Recombinant III-type humanized collagen and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant III-type humanized collagen and application thereof. Area fragments with good hydrophilicity in a human III-type collagen alpha chain are selected and sequentially connected to serve as expression protein, the amino acid sequence is 100% identical to that of a corresponding part of natural human III-type collagen, and immunological rejection cannot be generated when the human III-type collagen is applied to a human body. According to the invention, the screened new III-type humanized collagen sequence is transferred into pichia pastoris for recombinant expression, the prepared recombinant III-type humanized collagen has the advantages of strong stability, high expression quantity and good hydrophilicity, and meanwhile, the recombinant III-type humanized collagen also has obvious effects of promoting cell proliferation and cell adhesion.

Description

Technical Field

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

[0002] Collagen is a family of proteins that contains many types. It is the most abundant type of protein in the human body, accounting for 25-30% of the total protein. Collagen is distributed in the skin, tendons, cornea, cartilage, blood vessels and other parts, and can play a certain structural support role, thereby maintaining the normal morphology of various organs and tissues. Common collagens include type I, type III, type XVII, etc. Among them, type III collagen is composed of three α1 chains and is a type of fibrous collagen. It is most abundant in infants and young children, and then it decreases with age.

[0003] At present, the main sources of collagen include extraction from animal tissues and preparation using DNA recombinant technology. Collagen can be extracted from animal tissues by acid and alkali hydrolysis, but the collagen extracted from animal tissues contains a lot of collagen fibers that are difficult to dissolve in water, and the extraction process will inevitably cause the loss of some biological activity of collagen; in addition, collagen extracted from animal tissues is a heterologous protein, which may cause risks such as immune rejection, allergies, and diseases.

[0004] The development of recombinant technology has enabled the production of type III collagen with high purity and specificity without the use of animal sources, laying a solid foundation for its application in the fields of skin care products and pharmaceuticals. For example, Cai Size published a paper titled "Recombinant Expression Optimization and Property Research of Human Type III Collagen in Pichia Pastoris". The paper intercepted the amino acids 908 to 1137 of the α1 chain of human type III collagen as the expression protein, and recombinantly expressed it in Pichia pastoris to produce recombinant humanized collagen. This method uses DNA recombinant technology to select full-length or partial amino acid sequence fragments encoded by specific types of human collagen genes, or a combination of human collagen functional fragments to prepare recombinant humanized collagen, which overcomes the shortcomings of heterologous proteins.

[0005] Patent document CN114085284A discloses a recombinant type III humanized collagen, nucleic acid, carrier and implant. The recombinant type III humanized collagen is a peptide sequence 549-560aa, 597-613aa, 881-902aa, 648-662aa obtained through long-term screening from the α1 chain of natural human type III collagen, and the four peptide chains are spliced ​​in sequence to form monomers, and then the monomers are expressed in series. After fermentation and purification, the obtained recombinant type III humanized collagen has high stability, which effectively solves the problem of degradation of recombinant type III humanized collagen during clinical use, and can be prepared into a liquid preparation for storage and use.

[0006] Patent document CN117264047A discloses a recombinant type III humanized collagen amino acid sequence and its preparation method and application. The amino acid sequence includes n repeated sequences, each of which is directly connected. The engineered Escherichia coli high-density fermentation technology is used to efficiently express the recombinant type III humanized collagen; then the recombinant type III humanized collagen is modified with PEG of uniform molecular weight, and the obtained recombinant type III humanized collagen has the advantages of good hydrophilicity, high stability and good biocompatibility.

[0007] However, the recombinant collagen currently prepared by genetic engineering methods still has problems such as low expression level, low solubility or low activity of recombinant collagen. Summary of the invention

[0008] In order to solve the defects of the prior art, the present invention provides a recombinant type III humanized collagen and its application. The present invention designs a new type III humanized collagen sequence to ensure that the recombinant type III humanized collagen product has high stability and good solubility. The present invention transfers the screened new type III humanized collagen sequence to a Pichia yeast strain for recombinant expression, and the obtained recombinant type III humanized collagen has the advantages of strong stability, high expression level and good hydrophilicity. At the same time, the recombinant type III humanized collagen also has a significant effect of promoting cell proliferation and cell adhesion.

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

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

[0011] Furthermore, the present invention provides a gene encoding a recombinant humanized type III collagen, and the nucleotide sequence of the gene encoding the recombinant humanized type III collagen is shown in SEQ ID NO:2.

[0012] Furthermore, the present invention also provides a recombinant humanized type III collagen recombinant vector, wherein the recombinant vector comprises the coding gene of the above-mentioned recombinant humanized type III collagen.

[0013] Furthermore, the present invention also provides an engineered strain, which comprises the coding gene of the above-mentioned recombinant humanized type III collagen or the above-mentioned recombinant vector of recombinant humanized type III collagen.

[0014] In addition, the present invention also provides the use of the recombinant type III humanized collagen in the preparation of cosmetics or medical device products.

[0015] Furthermore, the recombinant humanized type III collagen is used in the preparation of anti-aging cosmetics, repair cosmetics or antioxidant cosmetics.

[0016] Furthermore, the recombinant humanized type III collagen is used in the preparation of a collagen dressing that promotes cell proliferation, adhesion and migration.

[0017] In addition, the present invention also provides a composition, which comprises the above-mentioned recombinant humanized type III collagen and an acceptable excipient.

[0018] The recombinant type III humanized collagen provided by the present invention is obtained by selecting the hydrophilic region fragments in the α chain of human type III collagen, connecting them in sequence, and preparing the recombinant type III humanized collagen as shown in SEQ ID NO.1. The recombinant type III humanized collagen prepared by the present invention has the advantages of strong stability, high expression level, and good hydrophilicity, and at the same time, it also has the significant effect of promoting cell proliferation and promoting cell adhesion. In addition, the amino acid sequence of the recombinant type III humanized collagen provided by the present invention is 100% identical to the corresponding part of natural human type III collagen, and will not cause immune rejection when applied to the human body.

[0019] In summary, compared with the prior art, the recombinant humanized type III collagen provided by the present invention has the following advantages:

[0020] (1) The amino acid sequence of the recombinant humanized type III collagen provided by the present invention is 100% identical to the corresponding part of natural human type III collagen, and will not cause immune rejection when applied to the human body.

[0021] (2) The recombinant type III humanized 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 relatively high, reaching 12.04 g / L.

[0022] (3) The recombinant humanized type III collagen provided by the present invention has strong solubility, and its solubility in saline solution can reach 100 g / L.

[0023] (4) The recombinant humanized type III 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 0.0625 mg / mL of recombinant humanized type III collagen is 122.01%, which is 1.17 times that of the 1 mg / mL bovine type I collagen standard.

[0024] (5) The recombinant humanized type III collagen provided by the present invention has a significant effect on promoting the adhesion of L929 cells. The cell adhesion rate of the recombinant humanized type III collagen at a concentration of 1 mg / mL is 82.01%, which is 2.80 times that of the 2 mg / mL bovine type I collagen standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the results of hydrophobicity analysis of recombinant type III humanized collagen.

[0026] Figure 2 Schematic diagram of the construction of recombinant type III humanized collagen plasmid pPICZαA-hcc2.

[0027] Figure 3 This is the SDS-PAGE electrophoresis diagram of recombinant type III humanized collagen expression in shake flasks (M: Marker; 1: negative control, 2: verification of recombinant type III humanized collagen expression in Pichia pastoris genetically engineered bacteria shake flasks).

[0028] Figure 4 This is the HPLC test result of the fermentation supernatant of recombinant type III humanized collagen.

[0029] Figure 5 The figure is the SDS-PAGE electrophoresis result of the purified recombinant type III humanized collagen, (1: recombinant type III humanized collagen; M: Marker).

[0030] Figure 6 This is a graph showing the HPLC detection results of the purified recombinant humanized type III collagen.

[0031] Figure 7 This is a graph showing the effect of recombinant humanized type III collagen on the proliferation rate of ESF cells (NC is a blank control, PC is a bovine type I collagen standard).

[0032] Figure 8 This is a graph showing the effect of recombinant humanized type III collagen on the proliferation rate of ESF cells and the results of observing the cell status.

[0033] Fig. 9 This is a graph showing the adhesion rate of recombinant humanized type III collagen to L929 cells (NC is a blank control, and the reference product is a bovine type I collagen standard product).

[0034] Fig.10 This is a graph showing the relative adhesion of recombinant humanized type III collagen to L929 cells (NC is a blank control, and the reference product is a bovine type I collagen standard product).

[0035] Fig.11 The figure is the result of cell adhesion of recombinant humanized type III 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 a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention. The experimental methods used in the following examples are 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 III collagen gene

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

[0039] The hydrophobicity of the α chain of human type III 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 III 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 III humanized collagen prepared by the present invention were as follows: Figure 1 As shown. Figure 1 It can be seen that the amino acid sequence of the recombinant type III humanized collagen prepared by the present invention has good hydrophilicity.

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

[0041] GARGNDGARGSDGQPGPPGPPGTAGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQG ESGRPGPPGSGPRGQPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGGAGPPGPEGGKGAAGPPGPPGAAGTPGMP GERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGAPGQNGEPGGKGERGAPGEKGEGGPPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGTAGEPGRDGNPGSDGLPGRDGSPGG KGDRGENGSPGAPGAPGHPGPPGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAP.

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

[0043] The amino acid sequence of the above-mentioned recombinant type III humanized collagen was designed to correspond to the nucleotide sequence according to the codon preference of Pichia pastoris, and the nucleotide sequence of the recombinant type III humanized collagen was optimized according to the codon preference of Pichia pastoris to obtain the nucleotide sequence of the recombinant type III humanized collagen as shown in SEQ ID NO. 2. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize it.

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

[0045]

[0046] 3. Construction of recombinant plasmid:

[0047] The nucleotide sequence of the above-synthesized recombinant type III humanized collagen was constructed into the pPICZαA vector to obtain a recombinant type III humanized collagen plasmid named pPICZαA-hcc2. The schematic diagram of the recombinant type III humanized collagen plasmid pPICZαA-hcc2 constructed by the present invention is as follows: Figure 2 shown.

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

[0049] 1. Preparation of linearized plasmid:

[0050] The plasmid was linearized by a single enzyme digestion method, and the recombinant type III humanized collagen plasmid pPICZαA-hcc2 prepared in Example 1 was extracted, digested with SacI enzyme, and then recovered by ethanol precipitation to obtain the recombinant type III humanized collagen linearized plasmid.

[0051] 2. Preparation of competent yeast:

[0052] Inoculate Pichia pastoris strain X-33 into YPD liquid medium, culture overnight at 28°C and 220rpm, and measure the OD600 value of the bacterial solution. Inoculate into 50mL YPD liquid medium according to the initial OD600=0.5 and culture until the OD600 is 1.3-1.5. Centrifuge at 4°C and 3000g for 5min and discard the medium. Add 50mL sterile water to resuspend the yeast cells, centrifuge at 4°C and 3000g for 5min and discard the supernatant. Add 25mL sterile water to resuspend the yeast cells, centrifuge at 4°C and 3000g for 5min and discard the supernatant. Add 1mL pre-cooled sorbitol to resuspend the yeast cells, centrifuge at 4°C and 3000g for 5min and discard the supernatant. Add 100μL pre-cooled sorbitol to resuspend the yeast cells, and dispense the bacterial solution into 80μL / tubes.

[0053] 3. Pichia pastoris transformation:

[0054] Take 10 μL of the prepared recombinant type III humanized collagen linearized plasmid and add it to the Pichia competent medium, mix well, and transfer it to the pre-cooled electroporation cup on ice, and place it on ice for more than 5 minutes. Wipe the outer wall of the electroporation cup dry, put it into the electroporator, add 1 mL of sorbitol to suspend the bacteria, and then transfer the liquid to a 1.5 mL centrifuge tube, incubate it at 28°C for 3 to 12 hours, then spread it on a YPD plate containing 0.1 mg / mL bleomycin, and incubate it at 28°C until colonies grow.

[0055] 4. Expression verification of recombinant humanized type III collagen gene engineering strain:

[0056] Pick a single colony in 10 mL of BMGY medium and culture at 28°C and 220 rpm until OD600 = 2-6. Centrifuge at 3000g for 5 min, resuspend the yeast cells in 10 mL of BMMY medium until OD600 = 1, continue to culture at 28°C and 220 rpm, and add methanol every 24 hours to a final concentration of 0.5%. After 72 hours of induction, centrifuge and take the supernatant, and send it to SDS-PAGE for electrophoresis detection.

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

[0058] Example 3: Preparation of recombinant humanized type III collagen

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

[0060] The recombinant type III humanized collagen expression strain prepared in Example 2 was inoculated into YPD medium and cultured at 28°C and 220rpm until the seed solution concentration OD600 was 15-25, and no foreign bacteria were found under microscopic examination. The seed solution was inoculated into a 7.5L fermenter (containing 3LBSM medium) at an inoculum of 5%, and the initial culture conditions were: a rotation speed of 100rpm, a tank pressure of 0.05MPa, a pH of 5.2, and a temperature of 30°C. The dissolved oxygen 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 feed 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. After 88 hours of induction, the fermentation supernatant is taken for HPLC detection. The HPLC detection results of the fermentation supernatant of the recombinant type III humanized collagen prepared by the present invention are as follows: Figure 4 shown.

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

[0063] The fermentation was terminated after 88 hours of induction 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 type III humanized collagen was eluted with an eluent (10 mmol / LPB buffer, pH 8.0). The purified recombinant type III humanized collagen was subjected to SDS-PAGE electrophoresis and HPLC detection.

[0064] The results of SDS-PAGE electrophoresis and HPLC detection of the purified recombinant type III humanized collagen of the present invention are as follows: Figure 5 and Figure 6 shown. Figure 5 The figure is the SDS-PAGE electrophoresis result of the purified recombinant type III humanized collagen, (1: recombinant type III humanized collagen; M: Marker). Figure 6 The figure is the HPLC test result of the purified recombinant humanized type III collagen. Figure 5 and Figure 6 It can be seen that the recombinant type III humanized collagen purified by the present invention has a single band and high purity.

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

[0066] 1. Experimental methods:

[0067] The recombinant humanized type III collagen prepared in Example 3 was dissolved in 15 mL of PBS (pH 7.4) solvent to prepare recombinant humanized type III 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 min.

[0068] 2. Experimental results:

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

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

[0071] Serial number Concentration of recombinant humanized type III collagen (g / L) Is it completely dissolved? 1 10 yes 2 15 yes 3 25 yes 4 50 yes 5 100 yes

[0072] As can be seen from Table 1, the recombinant type III humanized 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 III 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 Food and Drug Administration, and the detection concentration was 1 mg / mL. The experimental sample group was the recombinant type III humanized collagen prepared in Example 3 of the present invention, and the detection concentrations included 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 culture medium, added to a 96-well plate, and refrigerated for 24 hours; the culture medium in the wells was discarded, and human embryonic skin fibroblast (ESF) cells in good growth state were taken, digested and counted, and inoculated into a 96-well plate after adjusting the cell density, 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, cultured in the dark for 1 hour, and shaken for 30 minutes; the absorbance value of each well at a wavelength of 450 nm was measured in an ELISA instrument, 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 Figure 7-8 shown.

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

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

[0085] sample Proliferation rate (%) Remark NC 100％ / PC (1mg / mL bovine type I collagen standard) 104.04％ / 0.015625mg / mL recombinant humanized type III collagen 110.28％ The added value rate is 1.06 times that of PC 0.03125mg / mL recombinant humanized type III collagen 111.23％ The added value rate is 1.07 times that of PC 0.0625mg / mL recombinant humanized type III collagen 122.01％ The added value rate is 1.17 times that of PC 0.125mg / mL recombinant humanized type III collagen 118.07％ The added value rate is 1.13 times that of PC 0.25mg / mL recombinant humanized type III collagen 117.13％ The added value rate is 1.13 times that of PC 0.5mg / mL recombinant humanized type III collagen 117.44％ The added value rate is 1.13 times that of PC 1mg / mL recombinant humanized type III collagen 116.36％ The added value rate is 1.12 times that of PC 2mg / mL recombinant humanized type III collagen 120.92％ The added value rate is 1.16 times that of PC

[0086] 2.2. Effect of recombinant humanized type III collagen on the proliferation rate of ESF cells Figure 7 As shown:

[0087] Figure 7 This is the result graph of ESF cell proliferation rate of recombinant humanized type III collagen (NC is blank control, PC is bovine type I collagen standard).

[0088] 2.3. Effect of recombinant humanized type III collagen on the proliferation rate of ESF cells and observation of cell status Figure 8 As shown:

[0089] Figure 8 This is a graph showing the effect of recombinant humanized type III collagen on the proliferation rate of ESF cells and the results of observing the cell status.

[0090] From Table 2 and Figure 7 It can be seen that the cell proliferation rate of ESF cells of the recombinant humanized type III 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, 0.03125 mg / mL and 0.015625 mg / mL is higher than that of the blank control group (NC) and the positive control group (PC), and the cell proliferation rate of 0.0625 mg / mL recombinant humanized type III collagen is 1.17 times that of 1 mg / mL bovine type I collagen standard.

[0091] from Figure 8 It can be seen that from the cell number, it can be judged that the recombinant type III humanized 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, 0.03125 mg / mL and 0.015625 mg / mL.

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

[0093] 1. Experimental methods:

[0094] 1.1. Grouping:

[0095] The reference product was bovine type I collagen standard product of 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 III 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 96-well plate, incubate at 37℃ for 1h. Wash once with PBS. Block with 1% BSA at 37℃ for 1h, 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 1×105 cells / mL, and add Hoechst 33342, immediately add to 96-well plate, add 100μL to each well. Incubate cells at 37℃ for 1h. After filling with culture medium, seal with sealing film, turn the culture plate upside down and centrifuge at 350g for 5min. 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 sample / 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 9 to 11 shown.

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

[0106] Table 3 Results of adhesion rate and relative adhesion of recombinant humanized type III collagen to L929 cells

[0107]

[0108] 2.2. The results of the adhesion rate and relative cell adhesion of recombinant humanized type III collagen to L929 cells are as follows Figure 9-10 As shown:

[0109] Fig. 9 This is a graph showing the adhesion rate of recombinant humanized type III collagen to L929 cells (NC is a blank control, and the reference product is a bovine type I collagen standard product). Fig.10 This is a graph showing the relative adhesion of recombinant humanized type III collagen to L929 cells (NC is a blank control, and the reference product is a bovine type I collagen standard product).

[0110] 2.3. The results of cell adhesion of recombinant humanized type III collagen to L929 cells are as follows Fig.11 As shown:

[0111] Fig.11 The figure is the result of cell adhesion of recombinant humanized type III 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 Figure 9-10 It can be seen that the adhesion rates of the recombinant type III humanized 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 1 mg / mL recombinant type III humanized collagen is 2.80 times that of the 2 mg / mL bovine type I collagen standard.

[0113] from Fig.11 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 III humanized collagen sample groups all showed obvious adhesion and stretching.

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

Claims

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

1.

2. A gene encoding recombinant humanized type III collagen, characterized in that: The nucleotide sequence of the gene encoding the recombinant humanized type III collagen is shown in SEQ ID NO:

2.

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

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

5. Use of the recombinant humanized type III collagen as claimed in claim 1 in the preparation of cosmetics or medical device products.

6. The use according to claim 5, characterized in that The recombinant humanized type III collagen is used in the preparation of anti-aging cosmetics, repair cosmetics or antioxidant cosmetics.

7. The use according to claim 5, characterized in that The recombinant type III humanized collagen is used in the preparation of a collagen dressing capable of promoting cell proliferation, adhesion and migration.

8. A composition, characterized in that The composition comprises the recombinant humanized type III collagen according to claim 1 and an acceptable excipient.

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