Pair of alpha helical peptides capable of mutually recognizing and combining and application of alpha helical peptides

Abstract

The invention relates to the technical field of biology, in particular to a pair of mutually recognized and combined alpha helical peptides and application thereof. Comprising an alpha spiral peptide 1 and an alpha spiral peptide 2, the amino acid sequence of the alpha spiral peptide 1 is shown as SEQ ID No.1, and the amino acid sequence of the alpha spiral peptide 2 is shown as SEQ ID No.2. A pair of alpha helical peptides that can recognize and bind to each other bind to each other, whereby cells bind to each other, and the cells are arranged in a desired manner. Proper material concentration and action time are verified; the form of the formed cell mass block is observed through various microscopes; two models are successfully constructed; the improvement of the cell function through the addition of the DPH material is verified.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a pair of mutually recognizing and binding alpha helical peptides and applications thereof. Background Art

[0002] Current approaches to artificially manipulate cellular interactions include exploiting endogenous cell adhesion molecules (CAMs), using synthetic DNA molecules to interact with cell membranes, protein-based antigen-antibody binding systems, and using nanomaterials to synthesize Matrigel. However, all of these approaches have limitations: native CAMs, such as cadherins, are limited by cross-reactivity and dual signaling; DNA materials are unstable outside of cells and require chemical modification, which is incompatible with genetic regulation; protein-based systems, including nanobody-antigen pairs and synthetic receptors such as SynNotch, provide genetic encoding but face challenges in scalability and orthogonality; and nanomaterial-based Matrigel may not be universally effective. Recent advances in helical peptides, stable, modular protein interaction domains with predictable pairing rules, have shown promise in mediating specific interactions between synthetic surfaces. However, their applicability to dynamically control cell-cell and cell-surface interactions across diverse cell types, particularly in human systems, remains underexplored.

[0003] In order to address these limitations, the present invention has transformed and synthesized a pair of alpha helical peptides that can recognize and interact with each other, and one end is connected to DMPE that can be inserted into the cell membrane, ensuring that it has no limitations on the type of cells, and can be stably positioned on the cell membrane and form a stable coiled-coil structure. Using this pair of materials, the present invention can obtain cell clumps, so that cells that originally could not interact with each other can interact with each other, thereby achieving the purpose of artificially regulating the interaction between cells, and at the same time, the impact of the cells that produce the interacting cell clumps is explored. In addition, the present invention also constructs two cell interaction models, namely the "spherical model" and the "cell layer assembly model", which proves the controllability of the material in regulating the interaction between cells. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention provides a pair of α-helical peptides that recognize and bind to each other and their applications. The DPH1 / 2 material is a plug-and-play material. DMPE is inserted into the cell membrane, and through a pair of α-helical peptides that can recognize and bind to each other, the cells are bound to each other, thereby arranging the cells in the desired manner. The appropriate material concentration and action time were verified; the morphology of the formed cell clusters was observed using various microscopes; two models were successfully constructed; the improvement of cell function by the addition of DPH material was verified; and the mechanism of the DPH material previously verified by transcriptome sequencing analysis was further analyzed: the DPH material forms a coiled-coil structure, which brings the distance between cells closer, allowing more cells to bind to each other. As a result, due to the contact between cells, the expression of proteins on the cell surface changes, and the expression of genes related to these proteins also changes, thereby further strengthening the interaction between cells.

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

[0006] The present invention provides a pair of mutually recognizing and binding α-helical peptides, including α-helical peptide 1 and α-helical peptide 2, wherein the amino acid sequence of the α-helical peptide 1 is shown in SEQ ID No. 1, and the amino acid sequence of the α-helical peptide 2 is shown in SEQ ID No. 2.

[0007] The present invention also provides the use of the α-helical peptide described in the above technical solution in preparing cell cluster products.

[0008] The present invention also provides a pair of polypeptides for preparing cell clusters, including DPH1 and DPH2, wherein DPH1 is the α-helical peptide 1 in the α-helical peptide described in the above technical solution, to which PEG and DMPE are connected;

[0009] The DPH2 is the α-helical peptide 2 in the α-helical peptide described in the above technical solution, and PEG and DMPE are connected to it.

[0010] The present invention also provides the use of the polypeptide described in the above technical solution in preparing cell cluster products.

[0011] The present invention also provides a method for constructing a spherical model cell cluster, comprising the following steps:

[0012] 1) mixing DPH1 in the polypeptide described in the above technical solution with cells to obtain DPH1-cells;

[0013] 2) mixing DPH2 in the polypeptide described in the above technical solution with cells to obtain DPH2-cells;

[0014] 3) The DPH1 cells and DPH2 cells obtained in step 1) are mixed and cultured to obtain spherical model cell clusters.

[0015] Preferably, in step 1), DPH1 is mixed in the form of a DPH1 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a number of cells of 5×10 5 The volume ratio of the DPH1 solution to the cell suspension was 42:458;

[0016] The mixing time is 10 minutes.

[0017] Preferably, in step 1), DPH2 is mixed in the form of a DPH2 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a cell number of 5×10 5 The volume ratio of the DPH2 solution to the cell suspension was 42:458;

[0018] The mixing time is 10 minutes.

[0019] The present invention also provides a method for constructing a cell cluster of a layer-by-layer assembly model, comprising the following steps:

[0020] 1) After the cells are cultured on the wall, they are mixed with the DPH2 polypeptide described in the above technical solution and incubated.

[0021] 2) The mixture was mixed with DPH1 in the polypeptide described in the above technical solution and incubated for 10 minutes, and then mixed with cells to obtain a cell-layer assembly model cell cluster.

[0022] Preferably, the final concentration of DPH2 in step 1) is 25 μM, and the number of cells is 5×10 5 The incubation time is 10 min.

[0023] Preferably, the final concentration of DPH1 in step 1) is 25 μM, and the number of cells is 5×10 5 The mixing time with cells was 1 h.

[0024] Beneficial effects of the present invention:

[0025] This invention reports a method for rapidly preparing cell clusters using a pair of α-helical peptides that recognize and bind to each other. Specifically, the peptides SEQ ID No. 1: GEIAALEQENAALEQKIAALKWKNAALKQGGC and SEQ ID No. 2: CGGKIAALKQKNAAL-KYEIAALEQENAALEQG were designed and synthesized, then linked to form DPH1 and DPH2 using PEG and DMPE, which can insert into cell membranes. Two cell groups were incubated with 25 μM DPH1 and DPH2 for 10 minutes before mixing. The α-helical peptides recognized and bound to each other, thereby binding the two cell groups together to form cell clusters. Experimental results showed that the addition of DPH1 and DPH2 enabled the formation of cell clusters with human acute lymphoblastic leukemia (CEM) cells and pancreatic beta cells derived from human pluripotent stem cells. Exploration of their specific effects revealed that the addition of DPH1 and DPH2 enabled the assembled cell clusters to achieve similar functionalities to those obtained by induction. Multiple cell pathways and cell surface adhesion-related genes in cell clusters were significantly up- and down-regulated, such as the Hippo pathway, TGF-β pathway, and integrin genes.

[0026] The present invention modifies and transforms existing helical peptide sequences and applies them to artificially regulate cell interactions, thereby constructing a cell model. The advantages of the present invention are plug-and-play, ease of use, no requirements for cell type, and the ability to artificially regulate cell interactions.

[0027] The method disclosed in the invention has the following key points:

[0028] 1) Provides a simple method for constructing cell aggregates by using a pair of α-helical peptides that can insert into the cell membrane.

[0029] 2) Two cell assembly models were constructed, namely the cell spheroid model and the cell layer-by-layer assembly model. These two models can be used as tools to study the interactions between cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0031] Figure 1 For the structures of DPH1 and DPH2, Ac means acetyl;

[0032] Figure 2 LC-MS spectrum of DPH1 and 1 H-NMR spectrum;

[0033] Figure 3 LC-MS spectrum of DPH2 and 1 H-NMR spectrum;

[0034] Figure 4 Circular dichroism results of DPH1, DPH2 and the combination of DPH1 and DPH2;

[0035] Figure 5 Figure 3. Localization of DPH1 / 2 on the cell membrane. A: Localization of 25 μM DPH1 on the cell membrane at different times; B: Confocal fluorescence statistics; C: Distribution of materials after long-term interaction of DPH1 with cells; D: Flow cytometry results of 10-min interaction of cells with different concentrations of DPH1; E: Confocal fluorescence results of 10-min interaction of cells with different concentrations of DPH1; F: Confocal fluorescence results of localization of different materials on the cell membrane; G: Flow cytometry results of localization of different materials on the cell membrane.

[0036] Figure 6 Figure 3: The morphology and statistical data of cell clusters formed by DPH1 / 2-mediated cells under various microscopes. A: Cell aggregation of CEM cells and sp6 cells in the presence or absence of DPH1 / 2 materials was observed under an optical microscope; B: Cell aggregation of CEM cells and sp6 cells in the presence or absence of DPH1 / 2 materials was observed under a biological electron microscope; C: Cell aggregation of CEM cells and sp6 cells in the presence or absence of DPH1 / 2 materials was observed under a fluorescence confocal microscope; D: Statistical analysis of the aggregation rate of CEM cells and sp6 cells; E: Statistical analysis of the area of ​​CEM cells and sp6 cells.

[0037] Figure 7 To construct the cell spheroid model and the cell layer assembly model using DPH1 / 2, A: Characterization of the mCherry-IPS and GFP-IPS cells used in the model construction; B: The process of cell spheroid model formation; C: Cell spheroid model; D: Cell spheroid model control group; E: The process of cell layer assembly model formation; F: Cell layer assembly model; G: Cell layer assembly model control group;

[0038] Figure 8DPH1 / 2-mediated cell clusters can promote the differentiation of stem cells into pancreatic islet cells. A: Fluorescence confocal microscopy shows the effect of DPH1 / 2 on the secretion of insulin and glucagon by sp6 cells, and compares it with the expression of cell clusters induced by stem cells. B: Flow cytometry results reflect the effect of DPH1 / 2 on the secretion of insulin and glucagon by sp6 cells. C: Statistics of flow cytometry results. D: Effect of DPH1 / 2 on the expression of insulin and glucagon genes in sp6 cells. E: Effect of DPH1 / 2 on the expression of key genes in the process of sp6 cells being induced into SC-β cells. F: Glucose promotes insulin secretion.

[0039] Figure 9 To show the changes in gene expression caused by DPH1 / 2-mediated cell-cell interactions, A: GO enrichment histogram of CEM cell transcriptome sequencing; B: Volcano plot of differentially expressed genes in CEM cell transcriptome sequencing; C: KEGG enrichment scatter plot of differentially expressed genes in CEM cell transcriptome sequencing; D: Differentially expressed genes were screened from transcriptome sequencing results and verified by RT-qPCR in CEM cells; E: RT-qPCR results showed that the expression of surface cadherin genes in sp6 cells was different with or without the addition of DPH1 / 2; F: Immunofluorescence confocal microscopy results showed that the expression of surface cadherin genes in sp6 cells was different with or without the addition of DPH1 / 2; G: GO enrichment histogram of sp6 cell transcriptome sequencing; H: KEGG enrichment scatter plot of differentially expressed genes in sp6 cell transcriptome sequencing; I: Volcano plot of differentially expressed genes in sp6 cell transcriptome sequencing; J: Differentially expressed genes were screened from transcriptome sequencing results and verified by RT-qPCR in sp6 cells. DETAILED DESCRIPTION

[0040] The present invention provides a pair of mutually recognizing and binding α-helical peptides, including α-helical peptide 1 and α-helical peptide 2, wherein the amino acid sequence of the α-helical peptide 1 is shown in SEQ ID No. 1, and the amino acid sequence of the α-helical peptide 2 is shown in SEQ ID No. 2.

[0041] SEQ ID No.1: GEIAALEQENAALEQKIAALKWKNAALKQGGC;

[0042] SEQ ID No. 2: CGGKIAALKQKNAAL-KYEIAALEQENAALEQG.

[0043] The present invention also provides the use of the α-helical peptide described in the above technical solution in preparing cell cluster products.

[0044] The present invention also provides a pair of peptides for preparing cell clusters, comprising DPH1 and DPH2. DPH1 is the α-helical peptide 1 of the α-helical peptide described in the above technical solution, to which PEG and DMPE are attached; DPH2 is the α-helical peptide 2 of the α-helical peptide described in the above technical solution, to which PEG and DMPE are attached. The present invention does not specifically limit the method for attaching PEG and DMPE to α-helical peptide 1 or α-helical peptide 2; conventional methods employed by those skilled in the art can be employed. PEG: polyethylene glycol; DMPE: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine.

[0045] The present invention also provides the use of the polypeptide described in the above technical solution in preparing cell cluster products.

[0046] The present invention also provides a method for constructing a spherical model cell cluster, comprising the following steps:

[0047] 1) mixing DPH1 in the polypeptide described in the above technical solution with cells to obtain DPH1-cells;

[0048] 2) mixing DPH2 in the polypeptide described in the above technical solution with cells to obtain DPH2-cells;

[0049] 3) The DPH1 cells and DPH2 cells obtained in step 1) are mixed and cultured to obtain spherical model cell clusters.

[0050] The present invention mixes the DPH1 in the polypeptide of the above technical solution with cells to obtain DPH1-cells. In the present invention, the DPH1 is preferably mixed in the form of a DPH1 solution, 300 μM, and the cells are mixed in the form of a cell suspension, the number of cells in the cell suspension is 5×10 5 The volume ratio of the DPH1 solution to the cell suspension is 42:458; the mixing time is preferably 10 min. In the present invention, the DPH2 is preferably mixed in the form of a DPH2 solution, the concentration of the DPH2 solution is 300 μM, and the cells are mixed in the form of a cell suspension, the number of cells in the cell suspension is 5×10 5 The volume ratio of the DPH2 solution to the cell suspension is 42:458; the mixing time is preferably 10 min.

[0051] The present invention provides a method for constructing a cell cluster of a layer-by-layer assembly model, comprising the following steps:

[0052] 1) After the cells are cultured on the wall, they are mixed with the DPH2 polypeptide described in the above technical solution and incubated.

[0053] 2) The mixture was mixed with DPH1 in the polypeptide described in the above technical solution and incubated for 10 minutes, and then mixed with cells to obtain a cell-layer assembly model cell cluster.

[0054] In the present invention, cells are cultured on the wall and then mixed with the DPH2 in the polypeptide of the above technical solution and incubated. In the present invention, the final concentration of the DPH2 is 25 μM, and the number of the cells is 5×10 5 The incubation time is preferably 10 min.

[0055] The present invention is then mixed with the DPH1 in the polypeptide of the above technical solution and cells and incubated to obtain a cell layer assembly model cell cluster. In the present invention, the final concentration of the DPH1 is 25 μM, and the number of the cells is 5×10 5 The mixing time with cells is preferably 1 h.

[0056] The present invention has no particular limitation on the type of cells.

[0057] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1

[0059] Taking DPH1 as an example, the designed molecular synthesis route is as follows:

[0060] The specific steps are:

[0061] 1. Basic reaction process

[0062] 1. Resin activation: Take 0.91g 2-Cl resin in a clean and dry reaction tube, add 20ml DMF, and activate for about 30 minutes.

[0063] 2. Amino Acid Linking: Weigh the calculated amount of the C-terminal first amino acid Fmoc-Cys(Trt)-NH2 (protected) and 0.5ml of DIEA into a reaction tube. Add excess DMF as solvent for the reaction. Add different types of catalysts depending on the amino acid.

[0064] 3. Elution of Fmoc protection:

[0065] Step 1: Fmoc removal

[0066] 20% piperidine

[0067] DMF 5min, 1 time 15min, 1 time

[0068] Step 2 Wash with DMF×2, MeOH×2, DMF×2 for 1 min each time

[0069] Step 3: Coupling AA / HBTU / NMM for 30 minutes

[0070] Step 4: Wash with DMF × 2, MeOH × 2, and DMF × 2 for 1 min each time

[0071] 4. Detection: In solid-phase peptide synthesis, the connection efficiency is mainly determined by detecting the free amino groups on the resin. The detection method is called the Kaiser method. The detection results will show blue or reddish brown (Pro, Ser, His) when there are free amino groups.

[0072] Kaiser reagent includes: A, 6% ninhydrin in ethanol

[0073] B, 80% phenol in ethanol

[0074] C, 2% 0.001M KCN ​​in pyridine solution

[0075] Take a small amount of reacted resin, add 2-3 drops of A, B, and C respectively, and heat at 105-110℃ for 5 minutes. If the solution is blue, or the resin is blue or reddish brown, it indicates that there are free amino groups. Otherwise, the connection is complete.

[0076] 5. After successful detection, continue to link the second amino acid at the C-terminus. The method is the same as above, starting from step 3.

[0077] 6. Cutting: Cut with trifluoroacetic acid cutting solution for 3 hours. Filter the reaction solution to obtain a trifluoroacetic acid solution of the polypeptide.

[0078] 7. Precipitation: Use excess ether to precipitate and centrifuge. Elute the centrifuged sample with ether several times and centrifuge again to obtain the initial peptide sample.

[0079] 8. After the initial dissolution, add a certain concentration of iodine reagent, stir and oxidize for 5 hours, detect the oxidation status by mass spectrometry, and add citric acid to terminate the oxidation.

[0080] 9. Purification: The crude peptide was purified by HPLC.

[0081] 10. Mass spectrometry (detection)

[0082] 11. Freeze-drying: Rapidly cool with liquid nitrogen and then freeze-dry.

[0083] 2. Mass analysis (COA): High performance liquid chromatography (HPLC) and mass spectrometry (MS) are used to perform mass analysis of the target compound in terms of peptide sequence, molecular weight and chemical purity.

[0084] 3. Dissolve 80mg of the peptide in water and add acetonitrile until the peptide is completely dissolved. Dissolve 20mg of DMPE and the peptide solution in DMF and add acetonitrile until completely dissolved. Mix and adjust the pH to 7.5-7.8. Incubate overnight at room temperature.

[0085] 4. Filter through a filter membrane and freeze-dry to obtain DPH1.

[0086] DPH2 was prepared in the same manner as above.

[0087] Example 2

[0088] 1. DPH1 and DPH2 can be successfully located on the cell membrane and confirmed that the two bind to each other through the binding of α-helical peptides.

[0089] Select 1×10 5 The cells (CEM cells) were co-acted with DPH1 with green fluorescent molecules (DPH1-FITC) for 0min, 2min, 5min, 10min, and 15min, and the immunofluorescence and statistical results ( Figure 5 Middle AB) shows that 10 min is the most appropriate exposure time. Too long an exposure time will cause endocytosis of the material. In addition, 1×10 5 The cells (CEM cells) were treated with 0μM, 5μM, 10μM, 25μM, and 50μM DPH1 with green fluorescent molecules. The results of immunofluorescence and cell flow cytometry were ( Figure 5 Both C and D) show that 25 μM is the most suitable concentration. DPH1 with green fluorescent molecules: 1 mmol FITC was dissolved in 20 mL DMSO (dimethyl sulfoxide), and 1 mL DIEA (N,N-diisopropylethylamine) was added. The reaction was allowed to react with the synthesized DPH1 at room temperature for 2 hours.

[0090] DPH2 is the same as above.

[0091] In addition, this example also synthesized Rho B-helix peptide 2, which cannot be inserted into the cell membrane without DMPE, to verify that its binding with DPH1-FITC depends on the direct interaction between this pair of α-helical peptides. Flow cytometry and confocal microscopy results ( Figure 5 Figures E and F) both demonstrate that DPH1-FITC, with DMPE, can be inserted into the phospholipid bilayer of the cell membrane, while RhoB-H2 (peptide sequence 2 connected to rhodamine B, referred to as Rho B-H2) without DMPE can only be displayed on the cell membrane by binding to DPH1-FITC. RhoB-H2 alone cannot be localized on the cell membrane ( Figure 5 F and G).

[0092] 2. DPH1 / 2 mediates cell clustering

[0093] The results of optical microscopy, biological scanning electron microscopy, and fluorescence confocal microscopy all showed that materials with DPH1 / 2 could cause dispersed cells to aggregate into cell clumps. The cell surfaces of the cell clumps were pulled, generating intercellular forces. The number and size of the cell clumps also indicate that DPH1 / 2 has a significant effect on cell aggregation ( Figure 6 ).

[0094] 3. Artificially intervene in cell interactions by forming a coiled-coil structure through DPH1 / 2 to construct spherical models and cell-layer assembly models

[0095] In this example, the desired cell aggregates were obtained by adding cells and DPH1 / 2 materials in a predetermined order. In order to clearly distinguish the cells added in sequence and to better observe the different components of the cell clusters under a fluorescence confocal microscope, two IPS cell lines labeled with GFP and mCherry were used. 21 μl of 300 μM DPH1 and 229 μl containing 1×10 5 1×10 mCherry-IPS cell suspension was mixed; 21 μl of 300 μM DPH2 was mixed with 229 μl of 1×10 5 The cell suspension of GFP-IPS was mixed (final concentration 25 μM), and then the two were mixed. The whole process was carried out on a shaker in a CO2 37℃ incubator. After 1 hour of mixed culture, the mixed cells were photographed under a fluorescence confocal microscope. It can be seen that a cell sphere model with random mixing of red and green cells was formed as expected ( Figure 7 ).

[0096] First, plate the mCherry-IPS cells in a confocal dish. The next day, after they adhere to the wall, add 42 μl of a mixture of 300 μM DPH2 and 458 μl of culture medium (final concentration 25 μM). After incubation for 10 minutes, add 42 μl of a mixture of 300 μM DPH1 and 458 μl of culture medium (final concentration 25 μM).

[0097] Then, the digested GFP-IPS cells (500 μl containing 1×10 5 GFP-IPS cell suspension) and incubated for 1 hour before taking the photo. After taking the photos layer by layer under the fluorescence confocal microscope, it can be observed that the red fluorescent cells and green fluorescent cells are clearly layered, forming a cell layer assembly structure ( Figure 7 ).

[0098] 4. DPH1 / 2-mediated cell-cell interactions promote stem cell differentiation into pancreatic islet cells

[0099] During the generation of human pluripotent stem cell-derived pancreatic β cells, some cells were unable to aggregate into clusters, but were able to cluster well after adding DPH1 / 2 materials (refer to the literature Generation of insulin-producing pancreatic β cells from multiple human stem cell lines). Immunofluorescence, flow cytometry, and qPCR experiments were used to detect insulin expression levels. The experimental results showed that the cell clusters formed by adding DPH1 / 2 had similar insulin and glucagon expression levels as the induced cell clusters. The experimental group to which DPH1 / 2 materials were added to form a coiled-coil structure and the control sp6 cells without DPH1 / 2 were stimulated with high and low concentrations of glucose solutions for 1 hour. The results showed that cells with a coiled-coil structure responded better to high or low concentrations of glucose solutions, which means that the formation of a coiled-coil structure helps sp6 cells differentiate into SC-β cells. In addition, the expression levels of other important related genes in the process of stem cell differentiation into SC-β cells were also significantly increased ( Figure 8 ).

[0100] 5. Effects of DPH1 / 2-mediated cell-cell interactions on gene expression

[0101] A selection of genes commonly implicated in cell adhesion and interaction was initially validated by qPCR in CEM cell experimental and control groups. qPCR results showed that many genes involved in cell adhesion and interaction were significantly upregulated in the experimental group treated with DPH1 / 2 compared to the control group. Subsequently, transcriptome sequencing of the CEM cells in the experimental and control groups was performed. Gene oncology (GO) analysis of transcriptome sequencing revealed that coiled-coil-mediated CEM cluster formation significantly affected the expression of genes associated with the cell surface and plasma membrane. Furthermore, genes involved in common cellular pathways related to cell-cell interaction, such as the TGF-beta and Hippo signaling pathways, were also upregulated and downregulated. RT-qPCR was also performed to validate the upregulated genes, and the upregulation trend was consistent with the transcriptome sequencing results. Initial validation was also performed in sp6 cells, selecting genes commonly implicated in cell adhesion, including E-cadherins. Both immunofluorescence and qPCR results demonstrated upregulation of E-cadherin expression in cells treated with coiled-coil. Next, transcriptome sequencing was performed on sp6 cells. GO analysis results showed that the expression of genes related to the cell surface and plasma membrane changed significantly. Later, some upregulated genes were selected for RT-qPCR verification. The results of the upregulation trend were consistent with the results of transcriptome sequencing ( Figure 9 ).

[0102] Based on the above results, it is speculated that DPH1 / 2 forms a coiled-coil structure after inserting into the cell membrane, thereby shortening the distance between cells, allowing cells that were originally unable to contact to aggregate, and then changing the gene expression of cells by affecting their behavior.

[0103] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A pair of α-helical peptides that recognize and bind to each other, characterized in that: It comprises an alpha helical peptide 1 and an alpha helical peptide 2. The amino acid sequence of the alpha helical peptide 1 is shown in SEQ ID No. 1, and the amino acid sequence of the alpha helical peptide 2 is shown in SEQ ID No.

2.

2. Use of the α-helical peptide according to claim 1 in preparing cell pellet products.

3. A pair of polypeptides for preparing cell clusters, characterized in that: It comprises DPH1 and DPH2, wherein DPH1 is the α-helical peptide 1 in the α-helical peptide of claim 1 to which PEG and DMPE are connected; The DPH2 is the α-helical peptide 2 in the α-helical peptide according to claim 1, to which PEG and DMPE are connected.

4. Use of the polypeptide according to claim 3 in preparing a cell mass product.

5. A method for constructing a spherical model cell cluster, characterized in that: The following steps are involved: 1) mixing DPH1 in the polypeptide of claim 3 with cells to obtain DPH1-cells; 2) mixing DPH2 in the polypeptide of claim 3 with cells to obtain DPH2-cells; 3) The DPH1 cells and DPH2 cells obtained in step 1) are mixed and cultured to obtain spherical model cell clusters.

6. The method according to claim 5, characterized in that In step 1), DPH1 is mixed in the form of a DPH1 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a number of cells of 5×10 5 The volume ratio of the DPH1 solution to the cell suspension was 42:458; The mixing time is 10 minutes.

7. The method according to claim 5, characterized in that In step 1), DPH2 is mixed in the form of a DPH2 solution with a concentration of 300 μM, and the cells are mixed in the form of a cell suspension with a number of cells of 5×10 5 The volume ratio of the DPH2 solution to the cell suspension was 42:458; The mixing time is 10 minutes.

8. A method for constructing a cell cluster of a layer-by-layer assembly model, characterized in that: The following steps are involved: 1) culturing cells after adherence and mixing with the DPH2 polypeptide of claim 3 and incubating; 2) The mixture was mixed with DPH1 in the polypeptide of claim 3 and incubated for 10 minutes, and then mixed with cells to obtain a cell-layer assembly model cell cluster.

9. The method according to claim 8, characterized in that In step 1), the final concentration of DPH2 was 25 μM, and the number of cells was 5×10 5 The incubation time is 10 min.

10. The method according to claim 8, characterized in that In step 2), the final concentration of DPH1 was 25 μM, and the number of cells was 5×10 5 The mixing time with cells was 1 h.

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