Recombinant collagen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to recombinant collagen as well as a preparation method and application thereof. The amino acid sequence of the recombinant collagen is formed by randomly combining amino acid fragments (1), (2), (3) and (4), and the amino acid fragments are directly connected; the sequence of the amino acid fragment (1) is TRTLLLL, the sequence of the amino acid fragment (2) is FMRLL, the sequence of the amino acid fragment (3) is KHVWL, and the sequence of the amino acid fragment (4) is LLHPTII. According to the present invention, the sequence of the recombinant collagen is the first recombinant collagen containing hydroxyproline, and the recombinant collagen has characteristics of oxidation resistance, oxidation factor elimination, cell oxidation reduction state regulation, cell metabolism, growth and development regulation, collagen synthesis and growth promotion, fibroblast growth stimulation, and wound healing promotion.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is a key protein in the extracellular matrix of mammalian tissues, accounting for approximately 25% to 35% of total body protein. It is primarily found in fibrous tissues such as tendons, ligaments, cartilage, and skin. In the health supplement sector, collagen is rich in essential and non-essential amino acids and can be used as a nutrient in novel functional foods. Collagen peptides are already widely used in health foods in the United States. In the food industry, gelatin derived from collagen is widely used due to its excellent gelling, film-forming, and thickening properties. It can be used to increase the viscosity of juices and enhance the colloidal stability of jellies. In cosmetics, collagen's moisturizing, repairing, and supporting properties are utilized in creams, nutritional supplements, and fillers, offering moisturizing, repairing, anti-aging, and cosmetic benefits. In biomedical applications, collagen's fibrillogenicity, bioactivity, thermal stability, and degradation properties are utilized to create tissue guide materials, artificial skin substitutes, bone tissue scaffolds, artificial blood vessels, artificial tendons, intraocular lenses, and can be used to treat corneal injuries. Type I collagen is a fibrous collagen that is widely found in skin, bones, and tendons. It can enhance tissue strength and elasticity, provide cells with sufficient nutrients, and maintain normal physiological functions.

[0003] Type I collagen is approximately 300 nm long and 1.4–1.5 nm in diameter. Type I collagen is a fibrillar collagen, widely found in skin, tendons, and bone tissue, enhancing tissue strength and elasticity. However, studies have discovered additional functions. Within tissues, type III collagen fibers are smaller in diameter than type I collagen. When both type I and type III collagen are present in a single collagen fiber, type III collagen regulates the diameter of the collagen fibers. Type III collagen is also present in adult cartilage, and some studies suggest that during tissue healing, type III collagen acts as a modifier of the fibrous network composed of type II collagen and other small collagens. Furthermore, type III collagen is a major structural component of hollow organs such as large blood vessels, the uterus, and the intestine. As an extracellular matrix protein, it maintains the morphology and structure of skin, tissues, and organs. Type III collagen also interacts with platelets in the coagulation cascade and serves as an important signaling molecule for wound healing.

[0004] At present, domestic research is only focused on the Gly-Xaa-Yaa repeat region of collagen, or the splicing of an amino acid sequence of collagen or a specific amino acid sequence of a collagen molecule. There are currently no reports on the combined development of specific functional fragments of type I and type III collagen. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides a recombinant collagen protein. This protein utilizes three specific functional fragments of type I collagen—TRTLLLL, FMRLL, and KHVWL—and one specific functional fragment of type III collagen, LLHPTII, to form a novel recombinant collagen protein. This recombinant collagen protein sequence is the first recombinant collagen protein to contain hydroxyproline.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a recombinant collagen protein, the amino acid sequence of which is formed by a random combination of the following ①②③④ amino acid fragments, and the amino acid fragments are directly connected; the amino acid fragment ① sequence is shown in SEQ ID NO:4, the amino acid fragment ② sequence is shown in SEQ ID NO:5, the amino acid fragment ③ sequence is shown in SEQ ID NO:6, and the amino acid fragment ④ sequence is shown in SEQ ID NO:7.

[0008] Preferably, the amino acid sequence combination includes ①②③④, ①③④②, ①④③②, ②①③④, ②①④③, ③①②④, ③②④①, ③④②①, ④③②①, ④②①③, ④①③②, ④②③①.

[0009] Preferably, the amino acid sequence of the recombinant collagen is any one of the following:

[0010] a) the amino acid sequence shown in SEQ ID NO: 3;

[0011] b) an amino acid sequence that is 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3, and that retains the cell adhesion effect of the amino acid sequence of SEQ ID NO: 3;

[0012] c) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 3, and which retains the cell adhesion effect of the amino acid sequence of SEQ ID NO: 3.

[0013] In a second aspect, the present invention provides a polynucleotide encoding the recombinant collagen.

[0014] In a third aspect, the present invention provides an expression vector comprising a nucleotide sequence encoding the amino acid sequence of the recombinant collagen.

[0015] Preferably, the expression vector further contains a nucleotide sequence encoding proline hydroxylase.

[0016] Preferably, the expression vector contains the nucleotide sequence of proline hydroxylase P4Hα or / and P4Hβ.

[0017] Preferably, the amino acid sequence of P4Hα is shown in SEQ ID NO: 1, and the amino acid sequence of P4Hβ is shown in SEQ ID NO: 2.

[0018] In a fourth aspect, the present invention provides a host cell comprising the expression vector described in the third aspect.

[0019] In a fifth aspect, the present invention provides a method for preparing the recombinant collagen described in the first aspect, the method comprising the following steps:

[0020] (1) constructing the expression vector as described above;

[0021] (2) transforming the expression vector of step (1) into host cells, culturing the cells and collecting the cell culture fluid;

[0022] (3) Purifying the cell culture medium of step (2) to obtain the recombinant collagen.

[0023] In a sixth aspect, the present invention provides the use of the recombinant collagen described in the first aspect as a dressing in cell proliferation, wound healing, and scar repair.

[0024] In a seventh aspect, the present invention provides the use of the recombinant collagen described in the first aspect as a tissue engineering material in promoting cell metabolism, growth, and development; or in use as a hemostatic material.

[0025] The beneficial effects of the present invention are:

[0026] The present invention utilizes three specific functional fragments of type I collagen: ①TRTLLLL (SEQ ID NO: 4), ②FMRLL (SEQ ID NO: 5), and ③KHVWL (SEQ ID NO: 6), and one specific functional fragment of type III collagen: ④LLHPTII (SEQ ID NO: 7), in any combination to form a new recombinant collagen. Preferably, it can be LLHPTIITRTLLLLFMRLLKHVWL (④①②③ combination, SEQ ID NO: 3), wherein P is hydroxyproline. The present invention is the first recombinant collagen containing hydroxyproline. This recombinant collagen has a short amino acid sequence and a clear purpose. It has the effect of regulating cell metabolism, growth, and development, promoting collagen synthesis and growth, stimulating fibroblast growth, and promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 A schematic diagram of the pPICZαB vector used in the examples of the present invention;

[0029] Figure 2 A schematic diagram of the pPICZαA vector used in the embodiments of the present invention;

[0030] Figure 3 The target protein spectrum obtained in the embodiment of the present invention;

[0031] Figure 4 Schematic diagram of the effect of the recombinant collagen of the present invention on the elasticity of skin tissue;

[0032] Figure 5 This is a schematic diagram showing the effect of the recombinant collagen of the present invention on fibroblasts;

[0033] Figure 6 Schematic diagram of trypan blue staining of cell status after electrotransfection (cell magnification 40 times);

[0034] Figure 7 The figure is a flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0035] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0036] Example 1

[0037] This embodiment provides a method for preparing recombinant collagen, which adopts a chemical synthesis method. The specific steps are as follows:

[0038] First step chemical synthesis

[0039] 1. Resin swelling:

[0040] Weigh 5 g of 2-Chlorotrityl Chloride Resin with a degree of substitution of 0.5-1.0 mmol / g, place the resin in a reaction tube, add DCM (dichloromethane, 15 mL / g), and shake for 30 min.

[0041] 2. Connect the first amino acid:

[0042] Remove the solvent by suction through a sand core, add a 3-6-fold molar excess of Fmoc-Leu-OH amino acid (leucine), then a 10-fold molar excess of DIEA (N,N-diisopropylethylamine), and finally a small amount of DMF (dimethylformamide) to dissolve. Shake for 1-3 hours. Wash with DMF and DCM alternately six times.

[0043] 3. Deprotection:

[0044] Add 15-30 mL of 20% piperidine DMF solution (15 mL / g), stir for 5 min, remove the above piperidine DMF solution and add 15 mL of 20% piperidine DMF solution (15 mL / g), stir for 15 min.

[0045] 4. Testing:

[0046] Drain the piperidine solution, take a dozen resin pellets, wash them three to five times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃~110℃ for 5 minutes, and a dark blue color indicates a positive reaction.

[0047] 5. Washing:

[0048] The product was washed with DMF (10 mL / g) for two to five times, methanol (10 mL / g) for two to five times, and DMF (10 mL / g) for two to five times.

[0049] 6. Condensation:

[0050] A three-fold to five-fold excess of a protected amino acid (arginine, Fmoc-Arg(Pbf)-OH) and a three-fold excess of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) were dissolved in as little DMF as possible and added to the reaction tube. A ten-fold excess of DIEA was immediately added and the reaction was allowed to proceed for 40 minutes.

[0051] 7. Washing:

[0052] The product was washed with DMF (10 mL / g) one to three times, methanol (10 mL / g) twice, and DMF (10 mL / g) twice.

[0053] 8. Repeat steps 2 to 7 to connect the amino acids in the sequence from right to left (the amino acid sequence in this example is: LLHPTIITRTLLLLFMRLLKHVWL SEQ ID NO: 3).

[0054] 9. Testing:

[0055] The product was washed twice with DMF (10 mL / g), twice with DCM (10 mL / g), and twice with DMF (10 mL / g). The product was drained for 10 min and the ninhydrin test was negative.

[0056] 10. Remove the lysine protecting group (dde) in the sequence with DMF containing one or two drops of hydrazine hydrate. Add three times excess hexadecanoic acid and three times excess HBTU, both dissolved in as little DMF as possible, to the reaction tube. Immediately add ten times excess DIEA and react for 40 minutes.

[0057] 11. Washing: Wash three times with methanol (10 mL / g).

[0058] 12. Cleavage of peptides from resin:

[0059] Prepare a cutting solution (10 mL / g): 94.5% TFA, 2.5% water, 2.5% EDT, and 1% TIS. Place the resin in a flask or centrifuge tube at a ratio of 10 mL / g resin to cutting solution. Shake at a constant temperature for 120 min.

[0060] 13. Blow dry and wash:

[0061] The cleavage solution was blown dry with nitrogen as much as possible, chromatographed with ether, washed with ether six times, and then evaporated at room temperature to obtain the crude peptide sequence.

[0062] The second step is to purify the peptide by HPLC

[0063] Specific steps:

[0064] 1. Take 200-500 mg of crude peptide and place it in a container. Dissolve it with 2-15 mL of 50% acetonitrile aqueous solution. Ultrasonicate for 2-10 minutes to obtain a solution.

[0065] 2. Filter the solution with a 0.45 μm filter membrane.

[0066] 3. Analysis: Analyze the crude product by taking 3-6 μL of analytical-grade HPLC. The mobile phase is water and acetonitrile, and the elution time is 30 minutes. Equilibrate the HPLC with a starting gradient of 95% water and 5% acetonitrile for 5 minutes before injection. The starting gradient is 95% water and 5% acetonitrile, and the ending gradient is 5% water and 95% acetonitrile.

[0067] 4. Preparation: Prepare the dissolved sample for injection. Equilibrate the preparative HPLC for 10 minutes. Run a gradient of 95% water to 5% acetonitrile, with a final gradient of 25% to 50% water to 50% acetonitrile, over 40 minutes. Collect the sample from the detector.

[0068] 5. Identification: Take samples of the collected samples for purity and MS identification.

[0069] Example 2

[0070] This embodiment provides a method for preparing recombinant collagen, which adopts a biosynthesis method. The specific steps are as follows:

[0071] 1. Construct a recombinant plasmid for yeast transformation. The specific steps are as follows:

[0072] The human P4Hα gene, Gene ID: 5033, and the P4Hβ gene, Gene ID: 5034, were found. The P4Hα and P4Hβ gene sequences were optimized for Pichia pastoris preference by adjusting codon usage preferences, optimizing the 5' region (translation initiation efficiency), DNA repeat sequences, mRNA secondary structure, GC content, SD sequences, and excluding designated restriction enzyme sites. Restriction enzymes were selected based on the P4Hα and P4Hβ sequence information: EcoRI and KnpI, and NotI and BamHI, respectively, for full sequence synthesis.

[0073] According to the principle of codon preference, the expression vector pPICZαB (such as Figure 1 (As shown) The restriction site selection pPICZαB plasmid map, insertion site: between TEF1 promoter and promoter.

[0074] The optimized gene fragments encoding either or both of the amino acid sequence of the prolyl-4-hydroxylase α subunit (P4Hα) SEQ ID NO: 1 and the amino acid sequence of the prolyl-4-hydroxylase β subunit (P4Hβ) SEQ ID NO: 2 were double-enzyme digested and fused to the pPICZαB plasmid (the enzyme gene fragment insertion site was between the TEF1 promoter and the promoter), respectively obtaining recombinant plasmid 1 containing P4Hα (denoted as: pPICZαB-P4Hα), recombinant plasmid 2 containing P4Hβ (denoted as: pPICZαB-P4Hβ), and recombinant plasmid 3 containing both P4Hα and P4Hβ (denoted as: pPICZαB-P4Hα-P4Hβ); all three plasmids can be used to transform recombinant Pichia pastoris strains.

[0075] The amino acid sequence of P4Hα edited by the optimized P4Hα gene is shown in SEQ ID NO: 1, and the amino acid sequence of P4Hβ edited by the optimized P4Hβ gene is shown in SEQ ID NO: 2.

[0076] Amino acid sequence of SEQ ID NO: 1 (P4Hα):

[0077] MIWYILIIGILLPQSLAHPGFFTSIGQMTDLIHTEKDLVTSLKDYIKAEEDKLE

[0078] QIKKWAEKLDRLTSTATKDPEGFVGHPVNAFKLMKRLNTEWSELENLVLKD

[0079] MSDGFISNLTIQRQYFPNDEDQVGAAKALLRLQDTYNLDTDTISKGNLPGV

[0080] KHKSFLTAEDCFELGKVAYTEADYYHTELWMEQALRQLDEGEISTIDKVSV

[0081] LDYLSYAVYQQGDLDKALLLTKKLLELDPEHQRANGNLKYFEYIMAKEKD

[0082] VNKSASDDQSDQKTTPKKKGVAVDYLPERQKYEMLCRGEGIKMTPRRQKK

[0083] LFCRYHDGNRNPKFILAPAKQEDEWDKPRIIRFHDIISDAEIEIVKDLAKPRLR

[0084] RATISNPITGDLETVHYRISKSAWLSGYENPVVSRINMRIQDLTGLDVSTAEE

[0085] LQVANYGVGGQYEPHFDFARKDEPDAFKELGTGNRIATWLFYMSDVSAGG

[0086] ATVFPEVGASVWPKKGTAVFWYNLFASGEGDYSTRHAACPVLVGNKWVSN

[0087] KWLHERGQEFRRPCTLSELE

[0088] Amino acid sequence of SEQ ID NO:2 (P4Hβ):

[0089] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAP

[0090] WCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPT

[0091] IKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESS

[0092] EVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLF

[0093] KKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLF

[0094] LPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPA

[0095] VRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQ

[0096] PVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDH

[0097] ENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL.

[0098] 2. Acquisition of Pichia pastoris strains containing human prolyl hydroxylase (P4H)

[0099] (1) Amplification of P4H recombinant plasmids (P4Hα recombinant plasmid, P4Hβ recombinant plasmid, P4Hα and P4Hβ recombinant plasmids)

[0100] Take 2-5 μL of the three plasmids mentioned above and transform them into 30-60 μL of competent E. coli DH5α cells (placed on ice). Mix well and let it rest on ice for 30 minutes. Place the mixed suspension in a dry-well incubator set to 42°C for 90 seconds. Remove and place on ice for another 3 minutes. Add 500 μL of antibiotic-free LB medium and incubate at 37°C at 160-200 rpm in a constant temperature shaker for 2-3 hours. Take 20-40 μL of the bacterial solution and evenly spread it on an LB plate containing 5-20 μg / mL Zecion antibiotics. Incubate at 37°C in a constant temperature incubator for 16-18 hours. Pick a single colony on the plate and inoculate it into 10 mL of LB medium containing 10 μg / mL Zecion antibiotics. Incubate at 160-200 rpm at 37°C for 13-16 hours.

[0101] (2) Extraction, identification and linearization of P4H recombinant plasmid

[0102] Plasmids were extracted from the culture medium using a plasmid extraction kit to obtain recombinant plasmids 1, 2, and 3, respectively, and their concentrations were determined. Upstream and downstream primers were designed based on the gene sequence, and forward and reverse sequencing was performed three times for alignment.

[0103] If the identification is successful, the plasmid is double-digested with Not I and BamH I. The reaction system for each tube is as follows:

[0104]

[0105] The plasmid was linearized by digesting with BspHI. The enzyme digestion reaction system for each tube was as follows:

[0106]

[0107] Place in a PCR instrument and digest at 37°C for 1–3 hours. Then adjust the temperature to 60–80°C and inactivate the enzyme for 10 minutes. Purify the digested and linearized plasmid using the Wizard SV Gel and PCR Clean-Up System kit according to the manufacturer's instructions. Reserve a small sample for agarose gel electrophoresis and store the remaining sample at –20°C until further use.

[0108] (3) Preparation of competent yeast cells

[0109] Take 100-200 μL of X33 expression bacteria or GS115 expression strain or other yeast strains and inoculate them into 200 mL YPD medium (yeast extract peptone dextrose medium). Culture overnight at 200-250 rpm and 25-30 ° C until OD 600nm =1.2-1.5. The cell culture was centrifuged at 4°C, 1500 rpm for 5 minutes and resuspended in 20 mL of sterile water. After repeating twice, the resuspension was centrifuged at 4°C, 1500 rpm for 5 minutes, resuspended in 5 mL of sterile water, and 50-70 μL of DTT (DL-Dithiothreitol) was added, mixed, and allowed to stand at room temperature for 20 minutes. The resuspension was then centrifuged at 4°C, 1500 rpm for 5 minutes, and resuspended in 500 μL of 1 M sorbitol. Repeat twice.

[0110] (4) Electroporation of recombinant plasmids pPICZαB-P4Hα-P4Hβ, pPICZαB-P4Hβ, and pPICZαB-P4Hα was performed as follows:

[0111] a) Soak the rotating cup in 75% alcohol for more than 20 minutes, irradiate with UV light for 20 minutes, and blow dry for later use;

[0112] b) Add 20-100 μL of X competent cells and at least 4 μg of linearized recombinant plasmid to an electroporation cuvette. Place the cuvette in an ice bath for 10 minutes before electroporation. The electroporation conditions are set to: voltage 1.5 kV, capacitance 25 μF, resistance 200-400 W, and electroporation for 10 msec.

[0113] c) Add 2 mL of 4°C pre-cooled 1 M sorbitol solution, gently mix the cells with a pipette, and transfer to a 2.5 mL EP tube;

[0114] d) Spread the resuspension onto a YPD plate containing Zecion antibiotic and incubate at 25-30°C for 48 hours until single colonies appear.

[0115] (5) Expression and identification of recombinant plasmids in engineered bacteria:

[0116] The engineered yeast containing the recombinant plasmids pPICZαB-P4Hα-P4Hβ, pPICZαB-P4Hβ, and pPICZαB-P4Hα were cultured. The specific implementation plan is as follows:

[0117] a. Pick out the activated engineered bacteria containing the recombinant plasmids pPICZαB-P4Hα-P4Hβ, pPICZαB-P4Hβ, and pPICZαB-P4Hα and inoculate them into 20 mL of YPD medium. Incubate at 200-260 rpm and 25-30°C for 18-24 hours. Pipette the culture solution into 25 mL of YPG medium at a 2% inoculum volume. Incubate at 200-260 rpm and 25-30°C for 18-20 hours until the OD reaches 0. 600 =2.1.

[0118] b. Centrifuge the culture at 2000-4000 rpm for 10 minutes to collect the cells. Resuspend the cells in 20-50 mL of YPM and incubate in a 250 mL Erlenmeyer flask at 200-260 rpm and 25-30°C for 36-72 hours. Centrifuge the resulting fermentation broth at 10,000-15,000 rpm for 5 minutes. Collect the supernatant to obtain the crude rhP4H enzyme solution. Identify the enzyme by mass spectrometry.

[0119] 3. Expression and identification of recombinant collagen amino acid sequence in Pichia pastoris strain expressing recombinant human collagen prolyl hydroxylase.

[0120] The recombinant collagen amino acid sequence is: LLHPTIITRTLLLLFMRLLKHVWL (SEQ ID NO: 3), where P represents hydroxyproline. The DNA encoding this amino acid sequence was double-digested and ligated into a Pichia pastoris strain containing a human prolyl hydroxylase plasmid, specifically a yeast strain containing the pPICZαB-P4Hα-P4Hβ or pPICZαB-P4Hα or pPICZαB-4Hβ recombinant plasmid. The specific steps are as follows:

[0121] (1) Construction of rh C-P4H recombinant plasmids (i.e., recombinant plasmids containing recombinant collagen and human prolyl hydroxylase, denoted as: pPICZαB-C-P4Hα, pPICZαB-C-4Hβ, pPICZαB-C-4Hα-P4Hβ) was performed as follows:

[0122] The nucleotide fragment encoding the recombinant collagen amino acid sequence: LLHPTIITRTLLLLFMRLLKHVWL (SEQ ID NO: 3) is inserted into the TEF1 promoter and promoter site of the pPICZαB-P4Hα-P4Hβ or pPICZαB-P4Hα or pPICZαB-4Hβ recombinant plasmid to obtain the rh C-P4H recombinant plasmid containing the recombinant collagen DNA fragment, namely: pPICZαB-C-4Hα-P4Hβ or pPICZαB-C-P4Hα or pPICZαB-C-4Hβ recombinant plasmid.

[0123] (2) Obtaining rh C-P4H recombinant Pichia pastoris:

[0124] a. rh C-P4H recombinant plasmid amplification:

[0125] Take 2-5 μL of the rh C-P4H recombinant plasmid and transform it into 30-60 μL of competent E. coli DH5α cells (placed on ice). Mix thoroughly and place on ice for 30 minutes. Place the suspension in a dry-well incubator set to 42°C for 90 seconds. Remove and place on ice for another 3 minutes. Add 500 μL of antibiotic-free LB medium and incubate at 37°C in a shaker at 160-200 rpm for 2-3 hours. Spread 20-40 μL of the bacterial suspension evenly on an LB plate containing 5-20 μg / mL Zecion antibiotics. Incubate at 37°C for 16-18 hours. Pick a single colony from the plate and inoculate it into 10 mL of LB medium containing 10 μg / mL Zecion antibiotics. Incubate at 160-200 rpm at 37°C for 13-16 hours.

[0126] b. Extraction, identification and linearization of rh C-P4H recombinant plasmid

[0127] Plasmids were extracted from the culture medium using a plasmid extraction kit to obtain recombinant plasmids and measure their concentrations. Upstream and downstream primers were designed based on the gene sequence, and forward and reverse sequencing was performed three times for alignment.

[0128] If the identification is successful, the plasmid is double-digested with Not I and BamH I. The reaction system for each tube is as follows:

[0129]

[0130] The plasmid was linearized by digesting with BspHI. The enzyme digestion reaction system for each tube was as follows:

[0131]

[0132]

[0133] Place above the PCR plate and digest at 37°C for 1–3 hours. Then adjust the temperature to 60–80°C and inactivate the enzyme for 10 minutes. Purify the digested and linearized plasmid using the Wizard SV Gel and PCR Clean-Up System kit according to the manufacturer's instructions. Reserve a small sample for agarose gel electrophoresis and store the remaining sample at –20°C until further use.

[0134] c. Preparation of competent yeast cells

[0135] Take 100-200 μL of X33 expression bacteria or GS115 expression strain or other yeast strains and inoculate them into 200 mL YPD medium. Incubate at 200-250 rpm and 25-30°C overnight until OD 600nm =1.2-1.5. Centrifuge the cell culture at 1500 rpm at 4°C for 5 minutes and resuspend the cells in 20 mL of sterile water. Repeat twice. Centrifuge the resuspension at 1500 rpm at 4°C for 5 minutes. Resuspend the cells in 5 mL of sterile water, add 50-70 μL of DTT, mix well, and incubate at room temperature for 20 minutes. Then centrifuge the resuspension at 1500 rpm at 4°C for 5 minutes and resuspend the cells in 500 μL of 1 M sorbitol. Repeat twice.

[0136] d. Electroporation of recombinant plasmid rh C-P4H (pPICZαB-C-4Hα-P4Hβ, pPICZαB-C-P4Hα, pPICZαB-C-4Hβ) to prepare competent cells is as follows:

[0137] a) Soak the rotating cup in 75% alcohol for more than 20 minutes, irradiate with UV light for 20 minutes, and blow dry for later use;

[0138] b) Add 20-100 μL of X competent cells and at least 4 μg of linearized recombinant plasmid to an electroporation cuvette. Place the cuvette in an ice bath for 10 minutes before electroporation. The electroporation conditions are set to: voltage 1.5 kV, capacitance 25 μF, resistance 200-400 W, and electroporation for 10 msec.

[0139] c) Add 2 mL of 4°C pre-cooled 1 M sorbitol solution, gently mix the cells with a pipette, and transfer to a 2.5 mL EP tube;

[0140] d) Spread the resuspension onto a YPD plate containing Zecion antibiotic and incubate at 25-30°C for 48 hours until single colonies appear.

[0141] (4) Expression and identification of rh C-P4H in engineered bacteria

[0142] The pPICZαB-C-4Hα-P4Hβ, pPICZαB-C-P4Hα, and pPICZαB-C-4Hβ engineering bacteria containing the recombinant plasmids were cultured. The specific implementation plan is as follows:

[0143] a. Pick out the activated engineered bacteria containing the recombinant plasmids pPICZαB-C-4Hα-P4Hβ, pPICZαB-C-P4Hα, and pPICZαB-C-4Hβ and inoculate them into 20 mL of YPD medium. Incubate at 200-260 rpm and 25-30°C for 18-24 hours. Pipette the culture solution into 25 mL of YPG medium at a 2% inoculum volume. Incubate at 200-260 rpm and 25-30°C for 18-20 hours until the OD reaches 0. 600 =2.1.

[0144] b. Centrifuge the culture at 2000-4000 rpm for 10 minutes to collect the cells. Resuspend the cells in 20-50 mL of YPM and incubate in a 250 mL Erlenmeyer flask at 200-260 rpm and 25-30°C for 36-72 hours. Centrifuge the resulting fermentation broth at 10,000-15,000 rpm for 5 minutes. Collect the supernatant to obtain the recombinant collagen. Identify the protein by mass spectrometry.

[0145] Example 3

[0146] This embodiment provides a method for preparing recombinant collagen, which adopts a biosynthesis method. The specific steps are as follows:

[0147] This example uses a CHO (Chinese hamster ovary cell) expression system, and the specific steps are as follows:

[0148] 1. Construct plasmids and construct the gene sequences encoding SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 into mammalian cell vectors:

[0149] Use pPICZαA vector (vector plasmid map as shown in Figure 2 The gene sequences encoding SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 were respectively inserted into the corresponding positions between AOX1 PROMOTE and PROREGION to obtain four plasmid vectors: pPICZαA-4Hα-P4Hβ, pPICZαA-P4Hα, pPICZαA-4Hβ, and pPICZαA-C.

[0150] 2. Transform the above-mentioned pPICZαA-C plasmid and any of the other three plasmids into the CHO cell expression system at a ratio of 1:5 to 1:20.

[0151] 3. Perform CHO cell recovery, passage, transformation, culture, and purification. The specific steps are as follows:

[0152] recovery:

[0153] (1) Remove the cell cryovial from liquid nitrogen and quickly thaw it in a 37°C water bath until there are no crystals in the cryovial. Wipe the outer wall of the cryovial with 75% alcohol.

[0154] (2) Transfer the cells from the cryopreserved tube to a 15 mL centrifuge tube containing 6 mL of complete culture medium and centrifuge at 1000-2000 rpm for 5-10 minutes.

[0155] (3) Discard the supernatant, resuspend the precipitate in 6 mL of complete culture medium, and inoculate it in a 25 cm 2 Culture the cells in a culture flask at 37°C in a 5% CO2 cell culture incubator.

[0156] Passaging:

[0157] Adherent cells:

[0158] (1) When the cells grow to cover 80% to 90% of the culture flask, discard the 25cm 2 The culture medium in the culture flask was removed and the cells were washed once with PBS;

[0159] (2) Add about 1-3 mL of 0.25% trypsin digestion solution to the culture flask, invert it and observe it under a microscope. After the cells shrink and become round, add 5 mL of complete culture medium to terminate the digestion. Then gently blow the cells to remove them. Transfer the suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0160] (3) Discard the supernatant, resuspend the pelleted cells in 1–2 mL of complete culture medium, divide the cells into flasks and passage them at a ratio of 1:2, add culture medium, and culture in a 37°C, 5% CO2 cell culture incubator.

[0161] Suspension cells:

[0162] When the number of cells reaches 1×10 6 / mL~1×10 9 / mL, and the medium can be changed or subcultured according to any of the following methods.

[0163] Method ①: Collect cells, centrifuge at 1000-1500 rpm for 5 minutes, discard the supernatant, add 1-2 mL of culture medium and blow evenly, then divide the cell suspension into new bottles containing culture medium at a ratio of 1:2 to 1:5.

[0164] Method ②: Place the culture flask upright, discard the upper half of the culture medium after the cells settle, and divide the cell suspension into new bottles containing culture medium at a ratio of 1:2 to 1:5.

[0165] Conversion:

[0166] (1) CHO cells were cultured in DMEM medium containing 10% calf serum and passaged at a ratio of 1:3 to 1:10 48 h before electroporation. Fresh medium was replaced 24 h before electroporation. Cells were examined under a microscope before electroporation. Logarithmic growth phase cells that adhered to the wall in 80% to 90% of the field of view were selected and digested with trypsin. The cells were centrifuged at 1000 rpm for 10 min. The precipitate was washed with serum-free DMEM medium and centrifuged. The cells were counted and the number of cells in each experimental group was adjusted to 2 × 10 6 ~2×10 9 .

[0167] (2) CHO cell electrosensitivity test. A low ionic strength Tris-Cl buffer with a pH range of 6 to 8 was selected, the electric field strength was 500 to 800 V / cm, and the current was 25 to 100 μF. The cells were suspended in the electroporation buffer and placed on ice for 10 to 20 minutes. The number of pulses was 2 to 5, with an interval of 1 to 3 minutes of ice bathing. After the electroporation, the cells were placed on ice for 10 minutes. The cell suspension was diluted 10-fold with 10% calf serum DMEM medium and incubated at 37°C, 5% CO2 for 6 hours. The viable cells were counted by trypan blue staining. The results were analyzed by nested analysis of variance using SPSS 13.0 software. The medium was changed every 3 days. After the resistant clones appeared, the DMEM maintenance medium was changed, and the number of resistant clones was observed and counted daily.

[0168] nourish:

[0169] (1) After 2-3 days of shake flask culture, the cell density increases and the nutrients are exhausted. The expansion culture volume is calculated based on the cell density. The shake flask cells are inoculated into the shake flask in the clean bench. The inoculation shake flask pipeline and the reactor pipeline are aseptically welded with a sterile welding machine. The PBS in the tank is first discharged with a pump. During this process, pay attention to keep it sterile. After the PBS is discharged, a certain amount of culture medium is pumped in. The parameters of this batch of cell culture are adjusted. After the pH, temperature, dO2, Stirrer, and readings are stable, a certain amount of cells are inoculated into the bioreactor. Finally, the culture medium is added to the culture volume designed for this experiment. After the cells are inoculated, the bioreactor monitoring system BioXpert is turned on to monitor the culture status in real time until the cell culture is completed.

[0170] (2) Cells were cultured in a bioreactor at a density of 500,000 cells per milliliter in a total culture volume of 3 L. Dissolved oxygen was automatically controlled to maintain approximately 40% during the day. When the reactor was left overnight, air was aerated over the surface of the culture medium to prevent cell hypoxia. Throughout the culture process, cell counts were performed every 12 h, and 1 mL of the cell supernatant was collected for ELISA analysis to determine protein concentration until the expression of the recombinant protein decreased.

[0171] Purification of target protein:

[0172] (1) Install the 10kD membrane package on the ultrafiltration device, pass 0.1mol / L NaOH, and wash the membrane package for 20 minutes. Then change to pure water and wash for 10 minutes, then pass CHO cell supernatant, collect the filtered and unfiltered parts, and mark them. Then change to pure water and wash for 10 minutes, then change to 0.1mol / L NaOH and wash for 20 minutes. Finally, seal the pipeline to allow the entire membrane package to be filled with 0.1mol / L NaOH, otherwise the membrane package will be damaged. If the membrane package needs to be removed, it needs to be soaked in alkaline solution.

[0173] (2) Equilibrate the column with 50 mM Tris buffer, 20 mM imidazole, and 0.15 M NaCl equilibrator, with a volume of approximately 3 to 5 column volumes. Load the target protein with a sample volume of 3 column volumes. Rinse with 50 mM Tris buffer, 20 mM imidazole, and 0.15 M NaCl as a flushing solution for 3 to 5 column volumes. Elute with 50 mM Tris buffer, 0.15 M NaCl, and 200 mM imidazole eluent, with an elution volume of 3 to 5 column volumes. Activate the column with 100 mM EDTA and regenerate it with 100 mM NiSO4. Store the column in 25% ethanol. The resulting protein is analyzed by mass spectrometry.

[0174] The protein obtained by the above method was detected by mass spectrometry, and the experimental results were as follows Figure 3 As shown, it is shown that the target protein of the present invention is obtained.

[0175] Test example

[0176] 1. Cell Adhesion Test

[0177] (1) The concentration of the protein samples to be tested was detected by ultraviolet absorption method, and the protein concentration of all samples to be tested was adjusted to 0.5 mg / mL with PBS.

[0178] Protein samples include: commercial collagen (C3867, Sigma-Aldrich); synthetic amino acid fragments TRTLLLL, FMRLL, KHVWL, LLHPTII; and the recombinant collagen of the present invention (LLHPTIITRTLLLLFMRLLKHVWL).

[0179] (2) Add 100 μL of each protein solution and a blank PBS solution as a control to a 96-well plate and let it stand at room temperature for 60 min.

[0180] (3) Add about 1×10 6 3T3 cells in good culture condition were incubated at 37°C for 60 min and then washed with PBS.

[0181] (4) Collect adherent cells and measure the absorbance OD at 450 nm using LDH kit (Roche, 04744926001). 450nm The results are shown in the following table:

[0182] Table 1: Cell adhesion activity results

[0183] sample <![CDATA[OD 450nm ]]> PBS 0.4 Collagen C3867 1.2 TRTLLLL 0.8 FMRLL 0.7 KHVW 0.5 LLHPTII 0.6 Recombinant collagen of the present invention 1.8

[0184] As can be seen from the results in the table above, the recombinant collagen of the present invention has a higher absorbance value at 450nm, indicating that the number of adherent cells in the well is greater, and it can provide a high-quality external environment for the cells in a shorter time, helping the cells to adhere to the wall and promoting cell adhesion; while the absorbance value of the amino acid fragment group is smaller, indicating that the amino acid fragments do not have a cell adhesion effect.

[0185] 2. Antioxidant test

[0186] (1) The antioxidant effect of each protein sample was determined using the DPPH method.

[0187] Prepare 0.1 mM DPPH solution; prepare 0.5 mg / mL Vc solution as a positive control; prepare 0.5 mg / mL protein sample solution.

[0188] Protein samples include: commercial collagen (C3867, Sigma-Aldrich); synthetic amino acid fragments TRTLLLL, FMRLL, KHVWL, LLHPTII; and the recombinant collagen of the present invention (LLHPTIITRTLLLLFMRLLKHVWL).

[0189] (2) Plate the plates (keep the plates away from light. After loading, incubate in a dark place at room temperature for 30 minutes and measure the absorbance). Place the plates in three groups of 96-well plates, with three replicate wells in each group.

[0190] (3) Measure the absorbance at 517 nm, take the average value, and calculate the DPPH clearance rate.

[0191] Clearance % = (1-(A 样品 –A 空白 ) / A 阳性对照 )×100%. The test results are shown in the following table:

[0192] Table 2: Antioxidant test results

[0193] sample DPPH clearance rate (%) Collagen C3867 45.6 TRTLLLL 11.2 FMRLL 13.1 KHVW 11.3 LLHPTII 10.5 Recombinant collagen of the present invention 95.2

[0194] The results in the table above show that the recombinant collagen of the present invention has strong antioxidant properties, with a DPPH clearance rate of over 95%, far higher than commercially available collagen. However, the DPPH clearance rate of the amino acid fragments is low, indicating that they lack antioxidant properties.

[0195] 3. Stability test of recombinant collagen

[0196] (1) a Room temperature stability: Each protein sample was prepared into a protein solution with a protein concentration of 1 mg / mL using ddH2O, filtered through a 0.22 μm sterile filter, and then dispensed into sterile centrifuge tubes and sealed;

[0197] b. Acidic stability: Each protein sample was dissolved in acetate buffer solution at pH 3 to a protein concentration of 1 mg / mL. The sample was filtered through a 0.22 μm sterile filter and then dispensed into sterile centrifuge tubes and sealed.

[0198] The protein samples include: the recombinant collagen of the present invention (sequence: LLHPTIITRTLLLLFMRLLKHVWL) and the unhydroxylated recombinant protein (sequence: LLHPTIITRTLLLLFMRLLKHVWL, which can be prepared by chemical synthesis).

[0199] (2) Place at 25°C, take samples at 0 months, 6 months, 12 months, and 18 months, and use high performance liquid chromatography to determine the purity of the protein. The stability of the protein is determined based on the change in purity (test three times and take the average value). The test results are shown in the following table:

[0200] Table 3: 18-month stability test results of recombinant collagen solution

[0201]

[0202]

[0203] As can be seen from the results in the table above, compared to unhydroxylated recombinant collagen, the recombinant collagen of the present invention maintained a protein purity of over 95% in room temperature water and 88% in acidic environments after 18 months, demonstrating good stability without degradation. In contrast, the purity of unhydroxylated recombinant collagen was only 60% in room temperature water and 45% in acidic environments after 18 months, indicating that it had degraded by half. This demonstrates that hydroxylated proline significantly improves the protein's stability, imparting excellent hydrophilicity and acid stability.

[0204] 4. Effects of the recombinant collagen of the present invention on nude mouse skin

[0205] Six-week-old hairless mice (BALB / c nude mice, 6 years old, body weight (19±2) g, purchased from the Experimental Animal Science and Technology Center of Jiangxi University of Traditional Chinese Medicine) were used. All mice were housed in individual cages after surgery with free access to food and water. They were randomly divided into three groups: Group 1 was injected with phosphate-buffered saline (PBS) as a negative control group; Group 2 was injected with HA (hyaluronic acid) filler as a positive control group; Group 3 was injected with the target protein as the research group. All mice were exposed to ultraviolet light three times a week; tissue elasticity was measured using a Cutometer (MPA580, Courage Khazaka, Germany) six hours before injection and one, three, one, two, and four weeks after injection. Skin elasticity was tested by stretching the skin with a negative pressure of 50 mbar for one second and releasing it.

[0206] The test results are as follows Figure 4 As shown, the skin tissue elasticity of the test group was significantly improved on the 7th day compared with the control group, indicating that the recombinant collagen of the present invention has the effect of improving skin elasticity.

[0207] 5. Effect of the recombinant collagen of the present invention on stimulating mouse fibroblast regeneration

[0208] (1) Cell recovery:

[0209] Resuscitation steps:

[0210] ①Preheat the water bath to 37°C, prepare clean disposable PE gloves, and add 9 mL of sterile culture medium to a sterile centrifuge tube;

[0211] ②. Take the cells out of the liquid nitrogen tank and put them into PE gloves. Quickly immerse them in a water bath and shake the cryotubes to accelerate lysis. It is best to completely dissolve the cells within 1 minute.

[0212] ③. Add the revived cell solution to a centrifuge tube containing fresh culture medium in a clean bench and centrifuge at 1200 rpm for 3 minutes. Remove the supernatant after centrifugation.

[0213] ④. Resuspend the cells with an appropriate amount of complete culture medium corresponding to the cells, transfer them to a sterile container (culture flask or culture dish), add culture medium to an appropriate amount, and culture in an incubator;

[0214] Resuscitation precautions:

[0215] ① If the water bath is not in the same room as the liquid nitrogen tank, the cryovials need to be kept cold before being transferred to the water bath to prevent the cell surface from melting on the way.

[0216] ② During the cell lysis process, shake rapidly to accelerate the lysis;

[0217] ③. Store the thawed frozen cells at room temperature for as short a time as possible and centrifuge to remove DMSO as soon as possible.

[0218] ④Observe the adherent cells after 24 hours of recovery. If the density reaches 80%, subculture normally. If the density is less than 80%, continue culturing for 48 hours before changing the medium.

[0219] ⑤. It is not recommended to centrifuge and replace the medium within 3 days of suspension cell recovery;

[0220] 6. Cells insensitive to DMSO can be revived without centrifugation, reducing the number of steps and the risk of contamination. Transfer the thawed cell suspension directly to a T25 cell flask, add fresh culture medium, and culture in an incubator. However, after 12-24 hours of culture, fresh culture medium must be replaced to remove dead cells.

[0221] (2) Cell passage

[0222] Adherent cell passaging operation:

[0223] ①. Aspirate the original culture medium.

[0224] ②. Add about 2 mL of PBS, gently shake the culture flask to rinse the cells, and aspirate and discard the PBS.

[0225] ③. Add about 1 mL of trypsin and gently shake the culture flask to allow it to infiltrate all cells.

[0226] ④. Place the culture flask in the incubator for digestion (digestion time depends on cell characteristics). When the cells in the middle of the cells are obviously separated and rounded under the microscope, the process can be stopped. Do not tap the culture flask during the whole process.

[0227] ⑤. Add 3 mL of serum-containing culture medium to terminate digestion, blow the cells to detach them from the wall and blow them repeatedly in the liquid to make the cells as single-cell suspension as possible. At this time, you can observe them under a microscope.

[0228] ⑥. Collect the cell suspension and centrifuge at 1200 rpm for 3 minutes. After centrifugation, aspirate and discard the supernatant.

[0229] ⑦. Add fresh culture medium and mix the cells by pipetting a few times. Inoculate into new culture bottles as needed, replenish the culture medium, and loosen the bottle cap or use a breathable bottle cap for culture.

[0230] ⑧. Check the carbon dioxide, temperature and water tray of the incubator.

[0231] After the cells were passaged twice, cell proliferation assay was performed.

[0232] (3) Day 1:

[0233] ① Thaw trypsin and dispense 3 mL into 5 mL centrifuge tubes. Add approximately 1 mL of trypsin for digestion, preferably 0.25% trypsin and 0.01% EDTA (ethylenediaminetetraacetic acid). Digest in a 37°C CO2 incubator for approximately 2 minutes.

[0234] ② Transfer the cells to a 12 mL centrifuge tube, centrifuge at 200 g for 3 min, resuspend the cells in EME medium, and adjust the cell density to 1 × 10 5 Then, 100 μL of culture medium was added to a 96-well plate, the second and eleventh columns from the left were set as blank wells, and 100 μL of culture medium with a density of 1×10 cells was added to the remaining wells. 5 cells / mL of cell suspension (=1×10 4 The 96-well plate needs to be irradiated with UV light for 30 minutes.

[0235] The cells were incubated for 24 hours (5% CO2, 37°C, greater than 90% humidity), and the cell growth in each well was observed.

[0236] (4) The next day:

[0237] After incubating the cells for 24 hours, the culture medium was aspirated and 100 μL of the blank control group and the experimental group were added to the sample wells respectively.

[0238] Blank control group: 100 μL of culture medium was added; experimental group: 2 mg / mL of the recombinant collagen of the present invention;

[0239] The cells were incubated for 24 h (5% CO2, 37°C, humidity greater than 90%) and the cell density OD was measured. 600 value.

[0240] (5) Experimental results:

[0241] The experimental results of promoting the growth of mouse fibroblasts are shown in Table 4. Figure 5 As shown:

[0242] Table 4: Experimental results on promoting the growth of mouse fibroblasts

[0243]

[0244] The results show Figure 5 As shown, the recombinant collagen of the present invention can significantly improve the proliferation ability of fibroblasts.

[0245] It should also be noted that the recombinant collagen of the present invention can be a random combination of four amino acid fragments: ①TRTLLLL, ②FMRLL, ③KHVWL, and ④LLHPTII. Exemplarily, the amino acid sequence of the recombinant collagen can be ①②③④, ①③④②, ①④③②, ②①③④, ②①④③, ③①②④, ③②④①, ③④②①, ④③②①, ④②①③, ④①③②, etc.

[0246] The recombinant collagen has the effects verified above, which will not be described in detail here.

[0247] The recombinant protein of the present invention also has the functions of locking skin moisture, improving dullness, improving skin tone, whitening, and anti-wrinkle, and can be used in medical beauty, such as water light injections, fillers and other cosmetic materials.

[0248] The method of the present invention increases the degree of proline hydroxylation, solves the problem of failure to hydroxylate proline or low degree of hydroxylation, and allows collagen to have antioxidant properties and regulate cell metabolism, growth, and development, and can be used as a tissue engineering material.

[0249] The interaction between the recombinant collagen synthesized by the present invention and the cell surface receptor integrin plays a role in cell adhesion, migration, proliferation and differentiation. As a dressing, it can be used for wound healing, scar repair, and is used in the medical field.

[0250] The recombinant collagen synthesized by the present invention can also be used as a hemostatic material.

[0251] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A recombinant collagen, characterized in that, the amino acid sequence of the recombinant collagen is randomly formed by the following amino acid fragments ①②③④, and the amino acid fragments are directly connected; the amino acid sequence of the amino acid fragment ① is as shown in SEQ ID NO:4, the amino acid sequence of the amino acid fragment ② is as shown in SEQ ID NO:5, the amino acid sequence of the amino acid fragment ③ is as shown in SEQ ID NO:6, and the amino acid sequence of the amino acid fragment ④ is as shown in SEQ ID NO:

7.

2. The recombinant collagen according to claim 1, characterized in that, the combination of the amino acid sequence fragments includes ①②③④, ①③④②, ①④③②, ②①③④, ②①④③, ③①②④, ③②④①, ③④②①, ④③②①, ④②①③, ④①③②, ④②③①.

3. The recombinant collagen according to claim 2, characterized in that, the amino acid sequence of the recombinant collagen is any one of the following: a) the amino acid sequence shown in SEQ ID NO:3; b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:3, and retaining the cell adhesion effect of the amino acid sequence of SEQ ID NO:3; c) an amino acid sequence with one or more amino acid residues added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:3, and retaining the cell adhesion effect of the amino acid sequence of SEQ ID NO:

3.

4. A polynucleotide encoding the recombinant collagen according to any one of claims 1 to 3.

5. An expression vector, characterized in that, the expression vector contains a nucleotide sequence encoding the amino acid sequence of the recombinant collagen according to any one of claims 1 to 3.

6. The expression vector according to claim 5, characterized in that, the expression vector further contains a nucleotide sequence encoding prolyl hydroxylase.

7. A host cell, characterized in that, the host cell contains the expression vector according to claim 6.

8. The method for preparing the recombinant collagen according to any one of claims 1 to 3, characterized in that, the preparation method includes the following steps: (1) Construct the expression vector according to claim 5 or 6; (2) Transform the expression vector in step (1) into a host cell, culture the cells and collect the cell culture medium; (3) Purify the cell culture medium in step (2) to obtain the recombinant collagen.

9. The application of the recombinant collagen according to any one of claims 1 to 3 as a dressing in cell proliferation, wound healing or / and scar repair.

10. The application of the recombinant collagen according to any one of claims 1 to 3 as a tissue engineering material in promoting cell metabolism, growth and development; or the application as a hemostatic material.

Images