Recombinant human III-type collagen and application thereof

Abstract

The invention discloses recombinant human III-type collagen and application thereof, and belongs to the technical field of biochemical engineering. The method comprises the following steps: according to human III-type collagen, obtaining a target amino acid sequence through splitting, and integrating the target amino acid sequence into an expression vector to obtain plasmids; transforming the plasmid into a host, and screening positive clones to obtain an expression strain; and culturing the expression strain, and separating and purifying to obtain the high-yield and high-purity human III-type collagen. The obtained recombinant human III-type collagen has a collagen triple helix region characteristic sequence (Gly-X-Y) n, has good performances of promoting cell migration and adhesion and preventing light damage, and can be widely applied to the fields of medicines, biological materials, health care products, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and more specifically, to a recombinant human type III collagen and its applications. Background Technology

[0002] Collagen is the most abundant protein in mammals and a major component of the extracellular matrix. There are many types of collagen, commonly classified into type I, type II, type III, type V, and type XI. The molecular weight of collagen subunits is generally around 120 kDa, much larger than that of most bioactive protein subunits. Type III collagen, in particular, consists of three α chains forming a superhelical structure, acting as a homotrimer. It is abundant in blood vessels, the intestines, and skin, and participates in regulating basic cell activities such as adhesion, proliferation, migration, and differentiation. Collagen is now widely used in cosmetics, medical devices, and other fields to achieve tissue repair and regeneration.

[0003] Currently, collagen sources mainly include extraction from animal tissues and expression through genetic engineering. Extracting collagen from animal tissues presents significant challenges, including high difficulty, low purity, high cost, and poor batch-to-batch stability. Furthermore, due to its animal origin, it carries the risk of immunogenicity and potential pathogen infection. Recombinant collagen obtained through genetic engineering can alleviate pressure on animal husbandry and environmental protection. After genetic modification, it can be expressed in model organisms such as *E. coli*, yeast cells, and animal cells. High-density fermentation can yield large quantities of collagen products, effectively solving the bottleneck problem of large-scale collagen production. The produced collagen also boasts advantages such as good water solubility, no risk of viral transmission, low immune rejection, and good batch-to-batch consistency. Patent CN119350505A discloses a method for purifying proteins using self-aggregating peptides, effectively avoiding the disadvantages of introducing tags and requiring protease treatment. Therefore, designing, developing, and optimizing high-quality recombinant collagen is a pressing issue. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a recombinant human type III collagen and its applications. Through different sequence design and screening processes, the present invention obtains a highly expressed recombinant human type III collagen with good cellular activity, and simultaneously provides its preparation method and applications.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a recombinant human type III collagen, the amino acid sequence of which is:

[0007] (a) As shown in SEQ ID NO: 3 or SEQ ID NO: 4; or,

[0008] (b) An amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, which retains the cell migration, cell adhesion, and photodamage prevention effects of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or,

[0009] (c) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, which retains the cell migration, cell adhesion and photodamage prevention effects of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0010] Preferably, the recombinant human type III collagen is HC25, and its amino acid sequence is shown in SEQ ID NO: 3, or in SEQ ID NO: 1, segments 1023-1115aa; the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2, segments 2566-2844bp.

[0011] Preferably, the recombinant human type III collagen is HC26, and its amino acid sequence is shown in SEQ ID NO: 4, or in SEQ ID NO: 1, segments 483-569aa; the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2, segments 946-1206bp.

[0012] The present invention also provides a gene encoding the above-mentioned recombinant human type III collagen.

[0013] The present invention also provides a recombinant expression vector containing the above-mentioned coding gene.

[0014] The recombinant expression vector is based on the pET series, specifically pET-28a(+).

[0015] The recombinant expression vector also includes a self-aggregating peptide purification element for subsequent separation and purification; specifically, the self-aggregating peptide purification element includes a self-assembled peptide, a protein linker, a pH-induced C-terminal cleavage intron, and a subtilisin propeptide.

[0016] The present invention also provides a recombinant engineered bacterium containing the above-mentioned coding gene or recombinant expression vector.

[0017] Furthermore, the recombinant engineered bacteria uses Escherichia coli as the host, preferably Escherichia coli BL21(DE3).

[0018] The present invention also provides a method for constructing the above-mentioned recombinant engineered bacteria, comprising the following steps: transforming a recombinant expression vector containing a recombinant human type III collagen encoding gene into Escherichia coli BL21(DE3), culturing and screening positive transformants to obtain the recombinant engineered bacteria.

[0019] The present invention also provides a method for rapidly preparing recombinant human type III collagen, comprising the following steps: inoculating the recombinant engineered bacteria into the culture medium at an inoculation amount of 2-10%, adding IPTG for induction culture, collecting the bacterial cells, breaking them, collecting the lysed precipitate, cutting them, and collecting the supernatant, which is the recombinant human type III collagen.

[0020] Preferably, the induction culture conditions are 16-18℃, 200-300rpm for 20-28h; more preferably, 18℃, 250rpm for 24h.

[0021] The present invention also provides the application of the above-mentioned recombinant human type III collagen in the fields of medicine, biomaterials, health products, and cosmetics.

[0022] Specifically, the aforementioned recombinant human type III collagen has the properties of promoting cell adhesion, migration, and / or preventing photodamage.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] (1) Based on human type III collagen, the present invention obtains the target amino acid sequence by splitting and integrating it into an expression vector to obtain a plasmid; the plasmid is transformed into a host, positive clones are screened to obtain an expression strain; the expression strain is cultured and purified to obtain high-yield and high-purity recombinant human type III collagen; the obtained recombinant human type III collagen has the characteristic sequence (Gly-XY)n of the triple helix region of collagen, and has good properties of promoting cell migration, adhesion and preventing photodamage, and can be widely used in the fields of medicine, biomaterials, health products, cosmetics etc.

[0025] (2) This invention prepares high-purity recombinant human type III collagen using genetic engineering technology. It is not limited by source, does not carry pathogens, and the purity of recombinant human type III collagen can reach over 98% using self-aggregating peptide purification. The results of genetic engineering preparation and self-aggregating peptide purification are stable, and the results of multiple preparations are almost identical. Attached Figure Description

[0026] Figure 1 These are SDS-PAGE images of HC25 and HC26 after shake-flask fermentation purification. In these images, ES is the lysis supernatant, EP is the lysis precipitate, CP is the precipitate after cleavage reaction, CS is the supernatant after cleavage reaction, and M is the protein marker.

[0027] Figure 2 This is an HC25 and HC26 cytotoxicity assay.

[0028] Figure 3 This is an HC25 and HC26 cell adhesion experiment.

[0029] Figure 4 It is an HC25 cell scratch assay.

[0030] Figure 5 It is an HC26 cell scratch assay.

[0031] Figure 6 This is an experiment on the prevention of photodamage by HC25 and HC26; among them, HC26 compared with PBS, *p<0.05.

[0032] Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0034] In this invention, the α1 chain protein of natural human type III collagen (reference sequence number NP_000081.2) was obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and its amino acid sequence is shown in SEQ ID NO: 1. Based on the optimal effective sequence of mature human type III collagen peptide, functional fragment combination and optimization of physicochemical properties such as isoelectric point and protein stability were performed to obtain the target amino acid sequence of recombinant human type III collagen fragment as shown in SEQ ID NO: 3 and 4.

[0035] In one embodiment, the effective sequence of the above-mentioned preferred human type III collagen mature peptide was optimized according to the codon preference of Escherichia coli. The optimized gene (SEQ ID NO: 2) was synthesized into the pUC57 vector to obtain plasmid pUC57-rhC. The target gene was amplified by PCR, and a gene containing a self-assembled peptide, a protein linker, a pH-induced C-terminal cleavage intron, and a subtilisin propeptide was added before the target gene for subsequent isolation and purification. The plasmids pET28-HC25 and HC26 were obtained by seamlessly ligating them into the pET-28a(+) expression vector using GibsonAssembly.

[0036] In one embodiment, the isolation and purification of recombinant human type III collagen was performed with reference to patent document "CN119350505A, Protein Purification Method".

[0037] Example 1: Construction of a collagen fragment expression vector

[0038] Taking HC25 as an example, the oligonucleotide primers used in this embodiment are shown in Table 1.

[0039] Table 1

[0040]

[0041] The effective sequence was obtained by codon optimization of the E. coli system using the amino acid sequence of human type III collagen (SEQ ID NO: 1) to obtain the nucleotide sequence, as shown in SEQ ID NO: 2. Using pET-30a-L6KD-PT linker-PEP-Mtu ΔI-CM-LCB3 as a template, and primers cB2-F and cB2-R, a gene was amplified with the addition of a self-assembled peptide, a protein linker, a pH-induced C-terminal cleavage intron, and a subtilisin propeptide. Using pET-28a(+) as a template, primers cB1-F and cB1-R were used to amplify the backbone fragment. Using the nucleotide sequence of human type III collagen (SEQ ID NO: 2) as a template, primers HC25-F and HC25-R were used to amplify the human type III collagen fragment HC25, whose amino acid sequence is shown in SEQ ID NO: 3. The nucleotide sequence of HC25 is shown in SEQ ID NO: 2, bp 2566-2844. After the PCR reaction, the PCR products were separated and recovered using 1% agarose gel electrophoresis. The three recovered fragments were ligated using Gibson Assembly to obtain the plasmid pET28-HC25.

[0042] Among them, pET-30a-L6KD-PT linker-PEP-Mtu ΔI-CM-LCB3 is disclosed in the literature "Huang Y, Zhang Y, Yang X, et al. A high-performance protein preparation approach in a singlecolumn-free step[J].Trends in biotechnology:S0167-7799(24)00290-7.DOI:10.1016 / j.tibtech.2024.10.008."

[0043] Similarly, following the construction method of plasmid pET28-HC25 in this embodiment, plasmid pET28-HC26 was constructed. The amino acid sequence of the human type III collagen fragment HC26 is shown in SEQ ID NO: 4, and the nucleotide sequence is shown in SEQ ID NO: 2, bps 946-1206.

[0044] Example 2 Construction of engineered bacteria expressing recombinant human type III collagen fragment

[0045] pET28-HC25 and HC26 were transformed into Escherichia coli BL21(DE3) competent cells. Transformants were screened using kanamycin resistance as a selection marker and identified by sequencing. The engineered bacteria expressing recombinant human type III collagen fragments were successfully constructed.

[0046] Example 3: Verification of positive strain expression by shaking flask (100mL culture system)

[0047] This embodiment mainly describes the fermentation process of recombinant human type III collagen fragments in shake flasks (100mL culture system). The fermentation medium is LB medium, the fermentation volume is 100mL, and HC25 and HC26 are preferred for fermentation.

[0048] The specific steps are as follows:

[0049] 1. Preparation of fermentation seed liquid: The cryovial of the engineered bacterial strain expressing recombinant human type III collagen fragment constructed in Example 2 was streaked into LB solid medium containing 50 μg / mL kanamycin and activated at 37°C for 10 h.

[0050] 2. Select activated colonies and inoculate them into LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 220 rpm to obtain the seed culture.

[0051] 3. Inoculate the seed culture from step 2 into fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculation rate of 2% (v / v), and incubate at 37°C with a shaker at 220 rpm until OD. 600 =0.6-0.8, transfer to a shaker at 18℃ and 250rpm for 30min, then add IPTG to a final concentration of 0.2mM, and incubate at 18℃ and 250rpm for 24h.

[0052] 4. Take the bacterial cells expressing recombinant human type III collagen fragment produced by fermentation, resuspend them in buffer to 50 OD / mL, and sonicate them. Separate the lysed products by centrifugation to obtain the lysate and the lysate supernatant. For specific steps, refer to the patent document "CN119350505A, Protein Purification Method".

[0053] 5. After washing the lysed precipitate obtained in step 4 twice with buffer, add cleavage buffer and place in a constant temperature shaker at 25°C and 100 rpm for 24 h.

[0054] 6. Centrifuge to collect the reaction supernatant and precipitate. SDS-PAGE gel electrophoresis results are as follows: Figure 1 As shown in Table 2, the yield and purity of HC26 were the highest, at 25.16 mg / L with a purity of 91%; while the yield of HC25 was 24.11 mg / L with a purity of 94%.

[0055] Table 2

[0056]

[0057] Example 4: Small-scale fermentation of recombinant human type III collagen fragments (5L fermenter)

[0058] This embodiment mainly describes the small-scale fermentation process of recombinant human type III collagen fragments. In the fermentation process, glucose with a clearly defined composition is used as the carbon source for cell metabolism, and ammonia water (used for both pH adjustment and as a nitrogen source for cell metabolism) and diammonium hydrogen phosphate are used as the nitrogen source for cell metabolism. At the same time, in order to promote cell growth, shorten the fermentation cycle, and increase fermentation yield, yeast extract and peptone and other complex nutrients are added to the culture medium, with HC25 and HC26 being preferred for fermentation.

[0059] The specific steps are as follows:

[0060] 1. Preparation of fermentation seed liquid: The cryovial of the engineered bacterial strain expressing recombinant human type III collagen fragment constructed in Example 2 was streaked into LB solid medium containing 50 μg / mL kanamycin and activated overnight at 37°C.

[0061] 2. Select activated colonies and inoculate them into LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 220 rpm to obtain the seed culture.

[0062] 3. Transfer the seed culture from step 2 to 2L of fresh fermentation broth containing 50μg / mL kanamycin at an inoculation rate of 5-10% (v / v). The broth is formulated as follows: yeast extract 5g / L, potassium dihydrogen phosphate 8g / L, ammonium sulfate 2.5g / L, diammonium hydrogen phosphate 3.5g / L, citric acid 1.5g / L, magnesium sulfate 0.6g / L, defoamer 1 drop / L, glucose 10g / L (the defoamer is a polyether defoamer). Incubate in a 5L fermenter at 37℃ until OD reaches the target value. 600 Once the concentration reaches 20-30, IPTG is added for induction culture, and the temperature is lowered to 18℃. During the culture process, dissolved oxygen is controlled at 20%, and residual sugar is controlled at 0.1-1.5 g / L.

[0063] 4. Composition of the supplemental culture medium: glucose 600g / L, peptone 8g / L, magnesium sulfate heptahydrate 2g / L.

[0064] 5. Take the bacterial cells expressing recombinant human type III collagen fragment produced by fermentation, resuspend them in buffer to 50 OD / mL, and sonicate them. Separate the lysed products by centrifugation to obtain the lysate and the lysate supernatant. For specific steps, refer to the patent document "CN119350505A, Protein Purification Method".

[0065] 6. After washing the lysed precipitate obtained in step 5 twice with buffer, add cleavage buffer and place in a constant temperature shaker at 25°C and 100 rpm for 24 h.

[0066] 7. The reaction supernatant and precipitate were collected by centrifugation. The yield and purity of HC25 and HC26 were determined by protein content analysis, as shown in Table 3. The yield of HC25 was 1.0 g / L, the yield of HC26 was 0.8 g / L, and the purity of both HC25 and HC26 was 98%.

[0067] Table 3

[0068]

[0069] Example 5 Bioactivity test of recombinant human type III collagen fragment

[0070] (1) Cytotoxicity

[0071] In this embodiment, the cytotoxicity of recombinant human type III collagen fragments was detected using the CCK8 assay. NIH / 3T3 (mouse embryonic fibroblasts) of known origin were used in this test, and cell culture was performed in DMEM complete medium containing 10% fetal bovine serum. HC25 and HC26 were preferred for the test.

[0072] The specific steps are as follows:

[0073] a. Cell preparation: Prepare a cell suspension from well-cultured cells, perform cell counting, and take 5 × 10⁶ cells. 3 Cells were seeded per well in 96-well plates and cultured in a complete cell culture incubator (37°C, 5% CO2) until the cell density reached 40%-60%.

[0074] b. Drug treatment: 100 μL of culture medium containing different concentrations of recombinant human type III collagen fragments was added to each well of the experimental group. 100 μL of maintenance medium supplemented with PBS was added horizontally to the negative control group. No cells were seeded in the blank control group, but 100 μL of maintenance medium was added horizontally. Cells were cultured in a cell culture incubator (37℃, 5% CO2) for 24 h.

[0075] c. CCK8 assay: Prepare 10% CCK8 working solution, 100 μL / well, incubate at 37℃ for 2-3 hours, and measure absorbance A at 450 nm using a microplate reader. 450 ;

[0076] Cell viability calculation: Cell viability (%) = (Area A) 450 - Blank Hole A 450 ) / (Negative hole A) 450 - Blank Hole A 450 ) × 100%.

[0077] The results are as follows Figure 2 As shown, HC25 and HC26 did not exhibit significant cytotoxicity at any of the detection concentrations, demonstrating that both HC25 and HC26 possess good biocompatibility.

[0078] (2) Cell adhesion

[0079] This embodiment demonstrates that recombinant human type III collagen fragments possess cell adhesion-promoting activity. The assay was performed using cell adhesion experiments and crystal violet staining, employing NIH / 3T3 (mouse embryonic fibroblasts) of known origin. Cell culture was conducted using DMEM medium containing 10% fetal bovine serum and serum-free DMEM medium. HC25 and HC26 were preferentially used for testing.

[0080] a. Coating: Different concentrations of recombinant human type III collagen fragments were added to 24-well cell culture plates at 250 μL / well and incubated at 37 ℃ for 2 hours. PBS was used as a negative control, and Matrigel 1 mg / mL (standard Matrigel, Beyotime) was used as a positive control. Residual liquid was removed, 1% heat-denatured BSA was added, and the plates were blocked at 37 ℃ for 1 hour. The plates were then washed three times with serum-free medium.

[0081] b. Cell adhesion: NIH / 3T3 (mouse embryonic fibroblasts) in good culture condition were suspended in serum-free medium for cell counting. 1×10⁻⁶ cells were added to each well. 5 Cells were incubated in a cell culture incubator (5% CO2, 37 ℃) for 2 hours, and washed 3 times with PBS to remove non-adhering cells. Negative wells without added cells were set up as blank controls.

[0082] c. Crystal violet staining: After fixation with 4% paraformaldehyde, add 250 μL of crystal violet staining solution to each well and stain for 20-30 minutes at room temperature. After staining, wash three times with PBS. After photographing and recording, add 2% SDS solution to each well and incubate at room temperature for 20-30 minutes. Measure the absorbance A at 570 nm using a microplate reader. 570 ;

[0083] Relative adhesion activity calculation: Relative adhesion activity (%) = (Experimental well A) 570 - Blank Hole A 570 ) / (Positive hole A) 570 - Blank Hole A 570 ) × 100%.

[0084] The results are as follows Figure 3 As shown, HC25 at 1 mg / mL and 0.5 mg / mL, and HC26 at all three tested concentrations, exhibited significant cell adhesion-promoting activity. Among them, HC25 at 1 mg / mL showed the best cell adhesion-promoting activity, reaching the level of commercial matrix gel (1 mg / mL).

[0085] (3) Cell scratch

[0086] This embodiment illustrates the cell migration-promoting activity of recombinant human type III collagen fragments. The assay was performed using a cell scratch assay with a well-established NIH / 3T3 cell line (mouse embryonic fibroblasts). Cell culture was conducted using complete medium containing 10% fetal bovine serum (FBS) in DMEM and low-serum medium containing 0.5% FBS in DMEM. HC25 and HC26 were preferred for testing.

[0087] The specific steps are as follows:

[0088] a. Cell preparation: Prepare a cell suspension from well-cultured cells, perform cell counting, and take 1×10⁶ cells. 5 Cells were seeded per well in 24-well plates and cultured for 24 hours in a cell culture incubator (37°C, 5% CO2).

[0089] b. Starvation treatment: Under microscopic examination, when the cell density reaches more than 90%, replace the low serum culture medium and place the cell culture in a cell culture incubator (37℃, 5% CO2) for starvation treatment for 6 hours;

[0090] c. Cell scratching: Using a 200 μL pipette tip, make longitudinal and vertical scratches in a 24-well plate to form a cross. After scratching, wash three times with PBS to remove any loose cells.

[0091] d. Drug treatment: After washing, the cells were photographed to record the scratch state at time T0, and the photographed areas were marked. After photographing, different concentrations of HC25 and HC26 were added for drug administration, with 1 mL of drug per well. Each group had two replicates. The negative control group was PBS, and the positive control group was EGF (18 ng / mL). The cells were incubated in an incubator (37℃, 5% CO2) for further culture.

[0092] e. Photography: At two time points, 24 hours and 48 hours after drug administration, the marked regions of the cells (the same regions as at time T0) were photographed and recorded as time points T1 and T2, respectively;

[0093] f. Use ImageJ and GraphPad Prism Program software to perform analysis and plotting, analyze the scratch area at T0, T1 and T2, and calculate the cell wound healing rate;

[0094] The results are as follows Figure 4-5 As shown, both HC25 and HC26 exhibited significant cell migration-promoting activities. Among them, HC25 at 0.5 mg / mL and 0.2 mg / mL, and HC26 at 0.3 mg / mL and 0.1 mg / mL showed the best cell migration-promoting activities, reaching the level of EGF (18 ng / mL) under the same conditions.

[0095] (4) Anti-photodamage activity

[0096] This embodiment illustrates the anti-photoaging activity of recombinant human type III collagen fragments. The assay used the HDF-a cell line (human dermal fibroblasts) of known origin, and the cells were cultured in complete medium containing 10% fetal bovine serum (FBS) in DMEM / F12. HC25 and HC26 cells were preferred for the assay.

[0097] The specific steps are as follows:

[0098] a. Cell preparation: Prepare a cell suspension from well-cultured HDF-a cells, perform cell counting, and take 1×10⁶ cells. 4 Cells were seeded per well in 96-well plates and cultured in a complete cell culture incubator (37°C, 5% CO2) until the cell density reached 90%.

[0099] b. Pretreatment: 100 μL of complete culture medium containing 0.5 mg / mL recombinant human type III collagen fragment was added to each well of the experimental group, and 100 μL of maintenance culture medium supplemented with PBS was added horizontally to the negative control group. Cells were cultured in an incubator (37℃, 5% CO2) for 24-48 hours.

[0100] c. UVB illumination: Calculate the required illumination distance and duration based on the UVB lamp power to achieve a final illumination intensity of 200 mJ / cm². 2 After irradiation, detached cells were gently washed away with PBS; those not irradiated were treated as the normal group (NO UVB).

[0101] d. CCK8 assay: Prepare 10% CCK8 working solution, 100 μL / well, incubate at 37℃ for 2-3 hours, and measure absorbance A at 450 nm using a microplate reader. 450Calculate cell viability;

[0102] The results are as follows Figure 6 As shown, HC25 and HC26 can effectively prevent UVB-induced photodamage to HDF-a cells at a detection concentration of 0.5 mg / mL.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A recombinant human type III collagen, characterized in that: Its amino acid sequence is as follows: (a) As shown in SEQ ID NO: 3 or SEQ ID NO: 4; or, (b) An amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, which retains the cell migration, cell adhesion, and photodamage prevention effects of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or, (c) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, which retains the cell migration, cell adhesion and photodamage prevention effects of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

2. The gene encoding recombinant human type III collagen as described in claim 1.

3. The encoding gene according to claim 2, characterized in that: Its nucleotide sequence is shown as 2566-2844bp in SEQ ID NO: 2 or 946-1206bp in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the coding gene as described in claim 2 or 3.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contain the coding gene as described in claim 2 or 3 or the recombinant expression vector as described in claim 4.

6. The recombinant engineered bacteria according to claim 5, characterized in that: The recombinant engineered bacteria used Escherichia coli as the host.

7. A method for constructing the recombinant engineered bacteria according to claim 5 or 6, characterized in that: The recombinant expression vector described in claim 4 was transformed into Escherichia coli BL21(DE3), and positive transformants were cultured and screened to obtain the recombinant engineered bacteria.

8. A method for rapidly preparing the recombinant human type III collagen of claim 1, characterized in that, The process includes the following steps: inoculating the recombinant engineered bacteria of claim 5 or 6 into the culture medium at an inoculation rate of 2-10%, adding IPTG for induction culture, collecting the bacterial cells, breaking them, collecting the lysed precipitate, cutting them, and collecting the supernatant, which is the recombinant human type III collagen of claim 1.

9. The method according to claim 8, characterized in that: The induction culture conditions are 16–18℃, 200–300 rpm for 20–28 h.

10. The application of the recombinant human type III collagen according to claim 1 in the fields of medicine, biomaterials, health products, and cosmetics.

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