Keratinocyte growth factor-transdermal peptide fusion protein, and preparation and use thereof

Abstract

A keratinocyte growth factor-transdermal peptide fusion protein, the amino acid sequence of the protein being as shown in SEQ ID NO. 12, the nucleotide sequence of the gene encoding the fusion protein being shown in SEQ ID NO. 8, and a recombinant vector containing the gene, the recombinant vector using pGM3301. The fusion protein improves the biological safety and stability of KGF2 without affecting the biological activity of KGF2; KGF2 fused with TP1 significantly improves the transdermal rate of KGF2, and greatly improves the application prospects of the hair growth-promoting effect thereof. In addition, biological transdermal drug administration can reduce pain caused by microneedles, and the present fusion protein production system and gel preparation have no potential risks, and reduce damage to the skin.

Description

A keratinocyte growth factor-transdermal peptide fusion protein and its preparation and application Technical Field

[0001] The present invention relates to a fusion protein, in particular to a keratinocyte growth factor-transdermal peptide fusion protein and its preparation and application. Background Art

[0002] Keratinocyte growth factor-2 (KGF-2) is a key member of the fibroblast growth factor (FGF) family. In 1996, Yamasaki et al. isolated a novel cytokine from rat embryonic keratinocytes and named it KGF-2. KGF-2 is a protein molecule composed of 208 amino acid residues, with a 40-amino acid signal peptide at its N-terminus. KGF-2 promotes the repair of epidermal and corneal damage and participates in the entire process of embryonic lung development. Furthermore, KGF-2 significantly promotes hair growth. As a protein molecule, KGF-2 has fewer side effects than minoxidil, making it a promising candidate for hair growth drugs.

[0003] However, the key to developing a hair growth agent currently lies in the stability and transdermal efficiency of the drug. Therefore, improving the stability and transdermal penetration of KGF-2 is a primary issue for its application as a hair growth agent. Several approaches have been used to increase KGF-2's transdermal permeability, such as physically adsorbing KGF onto microneedles, which deliver KGF-2 directly to the dermis. Other approaches include fusing KGF-2 with polyethylene glycol (PEG) to increase its affinity for cell membranes and deliver it into the dermis. However, these methods irreversibly affect KGF-2's stability and also compromise the user experience.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a keratinocyte growth factor-transdermal peptide fusion protein and its preparation and application, which solves the problem that the existing technology causes irreversible impact on the stability of KGF-2 and also affects the user experience.

[0006] In order to achieve the above object, the present invention provides a keratinocyte growth factor-transdermal peptide fusion protein, the amino acid sequence of which is shown in SEQ ID NO.12.

[0007] The present invention provides a gene encoding the fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0008] The present invention provides a recombinant vector containing the gene.

[0009] Preferably, the recombinant vector is pGM3301.

[0010] The present invention provides a recombinant bacterium comprising the recombinant vector.

[0011] The present invention provides an application of the fusion protein in preparing a hair growth promoting agent.

[0012] The present invention provides a method for preparing an Arabidopsis thaliana transformed strain expressing the fusion protein, the method comprising:

[0013] (1) The TP-1 gene and the KGF-2 gene were ligated into the pGM3301 vector using T4 ligase, the ligation product was transformed into Escherichia coli, the gene containing the fusion protein was screened out, the constructed vector was named pGM3301-TP1-KGF2, and the pGM3301-TP1-KGF2 vector plasmid was extracted; wherein the nucleotide sequence of the TP-1 gene is as shown in SEQ ID NO.6, the nucleotide sequence of the KGF-2 gene is as shown in SEQ ID NO.7, and the nucleotide sequence of the pGM3301-TP1-KGF2 gene is as shown in SEQ ID NO.8;

[0014] (2) The extracted pGM3301-TP1-KGF2 vector plasmid was transferred into EHA105 competent Agrobacterium by freeze-thaw method, and the Agrobacterium containing the pGM3301-TP1-KGF2 plasmid was screened and identified by PCR method; the identified Agrobacterium was cultured at 28°C and 230 rpm until the absorbance of the Agrobacterium solution was OD 590 Reaching 1.0, the cultured Agrobacterium is collected by centrifugation; wherein, the nucleotide sequence of the first set of upstream primers used in the PCR method is such as SEQ ID NO.1, the nucleotide sequence of the downstream primer is such as SEQ ID NO.2, the nucleotide sequence of the second set of upstream primers is such as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is such as SEQ ID NO.4;

[0015] (3) The collected Agrobacterium is mixed into the transformation solution, the Arabidopsis inflorescence is immersed in the transformation solution, the transformed Arabidopsis is placed in a dark place, and the transformed Arabidopsis seed pods are collected, dried in the shade, shelled, and harvested. The seeds are sown and named T1. After sowing for two generations, a homozygous transformed strain is selected and named T3. The homozygous transformed strain T3 can express the fusion protein as described above.

[0016] The present invention provides a method for screening Arabidopsis thaliana transformed strains containing the fusion protein, the method comprising:

[0017] (1) using a genome extraction kit to extract the whole genome of Arabidopsis thaliana, separating the amplified products by PCR through agarose gel, and detecting them by ultraviolet imaging; wherein the nucleotide sequence of the first set of upstream primers used in the PCR method is such as SEQ ID NO.1, the nucleotide sequence of the downstream primers is such as SEQ ID NO.2, the nucleotide sequence of the second set of upstream primers is such as SEQ ID NO.3, and the nucleotide sequence of the downstream primers is such as SEQ ID NO.4;

[0018] (2) Grind the tissue of Arabidopsis thaliana identified as positive by PCR into powder, add protein extraction buffer to obtain a mixed solution, and place it in an ice bath; centrifuge the mixed solution after ice bath at 4°C, add loading buffer, and place it in a boiling water bath; separate the extracted total soluble protein of Arabidopsis thaliana by SDS-PAGE gel, transfer it to a PVDF membrane using a membrane transfer apparatus, add blocking solution and block at room temperature, wash away the blocking solution, add anti-KGF-2 mouse antibody, and incubate at 4°C overnight; wash away the antibody, add alkaline phosphatase-labeled goat anti-mouse antibody, and incubate at room temperature; wash away the goat anti-mouse antibody, add the color developer BCIP / NBT, and place it in an imager for detection; and screen out the transformed Arabidopsis thaliana strains that highly express the fusion protein as described by molecular biological detection.

[0019] The keratinocyte growth factor-transdermal peptide fusion protein of the present invention and its preparation and application solve the problem that the existing technology causes irreversible effects on the stability of KGF-2 and also affects the user experience, and has the following advantages:

[0020] 1. The Arabidopsis thaliana used in the present invention does not contain potentially pathogenic pyrogens and endotoxins, indicating that Arabidopsis thaliana is a highly safe expression system. Moreover, Arabidopsis thaliana is a eukaryotic organism with a complete protein post-translational modification function, which ensures that the expressed exogenous protein is soluble and biologically effective. At the same time, the planting, cultivation and transformation methods of Arabidopsis thaliana are simple to operate, which can significantly reduce the production cost of high-value-added proteins. Therefore, using Arabidopsis thaliana to produce KGF-2 protein has obvious advantages.

[0021] 2. The present invention can improve the biosafety and stability of KGF2 without affecting the biological activity of KGF2. KGF2 fused with TP1 significantly improves the transdermal rate of KGF2 and greatly enhances its application prospects for promoting hair growth. At the same time, biological transdermal administration can also reduce the pain caused by microneedles. This fusion protein production system and gel preparation have no potential risks and reduce damage to the skin.

[0022] 3. The fusion protein production system of the present invention is simple to prepare and has a significant transdermal effect. In addition, according to the results of animal experiments, the fusion expression of TP1 and KGF2 has no effect on their respective structures and no adverse effects on the test animals. Therefore, the fusion protein is a biosafe substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a partial schematic diagram of pGM3301-TP1-KGF2 of the present invention.

[0024] FIG2 is a diagram of the Agrobacterium-mediated transformation process of Arabidopsis thaliana according to the present invention.

[0025] FIG3 is a PCR detection diagram of the TP1-KGF2 Arabidopsis thaliana strain of the present invention.

[0026] FIG4 is a Western-blot detection diagram of the TP1-KGF2 Arabidopsis thaliana strain of the present invention.

[0027] FIG5 is a diagram showing the hair growth promoting effect of TP1-KGF2 of the present invention.

[0028] FIG6 is a diagram showing the detection of the transdermal release ability of TP1-KGF2 of the present invention (green represents the detected fluorescence). DETAILED DESCRIPTION

[0029] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0030] Example 1

[0031] A method for preparing a carrier of keratinocyte growth factor fused with a transdermal peptide, the method comprising:

[0032] (1) Construction of TP-1 and KGF-2 fusion gene expression vector

[0033] The TP-1 gene (its nucleotide sequence is shown in SEQ ID NO.6) on the cloning vector (the empty vector and Escherichia coli for cloning were purchased from Quanshijin Company) was cut out using Nde I and Kpn I endonucleases, and the FGF10 mRNA nucleic acid sequence of KGF-2 (sequence from GenBank, AB002097.1, its nucleotide sequence is shown in SEQ ID NO.7) stored in the cloning vector in our laboratory was cut out using Kpn I and BamH I endonucleases, and the FGF10 mRNA nucleic acid sequence was optimized according to the codon preference of Arabidopsis thaliana) gene was optimized using Nde I and BamH I endonucleases. The pGM3301 vector was cleaved with endonuclease I, and the excised TP-1 gene fragment, KGF-2 gene fragment, and cleaved pGM3301 vector were recovered using a DNA recovery kit. The TP-1 gene fragment, KGF-2 gene fragment, and cleaved pGM3301 vector were mixed in a molar ratio of 1:1:1, ligated with T4 ligase, and the ligated product transformed into Escherichia coli. Clones containing the desired vector were screened and named pGM3301-TP1-KGF2 (the nucleotide sequence of which is shown in SEQ ID NO. 8; the linker peptide is part of the "TP1-KGF2" fusion protein, the nucleotide sequence of which is shown in SEQ ID NO. 5, and is characterized by flexibility and low steric hindrance, serving to connect the two proteins without affecting the properties of the fusion protein). FIG1 is a partial schematic diagram of pGM3301-TP1-KGF2 of the present invention.

[0034] (2) Agrobacterium-mediated transformation of Arabidopsis thaliana

[0035] Extract the pGM3301-TP1-KGF2 vector plasmid (its amino acid sequence is shown in SEQ ID NO.12). The specific steps of extraction are as follows:

[0036] ① Extract 1-4 mL of bacterial suspension (pGM3301-TP1-KGF2) cultured overnight in LB medium, centrifuge at 12000×g for 1 min, and discard the supernatant.

[0037] ② Add 250μL Buffer S1 to suspend the bacterial pellet. The suspension should be uniform and no small bacterial clumps should be left.

[0038] ③ Add 250 μL of Buffer S2 and gently and thoroughly invert the tube 4-6 times to mix thoroughly to lyse the cells until a clear solution forms. This step should not exceed 5 minutes.

[0039] ④ Add 350 μL of Buffer S3, gently and thoroughly mix by inverting up and down 6 to 8 times, and centrifuge at 12,000 × g for 10 min.

[0040] ⑤ Aspirate the supernatant from step 4 and transfer it to a preparation tube. Centrifuge at 12,000 × g for 1 min and discard the filtrate.

[0041] ⑥ Place the preparation tube back into the centrifuge tube, add 500 μL Buffer W1, centrifuge at 12000×g for 1 min, and discard the filtrate.

[0042] ⑦ Return the preparation tube to the centrifuge tube, add 700 μL of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate. Wash once more with 700 μL of Buffer W2 using the same method. Discard the filtrate.

[0043] ⑧Place the preparation tube back into a 2 mL centrifuge tube and centrifuge at 12000×g for 1 min.

[0044] ⑨ Transfer the prepared tube to a new 1.5 mL centrifuge tube, add 150 μL of Eluent A to the center of the prepared tube membrane, let it stand at room temperature for 1 minute, and centrifuge at 12,000 × g for 1 minute.

[0045] The specific components of each substance are as follows:

[0046] Eluent A: deionized water;

[0047] Buffer W2: ethanol, Tris-HCl;

[0048] Buffer W1: isopropanol, Tris-HCl;

[0049] Buffer S3: NH4AC;

[0050] Buffer S2: NaOH, SDS (sodium dodecyl sulfate);

[0051] Buffer S1: Tirs-HCl, EDTA, glucose, pH 8.0;

[0052] LB: sodium chloride, tryptone, yeast powder, sucrose, pH 7.0.

[0053] The extracted plasmid was transferred into EHA105 competent Agrobacterium by freeze-thaw method, and the Agrobacterium containing pGM3301-TP1-KGF2 plasmid was screened and identified by PCR method (nucleotide sequence of TP1 upstream primer: SEQ ID NO.1, nucleotide sequence of TP1 downstream primer: SEQ ID NO.2; nucleotide sequence of EGF upstream primer: SEQ ID NO.3, nucleotide sequence of EGF downstream primer: SEQ ID NO.4); the identified Agrobacterium was placed in a 2L culture bottle on a shaker and cultured at 28°C and 230 rpm for 20 h until the absorbance of the Agrobacterium solution reached OD 590Reach 1.0; collect the cultured Agrobacterium by centrifugation, mix the collected Agrobacterium into 2L transformation solution (MS salt 4.3g, sucrose 100g, B vitamin solution 2mL, Slilwet L-77400μL, 1M NaOH 180μL, ddH2O 1980mL), and immerse the Arabidopsis inflorescence in the transformation solution for 7 minutes; transform the Arabidopsis again in the same way at an interval of 1 day (the transformation solution is the same as the previous time), and place the transformed Arabidopsis in the dark for 3 days; collect the transformed Arabidopsis seed pods successively until all the transformed Arabidopsis seed pods are collected, dry them in the shade, remove the shells, and sow them, named T1, and sow them for 2 generations and named T3 (continuous sowing and screening for 3 generations, the genetic traits are basically stable).

[0054] Figure 2 shows the Agrobacterium-mediated transformation of Arabidopsis thaliana according to the present invention. A shows bolting of wild Arabidopsis; B shows transformation of wild Arabidopsis; C shows sowing of transformed Arabidopsis; and D shows screening of transformed Arabidopsis. Figure 2 shows that resistant transformed strains were screened by applying a 1% glufosinate-ammonium screening agent. In Figure D, the green color represents successfully transformed resistant Arabidopsis seedlings, while the yellow color represents untransformed Arabidopsis seedlings.

[0055] (3) Screening of Arabidopsis strains containing TP1-KGF2 fusion protein

[0056] The whole genome of T3 Arabidopsis thaliana was extracted using a genome extraction kit. The nucleotide sequence of the upstream primer for TP1 was SEQ ID NO. 1, the nucleotide sequence of the downstream primer for TP1 was SEQ ID NO. 2; the nucleotide sequence of the upstream primer for EGF was SEQ ID NO. 3, the nucleotide sequence of the downstream primer for EGF was SEQ ID NO. 4. The PCR cycle was run as follows: 95°C for 5 min; (95°C for 30 sec; 60°C for 30 sec; 72°C for 70 sec) × 30 cycles; 72°C for 8 min. The amplified products were separated on agarose gel and detected using a UV imager.

[0057] The Arabidopsis tissue identified as positive by PCR was ground into powder, and protein extraction buffer was added (the ratio of the mass of Arabidopsis powder to the volume of protein extraction buffer was 1 kg: 2 L) to obtain a mixture, which was ice-bathed for 2 h; the ice-bathed mixture was centrifuged at 10,000 g for 20 min at 4°C, and protein loading was added. Buffer (the volume ratio of the mixed solution to protein loading buffer is 10:1), and the mixture was heated in a boiling water bath for 10 minutes. The extracted total soluble protein of Arabidopsis thaliana was separated by SDS-PAGE gel and transferred to a PVDF membrane using a transfer apparatus. Blocking solution was added and blocked at room temperature for 2 hours. The blocking solution was washed away and a 1:1000 diluted anti-KGF-2 mouse antibody was added, and the membrane was incubated at 4°C overnight; the antibody was washed away and a 1:10000 diluted alkaline phosphatase-labeled goat anti-mouse antibody was added, and the membrane was incubated at room temperature for 2 hours; the goat anti-mouse antibody was washed away and the color developer BCIP / NBT was added, and the membrane was placed in an imager for detection; three transformed Arabidopsis high-expression strains were screened out through molecular biological detection, namely NCF-365, NCF-669, and NCF-1470.

[0058] Figure 3 shows the PCR detection of the TP1-KGF2 Arabidopsis strain of the present invention, where A is the PCR detection of the TP1 gene fragment (approximately 100 bp); B is the PCR detection of the KGF2 gene fragment (approximately 500 bp). Figure 3 shows that the fusion gene TP1-KGF2 has been integrated into the Arabidopsis genome.

[0059] Figure 4 shows a Western blot of the TP1-KGF2 Arabidopsis strain of the present invention, with NCF-365, NCF-669, and NCF-1470 plotted on the abscissa and TSP% (total suspended particulate matter percentage) plotted on the ordinate. Figure 4 demonstrates that the TP1-KGF2 fusion gene can express the TP1-KGF2 protein in Arabidopsis.

[0060] Example 2: Verification of the hair growth promoting effect

[0061] (1) Establishment of animal model of hair loss

[0062] Hair plucking was used to induce hair loss and establish an animal model of hair loss: two groups of 7-week-old female C57BL / 6 mice (4 mice in each group) were anesthetized with a small animal gas inhalation anesthesia machine; rosin and paraffin were mixed in a mass ratio of 1:1, heated and melted, and evenly applied to the backs of the mice. After the depilatory wax solidified, it was peeled off; the above application, solidification, and peeling operations were repeated three times until the backs of the mice were smooth and hairless, resulting in two groups of hair loss mice.

[0063] (2) Preparation of TP1-KGF2 hair regeneration emulsion

[0064] 2 g of palmitic acid, 0.875 mL of ethanol, 2.5 mL of Arabidopsis extract (the Arabidopsis extract contains approximately 150 μg of TP1-KGF2 protein, and the components of Arabidopsis itself have no effect or synergistic effect on promoting hair growth) and 50 mg of benzethonium chloride are vigorously stirred and mixed at 50°C to obtain a uniform and stable cream. The loading value (hair growth promoting cream reagent) can make the resulting preparation contain 1.0 mg / mL to about 10.0 mg / mL.

[0065] (3) Detection of the hair growth promoting effect of TP1-KGF

[0066] Treatment of the first group of hair loss mice:

[0067] The prepared cream was applied to the backs of the first group of hair loss mice, with an interval of 24 hours between each application. Hair growth was measured every 3 days, serving as the experimental group.

[0068] The second group of hair loss mice were treated with:

[0069] The second group of hair loss mice did not apply the cream, and other conditions were the same as the first group, serving as the blank control group.

[0070] The two groups of mice were killed on day 15, and their skin was collected. The removed skin sample tissue was fixed on a section, and the section containing the skin sample was cut on a paraffin microtome to obtain a tissue sample with a thickness of approximately 5 to 10 μm. The tissue sample was placed in a basin of tap water and then soaked in 42°C warm water for approximately 5 to 10 seconds to allow the tissue to fully stretch. The soaked tissue sample was removed and naturally air-dried overnight, and then placed in a 60°C oven for 2 to 4 hours. Before use, the tissue sample was oven-dried for 20 minutes and xylene was added while hot to dewax the tissue sample. The specific steps are as follows: the tissue sample to be tested was placed in xylene and fully soaked for 15 minutes. After soaking, the xylene was replaced and soaked for another 15 minutes. The tissue sample to be tested, which had been soaked in xylene, was first soaked in anhydrous ethanol for 5 minutes, then removed and soaked in anhydrous ethanol for 5 minutes again to allow the xylene used in the dewaxing process to be washed out, allowing water to enter the tissue. The soaked tissue sample was then washed in 100%, 90%, 80%, and 70% ethanol in sequence. Pipette a pipette of pre-prepared hematoxylin staining solution and add 100 μL to each cleaned tissue sample. Stain thoroughly for 7–10 minutes. After staining, rinse with distilled water to remove excess hematoxylin staining solution. Then, use differentiation solution for 30 seconds and rinse the tissue sample with double-distilled water. Add eosin staining solution to the cleaned tissue sample and allow the tissue to fully stain for 3 minutes. After staining, dehydrate the tissue sample in a gradient manner, as follows: 80% ethanol dehydration for 5 seconds, 100% ethanol dehydration for 2 minutes, and anhydrous ethanol dehydration for 2 minutes. Soak the dehydrated tissue sample in xylene twice, each time for 15 minutes. Remove the xylene and seal the slide with neutral gum.

[0071] Figure 5 shows a graph demonstrating the hair growth-promoting effect of TP1-KGF. Figure A shows an H&E-stained tissue sample from a mouse not given TP1-KGF2; Figure B shows an H&E-stained tissue sample from a mouse given TP1-KGF2. Figure 5 demonstrates that hair follicles in the TP1-KGF2-treated mice regrow hair compared to the control group.

[0072] (4) Detection of TP1-KGF2 transdermal release ability

[0073] The tissue sample sealed with neutral gum was heated under a high pressure of 60-80 kPa, removed and cooled, washed with PBS, and blocked with 30 μL of 5% BSA (bovine serum albumin) for 1 hour; the blocking solution was washed off, and a 1:250 diluted anti-KGF2 antibody was added and incubated overnight at 4°C. The primary antibody on the tissue was washed off, and a 1:500 diluted TP1-KGF2 antibody was added and incubated at room temperature for 1 hour; the incubated tissue sample was air-dried, and an appropriate amount of DAB (diaminobenzidine) colorimetric solution was added. The tissue sample was examined under a microscope and staining was stopped when the tissue turned brownish yellow; the steps for cleaning the tissue sample were as follows: dehydration by immersion in 70%, 80%, 90%, and 100% ethanol in sequence, and after the tissue was completely dehydrated, the slide was mounted with neutral resin and observed and analyzed under a microscope.

[0074] Figure 6 shows the transdermal delivery of TP1-KGF2 according to the present invention (green represents detected fluorescence). Figure A shows immunofluorescence testing of hair follicles after administration of KGF-2, while Figure B shows immunofluorescence testing of hair follicles after administration of TP1-KGF2. As shown in Figure 6, the presence of white fluorescence in Figure B indicates that TP1-KGF2 has penetrated the hair follicle epidermis, while the absence of white fluorescence in Figure A indicates that KGF2 has not entered the hair follicles or has been licked away by the mice. Therefore, a clear fluorescence signal is detected after administration of TP1-KGF2, while a fluorescence signal is difficult to detect after administration of KGF-2.

[0075] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A keratinocyte growth factor-transdermal peptide fusion protein, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

12.

2. A gene encoding the fusion protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

8.

3. A recombinant vector containing the gene as claimed in claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector used was pGM3301.

5. A recombinant bacterium comprising the recombinant vector according to claim 3 or 4.

6. Use of the fusion protein as claimed in claim 1 in preparing a hair growth promoting agent.

7. A method for preparing an Arabidopsis thaliana transformed strain expressing the fusion protein according to claim 1, characterized in that: The method includes: (1) TP-1 gene and KGF-2 gene were connected into pGM3301 vector using T4 ligase, the ligation product was transformed into Escherichia coli, the gene containing the fusion protein as claimed in claim 2 was screened out, the constructed vector was named pGM3301-TP1-KGF2, and the pGM3301-TP1-KGF2 vector plasmid was extracted; Wherein, the nucleotide sequence of the TP-1 gene is as shown in SEQ ID NO.6, the nucleotide sequence of the KGF-2 gene is as shown in SEQ ID NO.7, and the nucleotide sequence of the pGM3301-TP1-KGF2 gene is as shown in SEQ ID NO.8; (2) The extracted pGM3301-TP1-KGF2 vector plasmid was transferred into the competent EHA105 Agrobacterium by freeze-thaw method, and the Agrobacterium containing the pGM3301-TP1-KGF2 plasmid was screened and identified by PCR method; the identified Agrobacterium was cultured at 28°C and 230 rpm until the absorbance OD of the Agrobacterium solution was 0. 590 When the value reaches 1.0, the cultured Agrobacterium is collected by centrifugation; The nucleotide sequence of the first set of upstream primers used in the PCR method is SEQ ID NO.1, the nucleotide sequence of the downstream primers is SEQ ID NO.2, the nucleotide sequence of the second set of upstream primers is SEQ ID NO.3, and the nucleotide sequence of the downstream primers is SEQ ID NO.4; (3) Mixing the collected Agrobacterium into the transformation solution, immersing the Arabidopsis inflorescence into the transformation solution, placing the transformed Arabidopsis in a dark place and collecting the transformed Arabidopsis seed pods, drying them in the shade, removing the shells and collecting the seeds, sowing them and naming them T1, continuing to sow for 2 generations to select a homozygous transformed strain named T3, and the homozygous transformed strain T3 can express the fusion protein as described in claim 1.

8. A method for screening Arabidopsis thaliana transformed strains containing the fusion protein according to claim 1, characterized in that: The method includes: (1) The whole genome of Arabidopsis thaliana was extracted using a genome extraction kit, the amplified products were separated by agarose gel using the PCR method, and detected using an ultraviolet imager; The nucleotide sequence of the first set of upstream primers used in the PCR method is SEQ ID NO.1, the nucleotide sequence of the downstream primers is SEQ ID NO.2, the nucleotide sequence of the second set of upstream primers is SEQ ID NO.3, and the nucleotide sequence of the downstream primers is SEQ ID NO.4; (2) Grind the tissue of Arabidopsis thaliana identified as positive by PCR into powder, add protein extraction buffer to obtain a mixed solution, and place it in an ice bath; centrifuge the mixed solution after the ice bath at 4°C, add loading buffer, and place it in a boiling water bath; separate the extracted total soluble protein of Arabidopsis thaliana by SDS-PAGE gel, transfer it to a PVDF membrane using a membrane transfer instrument, add blocking solution to block at room temperature, wash off the blocking solution, add anti-KGF-2 mouse antibody, and incubate at 4°C overnight; wash off the antibody, add alkaline phosphatase-labeled goat anti-mouse antibody, and incubate at room temperature; wash off the goat anti-mouse antibody, add the color developer BCIP / NBT, and place it in an imager for detection; and screen the transformed Arabidopsis thaliana strains that highly express the fusion protein as described in claim 1 through molecular biological detection.

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