Recombinant III-type humanized collagen as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses a recombinant III-type humanized collagen as well as a preparation method and application thereof. The amino acid sequence of the recombinant III-type humanized collagen is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The recombinant III-type humanized collagen has the advantages that the recombinant III-type humanized collagen can be used for preparing the recombinant III-type humanized collagen; the recombinant III-type humanized collagen provided by the invention has the activity of promoting cell adhesion, the amino acid sequence of the recombinant III-type humanized collagen is selected from the amino acid sequence of natural collagen, and the recombinant III-type humanized collagen does not generate immune response when being applied to a human body. The characteristic that corynebacterium glutamicum does not secrete endotoxin is utilized, the preparation method is simple, the product can meet the requirements of food, cosmetics, medical instruments and drugs without complex separation, and reference is provided for low-cost industrial production.

Description

Technical Field

[0001] The present application relates to the field of biological genetic engineering technology, and in particular to a recombinant humanized type III collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is biodegradable, biocompatible, and has low immunogenicity. It promotes cell proliferation and adhesion, and has functions such as tissue repair and hemostasis. Furthermore, collagen can form collagen fibers and possesses certain mechanical properties, making it an excellent biomaterial that is now widely used in food, cosmetics, biomedical materials, pharmaceuticals, and other fields.

[0003] Currently, 28 types of collagen have been discovered, each with distinct functions. They are generally divided into two categories: fibrillar collagen and non-fibrillar collagen. Fibrillar collagen includes types I, II, III, V, VI, and XXVI, while the rest are non-fibrillar 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 is found in cartilage, intervertebral discs, and the vitreous body. Type III is found in blood vessels, new skin, and scar tissue.

[0004] At present, the sources of collagen mainly include extraction from animal tissues and expression through genetic engineering technology. Naturally extracted collagen is a mixture of various collagens of different molecular weights. It is insoluble in water and has poor biocompatibility. Moreover, since it comes from animal tissues, there may be risks of immunogenicity and potential pathogen infection. In addition, because type III collagen coexists with other types of collagen and has a low content, extracting type III collagen from animal tissues presents serious problems such as difficulty, low purity, and high cost. The differences between individual animals also lead to poor batch stability of collagen. The recombinant collagen obtained by genetic engineering technology is similar to natural collagen and has the advantages of good water solubility, no risk of viral transmission, low immune rejection reaction, and good batch consistency.

[0005] Commonly used expression systems for recombinant collagen include Escherichia coli and Pichia pastoris. As a eukaryotic expression system, yeast can undergo post-translational modification and is non-pathogenic. However, the secreted collagen is mostly single-chain, and the fermentation cycle is long and the cost is high. Escherichia coli has the characteristics of short fermentation cycle, high expression level, and ease of genetic manipulation. However, due to the barrier effect of the E. coli outer membrane, exogenous proteins are mostly expressed intracellularly and easily form inclusion bodies. The product needs to be renatured, and the target protein is mixed with by-product endotoxins. The separation and purification process is cumbersome and prone to endotoxin quality risks.

[0006] Designing and developing excellent recombinant collagen and selecting a suitable expression system are issues that need to be urgently addressed. Summary of the Invention

[0007] In order to solve the existing technical problems, the present invention provides a recombinant humanized type III collagen and its preparation method and application. The technical solution is as follows:

[0008] In a first aspect, a recombinant humanized type III collagen is provided, wherein the amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0009] Furthermore, the amino acid sequence of the recombinant humanized type III collagen includes:

[0010] N repeated combinations of any one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, wherein N is an integer greater than 1; or

[0011] A combination of two or three of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0012] Furthermore, the amino acid sequence of the recombinant humanized type III 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.

[0013] Furthermore, the amino acid sequence has 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0014] In a second aspect, a nucleotide sequence encoding the recombinant humanized type III collagen as described in the first aspect is provided, wherein the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 is shown as SEQ ID NO.5;

[0015] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 is shown in SEQ ID NO.6;

[0016] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.3 is shown in SEQ ID NO.7;

[0017] The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.4 is shown in SEQ ID NO.8.

[0018] In a third aspect, an expression vector comprising the nucleotide sequence described in the second aspect is provided.

[0019] Furthermore, the expression vector includes p19, pET30a or pPIC9K.

[0020] In a fourth aspect, a host cell comprising the expression vector described in the third aspect is provided.

[0021] Furthermore, the host cell includes: Escherichia coli, Pichia pastoris or Corynebacterium glutamicum.

[0022] In a fifth aspect, a method for preparing recombinant humanized type III collagen is provided, comprising the following steps:

[0023] synthesizing a nucleotide sequence encoding recombinant humanized type III collagen;

[0024] The target DNA fragment was amplified by PCR using KOD-plus-neo polymerase. The PCR products were separated by agarose gel electrophoresis and recovered using the Gel Extraction Kit D2500. The DNA fragments were assembled using the Gibson Assembly Cloning Kit. The assembled products were transformed into DH5α or TOP10F competent cells, forming transformants the next day. The cells were then inoculated and cultured overnight, and the plasmid was extracted using the Plasmid Mini Kit ID6943.

[0025] Take the competent cells of Corynebacterium glutamicum, place them on ice to thaw, add the recombinant plasmid, mix thoroughly by gently pipetting, and let it stand on ice. Then transfer the competent cells to a -20°C frozen electroporation cup and use a BTX electroporator for electroporation. After electroporation, transfer the cells to an Eppendorf tube containing 1 mL of LBHis liquid medium and place them in a 46°C water bath for 6 minutes. Then, anneal and culture at 30°C for 1.5 hours at 200 rpm. Take the bacterial solution and evenly spread it on an LBHis agar plate containing the corresponding antibiotics. Culture it in a 30°C incubator overnight. Colonies will form on the plate containing chloramphenicol after 36 hours.

[0026] The seed solution was inoculated into LB medium and cultured at 30°C and 200 rpm for 16-24 h. The shake flask culture was terminated when the OD600 value was 5-8.

[0027] Inoculate with 10% inoculum, and ferment at 30°C, 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, and harvest when the protein content shows a downward trend.

[0028] The fermentation supernatant was filtered, affinity chromatography was performed using a HisTrap FF nickel column in a gradient elution manner, the eluate was collected, and the eluate was ultrafiltered to remove salt to obtain the product.

[0029] In a sixth aspect, a use of the recombinant humanized type III collagen described in the first aspect above in the preparation of foods, medicines, cosmetics, health products and medical devices is provided.

[0030] Furthermore, the medical device includes an artificial blood vessel, a hemostatic dressing, a skin wound repair material, a cartilage repair material or a medical cosmetic material.

[0031] In the seventh aspect, a composition is provided, comprising the recombinant humanized type III 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.

[0032] The beneficial effects of the technical solutions provided in the embodiments of the present application are as follows: the amino acid sequence of the recombinant humanized type III collagen of the present application is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The recombinant humanized type III collagen provided in the present application has the activity of promoting cell adhesion, and the amino acid sequence of the recombinant humanized type III collagen is selected from the natural collagen amino acid sequence, and will not produce an immune response when applied to the human body. The present application utilizes the characteristic that Corynebacterium glutamicum does not secrete endotoxins, and the preparation method is simple. The product can meet the requirements of food, cosmetics, medical devices and medicines without complex separation, providing a reference for low-cost industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0034] Figure 1 This is the SDS-PAGE electrophoresis diagram of the fermentation broth supernatant provided in Example 4 of the present application;

[0035] Figure 2 This is an SDS-PAGE electrophoresis diagram of the purified fermentation culture product provided in Example 6 of the present application;

[0036] Figure 3 This is a graph showing the cell proliferation activity of recombinant humanized type III collagen provided in Example 7 of the present application;

[0037] Figure 4 This is a graph showing the cell adhesion rate of recombinant humanized type III collagen provided in Example 8 of the present application;

[0038] Figure 5 This is a 6-hour cell migration graph of recombinant humanized type III collagen provided in Example 9 of the present application;

[0039] Figure 6 This is a 24h cell migration diagram of the recombinant humanized type III collagen provided in Example 9 of the present application. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of this application clearer, the implementation methods of this application will be further described in detail below with reference to the accompanying drawings.

[0041] It should be clear that the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0042] When the following description refers to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Instead, they are merely examples of devices and methods consistent with certain aspects of the present application, as detailed in the appended claims.

[0043] 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 precedence, nor should they be understood to indicate or imply relative importance. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0044] Example 1 Design of recombinant humanized type III collagen peptide fragment

[0045] Three groups of recombinant type III humanized collagen sequences were designed.

[0046] Polypeptide 1 (SEQ ID NO. 1)

[0047] GRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGK<A <A

[0048] Polypeptide 2 (SEQ ID NO.2)<A <A

[0049] GPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGIT<A <A

[0050] Polypeptide 3 (SEQ ID NO.3)<A <A

[0051] GRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPI<A <A

[0052] The three groups of amino acid sequences are sequentially linked to obtain Polypeptide 4 (SEQ ID NO.4)<A <A

[0053] GRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPG PQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGAR GLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPG HPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPI

[0054] Use the online codon optimization tool https: / / sg.idtdna.com / pages / tools / codon-

[0055]

[0056] select the desired expression host; select the enzyme cleavage sites that need to be avoided; and optimize and obtain the nucleotide sequence encoding each amino acid sequence.

[0057] (1) Nucleotide sequence encoding polypeptide 1 SEQ ID NO.5

[0058] GGGCGTGACGGTGTTCCGGGTGGTCCGGGAATGCGCGGAATGCCGGGCTCCCCGGGTGGACCAGGCTCTGATGGCAAGCCTGGTCCCCCTGGCTCCCAGGGTGAATCCGGACGCCCCGGACCGCCGGGGCCAAGTGGTCCTCGGGGCCAACCTGGCG2CATGGGATTTCCAGGTCCGAAGGGCAATGATGGTGCACCCGGTAAGAACGGCGAACGTGGCGGGCCAGGAGGCCCCGGGCCACAAGGACCACCGGGCAAGAATGGTGAAACCGGCCCCCAAGGTCCCCCCGGCCCAACAGGTCCCGGGGGAGACAAAGGGGACACCGGTCCGCCGGGACCTCAAGGTTTACAGGGACTCCCTGGTACGGGGGGTCCCCCCGGTGAGAACGGCAAACCAGGTGAGCCAGGCCCTAAGGGCGATGCAGGAGCCCCCGGTGCTCCTGGCGGCAAA

[0059] (2) The nucleotide sequence encoding polypeptide 2, SEQ ID NO. 6

[0060] GGGCCCCCCGGACCTGCAGGCTTTCCGGGCGCTCCTGGCCAAAATGGTGAACCCGGCGGCAAGGGTGAGCGCGGTGCGCCAGGAGAGAAGGGGGAAGGCGGCCCTCCGGGCGTGGCTGGGCCACCAGGGGGCAGCGGCCCAGCCGGTCCACCGGGCCCCCAGGGCGTTAAGGGTGAGCGCGGTTCACCGGGTGGACCAGGCGCTGCAGGATTTCCGGGTGCCAGAGGCCTGCCAGGCCCGCCGGGCTCTAATGGCAATCCTGGCCCACCTGGTCCAAGTGGCTCCCCAGGGAAAGACGGGCCACCCGGTCCGGCAGGCAATACGGGCGCACCGGGGAGTCCAGGGGTTTCGGGGCCAAAGGGCGATGCCGGCCAACCGGGTGAAAAAGGCTCCCCAGGTGCGCAGGGTCCGCCGGGTGCTCCAGGACCCTTGGGCATTGCAGGCATTACT

[0061] (3) Nucleotide sequence SEQ ID NO.7 encoding polypeptide 3

[0062] GGTCGCGACGGTAATCCCGGTTCAGATGGTCTTCCAGGGAGGGATGGCTCGCCTGGCGGCAAGGGCGATCGCGGCGAAAACGGTTCTCCTGGCGCCCCAGGTGCGCCTGGCCATCCTGGTCCACCTGGCCCAGTTGGCCCAGCTGGAAAATCCGGGGATCGCGGCGAGAGCGGCCCCGCCGGTCCAGCTGGTGCGCCGGGCCCCGCGGGTTCGAGAGGCGCCCCTGGTCCTCAGGGCCCCCGAGGCGACAAAGGAGAAACTGGTGAACGCGGTGCAGCTGGTATTAAAGGACACCGCGGGTTCCCGGGAAACCCTGGGGCGCCGGGTTCCCCAGGCCCCGCCGGACAGCAGGGTGCAATTGGTTCCCCTGGTCCAGCCGGACCACGGGGTCCCGTTGGTCCCTCCGGTCCACCAGGCAAAGATGGAACATCGGGGCATCCCGGACCGATC

[0063] (4) Nucleotide sequence SEQ ID NO.8 encoding polypeptide 4

[0064]

[0065] Example 2 Construction of recombinant humanized type III collagen expression plasmid

[0066] There are three main types of plasmid vectors used: the pBL1 replicon p19 series vectors, used to construct expression plasmids for recombinant proteins of Corynebacterium glutamicum; the pET-30a vector, used to construct expression plasmids for recombinant proteins of Escherichia coli BL21 (DE3); and the pPIC9K vector, used to construct expression plasmids for recombinant proteins of Pichia pastoris. Each cell plasmid was constructed according to the following process:

[0067] The target DNA fragment (any sequence among SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8) was amplified using KOD-plus-neo polymerase (Toyobo, Japan) according to conventional PCR procedures. The PCR products were separated by agarose gel electrophoresis and recovered using Gel Extraction Kit D2500 (Omega Bio-Tek, USA). The DNA fragments were assembled using Gibson Assembly Cloning Kit (tNEB, USA) at 50°C for 30 min. The assembled products were transformed into DH5α or TOP10F competent cells, and transformants were formed the next day. The cells were inoculated and cultured overnight, and the plasmids were extracted using Plasmid Mini Kit ID6943 (Omega Bio-Tek, USA) and verified by Sanger sequencing.

[0068] For p19 and pET-30a vector construction, E. coli DH5α was used as the cloning host for plasmid construction. LB medium (containing 1% peptone, 0.5% yeast extract, and 1% sodium chloride per liter) was used for transformation and bacterial growth. When needed, antibiotics were added at a concentration of 30 μg / mL chloramphenicol or 50 μg / mL kanamycin. For pPIC9K vector construction, E. coli TOP10F was used as the cloning host for plasmid construction, and the transformation method was the same as for E. coli DH5α.

[0069] Example 3 Construction of recombinant humanized type III collagen carrier

[0070] (1) Construction of recombinant bacteria of Corynebacterium glutamicum: Take out the competent cells of Corynebacterium glutamicum from the ultra-low temperature freezer, place them on ice to melt, add 100ng of the plasmid prepared in Example 2, gently pipette to mix thoroughly, let it stand on ice for 15 minutes, transfer the competent cells to a -20℃ frozen electroporation cup (0.1cm gap, Bio-Rad), and use a BTX electroporator for electroporation with parameters of 1.8kv, 200Ω, 25μF (5ms). The electroporated cells were transferred to an Eppendorf tube containing 1mL of LBHis liquid culture medium (containing 5g / L tryptone, 2.5g / L yeast extract, 18.5g / L brain heart infusion broth, 91g / L sorbitol and 5g / LNaCl.) (preheated at 46℃), water bathed at 46℃ for 6 minutes, and then annealed at 30℃ for 1.5 hours at 200rpm. Take an appropriate amount of bacterial solution (the electroporation efficiency is about 10 5 cfu / μg DNA) and evenly spread on LBHis agar plates (15 g / L) containing the corresponding antibiotic. Culture overnight at 30°C. Colonies will form on plates containing chloramphenicol after 36 hours. Single colonies will be cultured and plasmids will be extracted using the Plasmid Mini Kit ID6943 (Omega Bio-Tek, USA). Verification will be performed by Sanger sequencing. Strains with correct plasmid sequencing are considered positive recombinant strains.

[0071] (2) Construction of recombinant Escherichia coli: Take out the competent Escherichia coli cells BL21 (DE3) from the ultra-low temperature refrigerator, place them on ice to melt, add 100 ng of the plasmid prepared in Example 2, gently pipette to mix thoroughly, let it stand on ice for 5 minutes, place it in a 42°C water bath for 30 seconds, and then let it stand on ice for another 5 minutes. The transformation liquid was evenly spread on an LB agar (15 g / L) plate containing the corresponding antibiotic, cultured in a 37°C incubator overnight, and colonies formed in 24 hours. Pick a single clone for culture, use PlasmidMini Kit ID6943 (Omega Bio-Tek, USA) to extract the plasmid, and verify it by Sanger sequencing. Strains with correct plasmid sequencing are positive recombinant bacteria.

[0072] (3) Construction of recombinant Pichia pastoris: Boil 1 mL of 2 mg / L salmon sperm DNA for 5 min and quickly cool on ice; centrifuge competent GS115 cells at 12,000 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 vigorously mix in a vortex mixer for 1 min to thoroughly mix the cell pellet with the added solution; incubate at 30°C for 30 min. After heat shock in a 42°C water bath for 20 min, collect the cells by centrifugation at 8,000 rpm for 10 min, resuspend the cells in 200 μL of YPD medium, and culture at 30°C with shaking; directly add 20 μL of the resuspended cells to MD plates and culture at 30°C for 2-4 days until the colonies have a diameter of 1 mm. Single colonies were picked and cultured, and plasmids were extracted using Plasmid Mini Kit ID6943 (Omega Bio-Tek, USA). The plasmids were then sequenced and verified by Sanger sequencing. Strains with correct plasmid sequences were considered positive recombinant strains.

[0073] Taking polypeptide 4 (SEQ ID NO. 4) as an example, the expression identification, fermentation, purification and cell functional experiments of the recombinant humanized type III collagen of the present application are described below.

[0074] Example 4 Expression and Identification of Recombinant Humanized Type III Collagen

[0075] Corynebacterium glutamicum (expression plasmid p19 constructed using SEQ ID NO. 8, then reconstructed) was first inoculated into a small amount of LB medium in a shake flask, supplemented with 10 μg / mL chloramphenicol, and cultured as a primary seed solution. After 24 hours, the cells were transferred to secondary LB medium for fermentation. The culture temperature was 30°C for 24-36 hours. 25 μl of the fermentation supernatant was mixed with 5 μL of Loading buffer, treated at 98°C for 6 minutes, and then analyzed by SDS-PAGE (15% polyacrylamide gel). 20 μl of sample was added to each lane. Western blotting was performed according to standard experimental procedures. Image J software was used to analyze the grayscale value of the bands and detect protein expression. The results are shown in Figure 2. Figure 1 , where 1 is Protein marker; 2 is fermentation supernatant. The results show that the fermentation supernatant contains the above-mentioned recombinant type III humanized collagen (polypeptide 4, SEQ ID NO.4).

[0076] Example 5 Fermentation of recombinant humanized type III collagen

[0077] LB medium (g / L): tryptone 10, yeast powder 5, NaCl 10, pH 7.0.

[0078] Fermentation medium (g / L): tryptone 7, yeast powder 5, sodium chloride 15, glucose 40, ammonium sulfate 10, magnesium sulfate 0.25, sodium dihydrogen phosphate dihydrate 3, potassium hydrogen phosphate trihydrate 2.

[0079] Feed medium: glucose 300 g / L, ammonium sulfate 30 g / L, trace elements 96.8 mg / L.

[0080] Seed culture: After thawing the frozen tube, 200 μl of Corynebacterium glutamicum of Example 4 was inoculated into 200 mL of LB medium and cultured at 30° C. and 200 rpm for 16 to 24 h. Samples were taken to measure OD600 and microscopically examined for contamination. If there was no contamination and the OD600 was between 5 and 8, the shake flask culture was terminated.

[0081] Fermentation: Inoculate at 10% of the inoculum size, with fermentation parameters set at 30°C, 800-1000 rpm, 3-6 L / h aeration, and pH 7.0. Add feed medium after 10-12 hours of fermentation. Samples were taken at 32-48 hours to monitor protein expression. Harvest the culture if protein levels show a downward trend.

[0082] Example 6 Purification of recombinant humanized type III collagen

[0083] The fermentation supernatant of Example 5 was filtered through 0.45 μm and 0.22 μm filter membranes and affinity chromatography was performed using a HisTrap FF nickel column by gradient elution (equilibration solution: 0.5 mol / L NaCl, 20-30 mmol / L phosphate buffer, pH 7.4; pre-wash solution: 0.5 mol / L NaCl, 20-30 mmol / L phosphate buffer, 0.04 mol / L imidazole, pH 7.4; eluent: 0.5 mol / L NaCl, 20-30 mmol / L phosphate buffer, 0.5 mol / L imidazole, pH 7.4), and the eluate was collected. The eluate was ultrafiltered with 20-30 mmol / L phosphate buffer to remove salt. The purity of the eluate was determined by electrophoresis to be approximately 97%, as shown in FIG. Figure 2 , where 1 is Protein marker; 2 is sample (10μl); 3 is eluent (10μl). Figure 2 It can be seen that the target protein is present in the eluted sample, indicating that the collagen column has a good effect. The sample is concentrated and purified after passing through the nickel column.

[0084] Example 7 Proliferation Activity Test of Recombinant Humanized Type III Collagen

[0085] Remove the cultured L929 cells from the incubator, discard the old solution, add 1.5 ml of trypsin to digest, discard the trypsin after the cells shrink and become round, add 5 ml of complete medium (90% DMEM + 10% FBS) to resuspend the cells, and adjust the cell density to 1×10 after counting. 5 Cells were plated at 1000 cells / ml in a 96-well plate and cultured for 24 hours. The next day, the medium was replaced with 100 μl of recombinant type III humanized collagen solution (serum-free medium containing peptide 4, DMEM / F12 1:1 mixed to 1000 ml, HEPES 15 mM, soybean trypsin inhibitor 0.1%, insulin 10.0 μg / ml, transferrin 25.0 μg / ml, fibronectin 5.0 μg / ml) and cultured for another 48 hours. 10 μl of CCK-8 solution (10% of the culture medium volume) was added and incubated in an incubator for 3 hours. A450 was detected by microplate reader.

[0086] Affinity-purified samples were used to evaluate proliferation activity in L929 fibroblasts. Cells in the logarithmic growth phase were trypsinized to a single-cell suspension and seeded in complete culture medium in 96-well plates. The cells were cultured at a density of 1,000 cells / well in a 37°C, 5% CO2 incubator for 24 hours. Four mg of recombinant humanized type III collagen (peptide 4) was accurately weighed and diluted to 4 mg / ml in serum-free DMEM, then sterilized by filtration through a 0.25 μm filter. A sample with an initial concentration of 4 mg / ml was serially diluted using DMEM (Gibco, Cat. No. 11965092) to four concentrations: 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, and 4.0 mg / ml. The 96-well plate was removed, the supernatant discarded, and the diluted recombinant humanized type III collagen solution was added, with six replicates per well for each concentration. A group of cells receiving only DMEM served as a negative control. Another group of cells was added with complete culture medium only as a positive control. Culture was continued for 48 hours, the supernatant was discarded, and CCK-8 staining solution was added to detect cell proliferation. The control sample used recombinant type III humanized collagen sample; the positive control used 10% full serum culture medium (90% DMEM + 10% FBS); the negative control used serum-free culture medium (100% DMEM culture medium). The statistical results are all data on the proliferation activity of each sample compared with the basal culture medium. The results are shown in Tables 1-2 and Figure 3 .

[0087] Table 1 A450 test results after 30h of culture

[0088]

[0089] Table 2 Proliferative activity of recombinant humanized type III collagen

[0090] 0.5mg / ml 1mg / ml 2mg / ml 4mg / ml 30H 88％ 91％ 96％ 114％ 14H 86％ 85％ 99％ 106％

[0091] Cell experiments revealed that the recombinant humanized type III collagen sample (peptide 4) exhibited some cell proliferation-promoting activity at a concentration of 4 mg / ml. After 30 hours of culture, the proliferation effect increased by approximately 14% compared to the untreated control cells. It is anticipated that the addition of higher sample concentrations and longer treatment times may yield even greater activity.

[0092] Example 8 Cell Adhesion Assay of Recombinant Humanized Type III Collagen

[0093] 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 plate, and wash 1-3 times with washing solution.

[0094] Cell inoculation: After the cells are processed, trypsinize, wash with PBS, and then culture medium (Thermo Fisher molceular probes TM Resuspend in RPMI medium in the kit to prepare cell suspension. 4 -5×10 5 Cells / well were seeded into a 96-well plate. Incubated at 37°C for 30-120 min. The plate was removed, the culture medium was discarded, and the plate was washed 2-3 times. 100 μl of fresh culture medium containing peptide 4 (Thermo Fisher Scientific molceular probes) was added to each well. TM RPMI medium in the kit).

[0095] Adhesion rate detection: Add 10μl cell staining solution to each well of a 96-well plate and incubate at 37℃ for 30-240min. Measure the OD value of the sample wells and the absorbance value of each well at 450nm to calculate the adhesion rate. Figure 4 .

[0096] from Figure 4 It can be seen that compared with the blank control, as the concentration of the recombinant type III humanized collagen sample increases, its cell adhesion activity also gradually increases, indicating that the recombinant type III humanized collagen has good cell adhesion promoting activity.

[0097] Example 9 Cell migration activity test of recombinant humanized type III collagen

[0098] Take out the cultured L929 cells from the incubator, discard the old solution, add 1.5 ml of trypsin to digest, discard the trypsin after the cells shrink and become round, add 5 ml of complete culture medium to resuspend the cells, and adjust the cell density to 1×10 after counting. 5cell / ml, inoculate into 6-well plates, 2ml / well, so that 100% of the 6-well plates can be covered overnight. The next day, use the tip of a pipette to scratch against a ruler, wash the cells 3 times with PBS, and remove the scratched cells. Use DMEM medium containing polypeptide 4 (Gibco, product number 11965092) to prepare the purified sample into 0.5mg / ml, 1mg / ml, 2mg / ml, and 4mg / ml solutions, add 1ml to each well, place in a 37℃ CO2 incubator for 6h, observe and take pictures ( Figure 5 ).

[0099] In the 1-4 mg / ml sample concentration gradient set in this experiment, as the sample concentration increased, its effect on promoting cell migration also increased significantly. A second test was conducted on the 4 mg / ml concentration sample, which was placed in a 37°C CO2 incubator for 24 hours and observed and photographed ( Figure 6 ). From the results, the effect of promoting cell migration is significantly better than that of the negative control, and the sample concentration can be further increased to verify its biological activity.

[0100] The amino acid sequence of the recombinant humanized type III collagen of the present application is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The recombinant humanized type III collagen provided by the present application has the activity of promoting cell adhesion. The amino acid sequence of the recombinant humanized type III collagen is selected from the natural collagen amino acid sequence and does not produce an immune response when applied to the human body. The present application utilizes the characteristic that Corynebacterium glutamicum does not secrete endotoxins. The preparation method is simple, and the product can meet the requirements of food, cosmetics, medical devices and medicines without complex separation, providing a reference for low-cost industrial production.

[0101] The above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should be included within the scope of the claims of the present application.

Claims

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

3.

2. The recombinant humanized type III collagen according to claim 1, characterized in that The amino acid sequence of the recombinant humanized type III collagen includes: N repeated combinations of any one of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, wherein N is an integer greater than 1; or A combination of two or three of SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

3. The recombinant humanized type III collagen according to claim 2, characterized in that The amino acid sequence of the recombinant humanized type III 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.

4. The recombinant humanized type III collagen according to any one of claims 1 to 3, characterized in that The amino acid sequence has 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

5. A nucleotide sequence encoding the recombinant humanized type III collagen according to any one of claims 1 to 4, characterized in that: The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 is shown in SEQ ID NO.5; The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 is shown in SEQ ID NO.6; The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.3 is shown in SEQ ID NO.7; The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.4 is shown in SEQ ID NO.

8.

6. An expression vector comprising the nucleotide sequence according to claim 5, characterized in that The expression vector includes p19, pET30a or pPIC9K.

7. A host cell comprising the expression vector according to claim 6, characterized in that The host cells include: Escherichia coli, Pichia pastoris or Corynebacterium glutamicum.

8. A method for preparing recombinant humanized type III collagen, characterized in that: The following steps are involved: synthesizing a nucleotide sequence encoding recombinant humanized type III collagen; The target DNA fragment was amplified by PCR using KOD-plus-neo polymerase. The PCR products were separated by agarose gel electrophoresis and recovered using the Gel Extraction Kit D2500. The DNA fragments were assembled using the Gibson Assembly Cloning Kit. The assembled products were transformed into DH5α or TOP10F competent cells, forming transformants the next day. The cells were inoculated and cultured overnight, and the plasmid was extracted using the Plasmid Mini Kit ID6943. Take the competent cells of Corynebacterium glutamicum, place them on ice to thaw, add the recombinant plasmid, mix thoroughly by gently pipetting, and let it stand on ice. Then transfer the competent cells to a -20°C frozen electroporation cup and use a BTX electroporator for electroporation. After electroporation, transfer the cells to an Eppendorf tube containing 1 mL of LBHis liquid medium and place them in a 46°C water bath for 6 minutes. Then, anneal and culture at 30°C for 1.5 hours at 200 rpm. Take the bacterial solution and evenly spread it on an LBHis agar plate containing the corresponding antibiotics. Culture it in a 30°C incubator overnight. Colonies will form on the plate containing chloramphenicol after 36 hours. The seed solution was inoculated into LB medium and cultured at 30°C and 200 rpm for 16-24 h. The shake flask culture was terminated when the OD600 value was 5-8. Inoculate with 10% inoculum, and ferment at 30°C, 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, and harvest when the protein content shows a downward trend. The fermentation supernatant was filtered, affinity chromatography was performed using a HisTrap FF nickel column in a gradient elution manner, the eluate was collected, and the eluate was ultrafiltered to remove salt to obtain the product.

9. Use of the recombinant humanized type III collagen according to any one of claims 1 to 4 in the preparation of foods, medicines, cosmetics, health products and medical devices.

10. A composition, characterized in that The method comprises the recombinant humanized type III collagen according to claims 1 to 4, the nucleotide according to claim 5, the expression vector according to claim 6 or the host cell according to claim 7.

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