A VEGF-modified protein for repairing the skin barrier and its expression in plants.

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to a VEGF-modified protein (VEGFt) for repairing the skin barrier and a method for its expression in plants. This invention discloses a novel VEGF-modified protein. Through precise modification, the protein removes its binding regions to VEGFR1 and VEGFR2, thereby significantly reducing its risk of promoting tumor angiogenesis; simultaneously, it retains its key function of repairing the skin barrier and improves transdermal efficiency. Furthermore, this invention innovatively employs a plant bioreactor to express the VEGF-modified protein, using a plasmid vector containing the VEGFt gene fragment to transform recipient plants, and extracting VEGFt from successfully transgenic plant lines. This method not only improves biosafety and stability but also helps reduce production costs, thus having broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a VEGF modified protein for repairing the skin barrier and a method for its expression in plants. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a member of the platelet-derived growth factor (PDGF) family. VEGF can induce the regeneration of existing blood vessels (angiogenesis) or the growth of new blood vessels (angiogenesis), and is a key factor in embryonic development and vascular repair. Members of the VEGF family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factors 1 and 2 (PIGF-1 and PIGF-2). Among them, VEGF-A is the most effective factor for endothelial cell migration, proliferation, vasodilation, and angiogenesis. VEGF-A mRNA forms different allosteres due to different splicing methods. VEGF165 is the most abundant allosteric variant in vivo and has the strongest pro-angiogenic bioactivity, making it the main effector molecule among the VEGF-A subtypes and the most widely used in experimental and clinical applications.

[0003] VEGF165 is a polypeptide composed of 165 amino acids, rich in cysteine ​​residues, which form four pairs of disulfide bonds. These disulfide bonds primarily maintain the spatial structure of VEGF. The N-terminus of VEGF165 contains a receptor-binding domain, and the C-terminus contains a functional domain. The VEGF receptor-binding domain can bind to VEGFR1 and VEGFR2, respectively. When VEGF binds to its corresponding receptor, it triggers phosphorylation of the C-terminal functional domain, activating the MAPK signaling pathway and playing a role in promoting angiogenesis and keratinocyte proliferation and differentiation. VEGFR1 and VEGFR2 are highly expressed on the surface of tumor cells and are key receptors for VEGF's promotion of tumor angiogenesis.

[0004] VEGF plays a significant role in promoting angiogenesis and repair, accelerating wound healing and improving skin barrier function. However, due to the binding of VEGF to VEGFR1 and 2, its potential carcinogenic risk cannot be ignored. The biological characteristics of VEGF may cause unnecessary side effects and complications, which to some extent hinders its application in the field of medical biology. Summary of the Invention

[0005] In view of this, this invention proposes a novel VEGF-modified protein (VEGFt), which removes the VEGFR1 and VEGFR2 binding regions (100 amino acid residues at the N-terminus) of the VEGF sequence, but retains the 65 amino acid residues at the C-terminus, thus preserving all disulfide bonds that maintain the spatial conformation of VEGF. The modified VEGF retains its function of promoting keratinocyte (KC) proliferation and differentiation to repair the skin barrier, while reducing the risk of promoting tumor angiogenesis, decreasing its molecular weight, and improving transdermal efficiency, greatly broadening the application prospects of VEGF.

[0006] Furthermore, this invention also employs a plant bioreactor to express the VEGF-modified protein (VEGFt) provided by this invention. Plant bioreactors are eukaryotic expression systems, thus possessing complete post-translational modification capabilities. Compared to animal cell expression systems, they do not contain pyrogens or endotoxins, eliminating potential pathogenic risks. Moreover, plants do not contain sensitizing substances, improving safety and comfort. In addition, plant bioreactors offer advantages such as relatively low cost and ease of operation in preparing pharmaceutical proteins, making them an ideal system for preparing topical pharmaceutical proteins.

[0007] The technical solution of this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a VEGF modified protein, the amino acid sequence of which is shown in SEQ ID NO.1, and the VEGF modified protein is named VEGFt.

[0009] In a second aspect, the present invention provides a composition for repairing the skin barrier, the composition comprising the VEGF modified protein.

[0010] Thirdly, the present invention provides the use of the VEGF modified protein or the composition in the preparation of products for repairing skin barrier damage, the products including cosmetics, skin care products or pharmaceuticals.

[0011] Fourthly, the present invention provides a nucleic acid molecule, which is one of the following: (1) the nucleic acid molecule encodes the VEGF modified protein; (2) the sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0012] Fifthly, the present invention provides a plasmid vector containing the nucleic acid molecule.

[0013] Sixthly, the present invention provides a method for preparing the plasmid vector, the method comprising the following steps:

[0014] S1. Synthesize or clone the nucleic acid encoding VEGFt to obtain the VEGFt gene fragment;

[0015] S2. The VEGFt gene fragment obtained in step S1 is ligated to the expression vector, transformed and / or transfected into host cells, positive clones are screened and plasmids are extracted.

[0016] In a seventh aspect, the present invention provides a method for expressing VEGFt in plants, comprising the steps of: transforming a recipient plant with a plasmid vector, wherein the plasmid vector is prepared by any of the above-described preparation methods, and then screening and identifying successfully transgenic lines and extracting VEGFt. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is the verification result of agarose gel electrophoresis of PCR products;

[0019] Figure 2 This is the colony PCR verification result constructed from the plasmid vector pGM3301-VEGFt;

[0020] Figure 3 This is a schematic diagram of the T-DNA region of the plasmid vector pGM3301-VEGFt;

[0021] Figure 4 It involves Agrobacterium-mediated Arabidopsis thaliana culture, transformation, and screening;

[0022] Figure 5 These are the results of Western blot and Tricine-SDS-PAGE gel electrophoresis of VEGFt protein;

[0023] Figure 6 These are the skin lesion repair results of three groups of mice with skin barrier damage models; Figure A is the wild-type Arabidopsis thaliana extract treatment group; Figure B is the VEGF standard positive control group; Figure C is the transgenic Arabidopsis thaliana extract treatment group.

[0024] Figure 7 These are the tissue section examination results of three groups of mice with skin barrier damage models; Figure A is the wild-type Arabidopsis thaliana extract treatment group; Figure B is the VEGF standard positive control group; Figure C is the transgenic Arabidopsis thaliana extract treatment group.

[0025] Figure 8The results show the transdermal permeability of drug-loaded hydrogels prepared with VEGF and VEGFt.

[0026] Figure 9 This is the result of detecting the proliferative effect of VEGFt on mouse microvascular endothelial cells. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0028] This invention provides a novel modified VEGF protein (VEGFt) with an optimized structure that removes the binding regions for VEGFR1 and VEGFR2 while retaining its promoting effect on the proliferation and differentiation of keratinocytes (KCs), thereby enabling it to repair the skin barrier. Furthermore, this modified VEGFt not only reduces the potential risk of promoting tumor angiogenesis but also decreases its molecular weight and improves transdermal efficiency, thus demonstrating significant application potential.

[0029] Meanwhile, this invention utilizes a plant bioreactor to express VEGFt. Arabidopsis thaliana, as an expression system, boasts high safety, lacking potential pathogenic pyrogens and endotoxins, thus ensuring biocompatibility. As a eukaryote, Arabidopsis thaliana possesses well-developed post-translational protein modification capabilities, ensuring the expressed exogenous protein exhibits good solubility and bioactivity. Furthermore, the cultivation, planting, and transformation processes of Arabidopsis thaliana are simple and easy to implement, helping to reduce the production costs of high-value-added proteins. Therefore, utilizing Arabidopsis thaliana to produce VEGFt has significant advantages.

[0030] This invention not only improves the biosafety and stability of VEGF, but also increases its transdermal penetration rate, thereby reducing potential damage to the skin and greatly broadening its application prospects in the field of skin barrier repair.

[0031] Therefore, the embodiments of the present invention disclose at least the following technical solutions:

[0032] In a first aspect, the present invention provides a VEGF modified protein, the amino acid sequence of which is shown in SEQ ID NO.1, and the VEGF modified protein is named VEGFt.

[0033] In a second aspect, the present invention provides a composition for repairing the skin barrier, the composition comprising the VEGF modified protein.

[0034] Thirdly, the present invention provides the use of the VEGF modified protein or the composition in the preparation of products for repairing skin barrier damage, the products including cosmetics, skin care products or pharmaceuticals.

[0035] Fourthly, the present invention provides a nucleic acid molecule, which is one of the following: (1) the nucleic acid molecule encodes the VEGF modified protein; (2) the sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0036] Fifthly, the present invention provides a plasmid vector containing the nucleic acid molecule.

[0037] Sixthly, the present invention provides a method for preparing the plasmid vector, the method comprising the following steps:

[0038] S1. Synthesize or clone the nucleic acid encoding VEGFt to obtain the VEGFt gene fragment;

[0039] S2. The VEGFt gene fragment obtained in step S1 is ligated to the expression vector, transformed and / or transfected into host cells, positive clones are screened and plasmids are extracted.

[0040] In some preferred embodiments, in step S1, the upstream and downstream primers for the nucleic acid encoding VEGFt are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0041] In some preferred embodiments, in step S2, the pGM3301 vector and the VEGFt gene fragment obtained in step S1 are digested with restriction endonucleases Nco I and BstE II, respectively, to obtain the linearized pGM3301 vector and the digested VEGFt gene fragment. The vector is incubated overnight using a T4 DNA ligase system, and then transformed and / or transfected into host cells. Positive clones are screened and plasmids are extracted.

[0042] In a seventh aspect, the present invention provides a method for expressing VEGFt in plants, comprising the steps of: transforming a recipient plant with a plasmid vector, wherein the plasmid vector is prepared by any of the above-described preparation methods, and then screening and identifying successfully transgenic lines and extracting VEGFt.

[0043] In some preferred embodiments, the recipient plant is Arabidopsis thaliana, the plasmid vector transformation of the recipient plant is mediated by Agrobacterium, and the step of screening and identifying successfully transgenic lines specifically includes: selecting T0 generation plants that have successfully transgenic based on resistance markers to obtain T1 generation seeds, continuing to sow and propagate until T3 generation homozygous plants are screened out, and then performing PCR identification and protein expression level detection to screen out lines with high VEGFt expression.

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0045] Example 1: Construction of a plasmid vector containing nucleic acid encoding VEGF-modified protein (VEGFt).

[0046] Materials sourced from: pCAMBIA3301 expression vector purchased from Invitrogen; DNA recovery kit (Cat No: AP-GX-50) purchased from Axygen; and Escherichia coli (Trans1-T1 competent cells, Cat No: CD501-02) purchased from Beijing TransGen Biotech Co., Ltd.

[0047] This invention first designed specific primers for VEGFt and used VEGF (sequence shown in SEQ ID NO.5) as a template for PCR amplification. The PCR products were analyzed by agarose gel electrophoresis to verify whether the target fragment was amplified. The target fragment was then recovered and purified. Next, the pGM3301 vector and the VEGFt gene fragment obtained in step S1 were digested with restriction endonucleases Nco I and BstE II, respectively, to obtain the linearized pGM3301 vector and the digested VEGFt gene fragment. The fragments were incubated overnight using a T4 DNA ligase system, and then transformed and / or transfected into host cells. Positive clones were screened and plasmids were extracted.

[0048] The specific experimental steps and results are as follows:

[0049] (1) Amplify the target fragment

[0050] Design specific primers:

[0051] The upstream primer P1 (5'-AACAAGTGCGAA-3') is shown in SEQ ID NO.3;

[0052] The downstream primer P2 (5'-TTAGCGGCGCGG-3') is shown in SEQ ID NO.4.

[0053] PCR amplification was performed using VEGF (sequence shown in SEQ ID NO.5) as a template.

[0054] PCR reaction system 20 μL: PCR mix 10 μL; P1 and P2 primers 0.5 μL each; plasmid 1 μL; deionized water 8 μL.

[0055] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 65℃ annealing for 30 s; 72℃ extension for 1 min; 30 cycles; 72℃ final extension for 6 min; 4℃ to terminate the reaction.

[0056] The PCR products were analyzed by 0.1% agarose gel electrophoresis to verify whether the target fragment was amplified, and the target fragment was recovered and purified. The PCR product verification results are as follows: Figure 1 As shown.

[0057] (2) Constructing the pGM3301 vector:

[0058] The 35S promoter sequence was amplified by PCR, and Hind III and Nco I restriction sites were introduced into the upstream and downstream primers, respectively. The specific short sequence AGGAGGC was introduced into the 5'-UTR of the amplified 35S promoter sequence for modification.

[0059] The GUS fragment was amplified by PCR. The upstream primers introduced Nco I and Nde I restriction sites, respectively, and the downstream primers introduced BstE II and BamHI restriction sites, respectively. The pCAMBIA3301 plasmid was digested with Hind III and Nco I restriction enzymes. The modified 35S promoter sequence was then ligated into pCAMBIA3301 using T4 DNA ligase, yielding the pGM3301 vector.

[0060] (3) Connecting carrier

[0061] Then, the pGM3301 vector and the VEGFt gene fragment obtained in step S1 were digested with restriction endonucleases Nco I and BstE II, respectively. The VEGFt gene fragment and the cut pGM3301 linear vector were recovered using a DNA recovery kit, and the linearized pGM3301 vector and the digested VEGFt gene fragment were obtained. They were incubated overnight using a T4 DNA ligase system.

[0062] T4 DNA ligase system 10 μL: pGM3301 1 μL, VEGFt 3 μL, 10× buffer 1 μL, T4 DNA ligase 1 μL.

[0063] (4) Transformation and identification

[0064] Transformed *E. coli*, and selected using a selection medium (LB medium with 50 mg / mL kanamycin at a volume ratio of 2%). After colonies grew, resistant clones were picked and colony PCR was performed to verify the resistant clones. The PCR primers, reaction system, and reaction conditions were the same as in step (1) (verification results are as follows). Figure 2 As shown). The constructed plasmid vector containing nucleic acid encoding VEGFt was named pGM3301-VEGFt (as shown). Figure 3 (As shown).

[0065] Example 2: Expression of VEGFt in plants

[0066] In this embodiment, the plasmid vector constructed in Example 1 was used to transform EHA105 Agrobacterium competent cells, and then PCR was used to screen and identify Agrobacterium containing the pGM3301-VEGFt plasmid. The identified Agrobacterium culture was added to the transformation medium to transform Arabidopsis thaliana. Based on the glufosinate resistance marker, the successfully transgenic T0 generation plants were screened to obtain T1 generation seeds. The sowing and propagation continued until the T3 generation homozygous plants were screened. Then, PCR identification and protein expression level detection were performed to screen out lines with high VEGFt expression and extract VEGFt from them.

[0067] The specific experimental steps and results are as follows:

[0068] 1. Experimental Procedure

[0069] (1) Transformation

[0070] The pGM3301-VEGFt plasmid was extracted using the AxyGen plasmid extraction kit. The extracted plasmid was then transformed into EHA105 Agrobacterium competent cells using the freeze-thaw method. Agrobacterium species containing the pGM3301-VEGFt plasmid were screened and identified by PCR.

[0071] The steps for preparing EHA105 Agrobacterium competent cells are as follows:

[0072] a) Take the Agrobacterium tumefaciens strain from the -70℃ freezer and inoculate it into a 50mL Erlenmeyer flask containing 50μg / mL rifampicin YEB medium at a ratio of 1:50. Incubate at 28℃ and 200rpm for 18-24 hours.

[0073] b) Take 1 mL of activated bacterial culture and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of YEB medium with 50 μg / mL rifampicin. Incubate at 28℃ and 200 rpm for 6-7 hours. At this time, the OD600 value is about 0.4-0.6.

[0074] c) Pour the bacterial culture into a 50 mL centrifuge tube and incubate on ice for 30 min.

[0075] d) Centrifuge at 4.4℃ and 3000rpm for 10min, discard the supernatant, resuspend the precipitate in 10mL of ice-cold CaCl2 containing 15% glycerol (100mM), and collect the bacterial cells in a centrifuge tube.

[0076] e) Centrifuge at 5.4℃ and 3000rpm for 10min, discard the supernatant, resuspend the precipitate in 1mL of 100mM ice-cold CaCl2 containing 15% glycerol, aliquot 80μL into 1mL centrifuge tubes, freeze in liquid nitrogen, and store at -70℃.

[0077] (2) Identification of transformants

[0078] Agrobacterium species containing the pGM3301-VEGFt plasmid were screened and identified using PCR.

[0079] The upstream primer P1 (5'-AACAAGTGCGAA-3') is shown in SEQ ID NO.3;

[0080] The downstream primer P2 (5'-TTAGCGGCGCGG-3') is shown in SEQ ID NO.4.

[0081] PCR reaction system 20 μL: PCR mix 10 μL; P1 and P2 primers 0.5 μL each; plasmid 1 μL; deionized water 8 μL.

[0082] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 65℃ annealing for 30 s; 72℃ extension for 1 min; 30 cycles; 72℃ final extension for 6 min; 4℃ to terminate the reaction.

[0083] (3) Transformation of Arabidopsis thaliana

[0084] The identified Agrobacterium was placed in a 2L culture flask and cultured in a shaker at 28℃ and 180rpm for 20h until the absorbance OD590 of the Agrobacterium solution reached 1.2. The cultured Agrobacterium was collected by centrifugation and mixed into the transformation solution. Arabidopsis inflorescences were then immersed in the transformation solution for 5min.

[0085] Conversion solution ratio (per 1L conversion solution): MS 2.2g, sucrose 50g, surfactant 200μL.

[0086] (5) Identification of transgenic strains

[0087] Transformed Arabidopsis thaliana seeds were placed in the dark for 2 days. Seed pods were collected continuously until all transformed Arabidopsis thaliana seed pods were collected. The seeds were then air-dried, shelled, and harvested as the T1 generation. Positive clones were selected from the T1 generation seeds on MS solid medium containing glufosinate. Single-copy plants were selected from the T2 generation based on genetic characteristics. Homozygous plants were selected from the T3 generation seeds.

[0088] MS screening medium composition: MS 2.2 g / L, sucrose 10 g / L, glufosinate 11 mg / L, adjust pH to 5.7, then add plant agar 6.5 g - 7.0 g / L, sterilize and pour into plates.

[0089] (6) Detection of VEGFt protein expression

[0090] PCR-positive Arabidopsis thaliana plants were ground into powder and mixed with protein extraction buffer (the ratio of Arabidopsis thaliana plant mass to protein extraction buffer volume was 100 mg: 200 μL) and incubated on ice for 2 h. At 4 °C, 10000 g of the mixture was centrifuged for 20 min, and protein loading buffer (the volume ratio of the mixture to protein loading buffer was 10:1) was added. The mixture was then boiled for 10 min, and the extracted total soluble Arabidopsis thaliana protein was separated by Tricine-SDS-PAGE gel transfer and transferred to a PVDF membrane. Blocking buffer was added, and the membrane was blocked at room temperature for 2 h. After washing away the blocking buffer, 1:1000 diluted anti-VEGF mouse antibody was added, and the membrane was incubated overnight at 4 °C. After washing away the antibody, 1:10000 diluted alkaline phosphatase-labeled goat anti-mouse antibody was added, and the membrane was incubated at room temperature for 2 h. After washing away the goat anti-mouse antibody, BCIP / NBT chromogenic reagent was added, and the membrane was placed in an imaging system for detection. Three Arabidopsis thaliana high-expression lines were screened.

[0091] 2. Experimental Results

[0092] like Figure 4 As shown, the different states of Arabidopsis thaliana culture before and after transformation mediated by Agrobacterium tumefaciens in this invention present a stark contrast. Figure 5 As shown, the modified VEGFt protein was detected by gel electrophoresis and Western blotting. A protein band of approximately 72 kDa was visible in the gel electrophoresis image, with a molecular weight similar to the target protein, initially identified as VEGFt. Specific detection using an anti-VEGF antibody revealed a distinct specific protein band, indicating antibody binding to the target protein. This further confirms that the protein band is indeed VEGFt.

[0093] Example 3: Application of VEGFt in a skin barrier damage model

[0094] In this embodiment, the present invention first established a skin barrier damage model in C57BL / 6N male mice, then prepared a hydrogel containing VEGFt, and divided the model mice into three groups: blank control (Control group), wild-type Arabidopsis thaliana plant extract group, and transgenic Arabidopsis thaliana (high expression line screened in Example 2) plant extract group, and administered drug treatment accordingly. The damage changes of the mouse back skin before and after the modeling was completed and before and after treatment were observed and recorded. Tissue sections of mouse skin samples were prepared, and the tissue sections were stained with hematoxylin and eosin. The stained skin cells and tissue structures were observed under a microscope to evaluate the degree of skin barrier repair and pathological changes.

[0095] The specific experimental steps and results are as follows:

[0096] 1. Experimental Procedure

[0097] (1) Establishment of a skin barrier injury model in C57BL / 6N male mice

[0098] Three groups of 8-week-old male C57BL / 6N mice (n=8 per group) were shaved using a shaver. A suitable amount of depilatory cream was then applied to the shaved area on the mice's backs. After 2.5 minutes, the cream was gently wiped off with gauze. The next day, strong adhesive tape was repeatedly used to reattach the shaved area to establish an animal model of skin barrier damage. The extent of skin barrier damage in the mice was observed and recorded during the modeling process.

[0099] (2) Preparation of hydrogels containing VEGFt

[0100] First, weigh 0.3g of carbomer and add 50mL of ultrapure water. Stir the mixture with a magnetic stirrer until it becomes a homogeneous mixture. Then, add 0.3mL of triethanolamine to form a transparent gel. After sealing and autoclaving, store the gel in a refrigerator at 4°C for later use.

[0101] (3) Drug therapy

[0102] The blank control (wild-type Arabidopsis thaliana extract, i.e., Control group), VEGF+ gel group, and transgenic Arabidopsis thaliana (high-expression line screened in Example 2) extract+ gel group were administered to the corresponding groups for treatment.

[0103] The experimental group was prepared by adding 25 mL of total soluble protein (containing approximately 150 μg of VEGFt protein) to transgenic Arabidopsis thaliana extract to form a 0.2% hydrogel; the blank group (i.e., the control group) was prepared by adding 25 mL of total soluble protein to wild-type Arabidopsis thaliana extract to form a hydrogel; and the positive control group was prepared by using VEGF standard to form a 0.2% hydrogel.

[0104] After successful modeling, apply 0.2 mL of 0.2% hydrogel to the back of each mouse every 12 hours, twice a day. Discontinue administration after 84 hours. Photographs of the skin lesions on the back of the mice were taken at the end of modeling and before each 12-hour treatment to observe changes in the lesions.

[0105] (3) Preparation and observation of skin tissue sections

[0106] a) Drug administration and sample collection: Mice were sacrificed 5 days after drug administration. Skin samples were collected from the mice and fixed onto slides.

[0107] b) Sample Preparation: Sections containing skin samples were cut using a paraffin microtome to obtain tissue samples with a thickness of approximately 5 μm. The tissue samples were placed in a basin of tap water and then immersed in 42°C warm water for approximately 5 seconds to allow the tissue to fully expand. After immersion, the tissue samples were removed and air-dried overnight, then placed in a 60°C oven for 2 hours. Before use, the samples were oven-dried for 20 minutes, and xylene was added while still hot to dewax the tissue samples (the tissue samples to be tested were immersed in xylene for 15 minutes. After immersion, the xylene was replaced, and the samples were immersed for another 15 minutes). The xylene-immersed tissue samples were then immersed in anhydrous ethanol for 5 minutes, and then again in anhydrous ethanol for 5 minutes to wash away the xylene and allow water to enter the tissue. The immersed tissue samples were then washed sequentially in 100%, 90%, 80%, and 70% ethanol.

[0108] c) Staining: Pipette 100 μL of the pre-prepared hematoxylin staining solution onto each cleaned tissue sample and stain thoroughly for 7 minutes. After staining, wash away excess hematoxylin staining solution with distilled water. Differentiate with differentiation solution for 30 seconds, then rinse the tissue sample thoroughly with double-distilled water.

[0109] Add eosin staining solution to the cleaned tissue sample and stain thoroughly for 3 minutes.

[0110] d) Dehydration and mounting: Gradient dehydration: 80% ethanol for 5 seconds, 100% ethanol for 2 minutes, and anhydrous ethanol for 2 minutes. The dehydrated tissue samples were then soaked twice in xylene, each time for 15 minutes. The xylene was removed, and the samples were mounted with neutral resin.

[0111] e) Use a microscope to observe stained skin cells and tissue structures to assess the degree of skin barrier repair and pathological changes.

[0112] 2. Experimental Results

[0113] like Figure 6-7As shown in the comparison chart of skin damage repair capabilities among the three groups of male mice, the application of wild-type Arabidopsis thaliana extract significantly thickened the damaged epidermal barrier, indicating abnormal proliferation of keratinocytes and their inability to differentiate and reform the barrier normally. The transgenic group did not show significant thickening of the damaged barrier, indicating that keratinocytes could differentiate normally to form a new barrier, demonstrating a significant repair effect. Comparing truncated VEGFt with VEGF, the degree of skin barrier repair was not significantly different, indicating that the function of truncated VEGFt was not affected and its repair effect was consistent with that of VEGF.

[0114] Example 4: Testing the transdermal effect of VEGFt

[0115] In this embodiment, the present invention sets up three groups of mice: a negative control group, a VEGFt administration group, and a VEGF administration control group. Corresponding drug-loaded hydrogels were prepared for each group. After hair removal treatment on the back, skin tissue samples from the back were used to detect the transdermal permeability of the drug-loaded hydrogels in each group.

[0116] The specific experimental steps and results are as follows:

[0117] 1. Experimental Procedure

[0118] (1) Three male mice were divided into three groups: a blank control group, a VEGFt administration group, and a VEGF administration control group. First, the back hair of the mice was removed using a hair removal machine and hair removal cream.

[0119] (2) Preparation of three drug-loaded hydrogels. Weigh 0.3 g of carbomer and add 50 mL of ultrapure water. Stir with a magnetic stirrer to form a homogeneous mixture. Then add 0.3 mL of triethanolamine to form a transparent gel. After sealing and autoclaving, store in a refrigerator at 4°C for later use. For the experimental group, add 25 mL of total soluble protein from Arabidopsis thaliana extract (containing approximately 150 μg of VEGFt protein) (method as in Example 2) to prepare a 0.2% (V / V) hydrogel. For the negative control group, add 25 mL of total soluble protein from wild-type Arabidopsis thaliana extract to prepare a hydrogel. For the positive control group, add VEGF standard to prepare a 0.2% hydrogel.

[0120] (3) On the second day, take skin tissue from the back, the size of which can cover the mouth of the conical flask. Fill the conical flask with physiological saline so that the horizontal plane can contact the skin tissue. Apply drug-loaded hydrogel (0.2 mL, 0.2% hydrogel) to the outer surface of the skin tissue and let it penetrate into the physiological saline. Then, use the VEGF Elisa kit to detect the concentration of the physiological saline and judge the transdermal rate of different drugs over time.

[0121] 2. Experimental Results

[0122] like Figure 8As shown, the transdermal permeability of the VEGFt group was significantly higher than that of the VEGF-treated group.

[0123] Example 5: Determination of the effect of VEGFt on the proliferative activity of mouse microvascular endothelial cells (b.End3) using the Brd-U assay kit.

[0124] Materials source: Brd-U proliferation assay kit was purchased from Roche (Germany); b.End3 cells were purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.

[0125] The experimental steps are as follows:

[0126] (1) b. After digestion and collection of End3 cells, they were prepared into a density of 5×10⁻⁶. 4 b.End3 cell suspension was added at 100 μL / well to each well of a 96-well plate according to the number of wells. After mixing, the plate was placed in a cell culture incubator. When the cell density in the wells reached 50%–60%, the culture medium was replaced, and VEGF / VEGFt at 10 μg / mL was added for 24 h each, according to the experimental settings. The negative control (Con group) was treated with the corresponding volume of wild-type Arabidopsis thaliana extract.

[0127] (2) After 24 hours of treatment, aspirate the culture medium from the wells, take a 5 mL EP tube, add 2 mL of culture medium, add 200 μL of 10 × BrdU detection solution, mix well, and then add 100 μL / well to the wells treated with VEGF / VEGFt. Place the tube in an incubator. After 2 hours, remove the plate and aspirate the detection solution from the wells.

[0128] (3) Add 100 μL of Fix Denant to each well, wrap the 96-well plate with tin foil, and react at room temperature for 0.5 h. Then remove the plate from the tin foil and absorb the liquid in the well.

[0129] (4) Prepare 2 mL of 1× working solution Auti-BrdU-POD according to the instructions, and then add it to the treated wells at a rate of 100 μL / well. Wrap the 96-well plate with tin foil, and react at room temperature for 0.5 h. Then remove the plate from the tin foil and absorb the liquid in the wells.

[0130] (5) Dilute the 10× washing solution to 1× with ddH2O, and then add it to the treated wells at a rate of 200 μL / well. Place the wells flat on a shaker for washing, and repeat 3 times.

[0131] (6) Add the substrate solution to the wells at a rate of 100 μL / well, wrap the 96-well plate with aluminum foil, and react at room temperature for 20 min. Then remove the plate from the aluminum foil, remove the plate cap, and place it in an ELISA reader for detection at a wavelength of 370 nm.

[0132] Experimental results:

[0133] like Figure 9 As shown, an ELISA reader was used to measure OD... 370nm The assay revealed that the negative control group and VEGFt had no proliferative effect on b.End3 cells, while VEGF had a significant proliferative effect on b.End3 cells. This result indicates that VEGFt without the VFGFR1 / 2 receptor binding domain loses the proliferative effect of VEGF.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A VEGF engineered protein, characterized in that, The amino acid sequence of the VEGF modified protein is shown as SEQ ID NO. 1, and the VEGF modified protein is named as VEGFt.

2. A composition for repairing the skin barrier, characterized in that, The composition comprises the VEGF modified protein according to claim 1.

3. Use of the VEGF engineered protein of claim 1 or the composition of claim 2 for the preparation of a product for the repair of skin barrier impairment, characterized in that, The product comprises cosmetics or drugs.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule is one of the following: (1) the nucleic acid molecule encodes the VEGF modified protein according to claim 1; (2) the sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

5. A plasmid vector, characterized in that, The plasmid vector comprises the nucleic acid molecule according to claim 4.

6. The method of producing the plasmid vector according to claim 5, characterized in that, The method comprises the following steps: S1, artificially synthesizing or gene cloning a nucleic acid encoding VEGFt to obtain a VEGFt gene fragment; S2, connecting the VEGFt gene fragment obtained in step S1 with an expression vector, transforming and / or transfecting into a host cell, screening positive clones and extracting plasmids.

7. The method of claim 6, wherein the plasmid vector is prepared by the steps of: In step S1, the upstream and downstream primers for gene cloning a nucleic acid encoding VEGFt are shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

8. The method of claim 6, wherein the plasmid vector is prepared by the steps of: In step S2, restriction enzymes Nco I and BstE II are used to digest the pGM3301 vector and the VEGFt gene fragment obtained in step S1, respectively, to obtain a linearized pGM3301 vector after digestion and a VEGFt gene fragment after digestion, and then incubated overnight using a T4 DNA ligase system, and then transformed and / or transfected into a host cell, screened positive clones and extracted plasmids.

9. A method of expressing VEGFt in a plant, comprising, The method comprises the following steps: transforming a receptor plant with a plasmid vector prepared according to the preparation method of any one of claims 6-8, and then screening and identifying to obtain a transgenic successful strain and extracting VEGFt.

10. The method of expressing VEGFt in a plant according to claim 9, wherein, The receptor plant is Arabidopsis thaliana, the plasmid vector is transformed into the receptor plant by Agrobacterium mediation, and the step of screening and identifying to obtain a transgenic successful strain further comprises: screening T0 generation plants with successful transgenesis according to a resistance marker to obtain T1 generation seeds, continuing to sow and expand until T3 generation homozygous plants are screened, and then performing PCR identification and protein expression level detection, so as to screen a strain with high expression of VEGFt.

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