A recombinant humanized type III collagen and a preparation method and application thereof

Abstract

The application discloses a recombinant type III humanized collagen, a preparation method and application thereof, and an amino acid sequence of the recombinant type III humanized collagen is shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. The recombinant type III humanized collagen provided in the application has cell adhesion promoting activity, the amino acid sequence of the recombinant type III humanized collagen is selected from natural collagen amino acid sequences, and the application of the recombinant type III humanized collagen to human body does not cause an immune response. The application utilizes the characteristics that corynebacterium glutamicum does not secrete endotoxins, the preparation method is simple, and the product can meet the requirements of cosmetics, medical devices and medicines without complicated separation, and thus provides a reference for low-cost industrial production.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a recombinant type III humanized collagen, its preparation method, and its application. Background Technology

[0002] Collagen possesses characteristics such as biodegradability, good biocompatibility, and low immunogenicity. It can promote cell proliferation and adhesion, and has functions such as promoting tissue repair and hemostasis. In addition, collagen can form collagen fibers, which have certain mechanical properties, making it an excellent biomaterial that is now widely used in cosmetics, biomedical materials, pharmaceuticals, and other fields.

[0003] Currently, 28 types of collagen have been identified, each with different functions. They are generally divided into two main categories: fibroblastic collagen and non-fibroblastic collagen. Fibroblastic collagen includes types I, II, III, V, VI, and XXVI, while the rest are non-fibroblastic collagen. Type I collagen is the most abundant in the human body, accounting for over 85%. Type I collagen is found in high concentrations in bone, skin, tendons, and the cornea. Type II collagen is present in cartilage, intervertebral discs, and the vitreous humor, while type III collagen is found in blood vessels, new skin, and scar tissue.

[0004] Currently, collagen sources mainly include extraction from animal tissues and expression through genetic engineering. Naturally extracted collagen is a mixture of collagens with different molecular weights, insoluble in water, and has poor biocompatibility. Furthermore, because it originates from animal tissues, it may pose risks of immunogenicity and potential pathogen infection. In addition, type III collagen, due to its symbiotic relationship with other types of collagen, is present in low concentrations. Extracting type III collagen from animal tissues faces significant challenges, including difficulty, low purity, and high cost. Furthermore, variations between individual animals can lead to poor batch-to-batch stability. Recombinant collagen obtained through genetic engineering is similar to natural collagen, offering advantages such as good water solubility, no risk of viral transmission, low immune rejection, and good batch-to-batch consistency.

[0005] Commonly used expression systems for recombinant collagen include *Escherichia coli* and *Pichia pastoris*. Yeast, as a eukaryotic expression system, allows for post-translational modifications and is non-pathogenic; however, the secreted collagen is mostly single-chain, and the fermentation cycle is long and costly. *E. coli* has advantages such as a short fermentation cycle, high expression levels, and ease of genetic manipulation. However, due to the barrier effect of the *E. coli* outer membrane, exogenous proteins are mostly expressed intracellularly, easily forming inclusion bodies. The products require refolding treatment, and the target protein may contain endotoxins as a byproduct, making the separation and purification process cumbersome and posing a risk of endotoxin quality issues. Designing and developing superior recombinant collagen and selecting suitable expression systems are urgent problems to be solved. Summary of the Invention

[0006] To address the existing technical problems, this application provides a recombinant type III humanized collagen, its preparation method, and its application. The technical solution is as follows:

[0007] In a first aspect, a recombinant type III humanized collagen is provided, the amino acid sequence of which is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0008] Furthermore, the amino acid sequence of the recombinant type III humanized collagen includes:

[0009] N repeated combinations of any sequence from SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3, wherein N is an integer greater than 1; or

[0010] A combination of two or three sequences from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of the recombinant type III humanized collagen is SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 connected in sequence, and the amino acid sequence is shown in SEQ ID NO.4.

[0012] Furthermore, the amino acid sequence has 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4.

[0013] In a second aspect, a nucleotide sequence encoding recombinant type III humanized collagen as described in the first aspect above is provided, wherein the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 is shown in SEQ ID NO.5;

[0014] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 is shown in SEQ ID NO.6;

[0015] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.3 is shown in SEQ ID NO.7;

[0016] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.4 is shown in SEQ ID NO.8.

[0017] Thirdly, an expression vector comprising the nucleotide sequence described in the second aspect above is provided.

[0018] Furthermore, the expression vector includes p19, pET30a, or pPIC9K.

[0019] Fourthly, a host cell comprising the expression vector as described in the third aspect above is provided.

[0020] Furthermore, the host cells include: Escherichia coli, Pichia pastoris, or Corynebacterium glutamicum.

[0021] Fifthly, a method for preparing recombinant type III humanized collagen is provided, comprising the following steps:

[0022] Synthesize the nucleotide sequence encoding recombinant type III humanized collagen;

[0023] The target DNA fragment was amplified by PCR using KOD-plus-neo polymerase; the PCR products were separated by agarose gel electrophoresis, and the target DNA fragment was recovered from them using a Gel Extraction Kit D2500; the DNA fragment was assembled using Gibson Assembly Cloning Ki; the assembled product was transformed into DH5α or TOP10F competent cells, transformants were formed the next day, and the cells were inoculated and cultured overnight; plasmids were extracted using Plasmid Mini Kit I D6943.

[0024] Take competent Corynebacterium glutamicum cells, thaw them on ice, add recombinant plasmids, gently pipette to mix thoroughly, incubate on ice, then transfer competent cells to an electroporation cuvette stored at -20℃, electroporate using a BTX electroporator, transfer electroporated cells to Eppendorf tubes containing 1 mL of LBHis liquid medium, incubate at 46℃ for 6 minutes, then renature at 30℃ for 1.5 hours, at 200 rpm, take the bacterial suspension, spread it evenly on LBHis agar plates containing the corresponding antibiotic, incubate overnight at 30℃, and colonies will form on plates containing chloramphenicol after 36 hours;

[0025] Seed culture was inoculated into LB medium and cultured at 30℃ and 200rpm for 16-24h. The shake-flask culture was stopped when the OD600 reached 5-8.

[0026] Inoculate with a 10% inoculum, and set the fermentation parameters as follows: 30℃, 800-1000 rpm, 3-6 L / h aeration, and pH 7.0. Add feed medium after 10-12 hours of fermentation, take samples to monitor protein expression levels, and harvest the bacteria when the protein content shows a decreasing trend.

[0027] The fermentation supernatant was filtered and subjected to affinity chromatography using a HisTrap FF nickel column with gradient elution. The eluent was collected and then desalted by ultrafiltration to obtain the final product.

[0028] Sixthly, the application of the recombinant type III humanized collagen described in the first aspect above in the preparation of pharmaceuticals, cosmetics and medical devices is provided.

[0029] Furthermore, the medical device includes artificial blood vessels, hemostatic dressings, skin wound repair materials, cartilage repair materials, or medical aesthetic materials.

[0030] In a seventh aspect, a composition is provided comprising the recombinant type III humanized collagen as described in the first aspect, the nucleotide as described in the second aspect, the expression vector as described in the third aspect, or the host cell as described in the fourth aspect.

[0031] The beneficial effects of the technical solution provided in this application are as follows: The amino acid sequence of the recombinant type III humanized collagen of this application is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3. The recombinant type III humanized collagen provided in this application has cell adhesion-promoting activity. The amino acid sequence of the recombinant type III humanized collagen is selected from the natural collagen amino acid sequence, and its application in the human body will not produce an immune response. This application utilizes the characteristic that Corynebacterium glutamicum does not secrete endotoxins, the preparation method is simple, and the product can meet the requirements of cosmetics, medical devices, and pharmaceuticals without complex separation, providing a reference for low-cost industrial production. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is an SDS-PAGE electrophoresis image of the fermentation broth supernatant provided in Example 4 of this application;

[0034] Figure 2 This is an SDS-PAGE electrophoresis image of the purified fermentation culture product provided in Example 6 of this application;

[0035] Figure 3 This is a cell proliferation activity diagram of the recombinant type III humanized collagen provided in Example 7 of this application;

[0036] Figure 4 This is a cell adhesion rate diagram of recombinant type III humanized collagen provided in Example 8 of this application;

[0037] Figure 5 This is a 6-hour cell migration diagram of recombinant type III humanized collagen provided in Example 9 of this application;

[0038] Figure 6 This is a 24-hour cell migration diagram of recombinant type III humanized collagen provided in Example 9 of this application. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0040] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0041] In the following description, when referring to the accompanying drawings, the same numbers in different drawings denote the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0042] In the description of this application, it should be understood that the terms "first," "second," "third," etc., are used only to distinguish similar objects and are not necessarily used to describe a specific order or sequence, nor should they be construed as indicating or implying relative importance. In the description of this application, unless otherwise stated, "multiple" means two or more.

[0043] Example 1: Design of Recombinant Type III Humanized Collagen Peptide Fragments

[0044] Three sets of recombinant type III humanized collagen sequences were designed and obtained.

[0045] Polypeptide 1 (SEQ ID NO.1)

[0046] GRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGK

[0047] Polypeptide 2 (SEQ ID NO.2)

[0048] GPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGIT

[0049] Polypeptide 3 (SEQ ID NO.3)

[0050] GRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESPGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPI

[0051] The three amino acid sequences were sequentially linked to obtain polypeptide 4 (SEQ ID NO.4).

[0052] GRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPG PQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGAR GLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPG HPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPI

[0053] The nucleotide sequence was determined using the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-optimization-tool. The protein sequences of peptides 1-3 were entered into the webpage; the desired expression host was selected; restriction enzyme sites to be avoided were selected; and the nucleotide sequences encoding each amino acid sequence were optimized and obtained.

[0054] (1) The nucleotide sequence encoding polypeptide 1 is SEQ ID NO.5

[0055] GGGCGTGACGGTGTTCCGGGTGGTCCGGGAATGCGCGGAATGCCGGGCTCCCCGGGTGGACCAGGCTCTGATGGCAAGCCTGGTCCCCCTGGCTCCCAGGGTGAATCCGGACGCCCCGGACCGCCGGGGCCAAGTGGTCCTCGGGGCCAACCTGGCGTCATGGGATTTCCAGGTCCGAAGGGCAATGATGGTGCACCCGGTAAGAACGGCGAACGTGGCGGGCCAGGAGGCCCCGGGCCACAAGGACCACCGGGCAAGAATGGTGAAACCGGCCCCCAAGGTCCCCCCGGCCCAACAGGTCCCGGGGGAGACAAAGGGGACACCGGTCCGCCGGGACCTCAAGGTTTACAGGGACTCCCTGGTACGGGGGGTCCCCCCGGTGAGAACGGCAAACCAGGTGAGCCAGGCCCTAAGGGCGATGCAGGAGCCCCCGGTGCTCCTGGCGGCAAA

[0056] (2)The nucleotide sequence SEQ ID NO.6 encoding polypeptide 2

[0057] GGGCCCCCCGGACCTGCAGGCTTTCCGGGCGCTCCTGGCCAAAATGGTGAACCCGGCGGCAAGGGTGAGCGCGGTGCGCCAGGAGAGAAGGGGGAAGGCGGCCCTCCGGGCGTGGCTGGGCCACCAGGGGGCAGCGGCCCAGCCGGTCCACCGGGCCCCCAGGGCGTTAAGGGTGAGCGCGGTTCACCGGGTGGACCAGGCGCTGCAGGATTTCCGGGTGCCAGAGGCCTGCCAGGCCCGCCGGGCTCTAATGGCAATCCTGGCCCACCTGGTCCAAGTGGCTCCCCAGGGAAAGACGGGCCACCCGGTCCGGCAGGCAATACGGGCGCACCGGGGAGTCCAGGGGTTTCGGGGCCAAAGGGCGATGCCGGCCAACCGGGTGAAAAAGGCTCCCCAGGTGCGCAGGGTCCGCCGGGTGCTCCAGGACCCTTGGGCATTGCAGGCATTACT

[0058] (3) Nucleotide sequence SEQ ID NO.7 encoding polypeptide 3

[0059] GGTCGCGACGGTAATCCCGGTTCAGATGGTCTTCCAGGGAGGGATGGCTCGCCTGGCGGCAAGGGCGATCGCGGCGAAAACGGTTCTCCTGGCGCCCCAGGTGCGCCTGGCCATCCTGGTCCACCTGGCCCAGTTGGCCCAGCTGGAAAATCCGGGGATCGCGGCGAGAGCGGCCCCGCCGGTCCAGCTGGTGCGCCGGGCCCCGCGGGTTCGAGAGGCGCCCCTGGTCCTCAGGGCCCCCGAGGCGACAAAGGAGAAACTGGTGAACGCGGTGCAGCTGGTATTAAAGGACACCGCGGGTTCCCGGGAAACCCTGGGGCGCCGGGTTCCCCAGGCCCCGCCGGACAGCAGGGTGCAATTGGTTCCCCTGGTCCAGCCGGACCACGGGGTCCCGTTGGTCCCTCCGGTCCACCAGGCAAAGATGGAACATCGGGGCATCCCGGACCGATC

[0060] (4) Nucleotide sequence SEQ ID NO.8 encoding polypeptide 4

[0061]

[0062] Example 2 Construction of Recombinant Type III Humanized Collagen Expression Plasmid

[0063] Three main types of plasmid vectors were used: first, the p19 series vector containing the pBL1 replicon, used to construct expression plasmids for Corynebacterium glutamicum recombinant proteins; second, the pET-30a vector, used to construct expression plasmids for Escherichia coli BL21(DE3) recombinant proteins; and third, the pPIC9K vector, used to construct expression plasmids for Pichia pastoris recombinant proteins. Each cell plasmid was constructed according to the following procedure:

[0064] Using KOD-plus-neo polymerase (Toyobo, Japan), the target DNA fragment (any sequence from SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8) was amplified according to standard PCR procedures. The PCR products were separated by agarose gel electrophoresis, and the target DNA fragment was recovered using a Gel Extraction Kit D2500 (Omega Bio-Tek, USA). The DNA fragment was assembled using Gibson Assembly Cloning Ki (t NEB, USA) at 50°C for 30 min. The assembled product was transformed into DH5α or TOP10F competent cells, and transformants were formed the next day. The cells were inoculated and cultured overnight. Plasmids were extracted using Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA), and verified by Sanger sequencing.

[0065] In the construction of p19 and pET-30a vectors, *E. coli* DH5α was used as the cloning host for plasmid construction. Transformation and cell amplification were performed using LB medium (containing 1% peptone, 0.5% yeast extract, and 1% sodium chloride per liter). If necessary, antibiotics were added at a concentration of 30 μg / mL chloramphenicol or 50 μg / mL kanamycin. In the construction of pPIC9K vector, *E. coli* TOP10F was used as the cloning host for plasmid construction, and the transformation method was the same as for *E. coli* DH5α.

[0066] Example 3 Construction of Recombinant Type III Humanized Collagen Carrier

[0067] (1) Construction of recombinant *Corynebacterium glutamicum*: Competent *Corynebacterium glutamicum* cells were taken from an ultra-low temperature freezer, thawed on ice, and 100 ng of the plasmid prepared in Example 2 was added. The cells were gently pipetted and mixed thoroughly. The cells were then placed on ice for 15 minutes. The competent cells were transferred to an electroporation cuvette (0.1 cm gap, Bio-Rad) frozen at -20°C and electroporated using a BTX electroporator with parameters of 1.8 kV, 200 Ω, and 25 µF (5 ms). After electroporation, the cells were transferred to an Eppendorf tube containing 1 mL of LBHis liquid medium (containing 5 g / L tryptone, 2.5 g / L yeast extract, 18.5 g / L brain heart broth, 91 g / L sorbitol, and 5 g / L NaCl) (preheated to 46°C). The tube was incubated at 46°C for 6 minutes, followed by annealing at 30°C for 1.5 hours at 200 rpm. An appropriate amount of bacterial culture (electroporation efficiency approximately 10) was taken. 5 CFU / µg DNA was evenly spread onto LBHis agar (15 g / L) plates containing the corresponding antibiotic and incubated overnight at 30°C. On plates containing chloramphenicol, colonies formed after 36 hours. Single colonies were picked and cultured, and plasmids were extracted using the Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA) and verified by Sanger sequencing. Strains with correct plasmid sequencing were positive recombinant bacteria.

[0068] (2) Construction of recombinant Escherichia coli: BL21(DE3) competent Escherichia coli cells were removed from the cryogenic freezer and thawed on ice. 100 ng of the plasmid prepared in Example 2 was added, and the mixture was gently aspirated and mixed thoroughly. The mixture was then placed on ice for 5 min, then in a 42°C water bath for 30 s, and finally placed on ice for another 5 min. The transformation solution was evenly spread onto LB agar (15 g / L) plates containing the corresponding antibiotic and incubated overnight at 37°C. Colonies formed after 24 h. Single colonies were picked and cultured. Plasmids were extracted using the Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA), and verified by Sanger sequencing. Strains with correct plasmid sequencing were positive recombinant bacteria.

[0069] (3) Construction of Pichia pastoris recombinant strain: Boil 1 mL of salmon sperm DNA (2 mg / L) for 5 min and quickly cool on ice; centrifuge competent GS115 cells at 12000 rpm for 15 s and discard the upper LiCl layer; add 240 μL of 500 g / L PEG-3350, 36 μL of 1 mol / L LiCl, 25 μL of salmon sperm DNA and 50 μL of linearized recombinant plasmid DNA (5-10 μg) in sequence, and vortex vigorously for 1 min to ensure that the cell pellet is thoroughly mixed with the added solution; incubate at 30℃ for 30 min. After heat shock in a 42℃ water bath for 20 min, centrifuge at 8000 rpm for 10 min to collect the cells, resuspend the cells in 200 μL of YPD medium, and culture with shaking at 30℃; after resuspending, add 20 μL directly to an MD plate and culture at 30℃ for 2-4 days until the colony diameter reaches 1 mm. Single clones were selected and cultured. Plasmids were extracted using the Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA), and verified by Sanger sequencing. Strains with correct plasmid sequencing were positive recombinant bacteria.

[0070] The following describes the expression, identification, fermentation, purification, and cellular functional experiments of the recombinant type III humanized collagen of this application, using polypeptide 4 (SEQ ID NO.4) as an example.

[0071] Example 4: Expression and Identification of Recombinant Type III Humanized Collagen

[0072] Corynebacterium glutamicum (expression plasmid p19 constructed using SEQ ID NO. 8, then further constructed) was first inoculated into a shake flask containing at least a certain amount of LB medium, with 10 µg / mL chloramphenicol added for primary seed culture. After 24 hours, it was transferred to secondary LB medium for fermentation culture at 30℃ for 24-36 hours. 25 µl of fermentation supernatant was mixed with 5 µL of loading buffer and incubated at 98℃ for 6 minutes before SDS-PAGE analysis (15% polyacrylamide gel), with 20 µl of sample added to each lane. Western blotting was performed according to standard experimental procedures, and ImageJ software was used to analyze the band gray values ​​to detect protein expression. Results are shown below. Figure 1 1 is a protein marker; 2 is the fermentation broth supernatant. The results show that the fermentation supernatant contains the above-mentioned recombinant type III humanized collagen (peptide 4, SEQ ID NO.4).

[0073] Example 5 Fermentation of recombinant type III humanized collagen

[0074] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0.

[0075] Fermentation medium (g / L): tryptone 7, yeast extract 5, sodium chloride 15, glucose 40, ammonium sulfate 10, magnesium sulfate 0.25, sodium dihydrogen phosphate dihydrate 3, dipotassium hydrogen phosphate trihydrate 2.

[0076] Feeding medium: glucose 300 g / L, ammonium sulfate 30 g / L, trace elements 96.8 mg / L.

[0077] Seed culture: After thawing the cryovial, 200 µl of Corynebacterium glutamicum from Example 4 was inoculated into 200 mL of LB medium and cultured at 30 °C and 200 rpm for 16–24 h. Samples were taken to measure OD600 and examined under a microscope for contamination. If there was no contamination and OD600 was between 5 and 8, the shake-flask culture was terminated.

[0078] Fermentation culture: Inoculate with a 10% inoculum. Fermentation parameters are 30℃, rotation speed 800-1000 rpm, aeration rate 3-6 L / h, and pH 7.0. Add feed medium after 10-12 hours of fermentation. Sample and monitor protein expression levels after 32-48 hours of fermentation. When the protein content shows a decreasing trend, remove the culture tank and harvest the bacteria.

[0079] Example 6 Purification of recombinant type III humanized collagen

[0080] The fermentation supernatant from Example 5 was filtered through 0.45µm and 0.22µm filter membranes and subjected to affinity chromatography using a HisTrap FF nickel column with gradient elution (equilibration buffer: 0.5mol / L NaCl, 20-30mmol / L phosphate buffer, pH 7.4; pre-wash buffer: 0.5mol / L NaCl, 20-30mmol / L phosphate buffer, 0.04mol / L imidazole, pH 7.4; eluent: 0.5mol / L NaCl, 20-30mmol / L phosphate buffer, 0.5mol / L imidazole, pH 7.4). The eluent was collected. The eluent was desalted by ultrafiltration with 20-30mmol / L phosphate buffer. Electrophoresis showed the purity of the eluent to be approximately 97%, as shown in the figure. Figure 2 1 is the protein marker; 2 is the sample (10 µl); 3 is the eluent (10 µl). Figure 2 As can be seen, the target protein was present in the eluted sample, indicating that the collagen was well attached to the column. The sample was then concentrated and purified after passing through a nickel column.

[0081] Example 7 Proliferative Activity Assay of Recombinant Type III Humanized Collagen

[0082] Remove the cultured L929 cells from the incubator, discard the old medium, add 1.5 ml of trypsin to digest the cells, and discard the trypsin after the cells have shrunk and become rounded. Resuspend the cells in 5 ml of complete culture medium (90% DMEM + 10% FBS), count the cells, and adjust the cell density to 1×10⁻⁶. 5 Cells / ml were seeded into 96-well plates at 1000 cells / well and cultured for 24 hours. The next day, the medium was changed to 100µl of recombinant type III humanized collagen solution (serum-free medium containing peptide 4, DMEM / F12 1:1 mixed to 1000ml, HEPES 15mM, soybean trypsin inhibitor 0.1%, insulin 10.0µg / ml, transferrin 25.0µg / ml, fibronectin 5.0µg / ml), and cultured for another 48 hours. Then, 10µl of CCK-8 solution (10% of the medium volume) was added, and the plates were incubated for 3 hours. The A450 value was measured using a microplate reader.

[0083] Affinity-purified samples were used to evaluate the proliferation activity of L929 fibroblasts. Cells in logarithmic growth phase were trypsin-digested into single-cell suspensions and seeded into 96-well plates with complete culture medium at a density of 1000 cells / well. Cells were incubated at 37°C for 24 hours in a 5% CO2 incubator. 4 mg of recombinant humanized type III collagen (peptide 4) was accurately weighed and diluted to 4 mg / ml with serum-free DMEM, then filtered through a 0.25 μm filter for sterilization. The initial 4 mg / ml concentration sample was serially diluted with DMEM medium (Gibco, catalog number 11965092) to prepare four concentrations: 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, and 4.0 mg / ml. The 96-well plates were removed, the supernatant discarded, and the diluted recombinant type III humanized collagen solution was added, with six replicates per concentration. A group of cells was added to DMEM medium only as a negative control. Another group of cells was cultured in complete medium as a positive control. After 48 hours of further culture, the supernatant was discarded, and CCK-8 staining solution was added to detect cell proliferation. The control sample was recombinant type III humanized collagen; the positive control was in 10% whole serum medium (90% DMEM + 10% FBS); and the negative control was in serum-free medium (100% DMEM). All statistical results represent the proliferation activity of each sample compared to the basal medium, as shown in Tables 1-2 and 3. Figure 3 .

[0084] Table 1. A450 detection results after 30 hours of incubation.

[0085] Table 2. Proliferative Activity of Recombinant Type III Humanized Collagen

[0086] Cellular experiments showed that the recombinant type III humanized collagen sample (peptide 4) exhibited certain cell proliferation-promoting activity at a drug concentration of 4 mg / ml, with a proliferation rate approximately 14% higher than the untreated control cells after 30 hours of culture. It is anticipated that adding higher concentrations of the sample and processing for longer periods may yield even better activity.

[0087] Example 8 Cell adhesion assay of recombinant type III humanized collagen

[0088] Coating: Add 50-100 µl of coating solution to each well of a 96-well plate and incubate overnight at 2-8°C. Remove the coating solution, dry the culture dish, and wash 1-3 times with washing buffer.

[0089] Cell seeding: After the cells to be tested are processed, they are digested with trypsin, washed with PBS, and then cultured with Thermo Fisher molceular probes. TM Resuspend the cells in the RPMI medium (from the kit) to prepare a cell suspension. Add 5 × 10⁻⁶ cells per 100 ml solution. 4 -5×10 5 Seed cells / well in 96-well plates. Incubate at 37°C for 30-120 min. Remove the culture plate, discard the culture medium, wash 2-3 times, and add 100 µl of fresh culture medium containing peptide 4 (Thermo Fisher molceular probes) to each well. TM RPMI medium in the kit.

[0090] Adhesion rate assay: Add 10 µl of cell staining solution to each well of a 96-well plate and incubate at 37°C for 30–240 min. Measure the OD value of each well and the absorbance at 450 nm. Calculate the adhesion rate. Results are shown below. Figure 4 .

[0091] from Figure 4 As can be seen, compared with the blank control, the cell adhesion activity of the recombinant type III humanized collagen sample gradually increased with the increase of the concentration, indicating that the recombinant type III humanized collagen has good cell adhesion-promoting activity.

[0092] Example 9 Cell migration activity assay of recombinant III humanized collagen

[0093] Remove the cultured L929 cells from the incubator, discard the old medium, add 1.5 ml of trypsin to digest the cells, and discard the trypsin after the cells have shrunk and become rounded. Resuspend the cells in 5 ml of complete culture medium, count them, and adjust the cell density to 1×10⁻⁶. 5Cells / ml were seeded into 6-well plates at a rate of 2 ml / well, ensuring 100% coverage overnight. The next day, the cells were scored using a pipette tip along a ruler, and washed three times with PBS to remove the scored cells. The purified sample was then prepared into 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml solutions using DMEM medium (Gibco, catalog number 11965092) containing peptide 4. 1 ml of each solution was added to each well, and the samples were incubated at 37°C in a CO2 incubator for 6 hours. Observation and photography were then performed. Figure 5 ).

[0094] Within the 1-4 mg / ml sample concentration gradient set up in this experiment, the cell migration-promoting effect significantly increased with increasing sample concentration. A second experiment was conducted with the 4 mg / ml sample, which was incubated in a 37℃ CO2 incubator for 24 hours, and observations and photographs were taken. Figure 6 The results show that the cell migration-promoting effect is significantly better than that of the negative control, and further increases in sample concentration can be made to verify its biological efficacy.

[0095] The amino acid sequence of the recombinant type III humanized collagen of this application is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3. The recombinant type III humanized collagen provided in this application has cell adhesion-promoting activity. The amino acid sequence of the recombinant type III humanized collagen is selected from natural collagen amino acid sequences, and its application in the human body will not produce an immune response. This application utilizes the characteristic that Corynebacterium glutamicum does not secrete endotoxins, resulting in a simple preparation method. The product does not require complex separation and can meet the requirements of cosmetics, medical devices, and pharmaceuticals, providing a reference for low-cost industrial production.

[0096] The above are merely preferred embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A recombinant type III humanized collagen, characterized in that, The amino acid sequence of the recombinant type III humanized collagen is SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 connected in sequence, and the amino acid sequence is shown in SEQ ID NO.

4.

2. A nucleic acid molecule encoding the recombinant type III humanized collagen as described in claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

8.

3. An expression vector comprising the nucleic acid molecule as described in claim 2, characterized in that, The expression vectors include p19, pET30a, or pPIC9K.

4. A host cell comprising the expression vector as described in claim 3, characterized in that, The host cells include: Escherichia coli, Pichia pastoris, or Corynebacterium glutamicum.

5. The method for preparing recombinant type III humanized collagen as described in claim 1, characterized in that, Includes the following steps: Synthesize the nucleotide sequence encoding recombinant type III humanized collagen; The target DNA fragment was amplified by PCR using KOD-plus-neo polymerase; the PCR products were separated by agarose gel electrophoresis, and the target DNA fragment was recovered from them using a Gel Extraction Kit D2500; the DNA fragment was assembled using Gibson Assembly Cloning Ki; the assembled product was transformed into DH5α or TOP10F competent cells, transformants were formed the next day, and the cells were inoculated and cultured overnight; plasmids were extracted using Plasmid Mini Kit I D6943. Take competent Corynebacterium glutamicum cells, thaw them on ice, add recombinant plasmids, gently pipette to mix thoroughly, incubate on ice, then transfer competent cells to an electroporation cuvette stored at -20℃, electroporate using a BTX electroporator, transfer electroporated cells to Eppendorf tubes containing 1 mL of LBHis liquid medium, incubate at 46℃ for 6 minutes, then renature at 30℃ for 1.5 hours, at 200 rpm, take the bacterial suspension, spread it evenly on LBHis agar plates containing the corresponding antibiotic, incubate overnight at 30℃, and colonies will form on plates containing chloramphenicol after 36 hours; Seed culture was inoculated into LB medium and cultured at 30℃ and 200rpm for 16-24h. The shake-flask culture was stopped when the OD600 reached 5-8. Inoculate with a 10% inoculum, and set the fermentation parameters as follows: 30℃, 800-1000 rpm, 3-6 L / h aeration, and pH 7.

0. Add feed medium after 10-12 hours of fermentation, take samples to monitor protein expression levels, and harvest the bacteria when the protein content shows a decreasing trend. The fermentation supernatant was filtered and subjected to affinity chromatography using a HisTrap FF nickel column with gradient elution. The eluent was collected and then desalted by ultrafiltration to obtain the final product.

6. A composition, characterized in that, It includes the recombinant type III humanized collagen as described in claim 1, the nucleic acid molecule as described in claim 2, the expression vector as described in claim 3, or the host cell as described in claim 4.

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