Anti-aging recombinant collagen and its preparation method and application

Abstract

The present invention belongs to the field of cosmetics technology and discloses an anti-aging recombinant collagen protein, its preparation method, and application. The amino acid sequence of the anti-aging recombinant collagen protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The present invention constructs and expresses a recombinant collagen protein, CaI1, by recombining the effective sequences of human type I, type III, and type XVII collagen proteins. The recombinant collagen protein CaI1 is non-cytotoxic, free of endotoxin risks, and has excellent safety. Skin care products containing this recombinant collagen protein CaI1 can promote skin collagen absorption, inhibit skin aging, and improve skin elasticity, and have good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an anti-aging recombinant collagen and a preparation method and application thereof. Background Art

[0002] Collagen is the primary protein in the human extracellular matrix, accounting for one-third of human protein and three-quarters of the dry weight of skin. It maintains the structure and physiological functions of tissues and organs such as skin, cartilage, and internal organs. Currently, the most studied recombinant collagens are types I, II, and III, while other types of collagen are less studied. Type XV and II collagen are unique transmembrane proteins and key components of hemidesmosomes that connect epithelial cells to the basement membrane zone. They can improve the state of the basement membrane, achieve anti-aging effects on the skin, and play an important role in cell aging and skin differentiation.

[0003] At present, due to the low content of type XVII collagen in the human body and its complex molecular structure, direct extraction is very difficult and mass production is difficult to achieve. The extraction methods of type XVII collagen include acid extraction, enzyme extraction and neutral salt extraction. Existing type XVII collagen extraction methods are difficult to achieve low-cost large-scale production, and the stability of single-sequence type XVII collagen is poor, which limits its use conditions and effect. Therefore, how to prepare type XVII collagen without destroying the active ingredients of type XVII collagen and improving the extraction purity of collagen is a difficult problem that needs to be solved urgently. With the development of biotechnology, the production model of microbial directed production of collagen using DNA recombinant technology has been applied. Compared with collagen obtained by traditional extraction, it solves the problems of animal immunogenicity and viral risks, has low rejection reaction, and has the advantages of processability and stable quality. It has broad application prospects in the field of biomedical research.

[0004] Based on the above background, the present invention provides an anti-aging recombinant collagen and its preparation method and application, which has good safety and can be widely used in skin care products, biomaterials, artificial skin and other fields. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide an anti-aging recombinant collagen.

[0006] A second object of the present invention is to provide a method for preparing anti-aging recombinant collagen.

[0007] The third object of the present invention is to provide the use of the above-mentioned anti-aging recombinant collagen in the preparation of skin care products.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] An anti-aging recombinant collagen protein, the amino acid sequence of the anti-aging recombinant collagen protein is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO.2.

[0010] The present invention selects amino acids 261-410 and 573-677 of the functional region of natural type III collagen, and splices them end-to-end with the effective sequence of type XVII collagen, amino acids 594-700. The new collagen peptide is then modified with the amino acid sequence 1091-1114 of type I collagen at the end. By modifying the type I collagen at the end of the new collagen peptide, the hydrophilicity and stability of the recombinant collagen are improved. In addition, the recombinant collagen removes the full-length chain sequence at the C-terminus and N-terminus of the collagen coding region, effectively avoiding a series of antigenic immune reactions. The 386-amino acid recombinant protein structure is 100% identical to the corresponding protein sequences in native human collagens I, III, and XVII, and will not cause immune rejection when used in the human body.

[0011] The method for preparing the anti-aging recombinant collagen comprises the following steps:

[0012] (1) Connecting the nucleotide sequence encoding the anti-aging recombinant collagen to the vector to obtain a recombinant plasmid;

[0013] (2) The recombinant plasmid obtained in step (1) was transformed into competent cells, induced by IPTG, and bacterial precipitates were obtained;

[0014] (3) Separating and purifying the bacterial precipitate obtained in step (2) to obtain anti-aging recombinant collagen.

[0015] Furthermore, the specific operations of step (1) are:

[0016] S1. Splicing the effective sequences of human type I, type III, and type XVII collagen to obtain the amino acid sequence of recombinant collagen SEQ ID NO: 1;

[0017] S2. Reverse design the gene sequence based on the amino acid sequence of the recombinant collagen protein SEQ ID NO: 1, perform codon optimization, and obtain the nucleotide sequence encoding the recombinant collagen protein SEQ ID NO: 2;

[0018] S3. Based on the nucleotide sequence SEQ ID NO: 2 obtained in step S2, upstream and downstream primers for amplifying the corresponding nucleotide sequence of the recombinant collagen were designed, and the recombinant collagen sequence amplified by the primers was cloned into the pET30a vector to construct a recombinant plasmid.

[0019] Furthermore, the upstream primer sequence of step S3 is shown as SEQ ID NO.3; the downstream primer sequence is shown as SEQ ID NO.4.

[0020] Furthermore, the competent cells in step (2) are E. coli BL21 (DE3).

[0021] Furthermore, the concentration of IPTG-induced expression in step (2) is 0.3-0.5 mmol / L, and the time of IPTG-induced expression is 5-10 h.

[0022] The application of the above-mentioned anti-aging recombinant collagen in the preparation of skin care products.

[0023] Furthermore, the mass percentage of recombinant collagen in the skin care product is 5-8%.

[0024] The present invention has the following effects compared to the prior art:

[0025] The present invention constructs and expresses a recombinant collagen protein, Californium, by recombining effective sequences of human type I, type III, and type XVII collagen. Californium is non-cytotoxic, free of endotoxin risks, and exhibits excellent safety. Skin care products containing this recombinant collagen protein can promote collagen absorption, inhibit skin aging, and improve skin elasticity. The product is well-suited for industrial large-scale production and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the plasmid map of the recombinant plasmid pET30a-CaI1;

[0027] Figure 2 This is the expression diagram of the purified recombinant collagen Cal1 prepared by the present invention;

[0028] Figure 3 is the plasmid map of the recombinant plasmid pET30a-CaI2;

[0029] Figure 4 is the plasmid map of the recombinant plasmid pET30a-CaI3;

[0030] Figure 5 is the plasmid map of the recombinant plasmid pET30a-CaI4;

[0031] Figure 6 This is a graph showing the results of measuring the relative cell proliferation rate of the recombinant collagens CaI1, CaI2, CaI3, and CaI4 prepared in the present invention at 48 hours;

[0032] Figure 7 This is the percentage of wrinkle reduction of the skin care product prepared in the present invention. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0034] Example 1

[0035] An anti-aging recombinant collagen protein Cal1, the amino acid sequence of the anti-aging recombinant collagen protein Cal1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0036] The preparation method of the anti-aging recombinant collagen Cal1 specifically includes the following steps.

[0037] (1) The functional region sequences of natural type III collagen at amino acids 261-410 and 573-677 were selected and connected end-to-end with the effective sequence of type XVII collagen at amino acids 594-700 to form a new collagen peptide. The amino acid sequence of type I collagen at positions 1091-1114 was then used to modify the end of the new collagen peptide to form a new type III+XVII+III+I recombinant collagen sequence. The amino acid sequence of the obtained new type III+XVII+III+I recombinant collagen sequence is SEQ ID NO. 1, and is named CaI1.

[0038] (2) Based on the amino acid sequence of recombinant collagen protein Cal1, SEQ ID NO.1, the gene sequence was reverse-engineered and codon optimized to obtain the nucleotide sequence encoding recombinant collagen protein Cal1, SEQ ID NO.2. Based on the obtained nucleotide sequence of recombinant collagen protein Cal1, SEQ ID NO.2, upstream and downstream primers were designed to amplify the corresponding nucleotide sequence of recombinant collagen protein Cal1. The upstream primer sequence is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4. The Cal1 gene was amplified by PCR, and the target fragment was purified using a gel recovery kit.

[0039] (3) The plasmid pET30a was double-digested with restriction endonucleases Nde I and Hind Ⅲ. The double-digestion reaction system is shown in Table 1. The recombinant collagen CaI1 gene fragment recovered after amplification was ligated with the target fragment of the expression vector pET30a using T4 DNA ligase. The T4 DNA ligase reaction system is shown in Table 2. The constructed recombinant plasmid pET30a-CaI1 was transformed into E. coli DH5α competent cells and cultured on LB plates containing 100 μg / mL kanamycin. Monoclonal colonies were picked for bacterial liquid PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pET30a-CaI1. The map of the recombinant plasmid pET30a-CaI1 is shown in Figure 1 shown.

[0040] Table 1 Double enzyme digestion reaction system

[0041]

[0042] Table 2 T4 DNA ligase reaction system

[0043]

[0044] (4) The recombinant plasmid pET30a-CeI1 constructed in step (3) was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing 100 μg / mL kanamycin, and incubated inverted at 37°C overnight to obtain a recombinant strain named pET30a-CeI1 / BL21.

[0045] (5) Select positive monoclonal colonies and inoculate them into LB culture medium containing 100 μg / mL kanamycin. Culture them at 37°C with shaking overnight, and add 0.4 mmol / L IPTG to induce the expression of recombinant collagen CaI1 for 7 hours to obtain bacterial precipitates.

[0046] (6) The recombinant collagen protein CaI1 was purified by affinity chromatography to obtain the recombinant collagen protein CaI1. The purified recombinant collagen protein CaI1 was detected by SDS-PAGE gel. The results are as follows: Figure 2 As shown, M represents protein marker, and band 1 represents the purified recombinant collagen CaI1.

[0047] The obtained recombinant collagen protein CaI1 was tested for endotoxin levels using Limulus amebocyte lysate reagent, and the bacterial endotoxin content was 2.36 (EU / mL), which is less than 10 (EU / mL), indicating that the recombinant collagen protein CaI1 is highly safe and can be used in the field of skin care.

[0048] Comparative Example 1

[0049] An anti-aging recombinant collagen protein Cal12, the amino acid sequence of the anti-aging recombinant collagen protein Cal12 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0050] The preparation method of the above-mentioned anti-aging recombinant collagen CaI2 specifically includes the following steps.

[0051] (1) The functional region sequences of natural type III collagen at amino acids 261-410 and 573-677 were selected and spliced ​​end-to-end with the effective sequence of type XVII collagen at amino acids 707-808 to form a new type III+XVII+III recombinant collagen. The recombinant collagen removed the full-length chain sequence at the C-terminus and N-terminus of the collagen coding region, effectively avoiding a series of antigenic immune reactions. The recombinant protein structure consisting of 357 amino acids is 100% identical to the corresponding protein sequences in the original human collagen XVII and human collagen III. The recombinant collagen is named CaI2 and its sequence is SEQ ID NO. 5.

[0052] (2) Based on the amino acid sequence of recombinant collagen protein CaI2, SEQ ID NO.5, the gene sequence was reverse-engineered and codon optimized to obtain the nucleotide sequence encoding recombinant collagen protein CaI1, SEQ ID NO.6. Based on the nucleotide sequence of recombinant collagen protein CaI2, SEQ ID NO.6, upstream and downstream primers were designed to amplify the corresponding nucleotide sequence of recombinant collagen protein CaI2. The upstream primer sequence is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8. The CaI2 gene was amplified by PCR, and the target fragment was purified using a gel recovery kit.

[0053] (3) The plasmid pET30a was double-digested with restriction endonucleases Nde I and Hind Ⅲ. The recombinant collagen CaI2 gene fragment recovered after amplification was ligated with the target fragment of the expression vector pET30a using T4 DNA ligase. The constructed recombinant plasmid pET30a-CaI2 was transformed into E. coli DH5α competent cells and cultured on LB plates containing 100 μg / mL kanamycin. Monoclonal colonies were picked for bacterial liquid PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pET30a-CaI2. The map of the recombinant plasmid pET30a-CaI2 is shown in Figure 2. Figure 3 shown.

[0054] (4) The recombinant plasmid pET30a-CeI2 constructed in step (3) was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing 100 μg / mL kanamycin, and incubated inverted at 37°C overnight to obtain a recombinant strain named pET30a-CaI2 / BL21.

[0055] (5) Select positive monoclonal colonies and inoculate them into LB culture medium containing 100 μg / mL kanamycin. Culture them at 37°C with shaking overnight, and add 0.4 mmol / L IPTG to induce the expression of recombinant collagen CaI2 for 7 hours to obtain bacterial precipitates.

[0056] (6) The recombinant collagen protein CaI2 was purified by affinity chromatography to obtain the recombinant collagen protein CaI2.

[0057] Comparative Example 2

[0058] An anti-aging recombinant collagen protein Cal13, the amino acid sequence of the anti-aging recombinant collagen protein Cal13 is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.

[0059] The preparation method of the above-mentioned anti-aging recombinant collagen CaI3 specifically includes the following steps.

[0060] (1) The functional region sequences of natural type III collagen at amino acids 261-410 and 573-677 were selected and spliced ​​end-to-end with the effective sequence of type I collagen at amino acids 428-538 to form a new type III+I+III recombinant collagen. The recombinant collagen removed the full-length chain sequence at the C-terminus and N-terminus of the collagen coding region. The recombinant protein structure consisting of 366 amino acids was 100% identical to the corresponding protein sequences in the original human collagen I and human collagen III. The type III+I+III recombinant collagen was named CaI3 and its sequence is SEQ ID NO.9.

[0061] (2) Based on the amino acid sequence of recombinant collagen protein CaI3, SEQ ID NO.9, the gene sequence was reverse-engineered and codon optimized to obtain the nucleotide sequence encoding recombinant collagen protein CaI1, SEQ ID NO.10. Based on the obtained nucleotide sequence of recombinant collagen protein CaI3, SEQ ID NO.10, upstream and downstream primers were designed to amplify the corresponding nucleotide sequence of recombinant collagen protein CaI3. The upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.12. The CaI3 gene was amplified by PCR, and the target fragment was purified using a gel recovery kit.

[0062] (3) The plasmid pET30a was double-digested with restriction endonucleases Nde I and Hind Ⅲ. The recombinant collagen CaI3 gene fragment recovered after amplification was ligated with the target fragment of the expression vector pET30a using T4 DNA ligase. The constructed recombinant plasmid pET30a-CaI3 was transformed into E. coli DH5α competent cells and cultured on LB plates containing 100 μg / mL kanamycin. Monoclonal colonies were picked for bacterial liquid PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pET30a-CaI3. The map of the recombinant plasmid pET30a-CaI3 is shown in Figure 2. Figure 4 shown.

[0063] (4) The recombinant plasmid pET30a-CaI3 constructed in step (3) was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing 100 μg / mL kanamycin, and incubated inverted at 37°C overnight to obtain a recombinant strain named pET30a-CaI3 / BL21.

[0064] (5) Select positive monoclonal colonies and inoculate them into LB culture medium containing 100 μg / mL kanamycin. Culture them at 37°C with shaking overnight, and add 0.4 mmol / L IPTG to induce the expression of recombinant collagen CaI3 for 7 hours to obtain bacterial precipitates.

[0065] (6) The recombinant collagen protein CaI3 was purified by affinity chromatography to obtain the recombinant collagen protein CaI3.

[0066] Comparative Example 3

[0067] Disclosed is an anti-aging recombinant collagen protein Cal14, the amino acid sequence of which is shown in SEQ ID NO.13, and the nucleotide sequence of which is shown in SEQ ID NO.14.

[0068] The preparation method of the above-mentioned anti-aging recombinant collagen CaI4 specifically includes the following steps.

[0069] (1) The functional region sequences of natural type III collagen at amino acids 261-410 and 573-677 were selected and spliced ​​end-to-end with the effective sequence of type XVII collagen at amino acids 594-700 to form a new type III+XVII+III recombinant collagen. The recombinant collagen removed the full-length chain sequence at the C-terminus and N-terminus of the collagen coding region, effectively avoiding a series of antigenic immune reactions. The recombinant protein structure consisting of 362 amino acids is 100% identical to the corresponding protein sequences in the original human collagen XVII and human collagen III. The recombinant collagen is named CaI4 and its sequence is SEQ ID NO. 13.

[0070] (2) Based on the amino acid sequence of recombinant collagen protein CaI4, SEQ ID NO. 13, the gene sequence was reverse-engineered and codon optimized to obtain the nucleotide sequence encoding recombinant collagen protein CaI4, SEQ ID NO. 14. Based on the obtained nucleotide sequence of recombinant collagen protein CaI4, SEQ ID NO. 14, upstream and downstream primers were designed to amplify the corresponding nucleotide sequence of recombinant collagen protein CaI4. The upstream primer sequence is shown in SEQ ID NO. 15, and the downstream primer sequence is shown in SEQ ID NO. 16. The CaI4 gene was amplified by PCR, and the target fragment was purified using a gel recovery kit.

[0071] (3) The plasmid pET30a was double-digested with restriction endonucleases Nde I and Hind Ⅲ. The recombinant collagen CaI4 gene fragment recovered after amplification was ligated with the target fragment of the expression vector pET30a using T4 DNA ligase. The constructed recombinant plasmid pET30a-CaI4 was transformed into E. coli DH5α competent cells and cultured on LB plates containing 100 μg / mL kanamycin. Monoclonal colonies were picked for bacterial liquid PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pET30a-CaI4. The map of the recombinant plasmid pET30a-CaI4 is shown in Figure 2. Figure 5 shown.

[0072] (4) The recombinant plasmid pET30a-CaI4 constructed in step (3) was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing 100 μg / mL kanamycin, and incubated inverted at 37°C overnight to obtain a recombinant strain named pET30a-CaI4 / BL21.

[0073] (5) Select positive monoclonal colonies and inoculate them into LB culture medium containing 100 μg / mL kanamycin. Culture them at 37°C with shaking overnight, and add 0.4 mmol / L IPTG to induce the expression of recombinant collagen CaI4 for 7 hours to obtain bacterial precipitates.

[0074] (6) The recombinant collagen protein CaI4 was purified by affinity chromatography to obtain the recombinant collagen protein CaI4.

[0075] Test Example 1

[0076] Evaluation of recombinant collagens CaI1, CaI2, CaI3, and CaI4 in promoting cell proliferation:

[0077] Fibroblast L929 cells were cultured in DMEM + double anti-PS medium containing 10% fetal bovine serum at 37°C and 5% CO2 in a cell culture incubator to ensure that the fibroblast L929 cells were in the logarithmic growth phase for biological activity assay. 3 The cells were seeded into 96-well culture plates at a density of 100 μL / mL, and each well was inoculated with 100 μL. The plates were then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h. The original culture medium was discarded, and 100 μL of cell culture medium diluted with different concentrations of recombinant collagen CaI1, CaI2, CaI3, and CaI4 (0.5 g / L, 1 g / L, 5 g / L) was added to each well, which were respectively set as Example 1 group, Comparative Example group, Comparative Example 2 group, and Comparative Example 3 group. At the same time, a negative control group (DMEM containing 10% fetal bovine serum + double anti-PS culture medium) was set, and 3 replicates were set for each well; the test was performed at 48 h, 50 μL of MTT was added to each well, and the cells were cultured at 37 ° C for 2 h. The original culture medium was discarded, and 150 μL of dimethyl sulfoxide was immediately added to each well, and the cells were placed at room temperature and gently shaken for 20 min; the light absorbance (A) of each well was measured at a wavelength of 490 nm using a microplate reader, and the relative cell proliferation rate RGR (%) = (absorption value of the experimental group / absorption value of the negative control group) × 100%. The toxicity of recombinant collagens CaI1, CaI2, CaI3, and CaI4 was evaluated based on the relative cell proliferation rate and divided into six levels. When RGR (%) ≥ 100, there was no cytotoxicity; when RGR (%) was 75-99, it represented very low cytotoxicity; when RGR (%) was 50-74, it represented low cytotoxicity; when RGR (%) was 25-49, it represented moderate cytotoxicity; when RGR (%) was 1-24, it represented high cytotoxicity; when RGR (%) = 0, it represented extremely high cytotoxicity or lethality. The results of the relative cell proliferation rate determination of recombinant collagens CaI1, CaI2, CaI3, and CaI4 at 48h are shown in the figure. Figure 6 shown.

[0078] Depend on Figure 6 It can be seen that the cell proliferation rate of the experimental group added with recombinant collagen Cal, Cal, Cal, and Cal4 was improved, indicating that the anti-aging recombinant collagen Cal, Cal, Cal, and Cal prepared by the present invention have the effect of promoting cell proliferation, are non-cytotoxic, and have good safety, and can be used for the preparation and use of skin care products.

[0079] Test Example 2

[0080] Efficacy testing of anti-aging recombinant collagen in skin care products:

[0081] A skin care product is composed of the following raw materials in percentage by weight: 5% recombinant collagen, 5% glycerol, 2% butylene glycol, 3% caprylic / capric triglyceride, 5% sodium polyacrylate, 2% potassium cetyl phosphate, and the balance is water; the recombinant collagen is the recombinant collagen CaI1, CaI2, CaI3, and CaI4 prepared in Example 1 and Comparative Examples 1-3, respectively.

[0082] The skin care products prepared by adding the recombinant collagen of Example 1 and Comparative Examples 1-3 were tested for their skin elasticity-enhancing and anti-wrinkle effects. The specific process is as follows:

[0083] 40 female testers, aged between 18 and 50 years old, were selected and randomly divided into 4 groups, with 10 people in each group. Before the test, the measurement area was marked on the face of the tester, and the experimental area was at least 2cm×2cm. Before the start of the test, the probe of the skin elasticity tester was vertically touched to the test area, and the test was started until the test cycle was completed, and then the probe was removed. Three different points were taken in the test area for measurement, and the corresponding average value of each group was obtained as the skin elasticity result before use; a VISIA facial image analyzer was used to take facial photos of the tester, and the detection data of wrinkles on the face of the tester was recorded. The absolute scores obtained were statistically analyzed, and the corresponding average value of each group was obtained as the wrinkle detection data before use. After the start of the test, the tester used the skin care products made from the anti-aging recombinant collagen of Example 1 and Comparative Examples 1-3 at a rate of 3mL / cm 2 The amount of the product was applied to the test area once in the morning and evening. After 28 days, the skin elasticity of the test area was measured again using a skin elasticity tester, and the average value of each group was obtained. The results are shown in Table 3. After 28 days, the anti-wrinkle effect was measured again using a VISIA facial image analyzer. The absolute scores obtained were statistically analyzed, and the percentage of wrinkle reduction after 28 days of use was calculated. The results are shown in Table 3. Figure 7 shown.

[0084] Table 3 Improvement of skin elasticity

[0085]

[0086] From Table 3 and Figure 7 As can be seen, compared with Comparative Examples 1-3, the skin care product prepared with the anti-aging recombinant collagen protein CaI1 prepared in Example 1 can significantly improve skin elasticity and significantly improve wrinkles. This shows that the skin care product prepared with the anti-aging recombinant collagen protein CaI1 prepared in the present invention can promote the absorption of collagen in the skin, significantly improve skin elasticity, and improve wrinkles.

[0087] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. An anti-aging recombinant collagen, characterized in that: The amino acid sequence of the anti-aging recombinant collagen is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The method for preparing the anti-aging recombinant collagen according to claim 1, characterized in that: The following steps are involved: (1) Connecting the nucleotide sequence encoding the anti-aging recombinant collagen to the vector to obtain a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) was transferred into competent cells, induced by IPTG, and bacterial precipitates were obtained; (3) Separating and purifying the bacterial precipitate obtained in step (2) to obtain anti-aging recombinant collagen.

3. The method for preparing the anti-aging recombinant collagen according to claim 2, characterized in that: The specific operations of step (1) are: S1. Splicing the effective sequences of human type I, type III, and type XVII collagen to obtain the amino acid sequence of recombinant collagen SEQ ID NO.1; S2. Reverse design the gene sequence based on the amino acid sequence of the recombinant collagen protein SEQ ID NO.1, perform codon optimization, and obtain the nucleotide sequence encoding the recombinant collagen protein SEQ ID NO.2; S3. Based on the nucleotide sequence SEQ ID NO.2 obtained in step S2, upstream and downstream primers are designed to amplify the corresponding nucleotide sequence of the recombinant collagen protein, and the nucleotide sequence of the recombinant collagen protein amplified by the primers is cloned into the pET30a vector to construct a recombinant plasmid.

4. The method for preparing the anti-aging recombinant collagen according to claim 3, characterized in that: The upstream primer sequence of step S3 is shown as SEQ ID NO.3; the downstream primer sequence is shown as SEQ ID NO.

4.

5. The method for preparing the anti-aging recombinant collagen according to claim 2, characterized in that: The competent cells in step (2) are E. coli BL21 (DE3).

6. The method for preparing the anti-aging recombinant collagen according to claim 2, characterized in that: The concentration of IPTG-induced expression in step (2) is 0.3-0.5 mmol / L, and the time of IPTG-induced expression is 5-10 h.

7. Use of the anti-aging recombinant collagen according to claim 1 in the preparation of skin care products.

8. Use of the anti-aging recombinant collagen according to claim 7 in the preparation of a skin care product, wherein the mass percentage of the recombinant collagen in the skin care product is 5-8%.

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