Application of Siglec14 gene and expression substance in preparation of new coronavirus gene therapeutic drug

Abstract

The invention discloses application of a Siglec14 gene and an expression substance in preparation of a new coronavirus gene therapeutic drug, the expression substance comprises Siglec14 natural protein and Siglecl4 recombinant protein, and the Siglec14 gene and the expression substance inhibit expression and replication of a novel coronavirus by inducing a natural immune antiviral factor of an organism. A Siglec14 overexpression cell line is constructed, after overexpression of Siglec14 is verified through qRT-PCR, Westernblot and other methods, expression of an antiviral factor mRNA and protein is promoted, an innate immune pathway of a body is activated, and therefore the SARS-CoV-2 resisting effect is achieved, it is revealed that Siglec14 plays an important role in the new coronavirus infection resisting process, and new coronavirus infection can be effectively inhibited by enhancing the expression or activity of Siglec14 protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of research and development of novel coronavirus gene therapy drugs, and specifically relates to the application of Siglec14 gene and expression products in the preparation of novel coronavirus gene therapy drugs. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is the causative agent of the novel coronavirus infection (COVID-19).

[0003] The human body is generally susceptible to SARS-CoV-2. After exposure to SARS-CoV-2, the body's mechanisms for resisting and promoting viral replication and transmission are very complex. Different individuals have different resistance to SARS-CoV-2 and different rates of disease progression, which are closely related to factors such as the body's genetics, immunity, and health status. With the emergence of SARS-CoV-2 immune escape variants, the body's immune system is being challenged, especially the emergence of different mutant subtypes of the Omicron variant (XBB, BA.5.2, BF.7, etc.), which have caused the vaccine's protective effect to continue to weaken as the vaccination time increases. Whether the new coronavirus will continue to mutate is still a difficult problem facing this field.

[0004] Chinese patent CN117883501A (publication date: April 16, 2024) discloses the use of Qingyan drops in the preparation of anti-new coronavirus drugs, which relates to the field of traditional Chinese medicine technology. The Qingyan drops provided by the present invention, or a traditional Chinese medicine composition containing effective traditional Chinese medicine components of Qingyan drops or its preparation in the preparation of anti-new coronavirus drugs, the preparation of drugs for preventing, alleviating or treating new coronavirus infection, the preparation of new coronavirus protein inhibitors, the inhibition of new coronavirus for non-diagnostic and therapeutic purposes, or the inhibition of new coronavirus proteins for non-diagnostic and therapeutic purposes, alleviates the problem of lack of effective anti-new coronavirus means and drugs in the prior art.

[0005] Chinese patent CN117815242A (publication date: April 5, 2024) discloses the use of compound ATV014 or its pharmaceutically acceptable salt or its crystalline hydrate or its solvate in the preparation of products for preventing, alleviating or treating infection by the novel coronavirus Omicron strain, or the replication or reproduction of its homologous variant virus and the resulting cytopathic effects. It relates to the fields of pharmaceutical technology and viral infection disease technology.

[0006] Given that there are many types of mutant strains of the new coronavirus and their possible enhanced toxicity after mutation, research and development of gene therapy drugs for the new coronavirus based on antiviral targets is of great significance for the prevention and treatment of the new coronavirus.

[0007] Sialic Acid Binding Ig Like Lectins (Siglecs) are a family of cell surface transmembrane receptors that contain amino-terminal sialic acid binding sites and are widely expressed on immune cells. Siglecs can recognize sialic acid glycans of host cells, enabling host immune cells to distinguish between self and non-self. Siglec14 belongs to CD33-related Siglecs (CD33rSiglecs). Studies have shown that siglec14 can bind to the immune receptor tyrosine activation sequence (ITAM) through DAP12, induce immune activation signaling events, regulate innate immunity and adaptive immunity, and thus play a role in resisting foreign pathogens. Siglec14 can activate the NLRP3 inflammasome in macrophages and activate the innate immune pathway. In viral infections, especially in the infection of the new coronavirus, the role of Siglec14 has not been fully understood.

[0008] In summary, the present invention constructs a Siglec14 overexpression cell line, and analyzes the anti-new coronavirus infection effect of Siglec14 after overexpression by qRT-PCR, Western blot and other methods, in order to find new drug targets for the treatment of new coronavirus. Summary of the invention

[0009] The present invention provides the use of Siglec14 genes and expressions in the preparation of novel coronavirus gene therapy drugs.

[0010] The expressed products include Siglec14 natural protein and Siglecl4 recombinant protein.

[0011] The expression of the Siglec14 gene can inhibit the replication of the new coronavirus.

[0012] The Siglec14 gene and its expression products inhibit the expression and replication of the new coronavirus by inducing the body's natural immune antiviral factors.

[0013] The innate immune antiviral factors include one or more antiviral factors among SAMD9L, HERC5, ISG15, FIT1, IFIT2, RSAD2, and MX1.

[0014] Compared with the prior art, the advantages and effects of the present invention are as follows:

[0015] 1. The present invention studies the host factor Siglec14 in the human body and finds that Siglec14 is a target that can be used to treat novel coronavirus infection. It is found that the infection of novel coronavirus is significantly inhibited in the Siglec14 overexpressing cell line, which can activate the body's natural immune system and exert an antiviral effect.

[0016] 2. The present invention reveals that Siglec14 plays an important role in the process of resisting novel coronavirus infection, and enhancing the expression or activity of Siglec14 protein can effectively inhibit the infection of novel coronavirus.

[0017] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings as follows.

[0018] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0020] in:

[0021] Figure 1 Schematic diagram of fluorescence constructed by Siglec14 overexpression cell line in the present invention

[0022] Figure 1 A is the transfection effect of THP-1 cells infected with lentivirus under inverted fluorescence microscope

[0023] Figure 1 B is the transfection effect of THP-1 cells infected with lentivirus under inverted fluorescence microscope

[0024] Figure 2 Figure 1 is the expression result of Siglec14 protein in the Siglec14 overexpressing cell line of the present invention

[0025] Figure 3 Schematic diagram of the ability of Siglec14 to inhibit the replication of the novel coronavirus in the present invention

[0026] Figure 3 A is a statistical chart of the detection results of the novel coronavirus N gene in Siglec14 overexpressing cells and negative control cells by fluorescence quantitative PCR

[0027] Figure 3 B is a statistical graph of the detection results of the novel coronavirus ORF1ab gene in Siglec14-overexpressing cells and negative control cells by fluorescence quantitative PCR

[0028] Figure 4 The results of transcriptome sequencing analysis of total RNA in the cells of the Siglec14 overexpression group and the negative control group infected by SARS-CoV-2 in the present invention

[0029] Figure 4 A is a statistical diagram of the expression of related factors in the viral replication negative regulatory factor pathway

[0030] Figure 4 B is the ES score statistics of the viral replication negative regulator pathway

[0031] Figure 4 C is the statistical diagram of the difference in gene expression between the Siglec14 overexpression group and the negative control group

[0032] Figure 5 This is a comparative statistical diagram of the promoting effect of Siglec14 on antiviral factors after infection with SARS-CoV-2 in the present invention

[0033] Figure 5 A is the statistical graph of SAMD9L gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0034] Figure 5 B is the statistical graph of HERC5 gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0035] Figure 5 C is the statistical graph of ISG15 gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0036] Figure 5 D is the statistical graph of IFIT1 gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0037] Figure 5 E is the statistical graph of IFIT2 gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0038] Figure 5 F is the statistical graph of RSAD2 gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0039] Figure 5G is the statistical graph of MX1 gene expression in Siglec14 overexpressing cells and negative control cells detected by qRT-PCR

[0040] Figure 6 This is a statistical comparison of the results of the promotion of Siglec14 against antiviral factors after infection with SARS-CoV-2 in the present invention

[0041] Figure 6 A is the comparison of SAMD9L, IFIT1, GAPDH, and HERC5 protein expression levels in Siglec14 overexpressing cells and negative control cells detected by Wesrern Blot when MOI = 0.01

[0042] Figure 6 B is the comparison of SAMD9L, GAPDH, RSAD2 and HERC5 protein expression in Siglec14 overexpressing cells and negative control cells detected by Wesrern Blot when MOI=0.1

[0043] Figure 6 C is the comparison of IFIT1 and β-Actin protein expression levels in Siglec14 overexpressing cells and negative control cells detected by Wesrern Blot when MOI = 0.1

[0044] Figure 6 D is the comparison of the expression of SARS-CoV-2 N protein and GAPDH protein in Siglec14-overexpressing cells and negative control cells detected by Wesrern Blot when MOI=0.1 DETAILED DESCRIPTION

[0045] To make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all of the embodiments. In the following description, specific details such as specific configuration and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures is omitted in the embodiment.

[0046] It should be understood that the references to "one embodiment" or "this embodiment" throughout the specification mean that the specific features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present application. Therefore, the references to "one embodiment" or "this embodiment" appearing throughout the specification do not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0047] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity, and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0048] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the previous and next associated objects are in an "or" relationship.

[0049] The term "at least one" in this article is merely a description of the association relationship of associated objects, indicating that there may be three relationships. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0050] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusions.

[0051] The wild-type THP-1 cell line used in the experiment in the embodiment was preserved in the laboratory, and the novel coronavirus was the Delta strain, which was obtained by isolation and culture in the laboratory.

[0052] Example 1

[0053] This example is about the construction of Siglec14 overexpression cell line and the verification of the effect of Siglec14 expression at the protein level.

[0054] The wild-type THP-1 cell line used in the experiment was preserved in the laboratory, and the new coronavirus was the Delta strain, which was obtained by isolation and culture in the laboratory.

[0055] (1) Prepare wild-type THP-1 cells required for the experiment. Collect the cells and centrifuge at 900 rpm for 5 minutes at room temperature. Resuspend the cells in 1 mL of 1640 complete culture medium. Take an appropriate amount of the cell suspension, stain it with trypan blue, count it under a microscope, and inoculate it in a 12-well plate, with 300,000 cells in each well.

[0056] (2) According to the instructions, the required virus volume was calculated according to different infection multiplicities, virus volume = (MOI * number of cells) / virus titer. HiTranGA / P infection enhancement solution was added to each well, and 1640 complete medium without double antibody was added to each well to infect the cells. A negative control well was set up and placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours to observe the cell status.

[0057] (3) Replenish fluid during the process to maintain cell activity.

[0058] (4) After 48 hours, the cell infection efficiency (green fluorescence) was observed under a fluorescence microscope. The infection group with an infection efficiency higher than 80% and good cell growth status could be used as the subsequent experimental cell line. The cell lines were screened using 1640 complete medium containing puromycin (2 μg / mL) until the infection efficiency reached more than 90%.

[0059] Please refer to Figure 1 , Figure 1 A is the transfection effect picture of THP-1 cells after lentivirus infection under inverted fluorescence microscope. Figure 1 B is a picture of the transfection effect under a fluorescence inverted microscope after lentivirus infection of THP-1 cells; the results showed that cells successfully infected with the lentivirus exhibited green fluorescence under a fluorescence microscope, and the results showed that the fluorescence of the Siglec14 overexpression group was brighter, and the cell infection efficiency reached more than 90%, indicating that the cell infection was successful.

[0060] (5) Siglec14 recombinant protein was added to wild-type THP-1 cells, cultured for 48 hours, and the cell protein was extracted for detection.

[0061] Please refer to Figure 2 , are the Siglec14 protein expression results of the Siglec14 overexpressing cell line in the present invention. The results show that, after SDS PAGE electrophoresis, protein level verification shows that not only overexpression of Siglec14 at the gene level, or addition of Siglec14 recombinant protein, can effectively improve the expression of Siglec14.

[0062] Example 2

[0063] This example verifies the effect of Siglec14 against the novel coronavirus and its ability to inhibit the replication of the novel coronavirus.

[0064] (1) Siglec14-overexpressing THP-1 cell lines and negative control THP-1 cell lines were seeded in a 12-well cell culture plate at a rate of 500,000 cells per well, and stimulated with 50 ng / ml PMA for 48 hours to differentiate into macrophages. 1 mL of DMEM medium containing 10% FBS was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours until the cells grew to a confluence of 70% to 80%.

[0065] (2) The original culture medium was discarded, 300 μl of DMEM culture medium containing 2% FBS was added to the control group, and the experimental group was infected with SARS-CoV-2 strain (MOI = 0.1) and cultured at 37°C and 5% CO2 for 6 hours.

[0066] (3) Discard the virus solution, wash twice with PBS to remove uninfected virus, add 700 μl of DMEM medium containing 2% FBS to each well, and culture at 37°C, 5% CO2 for 24 hours.

[0067] (4) Discard the supernatant and add 500 μl of Trizol lysis buffer to each well of a 12-well plate. Perfuse thoroughly to lyse the cells for subsequent RNA extraction and gene expression detection.

[0068] (5) Add 100 μL of chloroform (Trizol: chloroform = 5:1) to each sample tube, vortex thoroughly, incubate at room temperature for 10 minutes, and centrifuge at 4°C 12000g / min for 15 minutes. After centrifugation, transfer the upper aqueous phase to a new 1.5 mL EP tube and add 250 μL of isopropanol (Trizol: isopropanol = 2:1). Incubate the mixture at room temperature for 10 minutes and centrifuge at 12000g / min for 10 minutes; discard the supernatant, wash the RNA precipitate with 500 μL of 75% alcohol, centrifuge at 7500g / min for 5 minutes, discard the supernatant, and repeat the washing once; discard the supernatant, dry the RNA precipitate in the air for about 5 to 10 minutes, then add 20 μL of DEPC to dissolve the RNA, and incubate in a 56°C metal bath for 10 minutes to further dissolve the RNA. The sample was then immediately placed in an ice box for 5 minutes and the purity ratio and concentration of the sample RNA were measured using a NanoDrop2000C spectrophotometer. The formula A260 reading × dilution factor × 40 = μg RNA / mL was used, and the A260 / A280 ratio was the purity. A ratio between 1.8 and 2.0 was considered to be of relatively high purity.

[0069] (6) RNA in the sample was reverse transcribed using the TAKARA PrimeScriptTM RT MasterMix-RR036A reverse transcription kit. Real-time fluorescence quantitative PCR was performed using the TaKaRa TB GreenTM Premix Ex TaqTMⅡ-RR820A kit. GAPDH expression level was used as the internal reference value. -ΔΔCT The mRNA expression levels of SARS-CoV-2 N and ORF1ab genes were calculated using the method.

[0070] Please refer to Figure 3 , is a schematic diagram of the function of Siglec14 in the present invention to inhibit the replication of the new coronavirus; wherein Figure 3 A is a statistical chart showing the results of fluorescence quantitative PCR detection of the SARS-CoV-2 N gene in Siglec14-overexpressing cells and negative control cells. Figure 3 B is a statistical chart of the detection results of the new crown ORF1ab gene in Siglec14 overexpressing cells and negative control cells by fluorescence quantitative PCR. The results showed that compared with the negative control group, the mRNA expression level of the SARS-CoV-2 gene in the Siglec14 overexpression group was 0.7 times that of the negative control cells, and the expression level was reduced. Siglec14 has an anti-SARS-CoV-2 effect.

[0071] Example 3

[0072] This example verifies the effect of Siglec14 in activating the innate immune pathway to exert an anti-SARS-CoV-2 effect.

[0073] (1) Siglec14-overexpressing THP-1 cell lines and negative control THP-1 cell lines were seeded in a 12-well cell culture plate at a rate of 500,000 cells per well, and stimulated with 50 ng / ml PMA for 48 hours to differentiate into macrophages. 1 mL of DMEM medium containing 10% FBS was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours until the cells grew to a confluence of 70% to 80%.

[0074] (2) The original culture medium was discarded, 300 μl of DMEM culture medium containing 2% FBS was added to the control group, and the experimental group was infected with SARS-CoV-2 strain (MOI = 0.1) and cultured at 37°C and 5% CO2 for 6 hours.

[0075] (3) Discard the virus solution, wash twice with PBS to remove uninfected virus, add 700 μl of DMEM medium containing 2% FBS to each well, and culture at 37°C, 5% CO2 for 24 hours.

[0076] (4) Discard the supernatant, wash once with PBS, add 500 μl of Trizol lysis buffer to each well of a 12-well plate, pipette thoroughly for lysis, and send for transcriptome sequencing.

[0077] (5) Analyze the transcriptome sequencing results.

[0078] Please refer to Figure 4 , is a graph showing the transcriptome sequencing analysis results of total RNA after SARS-CoV-2 infection of Siglec14 overexpression group and negative control group cells in the present invention, wherein, Figure 4 A is a statistical diagram of the expression of related factors in the viral replication negative regulatory factor pathway. Figure 4 B is the ES score statistics of the viral replication negative regulatory factor pathway. Figure 4 C is a statistical chart of the gene expression differences between the Siglec14 overexpression group and the negative control group. The results showed that the related expression factors activated by Siglec14 were enriched in the negative regulatory pathway of viral replication, and the expression levels of natural immune antiviral factors such as SAMD9L, HERC5, IFIT1, IFIT2, IFIT3, RSAD2 and MX1 increased.

[0079] Example 4

[0080] This example verifies the effect of Siglec14 in promoting the expression of mRNA of antiviral factors.

[0081] (1) Siglec14-overexpressing THP-1 cell lines and negative control THP-1 cell lines were seeded in a 12-well cell culture plate at a rate of 500,000 cells per well, and stimulated with 50 ng / ml PMA for 48 hours to differentiate into macrophages. 1 mL of DMEM medium containing 10% FBS was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours until the cells grew to a confluence of 70% to 80%.

[0082] (2) The original culture medium was discarded, 300 μl of DMEM culture medium containing 2% FBS was added to the control group, and the experimental group was infected with SARS-CoV-2 strain (MOI = 0.1) and cultured at 37°C and 5% CO2 for 6 hours.

[0083] (3) Discard the virus solution and wash twice with PBS to remove uninfected virus. Add 700 μl of DMEM medium containing 2% FBS to each well and incubate at 37°C, 5% CO2 for 24 hours.

[0084] (4) The supernatant was discarded, and 500 μl of Trizol lysis buffer was added to each well of a 12-well plate. The cells were lysed by pipetting, RNA was extracted, and gene expression was then detected. GAPDH was used as an internal reference and real-time fluorescence PCR was used to detect the expression levels of SAMD9L, HERC5, ISG15, IFIT1, IFIT2, RSAD2, and MX1 genes.

[0085] Please refer to Figure 5 , a comparative statistical diagram of the promoting effect of Siglec14 on antiviral factors after infection with SARS-CoV-2 in the present invention, wherein Figure 5 AG are statistical graphs of the expression of SAMD9L, HERC5, ISG15, IFIT1, IFIT2, RSAD2 and MX1 genes detected by qRT-PCR in Siglec14 overexpression cells and negative control cells. The results showed that compared with the negative control group, the mRNA expression levels of SAMD9L, HERC5, ISG15 and IFIT2 genes in the Siglec14 overexpression group increased by 2 times; compared with the negative control group, the mRNA expression levels of IFIT1 and MX1 genes in the Siglec14 overexpression group increased by 2.5 times; compared with the negative control group, the mRNA expression level of RSAD2 gene in the Siglec14 overexpression group increased by 7 times; it can be seen that the mRNA expression level of the Siglec14 overexpression group increased significantly, indicating that Siglec14 can promote the expression of antiviral factor mRNA.

[0086] Example 5

[0087] This example verifies the effect of Siglec14 in promoting the protein expression of antiviral factors.

[0088] (1) Siglec14-overexpressing THP-1 cell lines and negative control THP-1 cell lines were seeded in a 6-well cell culture plate at a rate of 1.2 million cells per well, and stimulated with 50 ng / ml PMA for 48 hours to differentiate into macrophages. 2 mL of DMEM medium containing 10% FBS was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours until the cells grew to a confluence of 70% to 80%.

[0089] (2) The original culture medium was discarded, and 500 μl of DMEM culture medium containing 2% FBS was added to the control group. The experimental group was infected with SARS-CoV-2 strain (MOI=0.01, MOI=0.1) and cultured at 37°C and 5% CO2 for 6 hours.

[0090] (3) Discard the virus solution and wash twice with PBS to remove uninfected viruses. Add 1 mL of DMEM medium containing 2% FBS to each well and culture at 37°C and 5% CO2 for 48 hours.

[0091] (4) Discard the supernatant, add 1 ml of PBS to a 6-well plate to wash the cells, add 1 ml of PBS, scrape the cells with a cell scraper, transfer the cell suspension to an EP tube, centrifuge at 3000 rpm for 5 minutes, discard the supernatant, lyse the cells on ice for 30 minutes with RIPA protein lysis buffer containing protease inhibitor (PMSF) (PMSF:RIPA=1:100), centrifuge at 12000 g / min for 10 minutes, collect the supernatant cell protein sample, and perform Western Blot to detect protein expression.

[0092] (5) The same procedure was used to collect protein samples 72 hours after SARS-CoV-2 infection.

[0093] (6) Add 5× protein loading buffer to the protein sample and boil to denature the protein. Perform SDS PAGE electrophoresis on the protein sample and transfer it to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked in 5% skim milk at room temperature for 1 hour, incubated in a specific primary antibody on a shaker at 4°C overnight, and then incubated with a horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. The results were observed using the OdysseyCLx dual-color infrared laser imaging system.

[0094] Please refer to Figure 6 , is a comparative statistical chart of the results of the promotion effect of Siglec14 on antiviral factors after infection with SARS-CoV-2 in the present invention, wherein, Figure 6 A is a comparison of the expression levels of SAMD9L, IFIT1, GAPDH, and HERC5 proteins in Siglec14-overexpressing cells and negative control cells detected by Wesrern Blot when MOI = 0.01. Figure 6 B is a comparison of the expression levels of SAMD9L, GAPDH, RSAD2, and HERC5 proteins in Siglec14-overexpressing cells and negative control cells detected by WesrernBlot when MOI=0.1. Figure 6 C is the comparison of IFIT1 and β-Actin protein expression levels in Siglec14 overexpressing cells and negative control cells detected by Wesrern Blot when MOI=0.1. Figure 6 D is a comparison of the expression levels of SARS-CoV-2 N protein and GAPDH protein in Siglec14-overexpressing cells and negative control cells detected by Wesrern Blot when MOI = 0.1.

[0095] The results showed that compared with the negative control group, the expression level of the new coronavirus N protein in the Siglec14 overexpression group was downregulated, and the protein expression levels of RSAD2, SAMD9L, IFIT1 and HERC5 were significantly increased. Siglec14 can promote the expression of antiviral factor proteins.

[0096] In summary, the present invention is the first to construct a Siglec14 overexpression cell line, and combines qRT-PCR, Western blot and other experiments to verify that the overexpressed Siglec14 promotes the expression of mRNA and protein of antiviral factors, activates the innate immune pathway and thus has an anti-SARS-CoV-2 effect. This invention is innovative.

[0097] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of Siglec14 gene and its expression product in the preparation of novel coronavirus gene therapy drugs, characterized in that: The expressed products include Siglec14 natural protein and Siglecl4 recombinant protein.

2. The use of the Siglec14 gene and expression product according to claim 1 in the preparation of a novel coronavirus gene therapy drug, characterized in that: The expression of the Siglec14 gene can inhibit the replication of the new coronavirus.

3. The use of the Siglec14 gene and expression product according to claim 2 in the preparation of a novel coronavirus gene therapy drug, characterized in that: The Siglec14 gene and its expression products inhibit the expression and replication of the new coronavirus by inducing the body's natural immune antiviral factors.

4. The use of the Siglec14 gene and expression product according to claim 3 in the preparation of a novel coronavirus gene therapy drug, characterized in that: The innate immune antiviral factors include one or more antiviral factors among SAMD9L, HERC5, ISG15, IFIT1, IFIT2, RSAD2, and MX1.

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