CN113881692A - Screening and evaluating method of host cell defense molecules against invasion of new coronavirus - Google Patents

Abstract

The application provides a host cell defense molecule screening and evaluating method for invasion of new coronavirus, wherein an interferon stimulating gene ISG is used for stably inducing an over-expression cell line and a pseudovirus expressing a new coronavirus envelope protein mutant based on an HIV framework for evaluation. The method is safe to operate, the detection result is stable and reliable, the interaction relation between the virus and the defense molecules is beneficially explored, the defense mechanism of the host for resisting virus infection is clarified, and a new target is provided for prevention and treatment of epidemic diseases.

Description

Screening and evaluating method of host cell defense molecules against invasion of new coronavirus

Technical Field

The application relates to the fields of molecular biology and infectious disease pathology, and particularly provides a screening and evaluating method for host cell defense molecules against invasion of new coronavirus.

Background

In recent decades, in the research of studying host-virus interaction, with the wide application of genetic screening technology with functional deficiency, it has been possible to study the functions of different genes in a plurality of model systems, but this technology is not suitable for mammalian cells. Later, the development and application of RNA interference (RNAi) technology has brought a new technical revolution for the study of virus-host interaction, but the technology has the disadvantages of high off-target rate, incomplete gene silencing, etc., and is therefore not suitable for explaining phenotypic changes. In recent years, the development of haploid embryonic stem cells has been greatly advanced, and various host factors relevant to regulating virus replication have been screened by using haploid technology. However, haploid embryonic stem cells are easy to be diploidized along with the increase of the number of passages, so the application of the technology in vitro gene screening of mammals is limited to a certain extent. The CRISPR/Cas9 technology which is started a few years ago leads the trend of functional gene screening due to the advantages of high excision rate, low off-target rate and the like, and is widely applied to the research of discovering host molecules related to virus replication, but the technology is only suitable for screening and identifying live virus defense molecules, so the research on highly pathogenic pathogens is very limited.

Screening the defense molecules such as interferon stimulating gene generated by the body after SARS-CoV-2 infects the host and evaluating the defense function level, the most direct method is to infect the target cell with the live virus, observe the cytopathic effect and calculate the number of the formed plaques. When there is high expression of certain defence molecules, cytopathic effects are not formed significantly. In contrast, cytopathic effect is severe and plaque formation is high; finally, the expression conditions of different genes in two cells causing the difference of cytopathic effects are analyzed by a high-throughput genome sequencing technology or the expression levels of different proteins in the cells are analyzed by an RNAseq sequencing technology, so that candidate defense molecules of host cells for resisting the invasion of the new coronavirus are preliminarily screened. However, the operation of the new coronavirus must be performed in a BSL-3 laboratory and is limited by factors such as experimental conditions and virus sources, so that the process of screening the defense molecules based on the infection of the live virus can not meet the research requirements of researchers, and can not screen the defense molecules in time to provide effective targets for epidemic situation prevention and control and vaccine research and development.

Disclosure of Invention

In order to solve the problem of limited research on live viruses, the research establishes a technology for stably inducing an over-expression cell line by an interferon stimulated gene ISG and packaging pseudovirions by combining HIV/Luc (NL 4-3R-E-Luc) reporter gene vectors, and carries out research on defense molecules of the new coronavirus according to a mechanism that the pseudovirions are mostly used in an enveloped virus invasion stage, and aims to screen and evaluate the host cell defense molecules against the invasion of the new coronavirus and evaluate the defense function of the host cell defense molecules. The interferon stimulated gene ISG stable induction overexpression cell line established in the research can effectively and stably express the interferon stimulated gene protein molecule under the condition of adding tetracycline inducer, and provides a mature infection target cell model for the research after transient transfection of hACE2 receptor. The pseudovirus detection system based on the HIV/Luc report vector established in the research successfully packages a plurality of pseudovirus particles such as new coronavirus and the like by using the lentiviral vector containing the report gene, thereby providing an effective virus test system for the research. The replication-critical genes Env and Vpr of the lentiviral vector have been mutated, thus packaging replication-defective pseudoviral particles. The expression of the luciferase gene (Luc) inserted into the Nef region can be accurately detected by a fluorescence determinator, so that the quantitative detection of the virus is realized, and the infection capacity of the pseudovirus is reflected. The pseudovirus detection system is safe to operate, stable and reliable in detection result and capable of effectively evaluating the function level of candidate protein molecules for resisting the invasion of new coronavirus. A fluorescent detection system is infected by pseudovirus and an ISG-induced over-expression cell system, after a target cell is transiently transfected with an hACE2 receptor to construct a mature cell infection model, SARS-CoV-2 pseudovirus particles are infected, a protein molecule LY6E expressed by an interferon stimulating gene is screened out by a fluorescent reporter gene detection system to be an important defense molecule for inhibiting an SARS-CoV-2 virus invasion link, and the functional level of LY6E for resisting SARS-CoV-2 infection is effectively evaluated.

In one aspect, the present application provides a method for screening and evaluating host cell defense molecules against invasion by new corona virus, wherein interferon-stimulated gene ISG is used to stably induce over-expressed cell lines and pseudoviruses expressing mutants of the envelope protein of new corona virus based on HIV backbone.

Furthermore, the cell line for stably inducing and over-expressing the interferon stimulated gene ISG is prepared by transfecting Flp-InTMT-RExTM-293 cells with an interferon stimulated gene protein molecule expression vector plasmid to be evaluated.

Further, the interferon-stimulated gene ISG stably induces the cell line over-expressed to express the interferon-stimulated gene protein molecule under the condition of adding tetracycline inducer, and further, the interferon-stimulated gene ISG stably induces the cell line over-expressed to be transiently transfected with hACE2 receptor.

Further, the HIV skeleton-based pseudovirus expressing the mutant of the novel coronavirus envelope protein is prepared by co-transfecting an HIV/Luc skeleton plasmid with an S protein or S protein mutant expression plasmid.

Further, the co-transfected subjects were 293T cells.

Further, the HIV/Luc backbone plasmid is pNL4-3.Luc. R-E-.

In another aspect, the application provides the use of LY6E in the manufacture of a medicament for the treatment or prevention of a disease caused by a novel coronavirus.

Further, LY6E inhibited the entry of new coronavirus.

The application also provides application of the interferon stimulating gene ISG in stably inducing an over-expression cell line and/or a pseudovirus expressing a new coronavirus envelope protein mutant based on an HIV framework in evaluating host cell defense molecules against invasion of new coronaviruses.

The methods and pseudoviruses of the present application can be used for diagnostic or non-diagnostic purposes, preferably, for non-diagnostic purposes such as research in science related to new coronaviruses, particularly in medicine and vaccine research.

The diseases caused by the new coronavirus in the application include but are not limited to pneumonia and complications thereof, asymptomatic infection state and the like.

Has the advantages that:

the method has the advantages of high safety, stable and reliable detection result and good repeatability. Specifically, the ISG protein molecule is proved to have important function of resisting virus infection by constructing a pseudovirus packaging technology and an ISG stable overexpression cell line, infecting the cell line overexpressing the ISG defense molecule by using pseudovirus particles and using a luciferase detection system. Search and identify the defense factor resisting virus invasion and effectively evaluate the defense function, which is helpful to clarify the host defense mechanism resisting virus infection and provide a new target for the prevention and treatment of epidemic diseases.

Drawings

FIG. 1 shows the result of Western blotting of LY6E tetracycline-induced expression;

FIG. 2 is a schematic diagram of the structure of an HIV/Luc reporter gene and a pseudoviral package based on an HIV/Luc reporter vector;

FIG. 3 shows the establishment of a pseudovirus in vitro infection model and the detection result of SARS-CoV-2 pseudovirus invading cells;

FIG. 4 is a graph showing the expression level of LY6E and the results of pseudovirion infection with the effective human coronavirus envelope protein.

Detailed Description

In order that those skilled in the art will more clearly and more readily understand the present invention, reference will now be made to the accompanying drawings.

Example 1 establishment and characterization of Interferon-stimulated Gene ISG Tetracycline-induced Stable overexpression cell lines

(1) Establishment of SARS-CoV-2 pseudovirus infected target cell model-ISG tetracycline induced over-expression cell line

ISG defense molecule gene sequence is cloned to an inducible expression vector pcDNA ^TM5/FRT/TO expression vector using human embryoKidney 293 cell line (Flp-In)^TM T-REx^TM-293) establishing a Flp-In T-REx-293-ISG tetracycline inducible expression cell line, and inducing the expression of the protein by using tetracycline. The expression level can be indirectly regulated and controlled by controlling the concentration of tetracycline. The ISG defense molecule is induced and expressed by tetracycline, and simultaneously, SARS-CoV-2 infection receptor hACE2 molecule is transiently transfected, thereby constructing a stable and reliable infection target cell model. (specific methods are described in detail in the examples below)

(2) Identification of ISG defense molecule inducible expression systems

The established Flp-In T-REx-293-ISG tetracycline induction expression cell line is used, after 24 hours of tetracycline induction, the cell is cracked to extract total protein, and the expression condition of ISG defense molecules is identified by a Western blotting method.

EXAMPLE 2 construction of SARS-CoV-2S protein expression plasmid and packaging of pseudovirus and establishment of in vitro infection model (shown in FIG. 2)

(1) Construction of S protein expression plasmid

The S protein of SARS-CoV-2(MN908947) with 19 amino acids deleted at C terminal is subjected to codon optimization without changing amino acid sequence, cloned into pSecTag2/HygroA vector, transformed, amplified, purified and recovered. (2) Packaging of SARS-CoV-2 pseudovirus

Eukaryotic expression plasmids containing the S protein were co-transfected with 293T cells with Env-deleted backbone plasmid pNL4-3.Luc. R-E-carrying luciferase reporter gene to obtain recombinant pseudoviruses. (specific methods are described in detail in the examples below)

(3) Establishment of SARS-CoV-2 pseudovirus in vitro infection model

The ISG defense molecule induced expression cell line was seeded into 6-well plates and hACE2 was transfected 12h later, resulting in cells that were approximately 80% abundant at the time of transfection. After the hACE2 receptor molecule is transfected for 24h, the hACE2 receptor molecule is inoculated to a 96-well cell culture plate, the next day, the expression of defense molecules is induced by tetracycline, tetracycline (the final concentration is 1 mu g/mL), SARS-CoV-2 pseudovirus particles are infected after 24h of induction, and the luciferase activity of the cells is detected at 48-72h after infection.

Example 3 establishment and characterization of the defense molecule LY6E Induction of an over-expressing cell line T Rex293-LY6E (1) establishment of Flp-In T-REx-293-LY6E Tetracycline Induction of an expressing cell line

1. Preparation of Flp-In^TM T-REx^TM-293 cells.

Resuscitating Flp-In good disc status^TM T-REx^TM293, seeded on 10cm cell culture dishes, rinsed once with 12mL PBS after the cells have grown, the residual medium serum is removed, and Flp-In is digested with 1mL of 0.05% trypsin^TM T-REx^TM293 cells for 3-5 min, resuspending the cells in 12mL System of 10% FBS-containing high glucose Medium DMEM at 4-8X 10⁵/cm²Was inoculated in a six-well plate, and a final volume of 2mL was made by supplementing fresh DMEM medium containing 10% FBS. Standing at 37 deg.C and 5% CO₂The cells were incubated overnight in the incubator.

2. And (4) transfection.

The next day, when the cell density reached 80% -90%, LY6E/pcDNA was co-transfected with the transfection method of Lipofectamine 2000 transfection kit ^TM5/FRT/TO expression vector plasmid and pOG44 plasmid, co-transfected with CAT/pcDNA^TMThe 5/FRT/TO expression vector plasmid and the pOG44 plasmid served as negative controls. After 6-8 hours, 2mL of fresh DMEM medium containing 10% FBS was supplemented.

3. And transferred to 10cm cell culture dishes.

24h after transfection, cells in the six-well plates were digested with 0.5mL of 0.05% trypsin for 3-5 min, and resuspended cells were neutralized with 1mL of fresh DMEM medium containing 10% FBS. And inoculated into a large dish with a diameter of 100mm to continue the culture, supplemented with fresh medium to 12 mL.

After the cells were attached for 24h, the medium in the dish was aspirated and 12mL of selective medium (DMEM high-sugar medium containing 10% FBS, 250. mu.g/mL hygromycin B, and 10. mu.g/mL blicidin) was added. The selective medium was changed every three days, the cell status was closely followed, after about 5-6 days, the cells began to die, after about 2-3 weeks, there was a clear clonal cell mass that grew stably in the selective medium with antibiotics, LY6E/pcDNA ^TM5/FRT/TO and CAT/pcDNA^TMThe 5/FRT/TO inducible expression cell strain is basically constructed, and after the cells are full, the cell strain is transmittedAnd (4) freezing and storing the cells at the temperature of-80 ℃.

(2) The expression of the target protein LY6E is detected by Western blotting experiment.

1. And (3) cell recovery: LY6E/pcDNA with good recovery state ^TM5/FRT/TO and CAT/pcDNA ^TM5/FRT/TO inducible expression cell strain, inoculated in 10cm cell culture dish, placed in 37 ℃ incubator overnight culture.

2. Dividing into 12 pore plates: LY6E/pcDNA from a 10cm cell culture dish ^TM5/FRT/TO and CAT/pcDNA ^TM5/FRT/TO-induced expression cell lines, cells rinsed with 12mL PBS, residual medium and serum washed, digested with 1.5mL 0.05% trypsin for 3-5 minutes, plated on 12-well plates by density, supplemented with fresh 10% FBS-containing high-glucose medium DMEM TO a volume of 1mL, and incubated overnight at 37 ℃ in an incubator.

TET tetracycline induction: 1 μ g/mL TET induction was required for 24h, first prepare 3 μ g/mL TET solution, add 50 μ l TET solution at 3 μ g/mL concentration in 12-well plate, and finally make the working concentration 1 μ g/mL TET.

4. Protein collection: a) after 24h of induction, the medium was aspirated, washed 3 times with PBS buffer, and the PBS was aspirated off the last time. b) mu.L of 2 XSDS (1% SDS, 2.5% beta-mercaptoethanol, 0.001% bromocresol blue, 5% glycerol, 0.25mmol/L Tris HCl, pH6.8) was added to each well and placed on a shaker at 80rpm and shaken at room temperature for 15 min. c) After the cells were lysed sufficiently, the mixture was pipetted by a micropipette and transferred to a clean 1.5ml lep tube. d) Boiling at 100 deg.C for 10min to denature protein. e) Centrifuging: 14680r/min for 5min (centrifugal radius r is 8.4 cm). And sucking the supernatant, namely the total cell protein (denatured).

Western blotting: a) preparing 1 XMOPS electrophoresis solution (MES buffer solution is adopted if small molecular protein is electrophoresed), and assembling the electrophoresis device according to the instruction. b) Electrophoresis: and taking 15 mu L of the denatured protein sample and 6 mu L of the protein Marker to carry out loading according to a preset sequence. And then setting 80V constant voltage electrophoresis, and stopping electrophoresis when the indicator bromophenol blue runs to the bottom of the precast gel. c) Film transfer (wet transfer). Preparing a film transfer liquid according to a film transfer liquid formula. Cutting the prefabricated glue. Taking out the precast gel after electrophoresis, cutting according to the range of the target protein,and (3) balancing in 1 × membrane transferring solution, and soaking sponge, filter paper and the like required in the membrane transferring process. Activating the PVDF film. And (3) soaking the cut PVDF membrane matched with the to-be-transferred glue in absolute methanol for 15s, then soaking in 1 x membrane transferring liquid for 2min, and washing off residual methanol on the membrane. And fourthly, assembling. Preparing 1 multiplied by membrane transferring liquid, placing the negative electrode surface of the membrane transferring clamp in the membrane transferring clamp, and assembling according to the sequence of 'membrane transferring clamp negative electrode surface-sponge-filter paper-prefabricated glue-PVDF membrane-filter paper-sponge-membrane transferring clamp positive electrode surface', wherein drying of the PVDF membrane and the prefabricated glue is avoided, and air bubbles are removed between each layer. Closing the film transferring clamp, putting the film transferring clamp into a film transferring groove according to the correct electrode sequence, and adding 1 x of film transferring liquid to ensure that the film transferring clamp is submerged at the top of the film transferring clamp. And fifthly, turning the film. And (3) placing the film rotating groove on an ice box, and carrying out constant current of 350mA and film rotating for 1h30 min. d) And (5) sealing. A blocking solution (TBS solution containing 5% skim milk) was prepared in advance, and then a PVDF membrane on which a protein Marker band was clearly observed was removed and transferred to the blocking solution. The mixture is placed on a shaking bed at room temperature and shaken for 1 hour. e) And (5) incubating the primary antibody. The rabbit-derived anti-LY6E antibody was diluted at a ratio of 1:200 and the mouse-derived anti-betaactin antibody at a ratio of 1:2000 using a primary antibody diluent, the blocked PVDF membrane was transferred thereto and incubated overnight in a shaker at 4 ℃ and washed 3 times with TBS for 5 minutes each time for repeated use of the primary antibody, and the primary antibody was incubated after removing the residual skim milk. f) And (5) washing the membrane. The primary antibody was recovered and washed 3 times for 5 min/time using 1 XTSST. g) And (5) incubating the secondary antibody. Selecting corresponding one according to one kind of anti-species source

800cm secondary antibody. It was diluted with blocking solution at a ratio of 1: 10000. The PVDF membrane was transferred to it and incubated for 1h at room temperature on a shaker in the dark. h) And (5) washing the membrane. The secondary antibody was recovered and washed with TBST 3 times for 5 min/time. Finally, it was immersed in 1 × TBS. i) The image was taken in an Odyssey near-infrared scanning imager (results are shown in FIG. 1).

Example 4 packaging of pseudovirions expressing the envelope protein of a novel coronavirus based on the HIV backbone and establishment of an infection model

(1) Construction of S protein expression plasmid

19 amino acids are removed from the C terminal of SARS-CoV-2(MN908947) S protein, codon optimization is carried out on the premise of not changing the amino acid sequence, the protein is cloned to pSecTag2/Hygro A vector, and transformation, amplification, purification and recovery are carried out.

(2) Packaging of pseudoviruses comprising the S protein

Eukaryotic expression plasmids containing the S protein were co-transfected with 293T cells with Env-deleted backbone plasmid pNL4-3.Luc. R-E-carrying luciferase reporter gene to obtain recombinant pseudoviruses.

At 5X 10⁶A10 cm cell culture dish was inoculated with 293T cells and the confluency reached 80% after overnight culture.

2.5. mu.g of expression plasmid containing S protein and the backbone plasmid pNL4-3.Luc. R-E-7.5. mu.g were added as described in 1: 3(w/w) into a 1.5mL centrifuge tube containing 500. mu.L of antibiotic-free serum-free DMEM.

Adding 30 μ L FuGENE HD (DNA and transfection reagent 1:3, w/v), mixing well, standing at room temperature for 15-30 min. The plasmid-transfection reagent mixture was added dropwise to a 10cm cell culture dish, and the mixture was mixed by gentle shaking. Standing at 37 deg.C for CO₂Culturing in an incubator.

Culture supernatants containing pseudoviruses were harvested after 48-72h of culture and supplemented with FBS to a final concentration of 20%. Filtering the virus solution with 0.45 μm filter, subpackaging, and freezing at-80 deg.C for storage.

(3) Establishment of SARS-CoV-2 pseudovirus in vitro infection model

a) Resuscitating cells and separating and transferring six-hole plates.

LY6E/pcDNA with good recovery state ^TM5/FRT/TO and CAT/pcDNA ^TM5/FRT/TO induction expression cell line, inoculating in 10cm cell culture dish, balancing one cell culture dish with 8-10mL collagen before inoculating, which is favorable for cell adherent and extended growth. When the cell growth density of 10cm cell culture dish is about 90%, rinsing the cells with 10mL PBS buffer solution containing calcium and magnesium, in order to remove residual serum and other culture medium components and facilitate digestion with trypsin, adding 1.5mL trypsin, standing at 37 deg.C and 5% CO₂Digesting in a cell incubator for 3-5 min, and then using 12mL of the bodyFresh DMEM medium containing 10% FBS of the line resuspended cells and at 4-8X 10⁵/cm²Density inoculation requires that LY6E/pcDNA is inoculated^TM5/FRT/TO and CAT/pcDNA ^TM5/FRT/TO induction expression cell line TO six-hole plate, the density of which reaches the transfection density requirement in the next day, the density is about 80% -90%, and the six-hole plate is placed at 37 ℃ and 5% CO₂The cells were incubated overnight in a constant temperature incubator.

b) The ACE2 receptor, the DPP4 receptor and the APN receptor were transfected.

The next day, when the cell density in the six-well plate reached 80% -90%, the ACE2/pcDNA3.1 plasmid, the DPP4/pcDNA3.1 plasmid and the APN/pcDNA3.1 plasmid were transfected according to the instructions of the Lipofectamine 2000 transfection reagent, while the pcDNA3.1 empty vector plasmid was transfected as a negative control. After 6-8 h of transfection, 2mL of fresh DMEM medium containing 10% FBS was supplemented.

c) And (4) transferring to a 96-well plate.

24h after transfection, cells were washed once with 1mL PBS, residual serum and other media components were removed, and the cells were incubated with 0.5mL trypsin at 37 ℃ in 5% CO₂After digesting for 3-5 minutes in a constant-temperature cell incubator, suspending cells by using 1mL of fresh DMEM medium containing 10% FBS, adding the cell suspension into a 15mL sterile centrifuge tube, supplementing 9.5mL of fresh DMEM medium containing 10% FBS, preparing into a 12mL system, gently blowing uniformly for 5-6 times, inoculating 100 mu L of the cell suspension into a 96-well black cell culture plate, placing the black cell culture plate at 37 ℃ and 5% CO₂The cells were cultured overnight in a cell incubator.

d) Infecting with pseudovirus.

When the cell density in the 96-well plate reaches 90%, throwing the plate, discarding the culture medium, and mixing the pseudovirus stock solution to be infected with a fresh DMEM culture medium containing 10% FBS according to the ratio of 1:1 dilution, after blowing and mixing, 100. mu.L of each well was added to the corresponding 96-well plate. 24 hours after infection, each well was supplemented with 100. mu.L of fresh DMEM medium containing 10% FBS.

e) Detecting luciferase activity.

After 48 hours of infection, the plates were spun off and the virus solution in the 96-well plates was discarded. According to the specification requirement of the luciferase reporter gene detection kit, 30 mu L of cell lysate is added into each hole, the hole is placed on a shaking table at room temperature and shaken for 15min, when the cells are fully lysed, 50 mu L of luciferase substrate is added into each hole, and the holes are uniformly mixed and then rapidly placed on a luciferin luminometer for reading. The luciferase activity measured in this experiment represents the ability of the pseudovirus to invade the cells (results are shown in FIG. 4).

The results show that:

the research utilizes the tetracycline-induced stable expression cell line Flp-In T-REx293-LY6E established by the method, combines with a SARS-CoV-2 pseudovirus infection system established by an HIV/Luc (NL 4-3R-E-Luc) reporter gene vector packaging pseudovirus particle technology, and has successfully screened and identified an important restriction factor LY6E for resisting SARS-CoV-2 invasion. The LY6E data are presented as follows: the LY6E protein can be stably expressed In the established Flp-In T-REx293-LY6E induction expression cell line under the induction of tetracycline (Tet) with different concentrations through immunoblotting experiments (FIG. 3, left). Moreover, by detecting the expression level of the luciferase reporter gene to realize the quantitative detection of the virus, the inhibition rate of LY6E on SARS-CoV-2 infection can be effectively evaluated to reach 70%, the inhibition rate on MERS-CoV, HCoV-229E and HCoV-NL63 can reach more than 80%, and the inhibition rate on HCoV-OC43 infection can even reach more than 90% (figure 3 right). Therefore, the technical platform which is invented by the research and is based on establishing a stable over-expression cell line and combined with a pseudovirus infection system into a whole can efficiently screen and identify host cell defense molecules against the invasion of new coronavirus and evaluate the defense function level of the host cell defense molecules.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.

Claims (10)

1. A method for screening and evaluating host cell defense molecules against invasion of a new coronavirus, wherein an overexpression cell line and a pseudovirus expressing a mutant of envelope protein of the new coronavirus based on HIV backbone are stably induced using an interferon-stimulated gene ISG.

2. The method according to claim 1, wherein the stable induction of the overexpression cell line by the interferon-stimulated gene ISG is achieved by Flp-In^TMT-REx^TM-293 cells are transfected with an interferon stimulated gene protein molecule expression vector plasmid to be evaluated.

3. The method of claim 2, wherein said stable ISG overexpression cell line expresses said ISG protein molecule with the addition of tetracycline-inducing agent.

4. The method of claim 2 or 3, wherein the cell line stably induced to over-express the interferon-stimulated gene ISG is transiently transfected with hACE 2.

5. The method according to any one of claims 1 to 4, wherein the HIV-backbone-based pseudovirus expressing a mutant of the envelope protein of the novel coronavirus is prepared by co-transfecting an HIV/Luc backbone plasmid with an S protein or S protein mutant expression plasmid.

6. The method of claim 5, wherein the co-transfected subject is 293T cells.

7. The method of claim 6, wherein the HIV/Luc backbone plasmid is pNL4-3.Luc.

Use of LY6E for the preparation of a medicament for the treatment or prevention of a disease caused by a new coronavirus.

9. The use of claim 8, wherein LY6E inhibits entry of a new coronavirus.

10. Use of the interferon stimulated gene ISG in any of claims 1 to 7 to stably induce an over-expressing cell line and/or a pseudovirus expressing a mutant of the envelope protein of a new coronavirus based on the HIV backbone, in the evaluation of host cell defense molecules against the invasion of a new coronavirus.

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