Recombinant collagen and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a recombinant collagen which is characterized by comprising one or more of the following characteristics: a, an amino acid sequence of the collagen is shown as any one of SEQ ID NO.1-4; and b. A, the collagen is added, substituted or deleted with one or more amino acids, and the protein has collagen activity. The recombinant collagen prepared by utilizing a genetic engineering technology has the remarkable advantages of stable quality, no virus transmission risk and the like, and has a good application prospect in the field of medical materials.

Description

TECHNICAL FIELD

[0001] The present specification relates to the field of bioengineering, in particular to a recombinant collagen and application thereof. BACKGROUND

[0002] Collagen is a family of proteins with a unique triple helix structure in the extracellular matrix, and is the most abundant structural and functional protein in the human body. Collagen self-assembles into a fibrous network structure in vivo, providing a biological scaffold for tissues and organs, and participating in the regulation of basic physiological activities such as cell proliferation, migration and differentiation. Collagen is widely used in the fields of tissue engineering and regenerative medicine due to its excellent biocompatibility, biodegradability and bioabsorbability.

[0003] Currently, 28 types of collagen have been found, and different types of collagen have different functions. Generally, they can be divided into two categories: one is fibrous collagen, and the other is non-fibrous collagen; fibrous collagen includes types I, II, III, V, VI and XXVI, and the rest belongs to non-fibrous collagen. The most abundant in the human body is type I collagen, which accounts for more than 85%, and type I collagen is high in bone, skin, tendon and cornea, type II is present in cartilage, intervertebral disc and vitreous body, and type III is present in blood vessels, newborn skin and scar tissue.

[0004] Currently, collagen is mainly extracted from animal tissues, but there are still the following limitations: (i) collagen structure is damaged, the extraction process may damage the integrity of the collagen structure and cause the destruction of tryptophan, serine and tyrosine; (ii) there is a large difference between batches of collagen products, due to the differences in age, strain and other factors of the tissue raw materials, there is a significant difference between different batches of collagen products; (iii) there is a risk of disease transmission when using bovine collagen, which may spread mad cow disease and foot-and-mouth disease and other zoonotic diseases, greatly limiting its clinical application. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a recombinant collagen, which comprises one or more of the following characteristics: a. the amino acid sequence of the collagen is as shown in any one of SEQ ID NO. 1-4; b. one or more amino acids are added, substituted or deleted in the collagen of a, and the protein has collagen activity.

[0006] The present application also provides an isolated polynucleotide encoding the above-mentioned collagen.

[0007] The present application also provides a construct comprising the above-mentioned isolated polynucleotide.

[0008] The present application also provides a host cell comprising the construct described above, or the genome of the cell is integrated with the polynucleotide described above.

[0009] The present application also provides a method for preparing recombinant collagen, which comprises culturing the cell described above to express recombinant collagen.

[0010] The present application also provides the use of the collagen described above in cosmetics, skin care products, tissue materials, or the preparation of drugs for promoting wound healing.

[0011] The present application also provides an active agent for promoting cell proliferation and / or migration, which comprises the collagen described above.

[0012] The recombinant collagen provided in the present application has the following beneficial effects, including but not limited to: 1) the E. coli engineering bacteria capable of inducible expression can achieve high-level expression of recombinant human collagen, and the culture is easy to scale up, and the high-density fermentation method can be used to produce the target protein, thereby reducing the production cost. 2) The collagen provided in the present application can significantly promote the proliferation and migration of cells in vitro through the scratch test, which indicates that the collagen of the present application has the characteristics of natural collagen for promoting wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0013] The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, in which:

[0014] FIG. 1 is a recombinant human type I collagen fermentation SDS-PAGE protein detection gel map according to some embodiments of the present application, Figure 1ALine 1 : BL21 (DE3)-pET28a-COL1-4 whole cell lysate, Line 2: BL21 (DE3)-pET28a-COL1-4 cell lysate supernatant, Line 3: BL21 (DE3)-pET28a whole cell lysate, Line 4: BL21 (DE3)-pET28a cell lysate supernatant, Line 5: Marker, Line 6: BL21 (DE3)-pET28a-COL1-8 whole cell lysate, Line 7: BL21 (DE3)-pET28a-COL1-8 cell lysate supernatant, Line 8: BL21 (DE3)-pET28a-COL1-9 whole cell lysate, Line 9: BL21 (DE3)-pET28a-COL1-9 cell lysate supernatant, Line 10: BL21 (DE3)-pET28a-COL1-10 whole cell lysate, Line 11 : BL21 (DE3)-pET28a-COL1-10 cell lysate supernatant, Line 12: BL21 (DE3)-pET28a-COL1-11 whole cell lysate, Line 13: BL21 (DE3)-pET28a-COL1-11 cell lysate supernatant, Line 14: BL21 (DE3)-pET28a-COL1-12 whole cell lysate, Line 15: BL21 (DE3)-pET28a-COL1-12 cell lysate supernatant; Figure 1B Line 1 : BL21 (DE3)-pET28a-COL1-1 whole cell lysate, Line 2: BL21 (DE3)-pET28a-COL1-1 cell lysate supernatant, Line 3: BL21 (DE3)-pET28a-COL1-2 whole cell lysate, Line 4: BL21 (DE3)-pET28a-COL1-2 cell lysate supernatant, Line 5: BL21 (DE3)-pET28a-COL1-3 whole cell lysate, Line 6: BL21 (DE3)-pET28a-COL1-3 whole cell lysate, Line 7: BL21 (DE3)-pET28a-COL1-5 cell lysate supernatant, Line 8: BL21 (DE3)-pET28a-COL1-5 whole cell lysate, Line 9: BL21 (DE3)-pET28a-COL1-6 cell lysate supernatant, Line 10: BL21 (DE3)-pET28a-COL1-6 whole cell lysate, Line 11 : BL21 (DE3)-pET28a-COL1-7 cell lysate supernatant, Line 12: BL21 (DE3)-pET28a-COL1-7 whole cell lysate, Line 13: Marker.

[0015] Figure 2 SDS-PAGE protein detection gel of purified recombinant human collagen shown according to some embodiments of the present application, Line 1: COL1-10 purified protein, Line 2: COL1-9 purified protein, Line 3: Marker, Line 4: COL1-4 purified protein, Line 5: COL1-11 purified protein.

[0016] Figure 3 Technical roadmap for evaluating the repair efficacy of samples by detecting cell migration shown according to some embodiments of the present application.

[0017] Figure 4 is a cell migration experiment of recombinant human collagen shown according to some embodiments of the present application, Figure 4A PC is 200 μg / mL bovine collagen type I, COL1-4, COL1-9, COL1-10, COL1-11 working concentration is 200 μg / mL, and BC is blank control; Figure 4B Migration rate histogram. DETAILED DESCRIPTION

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings required to be used in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some examples or embodiments of the present specification, and for those skilled in the art, the present specification can also be applied to other similar scenarios without creative labor. Unless the context clearly indicates otherwise or otherwise stated, the same reference numbers in the drawings represent the same structure or operation.

[0019] As shown in the specification and claims, unless the context clearly indicates otherwise or otherwise stated, "one", "a", "an", and / or "the" do not necessarily refer to a single number, but can also include a plurality. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements explicitly identified, and these steps and elements do not constitute an exclusive list, and the method or device can also include other steps or elements.

[0020] Flowcharts are used in the present specification to illustrate the operations performed by the system according to the embodiments of the present specification. It should be understood that the preceding or subsequent operations are not necessarily performed in sequence. On the contrary, each step can be processed in reverse order or simultaneously. At the same time, other operations can also be added to these processes, or one or more steps of operation can be removed from these processes.

[0021] The present application provides a recombinant collagen, which comprises one or more of the following features: a. the amino acid sequence of the collagen is shown in any one of SEQ ID NO. 1-4; b. the collagen has one or more amino acids added, substituted or deleted, and has the activity of collagen.

[0022] Unless the context requires otherwise, the term "sequence" herein is understood to include both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding the same, unless the context requires a more limited interpretation.

[0023] In some embodiments, the collagen is a recombinant collagen type I. In some embodiments, preferably, the collagen is a recombinant humanized collagen type I.

[0024] The present application also provides an isolated polynucleotide encoding the above-mentioned collagen.

[0025] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially pure form, or can exist in a non-native environment such as a host cell.

[0026] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single- or double-stranded form, and to polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0027] In some embodiments, the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO. 6-9.

[0028] The present application also provides a construct comprising the above-mentioned isolated polynucleotide.

[0029] A "construct" herein refers to a polynucleotide capable of carrying at least one polynucleotide segment. A construct can deliver a segment of nucleic acid, individual polynucleotides, into a host cell. It can comprise at least one expression cassette comprising regulatory sequences for proper expression of a polynucleotide incorporated therein. A polynucleotide to be introduced into a cell, such as a polynucleotide encoding a product of interest or a selection marker, can be inserted into an expression cassette of a vector for expression therefrom. A construct according to the present application can exist in a circular or linear (linearized) form, and also includes expression construct fragments. The term "construct" also encompasses artificial chromosomes or similar individual polynucleotides that allow for the transfer of exogenous nucleic acid segments.

[0030] The present application also provides a host cell comprising the above-mentioned construct, or the genome of which is integrated with the above-mentioned polynucleotide.

[0031] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include both primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present application, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as E. coli cells. Host cells include cells in culture as well as cells inside a transgenic animal, transgenic plant, or cultured plant tissue or animal tissue.

[0032] In some embodiments, the host cell is selected from a bacterial cell, a fungal cell, a plant cell, or an animal cell. In some embodiments, preferably, the host cell is selected from a bacterial cell. In some embodiments, more preferably, the host cell is selected from E. coli. In some embodiments, further preferably, the host cell is selected from E. coli BL21.

[0033] The present application also provides a method for preparing a recombinant collagen, comprising culturing the above-mentioned cell to express a recombinant collagen.

[0034] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0035] The present application also provides the use of the above-mentioned collagen in cosmetics, skin care products, tissue materials, or the preparation of a medicine for promoting wound healing.

[0036] The present application also provides an active agent for promoting cell proliferation and / or migration, comprising the above-mentioned collagen.

[0037] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0038] Example 1 - Construction and shake flask fermentation of collagen strains

[0039] Gene screening:

[0040] Based on human type I collagen alpha 1 chain and alpha 2 chain, 12 type I collagen fragments were designed and synthesized through bioinformatics analysis, and the integrated new proteins are the recombinant human type I collagen strains of the application, and the genes are named COL1-1~12, among which COL1-4, 9, 10, and 11 can obtain soluble expression, and the amino acid sequences are shown in SEQ ID NO. 1 / SEQ ID NO. 2 / SEQ ID NO. 3 / SEQ ID NO. 4.

[0041] SEQ ID No. 1 (COL1-4):

[0042] GVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGPKGAAGEPGKAGERGVPGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGPAGPPGEAGKPGEQGVPGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGARGSAGPPGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGPAGKSGDRGETGPA

[0043] SEQ ID No. 2 (COL1-9):

[0044] GVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGPKGAAGEPGKAGERGVPGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGPAGPPGEAGKPGEQGVPGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGPAGPPGEAGKPGEQGVPGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGPAGKSGDRGETGPA

[0045] SEQ ID No. 3 (COL1-10):

[0046] GVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGPKGAAGEPGKAGERGVPGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGPAGPPGEAGKPGEQGVPGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGPPGPTGPAGPPGEAGPQGPRGSEGPQGVRGEPGPPGPAGNPGADGQPGAKGANGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGPAGKSGDRGETGPA

[0047] SEQ ID No. 4 (COL1-11):

[0048] GVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPAGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGPAGKSGDRGETGPA

[0049] SEQ ID No. 5 (pET 28a):

[0050]

[0051] SEQ ID No. 6 (COL1-4):

[0052] GGTGTTGCTGGTCCTAAAGGCCCGGCCGGTGAACGCGGCTCCCCAGGTCCGGCAGGTC

[0053] CGAAAGGTTCCCCGGGTGAAGCAGGTCGTCCGGGCGAAGCGGGTCTGCCGGGTCTGAC

[0054] CGGCTCTCCGGGCTCCCCGGGCCCGGACGGTAAAACCGGTCCTCCGGGTCCAGCAGGT

[0055] CAGGACGGCCGCCCGGGCCCTCCGGGTCCGCCGGGTGCACGCGGTCCTAAAGGCGCCG

[0056] CAGGTGAACCGGGTAAAGCAGGTGAGCGTGGTGTACCGGGCCCGGCAGGTAAAGACG

[0057] GTGAAGCGGGCGCACAAGGCCCACCGGGCCCAGCTGGCCCGGCAGGTGAACGTGGCG

[0058] AACAGGGTCCTGCCGGTTCCCCGGGTCCGGCCGGTCCGCCGGGCGAAGCGGGTAAACC

[0059] GGGCGAACAGGGTGTACCAGGTGCTCCGGGTCCGTCCGGTGCTCGCGGTGAACGCGGT

[0060] TTTCCGGGTGAACGCGGTGTCCAGGGCCCACCGGGTCCGGCTGGTCCGCGTGGTGCCA

[0061] ACGGTGCACCAGGTAACGACGGCGCAAAGGGTGACGCGGGTGCGCCGGGCGCTCCGG

[0062] GCTCTCAGGGCGCTCCAGGTCTGCAGGGTATGCCAGGCGAACGCGGTGCTGCGGGTCT

[0063] GCCGGGTCCGAAAGGTGATCGTGGCGACGCGGGTCCGAAGGGCGCTGATGGCTCTCCG

[0064] GGCAAAGATGGTGTTCGTGGCCCTCCGGGTCCGGCAGGTGCACCAGGTGATAAGGGTG

[0065] AAAGCGGTCCTTCTGGTCCAGCTGGTCCGACTGGTGCCCGCGGTGCACCGGGCGATCG

[0066] CGGTGAGCCGGGCCCGCCGGGCGCGGACGGCCAGCCGGGCGCGAAGGGTGAACCAGG

[0067] TGACGCAGGTGCTAAAGGCGACGCGGGTCCGCCAGGTCCTGCAGGCCCGGCTGGTCCT

[0068] CCAGGCCCGATTGGTGCACGCGGTTCCGCAGGCCCGCCGGGTCGTGTTGGTCCGCCGG

[0069] GTCCATCTGGTAACGCGGGTCCACCGGGCCCGCCTGGCCCTGCGGGCAAGGAAGGTGG

[0070] TAAAGGCCCGCGTGGCGAAACCGGTCCGGCTGGCCGCCCGGGTGAAGTAGGTCCGCCT

[0071] GGCCCGCCAGGCCCAGCGGGTGAAAAAGGTTCTCCAGGCGCTGATGGCCCGGCAGGC

[0072] GCTCCGGGTACCCCGGGTCCGCAGGGTATCGCTGGCCAACGTGGTGAACGTGGTTTTC

[0073] CGGGCCTGCCGGGCCCAAGCGGCGAACCGGGTAAACAGGGTCCGTCTGGCGCTTCTGG

[0074] TGAACGTGGTCCGCCGGGCCCGATGGGTCCGCCTGGTGAAAGCGGTCGCGAGGGTGCA

[0075] CCGGGCGCAGAAGGCTCTCCTGGCCGTGACGGTTCTCCGGGTGCAAAAGGTGACCGCG

[0076] GCGAAACGGGCCCAGCTGGCCCGCCGGGCGCGCCGGGCGCTCCGGGTCCGGCTGGTAA

[0077] ATCCGGTGACCGTGGTGAAACCGGCCCTGCC

[0078] SEQ ID No. 7 (COL1-9):

[0079] GGTGTTGCAGGTCCAAAAGGTCCGGCAGGCGAACGTGGCAGCCCAGGCCCGGCTGGCC

[0080] CGAAAGGTTCTCCTGGTGAGGCGGGCCGTCCGGGTGAGGCTGGTCTGCCGGGTCTGAC

[0081] GGGTAGCCCAGGCTCTCCAGGCCCGGATGGTAAAACCGGCCCTCCGGGTCCAGCAGGC

[0082] CAGGACGGCCGTCCGGGTCCTCCGGGCCCACCAGGCGCCCGTGGTCCAAAAGGTGCTG

[0083] CGGGCGAGCCGGGCAAAGCAGGCGAACGCGGTGTACCTGGTCCGGCGGGCAAGGATG

[0084] GCGAAGCTGGTGCGCAGGGCCCGCCGGGTCCGGCTGGTCCGGCGGGTGAACGTGGCG

[0085] AACAGGGTCCGGCGGGCAGCCCGGGTCCGGCAGGCCCACCGGGTGAAGCAGGCAAAC

[0086] CGGGTGAGCAAGGTGTGCCGGGTCCGGCAGGCAAAGACGGTGAAGCAGGCGCACAGG

[0087] GTCCGCCAGGTCCGGCAGGTCCGGCAGGTGAGCGTGGCGAGCAGGGTCCGGCTGGCA

[0088] GCCCGGGTCCTGCGGGTCCGCCGGGTGAGGCTGGTAAACCGGGCGAACAGGGCGTTCC

[0089] GGGTGCGCCTGGTCCGTCCGGTGCGCGTGGTGAACGCGGTTTCCCGGGCGAACGTGGC

[0090] GTTCAGGGTCCGCCAGGTCCAGCTGGTCCGCGCGGTGCTAACGGTGCCCCGGGCAATG

[0091] ATGGCGCTAAAGGTGATGCAGGTGCACCAGGCGCACCGGGCTCCCAAGGTGCTCCGGG

[0092] CCTGCAGGGCATGCCAGGTGAACGTGGCGCAGCGGGTCTGCCAGGCCCGAAAGGTGAT

[0093] CGTGGCGACGCGGGTCCGAAAGGCGCCGACGGTTCCCCGGGTAAGGACGGCGTTCGTG

[0094] GTCCACCGGGTCCGGCAGGCGCACCGGGTGATAAAGGTGAATCCGGTCCGAGCGGCCC

[0095] GGCTGGCCCGACCGGTGCTCGTGGTGCTCCGGGTGACCGTGGTGAACCGGGTCCGCCG

[0096] GGTCGTGTCGGCCCGCCGGGTCCTAGCGGTAATGCTGGCCCGCCGGGCCCGCCGGGCC

[0097] CGGCAGGTAAAGAGGGTGGCAAAGGTCCGCGCGGCGAAACCGGTCCAGCTGGTCGTC

[0098] CAGGTGAAGTAGGTCCGCCGGGTCCGCCTGGTCCGGCTGGTGAGAAAGGTTCTCCAGG

[0099] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0100] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0101] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0102] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0103] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0104] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0105] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0106] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0107] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0108] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0109] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0110] CGCGGTGAGCGCGGTTTTCCGGGCCTGCCGGGTCCGTCCGGCGAACCGGGCAAACAGG

[0111] GTGAAGCGGGCGCTCAAGGTCCGCCTGGTCCAGCGGGTCCGGCTGGCGAACGTGGTGA

[0112] ACAGGGTCCAGCGGGCTCCCCAGGTCCAGCTGGCCCGCCGGGCGAAGCCGGTAAACCG

[0113] GGTGAGCAGGGCGTTCCGGGCGCTCCGGGCCCGTCTGGCGCTCGCGGCGAACGTGGTT

[0114] TTCCAGGCGAACGCGGCGTCCAGGGTCCGCCGGGTCCGGCTGGTCCGCGTGGTGCCAA

[0115] TGGCGCGCCAGGTAACGACGGCGCAAAAGGTGATGCGGGCGCACCGGGTGCACCGGG

[0116] TTCCCAGGGTGCACCGGGCCTGCAGGGCATGCCAGGTGAACGTGGTGCGGCGGGTCTG

[0117] CCGGGCCCGAAAGGCGATCGCGGCGACGCCGGCCCGAAAGGCGCGGACGGTTCTCCG

[0118] GGCAAAGACGGCGTTCGCGGCCCGCCGGGTCCGACCGGCCCGGCTGGCCCGCCGGGTG

[0119] AGGCAGGTCCTCAGGGTCCACGTGGCTCCGAGGGTCCGCAAGGTGTGCGTGGTGAACC

[0120] GGGCCCTCCAGGTCCGGCGGGTAACCCGGGTGCAGACGGCCAGCCGGGTGCGAAAGG

[0121] CGCTAACGGTGCTCGCGGTCCATCTGGTCCACAAGGTCCGGGTGGTCCGCCAGGTCCG

[0122] AAAGGTAACTCCGGTGAACCGGGCGCGCCGGGCTCTAAAGGTGACACCGGTGCAAAA

[0123] GGTGAACCGGGCCCGGTAGGCGTTCAGGGTCCGCCGGGTCCTGCGGGTGAAGAAGGTA

[0124] AACGTGGCGCCCGCGGCGAACCGGGTCCAACCGGCCTGCCGGGCCCTCCGGGTCCGGC

[0125] CGGCGCTCCGGGTGATAAAGGTGAGTCTGGTCCGAGCGGCCCGGCAGGTCCAACTGGT

[0126] GCTCGTGGCGCTCCGGGCGATCGCGGCGAACCTGGCCCTCCAGGCCGTGTTGGTCCAC

[0127] CGGGCCCGAGCGGTAACGCCGGTCCTCCGGGCCCGCCTGGTCCAGCCGGTAAAGAAGG

[0128] CGGTAAAGGTCCGCGTGGTGAGACTGGCCCGGCCGGCCGCCCGGGCGAAGTTGGCCCG

[0129] CCGGGTCCGCCAGGTCCGGCCGGTGAAAAAGGTAGCCCGGGCGCTGATGGCCCAGCCG

[0130] GCGCACCTGGTACCCCGGGTCCGCAAGGTATTGCCGGCCAACGTGGCGAACGCGGTTT

[0131] CCCGGGCCTGCCGGGCCCTTCCGGCGAACCGGGTAAGCAGGGTCCGTCCGGTGCATCC

[0132] GGTGAACGTGGTCCGCCGGGTCCGATGGGTCCGCCGGGTGAATCCGGTCGTGAAGGTG

[0133] CACCGGGTGCAGAGGGTTCTCCGGGTCGCGATGGCTCTCCAGGTGCGAAAGGCGACCG

[0134] TGGTGAAACCGGTCCAGCTGGCCCGCCTGGTGCGCCGGGTGCACCTGGTCCGGCTGGC

[0135] AAATCCGGTGATCGTGGTGAGACCGGTCCGGCA

[0136] SEQ ID No. 9 (COL1-11):

[0137] GGTGTAGCAGGTCCGAAAGGTCCGGCAGGCGAACGCGGTAGCCCGGGCCCGGCTGGCC

[0138] CTAAGGGTTCTCCAGGTGAGGCAGGTCGTCCTGGCGAAGCAGGTCTGCCGGGCCTGAC

[0139] TGGCTCCCCGGGTTCTCCGGGCCCGGATGGCAAAACCGGCCCGCCGGGCCCGGCTGGC

[0140] CAGGATGGTCGTCCGGGTCCACCGGGCCCGGCTGGTTTCCCGGGCGAACGTGGTGTTC

[0141] AGGGTCCGCCGGGCCCAGCCGGTCCACGTGGTGCCAATGGCGCCCCTGGTAATGATGGT

[0142] GCCAAAGGTGATGCAGGCGCGCCGGGTGCACCGGGTTCTCAGGGCGCTCCAGGTCTGC

[0143] AGGGTATGCCAGGCGAACGTGGTGCTGCTGGCCTGCCGGGTCCGAAAGGCGATCGCGG

[0144] TGATGCTGGTCCAAAAGGTGCGGACGGTAGCCCAGGCAAAGACGGTGTACGTGGTCTG

[0145] ACGGGTCCGATTGGCCCGCCGGGTCCGGCAGGTGCACCGGGCGACAAAGGTGAGTCCG

[0146] GCCCGTCCGGTCCAGCGGGTCCGACCGGTGCACGTGGTGCGCCGGGCGACCGTGGTGA

[0147] GCCGGGCCCGCCGGGTCCGGCTGGTTTCGCTGGTCCTCCGGGCGCGGATGGTCAGCCA

[0148] GGCGCAAAAGGTGAGCCGGGCGATGCCGGTGCGAAAGGCGATGCTGGCCCTCCGGGTC

[0149] CAGCGGGCCCTGCGGGTCCGCCGGGTCCGATCGGTAACGTTGGTGCGCCAGGTGCTAA

[0150] AGGTGCGCGTGGCAGCGCTGGTCCGCCGGGCGCGACTGGTTTTCCGGGCGCCGCGGGC

[0151] CGTGTTGGCCCGCCGGGTCCATCTGGCAACGCGGGCCCACCGGGCCCGCCGGGTCCAG

[0152] CCGGTAAAGAAGGCGGCAAAGGTCCGCGTGGTGAAACCGGCCCTGCTGGCCGTCCGGG

[0153] TGAAGTAGGCCCGCCAGGCCCGCCAGGTCCGGCTGGCGAAAAAGGTTCTCCAGGTGCC

[0154] GATGGCCCGGCTGGCGCGCCGGGTACCCCGGGTCCGCAAGGTATCGCAGGTCAGCGTG

[0155] GTGAACGTGGTTTCCCGGGCCTGCCGGGCCCATCCGGTGAGCCGGGCAAACAGGGTCC

[0156] GTCTGGCGCATCCGGTGAACGTGGCCCGCCTGGCCCGATGGGCCCTCCGGGTGAGTCTG

[0157] GCCGCGAAGGTGCGCCAGGTGCAGAAGGTTCTCCGGGCCGTGATGGCAGCCCGGGTGC

[0158] GAAGGGTGATCGTGGCGAAACGGGCCCGGCTGGCCCTCCGGGTGCTCCGGGTGCTCCA

[0159] GGTCCGGCGGGTAAATCCGGTGACCGTGGTGAAACCGGTCCGGCC

[0160] Construction of collagen expression host strain:

[0161] The coding genes of 12 sequences COL1-1~12 were synthesized by Jiangsu Saixuofei Biotechnology Co., Ltd., and were cloned into plasmid pET28a, wherein the nucleotide sequence of the pET28a plasmid is shown as SEQ ID NO. 5. The recombinant plasmid was transformed into a BL21 (DE3) host, and the corresponding engineering bacteria were obtained after antibiotic screening. The specific operation steps are as follows:

[0162] (1) CaCl2 method for transforming Escherichia coli BL21 (DE3)

[0163] Thaw the competent cells of Escherichia coli BL21 (DE3) on ice; add 1 μL of plasmid DNA, and place on ice for 30 min; heat shock at 42°C for 90 sec, and place on ice for 2 min; add 1 mL of LB liquid medium (formula as shown in Table 1), and incubate at 37°C, 220 rpm for 60 min; centrifuge at low speed to collect the bacterial cells, remove 900 μL of supernatant, resuspend the bacterial cells with the remaining medium, and spread on a solid LB (formula as shown in Table 2) plate containing kanamycin antibiotic for screening; culture at 37°C for 16 h, and identify as a positive clone by PCR.

[0164] Table 1

[0165] Material Ingredient Concentration Source Yeast Extract 5 g / L Oxoid Tryptone 10 g / L Oxoid NaCl 5 g / L Guojia

[0166] Table 2

[0167] Material Ingredient Concentration Source Yeast Extract 5 g / L Oxoid Tryptone 10 g / L Oxoid NaCl 5 g / L Guojia Agar powder 20 g / L Haibo

[0168] (2) Small-scale expression identification

[0169] Pick a single colony in LB liquid medium containing 50 μg / mL of kanamycin, and culture overnight at 37°C, 200 rpm. Then inoculate into LB medium at an inoculation amount of 1%, and culture at 37°C, 200 rpm until the OD600 is 0.6-0.8. Then add IPTG (isopropyl-B-D-thiogalactoside) at a final concentration of 0.1 mM for induction expression, and culture at 25°C for 20 h. Centrifuge to collect the bacterial cells, resuspend with ultrapure water, and then perform ultrasonic crushing. The whole cell crushing liquid and cell crushing supernatant are detected by SDS-PAGE. The protein expression results of the 4 strains are detected by SDS-PAGE, and the results are shown in FIG. 1.

[0170] (3) Sequence identification

[0171] The band corresponding to the protease enzyme was sent for amino acid identification, and the sequencing result was consistent with the expectation, confirming that the expressed band was collagen protein.

[0172] Example 2 - Purification and efficacy verification of collagen protein

[0173] (1) Purification of type I collagen

[0174] Experimental process: The COL1-4, 9, 10, 11 fermentation bacteria were ultrasonically broken, and the supernatant was purified by AKTA. The anion exchange column DEAE 6FF was used for multiple purification, and finally more than 90% pure product was obtained. The collagen protein pure product SDS-PAGE analysis is as follows Figure 2 .

[0175] (2) Efficacy verification

[0176] Test purpose: Cell migration, also known as cell crawling, cell movement or cell motion, refers to the movement of cells after receiving migration signals or sensing certain substance gradients. Cell migration is the alternation of cell head pseudopod extension, new adhesion establishment and cell body tail contraction in space and time. Cell migration is one of the basic functions of normal cells and is a physiological process of normal growth and development of the body. It is also a common form of movement of living cells. The most common method to detect cell migration ability is cell scratch test. Cell scratch test is a simple, economical and practical in vitro test method to study cell migration, which is similar to in vitro wound healing model and mainly detects the migration ability of cells in 2D space. In the single layer of adherent cells cultured in vitro dish or plate, a line is drawn in the central area of cell growth with a micro syringe needle or other hard objects, and the cells in the central part are removed, and then the cells are cultured for a certain time. According to the ability of the cells at the edge of the scratch to gradually enter the blank area and heal the scratch, the growth and migration ability of the cells is judged. It is also known as wound healing test because it is similar to the in vitro wound healing process. When the cells grow to a single layer, an empty area, called "scratch", is artificially created on the fused single layer of cells, and the cells at the edge of the scratch will gradually enter the blank area to heal the scratch, which simulates the in vivo cell migration process to some extent. The test takes human immortal keratinocytes (HaCaT) as the research object, and evaluates the repair efficacy of the sample by detecting cell migration, as shown in the technical route of Figure 3 .

[0177] The specific experimental method and data processing refer to the standard of "recombinant collagen protein" (YY / T 1849-2022).

[0178] The in vitro cell migration test can directly find that the recombinant type I collagen of the patent can significantly enhance cell migration, and the results are shown in Figure 4.

[0179] Having now described the fundamental concepts, it can be apparent to those with skill in the art that the above-described embodiments are only examples of the principles of the application, and that various modifications, improvements, and substitutions can be made by those skilled in the art without departing from the teachings of the present specification. Such modifications, improvements, and substitutions are considered to fall within the scope of the present specification. Accordingly, the application is not defined by the above description, but instead is defined by the claims that follow.

[0180] Also, the use of "adapted to" or "configured to," or "made to" herein is used to indicate that an element or structure is designed and prepared to implement a certain functionality. "Can" or "could" or "might" or "may" or "will" or "would" or "on the other hand" or "for example" are not used to convey an obligation, preclusion of, or other possibility of alternatives except where explicitly indicated by context.

[0181] Some embodiments use numerical terms to describe quantities of ingredients, properties, and the like. It is understood that such numerical terms are used to describe example embodiments and are not meant to limit the scope of embodiments of the present specification. Unless otherwise specified, "about" or "approximately" means ±20% of the value of the range that the term describes. Accordingly, the numerical parameters are approximations and the numerical parameters set forth in the specification and claims are approximations. Any numerical value, however, can inherently contain certain errors necessarily resulting from the standard deviation found in the respective testing measurements and / or the measurement of the properties. Also, it is understood that some of the best modes, preferred embodiments, and other information are given for purposes of disclosure, resulting in improvements over the art, and not necessarily having to meet local, domestic, and / or international requirements.

[0182] Finally, it should be understood that the embodiments described herein are merely exemplary of the principles of the application. Other variations having essentially the same structure and function are within the scope of the present application. Accordingly, the embodiments described herein are not limited to that precisely as shown and described.

Claims

1. A recombinant collagen protein, said recombinant collagen protein comprising one or more of the following characteristics: a. The amino acid sequence of the collagen is shown in any one of SEQ ID NO.1 to 4; The collagen protein described in ba has one or more amino acids added, substituted, or missing, and has collagen activity.

2. The recombinant collagen as described in claim 1, characterized in that, The collagen is recombinant type I collagen, preferably recombinant humanized type I collagen.

3. An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the collagen as described in claim 1 or 2.

4. The polynucleotide as described in claim 3, characterized in that, The nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO. 6 to 9.

5. A construct, characterized in that, The construct contains the isolated polynucleotides as described in claim 3 or 4.

6. A host cell comprising the construct of claim 5, or the host cell genome integrating the polynucleotides of claim 3 or 4.

7. The host cell as described in claim 6, characterized in that, The host cell is selected from bacterial cells, fungal cells, plant cells or animal cells. Preferably, the host cell is selected from bacterial cells. More preferably, the host cell is selected from Escherichia coli. Even more preferably, the host cell is selected from Escherichia coli BL21.

8. A method for preparing recombinant collagen, the method comprising culturing cells as described in any one of claims 6 to 8 to express recombinant collagen.

9. The use of the collagen as described in claim 1 or 2 in cosmetics, skin care products, tissue materials, or in the preparation of medicines that promote wound healing.

10. An active agent that promotes cell proliferation and / or migration, said active agent comprising collagen as described in claim 1 or 2.

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