Recombinant type III human collagen polypeptide with expansion joint-like structure, preparation method and use thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to a recombinant type III human collagen polypeptide with a quasi-expansion joint, a preparation method and its use. The present invention retains the activity of collagen by intercepting the amino acid residues at positions 268 to 828 of type III human collagen, shortens the length of the polypeptide to be expressed, and is more likely to obtain active high expression in Escherichia coli, and introduces a repeating sequence between the amino acid residues at positions 411 to 639 of type III human collagen to adjust the local steric hindrance, form a quasi-expansion joint structure, improve the ability of collagen to accommodate water molecules, slow down the absorption and metabolism of the human body, increase the half-life of collagen in the skin, can reduce the injection frequency, reduce the injection amount, and further reduce the side effects of collagen injection.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a recombinant type III human collagen polypeptide with an expansion joint-like substance, a preparation method and a use thereof. Background Art

[0002] Collagen is a biopolymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even up to 80% or more in some organisms. Animal tissues from livestock and poultry are the main way for people to obtain natural collagen and collagen peptides, but related livestock diseases limit people's use of terrestrial mammal collagen and its products.

[0003] According to their distribution and functional characteristics in the body, collagen can be divided into interstitial collagen, basement membrane collagen and cell periphery collagen. Interstitial collagen molecules account for the vast majority of collagen in the whole body, including collagen molecules of types I, II and III. Collagen is a natural protein in the body. It has a greater affinity for protein molecules on the surface of the skin, weaker antigenicity, good biocompatibility and biodegradation safety, is degradable and absorbable, and has good adhesion. Collagen has the effects of pure natural moisturizing, whitening, anti-wrinkle, and freckle removal, and can be widely used in beauty products.

[0004] Type III collagen is the main collagen in the skin of fetuses and infants (accounting for 60%). Collagen type III: mainly exists in infant skin or vascular endothelium and intestines. Type III collagen is the main component of reticular fibers in the skin. The higher the content, the thinner the fiber bundles. In the field of skin wrinkle removal, the higher the proportion of type III collagen, the finer and smoother the skin tissue. However, the fiber bundles formed by type III collagen are too thin and the distance between the fiber bundles is too small, which makes its water retention and anti-degradation ability relatively weak. It needs to be further modified to increase its half-life in the skin. At the same time, the existing recombinant type III collagen molecules are relatively long, with large molecular weight and complex structure. Yeast expression system is required, and the preparation cost is high.

[0005] In view of the above two shortcomings, the present invention is proposed. Summary of the invention

[0006] Problems to be solved by the invention:

[0007] 1. Transform type III human collagen to form fibrous micelles with expansion joint-like structures, which can be used to store and release water molecules, improve water retention capacity, and further extend its half-life in the skin.

[0008] 2. Truncating type III human collagen, retaining its active part, reducing its molecular weight to one-third of the original, and facilitating its expression in the E. coli expression system, thereby increasing expression activity and expression amount and reducing production costs.

[0009] Solutions used to resolve the problem include:

[0010] In a first aspect, the present invention provides a recombinant type III human collagen polypeptide for injection, wherein the recombinant type III human collagen polypeptide for injection comprises amino acid residues 268 to 828 of wild-type type III human collagen, and a portion of amino acid fragments between amino acid residues 411 to 639 of the wild-type type III human collagen are replaced by a plurality of repeated polypeptide fragments;

[0011] The amino acid sequence of the repeated polypeptide fragment is shown in SEQ ID No: 1 (GVVRLLGAVVGLLG);

[0012] The amino acid sequence of the wild-type type III human collagen is shown in SEQ ID No: 2:

[0013] .

[0014] In an optional embodiment, the number of substitutions of the repeated polypeptide fragment is 4-7 times.

[0015] In an optional embodiment, the number of amino acid residues between the repeated polypeptide fragments is not less than 7.

[0016] In an optional embodiment, the amino acid sequence of the recombinant type III human collagen polypeptide for injection is selected from any one of SEQ ID No: 3-6.

[0017] In a second aspect, the present invention provides any of the following biomaterials, including:

[0018] (i) a nucleic acid molecule encoding the recombinant human type III collagen for injection described in any one of the above embodiments;

[0019] (ii) a recombinant expression vector, a recombinant plasmid containing the nucleic acid molecule of (i);

[0020] (iii) Transformants, host cells containing the nucleic acid molecule (i) or the recombinant expression vector (ii).

[0021] In an optional embodiment, the original plasmid of the recombinant plasmid is pET-32a; and the recombinant expression vector contained in the host cell is linear.

[0022] In a third aspect, the present invention provides use of the recombinant type III human collagen polypeptide for injection as described in any one of the aforementioned embodiments in the preparation of a collagen gel or a collagen injection.

[0023] In an optional embodiment, the collagen injection is used to prepare a cosmetic product.

[0024] In a fourth aspect, the present invention provides a collagen injection, wherein the collagen in the collagen injection is the recombinant type III human collagen polypeptide for injection described in any one of the aforementioned embodiments.

[0025] In an optional embodiment, the collagen injection further contains active functional molecules; the active functional molecules include ferrous glucose and / or botulinum toxin type A.

[0026] Effects of the invention:

[0027] The present invention introduces multiple repeated leucine- and valine-rich polypeptide fragments into type III human collagen to increase the local spatial steric hindrance of the three monomers of type III human collagen in the process of assembling into fiber bundles, thereby forming a structure similar to an expansion joint, which is used to increase the load of water molecules, increase its water retention capacity and resistance to degradation, increase its half-life in the skin after injection, extend its effective period, reduce the injection frequency, and improve the medical compliance of patients / customers.

[0028] Furthermore, the present invention modifies the wild-type type III human collagen, truncates the type III human collagen, retains its active part, and reduces its molecular weight to one third of the original, making it more convenient to express it in an Escherichia coli expression system, thereby improving the expression activity and expression amount and reducing the production cost.

[0029] The modified type III human collagen is used to prepare collagen gel or recombinant type III human collagen polypeptide for injection, which can be used in the field of medical cosmetology to repair skin trauma, eliminate skin wrinkles, and keep the skin smooth and elastic for a long time. When it is made into an injection, under the same conditions, compared with the existing technology of once every 6-12 months, the injection period can be extended to once every 12-18 months, thereby reducing the injection frequency and improving patient / customer compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the pET-32a plasmid map;

[0031] Figure 2 The results are as follows: The effect of recombinant collagen 4 on the proliferation of human skin keratinocytes within 24 hours;

[0032] Figure 3 The results are as follows: The effect of recombinant collagen 4 on the proliferation of human skin keratinocytes within 48 hours;

[0033] Figure 4 This is the result of the scratch repair experiment using recombinant collagen 1;

[0034] Figure 5 This is the result of the scratch repair experiment using recombinant collagen 2;

[0035] Figure 6 This is the result of the scratch repair experiment using recombinant collagen 3;

[0036] Figure 7 These are the results of the scratch repair experiment using recombinant collagen 4. DETAILED DESCRIPTION

[0037] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0038] The term "and / or" means any one of the alternatives or two or more of the alternatives.

[0039] The term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, the situation consisting of the elements, integers or steps described is also covered.

[0040] The term "recombinant type III human collagen" refers to a full-length or partial amino acid sequence fragment encoded by a gene of a specific type of human collagen prepared by DNA recombinant technology, or a combination of functional fragments of human collagen.

[0041] Examples 1-4

[0042] Embodiment 1-4 is a method for preparing a recombinant type III human collagen polypeptide with an expansion joint-like structure, comprising the following steps:

[0043] (1) Construction of an expression vector containing a polynucleotide of a recombinant human type III collagen polypeptide for injection

[0044] In Examples 1-4, the synthesis of gene fragments was commissioned to Nanjing GenScript Biotechnology Co., Ltd. based on the corresponding nucleotide sequences of the amino acid sequences SEQ ID Nos: 3-6 as shown in SEQ ID Nos: 7-10.

[0045] SEQ ID No:3 is:

[0046] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvgllgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0047] SEQ ID No:4 is:

[0048] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvgllgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGgvvrllgavvgllgGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0049] SEQ ID No:5 is as follows:

[0050] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvgllgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGgvvrllgavvgllgGEPGRDGVgvvrllgavvgllgGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0051] SEQ ID No:6 is:

[0052] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGND

[0053] GARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPP

[0054] GPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvg

[0055] llgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGgvvrllgavvgllgGEPGRDG

[0056] VgvvrllgavvgllgGPGSDGKPgvvrllgavvgllgPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGG

[0057] PGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGE

[0058] PGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPG

[0059] AAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0060] SEQ ID No:7 is:

[0061]

[0062] SEQ ID No: 8 is as follows:

[0063] TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGC

[0064] GAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCG

[0065] CCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGAT

[0066] GGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGC

[0067] TTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGC

[0068] AACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAG

[0069] GGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGC

[0070] CCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCG

[0071] GCGGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCCGGGCAAAAAC

[0072] GGCGCGAAAGGCGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCGGC

[0073] GTGCCGGGCGCGAAAGGCGAAGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTG

[0074] CTGGGCCTGCCGGGCGCGGCGGGCGAACGCGGCGTGGTGCGCCTGCTGGGCGCGGTG

[0075] GTGGGCCTGCTGGGCCCGGGCGAAAAAGGCCCGGCGGGCGGCGTGGTGCGCCTGCTG

[0076] GGCGCGGTGGTGGGCCTGCTGGGCGGCGAACCGGGCCGCGATGGCGTGCCGGGCGGC

[0077] CCGGGCATGCGCGGCATGCCGGGCAGCCCGGGCGGCCCGGGCAGCGATGGCAAACCG

[0078] GGCCCGCCGGGCAGCCAGGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGAGCGGC

[0079] CCGCGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCG

[0080] CCGGGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCG

[0081] GGCAAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGC

[0082] GATAAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACC

[0083] GGCGGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGC

[0084] GGGCGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCG

[0085] GCCCGCCGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCC

[0086] CGGAAGGCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCC

[0087] CGGGCCTGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAG

[0088] GCGATAAAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCC

[0089] CGCGCGGCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATA

[0090] AAGGCGAAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGG

[0091] GCGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGGCGGCAAAGGCGAACGCGGC。

[0092] SEQ ID No:9 is:

[0093] TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGC

[0094] GAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCG

[0095] CCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGAT

[0096] GGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGC

[0097] TTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGC

[0098] AACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAG

[0099] GGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGC

[0100] CCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCG

[0101] GCGGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCCGGGCAAAAAC

[0102] GGCGCGAAAGGCGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCGGC

[0103] GTGCCGGGCGCGAAAGGCGAAGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTG

[0104] CTGGGCCTGCCGGGCGCGGCGGGCGAACGCGGCGTGGTGCGCCTGCTGGGCGCGGTG

[0105] GTGGGCCTGCTGGGCCCGGGCGAAAAAGGCCCGGCGGGCGGCGTGGTGCGCCTGCTG

[0106] GGCGCGGTGGTGGGCCTGCTGGGCGGCGAACCGGGCCGCGATGGCGTGGGCGTGGTG

[0107] CGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCGGCCCGGGCAGCGATGGCAAACCG

[0108] GGCCCGCCGGGCAGCCAGGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGAGCGGC

[0109] CCGCGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCG

[0110] CCGGGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCG

[0111] GGCAAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGC

[0112] GATAAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACC

[0113] GGCGGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGC

[0114] GGGCGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCG

[0115] GCCCGCCGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCC

[0116] CGGAAGGCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCC

[0117] CGGGCCTGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAG

[0118] GCGATAAAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCC

[0119] CGCGCGGCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATA

[0120] AAGGCGAAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGG

[0121] GCGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGGCGGCAAAGGCGAACGCGGC。

[0122] SEQ ID No:10 is as follows:

[0123] TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGC

[0124] GAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCG

[0125] CCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGAT

[0126] GGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGC

[0127] TTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGC

[0128] AACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAG

[0129] GGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGC

[0130] CCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCG

[0131] GCGGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCCGGGCAAAAAC

[0132] GGCGCGAAAGGCGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCGGC

[0133] GTGCCGGGCGCGAAAGGCGAAGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTG

[0134] CTGGGCCTGCCGGGCGCGGCGGGCGAACGCGGCGTGGTGCGCCTGCTGGGCGCGGTG

[0135] GTGGGCCTGCTGGGCCCGGGCGAAAAAGGCCCGGCGGGCGGCGTGGTGCGCCTGCTG

[0136] GGCGCGGTGGTGGGCCTGCTGGGCGGCGAACCGGGCCGCGATGGCGTGGGCGTGGTG

[0137] CGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCGGCCCGGGCAGCGATGGCAAACCG

[0138] GGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCCGGGCCCGAGCGGCC

[0139] CGCGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGC

[0140] CGGGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGG

[0141] GCAAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCG

[0142] ATAAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCG

[0143] GCGGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCG

[0144] GGCGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCGG

[0145] CCCGCCGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCCC

[0146] GGAAGGCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCCC

[0147] GGGCCTGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAGG

[0148] CGATAAAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCCC

[0149] GCGCGGCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATAA

[0150] AGGCGAAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGGG

[0151] CGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGGCGGCAAAGGCGAACGCGGC.

[0152] The synthesized gene fragment was inserted into the pET-32a expression vector (the original plasmid map is shown in Figure 1 ), and obtain the recombinant expression plasmid.

[0153] (2) Construction of genetically engineered Escherichia coli: The successfully constructed recombinant expression plasmid pET-32a was linearized by restriction enzyme digestion and then transformed into Escherichia coli competent cells BL21 (DE3).

[0154] The specific process is as follows: take out the competent E. coli cells BL21 (DE3) from the -80°C refrigerator and place them on ice. When they are half melted, take out 1 μL of the recombinant expression plasmid and add it to the competent E. coli cells BL21 (DE3) in the clean bench; place the mixture on ice for 5 minutes, then heat shock it in a 42°C water bath for 60 seconds, take it out and place it on ice for 5 minutes; add 500 μL of liquid LB culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 100 μg / ml ampicillin, 50 μg / ml kanamycin) in the clean bench, and revive it at 37°C and 220 rpm for 60 minutes; take out 200 μL of the revived bacterial solution and evenly spread it on the LB plate containing ampicillin and kanamycin; place the plate in a 37°C incubator overnight and wait for it to grow colonies of uniform size.

[0155] (3) Inducing Escherichia coli to express recombinant human type III collagen polypeptide with expansion joints

[0156] The specific process is as follows: pick the colonies on the plate to 10 ml LB liquid medium (100 μg / ml ampicillin, 50 μg / ml kanamycin), place in a 37°C shaker, culture at 220 rpm for about 12 h, transfer to 1 L culture medium, and culture at 37°C, 220 rpm until OD 600 When the pH was 0.6-0.8, 500 μL IPTG (final concentration 0.5 mM) was added and induced at 16°C and 160 rpm for 16-20 h.

[0157] (4) Protein purification

[0158] The specific process is as follows: the bacterial solution is divided into centrifuge bottles, centrifuged at 4000 rpm and 4°C for 20 minutes, and the bacterial cells are collected. The collected bacterial cells are resuspended in binding buffer (150 mM NaCl, 20 ml HEPES NaOH pH 7.4), and then ultrasonically disrupted. The bacterial solution after cell disruption is divided into centrifuge bottles, centrifuged at 16000 rpm and 4°C for 30 minutes, and the supernatant is collected.

[0159] The supernatant was added to a 1 mL Ni affinity resin gravity column. After the flow-through was completed, 8 times the column volume of binding buffer was added to wash the Ni column; then 8 times the column volume of washing buffer (150 mM NaCl, 20 ml HEPES NaOH pH 7.4, 20 mM imidazole) was added to wash the Ni column; finally, 8 times the column volume of elution buffer (150 mM NaCl, 20 ml HEPES NaOH pH 7.4, 300 mM imidazole) was used to elute the target protein to obtain a recombinant type III human collagen polypeptide monomer for injection, the amino acid sequence of which is shown in SEQ ID No: 3-6.

[0160] The endotoxin-removing column Detoxi-Gel was then used to further remove endotoxins and microorganisms.

[0161] The obtained recombinant humanized collagen monomer has a purity of 98.15-98.57% and an endotoxin limit test of 0.05EU / mg. The converted protein yield is 10-15g / L fermentation liquid (bacterial endotoxin test is carried out according to the method specified in GB / T14233.2).

[0162] The acidic solutions of collagen monomers of Examples 1-4 and Comparative Examples 1-2 were respectively dissolved in PBS buffer (pH 7.4). The instantaneous increase in pH value caused the collagen to self-assemble into a high-density fiber matrix, and then the collagen freeze-dried powder was prepared according to a conventional freeze-drying process.

[0163] Example 5

[0164] In this example, the recombinant collagen 4 obtained by inserting the fragment "gvvrllgavvgllg" 7 times in Example 4 was used as an example. Human skin keratinocytes (Human skin keratinocytes) in the G0 phase of the cell cycle were cultured, and different concentrations (g / mL) of recombinant collagen 4 (1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%) were added to investigate the effect of the period on the proliferation activity of human skin keratinocytes.

[0165] Human skin keratinocytes were seeded in an incomplete medium (DMEM medium lacking fetal bovine serum) plate, and after 24 hours, complete medium (DMEM medium containing 10% fetal bovine serum) was added to restart the cells from the G0 phase of the cell cycle. Recombinant collagen 4 (1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%) was added to the complete medium of the experimental group, and the complete medium with the corresponding concentration of marine collagen (MARINECOLLAGEN) was used as a positive control.

[0166] The culture medium of each experimental group was added to the wells containing cells in the G0 phase of the cell cycle, and the cells were incubated for 24h and 48h. At the end of the incubation period, MTT staining was performed to evaluate cell viability and proliferation index compared with the untreated control group (CT R, definition of 100% viability)

[0167] MTT staining method: 15 mg MTT (thiazolyl blue, CAS: 298-93-1) was added to 30 ml complete culture medium to prepare MTT complete culture medium. After the above cells were incubated, they were washed with 200 μL PBS. After removing the washing solution, 200 μL of MTT-complete culture medium was added to each culture well, and then incubated at 37°C and 5% CO 2 Incubate for 3 hours at 4 °C. At the end of the incubation period, remove the MTT medium and add 200 μL of isopropanol. Shake the plate on a rotating plate for 30 minutes to ensure that all crystals are dissolved from the cells and a homogenous solution is formed. The absorbance is measured at a wavelength of 540 nm using a microplate reader. The results are expressed as % cell viability compared to untreated control cell cultures. Figure 2 and Figure 3 As shown. It can be seen that both recombinant protein 4 and marine collagen can effectively increase cell proliferation in in vitro skin keratinocyte culture. In particular, under certain experimental conditions, recombinant protein 4 showed better performance than marine collagen.

[0168] Example 6

[0169] This example uses an in vitro experiment to detect the in vitro cell repair ability of the recombinant human collagen 1-4 obtained in Example 1.

[0170] In this example, the cells were cultured in a 12-well plate using Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37°C and 5% CO. 2 Human skin keratinocytes were cultured under the conditions of , and a human skin keratinocyte monolayer was obtained. Then, an artificial scratch was made on the monolayer by sliding the tip of a pipette. The scratch width was 1200μm±100μm. Then, DMEM cell culture medium containing 4mg / mL recombinant human collagen 1-4 was added to the artificial scratch, and the repair of the artificial scratch was detected at 24h, 48h, 72h and 96h. Untreated DMEM culture medium was used as a blank control group (CTR) in the experiment.

[0171] At each experimental time, the monolayer was visually inspected at 4x using a camera (OPTIKA, XDS-2), and camera pictures were collected (TiEsseLabPrimoCamHD 5.0 ​​with sensor Micron MT9P001, and the pictures were analyzed using Image J software ( Figures 4 to 7 As shown in the figure, it can be seen that recombinant collagens 1 to 4 can promote in vitro cell repair, and the promotion effect of recombinant collagen 4 is the most significant. The scratches are basically repaired at 72 hours, while the other recombinant proteins show that the repair is completed at 96 hours.

[0172] Test Example 1

[0173] The recombinant humanized collagen lyophilized powder prepared in Examples 1-4 was subjected to sterility testing.

[0174] According to the method specified in 1011 of the fourth volume of the 2015 edition of the Chinese Pharmacopoeia

[0175] The test sample was inoculated into 6 tubes of aerobic bacteria and anaerobic bacteria culture medium respectively by aseptic operation, of which 1 tube was inoculated with 1 ml of Staphylococcus aureus control bacterial solution as a positive control, and another 5 tubes of fungal culture medium were inoculated. Gently shake to mix the collagen freeze-dried powder obtained in the embodiment with the culture medium. The aerobic bacteria and anaerobic bacteria culture medium tubes were placed at 30-35°C, and the fungal culture medium tubes were placed at 20-25°C for 7 days. During the culture period, it should be observed and recorded daily whether there is bacterial growth. The positive control tube should have bacterial growth within 24 hours. If the culture medium becomes turbid after adding the collagen freeze-dried powder, after 7 days of culture, it is impossible to judge whether there is microbial growth from the appearance. The culture solution can be taken and transferred to the same fresh culture medium or the slant culture medium for continued culture. The bacteria were cultured for 2 days and the fungi were cultured for 3 days. Observe whether turbidity appears again or whether there is bacterial growth on the slant, or use an inoculation loop to take a smear of the culture solution, stain it, and observe whether there are bacteria under a microscope.

[0176] Results: The lyophilized collagen powder obtained in the examples and comparative examples is sterile and can be used for injection.

[0177] Test Example 2

[0178] Mechanical property test of the collagen gel prepared in Examples 1-4.

[0179] The freeze-dried powder prepared in each embodiment and comparative example was prepared into a hydrogel according to the following method, and the preparation method comprises the following steps:

[0180] At room temperature:

[0181] (1) Preparation of phase A: Add 2% collagen to 300 mM calcium chloride solution and mix thoroughly to obtain a collagen solution, i.e., phase A;

[0182] (2) Preparation of phase B: Disodium hydrogen phosphate and potassium dihydrogen phosphate were mixed at a molar ratio of 5:1, water was added to a total phosphate concentration of 0.04 M, and the pH value was adjusted to 7-8 with hydrochloric acid and sodium hydroxide to obtain a phosphate buffer solution, i.e., phase B.

[0183] Phase A and phase B were mixed in a volume ratio of 4:1 to obtain collagen hydrogel.

[0184] The viscoelasticity of the gel was measured using a Haake RS6000 (Thermo Fisher Scientific (China) Co., Ltd.) advanced rotational rheometer. Under the conditions of a test temperature of 25°C, a slit width of 1.00 mm, and a vibration frequency in the range of 0.1-100 Hz, the elasticity and viscosity of the injectable cross-linked recombinant collagen gel were dynamically measured, and the observation indicators were the elastic modulus (G') and the viscous modulus (G"). The elastic modulus (G') and the viscous modulus (G") were recorded at a frequency of 0.1 Hz.

[0185] As a result, the elastic modulus (G') of the hydrogels formed by the collagen prepared in Examples 1-4 was greater than 2000, and the viscosity modulus (G") was greater than 250, which was superior to the hydrogels prepared by the commercially available common wild type III human collagen and had similar performance.

[0186] Test Example 3

[0187] Sterilization stability of the collagen prepared in Example 1-4

[0188] The collagen gel prepared in Test Example 1 was subjected to a sterilization stability test.

[0189] The sterilization is 121°C high pressure steam sterilization.

[0190] According to the rotational viscometer method of viscosity determination method 0633 of the "Pharmacopoeia of the People's Republic of China (Volume IV)" (2020 edition), a rotor-type viscometer is used to immerse the rotor in the sample to be tested and rotate it at a constant angular velocity (ω) to measure the torque (M) generated by the motor rotation. The viscosity of the sample to be tested can be obtained according to the formula η=K*M / η (K is a constant). The results show that there is no obvious change in the viscosity of the sample before and after sterilization, indicating that moist heat sterilization can ensure the sterilization stability of the collagen gel sample.

[0191] Test Example 4

[0192] Wound healing test of the collagen prepared in Example 1-4 and the commercially available full-length human recombinant collagen injection (commercial control)

[0193] The collagen gel prepared in Test Example 1 was subjected to a wound healing test.

[0194] The guinea pigs were fasted for 18 h in advance and anesthetized with 10% chloral hydrate at a dose of 0.3 mL / 100 g by intraperitoneal injection. The rats were depilated on both sides of the back with 8% sodium sulfide solution. After disinfection, circular skin defect wounds with a diameter of 1.0 cm were made on both sides of the spine of the human and rats, reaching the fascia layer. After hemostasis, the wounds were disinfected and medication was given.

[0195] The guinea pigs with wounds were divided into model group, experimental group and control group, and normal guinea pigs without wounds were used as the normal group.

[0196] In the test group, the test sample was administered. Before administration, the drug was transferred onto a sulfuric acid paper to form a hydrogel. The sulfuric acid paper carrying the hydrogel was covered on the wound surface of the guinea pig and fixed with medical tape.

[0197] The dressing was changed once a day and the animals were allowed to eat freely for 14 consecutive days. One week after stopping the drug, the newly formed skin on the wound surface was collected as skin tissue samples.

[0198] Evaluation indicators

[0199] The appearance of the wound was observed with the naked eye and photographed, and the wound healing rate (%) = (original wound area - unhealed wound area) / original wound area × 100% was calculated 3d, 7d and 12d after the administration was completed.

[0200] The results show that the collagen prepared in Examples 1-4 of the present invention has better wound repair ability than the commercially available full-length human recombinant collagen injection and Comparative Example 1.

[0201] Test Example 5

[0202] Anti-aging performance test of the collagen prepared in Example 1-4 and the commercially available full-length human recombinant protein

[0203] 1. The collagen prepared in Example 1-4 is prepared into an injection, and the preparation method is as follows:

[0204] S1, take 2g of sodium hyaluronate powder and put it in 50ml of cold water for injection, swell naturally for 60min, heat and stir evenly at 80°C, the stirring speed is 800rpm, and the stirring time is 30min to obtain solution A1;

[0205] S2, after the solution A1 is cooled to 45°C, 1 g of collagen is added and stirred for 30 min, and then 0.4 g of glutathione glutamate is added and stirred for 20 min to obtain a solution B1;

[0206] S3, after the solution B1 is cooled to 25°C, 3.5 mg of sodium dihydrogen phosphate and 10 mg of sodium dihydrogen phosphate are added to the solution B1 to adjust the pH value to 7.2, and then sodium chloride is added to adjust the osmotic pressure to 300 mmol / L, and wet heat sterilization is performed to obtain solution C1,

[0207] S4, take another 50 ml of water for injection, add 3.5 mg of sodium dihydrogen phosphate and 10 mg of sodium dihydrogen phosphate at room temperature and adjust the pH value to 7.2, then add 0.45 g of sodium chloride to adjust the osmotic pressure to 300 mmol / L, to obtain solution A2;

[0208] S5, adding 30 mg of tranexamic acid to the solution A2 and dissolving it fully, filtering and sterilizing with a 0.2 μm microporous filter membrane to obtain a solution B2 for standby use;

[0209] S6, mixing the solution C1 and the solution B2 evenly, and performing ultrasonic vibration at 35° C. for 10 min at a frequency of 25 KHz to obtain a collagen injection solution.

[0210] 2. Effect of collagen injection on the contents of T-AOC, SOD and MDA in skin tissue of mice with UV skin photoaging model.

[0211] 2.1 The mice were randomly divided into 8 groups: a normal control group, a model control group, one group each of Examples 1-4, and one group of commercially available full-length human recombinant protein, with 10 mice in each group. The mice were injected with collagen injection (50 mg / kg), and the normal group and the model control group were injected with the same dose of normal saline. Each group of mice was irradiated with ultraviolet light from the third day after injection until the end of the experiment. The mice were depilated with 4% sodium sulfide before the first irradiation, and then depilated once every two weeks after the irradiation until the end of the irradiation. The depilated mice were placed under ultraviolet light (UVA+UVB, simulating sunlight) and irradiated three times a week. The irradiation dose in the first week was 2mJ / cm each time. 2 2 mJ / cm2 once a week 2 The dose was escalated to 8 mJ / cm per session in week 4. 2 . Continue irradiation at this dose until the 6th week.

[0212] 2.2 Determination of antioxidant indexes

[0213] Antioxidant enzyme activity and MDA content in serum After the experiment, blood was collected from the eyeballs. Serum was separated and the activities of SOD, GSH-Px, CAT and MDA content in serum were determined according to the instructions of the kit.

[0214] 2.3 Antioxidant enzyme activities and MDA and Hyp contents in skin tissue homogenates

[0215] Take about 0.5g of depilated skin tissue from the back, rinse with precooled saline, remove subcutaneous fat and other connective tissues, wipe dry with filter paper, and weigh. Pour into a beaker with tissue blocks, cut the skin into pieces, add 9 times the weight of precooled saline, and homogenize in an ice bath. Centrifuge at 4℃, 8000r / min for 15min, take the supernatant, and measure the SOD, GSH-Px, CAT activities and MDA, Hyp contents according to the instructions of the kit.

[0216] 2.4 Skin histological observation

[0217] Take 0.5cm of the back area 2 Skin tissues of different sizes were fixed with 4% neutral formaldehyde and frozen into sections with a thickness of 7 μm. The sections were stained with HE and Van Gieson respectively, and observed and photographed under an optical microscope.

[0218] Conclusion: The collagen prepared in Examples 1-4 of the present invention has better anti-aging ability than the commercially available full-length human recombinant collagen injection.

[0219] Test Example 6

[0220] Clinical trials, skin beauty anti-wrinkle tests

[0221] The injection was prepared according to the method of Test Example 5 and a clinical trial was conducted on volunteers. The observation indicators included: skin lesion area and color, transepidermal water loss (TEWL), epidermal oil content and epidermal water content.

[0222] 1. Skin lesion area and color

[0223] The injections prepared in Examples 1-4, the injections of Comparative Example 1, and the commercially available full-length human recombinant collagen injections were respectively injected for treatment, and the skin lesion area and skin lesion color before and after treatment were scored (scoring was performed at 1 month, 3 months, 6 months, 12 months, and 18 months after injection). The results showed that after treatment with the injections of Examples 1-4 of the present invention, the skin lesion area and skin lesion color were reduced.

[0224] 2. Furthermore, after treatment with the injections of Examples 1-4 of the present invention, transepidermal water loss (TEWL) was reduced, epidermal oil content was increased, and epidermal water content was increased, and the duration of the effects was longer than that of Comparative Example 1 and commercially available full-length human recombinant collagen injections.

[0225] 3. DLQI score and patient satisfaction evaluation: Patients were surveyed and scored on DLQI; then patients were asked to give satisfaction ratings based on their subjective feelings such as treatment comfort and perceived efficacy, with a total of four levels: very satisfied, satisfied, average, and dissatisfied. Satisfaction = (very satisfied + satisfied) cases / total cases × 100%. The results showed that after injection of the injection of Examples 1-4 of the present invention, the satisfaction and duration were longer than those of the commercially available full-length human recombinant collagen injection.

[0226] 4. Anti-wrinkle, comparison of wrinkle visual effects

[0227] The injections prepared in Examples 1-4 of the present invention were respectively used for injection treatment, and all of them could achieve the anti-wrinkle effect; and after the injection treatment of Examples 1-4 of the present invention, the subjective visual perception was that the skin was glossy and elastic, and the anti-wrinkle effect was better than the commercially available full-length human recombinant collagen injection.

[0228] It can be seen from the above embodiments and test examples that the present invention retains the activity of collagen and shortens the length of the polypeptide to be expressed by intercepting the amino acid residues 268 to 828 of type III human collagen, making it easier to obtain active high expression in Escherichia coli, and introduces multiple repeated polypeptide fragments rich in glycine, leucine and valine SEQ ID NO: 1 = GVVRLLGAVVGLLG between the amino acid residues 411 to 639 of type III human collagen, thereby increasing the local steric hindrance of the three monomers of injectable recombinant type III human collagen in the process of assembling into fiber bundles, improving the ability of collagen to accommodate water molecules, slowing down the absorption and metabolism of the human body, and increasing the half-life of collagen in the skin. It can not only be prepared into a conventional protein gel for external use, but also be made into an injection for skin anti-wrinkle and whitening. The injection can reduce the injection frequency and injection amount, and can further reduce the side effects of collagen injection.

[0229] The above are only a few preferred embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A recombinant type III human collagen polypeptide for injection, characterized in that: The amino acid sequence of the recombinant type III human collagen polypeptide for injection is selected from any one of SEQ ID No: 3-6.

2. Biological material, including any one of (i) to (iii): (i) a nucleic acid molecule encoding the recombinant human type III collagen polypeptide for injection according to claim 1; (ii) a recombinant expression vector, comprising a recombinant plasmid of the nucleic acid molecule of (i); (iii) Transformants, host cells containing the nucleic acid molecule (i) or the recombinant expression vector (ii).

3. The biomaterial according to claim 2, characterized in that The original plasmid of the recombinant plasmid is pET-32a; The recombinant expression vector contained in the host cell is linear.

4. Use of the recombinant type III human collagen polypeptide for injection according to claim 1 in the preparation of collagen gel or collagen injection.

5. The use according to claim 4, characterized in that: The collagen injection is used for preparing beauty products.

6. Collagen injection, characterized in that: The collagen in the collagen injection is the recombinant type III human collagen polypeptide for injection as described in claim 1.

7. The collagen injection according to claim 6, characterized in that: It also contains active functional molecules; the active functional molecules include ferrous gluconate and / or botulinum toxin type A.

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