A recombinant collagen and its preparation method and application

Abstract

The present invention belongs to the field of biological medicine, relates to the manufacture of biological drugs, and specifically relates to a recombinant collagen and its preparation method and application. The recombinant collagen includes CAIII81, CAIII82, and CAIII83, all of which contain 5 repeated amino acid sequences of human type III collagen from 387 to 467, and their C-terminals respectively contain amino acid fragments of human type III collagen from 1148 to 1159, 1189 to 1200, and 1316 to 1327. 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.

Description

Technical Field

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

[0002] Collagen is the most abundant structural and functional protein in the human body, accounting for about 1 / 3 of the total human protein. More than 75% of the components of the skin are composed of collagen. There are 3 kilograms of collagen in the human body, 2.2 kilograms in the skin, and only 15 grams of hyaluronic acid in the human body. Collagen is the raw material of the skin. There are currently 29 types in the collagen superfamily. Each peptide chain in collagen has a repeated Gly (glycine)-X-Y triplet sequence. The most common sequence is the "glycine-proline-hydroxyproline" sequence (gly-pro-hyp). Therefore, glycine accounts for about 1 / 3 of collagen. X and Y are mainly proline and hydroxyproline. This repeated sequence can form intermolecular hydrogen bonds and electrostatic interactions, enabling 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 loose reticular structure and mainly maintains and repairs skin elasticity. The acquisition of collagen is generally divided into extraction and preparation. Collagen extracted from animal sources still has multiple drawbacks such as immunogenicity, poor homogeneity, and the potential for infectious diseases. In contrast, recombinant collagen prepared by genetic engineering technology reduces the risks of rejection, immunogenicity, and viral infection compared to animal-derived collagen, and 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-terminals respectively contain amino acid fragments of human type III collagen 1148-1159, 1189-1200, and 1316-1327. 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 the raw material of a medical liquid dressing.

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

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

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

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

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

[0009] Preferably, the amino acid sequences of the recombinant collagens CAIII81, CAIII82, and CAIII83 are as 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 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0011] The present invention provides a method for preparing the recombinant collagen, which can be prepared using a prokaryotic expression system or a eukaryotic expression system:

[0012] Prokaryotic expression system, with the expression vector being pET28a(+), and expressed in Escherichia coli expression strains, including: 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.

[0013] Eukaryotic expression system, with the expression vector being pcDNA3.1(+), and expressed in eukaryotic cells, including: Expi293F, ExpiCHO, and Expi sf9.

[0014] Preferably, the expression vector contains an affinity tag and a TEV protease cleavage site. The affinity tag is one or more of His, MBP, GST, SUMO, HA, and Myc. The amino acid sequence and nucleotide sequence corresponding to the TEV protease cleavage site are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0015] Preferably, when preparing recombinant collagen using the prokaryotic expression system, the nucleotide sequence of recombinant collagen is first spliced and recombined onto the pET28a(+) expression vector, with the cleavage sites being BamHI and XhoI. Then, the nucleotide sequences of the affinity tag and the TEV protease cleavage site are spliced and recombined, with the cleavage sites being NcoI and BamHI.

[0016] Preferably, when preparing recombinant collagen using the eukaryotic expression system, the nucleotide sequence of recombinant collagen is first spliced and recombined onto the pcDNA3.1(+) expression vector, with the cleavage sites being BamHI and XhoI. Then, the nucleotide sequences of the affinity tag and the TEV protease cleavage site are spliced and recombined, with the cleavage sites being NheI and BamHI.

[0017] The present invention provides an application of recombinant collagen, specifically as follows:

[0018] The recombinant collagen can promote cell migration and can be used as a raw material for medical liquid dressings.

[0019] Advantages of the present invention: In the present invention, the amino acid sequence of human type III collagen at positions 387-467 is selected and repeated 5 times, and then ligated to the vectors pET28a(+) and pcDNA3.1(+). Recombinant collagen can be expressed in both the Escherichia coli expression system and the human cell expression system. The experimental results of the migration activity of mouse embryonic fibroblasts (BALB / c 3T3) show that the recombinant collagen CAIII81 expressed in the present invention has good activity in promoting cell migration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings described herein are provided to further understand the present application and form 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 to the present application. In the drawings:

[0021] Figure 1 It is the SDS-PAGE electrophoresis diagram of recombinant collagen CAIII82;

[0022] Figure 2 It is the experimental result diagram of recombinant collagen promoting cell migration;

[0023] Figure 3 It is the amino acid composition diagram of recombinant collagen;

[0024] Figure 4 It is the secondary structure prediction diagram of recombinant collagen;

[0025] Figure 5 It is the tertiary structure prediction diagram of recombinant collagen, corresponding to CAIII81, CAIII82, and CAIII83 from top to bottom;

[0026] Figure 6 It is the wound healing effect diagram of animal experiments;

[0027] Figure 7 It is the wound healing rate result diagram of animal experiments. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In order to more clearly illustrate the overall concept of the present application, the following will be described in detail by way of examples in conjunction with the drawings of the specification. In the following description, a large number of specific details are given 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.

[0029] Example 1: This example provides a method for preparing recombinant collagen in a prokaryotic expression system:

[0030] The recombinant collagen in this example is specifically CAIII82. Jiutian Gene Technology (Tianjin) Co., Ltd. synthesized the gene of recombinant collagen (SEQ ID NO: 2), spliced and recombined it onto the pET28a(+) vector, and the restriction enzyme sites were BamHI and XhoI. Jiutian Gene Technology (Tianjin) Co., Ltd. synthesized the gene of the affinity tag and the TEV protease cleavage site. The affinity tag consists of His and MBP, and the gene of the TEV protease cleavage site is shown as SEQ ID NO: 8. It was spliced and recombined onto the pET28a(+) vector already containing the recombinant collagen gene, and the restriction enzyme sites were NcoI and BamHI to obtain the expression vector. The expression vector was transferred into the expression strain, and the expression strain was the Escherichia coli competent cell BL21(DE3);

[0031] Single colony clones were selected from the transformation plate and cultured overnight at 37°C in 50 mL of LB medium (containing 100 mg / L kanamycin) (each liter of the medium contains 10 g of peptone, 5 g of yeast extract, and 5 g of sodium chloride). It was transferred at a ratio of 1:36 to 1.8 liters of LB medium (containing 100 mg / L kanamycin). It was cultured at 37°C until the OD600 reached 0.5, and 0.5 mM IPTG was added to induce protein expression at 16°C;

[0032] The bacterial solution was centrifuged at 7000 rpm for 10 min to collect the bacteria. The bacterial pellet was resuspended in 10 mM imidazole PBS (pH 7.5) solution, and the bacteria were disrupted by high pressure. It was centrifuged at 4°C and 11000 rpm for 15 min, and the supernatant was taken. TEV protease was used for hydrolysis. After completion, it was incubated with a balanced 5 mL nickel column (Thermo Fisher: 88221), eluted by gravity, and the miscellaneous proteins were washed away with 10 mM PBS solution (pH 7.5). Gradient elution was carried out with PBS solutions (pH 7.5) containing 30 mM, 50 mM, 100 mM, 200 mM, and 300 mM imidazole respectively. SDS-PAGE was used to detect the protein, and an ultrafiltration tube (Millipore, UFC9010) was used for ultrafiltration to concentrate and remove imidazole. The recombinant collagen CAIII82 was obtained for SDS-PAGE detection;

[0033] The results are as Figure 1 shown. It can be seen from Figure 1 that CAIII82 has obvious bands on the SDS-PAGE electrophoresis diagram, indicating that the above prokaryotic expression system achieved the expression of CAIII82.

[0034] Example 2: This example provides a method for preparing a recombinant collagen in a eukaryotic expression system:

[0035] The recombinant collagen in this example is also CAIII82. The gene for synthesizing recombinant collagen (SEQ ID NO: 2) was synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. and spliced and recombined onto the pcDNA3.1(+) vector with restriction enzyme sites BamHI and XhoI. The gene for the affinity tag and TEV cleavage site was synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. The affinity tag consists of His and GST and was spliced and recombined onto the pcDNA3.1(+) vector already containing the recombinant collagen gene with restriction enzyme sites NheI and BamHI to obtain the expression vector;

[0036] Plasmid extraction: The constructed expression vector was transformed into Escherichia coli DH5α, followed by ice bath, heat shock, ice bath, and then 1 ml of LB liquid medium was added and activated at 37°C and 200 rpm for 40 min. It was spread on an LB solid medium plate (0.1 mg / mL sodium ampicillin) and incubated upside down in an incubator at 37°C for 12 h. A single colony was picked and added to 100 mL of LB liquid medium (0.1 mg / mL sodium ampicillin), and cultured at 37°C and 200 rpm for 12 h. The plasmid was extracted using a plasmid large-scale extraction kit (Beyotime: D0025-3), and the concentration of the vector was detected using a Nanodrop instrument. It can be stored at 4°C for one month and is recommended to be used within one week;

[0037] Protein expression: Expi293F cells were pre-suspended and cultured in a shaker at 37°C, 110 rpm, humidity 80%, and carbon dioxide 7%. The medium used was a serum-free medium (GeneCreate: HE000-N012). When the cell density reached 3.0×10 6 cells / mL and the viable cell rate > 95%, plasmid transfection was carried out. A plasmid transfection kit (Beyotime: C0518) was used for Expi293F cell transfection (40 mL): 80 μL of transfection reagent was added to 1 mL of cell medium and gently mixed to obtain solution A; 40 μg of plasmid was added to 1 ml of cell medium and gently mixed to obtain solution B. Solution B was added to solution A, gently mixed, and allowed to stand for 15 min. The mixture of A and B was added to 40 mL of suspended Expi293F cells, and the cells were continuously cultured. The viable cell rate was detected every day. When the viable cell rate was lower than 50%, the cells were harvested;

[0038] Protein purification: The cell suspension was centrifuged at 5000 rpm for 5 min at 4°C. The supernatant medium was taken, hydrolyzed with TEV protease, and then incubated with 5 mL of nickel column (Thermo Fisher: 88221) after the hydrolysis. Gravity elution was used. The nickel column was washed with 50 mL of PBS solution containing 10 mM imidazole, and eluted with PBS solution (pH 7.5) containing 300 mM imidazole. The protein was detected by SDS-PAGE. The PBS eluate containing the target protein with good purity was combined, and ultrafiltration was performed using an ultrafiltration tube (Millipore, UFC9010) to concentrate and remove imidazole. The recombinant collagen CAIII82 was obtained for SDS-PAGE detection;

[0039] The results are as Figure 1 shown. It can be seen from Figure 1 the figure that CAIII82 has obvious bands on the SDS-PAGE electrophoresis diagram, indicating that the above eukaryotic expression system has achieved the expression of CAIII82.

[0040] Example 3: This example provides a method for preparing recombinant collagen in a prokaryotic expression system:

[0041] The difference between this example and Example 1 is that the recombinant collagen in this example is specifically CAIII81, the affinity tag consists of His and HA, and the expression strain used is BL21Star(DE3). Other steps are the same as those in Example 1, that is, the recombinant collagen CAIII81 of this example is obtained.

[0042] Example 4: This example provides a method for preparing recombinant collagen in a eukaryotic expression system:

[0043] The difference between this example and Example 2 is that the recombinant collagen in this example is specifically CAIII81, the affinity tag consists of His and SUMO, and the eukaryotic cell used is ExpiCHO. Other steps are the same as those in Example 2, that is, the recombinant collagen CAIII81 of this example is obtained.

[0044] Example 5: This example provides a method for preparing recombinant collagen in a prokaryotic expression system:

[0045] The difference between this example and Example 1 is that the recombinant collagen in this example is specifically CAIII83, the affinity tag consists of His and Myc, and the expression strain used is Rosetta(DE3). Other steps are the same as those in Example 1, that is, the recombinant collagen CAIII83 of this example is obtained.

[0046] Example 6: This example provides a method for preparing recombinant collagen in a eukaryotic expression system:

[0047] The difference between this example and Example 2 is that the recombinant collagen in this example is specifically CAIII83, and the affinity tag consists of His and MBP. The eukaryotic cell used is Expi sf9, and the other steps are the same as those in Example 2, that is, the recombinant collagen CAIII83 of this example is obtained.

[0048] Comparative Example 1: This comparative example provides a method for preparing recombinant collagen, and the specific content is as follows:

[0049] The recombinant collagen in 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 with reference to Example 1, that is, the recombinant collagen of this comparative example is obtained.

[0050] Experimental test:

[0051] I. Determination of the activity of recombinant collagen in promoting cell migration:

[0052] Use a marker pen to draw horizontal lines evenly on the back of a 12-well plate, about one line every 0.5 cm, and draw 3 lines in each well. Add about 5×10 4 BALB / c 3T3 cells to each well, and culture overnight to allow the cells to adhere. The next day, use a sterile pipette tip to scratch the cells along the ruler, wash 3 times with PBS to remove the scratched cells and the remaining serum-containing DMEM medium, add serum-free DMEM medium, and set up a control group, CAIII82 (Example 1), CAIII3010-2, CAIII-1-GFP, CAIII82 (Example 2), full-length type III collagen Col3a1, and an experimental group of commercial recombinant collagen (0.1 mg / mL). Among them, CAIII3010-2 and CAIII-1-GFP are recombinant collagens produced by the company. Return the cells to the incubator. Take samples and take pictures after 24 hours. Set 3 replicate wells for each group, and select 5 points for statistics in each well. The experimental results are as Figure 2 shown. As Figure 2 can be seen, after acting for 24 hours, among the 6 experimental groups at 0.1 mg / mL, CAIII82 (Example 1) (scratch recovery rate is 66%), CAIII3010-2 (scratch recovery rate is 50%), CAIII-1-GFP (scratch recovery rate is 61%), CAIII82 (Example 2) (scratch recovery rate is 70%), Col3a1 (scratch recovery rate is 56%), and commercial recombinant collagen (scratch recovery rate is 35%), compared with the control group (scratch recovery rate is 23%), showed obvious activity in promoting cell migration (with statistical differences). The protein CAIII82 expressed by eukaryotic cells has the best activity in promoting cell migration, and is significantly better than the activity of commercial recombinant collagen.

[0053] Prediction of Physicochemical Properties of Recombinant Collagen

[0054] The online software ProtParam was used to predict the physicochemical properties of recombinant collagens CAIII81, CAIII82, and CAIII83:

[0055] The amino acid proportions of recombinant collagens CAIII81, CAIII82, and CAIII83 are as Figure 3 shown, and it can be seen from Figure 3 that the C-terminal amino acid sequences of CAIII81, CAIII82, and CAIII83 are different, and have little effect on the amino acid proportion of the overall protein;

[0056] Table 1 Physicochemical Properties of Recombinant Collagen

[0057]

[0058] Note: a represents mammalian reticulocytes, in vitro; b represents yeast, in vivo; c represents Escherichia coli, in vivo

[0059] The physicochemical properties of recombinant collagens analyzed by ProtParam are shown in Table 1. It can be seen from Table 1 that the isoelectric points, molecular weights, predicted half-lives, aliphatic indices, etc. of CAIII81, CAIII82, and CAIII83 are relatively similar, and GRAVY indicates that CAIII81, CAIII82, and CAIII83 are all hydrophilic proteins. Thus, 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.

[0060] Prediction of Secondary and Tertiary Structures of Recombinant Collagen

[0061] The online software GOR4 was used to predict the secondary structures of recombinant collagens CAIII81, CAIII82, and CAIII83:

[0062] Table 2 Contents of α-helix, etc. in the Secondary Structure of Recombinant Collagen

[0063]

[0064] Figure 4 The secondary structure prediction diagrams of recombinant collagens CAIII81, CAIII82, and CAIII83 are shown, and from Figure 4It can be seen that all of CAIII81, CAIII82, and CAIII83 involve 5 β-sheets. The first four are related to 5 repeated basic units, and the last β-sheet is provided by the amino acid fragment at the C-terminus. That is, the recombinant collagen prepared in the present invention is mainly composed of 5 repeated units, and the amino acid fragment sequences at the C-terminus are different, but they show similarity in the overall recombinant collagen.

[0065] Using the SWISS-MODEL online software, the tertiary structures of recombinant collagens CAIII81, CAIII82, and CAIII83 were predicted, and the results are as Figure 5 shown. It can be Figure 5 seen that the tertiary structures of CAIII81, CAIII82, and CAIII83 are somewhat similar.

[0066] Therefore, recombinant collagens CAIII81, CAIII82, and CAIII83 have partially the same amino acid composition, specifically the amino acid sequences of 5 repeated human type III collagen from 387 to 467, and this part 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 one more β-sheet, which helps to stabilize the recombinant collagen.

[0067] Wound healing effect test:

[0068] Male SD rats weighing 250 - 350 g were used for the experiment. They were adaptively cultured for one week before the start of the experiment and raised in an SPF environment. The experiments were carried out using Example 1, Comparative Example 1, and commercial recombinant collagen. The rats were randomly divided into four groups, with 15 rats in each group, corresponding to the above three experimental groups + a blank group. The experimental groups were all configured as solutions 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 off, and then a sterile 15-mm puncture biopsy needle was used to puncture the wound. All rats were intraperitoneally injected with penicillin (20,000 U / kg / day) for 3 consecutive days. At each time point of 0 d, 7 d, 14 d, and 21 d, the rats were killed by CO 2 exposure and the samples were collected for histological examination. The wound healing situation was obtained by photographing and then imported into Image J software for processing. The wound healing rate was calculated as follows: Wound healing rate = wound area on the I-th day / wound area on the 0-th day × 100%, where I represents the time point.

[0069] The results are as Figure 6 and Figure 7 shown. Figure 6The wound healing conditions of SD rats in Example 1, Comparative Example 1, commercially available recombinant collagen, and the blank group were shown. Example 1 played a significantly better role. Further, to Figure 7 show all sample conditions. From Figure 7 it can be seen that the recombinant collagen obtained in Examples 1-6 had the best effect on promoting wound healing, while Comparative Example 1, which only involved 5 basic repeating units, had a poor effect. In addition, the recombinant collagen obtained by the eukaryotic expression system had 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 cooperate with other materials to promote wound healing synergistically.

[0070] The above are only several embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, makes some changes or modifications using the technical content disclosed above, which are all equivalent to equivalent implementation cases and fall within the scope of protection of the technical solution of the present invention.

Claims

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

2. A method for preparing the recombinant collagen according to claim 1, characterized in that: Preparation using prokaryotic expression system or 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) and 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.

3. The method for preparing recombinant collagen according to claim 2, 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.

4. The method for preparing recombinant collagen according to claim 3, 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.

5. The method for preparing recombinant collagen according to claim 4, 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.

6. A use of the recombinant collagen according to claim 1, characterized in that: The recombinant collagen is used as a raw material for medical liquid dressings.

Images