Gh625-h2a histone, chromatin thereof, and use thereof

Abstract

The present invention provides a gH625-H2A histone, a chromatin thereof, and use thereof. The amino acid sequence of the gH625-H2A histone is set forth in SEQ ID NO. 5. The gH625-H2A histone and the chromatin thereof provided by the present invention have strong cell penetration and skin penetration effects, and can be used to prepare drugs or drug delivery systems, as well as skincare products and cosmetics.

Description

A gH625-H2A histone, its chromatin, and its applications Technical Field

[0001] This invention relates to a gH625-H2A histone, its chromatin, and its applications, belonging to the field of biotechnology. Background Technology

[0002] DNA is considered a highly negatively charged polyanionic polymer that does not readily bind to or penetrate cell membranes without a specific uptake mechanism. Commercial transfection reagents are commonly used to introduce nucleic acids into mammalian cells. These reagents typically use cationic lipids to encapsulate DNA or concentrate nucleic acids through the formation of polycationic polymers. Membrane fusion and endocytosis are the primary mechanisms of intracellular DNA delivery for these reagents. For example, polyethyleneimine (PEI) is a widely used transfection reagent that enhances DNA delivery into cells by forming concentrated electrostatic PEI-DNA complexes. PEI-DNA polycationic polymers appear to promote interactions with negatively charged cell membranes, thereby enhancing endocytosis.

[0003] However, commercial transfection reagents still have limitations. These limitations include lower transfection efficiency compared to viral vectors and instability of the transfection reaction complex. This leads to aggregation, precipitation, or degradation over time, making the transfection reagents unsuitable for long-term storage and in vivo use.

[0004] Eukaryotic DNA is packaged within the cell nucleus through a multi-stage chromatin compaction process. This compaction begins with the assembly of 10 nm nucleosomes, in which the DNA is wound 1.67 times around a standard histone nucleus (two copies of H3, H4, H2A, and H2B). A single nucleosome contains approximately 146 base pairs of DNA, which binds to eight histones, resulting in an assembled molecular weight of approximately 179 kDa.

[0005] Chromatin structure can be viewed as a condensed polyanionic DNA polymer coated with cationic N-terminal histone tails. Four core histones carry protein transduction domains that facilitate membrane penetration. Furthermore, these core histones, individually or together, can condense DNA to facilitate DNA delivery into mammalian cells. More importantly, the reconstructed chromatin enables DNA delivery via a pathway independent of endocytosis. In summary, histones demonstrate great potential as nonviral DNA delivery carriers due to their ability to condense nucleic acids, form stable nanocomplexes, and target specific cell delivery through histone modifications. However, applying histones to mammalian DNA delivery via systemic administration faces several challenges, such as ensuring the stability of histone-DNA complexes, potential immunogenicity, and potential gene expression issues related to intracellular stability and histone-mediated gene repression.

[0006] The skin, the largest body organ in terms of surface area, is an attractive route for transdermal drug delivery due to its non-invasiveness and convenience. The transdermal route also minimizes non-specific systemic side effects, making it highly appealing for biopharmaceutical and skincare applications. However, the epidermis, particularly the stratum corneum, acts as a robust, impermeable barrier, making it difficult to deliver large biomolecules via passive local application. These high-molecular-weight biomolecules often require milder, more invasive delivery enhancers, such as microneedles and electroporation. Despite various physical and biochemical approaches, achieving efficient transdermal delivery remains a significant challenge. Summary of the Invention

[0007] This invention provides gH625-H2A histone, its chromatin, and its applications. The gH625-H2A histone and chromatin of this invention exhibit strong cell penetration and skin permeability, and are expected to promote the development of transdermal gene delivery systems, as well as biopharmaceuticals and skin care products. The biopharmaceuticals and skin care products prepared by this invention achieve passive skin delivery of nucleic acids, small peptides, growth factors, and enzymes without physical or chemical intervention.

[0008] To achieve this objective, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a gH625-H2A histone, wherein the gH625-H2A histone is histone H2A with the peptide gH625 added. Specifically, a cell-penetrating peptide [gH625:HGLASTLTRWAHYNALIRAF] derived from HSV glycoprotein H is inserted into the N-terminus of H2A.

[0010] Preferably, the amino acid sequence of gH625-H2A is shown in SEQ ID NO.5.

[0011] In this invention, the sequence SEQ ID NO.5 is:

[0012] This invention utilizes an artificial histone H2A variant fused with the gH625 peptide for the synthesis of chromatin or DNA-histone complex components to enhance specific functions of DNA and histone derivatives, including the synthesis of chromatin delivery systems (SCDS) and increased skin penetration.

[0013] A second aspect of the invention provides the use of gH625-H2A histone and its chromatin in the preparation of pharmaceuticals or pharmaceutical delivery systems.

[0014] Preferably, the drug is a gene therapy drug.

[0015] Preferably, the drug or drug delivery system can penetrate cell membranes and / or cells.

[0016] In this invention, the synthetic chromatin delivery system (SCDS) of this embodiment demonstrates the function of transporting chromatin from the extracellular to the intracellular space. This invention verifies that chromatin assembled with gH625-H2A has a higher nuclear localization capability compared to wild-type chromatin.

[0017] A third aspect of the present invention provides the use of gH625-H2A histone and its chromatin in the preparation of skin care products and cosmetics.

[0018] Preferably, the gH625-H2A histone and its chromatin can increase the skin penetration of skin care products and cosmetics.

[0019] In this invention, the Franz transdermal assay showed that the passive transdermal capacity of gH625-H2A chromatin was significantly higher than that of wild-type chromatin at all three time points in the experiment (24 hours, 48 ​​hours and 72 hours of exposure).

[0020] In summary, this invention demonstrates that human histone H2A modified with the gH625 peptide enhances the cell penetration ability of wild-type histone H2A. Chromatin assembled with gH625-H2A exhibits nuclear localization comparable to or superior to wild-type chromatin. More importantly, gH625-H2A enables significant chromatin delivery through the skin. Therefore, this invention holds promise for facilitating the development of transdermal gene delivery systems, as well as biopharmaceuticals and skincare products that enable passive skin delivery of nucleic acids, small peptides, growth factors, and enzymes without the need for physical or chemical intervention.

[0021] Compared with the prior art, the beneficial effects and significant progress of applying the technical solution of the present invention are as follows:

[0022] 1. The gH625-H2A histone provided by this invention enhances the cell penetration ability of human H2A by inserting a cell-penetrating peptide [gH625:HGLASTLTRWAHYNALIRAF] derived from HSV glycoprotein H into the N-terminus of H2A. Compared with wild-type H2A, the modified histone gH625-H2A exhibits a significant increase in cell permeability;

[0023] 2. The chromatin of histone gH625-H2A provided by the present invention has a higher nuclear localization ability compared with wild-type chromatin;

[0024] 3. The chromatin of histone gH625-H2A provided by the present invention has a higher skin penetration effect compared with wild-type chromatin. Attached Figure Description

[0025] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly described below.

[0026] Figure 1 shows the cell penetration experiment results of gH625-H2A in Example 2;

[0027] Figure 2 shows the results of a cell penetration experiment using biotin-labeled gH625-chromatin in Example 3.

[0028] Figure 3 shows the results of the cell penetration experiment using Cy5-labeled gH625-chromatin in Example 4.

[0029] Figure 4 shows the results of a skin penetration experiment using the Franz cell system of pig skin in Example 5.

[0030] Figure 5 shows the experimental results of the gH625-H2A chromatin epidermal delivery vector in Example 6. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0032] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0033] To better understand this invention, the technical terms used in this invention will be explained below.

[0034] The term “chromatin” as used herein, as is known in the art, refers to a complex of DNA and associated proteins, including histones, containing repeating nucleosome structures.

[0035] The terms “nucleosome” and “nucleosome structure” and their grammatical variants used in this article refer to structures produced in vitro by the assembly of DNA and four types of histones [H3, H4, H2A, H2B] that exhibit a “beaded” structure.

[0036] Those skilled in the art will be able to determine the appropriate use, application, and / or administration mode of the composition by reference and as described herein, and then formulate the synthetic chromatin described herein into a composition, particularly a pharmaceutical composition, by reference and as described herein, for such a mode of administration.

[0037] As used in this specification, the term "comprising" means "consisting of at least part of". When interpreting a statement in this specification that includes this term, the feature beginning with that term must be present in each statement, although other features may also be present. Related terms such as "comprising" and "included" will be interpreted in the same manner.

[0038] As used herein, the term "substantially composed of" refers to the specified materials or steps, as well as those that do not materially affect the essential and novel features of the claimed invention.

[0039] As used herein, the term "composed of" refers to the specific materials or steps of the claimed invention, excluding any elements, steps, or components not specifically described in the claims.

[0040] Example 1

[0041] Experimental methods

[0042] 1.1 Purification of histone octamers from inclusion bodies

[0043] The pET3 expression vector for human histones has been widely used for wild-type histone expression and purification. In this example, DNA synthesis (GenScript Biotech Corp.) was used to add peptide gH625 and EGF to the histone genes. Insoluble inclusion bodies were obtained by expressing each histone gene in BL21(DE3)CodonPlus staining (Stratagene) using twice concentrated Luria broth (Sigma) supplemented with 0.5% glucose (w / v).

[0044] The induction conditions for histone expression, inclusion body preparation, and two-step column purification using Sephacryl S200 (GE Healthcare) and SP Sepharose Fast Flow (GE Healthcare) are consistent with existing techniques (J. Rosenbluh, SK Singh, Y. Gafni, A. Graessmann, A. Loyter, Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells, Biochim Biophys Acta 1664 (2004) 230-240. KS Siu, D. Chen, X. Zheng, X. Zhang, N. Johnston, Y. Liu, K. Yuan, J. Koropatnick, ERGillies, W.P. Min, Non-covalently functionalized single-walled carbon nanotube for topical siRNA delivery into melanoma, Biomaterials). 35(2014)3435-3442.10.1016 / j.biomaterials.2013.12.079.DPWermeling,SLBanks,DAHudson,HSGill,J.Gupta,MRPrausnitz,ALStinchcomb,Microneedles permit transdermal delivery of a skin-impermeant medication to humans,Proc Natl Acad Sci USA 105(2008)2058-2063.10.1073 / pnas.0710355105.).

[0045] The purified recombinant core histone and its modified form were dissolved in unfolding buffer (8M guanidine hydrochloride, 20mM sodium acetate, pH 5.2, 10mM DTT) and reconstituted into histone octamers containing wild-type or hybrid wild-type and modified histones. The specific operation is the same as the existing technology (J. Rosenbluh, SKSingh, Y. Gafni, A. Graessmann, A. Loyter, Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: avel mechanism for the introduction of macromolecules into plant cells, Biochim Biophys Acta1664(2004)230-240.KSSiu, D.Chen, 35(2014)3435-3442.10.1016 / j.biomaterials.2013.12.079.DPWermeling,SLBanks,DAHudson,HSGill,J.Gupta,MRPrausnitz,ALStinchcomb,Microneedles permit transdermal delivery of a skin-impermeant medication to humans,Proc Natl Acad Sci USA 105(2008)2058-2063.10.1073 / pnas.0710355105.), histone octamers were refolded and purified using Superdex 200 gel filtration chromatography (Highload 16 / 600, GE Heathcare).

[0046] 1.2 Assembly of DNA and purified octamer salt gradient chromatin

[0047] Experimental methods

[0048] (1) DNA containing 16 repeats of the widom 601 sequence (pUC19 / 16x601 SEQ ID NO.7) was used for gene delivery detection, and pEGFP-C1 / Mlu16x601 (SEQ ID NO.8) supercoiled plasmid DNA was constructed.

[0049] The sequence of SEQ ID NO.7 is:

[0050] The sequence of SEQ ID NO.8 is:

[0051] (2) DNA was purified by conventional CsCl gradient ultracentrifugation;

[0052] (3) In order to obtain physiologically spaced nucleosome particles on synthetic chromatin fibers, the mass ratio of various histones to DNA was screened using a micro-salt dilution method.

[0053] (4) Large-scale assembly of up to 60 μg of chromatin in dialysis membranes using step gradient dialysis;

[0054] (5) The quality of the synthetic nucleosome array was evaluated using the assembled chromatin by micrococcal nuclease (MNase).

[0055] Experimental results

[0056] The biophysical properties of histones are shown in Table 1. The amino acid sequences of histones are shown in Table 2.

[0057] Table 1

[0058] Example 2: Cell penetration assay of free histone gH625-H2A

[0059] Experimental methods

[0060] 2.1 The gH625 peptide was fused to the N-terminus of the human H2A protein via gene synthesis, and recombinant gH625-H2A and wild-type canonical human histones were purified from an E. coli expression system. Both gH625-H2A and wild-type H2A were biotinylated via NHS (N-hydroxysuccinimide)-mediated biotin conjugation, and the labeled histones were further purified by gel filtration chromatography.

[0061] 2.2 To compare the cell penetration ability of gH625-H2A and wild-type H2A in HeLa cells, the same amount of histone was added directly to the cell culture medium, and the relative penetration efficiency was visualized using Avidin-Flur 488.

[0062] 2.3 To explore the penetration kinetics of gH625-H2A, treatment with gH625-H2A for up to 60 minutes at different time points was performed in intact growth medium containing 5% FBS.

[0063] Experimental results

[0064] The electrophoresis diagrams of recombinant gH625-H2A and wild-type canonical human histones are shown in Figure 1A.

[0065] The relative permeation efficiency results were visualized using Avidin-Flur 488, as shown in Figure 1B. While H2A wild-type treatment resulted in some visual fluorescence intensity, gH625-H2A treatment significantly increased the fluorescence signal in cells, indicating that more gH625-H2A histones could penetrate the entire cell membrane. gH-H2A was compared to WT H2A. Microplate readings of the fluorescence signal showed that gH625-H2A had approximately a 6-fold higher fluorescence signal compared to wild-type H2A.

[0066] The penetration kinetics of gH625-H2A are shown in Figure 1C. The results indicate that the gH625-H2A histone can penetrate cells to saturation levels within 30 minutes, suggesting a relatively faster transmembrane translocation rate than most commercial transfection reagents that rely on endophagy. Similar results were obtained using growth media containing 10% FBS. In summary, these results demonstrate that gH625 significantly enhances the intrinsic cell membrane penetration ability of H2A, regardless of the presence of FBS in the culture medium.

[0067] Example 3: Cell penetration assay using biotin-labeled gH625-chromatin

[0068] Experimental methods

[0069] 3.1 Histone biotinylation

[0070] The method is the same as in Example 1, using step gradient dialysis to perform large-scale assembly of up to 60 μg of chromatin in a dialysis membrane. The quality of the synthesized nucleosome array was evaluated using micrococcal nuclease (MNase) assays. Specific steps are as follows.

[0071] Partially purified single histone H2A and gH625-H2A were conjugated with biotin molecules using the commercially available EZ-Link pentamidine-biotin kit (Thermo Scientific). The specific method was as follows: Lyophilized histone powder was dissolved at a final concentration of 10 mg / mL in BupH MES (2-[N-morpholino]ethanesulfonic acid) buffer, pH 4.5–5 (Thermo Scientific Product 28390). EDC and Sulfo-NHS (Thermo Scientific Product Nos. 22980, 24510) were dissolved in MES buffer and then applied to the protein samples at final concentrations of 0.1 mM and 5 mM, respectively. After adjusting the reactants to 1x phosphate-buffered saline (PBS) by adding 10x concentrated PBS, EZ-Link pentamidine-biotin (Thermo Scientific Products 21345) was applied to the reactants at a final 20-fold molar excess of protein. The reaction was quenched by adding 1 M Tris (pH 8.0 or higher) to the final 50 mM Tris. The reactants were dialyzed in PBS to remove residual EDC and chemicals, and then further purified by applying the solution to a Superdex S200 gel filter column.

[0072] 3.2 Cell penetration assay using biotin-labeled gH625-chromatin

[0073] HeLa S3 cells were seeded at 10,000 cells / well in 96-well plates. Chromatin containing gH625-H2A or wild-type histones was assembled using biotinylated pUC19 / 16×601 plasmid DNA. The stained proteins were applied directly to the cell culture medium and incubated for 1 hour. The adherent cells were then fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. The cells were then blocked with 10% BSA in PBS containing 0.5% Tween-20. Avidin Alexa Fluor 488 (~488 / 525nm, Thermo Scientific, product number A21370) was applied to each well (0.5 μg / 100 μL PBS), followed by staining with DAPI (0.05 μg / 100 μL PBS). Cell fluorescence was observed quantitatively or qualitatively using a fluorescence microscope (Nikon inverted Ti-S microscope), a confocal fluorescence microscope (LSM 880, Zeiss), or a plate reader (Varioskan Lux, Thermo Scientific). Cell imaging was analyzed using ImageJ. The DAPI signal particle selection threshold was adjusted to 850 infinity (ImageJ parameter), and effective cell nuclei in the blue channel were selected. The effective intensity at the same location in the green channel was measured and statistically analyzed. The measurements were normalized to the mean (10). NS. Not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0074] Experimental results

[0075] The results of in vitro chromatin assembly to prepare recombinant octamers are shown in Figure 2A.

[0076] The MNase assay results for chromatin assembly quality are shown in Figure 2B. Although naked DNA mainly produced low molecular weight DNA fragments below 100 bp, chromatin assembled using wild-type or gH625-H2A containing octamers produced 200 bp DNA ladders, indicating that the chromatin is mainly composed of physiological spatial nucleosomes (Figure 2B, comparing lane 2 with lanes 4 and 6). The results show that biotin modification of the gH625 peptide at the H2AN terminus of histones or the plasmid DNA backbone does not affect the quality of chromatin assembly.

[0077] As shown in Figures 2C and 2D, the chromatin displayed by the gH625 peptide penetrates cells with greater efficiency than wild-type chromatin (Figure 2C, comparing WT H2A chromatin with gH625-H2A chromatin in Avidin-Flur 488). Given the intrinsic nuclear localization sequences of the four core histone 45s in humans, the chromatin displayed by gH625 appears to be predominantly localized in the nucleus (Figure 2C, see merged image of Avidin-Flur 488 and DAPI). Quantification of the images in Figure 2C using ImageJ software confirms that wild-type chromatin possesses intrinsic membrane permeability, and the presence of additional gH625 peptide on the chromatin significantly increases the transfer of plasmid DNA across the cell membrane to the nucleus (Figure 2D). However, there is a weakness in observing the translocation of biotin-labeled DNA through chromatin components because membrane permeability is required for visualization using secondary Avidin Flur 488 binding, which can lead to endogenous cytobiotin-mediated signaling and artifacts.

[0078] Example 4: Cell penetration assay using Cy5-labeled gH625-chromatin

[0079] To visualize the translocated plasmid DNA more directly, this embodiment pre-labeled the DNA with Cy5 using the Tag IT Nucleic Acid Labeling Kit before chromatin assembly.

[0080] Experimental methods

[0081] pUC19 / 16×601 plasmid DNA was labeled using the Label IT Tracker Intracellular Nucleic Acid Localization Kit (Mirus Bio, MIR7021). Excess dye was removed by ethanol precipitation. Following the manufacturer's protocol, 4 μg of DNA was used to transfect HeLa S cells using the Effectene Transfection Reagent (Qiagen, 301425). An equal volume of chromatin was added directly to the adherent cells in serum-free DMEM. After incubation at 37°C for 3 hours, the cells were washed once with PBS and further incubated for 1 hour with 1 mL of 5 U DNase in DMEM containing 15 mM MgCl2. Cells were collected, washed with PBS, and fixed by adding 4% paraformaldehyde. The collected cells were analyzed by FACS.

[0082] For fluorescence microscopy image analysis, HeLa S cells were grown on coverslips for 24 hours prior to treatment. Cell treatment was similar to that for FACS analysis. DAPI staining without synthetic chromatin treatment was used as a negative control. Cells were fixed on slides by immersing them in 4% paraformaldehyde in PBS at room temperature for 15 minutes, followed by immersion in PBS containing 1 mM CaCl2 and 0.5 mM MgCl2 for 10 minutes.

[0083] Infiltrate the cells with 0.1% Triton X-100 and stain with DAPI for 5 minutes. After thorough washing, fix the coverslip onto the slide with 15 μL Prolong Gold and secure with nail polish.

[0084] Experimental results

[0085] HeLa cells were treated with Cy5-labeled chromatin in growth medium for 3 hours, and Cy-5 positive cells were analyzed using a cytometer (Figure 3A). The results showed that more than 90% of the cells showed Cy5 positive signals after treatment with wild-type or gH625 chromatin, which was significantly higher than the control effect gene transfection method with a 3-hour latency period.

[0086] To further confirm plasmid translocation across the membrane, HeLa cells were similarly treated using cell penetration analysis conditions, and confocal fluorescence microscopy images were analyzed (Figure 3B). Consistent with FACS analysis, Cy5-tagged DNA from wild-type or gH625-displayed chromatin was found in both the cytoplasm and nucleus of the cells.

[0087] Example 5: Studying Skin Permeability Using the Franz Cell System Based on Pig Skin

[0088] Experimental methods

[0089] This experiment primarily investigated the skin penetration ability of Cy5-labeled pUC19 / 1×601 DNA in a piglet skin-Franz cell system. The distribution of DNA in pig skin was observed using fluorescence microscopy at time intervals of 24, 48, and 72 hours, and the penetration effect was evaluated by quantifying the fluorescence intensity.

[0090] Thaw the back skin stored at -20℃ with deionized water and repeatedly wash the skin with PBS buffer. Fix Franz cells between the donor and receiver chambers onto the porcine skin, ensuring the stratum corneum faces the donor chamber. Add 7.0 ml of solution to the receiver chamber, then add 1.0 ml of PBS through a sampler to ensure close contact with the dermis. Spread 0.5 μg of DNA sample onto the skin surface of the receiver chamber, covering an effective area of ​​approximately 3.14 cm². 2 Apply the mixture evenly from the center outwards. Maintain the water bath temperature at (321)℃ and set the stirrer speed to 300 rpm to promote absorption. Collect skin samples at specified time points (24h, 48h, and 2h) and wash five times with PBS. Fix the samples with 4% paraformaldehyde for at least 24 hours before cryosection analysis.

[0091] Experimental results

[0092] The results are shown in Figure 4. Cy5-labeled plasmid DNA and its derived chromatin were applied to porcine skin in triplicate at 24-hour intervals, and frozen thin sections were analyzed under a fluorescence microscope (Figures 4A and 4B). Significantly higher fluorescence was observed on the stratum corneum 24 hours after contact with gH625-H2A chromatin (Figure 4A, 24-hour column), indicating deeper penetration into the epidermal and dermal regions at 48 and 72 hours after contact. Wild-type chromatin also showed better penetration than naked DNA, but its efficiency was significantly lower than that of gH625-H2A chromatin (Figure 4B). These results demonstrate that the display of the gH625 peptide on the chromatin enhances chromatin penetration across the skin barrier.

[0093] Example 6: gH625-H2A Chromatin Epidermal Delivery Carrier Experiment

[0094] Skin penetration assays of gH625-H2A chromatin prompted us to investigate whether gH625-H2A chromatin could be used as a delivery carrier for collagen-stimulating bioactive peptides that reach the epidermis and stimulate cell growth and keratinocytes.

[0095] Experimental methods

[0096] 6.1. Human EGF peptide was fused to the C-terminus of human H2B histone via gene synthesis, and histone octamers containing modified histones gH625-H2A and H2B-EGF were purified from the E. coli expression system.

[0097] 6.2. Detection of in vitro chromatin assembly using modified histone octamers by MNase assay.

[0098] Experimental results

[0099] The results of histone octamers purified from the E. coli expression system containing modified histones containing gH625-H2A and H2B-EGF are shown in Figure 5A. The modified H2B-EGF carries an additional 6.8 kDa EGF at the C-terminus, and once incorporated into nucleosomes, it can display two EGF peptides per nucleosome in the chromatin structure.

[0100] The MNase assay results are shown in Figure 5B. Chromatin assembly was successfully performed using gH625-H2A / H2B-EGF octamers, although the assembly quality based on multiple DNA ladders appeared to be reduced (Figure 5B, lanes 1 and 2 versus lanes 3 and 4).

[0101] Furthermore, EGF is a growth factor that promotes cell division and stimulates the biosynthesis of collagen, elastin, and extracellular matrix, and has a short half-life due to its poor thermal stability. Since H2B-EGF undergoes a denaturation-renaturation cycle during purification, the bioactivity of EGF displayed on chromatin (EGF-chromatin) was evaluated using natural EGF peptides. Recommended doses of EGF peptides and EGF-displayed chromatin stimulated the growth of infinitely proliferating human keratinocytes, HaCa-T cells, reaching 50% compared to the simulated control in basal growth medium (Figure 5C). Assuming a maximum of 5% of chromatin comes from displayed EGF, 250 and 500 ng / ml of EGF-displayed chromatin were comparable in working concentrations of EGF peptides (10–100 ng / ml) in tissue culture cells.

[0102] It is known that EGF can slightly enhance collagen synthesis in type I collagen through sudden exposure. EGF peptides can increase the production of type I collagen, but this increase was not statistically significant compared to the simulated control (Fig. 5D). However, treatment of HaCa-T cells with EGF-displayed chromatin induced higher concentrations of type I collagen secreted into the culture medium (Fig. 5D). In summary, the results of this study demonstrate that EGF-displayed chromatin maintains its growth factor activity in cell proliferation and collagen synthesis.

[0103] The applicant declares that, in the process of describing the above-mentioned specification:

[0104] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0105] Finally, it should be noted that:

[0106] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0107] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A gH625-H2A histone, characterized in that, The gH625-H2A histone is histone H2A with the peptide gH625 added.

2. A gH625-H2A histone according to claim 1, wherein, The amino acid sequence of gH625-H2A is shown in SEQ ID NO.

5.

3. A synthetic chromatin comprising gH625-H2A histone, wherein the gH625-H2A histone is as described in any one of claims 1 or 2.

4. The use of the gH625-H2A histone as described in any one of claims 1 or 2, and the synthetic chromatin containing the gH625-H2A histone as described in claim 3, in the preparation of a drug or a drug delivery system.

5. The use according to claim 4, wherein the compound is ###0002### The drug in question is a gene therapy drug.

6. The use according to claim 4, wherein the compound is ###0002### The drug or drug delivery system can penetrate cell membranes and / or cells.

7. The application of the gH625-H2A histone as described in any one of claims 1 or 2, and the synthetic chromatin containing the gH625-H2A histone as described in claim 3, in the preparation of skin care products and cosmetics.

8. Use according to claim 7, wherein the compound is ###0002### gH625-H2A histone and synthetic chromatin containing gH625-H2A histone can increase the skin penetration of skin care products and cosmetics.

Images