Chromosome cross-species efficient transfer method and application thereof

Abstract

The invention provides a chromosome cross-species efficient transfer method and application thereof, and belongs to the technical field of synthetic biology. According to the method, the digestion and induction time of yeast and the effect-target ratio of co-incubation of the two cells are adjusted, a related signal channel inhibitor is added, and the chromosome transfer efficiency of a co-incubation system based on membrane fusion is optimized, so that the obtained method has practicability, universality and tolerance, the survival rate of fusion cells after chromosome transfer can be increased, and the survival rate of the fusion cells can be increased. The method has good portability and compatibility, can specifically inhibit death signal channels of bottom mammalian cells in a targeting manner, and improves the bearing degree of target cells to heterologous chromosomes. Based on the method, the invention further provides application of the method in cell fusion, gene synthesis and delivery.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a method for efficient chromosome cross-species transfer and its application. Background Art

[0002] Traditional methods of chromosome transfer mainly include microinjection, cationic liposome transfection, PEG-mediated cell fusion and microcell method. However, these technologies have great limitations, such as complex pretreatment steps, extremely low throughput or success rate (less than 5%), uncontrollable fusion process and results, non-selectivity for recipient cells and predictable cytotoxicity. Therefore, it is urgent to develop an efficient cross-species chromosome transfer technology to provide a theoretical basis for the subsequent development of synthetic biology.

[0003] At present, scientists have tried to use improved yeast spheroplasmic fusion instead of the traditional microcell method to transfer chromosomes synthesized in yeast. This method not only avoids the risk of exposing large DNA vectors to shear force by microinjection and liposome methods, but also is not affected by the size of DNA fragments. However, when animal cells fuse with yeast cells, the presence of fungi around the animal cells will activate the immune defense pathways in the cells. A series of cellular immunity and cell death events activated when a large number of yeast cells and mammalian cells are co-incubated will affect the efficiency of chromosome transfer, and the specific mode of cell death is still unclear. Therefore, optimizing the co-incubation conditions and how to avoid large-scale cell death are very important for the efficiency of cross-species chromosome transfer technology. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the object of the present invention is to provide a method for efficient chromosome transfer across species, which can optimize the chromosome transfer efficiency of the co-incubation system based on membrane fusion.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for efficient cross-species transfer of chromosomes, comprising the following steps: yeast cells are digested with yeast lytic enzymes to obtain yeast cell protoplasts, the yeast cell protoplasts are co-cultured with animal cells, and an apoptosis inhibitor Z-VAD-FMK or a necrosis inhibitor Necrostatin-1 is added during the co-culture; the yeast cells contain plasmid elements expressing human ACE2; and the animal cells contain plasmid elements expressing the S protein of SARS.

[0007] Preferably, the working concentration of the apoptosis inhibitor Z-VAD-FMK is 10-40 μM; the working concentration of the necrosis inhibitor Necrostatin-1 is 100-400 μM.

[0008] Preferably, the digestion time is 10 to 60 minutes, and the working concentration of the yeast cell wall-lytic enzyme is 60 to 300 U / mL.

[0009] Preferably, before the co-incubation culture, the yeast cell protoplasts are added with antifungal drugs for culture; the antifungal drugs are 0-25 ng / μL of caspofungin + 0-25 ng / μL of micafungin.

[0010] Preferably, the effector-target ratio of the yeast cell protoplasts to the animal cells is 1 to 100:1.

[0011] Preferably, the plasmid elements expressing human ACE2 also include an EGFP fluorescent reporter element, a GAL1 promoter element, a CYC1 terminator element and an IRPF fluorescent reporter element.

[0012] Preferably, the yeast cell comprises Saccharomyces cerevisiae BY4742.

[0013] Preferably, the animal cell is selected from any one of Hep3B, PLC, MCF7, HEK293T, Hela and U2OS.

[0014] The present invention also provides application of the method in fusion of yeast cells and animal cells.

[0015] The present invention also provides application of the above method in gene synthesis and gene delivery.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The present invention optimizes the chromosome transfer efficiency of the co-incubation system based on membrane fusion by adjusting the digestion and induction time of yeast, the effect-target ratio of the co-incubation of two cells, and adding relevant signal pathway inhibitors. The method of the present invention is practical and can increase the survival rate of fused cells after chromosome transfer; the method of the present invention is universal and has good transplantability and compatibility, and can be applied to various cells in theory; the method of the present invention is tolerable, and the necrosis and apoptosis inhibitors can specifically target and inhibit the death signal pathway of the underlying mammalian cells, thereby improving the tolerance of the target cells to the heterologous chromosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Protoplast wall removal at different digestion times;

[0019] Figure 2 : Fluorescence expression intensity and quantity of yeast at different induction times in different treatment groups;

[0020] Figure 3:Cell fusion effects of different effector-target ratios;

[0021] Figure 4 : Effects of death-related pathway inhibitors on cell fusion. DETAILED DESCRIPTION

[0022] The present invention provides a method for efficient chromosome cross-species transfer, comprising the following steps: yeast cells are digested with yeast lytic enzymes to obtain yeast cell protoplasts, yeast cell protoplasts and animal cells are co-incubated and cultured, and apoptosis inhibitor Z-VAD-FMK or necrosis inhibitor Necrostatin-1 is added during co-incubation and culture; the yeast cells of the present invention contain plasmid elements expressing human ACE2; the animal cells contain plasmid elements expressing SARS S protein, and the S protein is preferably a D614G point mutation S protein. As an optional embodiment, the vector of the plasmid element expressing human ACE2 of the present invention is pYES2, and the vector of the plasmid element expressing SARS S protein is pSecTag2-HygroA. The construction method of the plasmid element of the present invention is preferably obtained by a homologous recombination kit. When human ACE2 protein is expressed on the yeast cell membrane and D614G point mutation S protein is expressed on the animal cell membrane, the two proteins interact with each other, and based on this, the addition of related signal pathway inhibitors can reduce cell death events in a short time, and effectively optimize the chromosome transfer efficiency of the co-incubation system based on membrane fusion. Yeast cells and animal cells can complete membrane fusion in a relatively short period of time.

[0023] The working concentration of the apoptosis inhibitor Z-VAD-FMK of the present invention is 10-40 μM, preferably 20 μM; the working concentration of the necrosis inhibitor Necrostatin-1 is 100-400 μM, preferably 200 μM. The present invention adds the apoptosis inhibitor Z-VAD-FMK or the necrosis inhibitor Necrostatin-1.

[0024] As an optional embodiment, the yeast cells of the present invention are expanded and cultured in SD two-deficient medium containing ampicillin and kanamycin, the culture temperature is 29-31°C, and the culture time is 10-14h. The present invention performs a wall-breaking treatment on the cultured yeast cells to obtain yeast cell protoplasts, preferably, the yeast cells are digested with yeast wall-dissolving enzyme to obtain yeast cell protoplasts. The working concentration of the yeast wall-removing enzyme of the present invention is 60-300U / mL, and the digestion time is 10-60min, preferably 30-45min.

[0025] The present invention adds antifungal drugs to the yeast protoplast culture system to prevent the self-repair of the yeast cell wall. The antifungal drugs are caspofungin and micafungin. The dosage of caspofungin is 0-25 ng / μL, more preferably 12.5 ng / μL. The dosage of micafungin is 0-25 ng / μL, more preferably 12.5 ng / μL.

[0026] The effector-target ratio of the yeast cell protoplasts and animal cells of the present invention is 1 to 100:1, preferably 10:1 or 30:1.

[0027] Preferably, the plasmid elements expressing human ACE2 of the present invention further include an EGFP fluorescent reporter element, a GAL1 promoter element, a CYC1 terminator element and an IRPF fluorescent reporter element.

[0028] The yeast cell of the present invention includes Saccharomyces cerevisiae BY4742, and the animal cell is selected from any one of Hep3B, PLC, MCF7, HEK293T, Hela and U2OS.

[0029] The present invention also provides the application of the above method in the fusion of yeast cells and animal cells, gene synthesis and gene delivery.

[0030] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In a specific embodiment of the present invention, the nucleotide sequence of the plasmid element containing human ACE2 expression (denoted as pYES2CT-Y-ACE2-EGFP-CMV-IRFP) is shown in SEQ ID NO. 1; the plasmid element containing SARS S protein (S protein with D614G point mutation) is shown in SEQ ID NO. 2. Yeast lytic enzyme was purchased from Shanghai Maokang Biological Company; the yeast strain was BY4742, which is a commonly used Saccharomyces cerevisiae strain.

[0032] In a specific embodiment of the present invention, the relevant reagents are:

[0033] Buffer A: 1M sorbitol, 25mM ethylenediaminetetraacetic acid (EDTA), 50mM dithiothreitol (DTT), pH 8. 9.5mL H-Buffer C + 0.5mL 1M DTT to obtain 10ml H-Buffer A.

[0034] H-Buffer B: 1M sorbitol. Add 9.1g sorbitol to 50mL, dissolve and filter through a 0.22μm filter membrane.

[0035] H-Buffer C: 1M sorbitol, 25mM EDTA. That is, add 9.1g sorbitol to 50mL. Add 6.25mL mother solution (200mM EDTA) to every 50mL EDTA, adjust the pH to 8.0, and filter with a 0.22μm filter membrane.

[0036] Buffer D: Zymolyase in PB, 300 U / mL, 2.74 mg / mL. The enzyme buffer can be stored at 4°C for two weeks and at -20°C for several months.

[0037] Buffer E: 1M sorbitol, 1mM EDTA, 10mM sodium citrate. That is, add 9.1g sorbitol to 50mL. Add 250μL mother solution (200mM EDTA) to every 50mL of EDTA, add 5mL sodium citrate mother solution (100mM SC sodium citrate), adjust the pH to 5.8, and filter with a 0.22μm filter membrane.

[0038] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] 1. Optimization of yeast digestion conditions:

[0042] Take the yeast and add it to SD-Leu-Ura conditional deficiency medium (purchased from Solebao, catalog number S1090) containing ampicillin (working concentration of 50ng / μL) and kanamycin (working concentration of 50ng / μL) for expansion culture. The carbon source in the expansion culture medium is only glucose. The expansion culture is carried out in a 30°C incubator at 250rpm. After the bacterial solution becomes turbid (12h), the absorbance OD at 600nm is measured on a microplate reader. During the detection, the yeast is diluted to ensure that the monitoring value is within the range of 0.5 to 0.9 to reduce the detection error. The yeast is diluted 2 times, and the OD 600 The value is 0.88.

[0043] Take 4OD of yeast for subsequent cell wall breaking operation, and centrifuge at 1500g for 10 minutes at room temperature to collect the yeast. Resuspend the yeast cells in 3ml of pure water and centrifuge at 1500g for 5 minutes at room temperature, and discard the supernatant. Resuspend the precipitate in Buffer A and incubate at 30℃ for 45min to remove mannan. Then centrifuge at 1500g for 5min at room temperature. Resuspend the precipitate in 400 microliters of Buffer B, 1500g, 5min, room temperature. Resuspend the precipitate in Buffer E containing Buffer D (20μL of Buffer D was diluted with 1000μL of Buffer E), 30℃, set 30min, 45min, and 60min time points, digest with yeast cell wall enzyme, and then observe the cell wall and cytoplasm of the prepared protoplasts under a line scanning laser confocal microscope to observe the protoplast wall removal. The results are as follows Figure 1 The results showed that the best wall removal effect was achieved when the yeast wall-lytic enzyme was used for digestion for 45 min, and the formed protoplasts accounted for 74% of the total yeast.

[0044] 2. Optimization of yeast induced expression conditions:

[0045] Take the yeast and add it to SD two-deficient medium (purchased from Solebo) containing ampicillin (working concentration of 50ng / μL) and kanamycin (working concentration of 50ng / μL) for expansion culture. The carbon source in the expansion culture medium is only glucose, and the expansion culture is carried out in a 30°C incubator at 250rpm. After the bacterial solution becomes turbid (12h), the absorbance OD at 600nm is measured on an ELISA instrument. During the detection, the yeast is diluted to ensure that the monitoring value is within the range of 0.5 to 0.9 to reduce the detection error. The yeast is diluted 2 times, and the OD600 value is 0.88.

[0046] Take 4OD of yeast for subsequent cell wall breaking operation, centrifuge at 1500g for 10min at room temperature to collect yeast. Resuspend the yeast cells in 3ml of pure water and centrifuge at 1500g for 5min at room temperature, discard the supernatant. Resuspend the precipitate in BufferA and incubate at 30℃ for 45min to remove mannan. Then centrifuge at 1500g for 5min at room temperature. Resuspend the precipitate in 400 microliters of Buffer B, 1500g, 5min, room temperature. Resuspend the precipitate in Buffer E containing Buffer D (dilute 20μL of Buffer D with 1000μL of Buffer E) and incubate at 30℃ for 60min. Gently shake the centrifuge tube during this period. Collect protoplasts at 350g for 10min, resuspend the cells with 300 microliters of Buffer E, and wash once. The protoplasts were collected by centrifugation at 350 g for 5 min and added to 6 mL of SD medium containing raffinose (working concentration of 5 g / L) and galactose (working concentration of 10 g / L) as carbon sources, and antifungal drugs were added.

[0047] The treatment and induction dosing schemes of each group of yeast were:

[0048] NC group: yeast control group without any treatment;

[0049] Induce group: a yeast control group without wall removal, induced by raffinose and galactose, and without antifungal drugs;

[0050] ZI group: yeast control group with wall removal, no raffinose and galactose induction, and no antifungal drugs;

[0051] ZI+Mica 12.5 group: wall removal, raffinose and galactose induction, antifungal drug was Micafungin 12.5ng / μL;

[0052] ZI+Mica 25 group: wall removal, raffinose and galactose induction, antifungal drug was Micafungin 25ng / μL;

[0053] ZI+Mica 50 group: wall removal, raffinose and galactose induction, antifungal drug was Micafungin 50ng / μL;

[0054] ZI+Cas 12.5 group: wall removal, raffinose and galactose induction, antifungal drug is Caspofungin (Caspofungin) 12.5ng / μL;

[0055] ZI+Cas 25 group: wall removal, raffinose and galactose induction, antifungal drug is Caspofungin (Caspofungin) 25ng / μL;

[0056] ZI+Cas 50 group: wall removal, raffinose and galactose induction, antifungal drug was Caspofungin 50ng / μL;

[0057] ZI+Mica+Cas 12.5 group: wall removal, raffinose and galactose induction, antifungal drugs were Caspofungin 12.5ng / μL + Micafungin 12.5ng / μL;

[0058] ZI+Mica+Cas 25 group: wall removal, raffinose and galactose induction, antifungal drugs were Caspofungin 25ng / μL + Micafungin 25ng / μL.

[0059] When the yeast cells were induced and cultured for 0h, 12h, 24h, 36h, 48h, 60h, and 72h, the expression level of the element in the yeast was analyzed by flow cytometry. The EGFP fluorescence intensity was detected and the yeast cells were counted. The results are shown in Figure 2. Figure 2 The results show that the optimal induction time for yeast is 36h, and the addition of 12.5ng / μL caspofungin and 12.5ng / μL micafungin has the best effect.

[0060] Example 2

[0061] Optimization of the fusion ratio of yeast cells and animal cells:

[0062] 1. Bottom cell plating

[0063] Hep3B cells with 80-90% confluence were selected, digested and counted, and inoculated in a six-well plastic plate at 250,000 / well. The cells were given 12 hours of growth time. After the cells were stably attached to the wall, the D614G point mutation S protein expression plasmid (shown in SEQ ID NO.2) was transfected into the bottom cells using PEI transfection reagent (purchased from polyplus): For six-well plate cells, each empty transfection system was as follows: First, 1 μg of the functional plasmid—D614G point mutation S protein expression plasmid—was diluted with 200 μL buffer. Vortex for 10 seconds and spin for 1 second. Then add 2 μL of PEI reagent preparation and incubate for 15 minutes. Then add it directly to the cell culture medium. Change the medium after 8 hours.

[0064] 36 hours after cell transfection, the bottom cells were re-plated. The cells in the six-well plate were digested and counted, and 150,000 cells per well were plated in a 24-well glass bottom plate. The glass bottom plate was incubated with collagen in advance.

[0065] 2. Yeast wall removal

[0066] Yeast was taken and transformed using the Frozen-EZ Yeast Transformation II Kit. The plasmid used for transformation was pYES2CT-Y-ACE2-EGFP-CMV-IRFP (shown in SEQ ID NO.1).

[0067] Yeast was added to SD medium (purchased from Solebol) containing working concentrations of ampicillin (50 ng / μL) and kanamycin (50 ng / μL) for expansion culture. The carbon source in the expansion culture medium was glucose only, and the culture was expanded in a 30°C incubator at 250 rpm. After the bacterial solution became turbid (after 12 hours), the absorbance OD at 600 nm was measured using an ELISA reader.

[0068] Take 4OD of yeast for subsequent cell wall breaking operation, centrifuge at 1500g for 10min at room temperature to collect yeast. Resuspend the yeast cells in 3ml of pure water and centrifuge at 1500g for 5min at room temperature, discard the supernatant. Resuspend the precipitate in BufferA and incubate at 30℃ for 45min to remove mannan. Then centrifuge at 1500g for 5min at room temperature. Resuspend the precipitate in 400 microliters of Buffer B, 1500g, 5min, room temperature. Resuspend the precipitate in Buffer E containing Buffer D (dilute 20μL of Buffer D with 1000μL of Buffer E) and incubate at 30℃ for 60min. Gently shake the centrifuge tube during this period. Collect protoplasts at 350g for 10min, resuspend the cells with 300 microliters of Buffer E, and wash once. Collect protoplasts by centrifugation at 350g for 5min and add them to 6ml of SD medium with carbon sources of raffinose (working concentration of 5g / L) and galactose (working concentration of 10g / L). Antifungal drugs are added at the same time to induce yeast to express fusion protein while inhibiting yeast from repairing its own cell wall.

[0069] Yeast induction dosing regimen: Caspofungin and Micafungin, 12.5 ng / μL each;

[0070] 3. Co-incubation and observation

[0071] After 36 hours of yeast protoplast induction, the yeast OD was measured by microplate reader. 600 After the yeast quantification is completed, the protoplasts are collected by centrifugation at 350g for 10 minutes at room temperature, and the yeast protoplast cell suspension is obtained by resuspending with DMEM medium. The yeast suspension is inoculated into the glass plate according to the inoculation ratio of 1:1, 10:1, 30:1, and 100:1, respectively. The fluorescence is collected and photographed using a living cell workstation while maintaining the co-incubation system at 37°C and 5% CO2, with one frame taken every hour. The photography results and cell fusion rate results at 24 hours are statistically shown as follows: Figure 3 The results showed that the cell fusion rate was 57.2200% when the effector-target ratio was 30:1, the cell fusion rate was 50.6667% when the effector-target ratio was 100:1, the cell fusion rate was 37.0533% when the effector-target ratio was 1:1, and the cell fusion rate was 38.5667% when the effector-target ratio was 10:1. This shows that in this co-incubation system, the fusion effect is best when the yeast: cell effector-target ratio is 30:1.

[0072] Example 3

[0073] Optimization of inhibitors of death-related signaling pathways during fusion of yeast cells and animal cells:

[0074] 1. Bottom cell plating

[0075] Select MCF7 or HEK293T cells with 80-90% confluence, digest and count, and inoculate 250,000 / well in a plastic bottom six-well plate. Give the cells 12 hours of growth time, wait for the cells to stably adhere to the wall, and use PEI transfection reagent (purchased from polyplus) to transfect the bottom cells with D614G point mutation S protein expression plasmid (shown in SEQ ID NO.2): For six-well plate cells, each empty transfection system is as follows: First dilute 1μg of functional plasmid-D614G point mutation S protein expression plasmid with 200μL buffer. Vortex for 10s and spin for 1s. Then add 2μL PEI reagent preparation for 15min incubation. Then add directly to the cell culture medium. Change the medium after 8 hours.

[0076] 36 hours after cell transfection, the bottom cells were re-plated. The cells in the six-well plate were digested and counted, and 150,000 cells per well were plated in a 24-well glass bottom plate. The glass bottom plate was incubated with collagen in advance.

[0077] 2. Yeast wall removal

[0078] Yeast was taken and transformed using the Frozen-EZ Yeast Transformation II Kit. The plasmid used for transformation was pYES2CT-Y-ACE2-EGFP-CMV-IRFP (shown in SEQ ID NO.1).

[0079] Yeast was added to SD-Leu-Ura medium (purchased from Solebol) containing working concentrations of ampicillin (50 ng / μL) and cannabinoids (50 ng / μL) for expansion culture. The carbon source in the expansion culture medium was glucose only, and the culture was expanded in a 30°C incubator at 250 rpm. After the bacterial solution became turbid (about 12 to 16 hours later), the absorbance OD at 600 nm was measured using an ELISA reader.

[0080] Take 4OD of yeast for subsequent cell wall breaking operation, centrifuge at 1500g for 10min at room temperature to collect yeast. Resuspend the yeast cells in 3ml of pure water and centrifuge at 1500g for 5min at room temperature, discard the supernatant. Resuspend the precipitate in BufferA and incubate at 30℃ for 45min to remove mannan. Then centrifuge at 1500g for 5min at room temperature. Resuspend the precipitate in 400 microliters of Buffer B, 1500g, 5min, room temperature. Resuspend the precipitate in Buffer E containing Buffer D (dilute 20μL of Buffer D with 1000μL of Buffer E) and incubate at 30℃ for 60min. Gently shake the centrifuge tube during this period. Collect protoplasts at 350g for 10min, resuspend the cells with 300 microliters of Buffer E, and wash once. Collect protoplasts by centrifugation at 350g for 5min and add them to 6ml of SD medium with carbon sources of raffinose (working concentration of 5g / L) and galactose (working concentration of 10g / L). Antifungal drugs are added at the same time to induce yeast to express fusion protein while inhibiting yeast from repairing its own cell wall.

[0081] Yeast induction dosing regimen: Caspofungin and Micafungin, 12.5 ng / μL each.

[0082] 3. Co-incubation and observation

[0083] After 36 hours of yeast protoplast induction, the yeast OD was measured by microplate reader. 600 After the yeast quantification was completed, the protoplasts were collected by centrifugation at 350 g for 10 min at room temperature and resuspended in DMEM medium to obtain a cell suspension of yeast protoplasts.

[0084] The NC group was a co-incubation system with only yeast protoplasts added, the experimental group (Z-VAD-FMK) was a system in which yeast suspension and a normal working concentration of apoptosis inhibitor Z-VAD-FMK (20 μM) were added simultaneously, and the experimental group (Nec-1) was a system in which yeast suspension and a normal working concentration of necrosis inhibitor Necrostatin-1 (200 μM) were added simultaneously. In the co-incubation culture system, the ratio of the number of yeast to the number of bottom cells was 10:1. A living cell workstation was used to collect and photograph fluorescence under the culture conditions of 37°C and 5% CO2 in the co-incubation system. One frame was taken every hour. The shooting results and statistical results at the 24th hour are shown in the figure. Figure 4As shown. The results showed that for MCF7 cells, the chromosome transfer efficiency of the control group (NC group) was 52.3646%, the chromosome transfer efficiency of the Z-VAD-FMK group was 62.2692%, and the chromosome transfer efficiency of the Nec-1 group was 56.9222%; for HEK293T cells, the chromosome transfer efficiency of the control group (NC group) was 37.6480%, the chromosome transfer efficiency of the Z-VAD-FMK group was 40.7319%, and the chromosome transfer efficiency of the Nec-1 group was 37.9550%. This shows that by adding death-related pathway inhibitors, cell death events can be reduced in a short period of time.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for efficient chromosome cross-species transfer, characterized in that: The following steps are involved: Yeast cells are digested with yeast lytic enzyme to obtain yeast cell protoplasts, and the yeast cell protoplasts are co-cultured with animal cells, and apoptosis inhibitor Z-VAD-FMK or necrosis inhibitor Necrostatin-1 is added during the co-culture; The yeast cells contain plasmid elements for expressing human ACE2; the animal cells contain plasmid elements for expressing SARS S protein.

2. The method according to claim 1, characterized in that The working concentration of the apoptosis inhibitor Z-VAD-FMK is 10-40 μM; the working concentration of the necrosis inhibitor Necrostatin-1 is 100-400 μM.

3. The method according to claim 1, characterized in that The digestion time is 10 to 60 minutes, and the working concentration of the yeast lytic enzyme is 60 to 300 U / mL.

4. The method according to claim 1, characterized in that: Before the co-incubation culture, the yeast cell protoplasts are added with antifungal drugs for culture; the antifungal drugs are 0-25 ng / μL of caspofungin + 0-25 ng / μL of micafungin.

5. The method according to claim 1, characterized in that The effector-target ratio of the yeast cell protoplasts and the animal cells is 1-100:

1.

6. The method according to claim 1, characterized in that The plasmid elements expressing human ACE2 also include an EGFP fluorescent reporter element, a GAL1 promoter element, a CYC1 terminator element and an IRPF fluorescent reporter element.

7. The method according to claim 1, characterized in that The yeast cell includes Saccharomyces cerevisiae BY4742.

8. The method according to claim 1, characterized in that The animal cell is selected from any one of Hep3B, PLC, MCF7, HEK293T, Hela and U2OS.

9. Use of the method according to any one of claims 1 to 8 in the fusion of yeast cells and animal cells.

10. Use of the method according to any one of claims 1 to 8 in gene synthesis and gene delivery.

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