Recombinant collagen iii+x and preparation method and application thereof

Abstract

The application discloses a recombinant collagen III+X, a preparation method and application thereof, and belongs to the technical field of bio-chemical industry. The recombinant collagen III+X provided by the application has an amino acid sequence shown as SEQ ID NO:1, is composed of a human collagen III 387-467 amino acid fragment repeated twice, and is connected with a human collagen X 521-680 amino acid fragment at a C terminal. Experiments prove that the recombinant collagen III+X can form a stable triple helix structure by co-expression with a proline hydroxylase, has great advantages in cell migration and safety, and can be used as a raw material for preparation of wound dressings and skin care products.

Description

Technical Field

[0001] This application belongs to the field of biochemical technology, and in particular relates to a recombinant collagen III+X, its preparation method and application. Background Technology

[0002] Collagen, the most abundant structural and functional protein in the human body, accounts for about one-third of the total protein in the body and is the main component of skin tissue, making up more than 75% of the skin's dry weight. Studies have shown that an adult body contains about 3 kilograms of collagen, of which skin tissue accounts for as much as 2.2 kilograms, far exceeding the content of other components such as hyaluronic acid (only 15 grams). Due to its unique biological properties, collagen is known as "the skin's core" and is a key material for maintaining the integrity and function of skin structure.

[0003] The known collagen superfamily comprises 29 members, all sharing a common characteristic: a typical Glycine-XY triplet repeat sequence structure, where the X and Y sites are typically occupied by proline and hydroxyproline, respectively. This unique amino acid arrangement, through interchain hydrogen bonds and electrostatic interactions, promotes the formation of a stable triple helix structure in collagen molecules. Type III collagen, an important member, is a homotrimer composed of three α1(Ⅲ) chains (Col3A1), accounting for approximately 60% of the composition in fetal skin and 15-20% in adult skin. This type of collagen exhibits a loose network structure, playing a crucial role in maintaining skin elasticity and promoting tissue repair.

[0004] Traditional collagen is mainly obtained through extraction from animal tissues, but this method has significant limitations: firstly, animal-derived collagen may trigger immunogenic reactions; secondly, the extraction process makes it difficult to ensure product homogeneity and poses a risk of pathogen transmission. In contrast, recombinant collagen prepared using genetic engineering technology has significant advantages: by precisely controlling the amino acid sequence, immunogenicity can be effectively reduced; the production process is stable with minimal batch-to-batch variation; and the risk of contamination by animal-derived pathogens is completely avoided, making it the preferred solution to the collagen sourcing problem.

[0005] Type X collagen, as a representative of short-chain non-fibroblast collagen, exhibits unique anti-hydrolysis properties in its C-terminal non-helical region (NC1) due to its rich content of aromatic amino acids, enabling it to form a stable network structure. However, it lacks sufficient biological activity when used alone. While type III collagen functional fragments show significant activity, they suffer from short in vivo half-life and easy degradation. This contradiction between function and structure severely limits the clinical application of collagen products. Therefore, developing a recombinant collagen with both excellent biological activity and structural stability has become a key technical problem urgently needing to be solved by those skilled in the art. Summary of the Invention

[0006] To overcome the aforementioned deficiencies in the prior art, this application provides a recombinant collagen III+X, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0008] On the one hand, this application provides a recombinant collagen III+X, the amino acid sequence of which is shown in SEQ ID NO:1, consisting of two repetitions of the 387-467 amino acid fragment of human type III collagen, and the C-terminus linked with the 521-680 amino acid fragment of human type X collagen.

[0009] Secondly, this application provides a gene encoding the above-mentioned recombinant collagen III+X, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0010] Thirdly, this application provides an expression vector, genetically engineered bacteria, or transgenic cell line containing the aforementioned genes.

[0011] Fourthly, this application provides a recombinant collagen III+X expression vector containing the aforementioned genes.

[0012] Optionally, it also includes a proline hydroxylase gene;

[0013] The nucleotide sequence of the proline hydroxylase gene is shown in SEQ ID NO:3;

[0014] The amino acid sequence of the protein encoded by the proline hydroxylase gene is shown in SEQ ID NO:4.

[0015] Fifthly, this application provides a method for constructing the above-mentioned recombinant collagen III+X expression vector, comprising the following steps:

[0016] (1) The above-mentioned gene was ligated into the pRSFDuet-1 vector with the restriction site NCOI-EcoRI to obtain the initial expression vector;

[0017] (2) The proline hydroxylase gene is ligated into the initial expression vector with NdeI and XhoI restriction sites to obtain the expression vector of recombinant collagen III+X.

[0018] Sixthly, this application provides a method for preparing recombinant collagen III+X, comprising the following steps:

[0019] The above-mentioned recombinant collagen III+X expression vector was transformed into an Escherichia coli expression strain, and after induction of expression, the recombinant collagen III+X was purified to obtain recombinant collagen III+X.

[0020] Optionally, the *E. coli* 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, or BL21(DE3)+HSP60.

[0021] Seventhly, this application provides the application of the above-mentioned recombinant collagen III+X in the preparation of products that promote cell migration.

[0022] Optionally, the product may include wound dressings or skin care products.

[0023] Compared with the prior art, this application has the following advantages:

[0024] This application uses two repeats of the amino acid sequence 387-467 of human type III collagen, with an amino acid fragment from human type X collagen (521-680) linked to its C-terminus. This fragment is then ligated into the vector pRSFDuet-1 to synthesize a proline hydroxylase gene from *Chlamydomonas reinhardtii*, which is then spliced ​​and recombined into the pRSFDuet-1 vector containing a DNA fragment of recombinant collagen III+X. The *E. coli* expression system was used to express the triple-helix recombinant collagen III+X. Stability experiments showed that, after storage at 4°C for 10 days, the stability of the recombinant collagen III+X in this application was significantly better than that of commercially available recombinant type III collagen, indicating that the recombinant collagen III+X in this application has better stability. Recombinant collagen III+X was hydrolyzed, and the hydroxyproline content in the collagen was detected by high-performance liquid chromatography (HPLC). The results showed that the hydroxyproline content in recombinant collagen III+X was 10.35%, indicating that co-expression of recombinant collagen III+X and proline hydroxylase in the *E. coli* system can effectively obtain hydroxylated collagen, thus demonstrating the application prospects of this strategy in collagen production. The migration activity experiment of mouse embryonic fibroblasts (BALB / c 3T3) showed that the recombinant collagen III+X expressed in this application has excellent cell migration-promoting activity, significantly superior to the previously reported full-length type III collagen Col3a1 and commercial recombinant collagen. Animal wound healing experiments showed that the recombinant collagen III+X and commercial recombinant type III collagen significantly improved wound healing in SD rats, with recombinant collagen III+X showing the best wound healing effect. Based on this, the recombinant collagen obtained in this application can be used as a raw material for medical liquid dressings, and can be combined with other materials to synergistically promote wound healing. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is an SDS-PAGE electrophoresis image (protein trimer) of the recombinant collagen III+X of this application;

[0027] Figure 2 The circular dichroism chromatogram (protein triple helix structure) of the recombinant collagen III+X of this application is shown.

[0028] Figure 3The stability assay of recombinant collagen III+X in this application is shown in the figure (* indicates a significant difference, P<0.05; ** indicates P<0.01).

[0029] Figure 4 Figure 1 shows the experimental results of promoting cell migration by recombinant collagen III+X, full-length type III collagen Col3a1 and commercial recombinant collagen of this application;

[0030] Figure 5 The figure shows the statistical results of scratch recovery rates of recombinant collagen III+X, full-length type III collagen Col3a1, and commercial recombinant collagen in this application (Note: **** indicates significant difference, P<0.0001);

[0031] Figure 6 The figure shows the wound healing rate results of animal experiments using recombinant collagen III+X (* indicates a significant difference, P<0.05). Detailed Implementation

[0032] The present application is further illustrated below with reference to specific embodiments. The following descriptions are merely a few embodiments of the present application and are not intended to limit the present application in any way. Although the present application discloses preferred embodiments as follows, they are not intended to limit the present application. Any modifications or variations made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

[0033] Unless otherwise specified, the raw materials used in the embodiments of this application are all purchased commercially and used directly without any special treatment.

[0034] Unless otherwise specified, the analytical methods in the embodiments all adopt conventional instrument or equipment settings and conventional analytical methods.

[0035] Example 1

[0036] Expression and purification of recombinant collagen III+X in prokaryotic systems:

[0037]

[0038] Single-clone colonies were selected from transformation plates and cultured overnight at 37°C in 50 mL LB medium (containing 100 mg / L kanamycin, with each liter of medium containing 10 g peptone, 5 g yeast extract, and 5 g NaCl). The colonies were then transferred 1:36 to 1.8 L LB medium (containing 100 mg / L kanamycin). The culture was maintained at 37°C until an OD600 of 0.4–0.6 was reached, at which point protein expression was induced by adding 0.5 mM IPTG at 16°C.

[0039] The bacterial culture was centrifuged at 7000 rpm for 10 min, and the bacterial cells were collected. The bacterial pellet was resuspended in 10 mM imidazole PBS (pH 7.5), the bacteria were autoclaved, and centrifuged at 11000 rpm for 15 min at 4 °C. The supernatant was collected and incubated with a equilibrated 5 mL nickel column (Thermo Fisher Scientific: 88221). Gravity elution was performed, and contaminating proteins were washed away with 10 mM PBS (pH 7.5). Gradient elution was then performed with 30 mM, 50 mM, 100 mM, 200 mM, and 300 mM imidazole PBS (pH 7.5), respectively. SDS-PAGE was used to detect the protein. The protein was concentrated and the imidazole was removed by ultrafiltration (Millipore, UFC9010). Recombinant collagen III+X was obtained and its aggregates were detected by non-reducing SDS-PAGE. Figure 1 The results showed that recombinant collagen III+X expressed by pRSFDuet-1 could form a trimer. Circular dichroism spectroscopy showed ( Figure 2 The recombinant collagen CAⅢ+X has a negative peak in the range of 190nm to 216nm, with a trough around 205nm; and a positive peak in the range of 217nm to 234nm, with a peak around 222nm.

[0040] Example 2

[0041] PBS solutions of recombinant collagen III+X and commercial recombinant type III collagen at a concentration of 0.2 mg / mL were prepared as stock solutions and stored at 4°C. Samples were taken at 5, 10, 20 and 30 days for SDS-PAGE analysis, Coomassie brilliant blue staining, destaining and photographing, and protein band analysis was performed using ImageJ to detect changes in protein amount and position. BSA was used as a 2L control.

[0042] Results analysis: such as Figure 3As shown, when stored at 4℃ for 10 days, recombinant collagen III+X exhibited significantly better stability than commercially available recombinant type III collagen, indicating that recombinant collagen III+X possesses superior stability. After 30 days, both types of recombinant collagen showed significant degradation of over 50%. Recombinant collagen III+X was lyophilized into a powder and stored at -20℃. On day 60, the recombinant collagen III+X remained intact, demonstrating that lyophilization is beneficial for the preservation of recombinant collagen III+X. P < 0.05 was considered * (statistically significant difference), and P < 0.01 was considered **.

[0043] Example 3

[0044] Recombinant collagen III+X was hydrolyzed, and the hydroxyproline content in the collagen was determined using high-performance liquid chromatography (HPLC). The chromatographic column used was a C18 SHISEIDO (4.6 mm × 250 mm × 5 μm), with an injection volume of 10 μL, a column temperature of 40℃, and a wavelength of 214 nm. The mobile phases were: A - 0.1 M sodium acetate aqueous solution (pH 4.5); B - acetonitrile. The calculation formula was W = (C / C0) × V × N × 100 / m, where: W: hydroxyproline content in the sample (%); C: concentration of hydroxyproline in the sample solution, in mg / L; C0: concentration of hydroxyproline in the blank control, in mg / L; V: final volume, in L; N: dilution factor; m: sample volume, in mg. Based on the standard curve of the hydroxyproline standard and the calculation formula, the hydroxyproline content in recombinant collagen III+X was calculated to be 10.35%. These results demonstrate that co-expression of recombinant III+X collagen and proline hydroxylase in the E. coli system can effectively obtain hydroxylated collagen, thus proving the application prospects of this strategy in collagen production.

[0045] Example 4

[0046] Assay of the cell migration-promoting activities of recombinant collagen III+X, full-length type III collagen Col3a1, and commercially available recombinant collagen:

[0047] Using a marker, draw evenly spaced horizontal lines on the back of the 12-hole board, spaced approximately 0.5–1 cm apart, with 3 lines per hole. Add approximately 5 x 10 mm of marker to each hole. 4BALB / c 3T3 cells were cultured overnight to allow cell adhesion. The next day, cells were sliced ​​using a sterile pipette tip and a ruler. The cells were washed three times with PBS to remove the sliced ​​cells and residual serum-containing DMEM medium. Serum-free DMEM medium was added. Three experimental groups were set up: a control group (III+X), a group containing full-length type III collagen (Col3a1), and an experimental group containing commercially available recombinant collagen (0.1 mg / mL). Cells were returned to the incubator. Samples were taken and photographed after 24 hours. Each group had three replicate wells, and five points were selected from each well for statistical analysis. The representative scratch pattern is shown in the image. Figure 4 III+X showed the best activity in promoting cell migration. Statistical analysis of scratch recovery rate showed that ( Figure 5 After 24 hours of treatment (protein concentration 0.1 mg / mL), the three experimental groups III+X (70% scratch recovery rate), Col3a1 (56% scratch recovery rate), and commercial recombinant collagen (35% scratch recovery rate), compared with the control group (23% scratch recovery rate), showed significant cell migration-promoting activity (statistically significant difference). III+X showed the best cell migration-promoting activity, which was significantly better than that of commercial recombinant collagen. P < 0.05 was considered * (statistically significant difference), and P < 0.0001 was considered ****.

[0048] Example 5

[0049] Male SD rats weighing 250–350 g were used in the experiment. They were acclimatized for one week before the experiment and housed in an SPF environment. Recombinant collagen III+X and commercially available recombinant type III collagen were used in the experiment. Rats were randomly divided into three groups of 15 rats each, corresponding to the two experimental groups and a control group. The experimental groups received a 20 mg / mL solution, while the control group received Tegaderm. TM A transparent wound dressing (3587) was applied. After anesthetizing the rats, the hair on their backs was shaved, and a 15 mm sterile biopsy needle was used to puncture the wound. All rats were injected intraperitoneally with penicillin (20,000 U / kg / day) daily for 3 consecutive days. At each time point (0, 7, 14, and 21 days), rats were sacrificed by CO2 exposure, and samples were collected for histological examination. Wound healing was assessed by photographing the wounds and processing them using ImageJ software. The wound healing rate was calculated as: Wound healing rate = Wound area on day I / Wound area on day 0 × 100%, where I represents the time point. Results are as follows: Figure 6 As shown, recombinant collagen III+X and commercially available recombinant type III collagen significantly improved wound healing in SD rats, with recombinant collagen III+X exhibiting the best wound healing effect. Therefore, the recombinant collagen obtained in this application can be used as a raw material for medical liquid dressings, synergistically promoting wound healing in combination with other materials.

[0050] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. An expression vector for recombinant collagen III+X, characterized in that, The amino acid sequence of the recombinant collagen III+X is shown in SEQ ID NO:

1. It consists of two repetitions of the amino acid fragment of human type III collagen (387-467) and the linking of the amino acid fragment of human type X collagen (521-680) at the C-terminus. The expression vector contains a gene encoding the recombinant collagen III+X; The nucleotide sequence of the gene is shown in SEQ ID NO:2; The expression vector also contains a proline hydroxylase gene; The nucleotide sequence of the proline hydroxylase gene is shown in SEQ ID NO:3; The amino acid sequence of the protein encoded by the proline hydroxylase gene is shown in SEQ ID NO:

4.

2. The method for constructing the expression vector of recombinant collagen III+X according to claim 1, characterized in that, Includes the following steps: (1) The gene encoding the recombinant collagen III+X was ligated into the pRSFDuet-1 vector with the restriction enzyme site NCOI-EcoRI to obtain the initial expression vector; (2) The proline hydroxylase gene is ligated into the initial expression vector with NdeI and XhoI restriction sites to obtain the expression vector of recombinant collagen III+X.

3. A method for preparing recombinant collagen III+X, characterized in that, Includes the following steps: The expression vector of recombinant collagen III+X as described in claim 1 was transformed into an Escherichia coli expression strain, and after induction of expression, recombinant collagen III+X was obtained by purification.

4. The method for preparing recombinant collagen III+X according to claim 3, characterized in that, The *E. coli* 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, or BL21(DE3)+HSP60.

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