supernatant sample diluent and protein standard to the reaction wells in sequence, incubate, and then wash.

[0086] ② Add biotinylated detection antibody to each well, incubate, and then wash;

[0087] ③ Add avidin HRP to each well, incubate, and then wash;

[0088] ④ Add 3,3',5,5'-tetramethylbenzidine (TMB) to each well. It produces a blue product under the catalysis of HRP, which turns yellow after adding acidic stop solution.

[0089] ⑤ Using Sunrise TM The ELISA reader (Tecan) reads the reaction signal at 450 nm.

[0090] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0091] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of the ICAM3 gene or ICAM3 protein in inhibiting SARS-CoV-2 replication.

2. A method of inhibiting replication of SARS-CoV-2 in a cell of interest, characterized in that, The target cells were made to overexpress the ICAM3 gene.

3. The method of claim 2, wherein, The method further includes: The target cells were overexpressed by transfecting them with the ICAM3 gene expression vector.

4. Use of the ICAM3 gene or ICAM3 protein for the preparation of a medicament, characterized in that, The drug is used to inhibit SARS-CoV-2 replication or to prevent or treat COVID-19.

5. Use of the ICAM3 gene or ICAM3 protein for screening of drugs, characterized in that, The drug is used to inhibit SARS-CoV-2 replication or to prevent or treat COVID-19.

6. The use according to claim 5, characterized in that, The purpose is achieved through the following steps: (1) Contact the drug to be screened with mammalian cells infected with SARS-CoV-2; (2) Detect the expression level of the ICAM3 gene and / or the content of the ICAM3 protein in the mammalian cells before and after the addition of the drug to be screened. An increase in the expression level of the ICAM3 gene and / or an increase in the content of the ICAM3 protein and a decrease in the viral titer of SARS-CoV-2 after the addition of the drug to be screened are indicators that the drug to be screened is the target drug.

7. Use of the ICAM3 gene or ICAM3 protein in the preparation or screening of COVID-19 diagnostic reagents.

8. The use according to claim 7, characterized in that, The intended use is achieved through at least one of the following methods: (1) When used to prepare COVID-19 diagnostic reagents, a reagent that specifically detects the expression level of the ICAM3 gene is used to detect the expression of the ICAM3 gene in the sample to be tested, or a reagent containing the ICAM3 protein level in normal samples is used as a control, and the amount of ICAM3 protein in the sample to be tested is compared with the control. (2) When used to screen COVID-19 diagnostic reagents, the ICAM3 gene or ICAM3 protein is used as a target, and the diagnostic reagents are screened based on the ICAM3 gene or ICAM3 protein of the sample to be tested.

9. A COVID-19 diagnostic kit, characterized in that, This includes reagents capable of specifically detecting the content of the ICAM3 gene or ICAM3 protein. Optionally, the reagent capable of specifically detecting the content of the ICAM3 gene or ICAM3 protein is a primer that specifically amplifies the ICAM3 gene or an antibody that specifically targets the ICAM3 protein.

10. A pharmaceutical composition, characterized in that, It contains ICAM3 protein and / or ICAM3 gene expression vector.

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