Recombinant type i & iii fusion collagen, and preparation method and application thereof

Abstract

The present application belongs to the field of genetic engineering and biotechnology, and particularly relates to a recombinant type I & III fusion collagen protein, a preparation method and application thereof. The present application discloses constituent polypeptides of the protein and related sequences thereof. Meanwhile, the present application also constructs a recombinant strain capable of expressing the protein, and provides a method for producing the protein through a fermentation process. The results of examples show that the recombinant collagen protein provided by the present application has no toxicity to cells, and has a good cell proliferation promoting effect and a good cell migration promoting effect; the protein has certain anti-aging and anti-wrinkle effects and antioxidant effects, and it is proved through human skin patch tests that the protein does not cause adverse reactions to human skin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and biotechnology, and particularly relates to a recombinant type I & III fusion collagen as well as a preparation method and application thereof. BACKGROUND

[0002] Collagen, as an important structural protein in the human body, is widely distributed in various tissues such as skin, bone, tendon, etc. It interacts with cell surface receptor proteins to regulate the biological functions of various cells, and plays a key role in maintaining the integrity of tissue structure and normal physiological activities. Among them, type I collagen, as the most important collagen type, has excellent tensile strength and can provide mechanical support for tissues; while type III collagen mainly endows tissues with good elasticity and flexibility, which is particularly important in the repair process of skin healing and blood vessels.

[0003] Studies have shown that specific domains in collagen molecules have a variety of biological functions, such as promoting cell adhesion, differentiation, and immune regulation. The RGD sequence is a site that has been studied in depth, which can mediate cell adhesion with collagen and be specifically recognized by integrin receptors on the cell surface; in addition, this sequence can also interact with factors such as vascular endothelial growth factor (VEGF), thereby regulating its activity and distribution. The GER triad sequence plays an important role in promoting cell adhesion, platelet aggregation, and blood vessel repair and growth. The KGD sequence shows specific adhesion ability to different cell types such as nerve cells and immune cells. In addition to the above functional sequences, collagen also has good biocompatibility, biodegradability, low antigenicity, and low cytotoxicity, etc., thus showing broad application prospects in medical fields such as medical dressings, wound repair, bone scaffolds, tendon repair, and drug delivery systems. In addition, collagen-based materials are also widely used in the industrial fields of beauty and skin care, leather processing, sewage treatment, papermaking, and textiles.

[0004] Traditional collagen extraction methods have problems such as long production cycle, low yield, potential immunogenicity, and unstable quality between batches. Although chemical synthesis can avoid immune rejection and viral contamination risks, it is technically complex, high cost, and the obtained product usually lacks biological activity. In contrast, microbial expression systems have the advantages of low cost, short cycle, high expression level, good biocompatibility, and high safety. Compared with the Escherichia coli expression system, the Pichia pastoris expression system can perform post-translational modification and does not produce endotoxins, which can effectively reduce the immunogenicity risk in clinical application; at the same time, it can use methanol as the only carbon source for high-density fermentation, which is easy to realize industrialization and can further improve the expression level of target protein by optimizing fermentation conditions.

[0005] The conventional recombinant collagen sequence currently contains a large number of non-functional domains, and the proportion of hydrophobic amino acids in the natural collagen is high, which leads to low expression and poor solubility; in addition, the tag introduced for the purpose of facilitating purification may increase the risk of immunogenicity. Therefore, there is a need in the art for a collagen with higher water solubility and lower immunogenicity. SUMMARY

[0006] The purpose of the present application is to provide a recombinant I & III type fusion collagen and a preparation method and application, the recombinant I & III type fusion collagen provided by the present application not only has clear function but also has higher water solubility and lower immunogenicity. In addition, the preparation method provided by the present application helps to improve the production efficiency and product quality of collagen.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0008] The present application provides a recombinant I & III type fusion collagen, which comprises type I and type III collagen.

[0009] Preferably, the type I collagen is type I collagen alpha 1 chain; the type III collagen is type III collagen alpha 1 chain.

[0010] Preferably, the type I collagen comprises any one or more of COL1A, COL1B and COL1C, and the type III collagen comprises any one or more of COL3A, COL3B and COL3C.

[0011] The amino acid sequences of COL1A, COL1B, COL1C, COL3A, COL3B and COL3C are shown in SEQ ID NO. 5-10.

[0012] Preferably, the amino acid sequence of the recombinant I & III type fusion collagen is shown in SEQ ID NO. 3.

[0013] A nucleotide molecule encoding the above-mentioned amino acid sequence, the sequence of the nucleotide molecule is shown in SEQ ID NO. 4.

[0014] A recombinant plasmid comprising the above-mentioned nucleotide molecule.

[0015] A recombinant strain comprising the above-mentioned recombinant plasmid.

[0016] Preferably, the recombinant strain is Pichia pastoris or Escherichia coli.

[0017] A preparation method of a recombinant I & III type fusion collagen, the method comprising:

[0018] S1 fermenting and culturing the recombinant strain;

[0019] S2 inducing the expression of the target protein to obtain a fermentation broth;

[0020] S3 separating and purifying the fermentation broth to obtain the recombinant type I & III fusion collagen protein.

[0021] Preferably, the fermentation culture of S1 in the method is: inoculating the strain in a culture medium with a pH of 5, maintaining the DO value at more than 20%; when the DO value rises to 80%, adding the carbon source until the wet weight of the bacterial cells reaches 200 g / L; and when the DO value rises to 80% again, starving for 1-2 h.

[0022] Preferably, the induction of S2 in the method is: methanol induction.

[0023] Preferably, the separation and purification of S3 in the method comprises:

[0024] S31 preliminary purification of the protein: salting-out, and fractionated precipitation with saturated ammonium sulfate solution;

[0025] S32 ion exchange chromatography.

[0026] The application provides a polypeptide, which is any one of COL1A, COL1B, COL1C, COL3A, COL3B, COL3C repeated n times, wherein n is an integer greater than or equal to 1.

[0027] Preferably, n is an integer between 1 and 10.

[0028] The application provides a fusion collagen protein, which comprises two or more polypeptides in the above polypeptides in any combination, and at least one polypeptide is repeated more than twice.

[0029] The polypeptides in the application are connected by peptide bonds.

[0030] The application also provides application of the recombinant fusion collagen protein in preparing biomedical materials or cosmetics.

[0031] Beneficial effects:

[0032] The application provides a recombinant type I & III collagen protein, and designs a coding gene thereof. The gene is constructed in a recombinant expression vector, and after being transformed into Pichia pastoris GS115, the gene can express a recombinant collagen protein with a theoretical molecular weight of about 46 kDa. The collagen protein has high expression, high purity, no cytotoxicity, good effects of promoting cell proliferation and cell migration, good anti-aging and anti-wrinkle effects, good antioxidant capacity, and is safe and non-irritating to human skin, and has a wide application prospect in the fields of biomedical materials and beauty and skin care. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0034] Figure 1 The figure is a map of the recombinant expression vector pPIC9K-COL13 of the present application.

[0035] Figure 2 The figure is an agarose gel electrophoresis map of the recombinant expression vector pPIC9K-COL13 linearized by Sal I enzyme of the present application. (M: standard molecular weight marker; lane 1: recombinant plasmid linearized by Sal I enzyme.)

[0036] Figure 3 The figure is an SDS-PAGE electrophoresis verification map of 5 μL supernatant of fermentation liquor of the recombinant GS115 / pPIC9K-COL13 single colony on the YPD-G418 plate after methanol induction in a flask. (M: protein marker; 1: GS115 negative control; lanes 2-5: positive single colonies on the YPD-G418 plate, which are No. 1 strain, No. 2 strain, No. 3 strain and No. 4 strain respectively.)

[0037] Figure 4 The figure is a high-density fermentation cell wet weight and target protein expression curve of the present application. (A: glycerol feeding stage; B: methanol induction stage.)

[0038] Figure 5 The figure is an SDS-PAGE electrophoresis result of supernatant of fermentation liquor of the 7L fermentation tank after fermentation for 120 h of the present application. (M: protein marker; lanes 1-13: supernatant of fermentation liquor after fermentation for 48 h, 54 h, 60 h, 66 h, 72 h, 78 h, 84 h, 90 h, 96 h, 102 h, 108 h, 114 h and 120 h. The red dotted line on the right side indicates the start of induction; the loading amount of marker is 5 μL, and the loading amount of sample is 2 μL.)

[0039] Figure 6 The figure is a result of ammonium sulfate fractionation and precipitation of crude protein of the target protein of the present application. (M: marker; lanes 1: 10 μL of supernatant of fermentation liquor; lanes 2, 4 and 6: supernatant of 20%, 25% and 30% ammonium sulfate fractionation and precipitation; lanes 3, 5 and 7: crude protein of 20%, 25% and 30% ammonium sulfate fractionation and precipitation.)

[0040] Figure 7 The figure is a mass spectrum identification result of the target protein of the present application.

[0041] Figure 8 Cell toxicity experiment results of the protein for the purpose of the application.

[0042] Figure 9 Cell proliferation experiment results of the protein for the purpose of the application.

[0043] Figure 10 Cell migration experiment results of the protein for the purpose of the application.

[0044] Figure 11 Analysis of cell migration experiment results of the protein for the purpose of the application.

[0045] Figure 12 Anti-aging and anti-wrinkle gene detection experiment results of the protein for the purpose of the application.

[0046] Figure 13 Standard curve of ABTS+ free radical scavenging rate of glutathione in the application.

[0047] Figure 14 Standard curve of ABTS+ free radical scavenging rate of the protein for the purpose of the application. DETAILED DESCRIPTION

[0048] The application provides a recombinant type I & III fusion collagen, discloses an amino acid sequence and a coding gene of the recombinant type I & III fusion collagen, and further provides an engineering bacterium for generating the protein and a production method of the protein.

[0049] The application further provides a polypeptide, which is a repetition or combination of the amino acid sequence of the recombinant type I & III fusion collagen.

[0050] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0051] The production process, experimental method or detection method involved in the embodiments of the application are all conventional methods in the prior art if no special description is given, and the name and / or abbreviation thereof all belong to conventional names in the field, and are very clear and definite in the field of related uses, and the person skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement according to the conventional conditions or the conditions suggested by the manufacturer.

[0052] The various instruments, equipment, raw materials or reagents used in the embodiments of the application do not have special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, and can also be prepared according to the conventional methods well known to the person skilled in the art.

[0053] The determination method of the recombinant protein content in the embodiments of the application is as follows: The determination method of the recombinant protein content in the embodiments of the application is as follows:

[0054] (1) SDS-PAGE image using ImagJ software analysis of the purpose of the band and protein concentration and known quantitative pre-dye Marker gray value, by comparing the ratio of the two and the volume of the sample to calculate the content of the product.

[0055] (2) BCA protein content detection: the sample is appropriately diluted, add 20 μL to the sample well of 96-well plate. Each well is added with 200 μL of BCA working solution, 37℃ for 15-30 min. The absorbance at 562 nm is measured by microplate reader, and the protein concentration is calculated according to the standard curve.

[0056] The fermentation medium in the application is:

[0057] (1) The initial BMGY medium after shake flask optimization (g / L): yeast extract 10, proteose peptone 20, K2HPO43, KH2PO411.8, YNB 13.4, biotin 4×10 -4 , glycerol 10, adjust pH to 7;

[0058] (2) BMMY induction expression medium after shake flask optimization (g / L): yeast extract 10, proteose peptone 20, K2HPO43, KH2PO411.8, YNB 13.4, biotin 4×10 -4 , methanol 1%, adjust pH to 6;

[0059] (3) Trace salt solution PTM1 (g / L): 4.75 g PTM1 powder is added to 40 mL of distilled water, 0.25 mL of concentrated H2SO4 is added, and it is diluted to 50 mL, and then filtered with a 0.22 μm filter to remove bacteria.

[0060] (4) Fermentation medium BSM: 85% H3PO4106.8 mL, CaSO4·2H2O 3.72 g, K2SO472.8 g, MgSO4·7H2O 59.6 g, KOH 16.52 g, glycerol 160 g, PTM1 17.4 mL in (3).

[0061] The yeast nitrogen source (YNB), methanol, biotin, amino acids, glucose, PTM1 in the medium need to be filtered to remove bacteria, and the rest are sterilized at 121℃ for 20 min.

[0062] In the application, human type I and type III collagen sequences are selected for screening and optimization. The sequences of human type I collagen alpha 1 chain and type III collagen alpha 1 chain are as follows (SEQ ID NO. 1~2):

[0063] MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAA GEPGKAGERGV PGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGAR GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKG ADGSP GKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEV GPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSG EPGKQGPSGASGERGPP GPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGET GPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL (SEQ ID NO. 1)

[0064] MMSFVQKGSWLLLALLHPTIILAQQEAVEGGCSHLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPNGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAK GEPGPRGE RGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEP GRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQN GEPGGKGERG APGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGP PGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAP GPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAP GPQGPRGDKGETGERGAAGIKGHRGFP GNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVQLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL (SEQ ID NO. 2)

[0065] The underlined and bolded part in the above sequence is the selected amino acid sequence. The selected sequence has strong cell adhesion, high biocompatibility, good hydrophilicity and other advantages of biological materials. The sequence of the application does not introduce a tag, avoiding the risk of immunogenicity, and is suitable for use in the fields of biomedical materials, beauty and skin care, etc.

[0066] The selected amino acid sequence was spliced ​​together and named COL13.

[0067] Its amino acid sequence is: GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDA GAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKGADGSPGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPAGPAGPVGP VGARGPAGPQGPRGDKGETGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGEPGGKGERGAPGEKGEGGPPGVAG PPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO.3)

[0068]

[0069] In this invention, the sequences of specific amino acid regions were also tested:

[0070] (1) COL1A amino acid sequence: (168 Aa)

[0071] GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKGADGSP (SEQ ID NO.5)

[0072] (2) COL1B amino acid sequence: (75 Aa)

[0073] GPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPP (SEQ ID NO.6)

[0074] (3) COL1C amino acid sequence: (30Aa)

[0075] GPAGPAGPVGPVGARGPAGPQGPRGDKGET (SEQ ID NO.7)

[0076] (4) COL3A amino acid sequence: (87Aa)

[0077] GEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRD (SEQ ID NO.8)

[0078] (5) COL3B amino acid sequence: (126Aa)

[0079] GEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAP (SEQ ID NO.9)

[0080] (6) COL3C amino acid sequence: (27Aa)

[0081] GPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO.10)

[0082] In this invention, the polypeptide can be recombinant human collagen (COL1A). n， The sequence is an n-fold repeat of COL1A, consisting of 168 amino acids. n There are 1, and the basic repeating unit is:

[0083] GEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSP (SEQ ID NO.5) is a human collagen type I peptide.

[0084] Its amino acid sequence is an n-fold repetition of the sequence shown in SEQ ID NO.5.

[0085] Its nucleotide sequence is an n-fold repetition of the sequence shown in SEQ ID NO.11 below:

[0086] GGTGAACCTGGTAAAGCTGGTGAAAGAGGTGTTCCAGGTCCACCTGGTGCTGTTGGTCCAGCTGGTAAAGATGGTGAAGCTGGTGCTCAAGGTCCACCAGGTCCAGCTGGACCTGCTGGTGAAAGAG GAGAACAAGGTCCTGCTGGTTCTCCTGGTTTCCAAGGTTTGCCAGGTCCTGCTGGACCACCTGGTGAAGCTGGAAAACCTGGTGAACAAGGTGTTCCTGGTGACTTGGGTGCTCCAGGTCCTTCTGG TGCTAGAGGTGAAAGAGGTTTCCAGGTGAAAGAGGTGTCCAAGGTCCTCCTGGTCCAGCTGGTCCTAGAGGTGCTAACGGTGCTCCAGGAAATGATGGTGCTAAAGGTGACGCTGGTGCTCCTGGT GCTCCAGGTTCTCAAGGTGCTCCAGGTTTGCAAGGTATGCCTGGTGAAAGAGGTGCTGCTGGTTTGCCTGGTCCTAAAGGTGACAGAGGTGACGCTGGACCTAAGGGTGCTGATGGTTCTCCA (SEQ ID NO.11)

[0087] In this invention, the polypeptide can be recombinant human collagen (COL1B). n， The sequence is an n-fold repeat of COL1B, including amino acids (75). n There are 1, and the basic repeating unit is:

[0088] GPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPP (SEQ ID NO.6) is a type I peptide of human collagen.

[0089] Its amino acid sequence is an n-fold repetition of the sequence shown in SEQ ID NO.6.

[0090] Its nucleotide sequence is an n-fold repetition of the sequence shown in SEQ ID NO.12 below:

[0091] GGTCCACCAGGACCACCAGGTCCTGCCGGTGAAAAGGGTTCTCCTGGAGCTGATGGTCCAGCTGGTGCTCCAGGTACTCCTGGTCCACAAGGTATTGCTGGTCAAAGAGGTGTTGTTGGTTTGCCTGGACAAAGAGGTGAAAGAGGTTTTCCTGGTTTGCCTGGTCCATCTGGTGAACCTGGAAAGCAAGGTCCTTCTGGAGCTTCTGGTGAAAGAGGTCCACCT (SEQ ID NO.12)

[0092] In this invention, the polypeptide can be recombinant human collagen (COL1C). n， The n-fold repeat sequence of COL1C includes amino acids (30). n There are 1, and the basic repeating unit is:

[0093] GPAGPAGPVGPVGARGPAGPQGPRGDKGET (SEQ ID NO.7) is a type I peptide of human collagen.

[0094] Its amino acid sequence is an n-fold repetition of the sequence shown in SEQ ID NO.7.

[0095] Its nucleotide sequence is an n-fold repetition of the sequence shown in SEQ ID NO.13 below:

[0096] GGACCTGCTGGACCTGCCGGTCCAGTTGGTCCAGTTGGAGCTAGAGGTCCAGCTGGTCCACAAGGACCAAGAGGTGACAAAGGTGAAACT (SEQ ID NO.13)

[0097] In this invention, the polypeptide can be recombinant human collagen (COL3A). n， The nth repeat sequence of COL3A includes amino acids (87). n There are 1, and the basic repeating unit is:

[0098] GEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRD (SEQ ID NO.8) is a human collagen type III peptide.

[0099] Its amino acid sequence is an n-fold repetition of the sequence shown in SEQ ID NO.8.

[0100] Its nucleotide sequence is an n-fold repetition of the sequence shown in SEQ ID NO.14 below:

[0101] GGTGAACCTGGTCCAAGAGGTGAAAGAGGTGAAGCTGGTATTCCAGGTGTTCCTGGAGCTAAAGGTGAAGATGGTAAAGATGGATCTCCAGGTGAACCTGGTGCTAACGGATTGCCAGGTGCTGCTGGAGAA AGAGGTGCCCCTGGTTTCAGAGGTCCAGCCGGTCCAAATGGTATTCCAGGAGAAAAAGGTCCAGCTGGTGAAAGAGGTGCACCTGGTCCTGCTGGTCCTAGAGGAGCTGCTGGTGAACCAGGTAGAGAT (SEQ ID NO.14)

[0102] In this invention, the polypeptide can be recombinant human collagen (COL3B). n， The COL3B sequence is an n-fold repeat sequence, consisting of (126)n amino acids, with the basic repeating unit being:

[0103] GEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAP (SEQ ID NO.9) is a human collagen type III peptide.

[0104] Its amino acid sequence is an n-fold repetition of the sequence shown in SEQ ID NO.9.

[0105] Its nucleotide sequence is an n-fold repetition of the sequence shown in SEQ ID NO.15 below:

[0106] GGTGAACCAGGAGGTAAAGGTGAAAGAGGTGCTCCAGGTGAAAAGGGAGAAGGTGGTCCTCCAGGTGTTGCTGGTCCTCCAGGAGGTTCTGGTCCTGCTGGTCCACCAGGTCCCCAAGGTGTTAAGGGTGAAAGAGGTTCTCCTGGTGGTCCAGGTGCTGCCGGTTTTCCAGGTGCTAGAGGATTGCCAGG ACCACCTGGATCTAACGGTAACCCAGGTCCACCCGGTCCATCTGGATCTCCAGGAAAGGATGGTCCACCTGGTCCAGCCGGAAATACTGGTGCTCCTGGATCTCCTGGTGTTTCTGGTCCAAAGGGTGACGCTGGTCAACCAGGTGAAAAAGGTTCTCCTGGTGCTCAAGGACCTCCAGGTGCTCCA (SEQ ID NO.15)

[0107] In this invention, the polypeptide can be recombinant human collagen (COL3C). n， The nth repeat sequence of COL3C includes amino acids (27). n There are 1, and the basic repeating unit is:

[0108] GPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO.10) is a human collagen type III peptide.

[0109] Its amino acid sequence is an n-fold repetition of the sequence shown in SEQ ID NO.10.

[0110] Its nucleotide sequence is an n-fold repetition of the sequence shown in SEQ ID NO.16 below:

[0111] GGTCCACAAGGTCCTAGAGGTGACAAGGGTGAAACTGGAGAAAGAGGAGCTGCCGGTATTAAGGGTCATAGAGGTTTTCCA (SEQ ID NO.16)

[0112] In this invention, recombinant proteins may include any sequence from SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 repeated n times and freely combined.

[0113] In this invention, the number of repetitions is n, where n is an integer greater than or equal to 1. Preferably, n is an integer between 1 and 10.

[0114] Example 1: Construction of recombinant plasmid pPIC9K-COL13

[0115] The gene sequences of human type I and type III collagen α1 chains were obtained from the NCBI database. Protein sequence design was performed, and based on the codon bias of Pichia pastoris, the original gene sequences were optimized using specialized software to ensure efficient transcription and translation in Pichia pastoris and improve protein expression levels. PsiⅠ restriction sites were introduced at the 5' end and EcoRI restriction sites at the 3' end, respectively. The designed sequence was named COL13, with the amino acid sequence (SEQ ID NO. 3) and nucleotide sequence (SEQ ID NO. 4). Synthesis was performed by Nanjing GenScript Biotech Co., Ltd., and the recombinant plasmid pPIC9K-COL13 was obtained. The recombinant plasmid map is shown below. Figure 1 As shown, it was transformed into Escherichia coli DH5α.

[0116] Example 2: Construction of recombinant strain GS115 / pPIC9K-COL13

[0117] (1) Extraction of recombinant plasmid: The Escherichia coli DH5α strain containing the recombinant plasmid pPIC9K-COL13 synthesized by Nanjing Genscript Co., Ltd. was streaked on LB solid plates containing 50 μg / mL kanamycin and cultured overnight at 37℃. Single colonies were picked and inoculated into liquid LB medium containing kanamycin and cultured at 37℃ and 220 rpm for about 12 h. 1% of the culture was inoculated into 50 mL of fresh liquid LB medium and cultured until the OD600 was 0.4-0.6. The plasmid was extracted according to the ToloPrep Plasmid DNA MiniPreparation Kit column-based plasmid DNA mini-extraction kit to obtain a sufficient amount of plasmid for electroporation of Pichia pastoris.

[0118] (2) Plasmid linearization and ethanol precipitation: The recombinant plasmid (10-20 μg) was linearized using SalI enzyme. The linearization results are as follows: Figure 2 As shown; DNA was recovered and concentrated using ethanol precipitation, and the precipitate was dissolved in sterile water or TE for subsequent reactions (5-10 μL).

[0119] (3) Pichia pastoris GS115 electroporation: Pichia pastoris GS115 competent cells were prepared. 10-20 μg of linearized plasmid was added to 80 μL of yeast competent cells. The electroporation conditions were 1.5 KF and 0.5 ms. After the electroporation was completed, 1 ml of 1 M sorbitol solution in an ice bath was quickly added to the electroporation cup. The mixture was then transferred to a sterile centrifuge tube in a clean bench and incubated at 30°C for 1 h.

[0120] Example 3: Screening and Validation of Recombinant Strains GS115 / pPIC9K-COL13

[0121] (1) After incubation, spread the incubator on MD plates and incubate at 30°C inverted for 3-5 days until single clones grow.

[0122] (2) Screening of high-copy strains on YPD-G418 plates: High-copy strains were obtained, and the positive transformants grown on MD plates were transferred to G418-YPD plates with concentration gradients of 1.0, 2.0, 3.0 and 4.0 g / L. The plates were inverted at 30℃ and strains with good growth on each concentration plate were selected for further culture to obtain Pichia pastoris genetically engineered strains expressing recombinant type I & III fusion collagen.

[0123] (3) Verification of GS115 positive strains: Four single colonies from YPD-G418 plates were selected and inoculated into 50 mL of YPD liquid medium and cultured for 12-18 h. Then, 2% of the colonies were inoculated into 50 mL of BMGY medium and cultured for 24 h. The bacterial cells were collected, centrifuged at low temperature, and resuspended in fresh 50 mL of BMMY medium. 0.5% methanol was added to the medium every 24 h for induction for 120 h. Samples were taken every 24 h, and the culture medium was centrifuged at 12000 rpm for 6 min at room temperature. The supernatant was stored at -20℃. Protein expression was identified by SDS-PAGE, such as... Figure 3 As shown, strain 3 had the highest expression level.

[0124] Example 4: Fermentation culture and crude protein preparation of recombinant strain GS115 / pPIC9K-COL13

[0125] (1) Shake-flask fermentation culture: the same as the culture method for verifying positive strains.

[0126] Response surface methodology was used to determine that the target protein expression level was relatively high at an induction temperature of 28℃, a pH of 6, and a methanol addition of 1% / 24 h, with a protein expression level of approximately 0.464 g / L.

[0127] (2) Small-scale fermentation of GS115 / pPIC9K-COL13 recombinant strain in 7 L fermenter.

[0128] After sterilization of the BSM culture medium, the initial pH was adjusted to approximately 5 with ammonia. The culture was maintained at 30°C and an initial aeration rate of 450 rpm. As the cells grew and density increased, the aeration rate was gradually increased, raising the aeration rate to 650 rpm, maintaining a dissolved oxygen (DO) value above 20%. After inoculation, the DO value initially decreased and then increased. When the DO value reached approximately 80%, glycerol-fed medium was added until the cell wet weight reached approximately 200 g / L. Addition was then stopped, and the DO value was starved for 1–2 hours before methanol induction. The fermentation supernatant was sampled every 3 hours and analyzed by SDS-PAGE electrophoresis to determine collagen production. Changes in cell wet weight and target protein expression levels were observed after 120 hours of fermentation. Figure 4 A represents the glycerol feeding stage, and B represents the methanol induction stage. Analysis of the fermentation supernatant showed that the target protein was not expressed before methanol induction. Induction began from the fourth well (66 h of fermentation), at which point the target protein started to express. Methanol served as both an inducer and a carbon source. At 96 h of fermentation (42 h of induction), the expression level of the target protein began to decrease. Figure 5 Protein concentration analysis revealed that the expression level of this target protein was 2.43 g / L.

[0129] Example 5: Isolation and purification of recombinant type I & III fusion collagen

[0130] (1) Preliminary protein purification: Salting out, followed by fractional precipitation using saturated ammonium sulfate solution. Saturated ammonium sulfate was added drop by drop slowly to the supernatant of the fermentation broth at pH 7, while stirring, to explore the effect of the final concentration of saturated ammonium sulfate and pH on the fractional precipitation effect. The results were observed by SDS-PAGE band analysis. Figure 6 The final concentration of saturated ammonium sulfate was 25%. H2SO4 was added to adjust the pH of the solution to 3. The solution was stirred thoroughly on a magnetic stirrer for 2-3 hours. The solution was then centrifuged at 10,000 rpm for 10 minutes at 4°C. The precipitate was redissolved with 1 / 2 volume of 0.02 M PB, which was able to separate some of the impurities.

[0131] (2) Ion exchange chromatography:

[0132] The sample, after ammonium sulfate fractionation precipitation, was dissolved in 1 / 2 volume of 0.02 M PB and filtered through a 0.45 μm filter membrane. The sample was then subjected to cation exchange chromatography with a sodium chloride gradient of 0–500 mM. The elution peaks were collected and identified by SDS-PAGE. Through one-step purification, the purity of recombinant type I-III fusion collagen can reach over 95%.

[0133] Example 6: Characterization of recombinant type I & III fusion collagen

[0134] The recombinant collagen prepared above was desalted and freeze-dried to obtain pure recombinant type I-III fused collagen, which was then characterized structurally.

[0135] (1) Mass spectrometry analysis: Mass spectrometry analysis results ( Figure 7 The results showed that the relative molecular weight of the recombinant type I-III fusion collagen was 46 kDa, consistent with the theoretical molecular weight. However, SDS-PAGE results showed that the apparent molecular weight of the recombinant type I-III fusion collagen was approximately 72 kDa, significantly larger than the theoretical molecular weight. It is speculated that this may be because the recombinant type I-III fusion collagen contains hydrophilic amino acids, which weakens the protein's binding affinity to SDS, reducing the negative charge during electrophoresis and shortening the electrophoretic migration distance, thus increasing the apparent molecular weight.

[0136] Example 7: Functional analysis of recombinant type I & III fusion collagen

[0137] 7.1 Cytotoxicity assay of recombinant collagen COL13

[0138] 7.1.1 Experimental Methods

[0139] Cytotoxicity was detected using the MTT assay. Immortalized human skin fibroblasts (HSF) in the logarithmic growth phase were divided into groups of 2 × 10⁻⁶ cells. 5 Cells were seeded at 100 μL / well in 96-well plates and cultured for 24 h at 37℃, 5% CO2. When the cell deposition rate reached 40%–60%, the following treatments were administered: ① Sample group: 100 μL of culture medium containing different concentrations (5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.078 mg / mL) of COL13 protein was added to each well; ② Control group (NC): 100 μL of culture medium was added to each well; ③ Blank group (BC): 100 μL of culture medium was added to each well, containing no cells. Cultured for 24 h. The medium was then discarded and replaced with 100 μL / well of culture medium containing MTT (0.5 mg / mL), and cultured for another 4 h. The discarded medium was then replaced with 100 μL / well of DMSO, shaken thoroughly, and allowed to develop color. OD was then measured using a microplate reader. 490 Calculate the relative cell viability according to formula (1):

[0140] Relative cell viability (%) = ×100% ………(1)

[0141] Criteria for determining cytotoxicity: If the cell survival rate is greater than 70%, it is considered that there is no cytotoxic reaction; otherwise, it is considered that there is a cytotoxic reaction.

[0142] 7.1.2 Experimental Results

[0143] The results of the cytotoxicity experiment are shown in Table 1, and the generated bar chart is shown in [Table 1]. Figure 8 When COL13 protein was used in HSF cells at a concentration ≤5 mg / mL for 24 hours, the cell survival rate was greater than 70%, indicating that the recombinant collagen COL13 designed in this invention is not toxic to cells.

[0144] Table 1 Results of cytotoxic MTT assay

[0145]

[0146] 7.2 Cell proliferation experiment using recombinant collagen COL13

[0147] 7.2.1 Experimental Methods

[0148] Cell proliferation was detected using the MTT assay. Immortalized human skin fibroblasts (HSF) in logarithmic growth phase were divided into groups of 8 × 10⁻⁶ cells. 4 Cells were seeded at a concentration of 100 μL / well in 96-well plates and cultured for 24 h in an incubator (37℃, 5% CO2) to detect cell viability. The experiment included a blank group (BC), a control group (NC), a COL13 group, and a commercially available COL group. Drug administration and culture methods were the same as in the cytotoxicity assay. The COL13 group and the commercially available COL group were administered at a concentration of 0.156 mg / mL, with three replicate wells. Cell viability was measured at 0 h, 24 h, 48 h, and 72 h, and proliferation curves were plotted based on cell viability.

[0149] 7.2.2 Experimental Results

[0150] The results of the cell proliferation experiment are shown in Table 2, and the generated line graph is shown in Table 2. Figure 9 The results showed that the recombinant collagen COL13 designed in this invention had a better proliferative effect on HSF cells at a concentration of 0.156 mg / mL after 24 h of treatment, compared with commercially available COL.

[0151] Table 2. Cell proliferation detection results

[0152]

[0153] 7.3 Recombinant collagen COL13 promotes cell migration assay

[0154] 7.3.1 Experimental Methods

[0155] HSF cells in logarithmic growth phase were divided into groups of 1×10⁻⁶. 6Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight in an incubator (37°C, 5% CO2). Once the platelet coverage reached 70% or higher, a scratch assay was performed. Two vertical scratches were made in each well using a 10 μL pipette tip, with the vertical scratch serving as the baseline (the pipette tip perpendicular to the edge of the ruler), maintaining a 2 cm spacing between the scratches. After making the vertical scratches, horizontal scratches were made near the central axis of the 6-well plate, perpendicular to the baseline. Cells were washed three times with PBS after the scratch assay. The COL13 group and the commercially available COL group were administered at a concentration of 0.156 mg / mL, with 2 mL administered per well, and two replicates per group. Cells were incubated for another 24 hours in an incubator (37°C, 5% CO2). Cells were photographed using an inverted microscope to observe migration in each group. The migration rate of the negative control group was normalized, and the relative migration rate of each group was calculated.

[0156] 7.3.2 Experimental Results

[0157] The cell migration results are shown in Table 3 and Figure 10 The resulting bar chart is shown below. Figure 11 The results showed that the recombinant collagen COL13 designed in this invention, at a concentration of 0.156 mg / mL, had a better cell migration-promoting effect on HSF cells after 24 hours of treatment compared to commercially available COL protein.

[0158] Table 3 Cell migration results

[0159]

[0160] 7.4 Anti-aging and anti-wrinkle experiment of recombinant collagen COL13

[0161] 7.4.1 Administration

[0162] HSF cells in logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / well into 6-well plates and incubated overnight in an incubator (37°C, 5% CO2). When the cell deposition rate in the 6-well plates reached 40%–50%, the COL13 group and the commercially available COL group were administered drugs at a concentration of 0.156 mg / mL, with 2 mL administered per well, one well per group. After drug administration, the 6-well plates were incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0163] 7.4.2 Total RNA Extraction from Cells

[0164] 7.4.2.1 Sample preparation.

[0165] Cell samples: Collect approximately 1 million cells, aspirate the culture medium, wash three times with PBS, add 300 μL of lysis buffer, gently pipette 5-10 times until the suspension dissolves and the solution becomes clear, then transfer to a clean centrifuge tube.

[0166] 7.4.2.2 Add an equal volume of binding solution to the lysis buffer and gently invert to mix 3-5 times.

[0167] 7.4.2.3 Transfer the mixture (including the precipitate) to a new 1.5 ml centrifuge tube, add 20 µL of BeyoMag™ RNA magnetic bead suspension (be sure to mix well before use), mix gently, and incubate at room temperature for 3–5 minutes. Place the centrifuge tube in the magnetic field of the magnetic rack, and after the magnetic beads have completely aggregated, carefully aspirate any remaining liquid.

[0168] 7.4.2.4 Add 600µL of washing solution I, gently shake to disperse the magnetic beads, invert twice, and then place the centrifuge tube in the magnetic field of the magnetic rack. After the magnetic beads have completely gathered, try to remove as much residual liquid as possible.

[0169] 7.4.2.5 Add 600µL of washing solution II, gently shake to disperse the magnetic beads, invert twice, and then place the centrifuge tube in the magnetic field of the magnetic rack. After the magnetic beads have completely gathered, try to remove as much residual liquid as possible.

[0170] 7.4.2.6 Repeat step 7.4.2.5 once.

[0171] 7.4.2.7 Place the centrifuge tubes at room temperature for 5-10 minutes, or in a 37ºC oven for 5 minutes, to ensure that any residual ethanol or other trace amounts of liquid have completely evaporated.

[0172] 7.4.2.8 Add 50-100 μL of elution buffer, gently vortex to suspend the magnetic beads in the solution, and incubate at room temperature for 3-5 minutes, shaking the centrifuge tube 1-2 times during this period. Place the centrifuge tube in a magnetic field, and after the magnetic beads have completely aggregated, carefully aspirate the solution into a new centrifuge tube and store at -20ºC. The resulting solution is the extracted RNA.

[0173] The purity of the extracted RNA was determined using an ultra-micro spectrophotometer, OD. 260 / OD 280 The values ​​were all between 1.8 and 2.0, indicating that the next step of reverse transcription experiment could be carried out.

[0174] 7.4.3 Reverse Transcription Experiment

[0175] 7.4.3.1 Refer to Table 4 below to set up the reverse transcription reaction system.

[0176] Table 4. Preparation of Reverse Transcription Reaction System

[0177]

[0178] *To 12μL means adding DEPC-treated Water to a final volume of 12μL.

[0179] 7.4.3.2 Mix gently, then centrifuge to settle the liquid.

[0180] 7.4.3.3 Incubate at 42ºC for 60 min.

[0181] 7.4.3.4 Incubate at 80ºC for 10 min.

[0182] 7.4.3.5 The reverse transcription product can be used directly for subsequent real-time quantitative PCR experiments, or it can be frozen at -20ºC for later use.

[0183] 7.4.4 Real-time quantitative PCR experiment

[0184] The expression levels of h-COL1A1, h-COL3A1, and h-ELN, which are related to cellular anti-aging and anti-wrinkle effects, were detected.

[0185] 7.4.4.1 Primers used for gene detection are shown in Table 5.

[0186] Table 5 Primer information for quantitative real-time PCR

[0187]

[0188] 7.4.4.2 Reaction System

[0189] The PCR reaction system was prepared according to Table 6.

[0190] Table 6. Preparation of Real-Time Quantitative PCR Reaction System

[0191]

[0192] 7.4.4.3 Standard Procedure

[0193] The PCR reaction procedure is shown in Table 7.

[0194] Table 7 Real-time quantitative PCR reaction procedure

[0195]

[0196] 7.4.4.4 Results Analysis

[0197] Use 2 -△△CT The results were calculated using the following methods. T-tests were performed on the values ​​of the sample group and the NC group using software. A p-value < 0.05 was considered statistically significant compared to the SC group (denoted by *), and a p-value < 0.01 was considered highly statistically significant compared to the SC group (denoted by **).

[0198] Table 8 Results of Real-Time PCR Gene Detection

[0199]

[0200] The results of real-time PCR gene detection are shown in Table 8, and the generated bar chart is shown in Table 8. Figure 12 The results showed that the recombinant collagen COL13 designed in this invention increased the expression levels of h-ELN, h-COL1A1 and h-COL3A1 genes in HSF cells, indicating that the recombinant collagen COL13 designed in this invention has certain anti-aging and anti-wrinkle effects.

[0201] 7.5 Antioxidant Experiment of Recombinant Collagen COL13

[0202] 7.5.1 Solution Preparation

[0203] ABTS solution: Weigh 200.0 mg of ABTS and 34.4 mg of potassium persulfate, dissolve in 50.0 mL of distilled water, shake well, and let stand at room temperature in the dark for 24 h to obtain the ABTS stock solution. Take an appropriate amount of the ABTS stock solution and dilute with 95% ethanol until the absorbance value is within 0.70 ± 0.02 (OD). 734 This solution is used as the ABTS assay solution and should be prepared fresh for immediate use.

[0204] Recombinant collagen COL13 protein solution: Weigh 20.0 mg of sample, dilute to 1 mL with distilled water, mix thoroughly to prepare a 20 mg / mL stock solution. Dilute the stock solution with distilled water to different concentrations to obtain sample solutions of different concentrations.

[0205] Glutathione solution: Weigh 10.0 mg of L-reduced glutathione, dilute to 1 mL with distilled water, mix thoroughly to prepare a 10 mg / mL stock solution. Dilute the stock solution with distilled water to different concentrations to obtain glutathione solutions of different concentrations.

[0206] 7.5.2 Experimental Procedure

[0207] 1) Control group: Take two test tubes, numbered 1 and 2 respectively, and add reagents to each test tube according to the combination in Table 9. After mixing thoroughly, react at room temperature in the dark for 5 min, and measure the absorbance value at a wavelength of 734 nm using a UV spectrophotometer (zero calibration with sample solvent).

[0208] Table 9. ABTS Method Reagent Addition Amount

[0209]

[0210] 2) COL13 group: The sample solution was used instead of the glutathione solution, and other operations were the same as the control group.

[0211] Data processing

[0212] Calculate according to formula (2):

[0213] P = ×100% …………………(2)

[0214] In the formula:

[0215] P — clearance rate;

[0216] A b —Absorbance of the mixture of ABTS solution and sample solvent (test tube #2);

[0217] A s — (Test tube 1) Absorbance of the mixture of the test solution and ABTS solution;

[0218] A linear equation (R²) was established between the natural logarithm of the concentration of the test solution and the clearance rate, with the natural logarithm of the concentration of the test solution as the x-axis and the clearance rate as the y-axis. 2 (≥0.9500), calculate the half-maximal clearance rate (EC). 50 The antioxidant capacity AO value of the polypeptide sample is calculated according to formula (3).

[0219] AO= …………………(3)

[0220] In the formula:

[0221] AO – Antioxidant capacity;

[0222] EC 50 (S)——Half-clearance of peptide sample, in milligrams per liter (mg / L).

[0223] EC 50 (R) – Half-maximal clearance of glutathione, expressed in milligrams per liter (mg / L).

[0224] The calculation results are expressed as the arithmetic mean of parallel measurements, and are retained to three significant figures.

[0225] 7.5.3 Test Results

[0226] ABTS was conducted using glutathione solutions of five different concentrations. + The free radical scavenging test, and the standard curve plotted are shown in [reference needed]. Figure 13 The calculation results are shown in Table 10. ABTS was performed using recombinant collagen COL13 solutions of six different concentrations. + The free radical scavenging test, and the standard curve plotted are shown in [reference needed]. Figure 14 The calculation results are shown in Table 11.

[0227] Table 10 Results of Glutathione ABTS Method

[0228]

[0229] Table 11 Results of ABTS assay for recombinant collagen COL13 solution

[0230]

[0231] The test results showed that, according to the ABTS method, the half-maximal clearance (EC50) of glutathione was... 50 The half-maximal clearance (EC50) of recombinant collagen COL13 was 13.87 mg / L. 50 The concentration was 2446.46 mg / L, and its antioxidant capacity AO value was 176; that is, recombinant collagen COL13 has a certain antioxidant effect.

[0232] 7.6 Human skin patch test of recombinant collagen COL13

[0233] 7.6.1 Test Methods

[0234] Test substance: 10 mg / mL recombinant collagen COL13 solution.

[0235] A total of 30 participants were selected according to the inclusion criteria. A qualified plaster applicator was used, and the test substance was placed inside the applicator at a volume of approximately 0.020 ml to 0.025 ml. The control well served as a blank control (without any substance). The plaster applicator containing the test substance was applied to the flexor side of the participant's forearm using non-irritating adhesive tape, and the applicator was gently pressed with the palm of the hand to ensure even application to the skin. This process was continued for 24 hours.

[0236] The test substance was removed after 24 hours. Skin reactions were observed and recorded at 0.5 hours, 24 hours, and 48 hours after patch removal, according to the skin reaction grading standards in the "Cosmetic Safety Technical Specifications" (2020 edition) (see Table 12). The results are shown in Table 13. No adverse reactions were observed in any of the 30 participants after 48 hours.

[0237] Table 12 Grading Standards for Skin Reactions in Closed Patch Tests

[0238]

[0239] Table 13 Statistical Table of Results of Human Skin Occlusive Patch Experiment

[0240]

[0241] As demonstrated by the above embodiments, the recombinant type I-III fusion collagen provided by this invention is non-toxic to cells and exhibits good cell proliferation and migration promotion effects. This protein can increase the expression levels of h-ELN, h-COL1A1, and h-COL3A1 genes, thus possessing certain anti-aging and anti-wrinkle effects. This protein also has certain antioxidant properties, and human skin patch tests have proven that it does not produce adverse reactions on human skin.

[0242] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

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

3.

2. A nucleotide molecule encoding the amino acid sequence of claim 1, characterized in that, The sequence of the nucleotide molecule is shown in SEQ ID NO.

4.

3. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleotide molecule of claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the recombinant plasmid of claim 3.

5. A method for preparing recombinant type I & III fused collagen, characterized in that, The method includes: S1 ferments and cultivates the recombinant strain described in claim 4; S2 induces the expression of the target protein, and fermentation broth is obtained; S3 was used to separate and purify the fermentation broth to obtain recombinant type I & III fused collagen.

6. The use of the protein according to claim 1 in the preparation of biomedical materials.

7. The use of the protein according to claim 1 in the preparation of cosmetics.

Images