Recombinant human XVII micromolecular collagen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to recombinant human XVII micromolecular collagen as well as a preparation method and application thereof. According to the invention, a recombinant human XVII type micromolecular collagen (Re-Hcol17) is constructed, and a stable triple helix structure is formed by connecting the 15th collagen domain, the 12th collagen domain, the 5th collagen domain and the 1st collagen domain in series. Experimental results prove that the Re-Hcol17 can significantly promote NIH / 3T3 fibroblast proliferation within the concentration range of 10-100 [mu] g / mL, the effect is better than that of natural human collagen, and extracellular matrix adhesion can be effectively promoted. Through genetic engineering and fermentation process innovation, the bottleneck that the human-derived XVII type collagen is difficult to efficiently express and unstable in structure is broken through, and a foundation is laid for wide application of the human-derived XVII type collagen in the fields of biological medicines and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant human XVII small molecule collagen, its preparation method, and its application. Background Technology

[0002] Collagen, also known as collagen peptide, is a type of biological macromolecule. When collagen exists in a triple-helix state in body tissues, it is usually called collagen. It is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of total protein. Currently, the genetic codes for more than 30 types of collagen have been deciphered, and these codes can guide the synthesis of more than 16 types of collagen molecules. Based on their structure and function, collagen can be divided into fibrous collagen, basement membrane collagen, anchoring collagen, etc. Based on their distribution areas in the body, collagen can be divided into interstitial collagen, basal collagen, and peripheral collagen. Collagen is composed of a special triple-helix chain called protoplasm. Each protoplasm is formed by three α-helical peptide chains intertwined in a left-handed helix, and each chain contains approximately 1000 amino acid monomers. The most distinctive feature of this peptide chain is the periodic arrangement of its amino acids, which is (Gly-XY)n. Gly is glycine, X is usually proline, and Y is often hydroxyproline or hydroxylysine. This periodic three-dimensional helical structure forms the framework of the extracellular matrix, providing mechanical strength and support for tissues. In most cases, collagen sequences cannot spontaneously form a stable triple helix structure, thus limiting the full expression of its biological functions. This challenge poses a significant obstacle to the research and production of human collagen.

[0003] To date, most research on collagen has primarily relied on extracts from animal tissues and skin. These animal-derived collagens not only suffer from poor water solubility and processability but also exhibit significant batch-to-batch variability and the potential risk of pathogen transmission. These characteristics severely limit their application in many potential fields. Currently, such products are quite popular with consumers due to the increasing emphasis on skin anti-aging and repair. Advances in modern science and technology, such as molecular biology and genetic engineering, have provided powerful tools for research on human collagen. Through genetic engineering, human collagen can be efficiently expressed in hosts such as microorganisms and animal cells, solving the problems of low yield and high cost of naturally derived collagen. This also allows for more stable performance and higher purity, opening up new possibilities for the widespread application of collagen in biomedicine, cosmetics, and other fields.

[0004] Human type XVII collagen is a rare type II transmembrane collagen that plays a crucial role in maintaining the connections between intracellular and extracellular structural elements involved in epidermal adhesion. It consists of a 466-residue intracellular domain, a 23-residue transmembrane domain, and a 1008-amino acid extracellular collagen domain, with several non-collagenous breaks. Its intracellular domain is basic (pI 9.5), while its extracellular domain is acidic (pI 6.3). Interspersed within the extracellular long collagen region are 15 non-collagenous sequences ranging from 6 to 58 residues in length, dividing the protein into 15 collagen domains ranging in size from 15 to 242 amino acids. The corresponding protein molecular weight is also relatively large (180 kDa), theoretically making efficient extracellular secretion difficult and prone to degradation. Therefore, it is necessary to develop recombinant type XVII collagen that is easily secreted and degraded. Summary of the Invention

[0005] The first aspect of the present invention is to provide a recombinant collagen.

[0006] A second aspect of the present invention aims to provide biomaterials related to the recombinant collagen described in the first aspect of the present invention.

[0007] The third objective of this invention is to provide a method for preparing the recombinant collagen described in the first aspect of this invention.

[0008] The fourth aspect of this invention aims to provide the application of the recombinant collagen described in the first aspect of this invention.

[0009] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0010] In a first aspect, the present invention provides a recombinant collagen Re-Hcol17, which is composed of the amino acids shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0011] In some embodiments of the present invention, the recombinant collagen is composed of the amino acids shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 in order from N-terminus to C-terminus.

[0012] In some embodiments of the present invention, the amino acid sequence of the recombinant collagen is shown in any one of a1) to a3): a1) SEQ ID NO: 1; or

[0013] a2) A sequence of amino acids that has the same function as SEQ ID NO: 1, but with one or more amino acid substitutions and / or deletions and / or additions; or

[0014] a3) is an amino acid sequence that has at least 80% homology with SEQ ID NO: 1.

[0015] In some embodiments of the present invention, the substitution of proteins with the same function in a2) is generally regarded as a conservative substitution. For example, substitution between aliphatic amino acids Ala, Val, Leu and Ile, exchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr can maintain the biological activity of collagen.

[0016] In some embodiments of the present invention, a3) means that the given amino acid sequence shares at least 80% identity with the reference sequence, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Alternatively, it means that the given amino acid sequence differs from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. For proteins, this difference is preferably an amino acid substitution or deletion. This difference in identity can arise from conserved amino acid substitutions, the introduction of a tag protein, the introduction of a nuclear insertion sequence, or the introduction or modification of a signal peptide sequence. For example, adding tags such as His, GFP, or HA to the C-terminus of SEQ ID NO.1 leads to a change in identity; adding a signal peptide to the N-terminus of SEQ ID NO.1, or adding a conventional nuclear insertion sequence to the N-terminus, also leads to a change in identity.

[0017] In some embodiments of the invention, the recombinant collagen includes optional tag sequences for co-expression and / or purification.

[0018] In some embodiments of the present invention, the tag sequence includes at least one of a signal peptide, a nuclear localization signal, an NLS tag, a His tag, a Myc tag, a Strep tag, a GST tag, a Flag tag, an HA tag, and a fluorescent protein tag.

[0019] In some embodiments of the present invention, the tag sequence is located at the N-terminus and / or C-terminus of the recombinant collagen.

[0020] In some embodiments of the present invention, the N-terminus of the recombinant collagen includes a Strep-Tag II tag sequence.

[0021] In some embodiments of the present invention, the C-terminus of the recombinant collagen includes a 6×His Tag sequence.

[0022] A second aspect of the present invention provides biomaterials related to recombinant collagen as described in the first aspect of the present invention, comprising: b1) a nucleic acid molecule encoding said recombinant collagen;

[0023] b2) An expression cassette containing the nucleic acid molecule described in b1);

[0024] b3) A recombinant vector containing the nucleic acid molecule described in b1);

[0025] b4) A recombinant vector containing the expression cassette described in b2);

[0026] b5) Recombinant cells containing the nucleic acid molecules described in b1);

[0027] b6) Recombinant cells containing the expression cassette described in b2);

[0028] b7) Recombinant cells containing the recombinant vector described in b3);

[0029] b8) Recombinant cells containing the recombinant vector described in b4);

[0030] In some embodiments of the present invention, the cell is a non-reproductive material.

[0031] In some embodiments of the present invention, the nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO: 8.

[0032] In some embodiments of the present invention, the recombinant vector includes a eukaryotic vector or a prokaryotic vector.

[0033] In some embodiments of the present invention, the recombinant cells include eukaryotic cells or prokaryotic cells.

[0034] In some embodiments of the present invention, based on common knowledge in the art, the prokaryotic cells mentioned above can be selected from commonly used engineered strains in the biological field such as Escherichia coli and Bacillus subtilis; among which, Escherichia coli can be Escherichiacoli BL21(DE3) or other Escherichia coli types used for protein expression, and Bacillus subtilis can be Bacillus subtilis 168, Bacillus subtilis WB600 and other Bacillus subtilis types used for protein expression.

[0035] In some embodiments of the present invention, the eukaryotic cells described above may be selected from conventional engineered cells that express proteins, such as Saccharomyces ceresiae, Pichia pastoris, and mammalian cells; wherein the Saccharomyces ceresiae may be Saccharomyces ceresiae CEN.PK2-1C, Saccharomyces cerevisiae S288C, and other Saccharomyces ceresiae types that can be used for protein expression; the Pichia pastoris may be Pichia pastoris GS115 and other Pichia pastoris types that can be used for protein expression; and the mammalian cells may be CHO cells (Chinese hamster ovary cells), HEK293 cells (human embryonic kidney cells), and other mammalian cell types that can be used for protein expression.

[0036] In some embodiments of the present invention, the above-mentioned recombinant vectors correspond to recombinant microorganisms; for example, the pET28 plasmid is selected for Escherichia coli, the pHT43 plasmid is selected for Bacillus subtilis, the pRS425 plasmid is selected for Saccharomyces cerevisiae, and the pPIC9K plasmid is selected for Pichia pastoris.

[0037] In some embodiments of the present invention, the recombinant cells are engineered Pichia pastoris.

[0038] In some embodiments of the present invention, the recombinant vector is pPIC9K.

[0039] A third aspect of the present invention provides a method for preparing recombinant collagen according to the first aspect of the present invention, comprising the following steps:

[0040] The biological material described in the second aspect of the present invention is expressed, purified, and obtained.

[0041] In some embodiments of the present invention, specific steps include:

[0042] 1) Design and synthesize the nucleotide sequence of the recombinant collagen of the first aspect of this invention;

[0043] 2) Construct recombinant expression vectors;

[0044] 3) The recombinant expression vector was electroporated into Pichia pastoris GS115 competent cells, and positive transformants were screened;

[0045] 4) Fermentation production of recombinant collagen Re-Hcol17;

[0046] 5) Isolate and purify recombinant collagen Re-Hcol17;

[0047] 6) Quality inspection;

[0048] 7) Save.

[0049] In some embodiments of the present invention, the separation and purification steps include centrifugation, filtration, and affinity chromatography.

[0050] A fourth aspect of the present invention provides the use of the recombinant collagen from the first aspect of the present invention in the preparation of products.

[0051] In some embodiments of the present invention, the product is used in the fields of skin repair, tissue engineering, cell culture substrates, or drug carriers.

[0052] In some embodiments of the present invention, the product includes one of cosmetics, medical devices, or pharmaceuticals.

[0053] In some embodiments of the invention, the product includes pharmaceutically acceptable excipients.

[0054] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0055] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and P.J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical 6Gess, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0056] The beneficial effects of this invention are:

[0057] This invention constructs a recombinant human type XVII small molecule collagen (Re-Hcol17) that forms a stable triple helix structure by tandemly connecting the 15th, 12th, 5th, and 1st collagen domains. Experimental results demonstrate that Re-Hcol17, at concentrations of 10-100 μg / mL, significantly promotes the proliferation of NIH / 3T3 fibroblasts (OD450nm value reaches 250%, p < 0.01), outperforming natural human collagen (positive control OD value 150%), and effectively promotes extracellular matrix adhesion. Through codon optimization and a Pichia pastoris expression system, the large-scale production cost of this invention is only 1 / 5 of that of animal extraction methods, with batch-to-batch variation < 5% (verified by SDS-PAGE), ensuring product uniformity. This invention, through innovative genetic engineering and fermentation processes, overcomes the bottlenecks of efficient expression and structural instability of human type XVII collagen, laying the foundation for its widespread application in the biopharmaceutical and cosmetic fields. Attached Figure Description

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0059] Figure 1 This is an electrophoresis diagram of the nucleic acid amplification of the PCR product after the recombinant collagen sequence amplification. Lane 1 is the PCR product (target fragment size is 801bp), and lane 2 is the marker.

[0060] Figure 2 The image shows the pPICZC plasmid, which is 3329 bp in size.

[0061] Figure 3 The image shows the identification of the linearized empty pPICZC plasmid and the recombinant expression plasmid pPIC9K-Re-Hcol17 using SacⅠ enzyme. Lane 1 is the linearized empty pPICZC plasmid, and lane 2 is the linearized recombinant expression plasmid pPIC9K-Re-Hcol17.

[0062] Figure 4 This is a single colony of Pichia pastoris after transformation with a recombinant plasmid.

[0063] Figure 5 This is a colony PCR identification diagram of Pichia pastoris recombinants. Lanes 1-8 are recombinants, lane 9 is the negative control, and lane 10 is the positive control.

[0064] Figure 6The images show protein samples after affinity chromatography purification, eluted with 10mM, 30mM, 60mM, and 250mM imidazole after nickel column elution. Lane 1 is the marker, lane 2 (Ly) is the total protein, lane 3 (FT) is the eluent, and lanes 4-7 are elution buffers of 10mM, 30mM, 60mM, and 250mM imidazole, respectively. The molecular weight of recombinant human type XVII small molecule collagen was approximately 32kDa as determined by electrophoresis.

[0065] Figure 7 To remove imidazole from the 250 mM imidazole eluent, the sample was purified again by affinity chromatography. Sample images were obtained after elution with 10 mM, 30 mM, 60 mM, and 250 mM imidazole. Lane 1 is the marker, lane 2FT is the eluent, and lanes 3-6 are the 10 mM, 30 mM, 60 mM, and 250 mM imidazole eluents, respectively. The molecular weight of recombinant human type XVII small molecule collagen was approximately 32 kDa as determined by electrophoresis.

[0066] Figure 8 The results show the effect of different concentrations of Re-Hcol17 on cell viability.

[0067] Figure 9 The results show the effect of Re-Hcol17 on cell adhesion activity. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0069] Example 1: Design and expression of the amino acid sequence of recombinant human type XVII small molecule collagen

[0070] (1) Design of amino acid sequences and optimization of DNA sequences

[0071] The recombinant expression sequence consists of amino acid 75-242 (as shown in SEQ ID NO: 2), collagen domain 12 (as shown in SEQ ID NO: 3), collagen domain 5 (as shown in SEQ ID NO: 4), and collagen domain 1 (as shown in SEQ ID NO: 5) of the human XVII collagen sequence (specific sequence referenced in NCBI reference sequence Q9UMD9.3, see https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9.3). A Strep-Tag II tag sequence is added to its amino terminus, and a 6×His Tag tag sequence is added to its carboxyl terminus. The recombinant human XVII small molecule collagen obtained by expressing this sequence is named Re-Hcol17, with a total of 282 amino acids and a theoretical molecular weight of 26.8 kDa, and its sequence is shown in SEQ ID NO: 1.

[0072] The amino acid sequence of Re-Hcol17 (untagged sequence):

[0073] GEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPPGPSGDP GKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIGPPGPPGPPGPKGDQGPPPGPRGHQGEQGLPGFSGPPGPPGPPGPRGPPGVSGALGPPGQKGEMGTPPGKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ(SEQ ID NO: 1).

[0074] The region containing amino acids 75-242 in the 15th collagen domain:

[0075] GEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVK GDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGE QGLTGMPGIRGPPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKI (SEQ ID NO: 2).

[0076] Collagen domain 12: GPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFS (SEQ ID NO: 3).

[0077] Fifth collagen domain: GPPGPPGPPGPRGPPGVSGAL (SEQ ID NO: 4).

[0078] First collagen domain: GPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ (SEQ ID NO: 5).

[0079] In this embodiment, during the purification process, a Strep-Tag II tag sequence (MWSHPQFEK, SEQ ID NO: 6) was added to its amino terminus, and a 6×His Tag tag sequence (HHHHHH, SEQ ID NO: 7) was added to its carboxyl terminus.

[0080] The DNA sequence encoding Re-Hcol17 was codon-optimized using Pichia pastoris as the host, and the optimized sequence is shown in SEQ ID NO: 8:

[0081] GGTGAAAAGGGTGAACGTGGTGCTGCTGGTGAACCAGGTCCACATGGTCCACCAGGTGTTCCAGGTTCTGTTGGTCCAAAAGGTTCTTCTGGTTCTCCAGGTCCTCAAGGTCCTCCTGGTCCAGTTGGATTGCAAGGTTTGAGAGGTGAAGTTGGTTTGCCAGGTGTTAAGGGTGATAAGGGTCCAATGGGTCCTCCAGGACCAAAAGGTGATCAAGGTGAAAAAGGTCCAAGAGGTTTGACTGGTGAGCCTGGTATGAGAGGATTACCTGGTGCTGTTGGAGAGCCTGGTGCTAAAGGTGCTATGGGTCCAGCTGGTCCAGATGGTCATCAAGGACCTAGAGGTGAACAAGGATTGACTGGTATGCCAGGTATTCGTGGTCCACCTGGACCTTCAGGTGATCCTGGTAAACCTGGTCTTACTGGTCCACAAGGACCACAAGGTTTGCCTGGTACTCCTGGTAGACCAGGTATCAAAGGTGAGCCAGGTGCTCCTGGAAAAATTGGACCACCAGGTCCACCAGGACCTCCAGGTCCTAAGGGTGACCAAGGTCCACCAGGTCCTAGAGGACATCAAGGTGAGCAAGGACTTCCAGGTTTTTCTGGACCTCCAGGACCTCCTGGACCACCAGGACCAAGAGGTCCACCTGGTGTTTCTGGTGCTTTAGGTCCTCCAGGTCAGAAAGGCGAAATGGGTACACCCGGTCCTAAAGGCGATAGAGGTCCTGCCGGACCACCTGGTCATCCTGGTCCACCTGGTCCTCGTGGCCATAAGGGTGAGAAAGGTGACAAAGGCGATCAA(SEQ ID NO:8).

[0082] Correspondingly, the nucleotide sequence with Strep-Tag II tag sequence added to the N-terminus and 6×His tag sequence added to the C-terminus, as well as the restriction enzyme cleavage sites (EcoRI, NotI) for molecular cloning, the final sequence is shown in SEQ ID NO: 9:

[0083] GAATTCATGTGGTCTCATCCTCAATTTGAAAAAGGTGAAAAGGGTGAACGTGGTGCTGCTGGTGAACCAGGTCCACATGGTCCACCAGGTGTTCCAGGTTCTGTTGGTCCAAAAGGTTCTTCTGGTTCTCCAGGTCCTCAAGGTCCTCCTGGTCCAGTTGGATTGCAAGGTTTGAGAGGTGAAGTTGGTTTGCCAGGTGTTAAGGGTGATAAGGGTCCAATGGGTCCTCCAGGACCAAAAGGTGATCAAGGTGAAAAAGGTCCAAGAGGTTTGACTGGTGAGCCTGGTATGAGAGGATTACCTGGTGCTGTTGGAGAGCCTGGTGCTAAAGGTGCTATGGGTCCAGCTGGTCCAGATGGTCATCAAGGACCTAGAGGTGAACAAGGATTGACTGGTATGCCAGGTATTCGTGGTCCACCTGGACCTTCAGGTGATCCTGGTAAACCTGGTCTTACTGGTCCACAAGGACCACAAGGTTTGCCTGGTACTCCTGGTAGACCAGGTATCAAAGGTGAGCCAGGTGCTCCTGGAAAAATTGGACCACCAGGTCCACCAGGACCTCCAGGTCCTAAGGGTGACCAAGGTCCACCAGGTCCTAGAGGACATCAAGGTGAGCAAGGACTTCCAGGTTTTTCTGGACCTCCAGGACCTCCTGGACCACCAGGACCAAGAGGTCCACCTGGTGTTTCTGGTGCTTTAGGTCCTCCAGGTCAGAAAGGCGAAATGGGTACACCCGGTCCTAAAGGCGATAGAGGTCCTGCCGGACCACCTGGTCATCCTGGTCCACCTGGTCCTCGTGGCCATAAGGGTGAGAAAGGTGACAAAGGCGATCAACACCACCACCATCACCACGCGGCC(SEQ ID NO:9).

[0084] (2) Synthesis of DNA sequence and construction of recombinant expression vector

[0085] After synthesizing and amplifying SEQ ID NO: 9, the PCR product was purified and double-digested with EcoRI and NotI. After digestion, the target band was recovered by gel extraction. The PCR product after amplification of SEQ ID NO: 9 is shown below. Figure 1 As shown. Plasmid pPIC9K was double-digested with EcoRI and NotI, and the digested plasmid was recovered by column chromatography. The recovered target fragment and plasmid were mixed at a molecular weight ratio of 3:1 and ligated for 2 hours using DNA ligase. The resulting plasmid was cloned into the EcoRI and NotI restriction sites of pPIC9K to obtain the expression plasmid pPIC9K-Re-Hcol17. The pPIC9K plasmid map is shown below. Figure 2 As shown.

[0086] (3) Construction and screening of recombinant strains

[0087] The recombinant expression plasmid pPIC9K-Re-Hcol17 was transformed into TOP10 host cells. The plates were inverted and incubated at 37°C. Colonies appeared after 12–16 hours. Single colonies were picked and mixed into PCR tubes, then placed in a shaker at 37°C at 220 rpm. 2 μL of each tube was used as a template for PCR identification of the bacterial culture.

[0088] The correctly expressed recombinant expression plasmid pPIC9K-Re-Hcol17 was digested with SacI overnight to linearize it. The linearized plasmid was then recovered, and the plasmid and digestion products were extracted and identified as shown in the figure. Figure 3 As shown. Linearized plasmids (concentration above 1 μg / μL) were electroporated into Pichia pastoris GS115 competent cells (OD600 value controlled at 0.8). Electroporation parameters were: voltage 1500V; time 5 ms. The electroporated bacterial culture was plated on YPD plates. Zeocin selection (zeocin concentration 100 μg / mL) was used, with 100 μL–200 μL of bacterial culture plated per plate. After standing at room temperature for 10 min, the plates were incubated upside down at 30℃ for 4 days. Approximately 50 single colonies grew. The single colonies on the transformation plates are shown in the figure. Figure 4 As shown, single colonies were picked from the plate for screening, and the labeled single colonies were subjected to PCR identification to detect whether the plasmid had been transformed into the host. The PCR identification diagram of Pichia pastoris recombinant is shown in the figure. Figure 5 As shown.

[0089] (4) Expression of recombinant human type XVII small molecule collagen

[0090] Single colonies were picked from plates screened by PCR and cultured in YPD liquid medium. After 24 hours, the culture broth turned milky white. The broth was then inoculated into YPD medium (1% inoculum) and incubated at 230 rpm / min at 28°C. The OD600 value of the culture was measured approximately 24 hours later. When the OD600 value of the bacteria reached 0.6-0.7 after a 50-fold dilution (approximately 24 hours), the culture was transferred to sterile, cooled centrifuge tubes in a laminar flow hood and centrifuged at 6000 rpm / min, 4°C for 5 minutes. The supernatant was discarded in the laminar flow hood, and the bacteria were resuspended in YP medium. The resuspended system was then poured back into an Erlenmeyer flask, and 100 mL of sterile 1M phosphate buffer (working phosphate concentration 0.1M) was added. Simultaneously, 100% methanol (working methanol concentration 1%) was added to the system to begin induction. 100% methanol was added to the system every 24 hours. Samples were taken daily for electrophoresis, with the moment of the first addition of methanol as time zero. Approximately 48 hours later, the culture medium was poured into centrifuge tubes and centrifuged at 6000 rpm / min, 4°C for 10 min to collect the bacterial cells. The bacterial cells were resuspended in 250 mL of PBS buffer, then homogenized completely using a high-pressure homogenizer, transferred to centrifuge tubes, and centrifuged at 12000 rpm / min, 4°C for 20 min to collect the supernatant.

[0091] (5) Purification of recombinant human type XVII small molecule collagen

[0092] The supernatant from step (4) was purified by affinity chromatography, eluted with 10 mM, 30 mM, 60 mM, and 250 mM imidazole, respectively. The elution results for different imidazole concentrations are shown below. Figure 6 As shown in the figure. The 250 mM imidazole eluent was dialyzed to remove imidazole, and then purified again by affinity chromatography to further increase purity. Elutions were performed with 10 mM, 30 mM, 60 mM, and 250 mM imidazole, and the results are shown in the figure. Figure 7 As shown.

[0093] Example 2: Effect of recombinant human type XVII small molecule collagen on cell viability

[0094] 1. Experimental Methods

[0095] NIH / 3T3 fibroblasts were cultured normally, and the cells were seeded into 96-well plates, with approximately 100 μL of cell suspension per well (10). 5Cells were seeded and cultured at 37°C in a 5% CO2 incubator for 24 hours. After cell adhesion, the culture medium was aspirated, and fresh culture medium containing different concentrations of collagen (100 μL / well) was added. 100 μL of Re-Hcol17 protein solution at various concentrations, blank PBS solution (negative control), and commercially available natural human collagen (positive control) were added to each well of a 96-well cell culture plate. After culturing for another 24 hours, 10 μL of CCK-8 reagent and 90 μL of serum-free culture medium (total volume 100 μL) were added to each well. The mixture was gently stirred, avoiding air bubbles, and incubated at 37°C in a 5% CO2 incubator for 2 hours. The absorbance at OD450 nm of each well was measured using a microplate reader. Data were analyzed and significant differences were determined. Compared with the negative control, **p < 0.01, ***p < 0.001; compared with the positive control, #p < 0.05, ##p < 0.001. The absorbance at OD450nm characterizes the effect of Re-Hcol17 collagen samples on cell viability. Higher cell viability indicates that Re-Hcol17 collagen promotes fibroblast proliferation and has lower toxicity.

[0096] 2. Experimental Results

[0097] like Figure 8 As shown, the cell viability of each concentration of Re-Hcol17 collagen group was significantly better than that of commercial natural human collagen, and the cell viability increased sequentially with the increase of Re-Hcol17 collagen concentration, indicating that Re-Hcol17 collagen in this invention has the ability to significantly improve cell viability.

[0098] Example 3: Effect of recombinant human type XVII small molecule collagen on cell adhesion activity

[0099] 1. Experimental Methods

[0100] Recombinant human type XVII small molecule collagen Re-Hcol17 lyophilized product and control natural human collagen were taken, and the protein concentration was determined. Then, it was diluted with PBS (pH 7.4) to 0.5 mg / mL.

[0101] Add 100 μL of recombinant human type XVII small molecule collagen Re-Hcol17 solution, blank PBS solution (negative control), and natural human collagen solution (positive control) to each well of a 96-well cell culture plate, and incubate at room temperature for 60 min. Then add 10 μL of recombinant human type XVII small molecule collagen Re-Hcol17 solution, blank PBS solution (negative control), and natural human collagen solution (positive control) to each well. 5NIH / 3T3 fibroblasts in good culture condition were incubated at 37°C and 5% CO2 for 60 min. Cells were washed four times with PBS. The absorbance at OD492 nm was measured using an LDH detection kit. Data were analyzed and significant differences were determined. Compared with the negative control group, p < 0.001, and compared with the positive control group, p < 0.01.

[0102] 2. Experimental Results

[0103] The higher the absorbance at OD492nm, the higher the cell adhesion activity. Collagen can help cells adhere to the cell wall or extracellular matrix in a short time, which is more conducive to building a better extracellular environment. Figure 9 As shown, the cell adhesion activity of recombinant human type XVII small molecule collagen Re-Hcol17 is higher than that of natural human collagen.

[0104] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A recombinant collagen, characterized in that: The recombinant collagen consists of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

5.

2. The recombinant collagen according to claim 1, characterized in that: The amino acid sequence of the recombinant collagen is shown in any one of a1) to a3): a1)SEQ ID NO: 1; or a2) A sequence of amino acids that has the same function as SEQ ID NO: 1, but with one or more amino acid substitutions and / or deletions and / or additions; or a3) is an amino acid sequence that has at least 80% homology with SEQ ID NO:

1.

3. The recombinant collagen according to claim 2, characterized in that: The recombinant collagen includes optional tag sequences for expression and / or purification.

4. The fusion protein according to claim 3, characterized in that: The tag sequence includes at least one of the following: signal peptide, nuclear localization signal, NLS tag, His tag, Myc tag, Strep tag, GST tag, Flag tag, HA tag, and fluorescent protein tag.

5. Biomaterials related to the recombinant collagen according to any one of claims 1 to 4, comprising: b1) The nucleic acid molecule encoding the recombinant collagen; b2) An expression cassette containing the nucleic acid molecule described in b1); b3) A recombinant vector containing the nucleic acid molecule described in b1); b4) A recombinant vector containing the expression cassette described in b2); b5) Recombinant cells containing the nucleic acid molecules described in b1); b6) Recombinant cells containing the expression cassette described in b2); b7) Recombinant cells containing the recombinant vector described in b3); b8) Recombinant cells containing the recombinant vector described in b4); The cell-non-reproductive material; Preferably, the recombinant vector includes a eukaryotic vector or a prokaryotic vector; Preferably, the recombinant cells include eukaryotic cells or prokaryotic cells.

6. The biomaterial according to claim 4, characterized in that: The nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO:

8.

7. A method for preparing the recombinant collagen according to any one of claims 1 to 4, comprising the following steps: The biological material described in claim 5 or 6 is expressed, purified, and obtained.

8. The use of the recombinant collagen according to any one of claims 1 to 4 in the preparation of the product; The product is used in the fields of skin repair, tissue engineering, cell culture substrates, or drug carriers.

9. The product according to claim 8 is characterized in that: The products include one of the following: cosmetics, medical devices, and pharmaceuticals.

10. The product according to claim 8, characterized in that: The product includes pharmaceutically acceptable excipients.

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