Recombinant humanized type I collagen and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a recombinant humanized type I collagen and its application. The present invention designs a new humanized type I collagen sequence that can comprehensively improve the structural stability and solubility of humanized type I collagen, thereby improving the product quality of humanized type I collagen. The present invention transfers the screened new humanized type I collagen sequence into a Pichia pastoris strain for recombinant expression. The resulting recombinant humanized type I collagen has the advantages of strong stability, high expression level, and good hydrophilicity. At the same time, the recombinant humanized type I collagen also has the effect of significantly promoting cell proliferation and cell adhesion.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to a recombinant humanized type I collagen and an application thereof. Background Art

[0002] Type I collagen is an essential component of the extracellular matrix. It is a heterotrimer composed of two α1 chains and one α2 chain, with each α chain containing approximately 1,000 amino acid residues arranged in a helical structure. Type I collagen is primarily found in animal tissues, such as skin, bones, tendons, and blood vessels, where it plays a crucial role in maintaining tissue integrity and stability. Type I collagen also participates in physiological processes such as the synthesis, degradation, and repair of the extracellular matrix. As a medical material, it is widely used in clinical practice in areas such as hematopoietic stem cell transplantation, bone repair, and wound healing.

[0003] In recent years, studies have shown that combining type I collagen with other functional molecules or cells can achieve targeted gene and drug delivery, as well as construct biologically functional tissue engineering scaffolds. Therefore, type I collagen also holds significant application prospects in areas such as gene delivery, drug delivery, and tissue engineering. With the advancement of recombinant technology, type I collagen can be produced with high purity and specificity without the use of animal sources, overcoming the challenges of low bioactivity, poor solubility, and low bioavailability of type I collagen extracted using traditional methods.

[0004] Patent document CN117510618A discloses a synthetic humanized type I collagen, its preparation method, and its application. This synthetic humanized type I collagen is a nine-fold repeat of the peptide segment of amino acids 674-736 of the human type I collagen α1 chain. It contains nine GFPGER domains, which can bind to cellular integrin receptors. Increasing the number of these domains within a single protein molecule through tandem expression can enhance the collagen's affinity for cells and promote cell adhesion. The resulting humanized type I collagen can be used to prepare medical products such as dressings and repair solutions.

[0005] Patent document CN118047858A discloses a recombinant humanized type I collagen, its preparation method, and applications. This recombinant humanized type I collagen contains six amino acids that promote the growth of extracellular matrix components, including essential and semi-essential amino acids, and the collagen-specific amino acid hydroxyproline. This protein interacts with endothelial cells and osteoblasts, promoting the proliferation of skin fibroblasts, increasing skin elasticity, and facilitating the crawling and growth of osteoblasts. It can be widely used in orthopedic sports materials and has promising applications in medical cosmetology and sports medicine.

[0006] Patent document CN113621052A discloses a recombinant humanized type I collagen polypeptide, its preparation method, and uses. The N-terminus of this recombinant humanized type I collagen polypeptide contains an amino acid sequence that can be cleaved by TEV protease, demonstrating its ability to promote cell adhesion. Furthermore, the amino acid sequence of this recombinant protein is selected from the amino acid sequence of native collagen, and its application to the human body does not produce an immune response.

[0007] However, current research on recombinant type I collagen produced using genetic engineering methods primarily focuses on the functional aspects of type I protein, with limited research on its stability, solubility, and expression levels. Therefore, research and development of a type I collagen with high stability, good solubility, and high expression levels remains a challenge. Summary of the Invention

[0008] To address the deficiencies of the prior art, the present invention provides a recombinant humanized type I collagen and its application. The present invention designs a new humanized type I collagen sequence that can comprehensively enhance the structural stability and solubility of humanized type I collagen, thereby improving the product quality of humanized type I collagen. The present invention transfers the screened new humanized type I collagen sequence to a Pichia pastoris strain for recombinant expression. The resulting recombinant humanized type I collagen has the advantages of strong stability, high expression levels, and good hydrophilicity. Furthermore, the recombinant humanized type I collagen also significantly promotes cell proliferation and cell adhesion.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] The present invention provides a recombinant humanized type I collagen protein, the amino acid sequence of the recombinant humanized type I collagen protein is shown in SEQ ID NO: 1.

[0011] Furthermore, the present invention provides a recombinant humanized type I collagen encoding gene, which encodes the above-mentioned recombinant humanized type I collagen.

[0012] Furthermore, the nucleotide sequence of the recombinant humanized type I collagen encoding gene is shown in SEQ ID NO: 2.

[0013] Furthermore, the present invention also provides a recombinant humanized type I collagen recombinant vector, which contains the above-mentioned recombinant humanized type I collagen encoding gene.

[0014] Furthermore, the present invention also provides an engineered strain, which comprises the above-mentioned recombinant humanized type I collagen encoding gene or the above-mentioned recombinant humanized type I collagen recombinant vector.

[0015] In addition, the present invention also provides the use of the recombinant humanized type I collagen in the preparation of cosmetics or medical device products.

[0016] Furthermore, the recombinant humanized type I collagen is used in the preparation of anti-aging cosmetics, repair cosmetics or antioxidant cosmetics.

[0017] Furthermore, the recombinant humanized type I collagen is used in the preparation of a collagen dressing that promotes cell proliferation, adhesion and migration.

[0018] In addition, the present invention also provides a composition comprising the above-mentioned recombinant humanized type I collagen and an acceptable excipient.

[0019] The recombinant humanized type I collagen provided by the present invention is prepared by sequentially linking hydrophilic regions of the α1 chain of human type I collagen to produce the recombinant humanized type I collagen shown in SEQ ID NO. 1. The recombinant humanized type I collagen prepared by the present invention has the advantages of strong stability, high expression level, and good hydrophilicity. It also significantly promotes cell proliferation and cell adhesion. Furthermore, the amino acid sequence of the recombinant humanized type I collagen provided by the present invention is 100% identical to the corresponding portion of natural human type I collagen, and its application in the human body will not cause immune rejection.

[0020] In summary, compared with the prior art, the recombinant humanized type I collagen provided by the present invention has the following advantages:

[0021] (1) The amino acid sequence of the recombinant humanized type I collagen provided by the present invention is 100% identical to the corresponding portion of natural human type I collagen, and will not cause immune rejection when applied to the human body.

[0022] (2) The recombinant humanized type I collagen amino acid sequence provided by the present invention can be successfully expressed and secreted in a yeast expression system, and the expression level is high. The brightness of the electrophoresis band in the fermentation supernatant is about 3 times that of the 66.2KD standard.

[0023] (3) The recombinant humanized type I collagen provided by the present invention has strong solubility, and its solubility in saline solution can reach 100 g / L.

[0024] (4) The recombinant humanized type I collagen provided by the present invention has a significant effect on promoting the proliferation of human embryonic skin fibroblasts (ESF). The cell proliferation rate at 0.125 mg / mL of recombinant humanized type I collagen is 121.32%, which is 1.15 times that of the 1 mg / mL bovine type I collagen standard.

[0025] (5) The recombinant humanized type I collagen provided by the present invention has a significant promoting effect on the adhesion of L929 cells. The cell adhesion rate of the recombinant humanized type I collagen at 4 mg / mL is 77.76%, which is 2.66 times that of the 2 mg / mL bovine type I collagen standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the hydrophobicity analysis results of recombinant humanized type I collagen.

[0027] Figure 2 Schematic diagram of the construction of recombinant type I humanized collagen plasmid pPIC9K-hca1.

[0028] Figure 3 This is the SDS-PAGE electrophoresis diagram of recombinant humanized type I collagen expression in shake flasks (M: Marker; 1-2: verification of recombinant humanized type I collagen expression in Pichia pastoris genetically engineered bacteria shake flasks; 3: negative control).

[0029] Figure 4 This is the SDS-PAGE electrophoresis detection result of the fermentation supernatant of recombinant humanized type I collagen (M: Marker, 1-5: SDS-PAGE electrophoresis diagrams after 0h, 24h, 48h, 72h, and 88h of induction, respectively).

[0030] Figure 5 The figure shows the SDS-PAGE electrophoresis results of the purified recombinant humanized type I collagen (1: recombinant humanized type I collagen; M: Marker).

[0031] Figure 6 The graph shows the proliferation rate of ESF cells induced by recombinant humanized type I collagen (NC is the blank control, PC is the bovine type I collagen standard).

[0032] Figure 7 This is a graph showing the effect of recombinant humanized type I collagen on the proliferation rate of ESF cells and the observation of cell status.

[0033] Figure 8 The figure shows the adhesion rate of recombinant humanized type I collagen to L929 cells (NC is the blank control, and the reference product is the bovine type I collagen standard).

[0034] Figure 9 The figure shows the relative adhesion of recombinant humanized type I collagen to L929 cells (NC is the blank control, and the reference product is the bovine type I collagen standard).

[0035] Figure 10The figure shows the cell adhesion results of recombinant humanized type I collagen to L929 cells (NC is blank control, reference product is bovine type I collagen standard, PC is 0.1% gelatin). DETAILED DESCRIPTION

[0036] The present invention is further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art may make various modifications or improvements based on the basic concept of the present invention, but as long as they do not deviate from the basic concept of the present invention, they are all within the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] Example 1. Design and synthesis of recombinant humanized type I collagen gene

[0038] 1. Amino acid sequence of recombinant humanized type I collagen:

[0039] The hydrophobicity of the α1 chain of human type I collagen was analyzed, and regional fragments with good hydrophilicity of a certain length were selected and connected in sequence to obtain the amino acid sequence of recombinant type I humanized collagen as shown in SEQ ID NO.1. The hydrophobicity of SEQ ID NO.1 was analyzed, and the hydrophobicity analysis results of the recombinant type I humanized collagen prepared by the present invention are as follows: Figure 1 As shown. Figure 1 It can be seen that the amino acid sequence of the recombinant humanized type I collagen prepared by the present invention has good hydrophilicity.

[0040] The amino acid sequence of the recombinant humanized type I collagen is (SEQ ID NO: 1):

[0041] .

[0042] 2. Nucleotide sequence of recombinant humanized type I collagen:

[0043] The amino acid sequence of the recombinant humanized type I collagen was designed based on the codon preference of Pichia pastoris, and the nucleotide sequence of the recombinant humanized type I collagen was optimized based on the codon preference of Pichia pastoris to obtain the nucleotide sequence of the recombinant humanized type I collagen shown in SEQ ID NO. 2. Synthesis was commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0044] The nucleotide sequence of the recombinant humanized type I collagen is (SEQ ID NO: 2):

[0045]

[0046] 3. Construction of recombinant plasmid:

[0047] The nucleotide sequence of the above-synthesized recombinant type I humanized collagen was constructed into the pPIC9K vector to obtain a recombinant type I humanized collagen plasmid named pPIC9K-hca1. The schematic diagram of the recombinant type I humanized collagen plasmid pPIC9K-hca1 constructed by the present invention is shown in FIG. Figure 2 shown.

[0048] Example 2: Preparation of genetically engineered strains producing recombinant humanized type I collagen

[0049] 1. Preparation of linearized plasmid:

[0050] The plasmid was linearized using a single enzyme digestion method. The recombinant type I humanized collagen plasmid pPIC9K-hca1 prepared in Example 1 was extracted, digested with Sal I enzyme, and then recovered by ethanol precipitation to obtain the recombinant type I humanized collagen linearized plasmid.

[0051] 2. Preparation of competent yeast:

[0052] Inoculate Pichia pastoris GS115 into YPD liquid medium and culture overnight at 28°C and 220 rpm. Measure the OD600 value of the culture. At an initial OD600 of 0.5, inoculate 50 mL of YPD liquid medium and culture until the OD600 reaches 1.3-1.5. Centrifuge at 3000 g at 4°C for 5 minutes and discard the culture medium. Add 50 mL of sterile water to resuspend the yeast cells. Centrifuge at 3000 g at 4°C for 5 minutes and discard the supernatant. Add 25 mL of sterile water to resuspend the yeast cells. Centrifuge at 3000 g at 4°C for 5 minutes and discard the supernatant. Add 1 mL of pre-chilled sorbitol to resuspend the yeast cells. Centrifuge at 3000 g at 4°C for 5 minutes and discard the supernatant. Add 100 μL of pre-chilled sorbitol to resuspend the yeast cells and aliquot the culture medium into 80 μL tubes.

[0053] 3. Pichia pastoris transformation:

[0054] Add 10 μL of the prepared linearized recombinant humanized type I collagen plasmid to the Pichia pastoris competent culture, mix thoroughly, and transfer to a pre-chilled electroporation cuvette. Incubate on ice for at least 5 minutes. Wipe the outer wall of the cuvette dry and place it in an electroporator. After electroporation, add 1 mL of sorbitol to suspend the cells. Transfer the liquid to a 1.5 mL centrifuge tube and incubate at 28°C for 3–12 hours. Afterward, spread the plate onto a MD plate and incubate at 28°C until colonies form.

[0055] 4. Verification of expression of recombinant humanized type I collagen gene engineering strain:

[0056] Pick a single colony and culture in 10 mL of BMGY medium at 28°C and 220 rpm to an OD600 of 2-6. Centrifuge at 3000 g for 5 min. Resuspend the yeast cells in 10 mL of BMMY medium to an OD600 of 1. Continue incubating at 28°C and 220 rpm, adding methanol every 24 hours to a final concentration of 0.5%. After 72 hours of induction, centrifuge and remove the supernatant for SDS-PAGE analysis.

[0057] The results of SDS-PAGE electrophoresis of the recombinant humanized type I collagen produced by the present invention are as follows: Figure 3 shown. Figure 3 The SDS-PAGE electrophoresis diagram of recombinant type I humanized collagen protein shake flask expression (M: Marker; 1-2: recombinant type I humanized collagen protein Pichia pastoris genetic engineering bacteria shake flask expression verification, 3: negative control). Figure 3 It can be seen that the novel recombinant humanized type I collagen amino acid sequence provided by the present invention was successfully expressed and secreted in the Pichia pastoris expression system.

[0058] Example 3. Preparation of recombinant humanized type I collagen

[0059] 1. Fermentation of recombinant humanized type I collagen:

[0060] The recombinant humanized collagen type I expression strain prepared in Example 2 was inoculated into YPD medium and cultured at 28°C and 220 rpm until the seed solution concentration reached OD600 = 15-25, and no foreign bacteria were found under microscopic examination. The seed solution was inoculated into a 7.5 L fermenter (containing 3 L BSM medium) at a 5% inoculum. The initial culture conditions were: a rotation speed of 100 rpm, a tank pressure of 0.05 MPa, a pH of 5.2, and a temperature of 30°C. The dissolved oxygen content was controlled to be above 30% by adjusting the air flow and stirring speed.

[0061] When the carbon source in the initial fermentation medium is exhausted, the dissolved oxygen rises suddenly, and 50% glycerol is added to supplement the carbon source. When the carbon source (50% glycerol) is exhausted, the glycerol feeding is stopped, and methanol is added to start induction, and the dissolved oxygen (DO) is maintained above 5% by adjusting the rotation speed, air flow rate and methanol flow rate. The fermentation supernatant is taken at 0h, 24h, 48h, 72h, and 88h of induction for SDS-PAGE electrophoresis detection. The SDS-PAGE electrophoresis detection results of the fermentation supernatant of the recombinant type I humanized collagen prepared by the present invention are shown as follows: Figure 4 shown. Figure 4 This is the SDS-PAGE electrophoresis detection result of the fermentation supernatant of recombinant humanized type I collagen (M: Marker, 1-5: SDS-PAGE electrophoresis diagrams after 0h, 24h, 48h, 72h, and 88h of induction, respectively).

[0062] 2. Purification of recombinant humanized type I collagen:

[0063] After 88 hours of induction, the fermentation was terminated to obtain a fermentation product, which was centrifuged at 10°C and 9000 rpm for 20 minutes to obtain a fermentation filtrate. The fermentation filtrate was used to capture the protein using a cation exchange medium, and the recombinant humanized type I collagen was eluted with an eluent (10 mmol / LPB buffer, pH 8.0). The purified recombinant humanized type I collagen was detected by SDS-PAGE electrophoresis.

[0064] The results of SDS-PAGE electrophoresis of the purified recombinant humanized type I collagen of the present invention are as follows: Figure 5 . Figure 5 The figure shows the SDS-PAGE electrophoresis results of the purified recombinant humanized type I collagen (1: recombinant humanized type I collagen; M: Marker). Figure 5 It can be seen that the electrophoretic purity of the recombinant humanized type I collagen prepared in the present invention is 91.1%.

[0065] Example 4: Solubility test of recombinant humanized type I collagen

[0066] 1. Experimental methods:

[0067] The recombinant humanized type I collagen prepared in Example 3 was dissolved in 15 mL of PBS (pH 7.4) solvent to prepare recombinant humanized type I collagen solutions with concentrations of 10 g / L, 15 g / L, 25 g / L, 50 g / L, and 100 g / L, and the dissolution was observed within 30 minutes.

[0068] 2. Experimental results:

[0069] The experimental results are shown in Table 1.

[0070] Table 1 Solubility test results of recombinant humanized type I collagen

[0071] Serial number Recombinant humanized type I collagen concentration (g / L) Can it be completely dissolved 1 10 able 2 15 able 3 25 able 4 50 able 5 100 able

[0072] As shown in Table 1, the recombinant humanized type I collagen prepared in the present invention has strong solubility, and its solubility in saline solution can reach 100 g / L.

[0073] Example 5: Evaluation of cell proliferation activity of recombinant humanized type I collagen

[0074] 1. Experimental methods:

[0075] 1.1. Grouping:

[0076] The positive control group (PC) was the bovine type I collagen standard of the China National Institute for Food and Drug Control, with a detection concentration of 1 mg / mL. The experimental sample group was the recombinant type I humanized collagen prepared in Example 3 of the present invention, with detection concentrations including 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL.

[0077] 1.2. Cell proliferation rate detection:

[0078] Each group of samples was prepared with DMEM medium, added to a 96-well plate, and refrigerated for 24 hours. The medium in the wells was discarded, and well-growing human embryonic skin fibroblast (ESF) cells were obtained, digested and counted, and the cell density was adjusted before inoculation into a 96-well plate and cultured for 24 hours. The cells were observed under a microscope (100 μm) and photographed. 76 μL of CCK-8 working solution was added, incubated in the dark for 1 hour, and shaken for 30 minutes. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader, and the cell proliferation rate was calculated.

[0079] The cell proliferation rate was calculated as follows:

[0080] Cell proliferation rate = OD 450e / OD 450b (OD 450e is the absorbance of the experimental sample group and the positive control group; OD 450b is the absorbance of the blank control group).

[0081] 2. Experimental results:

[0082] The experimental results are shown in Table 2 and Figures 6 and 7 shown.

[0083] 2.1. The results of the test on the proliferation rate of ESF cells by recombinant humanized type I collagen are shown in Table 2:

[0084] Table 2 Results of the proliferation rate test of ESF cells by recombinant humanized type I collagen

[0085] sample Growth rate (%) Remark NC 100％ / PC (1 mg / mL bovine type I collagen standard) 105.91％ / 0.015625 mg / mL recombinant humanized type I collagen 100.67％ The added value rate is 0.95 times that of PC 0.03125mg / mL recombinant humanized type I collagen 113.14％ The added value rate is 1.07 times that of PC 0.0625mg / mL recombinant humanized type I collagen 117.07％ The added value rate is 1.11 times that of PC 0.125mg / mL recombinant humanized type I collagen 121.32％ The added value rate is 1.15 times that of PC 0.25mg / mL recombinant humanized type I collagen 120.19％ The added value rate is 1.13 times that of PC 0.5mg / mL recombinant humanized type I collagen 117.76％ The added value rate is 1.11 times that of PC 1mg / mL recombinant humanized type I collagen 116.84％ The added value rate is 1.10 times that of PC 2mg / mL recombinant humanized type I collagen 115.53％ The added value rate is 1.09 times that of PC

[0086] 2.2. The results of the effect of recombinant humanized type I collagen on the proliferation rate of ESF cells are as follows Figure 6 As shown:

[0087] Figure 6 The graph shows the proliferation rate of ESF cells induced by recombinant humanized type I collagen (NC is the blank control, PC is the bovine type I collagen standard).

[0088] 2.3. Effect of recombinant humanized type I collagen on the proliferation rate of ESF cells and observation of cell status Figure 7 As shown:

[0089] Figure 7 This is a graph showing the effect of recombinant humanized type I collagen on the proliferation rate of ESF cells and the observation of cell status.

[0090] From Table 2 and Figure 6 It can be seen that the cell proliferation rate of ESF cells of the recombinant humanized type I collagen prepared by the present invention at concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL and 0.03125 mg / mL is higher than that of the blank control group (NC) and the positive control group (PC), and the cell proliferation rate of 0.125 mg / mL recombinant humanized type I collagen is 1.15 times that of the 1 mg / mL bovine type I collagen standard.

[0091] from Figure 7 It can be seen from the cell number that the recombinant humanized type I collagen prepared by the present invention has a proliferation-promoting effect on ESF cells at concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL and 0.03125 mg / mL.

[0092] Example 6. Evaluation of cell adhesion activity of recombinant humanized type I collagen

[0093] 1. Experimental methods:

[0094] 1.1. Grouping:

[0095] The reference product was bovine type I collagen standard product of the China Food and Drug Administration, with a sample concentration of 2 mg / mL; the positive control group was sigma gelatin, with a sample concentration of 0.1%; the experimental sample group was the recombinant type I humanized collagen prepared in Example 3 of the present invention, with sample concentrations of 0.25 mg / mL, 1 mg / mL, and 4 mg / mL.

[0096] 1.2. Cell adhesion rate detection:

[0097] Sample coating: Add sample solution to a 96-well plate and incubate at 37°C for 1 hour. Wash once with PBS. Block with 1% BSA at 37°C for 1 hour and wash twice with PBS.

[0098] Cell plating, centrifugation and photography: Mouse skin fibroblasts (L929) were digested; resuspended and counted, and the cell density was adjusted to 1×10 5cells / mL, and add Hoechst 33342. Immediately add 100 μL to each well of a 96-well plate. Incubate the cells at 37°C for 1 hour. After filling with medium, seal the plate with sealing film, invert the plate, and centrifuge at 350g for 5 minutes. Wash with PBS.

[0099] The calculation formula of cell adhesion rate V is as follows:

[0100] Cell adhesion rate V: V = number of cells after centrifugation / number of cells before centrifugation × 100%

[0101] The relative cell adhesion P was calculated as follows:

[0102] Relative cell adhesion P: P = average adhesion rate of each replicate well of the experimental group samples / average adhesion rate of each replicate well of the negative control × 100%

[0103] 2. Experimental results:

[0104] The experimental results are shown in Table 3 and Figures 8 to 10 shown.

[0105] 2.1. The results of the adhesion rate test of recombinant humanized type I collagen to L929 cells are shown in Table 3:

[0106] Table 3 Adhesion rate and relative adhesion test results of recombinant humanized type I collagen to L929 cells

[0107]

[0108] 2.2. The results of the adhesion rate and relative cell adhesion of recombinant humanized type I collagen to L929 cells are as follows Figures 8 and 9 As shown:

[0109] Figure 8 The figure shows the adhesion rate of recombinant humanized type I collagen to L929 cells (NC is the blank control, and the reference product is the bovine type I collagen standard). Figure 9 The figure shows the relative adhesion of recombinant humanized type I collagen to L929 cells (NC is the blank control, and the reference product is the bovine type I collagen standard).

[0110] 2.3. The results of cell adhesion of recombinant humanized type I collagen to L929 cells are as follows Figure 10 As shown:

[0111] Figure 10 The figure shows the cell adhesion results of recombinant humanized type I collagen to L929 cells (NC is blank control, reference product is bovine type I collagen standard, PC is 0.1% gelatin).

[0112] From Table 3 and Figures 8 and 9It can be seen that the adhesion rates of the recombinant humanized type I collagen prepared by the present invention to L929 cells at concentrations of 4 mg / mL, 1 mg / mL and 0.25 mg / mL are significantly different from those of the blank group, and the cell adhesion rate of 4 mg / mL recombinant humanized type I collagen is 2.66 times that of the 2 mg / mL bovine type I collagen standard.

[0113] from Figure 10 It can be seen that some L929 cells in the blank control group adhered to the wall and stretched; cells in the 1 mg / mL reference product (bovine type I collagen standard) sample group did not adhere to the wall or stretch; cells in the 4 mg / mL, 1 mg / mL and 0.25 mg / mL recombinant type I humanized collagen sample groups all showed obvious adhesion and stretching.

[0114] The above are only further embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which fall within the scope of protection of the present invention.

Claims

1. A recombinant humanized type I collagen, characterized in that: The amino acid sequence of the recombinant humanized type I collagen is shown in SEQ ID NO:

1.

2. A recombinant humanized type I collagen encoding gene, characterized in that: The recombinant humanized type I collagen encoding gene encodes the recombinant humanized type I collagen according to claim 1.

3. The recombinant humanized type I collagen encoding gene according to claim 2, wherein: The nucleotide sequence of the recombinant humanized type I collagen encoding gene is shown in SEQ ID NO:

2.

4. A recombinant humanized type I collagen recombinant vector, characterized in that: The recombinant vector comprises the recombinant humanized type I collagen encoding gene according to claim 2 or 3.

5. An engineered strain, characterized in that The engineered strain comprises the recombinant humanized type I collagen encoding gene according to claim 2 or 3 or the recombinant humanized type I collagen recombinant vector according to claim 4.

6. Use of the recombinant humanized type I collagen according to claim 1 in the preparation of cosmetics or medical devices.

7. The use according to claim 6, characterized in that The application of the recombinant humanized type I collagen in the preparation of anti-aging cosmetics or repair cosmetics.

8. The use according to claim 6, characterized in that The recombinant humanized type I collagen is used in the preparation of a collagen dressing that promotes the proliferation and adhesion of skin fibroblasts.

9. A composition, characterized in that The composition comprises the recombinant humanized type I collagen according to claim 1.

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