CN113621651A

CN113621651A - Cell syncytium lesion model based on S-TET protein expression system and preparation method thereof

Info

Publication number: CN113621651A
Application number: CN202110812707.2A
Authority: CN
Inventors: 杨光; 周正; 黄啸; 陈雅丽; 郑黎
Original assignee: Nanchang Five Elements Bio Technology Co ltd
Current assignee: Nanjing Aorui Pharmaceutical Co.,Ltd.
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2021-11-09

Abstract

The invention relates to the technical field of biology, in particular to a cell syncytial lesion model based on a novel coronavirus spike protein (S) S-TET protein expression system and a preparation method thereof. The invention provides a stable cell line based on a Tet on system and expressing a novel coronavirus spike protein (S) by taking Vero E6 cells as backgrounds. The cell line was obtained by puromycin-containing screening. Under the condition of cell culture without an inducer, the S gene is in a silent state, and the cell line can grow normally and continue to passage stably. Upon addition of trace amounts of the tetracycline analogue doxycycline hydrochloride (DOX), the teton system is activated and the S gene is enhanced in transcription and produces large amounts of S protein. The establishment of the stable cell line of the S teton system provides an ideal research platform for research and development of novel coronavirus antibodies and related drugs and research of novel coronaviruses.

Description

Cell syncytium lesion model based on S-TET protein expression system and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a cell syncytium lesion model based on an S-TET protein expression system and a preparation method thereof.

Background

The coronavirus spike protein (S protein) plays an important role in the formation of syncytia of virus-invading cells and infected cells; the existing research methods and means are realized by pseudovirus infection or transient transfection; direct utilization of the new coronavirus itself was also performed in the BSL3 laboratory. At present, there is no report of successful research on coronavirus infection using stable cell lines expressing spike proteins.

All methods and techniques used in the current laboratory for studying the S protein do not achieve the goal of high throughput drug screening and therefore cannot be used for rapid screening of anti-neocoronavirus drugs.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a cell syncytium lesion model based on an S-TET protein expression system and a preparation method thereof.

Since the S protein of the new coronavirus (SARS CoV-2) causes severe cytopathic effect (CPE) and ultimately leads to cell death, the conventional transgenic methods cannot establish a cell line with both high and stable expression of the S protein, and moreover, the development of inhibitors of coronavirus invasion or fusion with cells faces more complicated challenges due to various complex ways such as receptor-mediated and non-receptor-mediated infection of cells by viruses and endocytosis. To simplify its complexity, screen for high throughput against invasion by new coronaviruses and inhibit infected cytopathies, we developed a cell model for tetracycline-regulated (Tet-On/Off) protein expression, i.e., S-Tet-On/Off, stable transgenic cell lines. The spike protein (S protein) can be expressed in large quantities only under the condition of tetracycline addition (Tet-On).

In order to achieve the purpose, the invention adopts the technical scheme that: the preparation method of the cell syncytium lesion model based on the S-TET protein expression system is characterized by comprising the following steps of:

s1, connecting a DNA sequence for coding a novel coronavirus S protein to a lentiviral expression vector containing a teton regulatory factor, and co-transfecting a cell by using the expression vector and a virus packaging auxiliary plasmid to obtain a recombinant lentivirus;

s2, transducing Vero E6 cells with the recombinant lentivirus, and screening to obtain a stable transgenic cell strain which expresses S protein under the regulation and control of teton, namely the syncytium lesion model of the cells;

the S protein DNA sequence is optimized and modified through a codon optimization strategy, a Kozak consensus sequence is added upstream, an alanine codon GCC is added after an initiation codon ATG, and the nucleotide sequence of the S protein DNA sequence is shown as SEQ ID NO: 2 or a functional equivalent thereof which is substantially identical to SEQ ID NO: 2 have at least 90% identity.

As a preferred embodiment of the preparation method of the present invention, the preparation method further comprises a step S3, wherein the step S3 is to add doxycycline hyclate to the culture medium for growing the syncytium lesion model of the cell, to induce the S protein to be expressed in a large amount, thereby generating the cell fusion lesion phenomenon.

As a preferred embodiment of the preparation method of the present invention, the nucleotide sequence of the tet on regulatory factor is as shown in SEQ ID NO: 5, respectively.

As a preferred embodiment of the preparation method of the present invention, the screening is: the slow virus expression vector contains puromycin resistance genes, and puromycin is added into a culture medium for screening.

As a preferred embodiment of the preparation method of the invention, the lentivirus expression vector is pLent-TRE3G-hPGK-rtTA-SV 40-Puro.

The invention also provides a cell syncytium lesion model prepared by the preparation method.

The invention also provides application of the cell syncytium lesion model in screening and identifying anti-new coronavirus medicines.

As a preferred embodiment of the application of the invention, the screening is to judge whether the screened drug has protective effect on cells in the cell fusion process caused by the binding of the S protein and the corresponding cell receptor protein ACE2 by observing the change of the degree of cell fusion.

As a preferred embodiment of the use of the present invention, the cell fusion is produced by rapid mass production of S protein under induction of doxycycline hyclate.

As a preferred embodiment of the use according to the invention, the doxycycline hydrochloride is present in a concentration of 0.1 to 1. mu.g/ml.

As a preferred embodiment of the use according to the invention, the S-TET protein expression system is also adapted to use a Tet off design such that S protein expression is only produced under the control of doxycycline hyclate removal. Since bovine serum is usually mixed with residual tetracycline, such as Tet-on regulated S protein, specially prepared tetracycline-free serum must be used. And the Tet off design can eliminate the interference of residual tetracycline in serum and reduce the implementation cost of a lesion model.

The invention has the beneficial effects that:

(1) the invention provides a stable cell line based on a Tet on system and expressing a novel coronavirus spike protein (S) by taking Vero E6 cells as backgrounds. The cell line can grow normally and passage stably in a basal culture medium containing puromycin. Once trace amounts of Doxycycline hyclate (DOX) are added to the medium, the teton system is activated and the S gene is transcribed and translated to produce a large amount of S protein. The S tet on stable cell line of the system provides an ideal research platform for research and development of novel coronavirus antibodies and related antiviral drugs and virology research.

(2) The invention verifies that the S tet on stable transfer cell line obtained by transducing Vero E6 cells with lentivirus can be cultured and proliferated in a large amount in a biosafety level 2 laboratory (BSL-2); the S tet on cell line is verified to rapidly express the spike protein (S) in a large amount under the induction of low-dose tetracycline analogue; the basic characteristics and immunogenicity of the spike protein of the S tet on cell line without pathogenicity are verified under specific culture conditions.

Drawings

FIG. 1: after the GFP tet on cells are induced by DOX for 24 hours, a large amount of GFP proteins excite green fluorescence under blue light, and the fact that the tet on system can effectively regulate and control the expression of the GFP proteins is shown.

FIG. 2: s tet on cells form a cytozygote after 8h, 16h, 24h and 32h of DOX induction.

FIG. 3: s tet on cells were induced by DOX to produce S protein.

FIG. 4: the S tet on cell is induced by DOX, when S protein is expressed in large quantity, the expression of cytoskeletal protein Tubulin is reduced rapidly and even disappears, which indicates that syncytia phenomenon appears between cells.

FIG. 5: map of basic slow virus expression vector pLent-TRE3G-hPGK-rtTA-SV 40-Puro.

FIG. 6: structure diagram of amplified target fragment.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

Example 1 construction of S tet on Stable cell lines

The construction of the S Tet on stable cell line (control group: GFP Tet on stable cell line) included: construction of a Tet on inducible S protein lentiviral expression vector, packaging of a lentivirus, transduction of Vero E6 cells by the lentivirus, screening of an S Tet on cell line, induction of GFP Tet on cell line expression GFP protein by doxycycline hydrochloride (DOX), induction of S Tet on cell expression S protein by DOX, induction of S Tet on monoclonal cell line expression S protein by DOX and establishment of a syncytium lesion model. The method comprises the following specific steps:

(1) obtaining DNA sequence information of the S protein from an NCBI database, and artificially synthesizing a DNA fragment of the S protein modified by codon optimization, wherein the nucleotide sequence of the DNA fragment of the S protein modified by codon optimization is shown as SEQ ID NO: 1 is shown. The codon optimised S protein nucleotide sequence was 72% identical to that of the New coronavirus Wuhan strain (NCBI database ACCESSION NC-045512 REGION: 21563..25384), while the protein sequence between the two remained 100% identical. The invention can optimize the codon of the virus spike protein gene to achieve the following purposes: firstly, the biological safety is improved, and the pollution of virus DNA generated by homologous recombination to human DNA is completely avoided; ② the expression of S protein in stable transfer cells is improved.

(2) In order to improve the expression efficiency of the S protein, a Kozak consensus sequence is added at the upstream of the DNA fragment of the S protein, and the DNA sequence of the Kozak consensus sequence is as follows: GCCACCATGG. Since the last G (+ G4) of the Kozak sequence plays an important role in the recognition of the mRNA reading frame start site by ribosomes during translation, an alanine codon GCC was added after the ATG start codon of the DNA fragment of the S protein. Alanine is one of the smallest aliphatic amino acids, and its methyl side chain is non-reactive and almost never directly involved in any protein function, thus eliminating or minimizing the possibility that newly added alanine will affect the protein function of the viral S protein product. The nucleotide sequence of the finally obtained target fragment is shown as SEQ ID NO: 2, respectively.

(3) And (3) connecting the target fragment obtained in the step (2) to a lentivirus expression vector containing a Tet on regulation and control system, and then co-transfecting the target fragment and a lentivirus packaging helper plasmid to obtain the recombinant lentivirus expressed by the Tet on inducible S protein.

In order to construct a transgenic vector which is regulated by a Tet-On system and can stably integrate a target DNA segment (encoding S protein) into a host cell genome to ensure long-term expression of the S protein, the invention adopts a lentivirus transduction design. The basic vector used is a lentivirus expression vector pLent-TRE3G-hPGK-rtTA-SV40-Puro (Virginia Biotechnology Co., Ltd., http:// www.weizhenbio.cn/clone erviceZtDetail. phpid ═ 122& pid ═ 11& cid ═ fk ═ 1).

The plasmid map of this basic vector is shown in FIG. 5, in which the elements directly relevant to the present invention are:

1) TRE3G (tetracycline responsive element 3G promoter, Genbank access number: MN044710.1), which can control the mRNA transcription level of the target protein S to change the expression level of the S protein from the very low basic expression to the maximum expression after induction. The nucleotide sequence of the element is shown as SEQ ID NO: 4 is shown in the specification;

2) rtTA (inverse tetracycline-controlled transactivator, i.e. tet on regulator, Genbank access number: u89930.1) having the nucleotide sequence set forth in SEQ ID NO: 5, respectively.

Other important elements contained in the basic vector are:

1) puro (puromycin) for use in the screening of resistant cells;

2) WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) for enhancing transgene expression; the following are the basic elements of a lentivirus transduction system:

3) cppt (central polypurine tract) which enhances integration and transduction efficiency of the vector;

4) psi (Psi) sequence), which is a high-level structure of the four-stem loop of RNA, plays a key role in improving the transgenic efficiency;

5) the 5 'LTR and 3' LTR (5 'and 3' long terminal repeats) facilitate integration of the transfer plasmid sequence into the host genome.

Designing a primer containing enzyme cutting sites BamHI and Mlu I, amplifying the target fragment obtained in the step (2), wherein the nucleotide sequence of the amplified product is shown as SEQ ID NO: 3 (see figure 6), then the amplified product and the vector pLent-TRE3G-hPGK-rtTA-SV40-Puro are respectively digested by BamHI and Mlu I enzymes, and then are connected to obtain the recombinant lentivirus expression vector.

The lentiviral packaging procedure is briefly described as follows: the mixture of the prepared S protein lentivirus expression vector containing Tet on regulation, a lentivirus packaging plasmid (psPAX2) and an envelope plasmid (pMD2G) is cotransfected with HEK293T cells, and after 72 hours of culture, lentivirus particles are collected from a supernatant culture medium and are used for virus titer determination. See http:// www.weizhenbio.cn/useFavoriteDetail. phpid 2& pid 20& cid 22.

(4) Vero E6 cells were plated on the day before lentivirus transduction into a 10cm cell culture dish and 1.5ml of a basal medium containing lentivirus (basal medium just passed over the cell surface layer) was added at an MOI of 1; taking 50ml as an example, the basic culture medium formula is as follows: 10% FBS, 1% P/S and DMEM/HG; (GFP tet on as control).

(5) After 8h, removing the basic culture medium containing lentivirus on the cell surface layer, adding 8ml of the basic culture medium, and adding 5% CO at 37 DEG C₂Culturing the cells;

(6) removing the basic culture medium after 2 days, adding a screening culture medium (the basic culture medium containing 16 mu g/ml puromycin), screening for about one week to obtain positive S tet on stably transformed cells, and replacing the culture medium every 2-3 days;

reagent	Manufacture	Cat.no.	Storage of	standard
					Puromycin	Solarbio	P8230	-20℃	25mg

(7) 1000S tet on cells were seeded in 10cm cell plates at 37 ℃ with 5% CO₂Culturing for about 1 month, and selecting monoclonal fineCarrying out cell amplification culture;

(8) adding inducer, namely 0.1-1 μ g/ml tetracycline analogue-doxycycline hydrochloride (DOX), into the screening culture medium, and inducing culture of S teton cells at 37 deg.C and 5% CO₂Culturing under the conditions of (1);

(9) after the cells are induced and cultured for 8h, observing the growth state of the S teton cells under a microscope;

(10) collecting total proteins of S tet on cells induced for different time periods, and verifying the expression of the S proteins by Western blot;

(11) and collecting total protein of S tet on monoclonal cells cultured for 24h in an induction way, and verifying the expression of the S protein by Western blot.

Example 2: GFP tet on cell line is induced and cultured for 24h by 0.1-1 mug/ml doxycycline hydrochloride (DOX), and GFP green fluorescent protein is expressed

Study subjects: GFP tet on cell line

The specific experimental steps are as follows:

(1) obtaining DNA sequence information of GFP and expression vector in the database;

(2) integrating a GFP sequence into a Tet on system, and packaging the modified Tet on system into a lentivirus;

(3) the GFP tet on stable cell screening method is the same as the S tet on stable cell strain screening method;

(4) removing the original culture medium of GFP teton cells in a six-hole plate in a biological safety cabinet, adding 2.5ml of pre-preheated DOX containing 0.1 mu g/ml or 1 mu g/ml into each hole, and recording the induction time at the moment as 0 h;

(5) observing whether the cells have green fluorescence signals of GFP tet on under a microscope when DOX is induced for 24h (GFP is excited by blue light to be green) (figure 1);

(6) and analyzing data and results.

The results are shown in FIG. 1, indicating that:

(1) tetracycline analogue doxycycline hydrochloride (DOX) (0.1-1 μ g/ml) can activate Tet on system, GFP protein is expressed, and cells excite green fluorescence under blue light.

(2) The addition of 1. mu.g/ml DOX as an inducer to the selection medium resulted in the production of a large amount of GFP protein at 24h, so the working concentration of DOX was defined as 1. mu.g/ml.

Example 3: the S tet on cell line undergoes cell fusion under the induction of 1 μ g doxycycline hyclate (DOX), i.e., syncytial is formed.

Study subjects: s tet on cell line

The experimental steps are as follows:

(1) six-well plate internal-plating S tet on cell 4.0X 10^5 cells/well, 2.5ml screening culture medium 37 deg.C, 5% CO₂Culturing overnight; in another experiment, cells S tet on and GFP tet on were plated in the same way, at a rate of 1: mixing at a ratio of 1.

(2) Removing the original screening culture medium of the S teton cells in the six-hole plate in the biological safety cabinet, adding 2.5ml of pre-preheated DOX containing 1 mu g/ml into each hole, and recording the induction time of 0 h;

(3) observing the growth state of the cells under the lens of 8h, 16h, 24h and 32h induced by adding DOX respectively, and photographing and recording; and analyzing data and results.

The results are shown in FIG. 2, indicating that:

(1) the fusion reaction of the cells is observed when the S Tet on cells are added into an induction culture medium for 8 hours, which indicates that 1 mu g/ml DOX activates the Tet on system to start expressing S protein, and then the S protein is combined with ACE2 on the cell surface to induce the fusion reaction of the cells.

(2) Along with the prolonging of the DOX induction time, the intercellular fusion phenomenon is intensified, cell membranes are ruptured, and a large number of plaques appear at the bottom of the culture dish; this provides a very intuitive index for drug screening using the cell model in the future: and (3) observing the degree change of cell fusion under a mirror, and judging whether the drug has a protective effect on the cells in the process of attacking the cells by the S protein.

(3) Along with the prolonging of the DOX induction time, the intercellular fusion phenomenon is intensified, cell membranes are ruptured, and a large number of plaques appear at the bottom of the culture dish; this provides a very intuitive index for drug screening using the cell model in the future: and (3) observing the degree change of cell fusion under a mirror, and judging whether the drug has a protective effect on the cells in the process of attacking the cells by the S protein.

Example 4: western blot proves that DOX induces S tet on cells to express S protein

Study subjects: s tet on cells

The specific experimental steps are as follows:

(1) six-well plate intracellular seeding 4.0X 10^5 cells/well, 2.5ml screening culture medium 37 ℃, 5% CO₂Culturing overnight;

(2) removing the original screening culture medium of the S teton cells in the six-hole plate in the biological safety cabinet, adding 2.5ml of pre-preheated DOX containing 1 mu g/ml into each hole, and recording the induction time as 0 h;

(3) collecting S tet on cells at 8h, 16h, 24h and 32h respectively after DOX induction;

(4) scraping off cells at the bottom of the pore plate by using a scraper, and then transferring the cell suspension into a 15ml centrifugal tube;

(5)3000g, centrifuging at 4 ℃ for 5 min;

(6) discarding the supernatant, adding 1ml of 1 XPBS, 3000g, centrifuging at 4 ℃ for 5 min;

(7) repeating the step (5) once;

(8) the supernatant was discarded and 40. mu.l RIPA buffer (containing 0.4. mu.l 100mM PMSF) was added;

(9) ice-cooling for 30min, and vortexing every 5min for 3 s;

(10) centrifuging at 14000g for 5min, and taking the supernatant in a clean 1.5ml centrifuge tube for later use;

(11) taking 20 mu l of a 5mg/ml BSA standard protein sample, adding 180 mu l of 1 XPBS, uniformly mixing by oscillation, and then diluting the mixture into standard proteins with different gradients for later use according to the table shown in the following;

0.5mg/ml BSA(μl)	1X PBS(μl)	BSA final concentration (. mu.g/ml)
			40	0	0.5
32	8	0.4
			24	16	0.3
16	24	0.2
			12	28	0.15
8	32	0.1
			4	36	0.05
0	40	0

(12) The extracted protein samples were diluted 4, 6 and 10 fold respectively (volume of dilution >10 μ l) for use;

(13) preparing a BCA working solution, and mixing the BCA solution and the Cu solution according to the weight ratio of 50: 1 to prepare a BCA working solution;

(14) a96-well plate with a transparent bottom is taken, and 100. mu.l of BCA working solution and 10. mu.l of a protein sample to be prepared are sequentially added into the 96-well plate (each gradient of a standard protein sample is prepared into 3 times);

(15) incubating at 37 ℃ for 30 min; measuring the absorbance at the wavelength of 562 nm; calculating the protein concentration of the sample to be detected according to the absorbance of the standard protein and the known protein concentration;

(16) preparing protein electrophoresis gel (8% of separation gel and 5% of concentrated gel); adding 20 μ g protein loading buffer (quantitative 28 μ l), denaturing at 100 deg.C for 5min, and sequentially adding into gel loading well; glue running, 90V 30min and 120V 2 h; membrane transfer, 200mM 150 min; sealing and standing overnight at 4 ℃;

(17) incubating the primary antibody for 1h at room temperature; wherein the dilution ratio of the antibody is as follows: anti-S (. about.180 kDa) antibody, 1:1000, parts by weight; anti-GAPDH (37KDa) antibody, 1:500, a step of;

(18) eluting primary antibody, adding eluent 1X TBST (0.05% TWEEN 20), washing with shaking for 10min, and repeating for 3 times;

(19) secondary antibody was incubated at room temperature for 1h, where for anti-S: using Anti-Mouse (1: 5000), the expression vector for Anti-GAPDH: Anti-Rabbit (1: 5000);

(20) eluting the secondary antibody, washing with 1 × TBST (0.05% TWEEN 20) for 10min with shaking, and repeating for 3 times;

(21) and (3) carrying out development detection, namely placing the PVDF film in a developing solution, oscillating and soaking for 2min, and exposing and developing in a developing instrument, wherein the developing solution is prepared as follows: solution I: solution II: water 1: 1: 2;

(22) and analyzing data and results.

A first antibody: SARS-CoV-2(2019-nCoV) nucleopacified Antibody, Rabbit PAb, Antibody Affinity Purified (yield: Sino Biological; Cat:40588-T62) (1:1000)

GAPDH antibody (Manual: Santa; Cat: SC32233) (1:500)

Secondary antibody:

Goat Anti-Rabbit IgG(H+L),HRP Conjugate(Manufacture：TRANS；Cat:HS101-01)(1:5000)

Goat Anti-Mouse IgG(H+L),HRP Conjugate(Manufacture：TRANS；Cat:HS201-01)(1:5000)

the results are shown in FIG. 3, indicating that:

(1)1 mu g/ml DOX induces S tet on cells to express S protein, and the experimental result is consistent with that of example 3;

(2) s tet on cells start to express S protein after being induced by DOX with the concentration of 1 mu g/ml for 8 hours, the expression quantity of the S protein is increased along with the increase of the induction time, and the expression quantity of the S protein is the highest after 24 hours; however, at 32h of DOX induction, the expression level of the full-length S protein is reduced, and the expression level of the S1 protein is up-regulated, which indicates that after 32h induction, a large amount of S protein is self-sheared, and a 110kDa S1 subfin fragment is generated.

Example 5: western blot confirmed that DOX induced S tet on monoclonal cells to express S protein.

Study subjects: s teton monoclonal cell line

The specific experimental steps are as follows:

(1) 1000S tet on cells are planted into a 10cm cell culture dish in a biosafety cabinet, and 8-10ml of screening culture medium (containing 5 mu M screening drug y27632) is added;

(2)37℃5％CO₂culturing;

(3) when the monoclonal cells in the plate were visible (about 1 month), the monoclonal was picked up in a 96-well plate and 200. mu.l of screening medium was added thereto at 37 ℃ with 5% CO₂Culturing;

(4) after the monoclonal cells are spread over more than 80% of the bottom area of the hole, transferring the digested cells to a 48-hole plate for culture, and then sequentially carrying out amplification culture to a 24-hole plate, a 12-hole plate, a 6-cm plate and a 10-cm plate;

(5) 6-well plate intracellular seeding 4.0X 10^5 cells/well, 2.5ml screening culture medium 37 ℃, 5% CO₂Culturing overnight;

(6) removing the original screening culture medium of S teton cells in a 6-well plate in a biological safety cabinet, adding 2.5ml of pre-preheated DOX containing 1 mu g/ml into each well, and recording the induction time as 0 h;

(7) when inducing for 24h, scraping the cells at the bottom of the pore plate by using a scraper, and then transferring the cell suspension into a 15ml centrifugal tube;

(8)3000g, centrifuging at 4 ℃ for 5 min;

(9) discarding the supernatant, adding 1ml of 1 XPBS, 3000g, centrifuging at 4 ℃ for 5 min;

(10) repeating the step (5) once;

(11) the supernatant was discarded and 40. mu.l RIPA buffer (containing 0.4. mu.l 100mM PMSF) was added;

(12) ice-cooling for 30min, and vortexing every 5 min;

(13)14000g of centrifugation is carried out for 5min, and the supernatant is transferred into a 1.5ml centrifuge tube for standby;

(14) taking 20 mu l of a 5mg/ml BSA standard protein sample, adding 180 mu l of 1 XPBS, uniformly mixing by oscillation, and then diluting the mixture into standard proteins with different gradients for later use according to the table shown in the following;

(15) Diluting the extracted protein sample by 4, 6 and 10 times respectively (the volume of the diluent is more than 10 mu l) for later use;

(16) preparing a BCA working solution, and mixing the BCA solution and the Cu solution according to the weight ratio of 50: 1 to prepare a BCA working solution;

(17) a96-well plate with a transparent bottom is taken, and 100. mu.l of BCA working solution and 10. mu.l of a protein sample to be prepared are sequentially added into the 96-well plate (each gradient of a standard protein sample is prepared into 3 times);

(18) incubating at 37 ℃ for 30 min;

(19) measuring the absorbance at the wavelength of 562 nm;

(20) and calculating the protein concentration of the sample to be detected according to the absorbance of the standard protein and the known protein concentration.

(21) Preparing protein electrophoresis gel (8% of separation gel and 5% of concentrated gel); adding 20 μ g protein loading buffer (quantitative 28 μ l), denaturing at 100 deg.C for 5min, and sequentially adding into gel loading well;

(23) glue running, 90V 30min and 120V 2 h; membrane transfer, 200mM 150 min; sealing and standing overnight at 4 ℃;

(24) incubating the primary antibody for 1h at room temperature; wherein the dilution ratio of the antibody is as follows: anti-S (. about.180 kDa) antibody, 1:1000, parts by weight; anti-GAPDH (37KDa) antibody, 1:500, a step of;

(25) eluting primary antibody, adding eluent 1X TBST (0.05% TWEEN 20), washing with shaking for 10min, and repeating for 3 times;

(26) secondary antibody was incubated at room temperature for 1h, with antibody to anti-S: using Anti-Mouse (1: 5000), the Anti-antibody GAPDH: Anti-Rabbit (1: 5000);

(27) eluting the secondary antibody, adding eluent 1X TBST (0.05% TWEEN 20), washing for 10min under shaking, and repeating for 3 times;

(28) and (3) carrying out development detection, namely placing the PVDF film in a developing solution, oscillating and soaking for 2min, and exposing and developing in a developing instrument, wherein the developing solution is prepared as follows: solution I: solution II: water 1: 1: 2;

(29) and analyzing data and results.

Anti-GAPDH antibody (manufacturing: Santa; Cat: SC32233) (1:500)

Anti-β-Tubulin Mouse Monoclonal Antibody(Manufacture：TRANS；Cat:HC101-02)(1:1000)

Secondary antibody:

the results are shown in FIG. 4, indicating that:

(1) culturing S tet on monoclonal cells for 24h by using an induction culture medium containing 1 mu g/ml DOX, and expressing a large amount of S protein;

(2) when the S protein in the cells is expressed in a large quantity, the expression of the cytoskeletal protein Tubulin is sharply reduced to disappear, which indicates that the syncytium phenomenon appears between the cells.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Reference to the literature

bioRxiv preprint doi:https://doi.org/10.1101/2020.03.16.994152；this version posted March 17,2020。

SEQUENCE LISTING

<110> five-membered Biotech Ltd of Nanchang

<120> cell syncytium lesion model based on S-TET protein expression system and preparation method thereof

<130> 2021.07.15

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 3822

<212> DNA

<213> Artificial sequence

<400> 1

atgttcgtgt tcctggtgct gctgccgctg gtgtcctccc aatgcgtgaa ccttaccact 60

cggacccagc tgcctcctgc ttacaccaat agctttacga gaggggtgta ctaccctgac 120

aaggtgttca gaagcagcgt gctgcacagc acccaagacc tgttcctgcc tttcttcagc 180

aatgtgacat ggttccacgc catccacgtg tctgggacta acggcaccaa gcgctttgac 240

aaccccgtgc tgcctttcaa cgatggcgtt tacttcgctt cgaccgaaaa gtccaatatc 300

atccgtggct ggattttcgg cacaaccctg gatagcaaga cccagagcct gctgatcgtg 360

aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccctttt 420

ctgggcgtgt attaccacaa aaacaacaag tcctggatgg aatcagaatt cagagtgtat 480

agcagcgcca acaactgcac gttcgagtac gtcagccagc cttttcttat ggacctggag 540

ggcaagcagg gcaacttcaa aaatctgaga gagttcgtgt tcaagaatat cgatggctac 600

ttcaagatct acagcaagca cacacctatt aacctggttc gggacctgcc ccagggcttt 660

agcgctctgg aacccctggt cgacctgcct atcgggatca acatcaccag atttcagacc 720

ctgctggccc tacacagaag ctacctgacc cctggcgact cttcttccgg atggaccgcc 780

ggcgctgccg cctactacgt gggctacctg cagcctagaa catttctgct gaaatacaac 840

gagaacggca ccatcaccga cgccgtggat tgtgccctgg accccctttc tgagacaaag 900

tgcaccctga aatctttcac cgtggagaag ggcatctacc agaccagcaa cttccgggtc 960

cagcctaccg agtctatcgt acggttccct aacatcacga acctctgtcc ttttggcgag 1020

gtgtttaacg ccacaagatt cgccagcgtg tacgcctgga accggaagag aatcagcaac 1080

tgcgtggctg attacagcgt gctgtacaac agcgcctctt tcagcacatt caagtgttac 1140

ggcgtgtctc ccaccaagct aaacgacctg tgcttcacca acgtgtatgc cgacagcttc 1200

gtgatcaggg gcgacgaggt gcgacagatc gctcctggcc aaacaggcaa gatcgccgac 1260

tacaattaca agctgccaga cgacttcaca ggctgcgtga tcgcctggaa cagcaacaac 1320

ctggacagca aggtgggcgg caactacaac tacctgtacc ggctgtttag aaagagcaac 1380

ctgaagccct tcgagagaga tatcagcacc gagatctacc aggctggcag caccccttgc 1440

aacggcgttg agggcttcaa ctgttatttc ccactccaat cttacggctt ccagcctaca 1500

aatggcgtgg gctaccagcc ttaccgggtg gtcgtgctca gctttgagct gctgcatgcc 1560

cctgccaccg tttgtggccc taagaagagc accaacctgg tgaagaataa atgcgtgaat 1620

ttcaatttca acggcctgac cggcaccggt gtcctgaccg aatccaacaa gaagttcctg 1680

cccttccaac agttcggcag agatatcgcc gacaccaccg atgctgttag agatccccag 1740

accctggaga tcctggacat cacaccctgc tctttcggtg gcgtcagtgt gatcactccc 1800

ggcacaaata ccagcaatca agtggccgtg ctttaccagg atgttaactg tacagaggtc 1860

cccgtggcca tacacgcgga ccagctgacc ccaacatggc gggtgtactc gacaggcagc 1920

aacgtgttcc agacccgcgc gggctgtctg atcggcgccg aacacgttaa caactcctac 1980

gagtgcgata tccctatcgg cgccggcatt tgcgccagct accagaccca gaccaacagc 2040

cctcggagag ctagatctgt ggccagccag tctatcatcg cctacaccat gagtctgggt 2100

gctgaaaact ctgtagcata tagcaataat agcatcgcca tccccaccaa tttcaccatc 2160

agcgtgacca cagaaatcct gcctgtgtct atgaccaaga ccagcgtgga ctgtaccatg 2220

tacatctgcg gcgattccac agaatgctct aacctgctgc tgcagtacgg ctccttttgc 2280

acacagctga acagagccct gaccggaatt gcagtggaac aagacaagaa cacacaggag 2340

gtgttcgctc aggtgaagca gatctacaaa acccctccta tcaaggactt cggcggtttc 2400

aatttcagcc agattctgcc tgatcctagc aaaccttcca agcggagctt catcgaggac 2460

ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcaata cggcgactgc 2520

ctgggagaca tcgccgctcg ggacctgatc tgcgcccaga agtttaacgg cctgaccgtg 2580

ctgcctccac tgttgaccga cgaaatgatc gctcagtaca ccagcgccct gctggccgga 2640

acaatcacca gcggatggac attcggcgcc ggcgccgccc tgcagattcc tttcgctatg 2700

cagatggcct atagattcaa cggaatcgga gtcacccaga acgtgctata cgagaaccag 2760

aaacttatcg ccaaccagtt taactccgcc atcggaaaga tccaggattc cctgagcagc 2820

acagcttctg ccctcggaaa actgcaggac gtggtgaacc aaaacgccca ggccctgaac 2880

accctggtga agcagctgag ctcaaacttc ggcgccatca gctccgtgct caacgacatc 2940

ctgtctagac tggacaaagt ggaagccgag gtgcagatcg accggctgat caccggaaga 3000

ctacagagcc tgcagacata cgtcacccag cagctgatca gagccgccga gattcgcgcc 3060

agcgctaatc tcgccgccac aaagatgagc gaatgtgtgc tgggccagag caagagagtg 3120

gacttctgcg gcaaaggcta ccacctgatg agcttccccc agtctgctcc ccacggcgtg 3180

gtgttcctgc atgtgaccta cgtgccagcc caggagaaga acttcactac agcccctgct 3240

atctgccacg acggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300

cactggttcg tgacccagag aaacttctac gagccccaga tcatcacaac agacaacacc 3360

ttcgtatctg gcaactgcga tgtggtgatc ggaatcgtga ataacaccgt gtacgacccc 3420

ctgcagcctg agctggatag ttttaaagag gaactggaca agtactttaa gaaccacacc 3480

tccccagacg tggacctggg cgatatcagc ggaataaatg cctcagtggt gaacatccag 3540

aaagaaatcg acagactgaa cgaggtcgcc aagaacctga acgagtccct gatcgacctg 3600

caagaactgg gcaagtacga gcagtacatc aagtggcctt ggtacatctg gctgggcttc 3660

atcgctggac tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gacaagctgc 3720

tgtagctgcc tgaagggctg ctgcagctgc ggatcttgtt gcaagttcga cgaagatgat 3780

tccgaacccg tgctgaaggg cgtgaaactc cactacacct ga 3822

<210> 2

<211> 3831

<212> DNA

<213> Artificial sequence

<400> 2

gccaccatgg ccttcgtgtt cctggtgctg ctgccgctgg tgtcctccca atgcgtgaac 60

cttaccactc ggacccagct gcctcctgct tacaccaata gctttacgag aggggtgtac 120

taccctgaca aggtgttcag aagcagcgtg ctgcacagca cccaagacct gttcctgcct 180

ttcttcagca atgtgacatg gttccacgcc atccacgtgt ctgggactaa cggcaccaag 240

cgctttgaca accccgtgct gcctttcaac gatggcgttt acttcgcttc gaccgaaaag 300

tccaatatca tccgtggctg gattttcggc acaaccctgg atagcaagac ccagagcctg 360

ctgatcgtga acaacgccac caacgtggtg atcaaggtgt gcgagttcca gttctgcaac 420

gacccttttc tgggcgtgta ttaccacaaa aacaacaagt cctggatgga atcagaattc 480

agagtgtata gcagcgccaa caactgcacg ttcgagtacg tcagccagcc ttttcttatg 540

gacctggagg gcaagcaggg caacttcaaa aatctgagag agttcgtgtt caagaatatc 600

gatggctact tcaagatcta cagcaagcac acacctatta acctggttcg ggacctgccc 660

cagggcttta gcgctctgga acccctggtc gacctgccta tcgggatcaa catcaccaga 720

tttcagaccc tgctggccct acacagaagc tacctgaccc ctggcgactc ttcttccgga 780

tggaccgccg gcgctgccgc ctactacgtg ggctacctgc agcctagaac atttctgctg 840

aaatacaacg agaacggcac catcaccgac gccgtggatt gtgccctgga ccccctttct 900

gagacaaagt gcaccctgaa atctttcacc gtggagaagg gcatctacca gaccagcaac 960

ttccgggtcc agcctaccga gtctatcgta cggttcccta acatcacgaa cctctgtcct 1020

tttggcgagg tgtttaacgc cacaagattc gccagcgtgt acgcctggaa ccggaagaga 1080

atcagcaact gcgtggctga ttacagcgtg ctgtacaaca gcgcctcttt cagcacattc 1140

aagtgttacg gcgtgtctcc caccaagcta aacgacctgt gcttcaccaa cgtgtatgcc 1200

gacagcttcg tgatcagggg cgacgaggtg cgacagatcg ctcctggcca aacaggcaag 1260

atcgccgact acaattacaa gctgccagac gacttcacag gctgcgtgat cgcctggaac 1320

agcaacaacc tggacagcaa ggtgggcggc aactacaact acctgtaccg gctgtttaga 1380

aagagcaacc tgaagccctt cgagagagat atcagcaccg agatctacca ggctggcagc 1440

accccttgca acggcgttga gggcttcaac tgttatttcc cactccaatc ttacggcttc 1500

cagcctacaa atggcgtggg ctaccagcct taccgggtgg tcgtgctcag ctttgagctg 1560

ctgcatgccc ctgccaccgt ttgtggccct aagaagagca ccaacctggt gaagaataaa 1620

tgcgtgaatt tcaatttcaa cggcctgacc ggcaccggtg tcctgaccga atccaacaag 1680

aagttcctgc ccttccaaca gttcggcaga gatatcgccg acaccaccga tgctgttaga 1740

gatccccaga ccctggagat cctggacatc acaccctgct ctttcggtgg cgtcagtgtg 1800

atcactcccg gcacaaatac cagcaatcaa gtggccgtgc tttaccagga tgttaactgt 1860

acagaggtcc ccgtggccat acacgcggac cagctgaccc caacatggcg ggtgtactcg 1920

acaggcagca acgtgttcca gacccgcgcg ggctgtctga tcggcgccga acacgttaac 1980

aactcctacg agtgcgatat ccctatcggc gccggcattt gcgccagcta ccagacccag 2040

accaacagcc ctcggagagc tagatctgtg gccagccagt ctatcatcgc ctacaccatg 2100

agtctgggtg ctgaaaactc tgtagcatat agcaataata gcatcgccat ccccaccaat 2160

ttcaccatca gcgtgaccac agaaatcctg cctgtgtcta tgaccaagac cagcgtggac 2220

tgtaccatgt acatctgcgg cgattccaca gaatgctcta acctgctgct gcagtacggc 2280

tccttttgca cacagctgaa cagagccctg accggaattg cagtggaaca agacaagaac 2340

acacaggagg tgttcgctca ggtgaagcag atctacaaaa cccctcctat caaggacttc 2400

ggcggtttca atttcagcca gattctgcct gatcctagca aaccttccaa gcggagcttc 2460

atcgaggacc tgctgttcaa caaggtgacc ctggccgatg ccggcttcat caagcaatac 2520

ggcgactgcc tgggagacat cgccgctcgg gacctgatct gcgcccagaa gtttaacggc 2580

ctgaccgtgc tgcctccact gttgaccgac gaaatgatcg ctcagtacac cagcgccctg 2640

ctggccggaa caatcaccag cggatggaca ttcggcgccg gcgccgccct gcagattcct 2700

ttcgctatgc agatggccta tagattcaac ggaatcggag tcacccagaa cgtgctatac 2760

gagaaccaga aacttatcgc caaccagttt aactccgcca tcggaaagat ccaggattcc 2820

ctgagcagca cagcttctgc cctcggaaaa ctgcaggacg tggtgaacca aaacgcccag 2880

gccctgaaca ccctggtgaa gcagctgagc tcaaacttcg gcgccatcag ctccgtgctc 2940

aacgacatcc tgtctagact ggacaaagtg gaagccgagg tgcagatcga ccggctgatc 3000

accggaagac tacagagcct gcagacatac gtcacccagc agctgatcag agccgccgag 3060

attcgcgcca gcgctaatct cgccgccaca aagatgagcg aatgtgtgct gggccagagc 3120

aagagagtgg acttctgcgg caaaggctac cacctgatga gcttccccca gtctgctccc 3180

cacggcgtgg tgttcctgca tgtgacctac gtgccagccc aggagaagaa cttcactaca 3240

gcccctgcta tctgccacga cggcaaggcc cacttcccta gagagggcgt gttcgtgagc 3300

aacggcaccc actggttcgt gacccagaga aacttctacg agccccagat catcacaaca 3360

gacaacacct tcgtatctgg caactgcgat gtggtgatcg gaatcgtgaa taacaccgtg 3420

tacgaccccc tgcagcctga gctggatagt tttaaagagg aactggacaa gtactttaag 3480

aaccacacct ccccagacgt ggacctgggc gatatcagcg gaataaatgc ctcagtggtg 3540

aacatccaga aagaaatcga cagactgaac gaggtcgcca agaacctgaa cgagtccctg 3600

atcgacctgc aagaactggg caagtacgag cagtacatca agtggccttg gtacatctgg 3660

ctgggcttca tcgctggact gatcgccatc gtgatggtga ccatcatgct gtgctgcatg 3720

acaagctgct gtagctgcct gaagggctgc tgcagctgcg gatcttgttg caagttcgac 3780

gaagatgatt ccgaacccgt gctgaagggc gtgaaactcc actacacctg a 3831

<210> 3

<211> 3843

<212> DNA

<213> Artificial sequence

<400> 3

ggatccgcca ccatggcctt cgtgttcctg gtgctgctgc cgctggtgtc ctcccaatgc 60

gtgaacctta ccactcggac ccagctgcct cctgcttaca ccaatagctt tacgagaggg 120

gtgtactacc ctgacaaggt gttcagaagc agcgtgctgc acagcaccca agacctgttc 180

ctgcctttct tcagcaatgt gacatggttc cacgccatcc acgtgtctgg gactaacggc 240

accaagcgct ttgacaaccc cgtgctgcct ttcaacgatg gcgtttactt cgcttcgacc 300

gaaaagtcca atatcatccg tggctggatt ttcggcacaa ccctggatag caagacccag 360

agcctgctga tcgtgaacaa cgccaccaac gtggtgatca aggtgtgcga gttccagttc 420

tgcaacgacc cttttctggg cgtgtattac cacaaaaaca acaagtcctg gatggaatca 480

gaattcagag tgtatagcag cgccaacaac tgcacgttcg agtacgtcag ccagcctttt 540

cttatggacc tggagggcaa gcagggcaac ttcaaaaatc tgagagagtt cgtgttcaag 600

aatatcgatg gctacttcaa gatctacagc aagcacacac ctattaacct ggttcgggac 660

ctgccccagg gctttagcgc tctggaaccc ctggtcgacc tgcctatcgg gatcaacatc 720

accagatttc agaccctgct ggccctacac agaagctacc tgacccctgg cgactcttct 780

tccggatgga ccgccggcgc tgccgcctac tacgtgggct acctgcagcc tagaacattt 840

ctgctgaaat acaacgagaa cggcaccatc accgacgccg tggattgtgc cctggacccc 900

ctttctgaga caaagtgcac cctgaaatct ttcaccgtgg agaagggcat ctaccagacc 960

agcaacttcc gggtccagcc taccgagtct atcgtacggt tccctaacat cacgaacctc 1020

tgtccttttg gcgaggtgtt taacgccaca agattcgcca gcgtgtacgc ctggaaccgg 1080

aagagaatca gcaactgcgt ggctgattac agcgtgctgt acaacagcgc ctctttcagc 1140

acattcaagt gttacggcgt gtctcccacc aagctaaacg acctgtgctt caccaacgtg 1200

tatgccgaca gcttcgtgat caggggcgac gaggtgcgac agatcgctcc tggccaaaca 1260

ggcaagatcg ccgactacaa ttacaagctg ccagacgact tcacaggctg cgtgatcgcc 1320

tggaacagca acaacctgga cagcaaggtg ggcggcaact acaactacct gtaccggctg 1380

tttagaaaga gcaacctgaa gcccttcgag agagatatca gcaccgagat ctaccaggct 1440

ggcagcaccc cttgcaacgg cgttgagggc ttcaactgtt atttcccact ccaatcttac 1500

ggcttccagc ctacaaatgg cgtgggctac cagccttacc gggtggtcgt gctcagcttt 1560

gagctgctgc atgcccctgc caccgtttgt ggccctaaga agagcaccaa cctggtgaag 1620

aataaatgcg tgaatttcaa tttcaacggc ctgaccggca ccggtgtcct gaccgaatcc 1680

aacaagaagt tcctgccctt ccaacagttc ggcagagata tcgccgacac caccgatgct 1740

gttagagatc cccagaccct ggagatcctg gacatcacac cctgctcttt cggtggcgtc 1800

agtgtgatca ctcccggcac aaataccagc aatcaagtgg ccgtgcttta ccaggatgtt 1860

aactgtacag aggtccccgt ggccatacac gcggaccagc tgaccccaac atggcgggtg 1920

tactcgacag gcagcaacgt gttccagacc cgcgcgggct gtctgatcgg cgccgaacac 1980

gttaacaact cctacgagtg cgatatccct atcggcgccg gcatttgcgc cagctaccag 2040

acccagacca acagccctcg gagagctaga tctgtggcca gccagtctat catcgcctac 2100

accatgagtc tgggtgctga aaactctgta gcatatagca ataatagcat cgccatcccc 2160

accaatttca ccatcagcgt gaccacagaa atcctgcctg tgtctatgac caagaccagc 2220

gtggactgta ccatgtacat ctgcggcgat tccacagaat gctctaacct gctgctgcag 2280

tacggctcct tttgcacaca gctgaacaga gccctgaccg gaattgcagt ggaacaagac 2340

aagaacacac aggaggtgtt cgctcaggtg aagcagatct acaaaacccc tcctatcaag 2400

gacttcggcg gtttcaattt cagccagatt ctgcctgatc ctagcaaacc ttccaagcgg 2460

agcttcatcg aggacctgct gttcaacaag gtgaccctgg ccgatgccgg cttcatcaag 2520

caatacggcg actgcctggg agacatcgcc gctcgggacc tgatctgcgc ccagaagttt 2580

aacggcctga ccgtgctgcc tccactgttg accgacgaaa tgatcgctca gtacaccagc 2640

gccctgctgg ccggaacaat caccagcgga tggacattcg gcgccggcgc cgccctgcag 2700

attcctttcg ctatgcagat ggcctataga ttcaacggaa tcggagtcac ccagaacgtg 2760

ctatacgaga accagaaact tatcgccaac cagtttaact ccgccatcgg aaagatccag 2820

gattccctga gcagcacagc ttctgccctc ggaaaactgc aggacgtggt gaaccaaaac 2880

gcccaggccc tgaacaccct ggtgaagcag ctgagctcaa acttcggcgc catcagctcc 2940

gtgctcaacg acatcctgtc tagactggac aaagtggaag ccgaggtgca gatcgaccgg 3000

ctgatcaccg gaagactaca gagcctgcag acatacgtca cccagcagct gatcagagcc 3060

gccgagattc gcgccagcgc taatctcgcc gccacaaaga tgagcgaatg tgtgctgggc 3120

cagagcaaga gagtggactt ctgcggcaaa ggctaccacc tgatgagctt cccccagtct 3180

gctccccacg gcgtggtgtt cctgcatgtg acctacgtgc cagcccagga gaagaacttc 3240

actacagccc ctgctatctg ccacgacggc aaggcccact tccctagaga gggcgtgttc 3300

gtgagcaacg gcacccactg gttcgtgacc cagagaaact tctacgagcc ccagatcatc 3360

acaacagaca acaccttcgt atctggcaac tgcgatgtgg tgatcggaat cgtgaataac 3420

accgtgtacg accccctgca gcctgagctg gatagtttta aagaggaact ggacaagtac 3480

tttaagaacc acacctcccc agacgtggac ctgggcgata tcagcggaat aaatgcctca 3540

gtggtgaaca tccagaaaga aatcgacaga ctgaacgagg tcgccaagaa cctgaacgag 3600

tccctgatcg acctgcaaga actgggcaag tacgagcagt acatcaagtg gccttggtac 3660

atctggctgg gcttcatcgc tggactgatc gccatcgtga tggtgaccat catgctgtgc 3720

tgcatgacaa gctgctgtag ctgcctgaag ggctgctgca gctgcggatc ttgttgcaag 3780

ttcgacgaag atgattccga acccgtgctg aagggcgtga aactccacta cacctgaacg 3840

cgt 3843

<210> 4

<211> 376

<212> DNA

<213> Artificial sequence

<400> 4

tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca gtgatagaga 60

acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt tactccctat 120

cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac gtatctacag 180

tttactccct atcagtgata gagaacgtat atccagttta ctccctatca gtgatagaga 240

acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg tttagtgaac 300

cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac 360

ttcctaccct cgtaaa 376

<210> 5

<211> 1008

<212> DNA

<213> Artificial sequence

<400> 5

atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60

ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc ttggtgtaga gcagcctaca 120

ctgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180

gataggcacc atactcactt ttgcccttta aaaggggaaa gctggcaaga ttttttacgc 240

aataacgcta aaagttttag atgtgcttta ctaagtcatc gcaatggagc aaaagtacat 300

tcagatacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360

tgccaacaag gtttttcact agagaacgcg ttatatgcac tcagcgctgt ggggcatttt 420

actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480

cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540

ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600

cttaaatgtg aaagtgggtc cgcgtacagc cgcgcgcgta cgaaaaacaa ttacgggtct 660

accatcgagg gcctgctcga tctcccggac gacgacgccc ccgaagaggc ggggctggcg 720

gctccgcgcc tgtcctttct ccccgcggga cacacgcgca gactgtcgac ggcccccccg 780

accgatgtca gcctggggga cgagctccac ttagacggcg aggacgtggc gatggcgcat 840

gccgacgcgc tagacgattt cgatctggac atgttggggg acggggattc cccgggtccg 900

ggatttaccc cccacgactc cgccccctac ggcgctctgg atatggccga cttcgagttt 960

gagcagatgt ttaccgatgc ccttggaatt gacgagtacg gtgggtag 1008

Claims

1. A preparation method of a cell syncytium lesion model based on an S-TET protein expression system is characterized by comprising the following steps:

2. The method of claim 1, further comprising a step S3 of adding doxycycline hyclate to the culture medium of the model for growing the syncytium disorder, and inducing mass expression of S protein, thereby generating the cytofusogenic disorder phenomenon.

3. The method of claim 1, wherein the nucleotide sequence of the tet on regulatory factor is as set forth in SEQ ID NO: 5, respectively.

4. The method of claim 1, wherein the screening is: the slow virus expression vector contains puromycin resistance genes, and puromycin is added into a culture medium for screening.

5. The method of claim 1, wherein the lentiviral expression vector is pLent-TRE3G-hPGK-rtTA-SV 40-Puro.

6. A cell syncytium lesion model prepared by the preparation method of claims 1-5.

7. Use of the cell syncytium lesion model of claim 6 for screening and identifying anti-neocoronavirus drugs.

8. The use of claim 7, wherein the screening is to determine whether the screened drug protects cells during cell fusion caused by binding of the S protein to its corresponding cellular receptor protein ACE2 by observing changes in the degree of cell fusion.

9. The use of claim 8, wherein the cell fusion is produced by rapid mass production of S protein induced by doxycycline hyclate.

10. Use according to claim 9, wherein the doxycycline hydrochloride is at a concentration of 0.1 to 1 μ g/ml.