Protein nano material as well as preparation method and application thereof

Abstract

The invention discloses a protein nano material as well as a preparation method and application thereof. The protein nano material comprises a disc-shaped self-assembly body formed by tobacco mosaic virus capsid protein and a peptide fragment containing RGD motif and presented on the surface of the disc-shaped self-assembly body, the peptide fragment containing the RGD motif is presented on the surface of the discoid self-assembly body in a multivalent form, and the peptide fragment containing the RGD motif contains at least two cysteine residues. According to the protein nanomaterial, the tobacco mosaic virus capsid protein disc-shaped self-assembly body is used as a support, stable multivalent presentation of the RGD motif peptide fragment is achieved on the surface of the support, and therefore stable and consistent adhesion sites can be provided on the culture surface; meanwhile, the material composition is clear, the preparation process is controllable, the batch difference is reduced, and the repeatability and reliability of cell culture and related application are improved.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical technology, and relates to a protein nanomaterial, its preparation method and application. Specifically, it relates to a protein nanomaterial with a disc-shaped self-assembled structure formed by tobacco mosaic virus (TMV) capsid protein as a scaffold, and multivalent RGD motif peptides on its surface, as well as the preparation method of the material and its application in promoting cell adhesion and / or cell spreading. Background Technology

[0002] Cell adhesion is one of the most fundamental and crucial initial steps in adherent cell culture. The ability of cells to quickly and stably adhere to the culture medium and form a good spreading morphology often determines subsequent proliferation, phenotypic maintenance, and the consistency of experimental or application results. Therefore, in applications such as cell culture, tissue engineering, and regenerative medicine, the culture medium substrate usually needs to be treated to enhance adhesion, providing sufficient "attachment sites" for cells to improve adhesion efficiency and spreading.

[0003] To improve cell adhesion and spread on culture surfaces or substrates, natural extracellular matrix components, matrix gels, or functional peptides containing adhesion motifs are often used to treat the culture surface. However, some existing materials suffer from problems such as complex composition or origin and significant batch-to-batch variations. Furthermore, the adsorption / immobilization mechanisms and spatial arrangement of matrix proteins or functional peptides on the surface are difficult to maintain consistently, leading to instability in the number and availability of effective adhesion sites, thus affecting the consistency and reproducibility of cell culture.

[0004] Therefore, there is an urgent need for a material system with a well-defined composition, batch consistency, and stable structure that can provide stable and consistent adhesion sites on the culture surface, especially enabling the multivalent presentation of adhesion ligands to enhance cell adhesion and spreading effects, and improve the reproducibility and reliability of cell culture and related applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a protein nanomaterial, its preparation method, and its application. The protein nanomaterial has a stable structure, excellent performance, is easy to prepare, and has a wide range of applications, effectively promoting cell adhesion and spreading.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a protein nanomaterial comprising: a disc-shaped self-assembly formed by tobacco mosaic virus capsid protein, and an RGD-containing motif peptide present on the surface of the disc-shaped self-assembly; wherein the RGD-containing motif peptide is presented on the surface of the disc-shaped self-assembly in a multivalent form, and the RGD-containing motif peptide contains at least two cysteine ​​residues.

[0007] In this invention, the protein nanomaterial uses the disc-shaped self-assembled body of tobacco mosaic virus capsid protein as a scaffold, enabling the stable multivalent presentation of RGD motif peptides on its surface, thereby providing relatively stable and consistent adhesion sites on the culture surface; at the same time, the material composition is well-defined and the preparation process is controllable, which helps to reduce batch differences and improve the reproducibility and reliability of cell culture and related applications.

[0008] Preferably, the RGD motif-containing peptide is covalently linked to the tobacco mosaic virus capsid protein subunit.

[0009] Preferably, the covalent linkage method includes: fusion expression of the RGD motif-containing peptide with the tobacco mosaic virus capsid protein.

[0010] Preferably, the RGD motif peptide is linked to the C-terminus or N-terminus of the tobacco mosaic virus capsid protein.

[0011] Preferably, the amino acid sequence of the RGD motif peptide includes the sequence shown in SEQ ID NO.1.

[0012] SEQ ID NO.1: CDCRGDCFC.

[0013] Preferably, the amino acid sequence of the fusion protein formed by the tobacco mosaic virus capsid protein and the RGD motif peptide includes the sequence shown in SEQ ID NO.2.

[0014] SEQ ID NO.2: SYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQVTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPCTAETLDATRRVDDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPATCDCRGDCFC.

[0015] Secondly, the present invention provides a method for preparing the protein nanomaterial described in the first aspect, the method comprising the following steps: (1) Construct a recombinant expression vector containing tobacco mosaic virus capsid protein and a gene encoding RGD motif peptide; (2) Transform the host and induce the expression of the fusion protein; (3) Purify the fusion protein; (4) Under in vitro assembly conditions, the fusion protein is self-assembled to form a disc-shaped self-assembled body.

[0016] Preferably, the purification includes at least two of the following: salting-out fractionation precipitation, ion exchange chromatography, or molecular sieve chromatography.

[0017] In some embodiments, salting-out fractionation precipitation may be performed under conditions of ammonium sulfate saturation of 15%-40% (e.g., 15%, 20%, 30%, or 40%); ion exchange chromatography may be anion exchange chromatography and / or cation exchange chromatography. As an example, but not limited to, the fusion protein may be expressed in an *E. coli* expression system, and purified by salting-out fractionation, ion exchange, and / or molecular sieve chromatography, followed by in vitro assembly in a suitable buffer system and further separation and enrichment of the disc-shaped components to obtain disc-shaped protein nanomaterials.

[0018] Preferably, the in vitro assembly conditions include: preparing the purified fusion protein at a concentration of 0.01-5 mg / mL (e.g., 0.01 mg / mL, 0.1 mg / mL, 1 mg / mL, 3 mg / mL or 5 mg / mL) in an assembly buffer and incubating it at 4-37°C (e.g., 4°C, 10°C, 20°C, 30°C or 37°C) for 0.5-48 h (e.g., 0.5 h, 1 h, 10 h, 20 h, 30 h or 40 h) to complete self-assembly.

[0019] Preferably, the assembly buffer includes phosphate buffer, Tris buffer, or HEPES buffer.

[0020] Preferably, the concentration of the assembly buffer is 10-200 mM (e.g., 10 mM, 20 mM, 50 mM, 100 mM or 200 mM) and the pH is 6.5-8.5 (e.g., 6.5, 7, 7.5, 8 or 8.5).

[0021] In some embodiments, the assembly buffer may or may not contain inorganic salts, with a final concentration of 0-300 mM, such as 0 mM, 10 mM, 100 mM, 200 mM, or 300 mM.

[0022] Thirdly, the present invention provides the application of the protein nanomaterials described in the first aspect in promoting cell adhesion and / or cell spreading.

[0023] Preferably, the application includes: using the protein nanomaterial to form a coating on the cell culture surface or cell culture substrate.

[0024] Preferably, the application includes: preparing the protein nanomaterial into a solution with a concentration of 0.1-500 μg / mL (e.g., 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 300 μg / mL or 500 μg / mL), contacting the solution with the cell culture surface or cell culture substrate for 0.1-24 h (e.g., 0.1 h, 1 h, 5 h, 10 h, 20 h or 24 h), removing unbound protein nanomaterials, and then inoculating and culturing cells.

[0025] Preferably, the cell culture surface or cell culture substrate is pretreated with poly-D-lysine and / or poly-L-lysine before the coating is formed.

[0026] Preferably, the cells include any one or a combination of at least two of adherent cells, primary cells, or stem cells.

[0027] Preferably, the stem cells include any one or a combination of at least two of neural stem cells, intestinal stem cells, or induced pluripotent stem cells.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention utilizes the disc-shaped structure formed by the in vitro self-assembly of tobacco mosaic virus capsid protein as a scaffold, which is structurally stable and uniform in size, making it easy to achieve spatially controllable arrangement of adhesion ligands. (2) The present invention significantly increases the local density of adhesion ligands by presenting peptides containing RGD motifs on the surface of the disc scaffold, thereby enhancing the binding ability with cell surface integrins and effectively promoting cell adhesion and spreading. (3) The protein nanomaterial of the present invention is composed of a single recombinant protein. The preparation process is standardized, the composition is clear and the structure is controllable, which avoids the batch difference problem of traditional matrix gel or natural matrix components and improves the repeatability and reliability of experiments and applications. (4) This invention uses a common E. coli expression system to produce fusion proteins, combined with conventional protein purification and in vitro self-assembly technology. It is simple to operate, low in cost, and easy to achieve large-scale preparation. (5) This invention is applicable to a variety of adherent cells, primary cells and stem cells (such as neural stem cells, intestinal stem cells, induced pluripotent stem cells, etc.), and has broad application potential in cell culture, tissue engineering, regenerative medicine and other fields. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the recombinant plasmid. Figure 2 SDS-PAGE image of the purified fusion protein; Figure 3Transmission electron microscope image of discoid protein nanomaterials; Figure 4 Morphological images and quantitative results of neural stem cell adhesion and spreading promoted by different treatment groups; Figure 5 Morphological images and quantitative results of different treatment groups promoting intestinal stem cell adhesion and spreading; Figure 6 Morphological images and quantitative results of different treatment groups promoting the adhesion and spread of induced pluripotent stem cells. Detailed Implementation

[0030] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0031] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0032] Example 1 This embodiment describes the construction, prokaryotic expression, and isolation and purification of the fusion protein expression plasmid, as detailed below: Based on the cDNA nucleotide sequence of the wild-type tobacco mosaic virus (TMV) capsid protein monomer, the target gene (SEQ ID NO.3) encoding the TMV capsid protein and fused with an RGD motif peptide at its C-terminus was inserted into the pET32a vector using molecular biology techniques to construct the recombinant expression vector pET32a-TMV-RGD4C. Figure 1 After confirming the correctness of the inserted sequence via DNA sequencing, the recombinant expression vector was transformed into E. coli BL21 competent cells using the CaCl2 method, plated on LB agar plates, and incubated overnight at 37°C. Single clones were picked and inoculated into 5 mL of LB medium, and ampicillin (final concentration 50 μg / mL) was added. The cells were then incubated at 37°C and 200 r / min for 16 h.

[0033] Transfer 0.5% inoculum to 500 mL LB medium, add ampicillin (final concentration 50 μg / mL), and incubate at 37℃ with shaking at 200 r / min for 3 h until OD is reached. 600 When the concentration of the culture medium was 0.4-0.6, IPTG inducer (final concentration 1 mM) was added, and the culture was continued at 27℃ and 200 r / min for 14 h. After the induction was completed, the cells were collected by centrifugation at 8000 rpm for 5 min.

[0034] The collected bacterial cells were resuspended in lysis buffer and sonicated on ice (400 W, 4 s on, 4 s off, total 60 min). The supernatant was collected by centrifugation at 12000 rpm for 30 min. Solid ammonium sulfate was then slowly added to the supernatant until a final concentration of 40% saturation was reached. After standing at 4°C for 2 h, the precipitate was collected by centrifugation at 12000 rpm for 30 min at 4°C. The precipitate was resuspended in pH 9.5, 50 mM sodium carbonate-sodium bicarbonate buffer, and 20 mM DTT was added. The solution was slowly dissolved by shaking overnight at 4°C. The supernatant was collected by centrifugation at 12000 rpm for 15 min at 4°C.

[0035] The supernatant was dialyzed into anion exchange chromatography loading buffer and filtered through a 0.22 μm syringe filter to remove impurities. The HiTrap Q HP pre-packed column was equilibrated with loading buffer and then loaded with the sample. The column was loaded into an AKTA system, and elution was performed using a 0-1 M NaCl gradient at a flow rate of 2 mL / min. Elution peaks were collected, and the column was dialyzed at 4°C to sodium carbonate-sodium bicarbonate buffer (50 mM, pH 9.5; 20 mM DTT). The purity of the target protein was determined by SDS-PAGE (e.g., [missing information]). Figure 2 As shown in the figure, the high-purity protein solution is stored at -80℃ for later use.

[0036] SEQ ID NO. 3: TGTGATTGTCGCGGGGATTGCTTTTGC.

[0037] Example 2 This embodiment describes the in vitro self-assembly, fine separation, and characterization of discoid protein nanomaterials, as detailed below: The high-purity TMV-RGD4C monomeric protein obtained in Example 1 was dialyzed and replaced in the assembly buffer, which was a pH 7.4, 50 mM phosphate buffer; then it was incubated overnight at 4°C to promote the in vitro self-assembly of the fusion protein.

[0038] The self-assembled sample was further separated by gel chromatography. The chromatography column used was a HiPrep 16 / 60 Sephacryl S-400 HR, and the mobile phase was pH 7.4, 50 mM phosphate buffer. The column volume was fully equilibrated with the phosphate buffer before chromatography, and then the sample was loaded and eluted at a constant flow rate of 1 mL / min. The target peak was collected in fractions according to the elution volume.

[0039] The separated target components were observed using transmission electron microscopy to confirm the formation of the disk-like structure (e.g. Figure 3 (As shown). Finally, the self-assembled product was concentrated by ultrafiltration to obtain disc-shaped protein nanomaterials, which were stored in a 4°C freezer for subsequent cell experiments.

[0040] The discoid protein nanomaterial described in this embodiment can maintain morphological stability at 4°C and meet the requirements for use in cell experiments.

[0041] Example 3 This embodiment provides the application of the discoidal protein nanomaterials obtained in Example 2 in promoting cell adhesion and spreading, as detailed below: 96-well plates were used for surface treatment during culture. First, the 96-well plates were pretreated with poly-D-lysine (PDL) to make their surface positively charged and form an adsorption layer. Then, a solution of disc-shaped protein nanomaterials obtained in Example 2 (100 μg / mL) was added, allowing it to adsorb onto the bottom of the wells and form a protein coating. After adsorption, the solution was discarded, and the plates were gently washed with sterile PBS to remove unadsorbed components. Blank control group: treated with only poly-D-lysine (PDL).

[0042] The cells to be tested, including neural stem cells, intestinal stem cells, and induced pluripotent stem cells, were prepared as single-cell suspensions and seeded into wells in each group for culture. Cell adhesion and spreading were recorded under a microscope at a predetermined time point (6 h post-seeding), and morphological quantitative analysis was performed. The quantitative indicators included cell spreading area and parameters reflecting cell morphological complexity. Results are as follows: Figures 4-6 As shown, compared with the poly-D-lysine (PDL)-only treatment group, the discoid protein nanomaterial treatment group (TMV-RGD4C) of the present invention can promote the adhesion of various cells and significantly improve cell spreading morphology.

[0043] In summary, the protein nanomaterials of this invention use the disc-shaped self-assembled capsid protein of tobacco mosaic virus as a scaffold, enabling stable multivalent presentation of RGD motif peptides on its surface, thereby providing relatively stable and consistent adhesion sites on the culture surface. At the same time, the material composition is well-defined and the preparation process is controllable, which helps to reduce batch differences and improve the reproducibility and reliability of cell culture and related applications.

[0044] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A protein nanomaterial, characterized in that, The protein nanomaterial comprises: a disc-shaped self-assembly formed by tobacco mosaic virus capsid protein, and an RGD-containing motif peptide present on the surface of the disc-shaped self-assembly; the RGD-containing motif peptide is presented on the surface of the disc-shaped self-assembly in a multivalent form, and the RGD-containing motif peptide contains at least two cysteine ​​residues.

2. The protein nanomaterial according to claim 1, characterized in that, The RGD motif peptide is covalently linked to the tobacco mosaic virus capsid protein subunit. Preferably, the covalent linkage method includes: fusion expression of the RGD motif-containing peptide with the tobacco mosaic virus capsid protein; Preferably, the RGD motif peptide is linked to the C-terminus or N-terminus of the tobacco mosaic virus capsid protein; Preferably, the amino acid sequence of the RGD motif peptide includes the sequence shown in SEQ ID NO.

1.

3. The protein nanomaterial according to claim 1 or 2, characterized in that, The amino acid sequence of the fusion protein formed by the tobacco mosaic virus capsid protein and the RGD motif peptide includes the sequence shown in SEQ ID NO.

2.

4. A method for preparing the protein nanomaterial according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) Construct a recombinant expression vector containing tobacco mosaic virus capsid protein and a gene encoding RGD motif peptide; (2) Transform the host and induce the expression of the fusion protein; (3) Purify the fusion protein; (4) Under in vitro assembly conditions, the fusion protein is self-assembled to form a disc-shaped self-assembled body.

5. The method according to claim 4, characterized in that, The purification includes at least two of the following: salting-out fractionation precipitation, ion exchange chromatography, or molecular sieve chromatography.

6. The method according to claim 4 or 5, characterized in that, The in vitro assembly conditions include: preparing the purified fusion protein in assembly buffer at a concentration of 0.01-5 mg / mL and incubating it at 4-37℃ for 0.5-48 h to complete self-assembly; Preferably, the assembly buffer includes phosphate buffer, Tris buffer, or HEPES buffer; Preferably, the concentration of the assembly buffer is 10-200 mM and the pH is 6.5-8.

5.

7. The use of the protein nanomaterials according to any one of claims 1-3 in promoting cell adhesion and / or cell spreading.

8. The application according to claim 7, characterized in that, The applications include: using the protein nanomaterials to form a coating on the surface of cell culture or the substrate of cell culture.

9. The application according to claim 8, characterized in that, The application includes: preparing the protein nanomaterial into a solution with a concentration of 0.1-500 μg / mL, contacting the solution with the cell culture surface or cell culture substrate for 0.1-24 hours, removing unbound protein nanomaterials, and then inoculating and culturing cells.

10. The application according to claim 9, characterized in that, The cell culture surface or cell culture substrate is pretreated with poly-D-lysine and / or poly-L-lysine before the coating is formed. Preferably, the cells include any one or a combination of at least two of adherent cells, primary cells, or stem cells; Preferably, the stem cells include any one or a combination of at least two of neural stem cells, intestinal stem cells, or induced pluripotent stem cells.

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