Recombinant type III human collagen polypeptide with disulfide bonds introduced, preparation method and uses thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to a recombinant type III human collagen polypeptide with introduced disulfide bonds, a preparation method thereof, and uses thereof. By truncating the amino acid residues at positions 268 to 828 of type III human collagen, the present invention retains the activity of collagen, shortens the length of the polypeptide to be expressed, and makes it easier to obtain highly expressed and active products in Escherichia coli. Additionally, multiple repeated polypeptide fragments rich in glycine, leucine, and valine, namely CVVRLLGAVVGLLC, are introduced between the amino acid residues at positions 411 to 639 of type III human collagen, increasing the local steric hindrance during the assembly of the three monomers of the recombinant type III human collagen for injection into fiber bundles, enhancing the ability of collagen to accommodate water molecules. Meanwhile, cysteines introduced at both ends can couple to form disulfide bonds, thereby improving the stability of the polymer and further increasing the half-life of the collagen in the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a recombinant type III human collagen polypeptide into which a disulfide bond is introduced, a preparation method thereof, and uses thereof. Background Art

[0002] Collagen is a biological macromolecule, the main component of animal connective tissue, and also the most abundant and widely distributed functional protein in mammals, accounting for 25% - 30% of the total protein, and even up to more than 80% in some organisms. Animal tissues of livestock and poultry origin are the main sources for people to obtain natural collagen and its collagen peptides. However, due to related livestock diseases, the use of collagen and its products from terrestrial mammals is restricted.

[0003] According to their distribution and functional characteristics in the body, collagens can be divided into interstitial collagens, basement membrane collagens, and pericellular collagens. Interstitial collagen molecules account for the vast majority of the collagens in the whole body, including type I, II, and III collagen molecules. Collagen is a natural protein of the body, has a large affinity for protein molecules on the skin surface, weak antigenicity, good biocompatibility, and biodegradation safety, can be degraded and absorbed, and has good adhesion. Collagen has functions such as 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 the skin of infants, the inner membrane of blood vessels, and the intestine. Type III collagen is the main component of reticular fibers in the skin. The higher the content, the finer the fiber bundles. In the field of skin anti-wrinkle, when the proportion of type III collagen is high, the skin tissue is delicate and smooth. However, the fiber bundles formed by type III collagen are too thin and the spacing between the fiber bundles is too small, making its water retention ability and anti-degradation ability relatively weak, and it needs to be further modified to improve its half-life in the skin. At the same time, the existing recombinant type III collagen molecules are long in length, large in molecular weight, and complex in structure, and a yeast expression system needs to be used, resulting in high preparation costs.

[0005] In the existing daily chemical industry, the collagen contained in collagen supplement products is usually a degraded small molecule, omitting the in vivo degradation process of collagen and being able to be absorbed by the skin faster. However, the aggregates formed by self-assembly of small molecule collagen have poor stability and are easily decomposed into monomers, losing the collagen function. It has been reported that type III human collagen is modified to form a fiber micelle with a structure similar to an expansion joint, which can be used to store and release water molecules and improve the water retention ability. However, although the water storage ability of the conventionally modified expansion joint structure increases, it has not completely solved the instability problem and has a short half-life, resulting in a great reduction in practicality.

[0006] In view of the above two drawbacks, the present invention is specifically proposed. Summary of the Invention

[0007] Problems to be Solved by the Invention:

[0008] 1. Modify type III human collagen to form a fiber micelle with a structure similar to an expansion joint, which can be used to store and release water molecules, improve water retention ability, and further extend its half-life in the skin.

[0009] 2. Truncate type III human collagen, retain its active part, reduce its molecular weight to one-third of the original, facilitate its expression in the Escherichia coli expression system, improve the expression activity and expression level, and reduce the production cost.

[0010] 3. Introduce cysteine at both ends of the structure similar to an expansion joint, so that while the structure similar to an expansion joint is formed, there are disulfide bonds for monomer coupling at both ends of the structure similar to an expansion joint, thereby improving the stability of the polymer.

[0011] Solutions for Solving the Problems Include:

[0012] In a first aspect, the present invention provides a recombinant type III human collagen polypeptide for injection, which comprises amino acid residues 268 to 828 of wild-type type III human collagen, and a partial amino acid fragment between amino acid residues 411 to 639 of the wild-type type III human collagen is replaced by a plurality of repetitive polypeptide fragments;

[0013] The amino acid sequence of the repetitive polypeptide fragment is as shown in SEQ ID No: 1 (CVVRLLGAVVGLLC);

[0014] The amino acid sequence of the wild-type type III human collagen is as shown in SEQ ID No: 2:

[0015] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0016] In an alternative embodiment, the number of substitutions of the repetitive polypeptide fragment is 4 - 7 times.

[0017] In an alternative embodiment, the number of amino acid residues between the repetitive polypeptide fragments is not less than 7.

[0018] In an alternative embodiment, the amino acid sequence of the recombinant human type III collagen polypeptide for injection is selected from any one of SEQ ID No: 3 - 6.

[0019] In a second aspect, the present invention provides any one of the following biomaterials, including:

[0020] (i) A nucleic acid molecule encoding the recombinant human type III collagen for injection according to any one of the above embodiments;

[0021] (ii) A recombinant expression vector, a recombinant plasmid containing the nucleic acid molecule of (i);

[0022] (iii) A transformant, a host cell containing the nucleic acid molecule of (i) or the recombinant expression vector of (ii).

[0023] In an alternative embodiment, the original plasmid of the recombinant plasmid is pET-32a; the recombinant expression vector contained in the host cell is linear.

[0024] Thirdly, the present invention provides the use of the recombinant human type III collagen polypeptide for injection according to any one of the foregoing embodiments in the preparation of a collagen gel or a collagen injection.

[0025] In an alternative embodiment, the collagen injection is used for the preparation of a beauty product.

[0026] Fourthly, the present invention provides a collagen injection, wherein the collagen in the collagen injection is the recombinant human type III collagen polypeptide for injection according to any one of the foregoing embodiments.

[0027] In an alternative embodiment, the collagen injection further contains active functional molecules; the active functional molecules include ferrous gluconate and / or botulinum toxin type A.

[0028] Effects of the invention:

[0029] In the present invention, by introducing multiple repeated polypeptide fragments rich in leucine and valine into type III human collagen, the local steric hindrance during the assembly of the three monomers of type III human collagen into fibrils is increased, thereby forming a structure similar to an expansion joint, which can be used to increase the water molecule load, enhance its water retention ability and resistance to degradation, increase its half-life in the skin after injection, extend its validity period, reduce the injection frequency, and improve the medical compliance of patients / customers.

[0030] Furthermore, in the present invention, by modifying wild-type type III human collagen, truncating type III human collagen and retaining its active part, its molecular weight is changed to one-third of the original, which is more convenient for its expression in the Escherichia coli expression system, improves the expression activity and expression level, and reduces the production cost.

[0031] Using the modified type III human collagen to prepare a collagen gel or a recombinant human type III collagen polypeptide for injection can be used in the field of medical beauty, repair skin wounds, eliminate skin wrinkles, and keep the skin smooth and elastic for a long time. When made into an injection, compared with the prior art that requires injection once every 6-12 months under the same conditions, it can be extended to once every 12-18 months, reducing the injection frequency and improving the compliance of patients / customers.

[0032] Compared with the substituted fragment without the introduction of disulfide bonds, the present invention introduces cysteine ​​at both ends of the expansion joint-like structure, so that while the expansion joint-like structure is formed, there are disulfide bonds coupling between monomers at both ends of the expansion joint-like structure, thereby improving the stability of the polymer and further increasing the half-life of the collagen in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the pET-32a plasmid map;

[0034] Figure 2 The effect of recombinant collagen 4 on the proliferation of human skin keratinocytes;

[0035] Figure 3 The results of the recombinant collagen 4 scratch test. DETAILED DESCRIPTION

[0036] 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.

[0037] The term "and / or" means any one of the alternatives or two or more of the alternatives.

[0038] 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.

[0039] The term "recombinant human type III collagen" refers to a specific type of human collagen gene produced by DNA recombinant technology.

[0040] The protein may be a fragment of the full-length or partial amino acid sequence encoded by the protein, or a combination of functional fragments of human collagen.

[0041] Examples 1-4

[0042] Example 1-4 is a method for preparing a recombinant type III human collagen polypeptide for injection, 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] Examples 1-4 were respectively commissioned by Nanjing Genscript Biotech Co., Ltd. to synthesize gene fragments according to the corresponding nucleotide sequences of amino acid sequences SEQ ID No: 3-6 as shown in SEQ ID No: 7-10.

[0045] SEQ ID No: 3 is:

[0046] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAcvvrllgavvgllcPGKNGAKGcvvrllgavvgllcGVPGAKGEcvvrllgavvgllcLPGAAGERcvvrllgavvgllcPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG.

[0047] SEQ ID No: 4 is:

[0048] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAcvvrllgavvgllcPGKNGAKGcvvrllgavvgllcGVPGAKGEcvvrllgavvgllcLPGAAGERcvvrllgavvgllcPGEKGPAGcvvrllgavvgllcGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

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

[0050] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAcvvrllgavvgllcPGKNGAKGcvvrllgavvgllcGVPGAKGEcvvrllgavvgllcLPGAAGERcvvrllgavvgllcPGEKGPAGcvvrllgavvgllcGEPGRDGVcvvrllgavvgllcGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0051] SEQ ID No:6 is:

[0052] FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAcvvrllgavvgllcPGKNGAKGcvvrllgavvgllcGVPGAKGEcvvrllgavvgllcLPGAAGERcvvrllgavvgllcPGEKGPAGcvvrllgavvgllcGEPGRDGVcvvrllgavvgllcGPGSDGKPcvvrllgavvgllcPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERG。

[0053] SEQ ID No:7 is: (corresponding to 3)

[0054]

[0055] SEQ ID No: 8 is: (corresponding to 4)

[0056] TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGC

[0057] GAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCG

[0058] CCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGAT

[0059] GGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGC

[0060] TTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGC

[0061] AACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAG

[0062] GGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGC

[0063] CCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCG

[0064] GCGTGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCCCGGGCAAAAACG

[0065] GCGCGAAAGGCTGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCGGCGT

[0066] GCCGGGCGCGAAAGGCGAATGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCT

[0067] GTGCCTGCCGGGCGCGGCGGGCGAACGCTGCGTGGTGCGCCTGCTGGGCGCGGTGGTG

[0068] GGCCTGCTGTGCCCGGGCGAAAAAGGCCCGGCGGGCTGCGTGGTGCGCCTGCTGGGC

[0069] GCGGTGGTGGGCCTGCTGTGCGGCGAACCGGGCCGCGATGGCGTGCCGGGCGGCCCG

[0070] GGCATGCGCGGCATGCCGGGCAGCCCGGGCGGCCCGGGCAGCGATGGCAAACCGGGC

[0071] CCGCCGGGCAGCCAGGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGAGCGGCCCG

[0072] CGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGCCG

[0073] GGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGGGC

[0074] AAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCGAT

[0075] AAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCGGC

[0076] GGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCGGG

[0077] CGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCGGCCC

[0078] GCCGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCCCGGA

[0079] AGGCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCCCGGG

[0080] CCTGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAGGCGA

[0081] TAAAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCCCGCG

[0082] CGGCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATAAAGG

[0083] CGAAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGGGCGA

[0084] ACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGGCGGCAAAGGCGAACGCGGC。

[0085] SEQ ID No:9 is: (corresponding to 5)

[0086] TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGC

[0087] GAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCG

[0088] CCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGAT

[0089] GGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGC

[0090] TTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGC

[0091] AACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAG

[0092] GGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGC

[0093] CCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCG

[0094] GCGTGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCCCGGGCAAAAACG

[0095] GCGCGAAAGGCTGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCGGCGT

[0096] GCCGGGCGCGAAAGGCGAATGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCT

[0097] GTGCCTGCCGGGCGCGGCGGGCGAACGCTGCGTGGTGCGCCTGCTGGGCGCGGTGGTG

[0098] GGCCTGCTGTGCCCGGGCGAAAAAGGCCCGGCGGGCTGCGTGGTGCGCCTGCTGGGC

[0099] GCGGTGGTGGGCCTGCTGTGCGGCGAACCGGGCCGCGATGGCGTGTGCGTGGTGCGCC

[0100] TGCTGGGCGCGGTGGTGGGCCTGCTGTGCGGCCCGGGCAGCGATGGCAAACCGGGCC

[0101] CGCCGGGCAGCCAGGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGAGCGGCCCGC

[0102] GCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGCCGG

[0103] GCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGGGCA

[0104] AAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCGATA

[0105] AAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCGGCG

[0106] GCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCGGGC

[0107] GCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCGGCCCG

[0108] CCGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCCCGGAA

[0109] GGCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCCCGGGC

[0110] CTGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAGGCGAT

[0111] AAAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCCCGCGC

[0112] GGCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATAAAGGC

[0113] GAAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGGGCGAA

[0114] CGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGGCGGCAAAGGCGAACGCGGC。

[0115] SEQ ID No: 10 is: (corresponding to 6)

[0116] TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGC

[0117] GAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCG

[0118] CCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGAT

[0119] GGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGC

[0120] TTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGC

[0121] AACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAG

[0122] GGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGC

[0123] CCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCG

[0124] GCGTGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCCCGGGCAAAAACG

[0125] GCGCGAAAGGCTGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCGGCGT

[0126] GCCGGGCGCGAAAGGCGAATGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCT

[0127] GTGCCTGCCGGGCGCGGCGGGCGAACGCTGCGTGGTGCGCCTGCTGGGCGCGGTGGTG

[0128] GGCCTGCTGTGCCCGGGCGAAAAAGGCCCGGCGGGCTGCGTGGTGCGCCTGCTGGGC

[0129] GCGGTGGTGGGCCTGCTGTGCGGCGAACCGGGCCGCGATGGCGTGTGCGTGGTGCGCC

[0130] TGCTGGGCGCGGTGGTGGGCCTGCTGTGCGGCCCGGGCAGCGATGGCAAACCGTGCGT

[0131] GGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGTGCCCGGGCCCGAGCGGCCCGCGC

[0132] GGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGCCGGGC

[0133] AAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGGGCAA

[0134] AAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCGATAA

[0135] AGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCGGCGG

[0136] CCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCGGGCG

[0137] CGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCGGCCCGC

[0138] CGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCCCGGAAG

[0139] GCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCCCGGGCC

[0140] TGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAGGCGATA

[0141] AAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCCCGCGCG

[0142] GCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATAAAGGCG

[0143] AAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGGGCGAAC

[0144] GCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGGCGGCAAAGGCGAACGCGGC。

[0145] Insert the synthesized gene fragment into the pET-32a expression vector through the restriction enzyme sites of KpnI and BamHI (the original plasmid map is as Figure 1 shown) to obtain the recombinant expression plasmid.

[0146] (2) Construct an Escherichia coli genetic engineering bacterium: After linearizing the successfully constructed recombinant expression plasmid pET-32a by restriction enzyme digestion, transform it into the Escherichia coli competent cell BL21(DE3).

[0147] The specific process is as follows: Take out Escherichia coli competent cell BL21(DE3) from an -80°C refrigerator and place it on ice. When it is half-thawed, take 1 μL of the recombinant expression plasmid in a sterile bench and add it to Escherichia coli competent cell BL21(DE3); place the mixture on ice for an ice bath for 5 min, then perform heat shock in a 42°C water bath for 60 s, take it out and place it on ice for an ice bath for 5 min; add 500 μL of liquid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 100 μg / ml ampicillin antibiotic, 50 μg / ml kanamycin antibiotic) in a sterile bench, and place it at 37°C and 220 rpm for 60 min of recovery; take 200 μL of the recovered bacterial solution and evenly spread it on an LB plate containing ampicillin antibiotic and kanamycin antibiotic; place the plate in an incubator at 37°C for overnight culture until colonies of uniform size grow.

[0148] (3) Induce the expression of recombinant human type III collagen polypeptide for injection in genetically engineered Escherichia coli

[0149] The specific process is as follows: Pick colonies on the plate into 10 ml of LB liquid medium (100 μg / ml ampicillin antibiotic, 50 μg / ml kanamycin antibiotic), place it in a shaker at 37°C, culture at 220 rpm for about 12 h and then transfer it to 1 L of medium, culture at 37°C and 220 rpm until the OD600 reaches 0.6 - 0.8, add 500 μL of IPTG (final concentration 0.5 mM), and induce at 16°C and 160 rpm for 16 - 20 h.

[0150] (4) Purification of the protein

[0151] The specific process is as follows: Aliquot the bacterial solution into centrifuge bottles, centrifuge at 4000 rpm and 4°C for 20 min to collect the bacterial cells. Resuspend the collected bacterial cells with binding buffer (150 mM NaCl, 20 ml HEPES NaOH pH 7.4), then perform ultrasonic disruption. Aliquot the disrupted bacterial solution into centrifuge bottles, centrifuge at 16000 rpm and 4°C for 30 min to collect the supernatant.

[0152] Add the supernatant to a 1 mL Ni affinity resin gravity column. After the flow-through is completed, add 8 column volumes of binding buffer to wash the Ni column; then add 8 column volumes of washing buffer (150 mM NaCl, 20 ml HEPES NaOH pH 7.4, 20 mM imidazole) to wash the Ni column; finally, elute the target protein with 8 column volumes of elution buffer (150 mM NaCl, 20 ml HEPES NaOH pH 7.4, 300 mM imidazole) to obtain the monomer of recombinant human type III collagen polypeptide for injection, and the amino acid sequence is as shown in SEQ ID No: 3 - 6.

[0153] Subsequently, a Detoxi-Gel endotoxin removal column was used for further removal of endotoxins and microorganisms.

[0154] Recombinant humanized collagen monomers with a purity of 98.30 - 99.25% and a qualified endotoxin limit detection of 0.05 EU / mg were obtained. After conversion, the pure protein yield was 7 - 9 g / L of the fermentation broth (for bacterial endotoxin detection, the method specified in GB / T 14233.2 was used).

[0155] The acidic solutions of the collagen monomers in Examples 1 - 4 were respectively mixed into PBS buffer (pH 7.4). The self-assembly of collagen into a high-density fiber matrix was induced by a transient increase in pH value, and then the collagen freeze-dried powder was prepared according to the conventional freeze-drying process.

[0156] Example 5

[0157] In this example, a cell viability detection kit (CCK-8) was used to detect the effect of the recombinant collagen 4 obtained in Example 4 on the proliferation of human skin keratinocytes. The basic principle is as follows: This kit contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan product under the action of the electron carrier 1-methoxy-5-methylphenazinium sulfate (1-MethoxyPMS). The amount of formazan produced is proportional to the number of living cells. Therefore, this property can be used directly for cell proliferation and toxicity analysis. The CCK-8 method is a highly sensitive, non-radioactive colorimetric detection method for determining the number of living cells in cell proliferation or toxicity experiments and can replace the traditional MTT method.

[0158] The experimental steps are as follows:

[0159] 1. Add 100 μL of cells to each well of a 96-well plate (leave 2 blank groups without adding cells and add the same volume of medium), approximately containing 2000 cells. The cells were cultured in a 5% CO 2 incubator at 37°C for 24 h.

[0160] 2. Add 10 μL of 4 mg / mL recombinant collagen 4 to each well, and add the same volume of medium to the control group.

[0161] 3. Incubate the 96-well plate in a cell incubator at 37°C with 5% CO 2 air and 100% humidity for 36 h.

[0162] 4. Add 10 μL of CCK-8 solution to each well. Incubate at 37 °C in a 5% CO 2 incubator for 1 - 4 h.

[0163] 5. Measure the absorbance at 450 nm using a microplate reader.

[0164] 7. Result analysis:

[0165] A. Cell viability: Subtract the OD value of the background (blank group) from the OD value of each test well, and take the average ± SD of the OD values of each replicate well.

[0166] Cell viability % = (OD of drug-treated cells / OD of control cells) × 100%.

[0167] B. Calculate the drug concentration (IC50) when T / C = 50% and the drug concentration (IC90) when T / C = 10%.

[0168] The experimental results are as Figure 2 shown. It can be seen that the recombinant collagen 4 provided in Example 4 of the present invention can significantly promote the proliferation of human skin keratinocytes.

[0169] Example 6

[0170] In this example, an in vitro experiment was used to detect the in vitro cell repair ability of the recombinant human-derived collagen 4 obtained in Example 1.

[0171] In this example, in a 12-well plate, human skin keratinocytes were cultured in DMEM medium (Dulbecco's Modified Eagle’s medium) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO 2 condition to obtain a single cell layer of human skin keratinocytes. Then, a pipette tip was slid on the single cell layer to create an artificial scratch with a width of 1200 μm ± 100 μm. Then, DMEM cell culture medium containing 4 mg / mL of recombinant human-derived collagen 4 was added to the artificial scratch, and the repair of the artificial scratch was detected at 24 h, 48 h, 72 h, and 96 h later. In the experiment, untreated DMEM medium was used as the blank control group (CTR).

[0172] At each experimental time, the single cell layer was visually inspected at 4× magnification using a camera (OPTIKA, XDS-2), and camera pictures were collected (TiEsseLab PrimoCamHD 5.0 with a sensor micron MT9P001, and the pictures were analyzed using Image J software ( Figure 3 as shown). It can be seen that recombinant collagen 4 can promote in vitro cell repair and has completed the repair at 96 h.

[0173] As can be seen from the above embodiments and test examples, the present invention intercepts the amino acid residues at positions 268 to 828 of type III human collagen, retains the collagen activity, shortens the length of the polypeptide to be expressed, and is more likely to obtain highly expressed and active products in Escherichia coli. Moreover, multiple repeated polypeptide fragments rich in glycine, leucine, and valine, SEQ ID NO: 1 = CVVRLLGAVVGLLC, are introduced between the amino acid residues at positions 411 to 639 of type III human collagen, increasing the local steric hindrance during the assembly of the three monomers of the recombinant type III human collagen for injection into fibrotic bundles, improving the ability of collagen to accommodate water molecules, slowing down the absorption and metabolism of the human body, 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, reduce the injection volume, and further reduce the side effects of collagen injection. At the same time, the introduction of disulfide bonds replacing both ends of the amino acid fragment helps to improve the stability of the collagen polymer, further enhancing the stability and half-life of the collagen of the present invention, greatly increasing its practicality.

[0174] The above are only a limited number of preferred embodiments of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these 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 shown in SEQ ID No:

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.

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.

Images