Chimeric collagen and method for expressing the same

Abstract

This invention belongs to the fields of genetic engineering and collagen technology, specifically relating to a chimeric collagen and its expression method. It provides a chimeric collagen HC92 that can be industrially produced, is non-cytotoxic, and exhibits superior cell proliferation and migration-promoting activity, showing promise for applications in pharmaceutical products, medical devices, biomaterials, and tissue engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and collagen protein, and particularly relates to a chimeric collagen protein and an expression method thereof. BACKGROUND

[0002] Collagen is the most abundant protein in mammals, accounting for about 30% of the total protein, and is also an extracellular matrix protein like elastin. It is closely related to beauty care, skin care and tissue regeneration. At present, 28 types of collagen (I-XXVIII) have been found, of which I, II, III, IV, V and XVII are the most studied. Among them, I, II and III belong to interstitial collagen, IV and XVII belong to basement membrane collagen, and V belongs to extracellular peripheral collagen. Although there are many types of collagen with complex structures, their basic compositions are similar, and they all contain (Gly-X-Y) amino acid sequence repeats. These repeats are the basis of the triple helix structure, in which X is usually proline (Pro) and Y is usually hydroxyproline (HyPro) or hydroxylysine (Hy Lys), which are rarely seen in other proteins.

[0003] Type III collagen is one of the most important and abundant proteins in mammals, and is a structural protein found in the skin, connective tissue and bone of the human body and other tissues, which is twisted into a triple helix by three peptide chains. Type IV collagen is less abundant in animals, making it difficult to extract a single component. Col17a1 is a homogeneous trimer composed of three identical α1 chains, which contains a 50 kDa intracellular domain (ICD), a transmembrane domain and a 120 kDa extracellular domain. Studies have shown that Col17a1 is an important component of the epidermal stem cell hemidesmosome, an important component of the hair follicle stem cell and skin stem cell homeostasis, and plays an important role in basement membrane adhesion, skin aging mechanism and maintenance of hair follicle stem cells. It is a potential focus for future anti-aging and hair loss prevention skin care products. Collagen is one of the most widely used protein materials in the fields of biomaterials and regenerative medicine due to its excellent biological functions, biocompatibility and biodegradability.

[0004] In addition to type I, II and III collagen, which are relatively abundant in the human and animal body, other types of collagen are very low in content, which is not conducive to extraction and application. At the same time, the existing biosynthetic collagen is mainly a single type of recombinant expression, and the combined use of multiple types of collagen is required for some scenarios. To simplify the product application formula, it is of great significance to design a chimeric collagen with different type collagen characteristics. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a chimeric collagen and its expression method.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] Technical Topic 1:

[0008] This invention provides a chimeric collagen protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Technical Topic Two:

[0010] The present invention provides a gene encoding the chimeric collagen described in Technical Subject 1, the gene sequence of which is shown in SEQ ID NO.2.

[0011] Technical Topic 3:

[0012] The present invention provides a vector comprising the gene described in Technical Subject 2.

[0013] Technical Topic 4:

[0014] The present invention provides a host cell comprising the gene described in Technical Subject 2 or the vector described in Technical Subject 3.

[0015] Technical Topic 5:

[0016] This invention provides a method for expressing the chimeric collagen described in Technical Subject 1, comprising the following steps:

[0017] S1 protein sequence design: Based on the amino acid sequences of human type III, IV and XVII collagen, design the amino acid sequence of chimeric collagen;

[0018] S2 gene design and synthesis: The DNA sequence is reverse-engineered based on the amino acid sequence described in S1, and codon optimization is performed to obtain the gene sequence, and nucleic acid fragments are synthesized.

[0019] Construction of S3 expression vector: The nucleic acid fragment obtained in step S2 is ligated to the plasmid through a multiple cloning site to obtain the expression plasmid;

[0020] Construction and screening of S4 expression strains: The expression plasmid obtained from S3 was transformed into host cells, plated on a medium containing antibiotics, and cultured to obtain the expression strains;

[0021] S5 Induction of expression: The expression strain obtained in step S4 was induced to express the bacterial culture, and the bacterial culture was collected;

[0022] S6 Purification: The bacterial culture obtained in step S5 is broken, centrifuged, purified, and desalted to obtain chimeric collagen.

[0023] Furthermore, the plasmid described in S3 is pET30a(+).

[0024] Furthermore, the multiple cloning sites described in S3 are Nde I and Kpn I.

[0025] Furthermore, the host cell described in S4 is Escherichia coli BL21 (DE3).

[0026] Furthermore, the purification described in S6 is performed by affinity chromatography and ion exchange chromatography.

[0027] Technical Topic Six:

[0028] Use of a chimeric collagen as described in Technical Subject 1 in pharmaceutical products, medical devices, biomaterials, and tissue engineering.

[0029] The beneficial effects of adopting the above technical solution are as follows:

[0030] (1) The present invention selects partial sequences of human type III, IV and XVII collagen and optimizes them to obtain target chimeric collagen molecules. After testing, the obtained chimeric collagen has better biocompatibility than natural collagen and better effects in promoting cell proliferation and cell migration, and has good application prospects.

[0031] (2) The fusion collagen provided by the present invention has no animal-derived infectious agents, has high biosafety, and the preparation method of the fusion collagen is suitable for large-scale industrial production. Attached Figure Description

[0032] Figure 1 These are electrophoretic images of bacterial cells obtained at different induction times in Example 1 of the present invention; where M is the marker, 1 is before induction, and 2 is after 12 hours of induction.

[0033] Figure 2 This is an electrophoresis image of a sample from the affinity chromatography process in Example 1 of the present invention; where 1 is a marker, 2 is the bacterial cells obtained from induced expression, 3 is the supernatant, and 4 is the elution buffer;

[0034] Figure 3 This is an electrophoresis image of a sample from the ion chromatography process in Example 1 of the present invention; where 1 is the marker, 2 is the replacement solution, and 3 is 25% elution.

[0035] Figure 4 This is a statistical chart of the cytotoxicity experimental data of this invention;

[0036] Figure 5 This is a statistical chart of cell proliferation test data from the present invention;

[0037] Figure 6This is a statistical chart of cell migration promotion test data from the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless otherwise specified, the experimental or testing methods involved in the embodiments of this invention are conventional methods in the prior art, and their names and / or abbreviations are conventional names in the art, clearly defined in their respective fields of application. Those skilled in the art can understand the conventional process steps based on these names and apply the corresponding equipment, implementing them under conventional conditions or conditions recommended by the manufacturer. The various instruments, equipment, raw materials, or reagents used in the embodiments of this invention are not subject to any special restrictions on their source; they are all conventional products that can be purchased through legitimate commercial channels and can be prepared according to conventional methods well known to those skilled in the art.

[0040] The collagen sequences used in the embodiments of this application are as follows:

[0041] The recombinant human type III collagen was prepared according to Examples 1-5 of Chinese Patent Application No. CN111087464B, entitled “A recombinant human type III collagen with a functional structure and its expression method thereof”, and the amino acid sequence is shown in SEQ ID NO:1.

[0042] The recombinant human type XVII collagen was prepared according to the technical solution described in Chinese Patent Application No. CN117801096B, entitled "A water-soluble recombinant human type XVII collagen and its preparation method and application", and its amino acid sequence is shown in SEQ ID No.1.

[0043] The recombinant human type IV collagen was prepared according to the technical solution described in Chinese Patent Application No. CN119039425A, entitled "A Recombinant Human Type IV Collagen and Its Preparation Method and Application", and its amino acid sequence is shown in SEQ ID No. 1.

[0044] Example 1

[0045] 1. Gene design and synthesis

[0046] Active fragments of human type III, IV and XVII collagen were selected: type III (267-320), type IV (958-1019), and type XVII (1316-1378). They were linked in the order of type IV-type III-XVII, and a His tag was added to the C-terminus to obtain the amino acid sequence of chimeric collagen HC92, as shown in SEQ ID NO.1.

[0047] SEQ ID NO.1:

[0048] MGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGGAGSLGAGGAFGEAAGDRGPYGTDIGPGGGYGAAAEGGMYAGNGGLLGADFAGDLDYNELAHHHHHHH

[0049] The coding nucleic acid sequence was designed using the online design tool Jcat (http: / / www.jcat.de / ), and codons were optimized according to the codon preferences of the host cell *E. coli*, resulting in the gene sequence of chimeric collagen HC92, as shown in SEQ ID NO.2. The gene fragment of chimeric collagen HC92 of this invention was synthesized by Genscript Biotech Inc.

[0050] SEQ ID NO.2:

[0051] .

[0052] Construction of expression vector pET30a(+)-HC92

[0053] The gene fragment obtained in step 1 and the pET30a(+) plasmid were ligated through Nde I and Kpn I multiple cloning sites to obtain the recombinant plasmid pET30a(+)-HC92.

[0054] Construction of expression strain BL21(DE3) / pET30a(+)-HC92

[0055] The expression strain was constructed according to the method described in *Molecular Cloning: A Laboratory Manual (3rd Edition)* (by J. Sambrook et al.), and the specific steps are as follows:

[0056] (1) Pick a single colony of Escherichia coli BL21(DE3) and inoculate it into LB liquid medium. After inoculation, culture overnight at 37°C and 200 rpm with shaking. Add 0.5 mL of overnight culture medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0-7.5) to an Erlenmeyer flask containing 50 mL of LB liquid medium and culture overnight at 37°C. Incubate the cells at 200 rpm with vigorous shaking for 2 hours to allow them to grow to the prelog phase. Under aseptic conditions, transfer the cells to a 50 mL pre-chilled polypropylene tube and place on ice for 10 minutes. Centrifuge at 4°C and 4000 rpm, discard the supernatant, and invert the tube to allow as much residual liquid as possible to drain. Resuspend the precipitate in 6 mL of pre-chilled 0.1 mol / L CaCl2 solution and place on ice for 30 minutes. Centrifuge at 4°C and 3000 rpm, discard the supernatant, and invert the tube to allow as much residual liquid as possible to drain. Resuspend the precipitate in 1.2 mL of pre-chilled 0.1 mol / L CaCl2 solution (if preparing competent cells for storage at -70°C, resuspend the cells in 0.1 mol / L CaCl2 solution containing 20% ​​glycerol). Incubate at 4°C for 24 hours to obtain a suspension of competent Escherichia coli BL21(DE3) cells.

[0057] (2) Take 200 μL of competent Escherichia coli suspension, add the recombinant plasmid pET30a(+)-HC92 (volume is 2 μL, DNA < 50 ng) prepared in step 2, mix gently, and place on ice for 30 minutes; perform static heat shock in a 42℃ water bath for 90 seconds, and immediately place on ice to cool; add 500 μL of LB liquid culture medium, mix well, and place in a 37℃ shaker at 200 rpm for 45 minutes to obtain the transformation solution; take the transformation solution and spread it on a solid LB medium plate containing 50 μg / mL kanamycin, and place it in an incubator at 37℃ to invert and culture to obtain the expression strain BL21(DE3) / pET30a(+)-HC92.

[0058] Induced expression

[0059] Single colonies of the expression strain BL21(DE3) / pET30a(+)-HC92 obtained in step 3 were picked and cultured overnight at 37°C and 200 rpm in LB liquid medium containing 50 μg / mL kanamycin to obtain activated seeds. The activated seeds were then inoculated at a rate of 3% into a 5L fermenter containing 3L of complete culture medium (1 g / 100mL peptone, 0.5 g / 100mL beef extract, and 0.5 g / 100mL sodium chloride). Fermentation was carried out at a controlled temperature of 37°C, dissolved oxygen of 30%, and pH of 7.0. When OD... 600When the concentration reaches 60, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.5 mM, continue culturing for 12 hours, and collect the bacterial cells by centrifugation.

[0060] ,purification

[0061] (1) Sterilization: The bacterial cells obtained in step 4 were resuspended in a lysis buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5). The ratio of bacterial cells to lysis buffer (m / v) was 1:5~20, and 1:10 was used in this example. The bacterial cells were lysed using a high-pressure homogenizer. The pH of the lysed bacterial solution was adjusted to 3 with hydrochloric acid, and centrifuged at 12000g for 0.5 h. The supernatant was filtered through a 0.45 μm filter membrane to obtain the whole bacterial solution.

[0062] (2) Affinity chromatography

[0063] 1) Column equilibration: The Ni Focurose FF (IMAC) chromatography column was equilibrated with affinity chromatography solution A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) until the baseline was stable.

[0064] 2) Loading: Load the whole bacterial solution obtained in step (1) into the chromatography column and control the column retention time to be no less than 5 min.

[0065] 3) Column washing: Wash the chromatography column with affinity chromatography solution A until the baseline is stable and not less than 5 column volumes.

[0066] 4) Target protein elution: Elute with a mixture of 75% affinity chromatography solution A and 25% affinity chromatography solution B (20mM Tris, 500mM NaCl, 500mM imidazole, pH 7.5), collect the eluent, and obtain an affinity chromatography purification solution containing chimeric collagen.

[0067] (3) Ion exchange chromatography

[0068] 1) Sample preparation: Replace the affinity chromatography purification solution with ion exchange solution A (20mM Tris, pH7.5) by ultrafiltration until the conductivity is less than 4mS / cm to obtain the replacement solution.

[0069] 2) Column equilibration: The Q Sepharose FF column was equilibrated with ion exchange chromatography solution A until the baseline was stable.

[0070] 3) Sample loading: Load the replacement solution into the chromatography column and control the column retention time to be no less than 5 minutes.

[0071] 4) Column washing: Wash the chromatography column with ion exchange chromatography solution A until the baseline is stable and not less than 5 column volumes.

[0072] 5) Target protein elution: Elute with a mixture of 75% ion exchange chromatography solution A and 25% ion exchange chromatography solution B (20 mM Tris, 1 M NaCl, pH 7.5), collect the eluent, and obtain an ion exchange chromatography solution containing chimeric collagen.

[0073] (4) Desalting: The above ion exchange chromatography solution was replaced with purified water using a 5kDa ultrafiltration membrane device in an equal volume replacement manner to remove the salt in the solution. The solution was then filtered with a 0.45μm filter membrane and freeze-dried to obtain the chimeric collagen HC92 freeze-dried product.

[0074] Test Example 1: SDS-PAGE Protein Electrophoresis Detection

[0075] 1. Sample preparation:

[0076] Collect the bacterial cells obtained after induction expression in step 4 of Example 1 and the lyophilized HC92 product obtained in Example 1, add protein loading buffer, mix well, boil in a water bath for 10 min, and cool naturally for later use. Use GenScript SurePAGE™ precast gel (4-12%) for loading, and electrophoresis at 140V for 50±5 minutes until the bromophenol blue band reaches the bottom of the gel.

[0077] 2. Microwave staining with Coomassie Brilliant Blue R-250:

[0078] (1) Preparation of staining solution: Dissolve Coomassie Brilliant Blue R250 in 40% ethanol and 10% acetic acid solution to a final concentration of 0.1% (W / V).

[0079] (2) Preparation of decolorizing solution: Dissolve 10% (V / V) ethanol and 7.5% (V / V) acetic acid together.

[0080] (3) After electrophoresis, pry open the gel plate, remove the gel, and put it into a staining container containing 100 mL of staining solution.

[0081] (4) Cover the container and heat it in the microwave on high for 8 minutes. To avoid danger, please do not let the solution boil.

[0082] (5) Remove the dyeing container from the microwave oven and place it on a decolorizing shaker and shake gently at room temperature for 5 minutes.

[0083] (6) Discard the staining solution and carefully wash the gel with deionized water.

[0084] (7) Discard the deionized water and add 100 mL of decolorizing solution.

[0085] (8) Cover the microwave and heat on high for 8 minutes.

[0086] (9) Discard the decolorizing solution, add new decolorizing solution, and repeat step (8).

[0087] (10) Remove from microwave oven and place on a decolorizing shaker at room temperature and gently shake until the background is clear.

[0088] Example of effect 1: Cytotoxicity test

[0089] The cytotoxicity of the HC92 protein obtained in this invention was evaluated according to the method specified in Appendix C: MTT cytotoxicity test of GB / T 16886.5-2017 Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test.

[0090] The experiment used L929 (mouse fibroblast) cells as the experimental subjects and was divided into four groups: experimental group, blank group, negative control group and blank control group, with 5 replicates in each group;

[0091] Among them, experimental group 1~10 samples: HC92 lyophilized solutions obtained in Example 1 were dissolved in MEM medium containing 10% fetal calf serum at final concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL and 10 mg / mL respectively.

[0092] Blank group samples: MEM culture medium containing 10% fetal calf serum;

[0093] Negative control group sample: High-density polyethylene (HDPE) film was extracted at 37℃±1℃ for 24±2h using 0.2g / ml cell culture medium (MEM medium containing 10% fetal bovine serum).

[0094] Positive control group sample: Cell culture medium extract of polyurethane film (ZDEC Polyurethane Film) at a ratio of 0.2 g / ml cell culture medium (MEM medium containing 10% fetal bovine serum) at 37℃±1℃ for 24±2 h.

[0095] L929 cells were divided into 1×10 4 Cells were seeded in 96-well plates, with 100 μL of MEM medium added to each well. The plates were incubated at 37°C and 5% CO2 for 24 h. After removing the medium, 100 μL of sample was added, and the plates were incubated at 37°C and 5% CO2 for 24 h. Cell morphology was observed under a microscope. After removing the medium, 50 μL of MTT solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 2 h. After removing the MTT solution, 100 μL of isopropanol was added to each well, and the absorbance (OD) at 570 nm was measured.570 ), calculate cell viability.

[0096] Cell viability (%) = (OD) 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )*100%

[0097] like Figure 4 As shown, cell viability was greater than 100% in the concentration range of 0.1 mg / mL to 10 mg / mL, indicating that the chimeric collagen HC92 obtained in this invention has no cytotoxicity.

[0098] Example 2: Cell proliferation assay

[0099] Cell proliferation assays were performed using the lyophilized chimeric collagen HC92 obtained in Example 1. An experimental group and a control group were set up, with 5 replicates per group.

[0100] Among them, the experimental group 1 sample was a 3 mg / mL chimeric collagen HC92 solution dissolved in DMEM medium.

[0101] Sample 2 in experimental group: Recombinant human type III collagen solution dissolved in DMEM medium with a final concentration of 3 mg / mL;

[0102] Sample 3 in experimental group: Recombinant human type IV collagen solution dissolved in DMEM medium with a final concentration of 3 mg / mL;

[0103] Sample 4 in experimental group: Recombinant human type XVII collagen solution dissolved in DMEM medium to a final concentration of 3 mg / mL;

[0104] Control group sample: DMEM medium.

[0105] Human skin keratinocytes HaCaT were used at a dose of 1×10 4 The samples were seeded in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was discarded, and 100 μL of sample was added. The plates were incubated for another 24 hours, then the supernatant was discarded. 50 μL of MTT solution was added, and the plates were shaken well and incubated for another 2 hours. After incubation, 100 μL of DMSO was added, and the mixture was shaken for 15 minutes before measuring the OD. 570 ), calculate cell viability.

[0106] Cell viability (%) = (OD) 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )*100%

[0107] like Figure 5As shown, the experimental results indicate that, compared with the control group, all experimental group samples promoted HaCaT cell proliferation at the same concentration; HC92 was more effective than recombinant human type III, IV, and XVII collagen.

[0108] Example 3: Cell migration promotion assay

[0109] This efficacy example was conducted according to Appendix C of the "Pharmaceutical Industry Standard of the People's Republic of China" YY / T1849-2022: Recombinant Collagen – Cell Migration Assay – Cell Scratch Test. The chimeric collagen HC92 obtained in Example 1 was used for the cell migration assay. Experimental groups 1-4 and a control group were set up, with 5 replicates per group.

[0110] Among them, the experimental group 1 sample was a 3 mg / mL chimeric collagen HC92 solution dissolved in DMEM medium.

[0111] Sample 2 in experimental group: Recombinant human type III collagen solution dissolved in DMEM medium with a final concentration of 3 mg / mL;

[0112] Sample 3 in experimental group: Recombinant human type IV collagen solution dissolved in DMEM medium with a final concentration of 3 mg / mL;

[0113] Sample 4 in experimental group: Recombinant human type XVII collagen solution dissolved in DMEM medium to a final concentration of 3 mg / mL;

[0114] Control group sample: DMEM medium.

[0115] L929 cells were fed at a rate of 1×10 6 The cells were seeded in 6-well plates, with 2 mL of DMEM medium added to each well. The cells were cultured at 37°C and 5% CO2 for 24 h. After 24 h of culture, the cells were streaked vertically (or horizontally) with a 10 μL pipette tip to create scratches. The cells were washed three times with PBS to remove the streaked cells, and 2 mL of sample was added. The cells were then cultured again at 37°C and 5% CO2. At 0 h, 24 h, 48 h, and 72 h, photographs were taken under a 40x microscope, with the intersection of the horizontal and vertical streaks as the focal point.

[0116] Cell migration rate (%) = (0h scratch area - current scratch area) / 0h scratch area * 100%.

[0117] Note: The sample detection concentration is based on the sample preparation concentration specified in YY / T 16886.12-2022. If there are special requirements, the sample should be prepared according to the concentration requirements for submission.

[0118] like Figure 6As shown in the figure, the experimental results indicate that, compared with the control group, all samples at the same concentration promoted the migration of L929 cells. HC92 showed stronger effects than recombinant human type III, IV, and XVII collagen and had a time-dependent effect.

[0119] In summary, this invention provides a chimeric collagen HC92 that can be industrially produced. This chimeric collagen HC92 is non-cytotoxic and exhibits superior cell adhesion and migration-promoting activity compared to single-type recombinant collagen.

[0120] Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chimeric collagen protein, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the chimeric collagen of claim 1, characterized in that, The gene sequence is shown in SEQ ID NO.

2.

3. A carrier, characterized in that, It contains the gene described in claim 2.

4. A host cell, characterized in that, It contains the gene as described in claim 2 or the vector as described in claim 3.

5. A method for expressing the chimeric collagen according to claim 1, characterized in that, The steps are as follows: S1 protein sequence design: Based on the amino acid sequences of human type III, IV and XVII collagen, design the amino acid sequence of chimeric collagen; S2 gene design and synthesis: The DNA sequence is reverse-engineered based on the amino acid sequence described in S1, and codon optimization is performed to obtain the gene sequence, and nucleic acid fragments are synthesized. Construction of S3 expression vector: The nucleic acid fragment obtained in step S2 is ligated to the plasmid through a multiple cloning site to obtain the expression plasmid; Construction and screening of S4 expression strains: The expression plasmid obtained from S3 was transformed into host cells, plated on a medium containing kanamycin, and cultured to obtain the expression strains; S5 Induction of expression: The expression strain obtained in step S4 was induced to express the bacterial culture, and the bacterial culture was collected; S6 Purification: The bacterial culture obtained in step S5 is broken, centrifuged, purified, and desalted to obtain chimeric collagen.

6. The expression method according to claim 5, characterized in that, The plasmid described in S3 is pET30a(+).

7. The expression method according to claim 5, characterized in that, The multiple cloning sites mentioned in S3 are Nde I and Kpn I.

8. The expression method according to claim 5, characterized in that, The host cell described in S4 is Escherichia coli BL21 (DE3).

9. The expression method according to claim 5, characterized in that, The purification described in S6 is performed by affinity chromatography and ion exchange chromatography.

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