Recombinant collagen as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to biological medicine manufacturing, in particular to recombinant collagen as well as a preparation method and application thereof. The recombinant collagen contains CAIII81, CAIII82 and CAIII83, each of the CAIII81, CAIII82 and CAIII83 contains five repeated amino acid sequences of human III-type collagen 387-467, C terminals of the CAIII81, CAIII82 and CAIII83 respectively contain amino acid fragments of human III-type collagen 1148-1159, 1189-1200 and 1316-1327, and after the recombinant collagen is prepared by a prokaryotic expression system or eukaryotic expression system, the obtained recombinant collagen has the activity of promoting cell migration and can be used as a raw material of a medical liquid dressing.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the manufacture of biopharmaceuticals, and specifically relates to a recombinant collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant structural and functional protein in the human body, accounting for about 1 / 3 of the total protein in the human body. More than 75% of the skin is composed of collagen. The human body has 3 kg of collagen, the skin has 2.2 kg, and the human body has only 15 grams of hyaluronic acid. Collagen is the raw material of the skin. There are currently 29 types of collagen superfamily. Each peptide chain in collagen has a repeated Gly (glycine)-XY triplet sequence. The most common sequence is the "glycine-proline-hydroxyproline" sequence (gly-pro-hyp). Therefore, glycine accounts for about 1 / 3 of collagen, and X and Y are mainly proline and hydroxyproline. This repeating sequence can form interchain hydrogen bonds and electrostatic interactions, allowing collagen to form a stable triple helix structure. Type III collagen is a homotrimer formed by three α1 (III) chains (Col3A1). Type III collagen is mainly distributed in the skin and vascular system, accounting for about 60% in normal fetal skin and 15-20% in adult skin. Type III collagen has a sparse reticular structure and is mainly used to maintain and repair skin elasticity. The acquisition of collagen is generally divided into extraction and preparation. Collagen extracted from animal sources still has many disadvantages such as immunogenicity, poor uniformity, and hidden dangers of infectious diseases. Compared with animal-derived collagen, recombinant collagen prepared by genetic engineering technology reduces the risk of rejection, immunogenicity, and viral infection. It is currently the most promising method to solve the problem of collagen sources. Summary of the invention

[0003] Based on the above problems, the present invention provides a recombinant collagen, which includes CAIII81, CAIII82, and CAIII83, all of which contain 5 repeated amino acid sequences of human type III collagen 387~467, and their C-termini contain amino acid fragments of human type III collagen 1148~1159, 1189~1200, and 1316~1327, respectively. After being prepared by a prokaryotic expression system or a eukaryotic expression system, the obtained recombinant collagen has the activity of promoting cell migration and can be used as a raw material for medical liquid dressings.

[0004] The present invention provides a recombinant collagen comprising CAIII81, CAIII82 and CAIII83. The recombinant collagen comprises 5 repeated basic repeating units, wherein the basic repeating unit is GINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKD, and the basic repeating unit is derived from the amino acid sequence 387-467 of human type III collagen.

[0005] Preferably, the C-termini of the recombinant collagens CAIII81, CAIII82 and CAIII83 contain the amino acid sequence of 1148-1159 of human type III collagen, the amino acid sequence of 1189-1200 of human type III collagen and the amino acid sequence of 1316-1327 of human type III collagen, respectively.

[0006] Preferably, the amino acid sequence of 1148-1159 of human type III collagen is PGKDGTSGHPGP.

[0007] Preferably, the amino acid sequence of 1189-1200 of human type III collagen is PPGAPGPCCGGV.

[0008] Preferably, the amino acid sequence of 1148-1159 of human type III collagen is WTDSSAEKKHVW.

[0009] Preferably, the amino acid sequences of the recombinant collagens CAIII81, CAIII82 and CAIII83 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0010] Preferably, the nucleotide sequences corresponding to the recombinant collagens CAIII81, CAIII82 and CAIII83 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0011] The present invention provides a method for preparing recombinant collagen, which can be prepared using a prokaryotic expression system or a eukaryotic expression system: Prokaryotic expression system, the expression vector is pET28a(+), and the Escherichia coli expression strain is used for expression. The expression strains include: BL21(DE3), BL21(DE3)pLysS, BL21Star(DE3), BL21Star(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, OrigamiB(DE3), OrigamiB(DE3) pLysS, SHuffle T7, BLR(DE3), BLR(DE3)pLysS, B834(DE3), B834(DE3)pLysS BL21(DE3)+HSP60.

[0012] Eukaryotic expression system, the expression vector is pcDNA3.1(+), and the eukaryotic cells are used for expression. Eukaryotic cells include: Expi293F, ExpiCHO and Expi sf9.

[0013] Preferably, the expression vector contains an affinity tag and a TEV cleavage site, the affinity tag is one or more of His, MBP, GST, SUMO, HA, and Myc, and the amino acid sequence and nucleotide sequence corresponding to the TEV cleavage site are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0014] Preferably, the recombinant collagen is prepared using a prokaryotic expression system by first splicing and recombining the nucleotide sequence of the recombinant collagen into a pET28a(+) expression vector with restriction sites of BamHI and XhoI, and then splicing and recombining the nucleotide sequence of the affinity tag and the TEV restriction site with restriction sites of NcoI and BamHI.

[0015] Preferably, the recombinant collagen is prepared using a eukaryotic expression system by first splicing and recombining the nucleotide sequence of the recombinant collagen into a pcDNA3.1(+) expression vector with restriction sites of BamHI and XhoI, and then splicing and recombining the nucleotide sequence of the affinity tag and the TEV restriction site with restriction sites of NheI and BamHI.

[0016] The present invention provides an application of recombinant collagen, which is as follows: The recombinant collagen can promote cell migration and can be used as a raw material for medical liquid dressings.

[0017] Beneficial effects of the present invention: The present invention selects 5 repeats of the amino acid sequence of 387-467 of human type III collagen and connects it to the vector pET28a(+) and pcDNA3.1(+). Both the Escherichia coli expression system and the human cell expression system can achieve the expression of recombinant collagen. The results of the activity experiment of the migration of mouse embryonic fibroblasts (BALB / c 3T3) show that the recombinant collagen CAIII81 expressed by the present invention has a good activity of promoting cell migration. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 It is the SDS-PAGE electrophoresis diagram of recombinant collagen CAIII82; Figure 2 This is the result of the experiment of recombinant collagen promoting cell migration; Figure 3 This is the amino acid composition diagram of recombinant collagen; Figure 4 This is the predicted secondary structure of recombinant collagen; Figure 5 This is the predicted tertiary structure of recombinant collagen, which corresponds to CAIII81, CAIII82, and CAIII83 from top to bottom; Figure 6 This is a picture of wound healing effect in animal experiments; Figure 7 This is a graph showing the wound healing rate results of animal experiments. DETAILED DESCRIPTION

[0019] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0020] Example 1: This example provides a method for preparing recombinant collagen in a prokaryotic expression system: The recombinant collagen described in this embodiment is specifically CAIII82. The gene of recombinant collagen (SEQ ID NO: 2) is synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. and spliced ​​and recombined into the pET28a (+) vector. The restriction sites are BamHI and XhoI. The genes of affinity tag and TEV restriction site are synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. The affinity tag is composed of His and MBP. The gene of TEV restriction site is shown in SEQ ID NO: 8. It is spliced ​​and recombined into the pET28a (+) vector containing the recombinant collagen gene. The restriction sites are NcoI and BamHI to obtain an expression vector. The expression vector is transferred into the expression strain, and the expression strain is the competent Escherichia coli cell BL21 (DE3); Single-spot clones were selected from the transformation plate and cultured overnight at 37°C in 50 mL LB medium (containing 100 mg / L kanamycin) (each liter of medium contains 10 g peptone, 5 g yeast powder and 5 g sodium chloride). Transferred to 1.8 liters LB medium (containing 100 mg / L kanamycin) at a ratio of 1:36. Cultured at 37°C until OD600 was 0.5, and 0.5 mM IPTG was added to induce protein expression at 16°C; The bacterial liquid was centrifuged at 7000rpm for 10min to collect the bacteria. The bacterial precipitate was resuspended in 10mM imidazole PBS (pH7.5) solution, the bacteria were crushed under high pressure, centrifuged at 4℃, 11000rpm for 15min, the supernatant was taken, and TEV protease was hydrolyzed. After the end, it was incubated with a balanced 5mL nickel column (Thermo Fisher: 88221), gravity eluted, and the impurities were washed with 10mM PBS solution (pH7.5). Gradient elution was performed with 30mM, 50mM, 100mM, 200mM and 300mM imidazole PBS solution (pH7.5), respectively, and the protein was detected by SDS-PAGE. Ultrafiltration tube (Millipore, UFC9010) was used for ultrafiltration to concentrate and remove imidazole. The recombinant collagen CAIII82 was obtained for SDS-PAGE detection; The results are as follows Figure 1 As shown by Figure 1 It can be seen that CAIII82 has obvious bands on the SDS-PAGE electrophoresis diagram, indicating that the above-mentioned prokaryotic expression system achieves the expression of CAIII82.

[0021] Example 2: This example provides a method for preparing recombinant collagen in a eukaryotic expression system: The recombinant collagen described in this example is also CAIII82. The gene of recombinant collagen (SEQ ID NO: 2) is synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. and spliced ​​and recombined into the pcDNA3.1(+) vector, and the restriction sites are BamHI and XhoI. The gene of affinity tag and TEV restriction site is synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd., and the affinity tag is composed of His and GST, and spliced ​​and recombined into the pcDNA3.1(+) vector containing the recombinant collagen gene, and the restriction sites are NheI and BamHI to obtain the expression vector; Plasmid extraction: Transform the above-constructed expression vector into E. coli DH5α, perform ice bath, heat shock, ice bath, then add 1ml LB liquid culture medium to activate at 37℃, 200rpm for 40min, apply LB solid culture medium plate (0.1mg / mL ampicillin sodium), incubate inverted at 37℃ for 12h, pick a single colony and add it to 100mL LB liquid culture medium (0.1mg / mL ampicillin sodium), 37℃, 200rpm, culture for 12h. Use plasmid large-scale extraction kit (Biyuntian: D0025-3) to extract plasmid, Nanodrop instrument to detect the concentration of vector, it can be stored for one month at 4℃, and it is recommended to use within one week; Protein expression: Expi293F cells were pre-suspended and cultured in a shaker at 37°C, 110 rpm, 80% humidity, and 7% carbon dioxide. The culture medium used was serum-free culture medium (Gitex: HE000-N012). When the cell density reached 3.0×10 6 / mL, and when the viable cell rate is >95%, plasmid transfection is performed. Use the plasmid transfection kit (Biyuntian: C0518) for Expi293F cell transfection (40mL): 80μL of transfection reagent is added to 1mL of cell culture medium and gently mixed to obtain solution A; 40μg of plasmid is added to 1ml of cell culture medium and gently mixed to obtain solution B. Solution B is added to solution A, gently mixed, and allowed to stand for 15 minutes. Add the mixture of A and B to 40mL of suspended Expi293F cells, continue culturing, detect the viable cell rate every day, and harvest the cells when the viable cell rate is lower than 50%; Protein purification: The cell suspension was centrifuged at 4°C, 5000rpm for 5min, the supernatant medium was taken, and hydrolyzed with TEV protease. After completion, it was incubated with 5mL nickel column (Thermo Fisher: 88221), and gravity eluted. The nickel column was washed with 50mL PBS solution containing 10mM imidazole, and eluted with 300mM imidazole PBS solution (pH7.5). The protein was detected by SDS-PAGE, and the PBS eluates containing the target protein and good purity were combined, and ultrafiltration was concentrated and imidazole was removed by ultrafiltration (Millipore, UFC9010). The recombinant collagen CAIII82 was obtained for SDS-PAGE detection; The results are as follows Figure 1 As shown by Figure 1 It can be seen that CAIII82 has obvious bands on the SDS-PAGE electrophoresis diagram, indicating that the above-mentioned eukaryotic expression system achieves the expression of CAIII82.

[0022] Example 3: This example provides a method for preparing recombinant collagen in a prokaryotic expression system: The difference between this embodiment and embodiment 1 is that the recombinant collagen described in this embodiment is specifically CAIII81, the affinity tag is composed of His and HA, the expression strain used is BL21Star (DE3), and the other steps are consistent with embodiment 1, that is, the recombinant collagen CAIII81 of this embodiment is obtained.

[0023] Example 4: This example provides a method for preparing recombinant collagen in a eukaryotic expression system: The difference between this embodiment and embodiment 2 is that the recombinant collagen described in this embodiment is specifically CAIII81, the affinity tag is composed of His and SUMO, the eukaryotic cell used is ExpiCHO, and the other steps are consistent with embodiment 2, that is, the recombinant collagen CAIII81 of this embodiment is obtained.

[0024] Example 5: This example provides a method for preparing recombinant collagen in a prokaryotic expression system: The difference between this embodiment and embodiment 1 is that the recombinant collagen described in this embodiment is specifically CAIII83, the affinity tag is composed of His and Myc, the expression strain used is Rosetta (DE3), and the other steps are consistent with embodiment 1, that is, the recombinant collagen CAIII83 of this embodiment is obtained.

[0025] Example 6: This example provides a method for preparing recombinant collagen in a eukaryotic expression system: The difference between this example and Example 2 is that the recombinant collagen described in this example is specifically CAIII83, the affinity tag is composed of His and MBP, the eukaryotic cell used is Expi sf9, and the other steps are consistent with Example 2, that is, the recombinant collagen CAIII83 of this example is obtained.

[0026] Comparative Example 1: This comparative example provides a method for preparing recombinant collagen, the specific contents are as follows: The recombinant collagen of this comparative example is a recombinant collagen composed of only 5 repeated basic repeating units, and the C-terminus does not involve other amino acid sequences. The preparation method is carried out by referring to Example 1 to obtain the recombinant collagen of this comparative example.

[0027] Experimental test: 1. Determination of the activity of recombinant collagen in promoting cell migration: Use a marker pen to draw horizontal lines evenly on the back of the 12-well plate, with a ruler, about 0.5 cm apart, and draw 3 lines in each well. Add about 5×10 4 BALB / c 3T3 cells were cultured overnight to allow the cells to adhere to the wall. On the second day, the cells were scratched with a ruler using a sterile pipette tip, washed three times with PBS, the scratched cells and the remaining DMEM medium with serum were removed, serum-free DMEM medium was added, and a control group, CAIII82 (Example 1), CAIII3010-2, CAIII-1-GFP, CAIII82 (Example 2), full-length type III collagen Col3a1 and commercial recombinant collagen experimental groups (0.1 mg / mL) were set up, of which CAIII3010-2 and CAIII-1-GFP are recombinant collagens produced by the company, and the cells were returned to the incubator. Samples were taken and photographed after 24 hours. Three replicate wells were set up for each group, and 5 points were selected in each well for statistics. The experimental results are shown in the figure. Figure 2 As shown by Figure 2 It can be seen that after 24 hours of action, the six experimental groups of CAIII82 (Example 1) (scratch recovery rate of 66%), CAIII3010-2 (scratch recovery rate of 50%), CAIII-1-GFP (scratch recovery rate of 61%), CAIII82 (Example 2) (scratch recovery rate of 70%), Col3a1 (scratch recovery rate of 56%) and commercial recombinant collagen (scratch recovery rate of 35%) at 0.1 mg / mL showed obvious activity in promoting cell migration (with statistical difference) compared with the control group (scratch recovery rate of 23%). The protein CAIII82 expressed by eukaryotic cells has the best activity in promoting cell migration, and is significantly superior to the activity of commercial recombinant collagen.

[0028] Prediction of the physicochemical properties of recombinant collagen: ProtParam online software was used to predict the physicochemical properties of recombinant collagen CAIII81, CAIII82, and CAIII83: The amino acid ratios of recombinant collagen CAIII81, CAIII82, and CAIII83 are as follows: Figure 3 As shown by Figure 3 It can be seen that the C-terminal amino acid sequences of CAIII81, CAIII82, and CAIII83 are different, which has little effect on the amino acid proportion of the overall protein; Table 1 Physicochemical properties of recombinant collagen

[0029] Note: a represents mammalian reticulocytes, in vitro; b represents yeast, in vivo; c represents Escherichia coli, in vivo The physicochemical properties of recombinant collagen analyzed by ProtParam are shown in Table 1. As can be seen from Table 1, the isoelectric point, molecular weight, estimated half-life, aliphatic index, etc. of CAIII81, CAIII82, and CAIII83 are relatively similar. GRAVY indicates that CAIII81, CAIII82, and CAIII83 are all hydrophilic proteins. It can be seen that CAIII81, CAIII82, and CAIII83 all contain 5 repeated basic repeating units, which play an important role in the physicochemical properties of recombinant collagen.

[0030] Prediction of secondary and tertiary structures of recombinant collagen: GOR4 online software was used to predict the secondary structures of recombinant collagen CAIII81, CAIII82, and CAIII83: Table 2 Content of α-helix and other components in the secondary structure of recombinant collagen

[0031] Figure 4 The secondary structure prediction diagrams of recombinant collagens CAIII81, CAIII82, and CAIII83 are shown. Figure 4 It can be seen that CAIII81, CAIII82, and CAIII83 all involve 5 β-folds, and the first four are related to 5 repeated basic units, and the last β-fold is provided by the amino acid fragment at the C-terminus. That is, the recombinant collagen prepared by the present invention is mainly composed of 5 repeated units, and the amino acid fragment sequence at the C-terminus is different, but the performance is similar in the overall recombinant collagen.

[0032] The tertiary structures of recombinant collagens CAIII81, CAIII82, and CAIII83 were predicted using SWISS-MODEL online software. Figure 5As shown by Figure 5 It can be seen that the tertiary structures of CAIII81, CAIII82, and CAIII83 have certain similarities.

[0033] Therefore, the recombinant collagens CAIII81, CAIII82, and CAIII83 have partially identical amino acid compositions, specifically five repeats of the amino acid sequence of 387 to 467 of human type III collagen, which has the function of promoting cell migration. In addition, the differences in the C-terminal amino acid sequences of CAIII81, CAIII82, and CAIII83 can provide an additional β-fold, which helps stabilize the recombinant collagen.

[0034] Wound healing effect test: The experiment was conducted using male SD rats weighing 250-350g. They were cultured for one week before the experiment and raised in an SPF environment. Example 1, Comparative Example 1, and commercial recombinant collagen were used for the experiment. The rats were randomly divided into four groups, each with 15 rats, corresponding to the above three experimental groups + a blank group. The experimental groups were all configured with a solution with a concentration of 20 mg / mL, and the blank group was covered with Tegaderm™ transparent wound dressing 3587. After the rats were anesthetized, the hair on the back surface was shaved, and then the wound was punctured with a sterile 15 mm puncture biopsy needle. All rats were injected intraperitoneally with penicillin (20,000U / kg / day) every day for 3 consecutive days. The rats were killed by CO2 exposure after each time point of 0d, 7d, 14d, and 21d, and samples were collected for histological examination. The wound healing status was obtained by photographing and then imported into Image J software for processing, and the wound healing rate was calculated: wound healing rate = wound area on day I / wound area on day 0 × 100%, where I represents the time point.

[0035] The results are as follows Figure 6 , Figure 7 As shown, Figure 6 The wound healing of SD rats in Example 1, Comparative Example 1, commercial recombinant collagen and blank group was shown. Example 1 obviously played a better role. Figure 7 Display all samples, by Figure 7 It can be seen that the recombinant collagen obtained in Examples 1-6 has the best effect in promoting wound healing, while the comparative example 1 involving only 5 basic repeating units has a poor effect. In addition, the recombinant collagen obtained by the eukaryotic expression system has a slightly better effect. Based on this, the recombinant collagen obtained in the present invention can be used as a raw material for medical liquid dressings, and combined with other materials to synergistically promote wound healing.

[0036] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant collagen, characterized in that: Recombinant collagen contains CAIII81, CAIII82, and CAIII83. Recombinant collagen contains 5 repeated basic repeating units. The basic repeating units are derived from the amino acid sequence of 387 to 467 of human type III collagen, as follows: GINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKD.

2. The recombinant collagen according to claim 1, characterized in that: The C-termini of the recombinant collagens CAIII81, CAIII82 and CAIII83 contain the amino acid sequence of 1148-1159 of human type III collagen, the amino acid sequence of 1189-1200 of human type III collagen and the amino acid sequence of 1316-1327 of human type III collagen, respectively.

3. The recombinant collagen according to claim 2, characterized in that: The amino acid sequences of the recombinant collagens CAIII81, CAIII82 and CAIII83 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the corresponding nucleotide sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:

6.

4. A method for preparing the recombinant collagen according to any one of claims 1 to 3, characterized in that: It can be prepared using a prokaryotic expression system or a eukaryotic expression system: Prokaryotic expression system, the expression vector is pET28a(+), and the Escherichia coli expression strain is used for expression. The expression strains include: BL21(DE3), BL21(DE3)pLysS, BL21Star(DE3), BL21Star(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, OrigamiB(DE3), OrigamiB(DE3) pLysS, SHuffle T7, BLR(DE3), BLR(DE3)pLysS, B834(DE3), B834(DE3)pLysS BL21(DE3)+HSP60; Eukaryotic expression system, the expression vector is pcDNA3.1(+), and the eukaryotic cells are used for expression. Eukaryotic cells include: Expi293, ExpiCHO and Expi sf9.

5. The method for preparing recombinant collagen according to claim 4, characterized in that: The expression vector contains an affinity tag and a TEV restriction site, the affinity tag is one or more of His, MBP, GST, SUMO, HA, and Myc, and the amino acid sequence and nucleotide sequence corresponding to the TEV restriction site are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

6. The method for preparing recombinant collagen according to claim 5, characterized in that: The recombinant collagen was prepared by using a prokaryotic expression system. The nucleotide sequence of the recombinant collagen was first spliced ​​and recombined into the pET28a(+) expression vector, with restriction sites of BamHI and XhoI, and then the nucleotide sequence of the affinity tag and the TEV restriction site were spliced ​​and recombined, with restriction sites of NcoI and BamHI.

7. The method for preparing recombinant collagen according to claim 6, characterized in that: The eukaryotic expression system is used to prepare recombinant collagen. The nucleotide sequence of the recombinant collagen is first spliced ​​and recombined into the pcDNA3.1(+) expression vector, with restriction sites of BamHI and XhoI, and then the nucleotide sequence of the affinity tag and the TEV restriction site is spliced ​​and recombined, with restriction sites of NheI and BamHI.

8. A use of the recombinant collagen according to claim 3 or 7, characterized in that: The recombinant collagen promotes cell migration and can be used as a raw material for medical liquid dressings.

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