Recombinant GHK-CL2 collagen as well as preparation method and application thereof

Abstract

The invention provides a recombinant GHK-CL2 collagen. GHK is located at an amino terminal or a carboxyl terminal of CL2. The recombinant GHK-CL2 collagen obtained through design, expression and purification is uniform in molecular weight and non-toxic, and has good biological activity of promoting cell adhesion, proliferation and migration; and the recombinant GHK-CL2 collagen has a good property of resisting trypsin hydrolysis, which indicates that the protein has a complete three-chain triple-helix structure.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a recombinant GHK-CL2 collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is a type of functional biopolymer protein widely distributed in animal connective tissues. It is the most abundant and widely distributed in mammals. Collagen accounts for about 25% to 30% of the total protein in the human body and is an important component of the extracellular matrix. At present, at least 28 different types of collagen have been found in vertebrates. Different types of collagen have different biological functions and physical properties, but they are all composed of three polypeptide chains composed of repeated Gly-Xaa-Yaa sequences in the form of right-handed helices. Collagen is widely used in cosmetics, food and health products, medical supplies, etc. As a medical material, collagen has broad potential value in drug delivery, tissue transplantation, etc.

[0003] The animal-derived collagen currently under study has the disadvantages of poor biocompatibility, immunogenicity and cytotoxicity. It is necessary to research and develop collagen-like proteins with good biocompatibility, non-immunogenicity and non-cytotoxicity.

[0004] Bacterial collagen region (CL-2) generally does not have the biological activity of collagen. Tripeptide-1 and blue copper peptide are commonly used anti-aging repair peptides in cosmetics. They are composed of glycine, histidine and lysine, and the sequence is GHK. There is currently no research on the fusion of GHK with bacterial collagen region. Summary of the invention

[0005] The object of the present invention is to provide a recombinant GHK-CL2 collagen protein in view of the above shortcomings, wherein GHK is located at the amino end or carboxyl end of CL2.

[0006] Furthermore, the amino acid sequence of the recombinant GHK-CL2 collagen is as follows: GHK is located at the amino terminus of CL2: GHKGSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGE PGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKY; GHK is located at the carboxyl terminus of CL2: GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGT QGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGHK.

[0007] The second aspect of the present invention provides a method for preparing the above-mentioned recombinant GHK-CL2 collagen, comprising the following steps: Step 1, amino acid sequence design: recombinant GHK-CL2 collagen is formed by connecting the GHK sequence with the CL2 collagen; Step 2, constructing an expression vector of recombinant GHK-CL2 collagen: designing and synthesizing a gene fragment encoding GHK-CL2 collagen, cloning it into an expression vector, optimizing the sequence according to the codon preference of Escherichia coli, inserting the synthesized gene sequence into the expression vector, and obtaining an expression vector of recombinant GHK-CL2 collagen; Step 3: Protein expression: Transform the constructed recombinant GHK-CL2 collagen-like protein expression vector into BL21 (DE3) competent cells, screen out positive clones, culture and induce protein expression, and collect the expressing bacteria; Step 4. Protein acquisition and purification: The collected expression bacteria are lysed, centrifuged, and filtered to obtain the supernatant, i.e., crude protein. The GHK-CL2 collagen solution is prepared by nickel ion column chromatography, and freeze-dried. The freeze-dried fibers are collected to obtain the recombinant GHK-CL2 collagen.

[0008] Furthermore, in the step 1, a His-SUMO tag is added to the amino terminus of the recombinant GHK-CL2 collagen, and the sequence is as follows: MGHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.

[0009] Furthermore, the method for constructing the expression vector of GHK-CL2 collagen in step 2 is specifically as follows: inserting the gene fragment of synthetic recombinant GHK-CL2 collagen into the pET28a vector through the NdeI / XhoI double restriction site, and marking it as: pET28a-6xHis-SUMO-GHK-CL2.

[0010] Furthermore, the protein expression method in the step three is: transforming pET28a-6xHis-SUMO-GHK-CL2 into the competent Escherichia coli BL21 (DE3) strain, culturing on LB plate at 37°C overnight, selecting single clones, and further culturing in 2xYT medium until OD600 reaches 0.6-0.8, reducing the culture temperature to 20°C, adding IPTG, continuing to culture for 16 hours, and collecting the expressing bacteria by centrifugation.

[0011] Furthermore, the protein acquisition and purification method in step 4 is as follows: protein extract, lysozyme and DNase I are added to the collected bacteria, and the mixture is allowed to stand on ice for 1 hour to obtain a protein solution. After homogenization and lysis, the supernatant is collected by centrifugation or filtration to obtain crude protein; the obtained crude protein is purified using a nickel-loaded gravity column; ULP-1 enzyme is added to the obtained purified protein solution for overnight enzymatic digestion and dialysis; the obtained protein solution is purified again by a nickel column to remove the label; the obtained protein is dialyzed to obtain recombinant GHK-CL2 collagen.

[0012] The third aspect of the present invention provides a recombinant GHK-CL2 collagen having good biological activity and structural stability, specifically: biological activity verification and resistance to trypsin hydrolysis.

[0013] Furthermore, the biological activity verification includes evaluation of promoting cell proliferation activity, promoting cell adhesion activity and promoting cell migration activity.

[0014] The present invention also provides a use of the above-mentioned recombinant GHK-CL2 collagen, specifically for use in medical devices, biomaterials and cosmetic products.

[0015] The recombinant GHK-CL2 collagen provided by the present invention belongs to collagen-like proteins. Bacterial collagen-like proteins are usually composed of a variable region (V) and a collagen-like region; its collagen-like region has the same Gly-XY domain characteristics as collagen and can form a triple-chain triple helix structure. Compared with animal-derived collagen, the collagen-like domain of bacterial collagen has the advantages of good biocompatibility, non-immunogenicity and non-cytotoxicity.

[0016] Tripeptide-1 and blue copper peptide are commonly used anti-aging repair peptides in cosmetics. They are composed of glycine, histidine and lysine, and the sequence is GHK, which has the same characteristics as the Gly-XY domain of collagen. Therefore, the fusion expression of GHK and collagen or collagen-like can be achieved, and the fusion protein has the characteristics of both GHK and collagen.

[0017] Beneficial effects of the present invention: The recombinant GHK-CL2 collagen provided by the present invention has the following advantages (1) The present invention constructs GHK-CL2 collagen and uses the common Gly-Xaa-Yaa of GHK and collagen-like to achieve fusion.

[0018] (2) The recombinant GHK-CL-2 designed by the present invention achieves large-scale soluble expression without a variable region, and the recombinant protein finally obtained has the anti-trypsin hydrolysis characteristics of triple-chain triple-helical collagen.

[0019] (3) GHK-CL2 collagen has good biological activity and can effectively promote cell proliferation, migration and adhesion. It can be widely used in the biopharmaceutical and cosmetic industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment: Figure 1 It is a schematic diagram of the structure of recombinant GHK-CL2; Figure 2 This is the electrophoresis diagram of the purification results of recombinant GHK-CL2; Figure 3 This is the electrophoresis diagram of the recombinant GHK-CL2 anti-trypsin hydrolysis; Figure 4 This is the result of the cell proliferation experiment; Figure 5 Figure 2 is the result of cell adhesion experiment; Figure 6 This is the result of the cell scratch healing experiment; Figure 7 The figure is a migration bar graph of the cell scratch healing assay results. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification. The methods described are all conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0022] The term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0023] A recombinant GHK-CL2 collagen, wherein GHK is located at the amino terminal or carboxyl terminal of CL2.

[0024] GHK is located at the amino terminus of CL2: GHKGSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGE PGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKY; GHK is located at the carboxyl terminus of CL2: GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGT QGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGHK.

[0025] Example 1 Step 1: Amino acid sequence design: refer to Figure 1 Based on the amino acid sequence of GHK-CL2, a gene sequence encoding GHK-CL2 collagen was designed and synthesized, and the 6xHis-SUMO expression gene was fused to the 5' end of the GHK-CL2 collagen gene by overlapping extension PCR to form the 6xHis-SUMO-GHK-CL2 expression gene.

[0026] Step 2: Construct the expression vector of recombinant GHK-CL2 collagen: clone and amplify the DNA fragment by PCR, add Nde I and Xho I restriction sites by primers during amplification, and insert the target fragment into the pET28a vector by restriction digestion and ligation. After clone screening and sequencing verification, the recombinant vector pET28a-6xHis-SUMO-GHK-CL2 recombinant expression vector was obtained.

[0027] Step 3, protein expression: The pET28a-6xHis-SUMO-GHK-CL2 plasmid was transformed into the Escherichia coli expression strain BL21 (DE3), and single clones were obtained by kanamycin resistance screening. Single clones were picked and cultured overnight in 5 ml 2xYT medium, inoculated at a ratio of 1:100, and cultured at 37°C with shaking until OD600 was 0.6-0.8. After the culture temperature was reduced to 20°C, IPTG was added to a final concentration of 0.2 mM, and the culture was continued for 16 hours. The bacteria were collected by centrifugation for the next purification step.

[0028] Step 4: Purification of recombinant GHK-CL2 collagen (1) Bacterial lysis: Resuspend the bacteria in 20 mM phosphate at a ratio of 1:15, lyse the bacteria using a homogenizer under cold water, and collect the supernatant by centrifugation or filtration after lysis; (2) The supernatant was passed through Ni-NTA, washed with a solution containing 20 mM imidazole to remove impurity proteins, and then the target protein was eluted with a solution containing 250 mM imidazole; (3) Ulp-1 enzyme digestion: Add 1 / 50000 Ulp1 enzyme to the clear solution after elution in the previous step, mix well, and dialyze using a dialysis bag with a molecular weight cutoff of 3 kDa to remove salt and obtain a protein solution; (4) Anion exchange chromatography: The protein solution obtained in the previous step is subjected to anion exchange chromatography to remove impure proteins and nucleic acids to obtain high-purity recombinant GHK-CL2 collagen.

[0029] The purified recombinant GHK-CL2 collagen was subjected to SDS-PAGE electrophoresis detection. Figure 2As shown, lane 1 is the marker and lane 2 is the purified recombinant GHK-CL2 collagen. The electrophoresis results show that the purity of the purified recombinant GHK-CL2 collagen is greater than 95%, and the molecular weight is around 25kDa (theoretical molecular weight is 23.3kDa). The deviation of the molecular weight is related to its structure.

[0030] Experimental Example 1 Trypsin digestion experiment Recombinant GHK-CL2 collagen and commercially available recombinant type III collagen were prepared into a 1 mg / ml protein solution with PBS. A 5% trypsin solution (trypsin concentration was 200 ug / ml) was added to the protein solution, mixed well, and placed in a 4°C refrigerator for 16 hours. Samples were taken for SDS-PAGE analysis of the enzyme cleavage of the protein by trypsin. Figure 3 As shown, the results show that after 16 hours of trypsin treatment, there is no particularly obvious change in the bands of the recombinant GHK-CL2 collagen (lane 2) and the control (lane 1), indicating that the recombinant GHK-CL2 collagen has good resistance to trypsin hydrolysis. After 16 hours of trypsin treatment, most of the recombinant type III collagen (lane 4) and the recombinant GHK-CL2 collagen (lane 3) are cut into small fragments by trypsin, indicating that the commercially available recombinant type III collagen does not have the property of resisting trypsin hydrolysis.

[0031] Experimental Example 2 Bioactivity verification Cell proliferation assay, cell adhesion assay and cell scratch healing assay 1. Evaluation of the cell proliferation activity of recombinant GHK-CL2 collagen BALB / c 3T3 cells (mouse embryonic fibroblasts) in good logarithmic growth phase were selected, digested with 0.25% trypsin, centrifuged and resuspended in DMEM medium containing 10% serum and counted; BALB / c 3T3 cells were inoculated into 96-well culture plates at 3000 cells per well, and blank controls without cells were set up at the same time. After culturing for 16 hours at 37°C and 5% CO2, the culture medium was changed to DMEM medium containing 2% serum and continued to be cultured for 16 hours until the cells adhered well. The original culture medium was aspirated, and a culture medium containing 200μg / ml and 400μg / ml recombinant GHK-CL2 collagen was added. The cell control group was DMEM medium containing 2% serum, and the positive control group was 200pg / ml epidermal growth factor (EGF), and the culture was continued for 48 hours. 4 h before the end of the culture, add 5 g / L MTT solution, continue to culture for 4 h and then terminate the culture; discard the liquid in the wells, wash once with PBS, add DMSO to dissolve the crystals, and measure the absorbance value of each well at a wavelength of 570 nm.

[0032] Cell proliferation rate = (absorbance value of the assay well - absorbance value of the blank control group) / (absorbance value of the cell control group - absorbance value of the blank control group) × 100%.

[0033] In this example, the positive control group was a solution of epidermal growth factor (EGF), which was added to the cell culture medium at 200 pg / ml, with 3 parallel samples in each group. Figure 4 As shown, the experimental results show that at a concentration of 400 μg / ml, the proliferation rates of the control group and the recombinant GHK-CL2 collagen of the present application on BALB / c 3T3 cells were 112.8% and 122.1%, respectively, indicating that the recombinant GHK-CL2 collagen prepared by the present invention can significantly promote the proliferation of fibroblasts and has the biological activity of promoting cell proliferation.

[0034] 2. Evaluation of the cell adhesion promoting activity of recombinant GHK-CL2 collagen The prepared recombinant GHK-CL2 collagen was dissolved in PBS and added to a 96-well cell culture plate at a final concentration of 200 μg / ml and 500 μg / ml, respectively. 100 μL of recombinant GHK-CL2 collagen solution was added to each well and placed at 37°C for 16 h. PBS was added to the negative control well. BALB / 3T3 cells were digested with 0.25% trypsin, counted, and 5×10 4 Cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. PBS was washed three times to remove non-adherent cells, and then 200 μL DMEM medium was added; 5 g / L MTT solution was added, and the cells were incubated in a 37°C, 5% CO2 cell incubator for 2 hours before removal. The absorbance values ​​of the 96-well plate at 450 nm and 630 nm were read with an ELISA reader, and the absorbance was measured at 450 nm with 630 nm as the reference wavelength, and the measurement results were recorded.

[0035] Cell adhesion promotion rate = (450nm absorbance of the experimental group - 450nm absorbance of the negative control group) / 450nm absorbance of the negative control group × 100%.

[0036] In this example, the control group was a commercially available recombinant type III collagen sample solution, which was added to the cell culture medium at 200 μg / ml, with 3 parallel samples in each group. Figure 5 As shown, the results show that at a concentration of 200 μg / ml and 500 μg / ml, the cell adhesion promoting rates of the recombinant GHK-CL2 collagen of the present application on BALB / c 3T3 cells were 23.61% and 25.96%, respectively, and both were better than 22.72% of the commercially available recombinant type III collagen at a concentration of 200 μg / ml, indicating that the recombinant GHK-CL2 collagen prepared by the present invention has good cell adhesion promoting activity.

[0037] 3. Evaluation of the cell migration-promoting activity of recombinant GHK-CL2 collagen Take recombinant GHK-CL2 collagen and commercially available recombinant type III collagen as a reference, and dilute the samples to 0.5 mg / mL with DMEM 2% serum medium, pH 7.0-7.4. Culture and passage BALB / c 3T3 cells normally. Inoculate cells in good condition into 6-well plates, and place 5×10 cells per well. 5 2mL cell suspension was inoculated per well and cultured for 24h. A 200ul pipette tip was placed perpendicular to the surface of the 6-well plate to make a scratch perpendicular to the bottom line of the 6-well plate in the monolayer of cells. The width of the scratch was kept consistent as much as possible during the process. The cells were washed with PBS for 3 times to remove the free cells and cell fragments caused by the scratch. A protein solution diluted with 2% serum DMEM medium was added to the wells and the cells were cultured in a 37℃, 5% CO2 incubator. Samples were taken at 0h and 24h and photos were taken. The results are shown in the table. Figure 6 Image J software was used to obtain the initial scratch area and the area of ​​the blank area without cells, and the migration rate was calculated. The migration rate = (initial scratch area - blank area after cell migration / initial scratch area) * 100%. The experimental results are shown in Figure 7 The migration rate of the recombinant GHK-CL2 collagen was 28.59% at a concentration of 200 μg / ml, while the migration rate of commercially available recombinant type III collagen was 21.62%, indicating that the recombinant GHK-CL2 collagen had good biological activity in promoting cell migration.

[0038] Based on the above embodiments and experimental examples, the recombinant GHK-CL2 collagen designed, expressed and purified by the present invention has uniform molecular weight, is non-toxic, and has good biological activity in promoting cell adhesion, proliferation and migration; and the recombinant GHK-CL2 collagen has good resistance to trypsin hydrolysis, indicating that the protein has a complete triple-chain triple-helix structure. The recombinant GHK-CL2 collagen can be applied to a variety of fields, such as pharmaceutical compositions, medical devices, tissue engineering products or cosmetics.

Claims

1. A recombinant GHK-CL2 collagen, characterized in that: GHK is located at the amino-terminus or carboxyl-terminus of CL2.

2. The recombinant GHK-CL2 collagen according to claim 1, characterized in that: The amino acid sequence of the collagen is as follows: GHK is located at the amino terminus of CL2 GHKGSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGE PGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKY; GHK is located at the carboxyl terminus of CL2 GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGT QGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGHK.

3. A method for preparing the recombinant GHK-CL2 collagen according to any one of claims 1 to 2, characterized in that: The steps include: Step 1, amino acid sequence design: the recombinant GHK-CL2 collagen is formed by connecting the GHK sequence with the CL2 collagen; Step 2, constructing an expression vector of recombinant GHK-CL2 collagen: designing and synthesizing a gene fragment encoding GHK-CL2 collagen, cloning it into an expression vector, optimizing the sequence according to the codon preference of Escherichia coli, inserting the synthesized gene sequence into the expression vector, and obtaining an expression vector of recombinant GHK-CL2 collagen; Step 3: Protein expression: Transform the constructed recombinant GHK-CL2 collagen-like protein expression vector into BL21 (DE3) competent cells, screen out positive clones, culture and induce protein expression, and collect the expressing bacteria; Step 4. Protein acquisition and purification: The collected expression bacteria are lysed, centrifuged, and filtered to obtain the supernatant, i.e., crude protein. The GHK-CL2 collagen solution is prepared by nickel ion column chromatography, and freeze-dried. The freeze-dried fibers are collected to obtain the recombinant GHK-CL2 collagen.

4. The method for preparing the recombinant GHK-CL2 collagen according to claim 3, characterized in that: In the step 1, a 6xHis-SUMO tag is added to the amino terminus of the GHK-CL2 collagen, and the sequence is as follows: MGHHHHHHGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKK TTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHRE QIGG.

5. The method for preparing the recombinant GHK-CL2 collagen according to claim 4, characterized in that: The method for constructing the expression vector of GHK-CL2 collagen in step 2 is specifically as follows: inserting the gene fragment of synthetic recombinant GHK-CL2 collagen into the pET28a vector through the NdeI / XhoI double restriction site, and marking it as: pET28a-6xHis-SUMO-GHK-CL2.

6. The method for preparing the recombinant GHK-CL2 collagen according to claim 4, characterized in that: The protein expression method in the step three is as follows: pET28a-6xHis-SUMO-GHK-CL2 is transformed into the competent BL21 (DE3) strain of Escherichia coli, cultured on LB plate at 37°C overnight, single clones are selected, and further cultured in 2xYT medium until OD600 reaches 0.6-0.8, the culture temperature is reduced to 20°C, IPTG is added, and the culture is continued for 16 hours, and the expression bacteria are collected by centrifugation.

7. The method for preparing the recombinant GHK-CL2 collagen according to claim 4, characterized in that: The protein acquisition and purification method in step 4 is as follows: adding protein extract, lysozyme and DNase I to the collected bacteria, standing on ice for 1 hour to obtain a protein solution, after homogenization and lysis, collecting the supernatant by centrifugation or filtration to obtain crude protein; purifying the obtained crude protein using a nickel-loaded gravity column; adding ULP-1 enzyme to the obtained purified protein solution for overnight enzymatic digestion and dialysis; the obtained protein solution is purified again by a nickel column to remove the label; the obtained protein is dialyzed to obtain recombinant GHK-CL2 collagen.

8. A method for detecting the recombinant GHK-CL2 collagen according to any one of claims 1 to 2, characterized in that: Specifically: biological activity verification and anti-trypsin hydrolysis.

9. The method for detecting recombinant GHK-CL2 collagen as claimed in claim 8, characterized in that: The biological activity verification includes evaluation of promoting cell proliferation activity, promoting cell adhesion activity and promoting cell migration activity.

10. A medical device, a biomaterial and a cosmetic product, characterized in that: The product comprises the recombinant GHK-CL2 collagen described in any one of claims 1 to 2.

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