Use of fusion proteins and methods of cell fusion

Abstract

The application relates to the field of biotechnology, in particular to the application of a fusion protein and a cell fusion method. The application provides application of DENV2E protein and VSV G protein in fusion of a Saccharomyces cerevisiae protoplast and a mammalian cell, wherein the mammalian cell includes HEK293T. Compared with PEG-mediated fusion, the fusion efficiency of FUS1 prepared by the application and HEK293T is respectively increased by 5.25 times and 5.83 times; the fusion efficiency of FUS2 prepared by the application and HEK293T is respectively increased by 1.31 times and 2.78 times.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of fusion proteins and methods for cell fusion. Background Technology

[0002] Efficient transfer of large DNA fragments is a prerequisite and foundation for their function in target cells. The cell membrane, composed of a phospholipid bilayer and embedded proteins, carries a net negative charge, preventing large molecules like DNA from passing directly. DNA transfer can be achieved using physical, chemical, or biological methods. 1) Physical methods: Nucleic acids are directly introduced into the cytoplasm or nucleus through electroporation, gene guns, microinjection, etc.; 2) Chemical methods: DNA is encapsulated using liposomes, PEG-mediated transfer, etc., to allow it to pass through the cell membrane; 3) Biological methods include the transfer of non-viral genes into cells using genetically modified viruses (also known as transduction), cell-cell fusion, etc.

[0003] Traditional DNA transfer methods all require in vitro manipulation of DNA molecules. However, large DNA fragments are easily broken by the shearing forces exerted during in vitro manipulation, resulting in the loss of their integrity. Currently, for Mb-level DNA integration, multiple introductions are generally required. Existing DNA delivery methods do not yet meet the requirements for mammalian artificial chromosomes in terms of transfer scale, efficiency, and universality. Research on animal artificial chromosome transfer includes: 1) chromosome transfer technology between Saccharomyces cerevisiae and animal cells; 2) polymer-mediated artificial chromosome transfer technology; and 3) mammalian cell-to-cell artificial chromosome transfer technology.

[0004] Saccharomyces cerevisiae has strong homology. Group This technology has the capability to de novo assemble synthetic chromosomes at the Mb level. Large DNA fragments can be transferred via PEG-mediated yeast protoplast-mammalian cell fusion. In 2013, Li et al. used PEG-mediated yeast protoplast-mammalian cell fusion to transfer a 1.4 Mb human T-cell receptor gene assembled in Saccharomyces cerevisiae into mouse embryonic stem cells, generating humanized T-cell receptor mice. However, the efficiency of PEG-mediated yeast-to-mammalian cell transfer of large-scale DNA is extremely low (1 × 10⁻⁶). -6 ~1×10 -5). One of the reasons for its low efficiency is that most of the large fragments of DNA transferred by fusion are located in the cytoplasm of animal cells and cannot be stably transported into the nucleus to function. In order to improve the efficiency of DNA transfer to the nucleus, Brown et al. used mitotic microtubule formation inhibitors such as colchicine and Nocodazole to treat recipient cells in 2017, so that the cells were in M phase, at which time the nuclear membrane disappeared, and after fusion the cells re-entered the cycle, the YAC in the cytoplasm was wrapped by the newly formed nuclear membrane and entered the nucleus, and the transfer efficiency was improved by nearly 300 times. The transfer efficiency of different cell lines varies greatly, and the highest transfer efficiency is still about one thousandth.

[0005] High PEG concentration and long contact time can significantly improve the transfer efficiency, but at the same time the toxicity of PEG to cells increases. Since the mechanism of PEG-mediated fusion is not clear, it is not possible to improve it with a theoretical guide. Membrane fusion in physiological processes, such as viral infection, synaptic vesicle fusion, skeletal muscle cell fusion, sperm-egg fusion, and other processes, are expected to provide theoretical guidance for the fusion of S. cerevisiae protoplasts and mammalian cells. Membrane fusion-mediated transfer not only bypasses the in vitro manipulation steps of large fragment DNA, but more importantly, it directly transfers synthetic animal chromosomes synthesized from scratch to cells, greatly simplifying the operation steps. Optimization of the transfer efficiency of membrane fusion-mediated transfer is expected to bridge the gap between the synthesis and assembly of synthetic animal chromosomes and functional research, and to provide key research tools for animal synthetic genomics.

[0006] Viral infection and transmission involve viral fusion processes, which are mainly mediated by viral fusion proteins. Most viral fusion proteins undergo dramatic conformational changes under special stimulus conditions (such as binding to host cell surface receptors), and the fusion loop or fusion peptide is exposed and inserted into the host cell membrane, causing a decrease in the stability of the lipid bilayer, accompanied by energy release, thereby achieving fusion. Viral fusion proteins can be divided into four types according to their structural characteristics and fusion mechanisms: type I viral fusion protein, such as the hemagglutinin protein of influenza virus; type II viral fusion protein, such as the envelope protein of Dengue fever type 2 virus (DENV2 E); type III viral fusion protein, such as the vesicular stomatitis virus glycoprotein (VSV G); and type IV viral fusion protein, such as the P15 fusion-related small transmembrane protein.

[0007] Viruses fusion proteins not only participate in the process of virus infection of cells in nature, some virus fusion proteins are also used by researchers in biomedical research and identification of cell surface receptors that mediate virus infection of cells and other related researches. Han Lin et al. have successfully fused 293A cells expressing DENV E protein with various cell lines, such as 293A, CHO-K1, Vero and BHK-21; VSV G is the first and widely used glycoprotein for pseudotyped lentiviral vectors, and human immunodeficiency virus type 1 retroviral vector pseudotyped with VSV G can efficiently transfer genes into CD34+ cells Figure 1A 、 Figure 1B The fusion scores of other fusion proteins are shown.

[0008] However, the current method of cell fusion mediated by PEG has the problems of low fusion efficiency, strong cytotoxicity, and weak interspecies specificity and specificity. SUMMARY

[0009] Therefore, the application provides the application of fusion protein and the method of cell fusion, which has high fusion efficiency, low cytotoxicity, and strong interspecies specificity and specificity.

[0010] In order to achieve the above application purposes, the application provides the following technical solutions.

[0011] The application provides the application of DENV2 E protein and VSV G protein in the fusion of Saccharomyces cerevisiae protoplast and mammalian cells.

[0012] The mammalian cells include HEK293T.

[0013] The application also provides a nucleic acid molecule, which has:

[0014] (1) the nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No. 4; or

[0015] (2) the nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (1), and the function is the same or similar to (1); or

[0016] (3) the nucleotide sequence having at least 80% homology with the nucleotide sequence shown in (1) or (2);

[0017] The number is 2 to 150.

[0018] The application also provides an expression vector, which has the above nucleic acid molecule and acceptable genetic elements.

[0019] In some specific embodiments of the application, the expression vector has a backbone including pRS426.

[0020] The present application also provides a host cell, comprising:

[0021] the nucleic acid molecule and the expression product thereof;

[0022] or

[0023] the expression vector and the expression product thereof.

[0024] In some embodiments of the present application, the host cell comprises a S. cerevisiae protoplast.

[0025] The present application also provides the use of the nucleic acid molecule, the expression vector and / or the host cell in:

[0026] (I) fusion of S. cerevisiae protoplasts with mammalian cells; and / or

[0027] (II) improving the efficiency of fusion of S. cerevisiae protoplasts with mammalian cells; and / or

[0028] (III) avoiding the toxic effect of PEG on cells during the fusion process.

[0029] The mammalian cells comprise HEK293T.

[0030] The present application also provides a method for fusing S. cerevisiae protoplasts with mammalian cells, comprising expressing the nucleic acid molecule in the S. cerevisiae protoplasts to obtain a recombinant S. cerevisiae protoplast, contacting the recombinant S. cerevisiae protoplast with the mammalian cells to achieve the purpose of fusion.

[0031] without involving a PEG-mediated step.

[0032] The mammalian cells comprise HEK293T.

[0033] In some embodiments of the present application, the method further comprises expressing the expression vector in the S. cerevisiae protoplasts to obtain a recombinant S. cerevisiae protoplast, contacting the recombinant S. cerevisiae protoplast with the mammalian cells to achieve the purpose of fusion.

[0034] The mammalian cells comprise HEK293T.

[0035] In some embodiments of the present application, the method further comprises contacting the host cell with the mammalian cells to achieve the purpose of fusion.

[0036] The mammalian cells comprise HEK293T.

[0037] The method of the present application has the following effects:

[0038] Compared with PEG-mediated fusion, the fusion efficiency of FUS1 with HEK293T was increased by 5.25 times and 5.83 times, respectively, and the fusion efficiency of FUS2 with HEK293T was increased by 1.31 times and 2.78 times, respectively. It is proved that the technical method provided by the present application is superior to the conventional PEG-mediated fusion of Saccharomyces cerevisiae protoplast and mammalian cells, and at the same time, the use of PEG is avoided in the present application, reducing the cytotoxicity. In addition, the present application utilizes the interaction of the fusion protein and its corresponding receptor to promote membrane fusion, which is more specific than the conventional PEG fusion method. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0040] Figure 1A Fusion scores of different fusion proteins are shown;

[0041] Figure 1B Fusion scores of different fusion proteins are shown;

[0042] Figure 2 Construction of vector plasmid pLJW1 is shown; wherein, A shows the plasmid structure; B shows the electrophoresis map, lane 1 is pLJW1 plasmid PCR verification 1, lane 2 is pLJW1 plasmid PCR verification 2;

[0043] Figure 3 Construction of vector plasmid pLJW2 is shown;

[0044] Figure 4 A schematic diagram of Saccharomyces cerevisiae-mammalian cell fusion detection is shown;

[0045] Figure 5 Flow cytometry identification of expression of fusion protein is shown; wherein, A is the control group; B is the FUS2 group; C is the FUS1 group;

[0046] Figure 6 Localization of fusion protein is shown (the left two pictures are taken under fluorescence; the middle two pictures are taken under bright field; the right two pictures are taken under fluorescence and bright field); wherein, A is the FUS2 group; B is the FUS1 group;

[0047] Figure 7 PEG-mediated cell fusion efficiency is shown; wherein, A is the control group; B is the FUS1 group; C is the FUS2 group;

[0048] Figure 8 Cell fusion efficiency under co-culture of yeast-mammalian cells (n=2) is shown; wherein, A is the control group; B and C are the FUS2 groups; D and E are the FUS1 groups. DETAILED DESCRIPTION

[0049] The application of fusion protein and the method of cell fusion are disclosed in the present application, and those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0050] The present application aims to solve the problem of low fusion efficiency of Saccharomyces cerevisiae protoplast and mammalian cells. Further, to bypass the effect of PEG on cells and expect to further improve the fusion efficiency of Saccharomyces cerevisiae protoplast and mammalian cells.

[0051] In order to bypass the effect of PEG on cells and expect to further improve the fusion efficiency of Saccharomyces cerevisiae protoplast and mammalian cells, and inspired by the membrane fusion process of virus infection of cells, the present application heterologously expresses selected viral fusion proteins on the cell membrane of Saccharomyces cerevisiae, removes the cell wall by enzymolysis to form protoplast, and occurs membrane fusion with the target cells under the interaction of the fusion protein and its corresponding receptor, so as to transfer large fragments of DNA to the target mammalian cells.

[0052] The specific technical solution of the present application is: the application of two fusion proteins in the fusion of Saccharomyces cerevisiae protoplast and mammalian cells, the two fusion proteins are DENV2 E and VSV G. The amino acid sequences of the two fusion proteins are shown in SEQ ID No. 1 and SEQ ID No. 2. DENV2 E can interact with a variety of receptor molecules, including Tyro3, Axl and Mer (Tyro3 / Axl / Mer, TAM) receptors, T cell immunoglobulin mucin receptors and C-type lectins; the receptor of VSV G is phosphatidylserine widely distributed on the cell membrane. Since the TAM receptor of DENV2 E and the receptor of VSV G are expressed in the human embryonic kidney cell HEK-293T cell line, the HEK-293T cell line is selected as the target receptor cell for fusion experiment.

[0053] The medium formula in the example is as follows:

[0054] LB liquid medium: 10 g / L NaCl, 10 g / L peptone and 5 g / L yeast powder, the rest is water, sterilized at 0.1 MPa pressure for 20 min at 121℃. The solid medium can be prepared by adding 1.5 g / 100 mL agar powder.

[0055] YPD liquid medium is prepared as follows: glucose 20 g / L, tryptone 20 g / L, yeast extract 10 g / L. YPD solid medium is prepared by adding an additional 2% (w / v) agar. After preparation, the pH is adjusted to 6.5 with 10 M HC1 solution and sterilized at 0.1 MPa for 15 min at 115 °C.

[0056] SC selective medium: glucose 20 g / L, yeast nitrogen base without amino acids 6.7 g / L, amino acid dropout powder mix 2 g / L (see Table 1 for formulation). SC solid medium is prepared by adding an additional 2% (w / v) agar. After preparation, the pH is adjusted to 6.5 with 10 M NaOH solution and sterilized at 0.1 MPa for 15 min at 115 °C. The SC medium is supplemented with the required amino acid stock solutions before use, with final concentrations of 20 mg / L uracil (URA), 20 mg / L tryptophan (TRP), 20 mg / L histidine (HIS), and 100 mg / L leucine (LEU).

[0057] Table 1. Amino acid dropout powder mix formulation

[0058] Ingredients Mass (g) Ingredients Mass (g) Adenine 0.5 Valine 2.0 Myo-inositol 2.0 Lysine 2.0 Arginine 2.0 Methionine 2.0 Proline 2.0 Isoleucine 2.0 Serine 2.0 Phenylalanine 2.0 Threonine 2.0 Aspartic acid 2.0 Tyrosine 2.0 Asparagine 2.0 Glutamic acid 2.0 Cysteine 2.0 Alanine 2.0 Glutamine 2.0 Glycine 2.0 p-Aminobenzoic acid 2.0

[0059] The solutions used in the examples are prepared as follows:

[0060] Sorbitol solution (1 M): To 700 mL of distilled water, add 182 g of sorbitol and stir until dissolved. Bring to 1,000 mL with distilled water. Transfer the solution to a glass storage bottle and autoclave for 15 min. The sorbitol solution can be stored at room temperature for 6 months.

[0061] Tris-HCl solution (1 M; pH 7.5): To 800 mL of distilled water, add 121.1 g of Tris and 70 mL of concentrated hydrochloric acid to adjust the pH to 7.5. Bring to 1,000 mL with distilled water and mix well. The Tris-HCl solution can be stored at room temperature for 12 months.

[0062] CaCl2solution (1 M): Dissolve 14.7 g of solid CaCl2-2H2O in 100 mL of distilled water. The CaCl2solution can be stored at room temperature for up to 12 months.

[0063] EDTA solution (0.5 M; pH 7.5): To 800 mL of distilled water, add 186.1 g of Na2EDTA-2H2O and mix vigorously with a magnetic stirrer until dissolved. Adjust the pH to 7.5 with 10 M NaOH and autoclave. The EDTA solution can be stored at room temperature for 12 months.

[0064] STC solution: Take 400 mL distilled water, add 91 g sorbitol, 5 mL 1 M Tris-HCl (pH 7.5), 5 mL 1 M CaCl2, stir to dissolve, and dilute to 500 mL with distilled water. The solution is transferred to a glass storage bottle and autoclaved for 15 minutes. The STC solution can be stored at room temperature for 6 months.

[0065] SPE solution: Take 400 mL distilled water, add 91 g sorbitol, 1.04 g Na2HPO4·7H2O, 0.16 g NaH2PO4·1H2O, 10 mL 0.5 M EDTA (pH 7.5), stir to dissolve, and dilute to 500 mL with distilled water. The solution is transferred to a glass storage bottle and autoclaved for 15 minutes. The SPE solution can be stored at room temperature for 6 months.

[0066] Yeast lysing enzyme solution: Take 9 mL distilled water, add 200 mg Zymolyase 20T, 1 mL Tris-HCl, pH 7.5, stir to dissolve. Add 10 mL 50% (w / v) glycerol, stir evenly, and divide into 500 μL, which can be stored at -20°C for 6 months.

[0067] 2% SDS solution: Take 80 mL distilled water, add 2 g SDS, stir to dissolve, and dilute to 100 mL with distilled water.

[0068] Fusion buffer: Add 20 μL 1 M CaCl2, 200 μL DMSO, and 4 μL 50 mM β-mercaptoethanol to 4 mL 50% (w / v) PEG1500.

[0069] The detection method involved in the examples is as follows:

[0070] To prepare the sample for flow cytometry, the fused cells are digested with Tryple Express enzyme. The cells are washed twice with flow cytometry staining buffer (eBioscience) and finally resuspended in PBS. Flow cytometry detection is performed using BD Aria III, and data analysis is performed using FlowJo (Treestar).

[0071] The sequence information involved in the present application is as follows:

[0072] SEQ ID No. 1:

[0073] MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA

[0074] SEQ ID No. 2:

[0075] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0076] SEQ ID No. 3:

[0077]

[0078] SEQ ID No.4

[0079]

[0080] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present application are ordinary commercially available products, which can be purchased from the market.

[0081] The present application is further described below in conjunction with examples:

[0082] Example 1: Construction of expression vector and recombinant strain of fusion protein

[0083] According to the amino acid sequence of DENV2 E (NP_739583.2), combined with the preference of Saccharomyces cerevisiae for codons, and removing commonly used enzyme cutting sites, a full-length DENV2 E gene was designed, the nucleotide sequence of which is shown in SEQ ID No. 3, and (GGGGS)3 was used as a linker to connect the DENV2 E gene and the green fluorescent protein (GFP), and the gene was connected to the pRS426 plasmid, and the obtained vector plasmid was named pLJW1, the structure of which is shown in Figure 2 .

[0084] The VSV G gene was connected to the pRS426 plasmid according to the above method, and the plasmid pLJW2 was constructed, the structure of which is shown in Figure 3 The nucleotide sequence of the VSVG gene is shown in SEQ ID No. 4.

[0085] The plasmid pLJW1 and the plasmid pDelivery-YA were sequentially transformed into SY14 yeast by lithium acetate transformation method, and the yeast was cultured on SC-His-Ura solid plate for 3-4 days, and positive clones were screened, and the strain was named FUS1, and was stored for later use.

[0086] The plasmid pLJW2 and the plasmid pDelivery-YA were sequentially transformed into SY14 yeast by lithium acetate transformation method, and the yeast was cultured on SC-His-Ura solid plate for 3-4 days, and positive clones were screened, and the strain was named FUS2, and was stored for later use.

[0087] Example 2: Characterization of fusion efficiency

[0088] Saccharomyces cerevisiae expressing VSV G and DENV2 E proteins was fused with HEK-293T, and the delivered plasmid carried a mammalian cell promoter and a red fluorescent protein (mCherry) gene, and the proportion of red cells was detected by flow cytometry to characterize the fusion efficiency, Figure 4 which is a schematic diagram.

[0089] Example 3: Expression identification of fusion protein

[0090] Strains FUS1 and FUS2 were inoculated into liquid medium and cultured on a shaker for 48 h, and then the expression of the fusion protein was identified by flow cytometry, and the results are shown in Figure 5

[0091] The proportion of GFP-positive yeast cells in the control group was 0.15%, indicating that there was a very small amount of leaky expression. The proportion of GFP-positive cells of strain FUS2 expressing VSV G was 18.7%, and the proportion of GFP-positive cells of strain FUS1 expressing DENV2 E was 10.9%. Not all yeast cells were GFP-positive, which might be due to insufficient protein abundance, failing to reach the lower limit of flow cytometry detection. Under a microscope, it was observed that part of the yeast with viral envelope protein VSV G and DENV2 E plasmids was in a string of beads shape, indicating that it could not divide correctly, which might be caused by potential interaction between the envelope protein and the protein on the yeast cell membrane or other endogenous proteins, ultimately leading to the death of yeast with high expression of viral envelope protein due to its inability to divide correctly.

[0092] The expression and localization of the fusion protein in strains FUS1 and FUS2 were observed by fluorescence microscopy, and the results are shown in Figure 6

[0093] It can be observed from Figure 6 that some yeast cells were green, indicating that viral envelope protein was indeed expressed in the yeast cells. The green fluorescence around the yeast cell outline suggested that DENV2 E and VSV G proteins were localized on the yeast cell membrane.

[0094] Example 4: Preparation of Saccharomyces cerevisiae protoplasts

[0095] Strains FUS1 and FUS2 were inoculated into liquid medium corresponding to the corresponding nutritional deficiency, and cultured overnight at 30°C and 200 rpm, until the OD 600 = 2.0-3.0. The yeast culture was transferred to a 50 mL centrifuge tube and centrifuged at 4°C for 5 min at 3,000 g, and the supernatant was removed; the cell pellet was resuspended with 30 mL of sterile water, then centrifuged under the above conditions, and the supernatant was removed; the cell pellet was resuspended with 20 mL of sterilized 1M sorbitol, centrifuged under the above conditions, and the supernatant was removed; the cell pellet was resuspended with 20 mL of SPE solution, 20 μL of yeast lytic enzyme solution and 40 μL of β-mercaptoethanol were added, mixed thoroughly, and incubated at 30°C with gentle shaking for about 20 minutes; the preparation of protoplasts was checked by comparing the optical density of the cell suspension in 1M sorbitol solution and 2% SDS solution, and the OD 600 ​​The OD ratio of sorbitol and SDS was 3-5 times, and the protoplast preparation was confirmed to be complete. Centrifugation was performed at 4°C for 10 min at 570 g, and the supernatant was removed. 50 mL of 1M sorbitol solution was added, and then the precipitate was resuspended by gentle shaking. Centrifugation was performed at 4°C for 10 min at 300-600 g; the final precipitate was resuspended in 2 mL of STC solution after repeated washing with 50 mL of 1M sorbitol solution once, and stored at room temperature for 10-60 min for subsequent fusion experiments.

[0096] Example 5: PEG-mediated cell fusion compared with the present application

[0097] (I) Control group: PEG-mediated fusion of Saccharomyces cerevisiae protoplasts and mammalian cells

[0098] 12 h before fusion, mitotic microtubule inhibitors (Nocodazole, 100 nM) were added to the cells to block the cells in the M phase of the cell cycle, at which time the cells had no nuclear membrane, which was conducive to the delivery of DNA in yeast into the mammalian cell nucleus. The adherent cells were digested with Tryple Express enzyme and transferred to a centrifuge tube, centrifuged at 300 g at 4°C for 5 min, and the supernatant was discarded after centrifugation. The Saccharomyces cerevisiae protoplasts were added to PBS and mixed with HEK-293T cells to make the ratio of Saccharomyces cerevisiae protoplasts to HEK-293T cells 100:1. Centrifugation was performed according to the procedure described above, the supernatant was discarded, and the Fusion Buffer was prepared during centrifugation. 2 mL of fusion buffer was added, and the HEK-293T cells and protoplasts were mixed gently, and blown and sucked three times. Incubation was performed at room temperature for 1 min, and the cells were washed gently with PBS. Centrifugation was performed according to the procedure described above, and the cells were washed with PBS 1-2 times, the supernatant was removed, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum and inoculated in a culture dish of appropriate size, and then placed in a 37°C CO2 incubator for 12 h. The culture medium was replaced after 48 h of fusion, and the fusion efficiency was detected by flow cytometry.

[0099] The fusion efficiency of FUS1 with HEK293T was 0.24%, and the fusion efficiency of FUS2 with HEK293T was 0.51% (see Figure 7 ).

[0100] (II) Experimental group: fusion of Saccharomyces cerevisiae protoplasts and mammalian cells under co-culture

[0101] 12h before fusion experiment, mitotic microtubule inhibitor (Nocodazole, 100nM) was added to the cells. The prepared S. cerevisiae protoplasts were added into the mammalian cells at a ratio of 100:1, and 50 μL of yeast lytic enzyme solution was added. After the formation of yeast protoplasts, the yeast protoplasts were immediately fused with HEK-293T cells. After the fusion of S. cerevisiae protoplasts with mammalian cells, the samples were placed in a culture dish of appropriate size, and the S. cerevisiae and mammalian cells HEK-293T were co-cultured for 48h, and the fusion efficiency of each group was detected.

[0102] Through flow cytometry detection, the fusion efficiency of FUS1 with HEK293T was 1.50%, 1.64%, and the fusion efficiency of FUS2 with HEK293T was 1.18%, 1.93% (see Figure 8 ). It is shown that the interaction of the fusion protein with its corresponding receptor can mediate cell fusion without the presence of PEG. Compared with the addition of PEG (control group), the fusion efficiency of FUS1 with HEK293T was increased by 5.25 times and 5.83 times, respectively; the fusion efficiency of FUS2 with HEK293T was increased by 1.31 times and 2.78 times, respectively.

[0103] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of DENV2 E protein or VSV G protein in fusion of Saccharomyces cerevisiae protoplasts with mammalian cells; the amino acid sequence of the DENV2 E protein or the VSV G protein is shown in SEQ ID No. 1 or SEQ ID No. 2, respectively; the mammalian cells are HEK293T.

2. Use of nucleic acid molecule, expression vector or host cell in: (I) fusion of Saccharomyces cerevisiae protoplasts with mammalian cells; and / or (II) improving the efficiency of fusion of Saccharomyces cerevisiae protoplasts with mammalian cells; and / or (III) avoiding the toxic effect of PEG on cells during the fusion process; the nucleic acid molecule has the nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No. 4; the expression vector comprises the nucleic acid molecule and acceptable genetic elements; the host cell has the nucleic acid molecule and its expression product; or the host cell has the expression vector and its expression product; the mammalian cells are HEK293T.

3. A method of fusing a yeast protoplast with a mammalian cell, characterized in that, comprises expressing the nucleic acid molecule in the yeast protoplasts to obtain recombinant yeast protoplasts, contacting the recombinant yeast protoplasts with the mammalian cells to achieve the fusion; the nucleic acid molecule has the nucleotide sequence shown in SEQ ID No. 3 or SEQ ID No. 4; does not comprise a PEG-mediated step; the mammalian cells are HEK293T.

4. The method of claim 3, wherein, comprises expressing the expression vector in the yeast protoplasts to obtain recombinant yeast protoplasts, contacting the recombinant yeast protoplasts with the mammalian cells to achieve the fusion; the expression vector comprises the nucleic acid molecule and acceptable genetic elements; the mammalian cells are HEK293T.

5. The method of claim 3, wherein, comprises contacting the host cell with the mammalian cells to achieve the fusion; the host cell has the nucleic acid molecule and its expression product; or the host cell has the expression vector and its expression product; the mammalian cells are HEK293T.

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