Expression system for mediating fusion of yeast cells and animal cells and application of expression system

Abstract

The invention provides an expression system for mediating fusion of yeast cells and animal cells and application of the expression system, and belongs to the technical field of synthetic biology. The expression system comprises a plasmid element A and a plasmid element B. The plasmid element A is a plasmid element for expressing human-derived ACE2, and the nucleotide sequence of the human-derived ACE2 is as shown in SEQ ID NO. 1. The plasmid element B is a plasmid element for expressing S protein of the SARS; the S protein is an S protein with D614G point mutation, and the nucleotide sequence of the S protein is shown as SEQ ID NO. 2. The expression system has high efficiency, universality, usability, controllability, tolerance and targeting, the invention further provides a method for transferring the yeast chromosome to the animal cell by using the expression system, and the fusion rate of the yeast cell and the animal cell can reach more than 50%.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to an expression system for mediating the fusion of yeast cells and animal cells and an application thereof. Background Art

[0002] With the development in the past 20 years, synthetic biology has made great progress in the field of gene sequence synthesis. Synthesis as small as a few thousand base pairs and sequence assembly as large as tens of millions of base pairs have been reported, and synthetic genomics is entering the era of artificial total chromosome synthesis. However, as the length of gene sequences that can be assembled by humans gradually approaches the level of human chromosomes, more theoretical gaps and technical difficulties begin to emerge. Due to the slow growth rate of human cells, insufficient DNA recombination ability, and low transformation efficiency, it is difficult to assemble the genome directly in the target cells. Yeast is currently the main model organism for artificial chromosome assembly due to its powerful recombination ability and amazing tolerance to chromosome length. The mammalian chromosomes assembled and synthesized in yeast ultimately need to be transferred into mammalian cells for functional verification and genetic manipulation. Therefore, the delivery of large DNA vectors (>100,000 bp) is still a limiting step in the fields of mammalian cell engineering and human artificial chromosomes (HACs) development.

[0003] At present, the 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 flux or success rate (less than 5%), uncontrollable fusion process and results, non-selectivity for recipient cells and predictable cytotoxicity. Therefore, the development of efficient yeast chromosome cross-species transfer technology plays an important role in the subsequent development of synthetic biology. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide an expression system for mediating the fusion of yeast cells and animal cells, which has high efficiency, versatility, ease of use, controllability, tolerance and targeting.

[0005] The present invention also aims to provide a method for transferring yeast chromosomes to animal cells using the expression system, and the fusion rate of yeast cells and animal cells can reach more than 50%.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an expression system for mediating the fusion of yeast cells and animal cells, comprising a plasmid element A and a plasmid element B, wherein the plasmid element A is a plasmid element for expressing human ACE2, and the nucleotide sequence of the human ACE2 is shown in SEQ ID NO.1; the plasmid element B is a plasmid element for expressing the S protein of SARS; the S protein is an S protein with a D614G point mutation, and the nucleotide sequence is shown in SEQ ID NO.2.

[0008] Preferably, the vector of the plasmid element A is pYES2, and the vector of the plasmid element B is pSecTag2-HygroA.

[0009] Preferably, in the plasmid element A, the 3' end of human ACE2 is connected to the fluorescent reporter gene EGFP, and the nucleotide sequence of the ACE2-EGFP fragment is shown in SEQ ID NO.3.

[0010] Preferably, the plasmid element A further comprises a GAL1 promoter element, a CYC1 terminator element and an IRPF fluorescent reporter element.

[0011] The present invention also provides the application of the above expression system in mediating the fusion of yeast cells and animal cells.

[0012] The present invention also provides a method for transferring yeast chromosomes to animal cells. The above expression system is used to transfect plasmid element A in yeast cells and plasmid element B in animal cells. The transfected yeast cells are used to prepare yeast protoplasts. The yeast protoplasts are co-incubated with the transfected animal cells to complete the transfer of chromosomes from yeast cells to animal cells.

[0013] Preferably, before co-incubation, the yeast protoplasts are cultured with galactose and raffinose as carbon sources, the working concentration of galactose is 2.5-7.5 g / L, and the working concentration of raffinose is 5-15 g / L.

[0014] Preferably, an antifungal drug is added during the yeast protoplast culture.

[0015] Preferably, the co-incubation time is 12 to 36 hours.

[0016] The present invention also provides the application of the above expression system or the above method in gene synthesis and gene delivery.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The S protein derived from the new coronavirus (SARS-COV-2) can effectively mediate cell fusion after recognizing the ACE2 receptor. This process can occur directly on the cell surface and does not rely on the endosome-lysosome pathway. After the present invention introduces the ACE2 receptor protein and the S protein after point mutation modification into yeast cells and animal cells respectively, the two cells can complete membrane fusion in a relatively short time, and the fusion rate between cells can reach up to 60%, which is highly efficient.

[0019] The expression system provided by the present invention is universal, has good transplantability and compatibility, and can theoretically target various cells; the expression system provided by the present invention is easy to use, and the transfer system only requires the expression of a pair of proteins, with little impact on other components; the expression system provided by the present invention is controllable, and the fusion time can be controlled by adding a carbon source to the culture medium; the expression system provided by the present invention is tolerant, and cell-to-cell fusion is not affected by the transferred substance, and can be competent for multiple or even full sets of chromosome transfer; the expression system provided by the present invention is targeted, and by selecting and modifying the envelope protein, the recognition of the specific receptor protein is realized, and the selective transfer of chromosomes targeting specific cells is realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Sequencing results of pYES2CT-Y-ACE2-EGFP-CMV-IRFP plasmid;

[0021] Figure 2 : Sequencing results of D614G point mutation S protein expression plasmid;

[0022] Figure 3 :Staining results of yeast protoplasts;

[0023] Figure 4 :Fluorescence characterization after fusion of yeast cells and animal cells, a. Microscopic fluorescence imaging of chromosomes in yeast cells delivered to different target cells, namely HEK293T, MCF7 and MCF10A; b. Quantitative results of chromosome transfer efficiency. DETAILED DESCRIPTION

[0024] The present invention provides an expression system for mediating the fusion of yeast cells and animal cells, including plasmid elements A and plasmid elements B, wherein the plasmid element A is a plasmid element for expressing human ACE2, and the nucleotide sequence of the human ACE2 is shown in SEQ ID NO.1; the plasmid element B is a plasmid element for expressing the S protein of SARS; the S protein is a D614G point mutated S protein, and the nucleotide sequence is shown in SEQ ID NO.2. The nucleotide sequence of the human ACE2 of the present invention is obtained by codon optimization of the CDS region of the humanized ACE2 element, the amino acid sequence of the human ACE2 is shown in SEQ ID NO.4, and the amino acid sequence of the D614G point mutated S protein is shown in SEQ ID NO.5. The present invention performs a point mutation (D614G) on the fusion element S protein to obtain a fusion system with stronger fusion ability and wider targeting. In the expression system of the present invention, the position of humanized ACE2 expression is on the cell membrane of yeast, and the position of S protein expression is on the cell membrane of animal cells. Through the interaction of the two proteins, the yeast cells and animal cells complete membrane fusion in a relatively short time.

[0025] The vector of the plasmid element A of the present invention is pYES2, and the vector of the plasmid element B is pSecTag2-HygroA.

[0026] In the plasmid element A of the present invention, the 3' end of human ACE2 is connected to the fluorescent reporter gene EGFP, the nucleotide sequence of the ACE2-EGFP fragment is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.6. Preferably, the plasmid element A of the present invention also includes a GAL1 promoter element, a CYC1 terminator element and an IRPF fluorescent reporter element. In the present invention, the inducible promoter GAL1 is added to the front end of the fusion element ACE2 accessed in yeast, and the expression or stop of ACE2 can be regulated by switching the carbon source of yeast culture, and the fusion of yeast cells and animal cells can be controlled in time. The present invention constructs different reporter genes connected to yeast and mammalian cell specific promoters, respectively, and uses the reporter gene EGFP to carry the yeast inducible specific promoter GAL1 to mark the expression of the fusion element in yeast, and uses the reporter gene IRFP to carry the mammalian cell specific strong promoter CMV to mark the fusion event, and the occurrence and progress of the fusion event between cells are determined by the temporal expression of different reporter genes.

[0027] The present invention also provides the use of the above expression system in mediating the fusion of yeast cells and animal cells. The yeast cells of the present invention are preferably cerevisiae cells, and the animal cells are preferably mammalian cells.

[0028] The present invention also provides a method for transferring yeast chromosomes to animal cells. The above expression system is used to transfect plasmid element A in yeast cells and plasmid element B in animal cells. The transfected yeast cells are used to prepare yeast protoplasts. The yeast protoplasts are co-incubated with the transfected animal cells to complete the transfer of chromosomes from yeast cells to animal cells.

[0029] The present invention transfects plasmid element A in yeast cells, and the transfection method of plasmid element A can be conventional methods in the art. The yeast cells contain gene sequences that need to be transferred to animal cells. The transfer of plasmid element A in the present invention has almost no effect on other components of yeast cells, and is only used to express human ACE2 for subsequent cell fusion. The present invention transfects plasmid element B in animal cells, and the transfection method of plasmid element B can be conventional methods in the art. The transfer of plasmid element B in the present invention has almost no effect on other components of animal cells, and is only used to express S protein for subsequent cell fusion.

[0030] The present invention removes the cell wall and induces expression of yeast cells after transfection of plasmid element A. Glucose is used as a carbon source to maintain the basic growth of yeast; sorbitol, yeast cell wall removing enzyme and other reagents are used to remove the yeast cell wall to eliminate the interference of the cell wall on the fusion process; the carbon source is replaced with raffinose and galactose to activate the inducible promoter GAL1 to start the membrane-localized fusion expression of the fusion element ACE2 and the fluorescent reporter gene, and fungal inhibitors are added to prevent the self-repair of the yeast cell wall.

[0031] Preferably, the culture medium of the present invention is a double-deficient SD culture medium, the working concentration of galactose in the culture medium is 2.5-7.5 g / L, preferably 5 g / L, the working concentration of raffinose is 5-15 g / L, preferably 10 g / L; the fungal inhibitor includes caspofungin and / or micafungin; the added concentration of the fungal inhibitor is 0-25 ng / μL, preferably 12.5-15 ng / μL. The co-incubation time is 12-36 h, preferably 24 h.

[0032] The present invention also provides the application of the above expression system or the above method in gene synthesis and gene delivery.

[0033] 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.

[0034] In the specific embodiment of the present invention, vectors pYES2, pQCXIP-IRFP, and pSecTag2-Hygro A were purchased from Youbao Biotechnology Co., Ltd.; plasmid WHC2115669-ACE2-EGFP_in_pYES2_CT_BamHI_EcoRI was purchased from Qingke Co., Ltd., and the sequence is shown in SEQID NO.7; restriction endonucleases BamHI and XhoI were purchased from NEB Co., Ltd. Live cell dynamic microscopic imaging and fluorescence imaging were performed on a live cell workstation (NIS elements, Nikon), a spinning disk laser confocal microscope (UltraVIEWVoX, Perkin Elmer), and a line scanning laser confocal microscope (AR1+SIM, Nikon), respectively. Flow cytometry was performed on a 3-laser 11-channel flow cytometer (NovoCyte flow cytometer, Agilent, USA), and data analysis and statistics were performed using Novoexpress software. The primer sequences were generated by snapgene software. Lowercase letters indicate that the end sequence can complement the vector, and uppercase sequences indicate free end nucleotides that cannot bind to the vector.

[0035] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1

[0038] 1. Construction of fluorescent reporter system plasmid:

[0039] (1) Linearizing the yeast recombinant expression vector pYES2 by PCR: Plasmid pYES2 was used as a template, and the primers were as shown in Table 1. PCR was performed using the 2×Phanta Max Master Mix PCR system of Novazonics, the PCR system was as shown in Table 2, and the PCR program was as shown in Table 3. The PCR product obtained by the PCR reaction was the linearized recombinant expression vector pYES2.

[0040] Table 1 Primers for linearization of vector pYES2

[0041]

[0042] Table 2 PCR system for linearized vector pYES2

[0043]

[0044]

[0045] Table 3 PCR program for linearized vector pYES2

[0046]

[0047] (2) Obtaining the DNA fragment of ACE2-EGFP after codon optimization: Plasmid WHC2115669-ACE2-EGFP_in_pYES2_CT_BamHI_EcoRI was used as a template, and the primers were shown in Table 4. PCR was performed using the 2×Phanta MaxMaster Mix PCR system of Novagen, the PCR system was shown in Table 5, and the PCR program was shown in Table 6. The PCR product obtained by the PCR reaction was the DNA fragment of ACE2-EGFP after codon optimization.

[0048] Table 4 Primers for obtaining DNA fragments of ACE2-EGFP

[0049]

[0050] Table 5 PCR system for obtaining DNA fragments of ACE2-EGFP

[0051] Components volume WHC2115669-ACE2-EGFP_in_pYES2_CT_BamHI_EcoRI 1μL Upstream primer 2μL Downstream primer 2μL 2 × PhantaMaxMasterMix 25μL <![CDATA[ddH2O]]> 20μL

[0052] Table 6 PCR program for obtaining DNA fragments of ACE2-EGFP

[0053]

[0054] (3) Obtaining the DNA fragment of terminator CYC1: Plasmid pYES2 was used as a template, and the primers were as shown in Table 7. PCR was performed using the PCR system of 2×Phanta Max Master Mix from Novazonics, the PCR system was as shown in Table 8, and the PCR program was as shown in Table 9. The PCR product obtained by the PCR reaction was the terminator CYC1 fragment.

[0055] Table 7 Primers for obtaining terminator CYC1 fragment

[0056]

[0057] Table 8 PCR system for obtaining terminator CYC1 fragment

[0058]

[0059]

[0060] Table 9 PCR program for obtaining the terminator CYC1 fragment

[0061]

[0062] (4) Obtaining a DNA fragment of far-red fluorescent IRFP connected to CMV promoter and enhancer: plasmid pQCXIP-IRFP was used as a template, and the primers were as shown in Table 10. PCR was performed using the 2×PhantaMax Master Mix PCR system of Novagen, the PCR system was as shown in Table 11, and the PCR program was as shown in Table 12. The PCR product obtained by the PCR reaction was a DNA fragment of far-red fluorescent IRFP connected to CMV promoter and enhancer.

[0063] Table 10 Primers for obtaining IRFP fluorescent reporter elements

[0064]

[0065] Table 11 PCR system for obtaining IRFP fluorescent reporter element

[0066] Components volume pQCXIP-IRFP 1μL Upstream primer 2μL Downstream primer 2μL 2 × PhantaMaxMasterMix 25μL ddH2O 20μL

[0067] Table 12 PCR program for obtaining IRFP fluorescent reporter element

[0068]

[0069] (5) The obtained linearized recombinant expression vector pYES2, the DNA fragment of ACE2-EGFP after codon optimization, the DNA fragment of the terminator CYC1, and the DNA fragment of the far-red fluorescent IRFP connected to the CMV promoter and enhancer were homologously recombined. The homologous recombination kit CloneSmarter was purchased from Sino-US Taihe Company. The homologous recombination system is shown in Table 13.

[0070] Table 13 Homologous recombination system

[0071]

[0072]

[0073] After adding the above system, mix well. Incubate at 50℃ for 15min to complete homologous recombination. The constructed fluorescent reporter system recombinant plasmid is named pYES2CT-Y-ACE2-EGFP-CMV-IRFP. (The CYC1 terminator element does not appear in the name of this plasmid, and CMV is a promoter that only mammals can recognize in this system). The plasmid sequencing results are as follows: Figure 1 shown.

[0074] 2. Construction of humanized D614G point mutation S protein expression plasmid

[0075] (1) Obtain the linearized original vector pSecTag2-HygroA by enzyme digestion

[0076] The vector pSecTag2-HygroA was double-digested with restriction endonucleases BamHI and XhoI. The double-digestion system is shown in Table 14, and the digestion was performed at 37°C for 2h to achieve linearization of the vector.

[0077] Table 14 Double restriction enzyme digestion system of vector pSecTag2-HygroA

[0078] Element volume pSecTag2-HygroA 5μL B H 1μL XOt 1μL 10 × rCutSmart 5μL <![CDATA[ddH2O]]> 38μL

[0079] (2) Obtaining the sequence of the S protein point mutation (D614G)

[0080] PCR was used, and the template was pSec-tag-HA-SARI-HIS donated by Lu Lu's research group at Fudan University. The original vector backbone of the plasmid was pSecTag2 / HygroA, and the functional segment of the S protein containing SARI was inserted in the middle. The sequence is shown in SEQ ID NO.16. Two pairs of primers were designed using snapgene software, and point mutations were achieved by homologous recombination using two pairs of primers. The sequences of the two pairs of primers are shown in Table 15. The two pairs of primers were used for PCR respectively, and the PCR system of 2×Phanta MaxMaster Mix of Novozyme was used. The PCR system is shown in Table 16, and the PCR program is shown in Table 12. The two PCR products are the upper half (fragment 1) and the lower half (fragment 2) of the point mutation of the S protein.

[0081] Table 15 Primers for obtaining the sequence of the S protein point mutation (D614G)

[0082]

[0083] Table 16 PCR system for obtaining S protein point mutation (D614G)

[0084] Components volume pSec-tag-HA-SARI-HIS 1μL Upstream primer 2μL Downstream primer 2μL 2 × PhantaMaxMasterMix 25μL <![CDATA[ddH2O]]> 20μL

[0085] (3) The obtained pSecTag2-HygroA linearized vector and the sequence of the S protein point mutation (D614G) were homologously recombined, and the homologous recombination kit CloneSmarter was purchased from Sino-US Taihe Company. The homologous recombination system is shown in Table 17.

[0086] Table 17 Homologous recombination system

[0087]

[0088]

[0089] After adding the above system, mix well. Incubate at 50℃ for 15min to complete homologous recombination. The D614G point mutation S protein expression plasmid was constructed, and the plasmid sequencing results were as follows: Figure 2 shown.

[0090] Example 2

[0091] Method for transferring chromosomes of Saccharomyces cerevisiae BY4742 into HEK293T, MCF7 or MCF10A cells:

[0092] Day 1: Bottom cell plating. HEK293T, MCF7 and MCF10A cells with 80-90% confluence were selected, digested and counted, and 300,000 cells were seeded per well in a six-well plate with a plastic bottom. After they adhered to the wall and stabilized for a period of time, they were used for transfection.

[0093] Day 2: Cell transfection. After the cells deposited on the first day have stably adhered to the wall, transfection reagent PEI ( The cells were transfected with a genetically modified plasmid, D614G point mutation S protein expression plasmid (prepared in step 2 of Example 1), and the cell culture medium was replaced after 8 hours.

[0094] Yeast cell culture. Saccharomyces cerevisiae BY4742 (containing artificial chromosomes, which contain mcherry red fluorescence for tracing) containing genetically modified plasmids was expanded using SD-Leu-Ura conditional deficiency medium (purchased from Solebo, item number S0190) containing 20 g / L glucose. At this time, copies that do not meet the requirements will not grow to complete the bacterial screening. The OD number of the bacterial solution at 600 nm was measured using an ELISA instrument, and the experiment was carried out when the OD number reached 0.8.

[0095] Yeast transformation: The yeast transformation was performed using the Frozen-EZ Yeast Transformation II Kit, and the plasmid used for transformation was pYES2CT-Y-ACE2-EGFP-CMV-IRFP (prepared in step 1 of Example 1).

[0096] Day 3: Preparation and quality control of yeast protoplasts.

[0097] (1) Yeast wall removal enzyme related reagents are:

[0098] 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.

[0099] H-Buffer B: 1M sorbitol. Add 9.1g sorbitol to 50mL, dissolve and filter through a 0.22μm filter membrane.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] (2) Yeast wall removal

[0104] Inoculate yeast, shake the bacteria in a 30℃ incubator, and then measure OD on a microplate reader. 600 . Take the yeast solution containing 4OD and centrifuge it at 1500g for 10 minutes at room temperature to collect the yeast. Resuspend the cells in 5mL of sterile double distilled water and centrifuge at 1500g for 5min at room temperature, and discard the supernatant. Resuspend the cells in 1mL of BufferA and incubate at 30℃ for 45min to remove mannan. Then centrifuge at 1500g for 5min at room temperature and discard the supernatant. Resuspend the cells in 1mL of Buffer B and centrifuge at 1500g for 5min at room temperature, and discard the supernatant. Take 20μL of enzyme working solution with an enzyme concentration of 300U / mL and dilute it with 1mL of BufferE. Resuspend the cells with the liquid obtained in the previous step and incubate at 30℃ for 15min. Gently shake the centrifuge tube during this period. Use a horizontal rotor centrifuge to collect protoplasts by centrifugation at 350g for 5min at room temperature, resuspend the cells with 1mL of Buffer E, and wash once. Protoplasts were collected by centrifugation at 350 g for 3 min at room temperature, and galactose (working concentration 5 g / L) and raffinose (working concentration 10 g / L) were added to the di-deficient SD medium (purchased from Solebow) to induce the yeast protoplasts. At the same time, two antifungal drugs (12.5 ng / μL caspofungin and 12.5 ng / μL micafungin) were added to prevent the resynthesis of the yeast cell wall.

[0105] (3) Quality testing of yeast protoplasts

[0106] Take 500 μL of yeast protoplast suspension and centrifuge at 350g for 5 minutes at room temperature to collect the protoplasts (it is best to use a horizontal rotor centrifuge). Add 1 mL of buffer E, add the live cell tracer probe (orange) at a dilution ratio of 1:2000, mix well, and stain for 30 minutes. When there are 5 minutes left for staining, add fluorescent brightener 28 (purchased from Shanghai Maokang Biological Company, product number MS4040-1G) at a dilution ratio of 1:500 and blow well for 5 minutes of staining. Centrifuge at 350g for 5 minutes at room temperature to collect the protoplasts. Resuspend with 1 mL of buffer E to obtain a cell suspension, make a slide and observe it under an online scanning confocal microscope, and select DAPI, FITC, and mCherry as the fluorescent channels. The staining results were quantitatively detected by line scanning confocal microscopy and flow cytometry, and the results are as follows. Figure 3 shown. Figure 3 In the figure, the Merge group shows the result of superimposing all fluorescent channels; the Cell Wall group shows the yeast cell wall structure labeled with fluorescent brightener 28; the Phalloidin group shows the cytoplasmic structure labeled with phalloidin staining; the Cell Tracker group shows the cytoplasmic structure labeled with live cell tracking probe (orange), and the mCherry channel is collected. The first row shows yeast that has not been completely wall-removed, with obvious cell wall structure under the staining of fluorescent brightener 28. The second row shows yeast that can be co-incubated. In this environment, the yeast has sufficient cell membrane surface to contact the underlying mammalian cells. The only remaining cell wall is the bud scar structure of the yeast cell, where the cell wall is thicker. The third row shows that the contents of the yeast cells have slipped out of the damaged cell wall structure caused by digestion. The cytoplasmic staining shows a clear outline, proving that the cell membrane in the yeast remains intact. The fourth row shows the ideal state of yeast after wall removal, that is, there is no obvious cell wall structure and the cytoplasm remains intact. The fifth row shows the over-digestion situation, where darker cytoplasm and no obvious cell wall structure can be observed.

[0107] For HEK293T, MCF7 or MCF10A cell lines, 120,000 cells were seeded per well in a 24-well glass-bottom plate, and yeast and cells were co-incubated after 36 hours of yeast protoplast induction.

[0108] Day 4: Co-incubation and observation. After resuspending the yeast and counting by centrifugation, co-incubate at a yeast cell ratio of 100:1. At this time, resuspend the yeast in the cell culture medium and add it to the glass well plate. 12 hours after adding the yeast, discard the old culture co-incubation system, rinse off the yeast with PBS, replace with fresh cell culture medium and continue to culture for 12 hours and observe the fluorescence of the cells.

[0109] Fluorescence characterization of cells. When yeast and cells fuse, the CMV promoter in the genetically modified plasmid containing far-red fluorescence in the yeast is specifically recognized by the underlying cells, and the expression of IRFP begins. When the artificial chromosome in the yeast enters the cell, the mcherry red fluorescence contained in the artificial chromosome itself begins to express. By counting the proportion of cells containing the two fluorescences, the working efficiency of the chromosome transfer technology is quantified.

[0110] The results are as follows Figure 4 As shown. In the figure, a. Microscopic fluorescence imaging of chromosome delivery to different target cells, namely HEK293T, MCF7 and MCF10A. b. Quantitative results of chromosome transfer efficiency. The results show that the efficiency of chromosome transfer in the HEK293T group is 37.6480%; the efficiency of chromosome transfer in the MCF7 group is 28.4720%; and the efficiency of chromosome transfer in the MCF10A group is 53.8842%. This shows that the method of the present invention has a high chromosome transfer efficiency.

[0111] 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. An expression system for mediating fusion of yeast cells and animal cells, characterized in that: It includes plasmid element A and plasmid element B, wherein plasmid element A is a plasmid element for expressing human ACE2, and the nucleotide sequence of human ACE2 is shown in SEQ ID NO.1; plasmid element B is a plasmid element for expressing the S protein of SARS; the S protein is an S protein with a D614G point mutation, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The expression system according to claim 1, characterized in that The vector of the plasmid element A is pYES2, and the vector of the plasmid element B is pSecTag2-HygroA.

3. The expression system according to claim 1, characterized in that In the plasmid element A, the 3' end of human ACE2 is connected to the fluorescent reporter gene EGFP, and the nucleotide sequence of the ACE2-EGFP fragment is shown in SEQ ID NO.

3.

4. The expression system according to claim 1, characterized in that The plasmid element A also includes a GAL1 promoter element, a CYC1 terminator element and an IRPF fluorescent reporter element.

5. Use of the expression system according to any one of claims 1 to 4 in mediating the fusion of yeast cells and animal cells.

6. A method for transferring yeast chromosomes into animal cells, characterized in that: The expression system according to any one of claims 1 to 4 is used to transfect plasmid element A in yeast cells and plasmid element B in animal cells, the transfected yeast cells are used to prepare yeast protoplasts, and the yeast protoplasts are co-incubated with the transfected animal cells to complete the transfer of chromosomes from yeast cells to animal cells.

7. The method according to claim 6, characterized in that Before co-incubation, the yeast protoplasts are cultured with galactose and raffinose as carbon sources, the working concentration of the galactose is 2.5-7.5 g / L, and the working concentration of the raffinose is 5-15 g / L.

8. The method according to claim 7, characterized in that Antifungal drugs were added during yeast protoplast culture.

9. The method according to claim 6, characterized in that The co-incubation time is 12 to 36 hours.

10. Use of the expression system according to any one of claims 1 to 4 or the method according to any one of claims 6 to 9 in gene synthesis and gene delivery.

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