Recombinant human III-type collagen and preparation method thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to recombinant humanized collagen and a preparation method thereof. The recombinant humanized III-type collagen provided by the invention can be efficiently and stably expressed under three expression systems of yeast, escherichia coli and CHO cells, and has biological activity superior to that of commercially available collagen products. The recombinant humanized III-type collagen disclosed by the invention has structural characteristics and functional activity similar to those of natural collagen, so that the recombinant humanized III-type collagen has wide application prospects in the aspects of biomedical materials, tissue engineering products, cosmetics, foods, health care products or medicines, medical instruments, medical cosmetology and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant human type III collagen and a preparation thereof. Background Art

[0002] Collagen is a natural protein essential for connective tissue, accounting for 25% to 30% of the total protein content. Structurally, collagen uses tropocollagen as the basic structural unit to form a special triple helical structure chain. Tropocollagen is composed of a peptide chain with (Gly-XY)n repeating units, where X and Y can be any amino acid, usually proline and hydroxyproline (Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem. 2009; 78: 929-58.).

[0003] Collagen can be divided into many types, including type I collagen, type II collagen, type III collagen, and type IV collagen. Type I and type III collagen are mainly found in connective tissues such as skin, tendons, ligaments, and joints, and are the two types of collagen with the highest content in the skin. Type I collagen is the main body of the skin, accounting for 80-85% of the total collagen in the skin, providing a strong supporting structure and support for the skin. The loss of type I collagen will cause facial wrinkles and depressions; type III collagen is loose and mesh-like, relatively small, accounting for 10-15% of the total collagen in the skin, providing elasticity and stress resistance for the skin, and has the effect of promoting elastic repair.

[0004] At present, the collagens applied in commercialization mainly include type I and type III. Type I is mainly used in the facial mask field, while type III is used in the fields of medical aesthetics filling and medical devices. The raw material sources of collagen products on the market are mainly extracted from the connective tissues of animals such as pigs, cows, and fish through acid, alkali, and enzymatic methods. However, there are certain problems with collagen extracted from animals, such as the risk of virus infection, human rejection reaction, and the weakening of efficacy and poor stability caused by the destruction of the collagen structure during the extraction process (Zhou N, et al. Pharmacological Functions, Synthesis, and Delivery Progress for Collagen as Biodrug and Biomaterial. Pharmaceutics. 2023 May 9; 15(5): 1443.). Recombinant collagen overcomes these problems. Compared with traditional animal collagen, recombinant collagen has the characteristics of no viral hidden danger, excellent biocompatibility and efficacy, and low immunogenicity, effectively avoiding the viral hidden danger and rejection reaction of traditional animal-derived collagen, and overcoming the drawbacks of inaccurate clinical efficacy and unstable quality of terminal products caused by animal age differences and species differences in traditional animal collagen.

[0005] Currently, due to the low cost, short cycle, and easy cultivation of the microbial fermentation system, Escherichia coli and Pichia pastoris are mainly used for the production and expression of recombinant collagen at home and abroad. However, since Escherichia coli and yeast lack prolyl hydroxylase themselves, when expressing collagen alone, they cannot obtain hydroxylated collagen, and cannot effectively form a triple helix structure, thus inhibiting the self-assembly of natural structural collagen molecules into collagen fibers (Ramshaw JA. Biomedical applications of collagens. J Biomed Mater Res B Appl Biomater. 2016 May; 104(4): 665-75.).

[0006] Therefore, to solve the current problems in the production of recombinant collagen, there is an urgent need for a simple and efficient method for preparing recombinant collagen, and to obtain recombinant human-like collagen with better functional characteristics and a conformation closer to the natural conformation. Summary of the Invention

[0007] In view of the above production problems of recombinant collagen, the present invention provides a recombinant humanized collagen and a preparation method thereof.

[0008] In the first aspect, the present invention provides a recombinant humanized collagen, and the sequence of the recombinant humanized collagen includes:

[0009] (1) Core sequence 1, where the core sequence 1 is the sequence shown in SEQ ID NO: 1 (reference sequence) or its variant sequence, and

[0010] (2) A sequence selected from core sequence 2, core sequence 3, core sequence 4, or a combination thereof, where the core sequence 2 is the sequence shown in SEQ ID NO: 2 (reference sequence) or its variant sequence, the core sequence 3 is the sequence shown in SEQ ID NO: 3 (reference sequence) or its variant sequence, and the core sequence 4 is the sequence shown in SEQ ID NO: 4 (reference sequence) or its variant sequence;

[0011] Preferably, the variant sequence of SEQ ID NO: 1 is mutated or deleted by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus or C-terminus based on SEQ ID NO: 1. More preferably, 6 amino acids are deleted at the N-terminus of SEQ ID NO: 1;

[0012] Preferably, the variant sequence of SEQ ID NO: 2 is mutated or deleted by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus or C-terminus based on SEQ ID NO: 2. More preferably, 6 amino acids are deleted at the N-terminus of SEQ ID NO: 2;

[0013] Preferably, the variant sequence of SEQ ID NO: 3 is mutated or deleted by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus or C-terminus based on SEQ ID NO: 3. More preferably, 6 amino acids are deleted at the N-terminus of SEQ ID NO: 3;

[0014] Preferably, the variant sequence of SEQ ID NO: 4 is mutated or deleted by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus or C-terminus based on SEQ ID NO: 4. More preferably, 6 amino acids are deleted at the N-terminus of SEQ ID NO: 4.

[0015] Furthermore, the recombinant humanized collagen optionally contains a signal peptide at the N-terminus, and contains the amino acid sequence shown in SEQ ID NO: 5 and / or a histidine tag at the C-terminus. The signal peptide is preferably the amino acid sequence shown in SEQ ID NO: 13, and the histidine tag is preferably the amino acid sequence shown in SEQ ID NO: 14.

[0016] Even further, the recombinant humanized collagen contains the following sequence combinations:

[0017] (1) Core sequence 1 and core sequence 2; or

[0018] (2) Core sequence 1, core sequence 3 and core sequence 4; or

[0019] (3) Core sequence 1, core sequence 2, core sequence 3 and core sequence 4;

[0020] wherein core sequence 1, core sequence 2, core sequence 3 and core sequence 4 are directly or indirectly connected, and the indirect connection is preferably carried out using a linker.

[0021] The linker is any linker peptide commonly used in the art (such as flexible linker peptide, rigid linker peptide, semi-rigid linker peptide), including but not limited to G n or the amino acid sequence of GSGGGG or (EAAAK)n or GGSGGEAAAK, where n can be an integer greater than 0 and less than or equal to 10, preferably n is 1, 2, 3 or 4. Specifically, the recombinant humanized collagen sequence containing the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2 is SEQ ID NO: 6, or a sequence having 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 6; the recombinant humanized collagen sequence containing the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 2 and the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 is SEQ ID NO: 7, or a sequence having 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 7.

[0022] In one embodiment, the recombinant humanized collagen contains repeating unit 1 composed of core sequence 1 and core sequence 2, or repeating unit 2 composed of core sequence 1, core sequence 3 and core sequence 4, and the number of repetitions of repeating unit 1 or repeating unit 2 is 2-20, preferably 2, 3, 4, 5, 6, 7 or 8.

[0023] Specifically, the recombinant humanized collagen having repeating unit 1 contains 2, 4 or 6 repeating units 1, and the specific sequence is preferably SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence having 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0024] Specifically, the recombinant humanized collagen containing repeat unit 2 contains 3 or 6 repeat units 2, and the specific sequence is preferably SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence having 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 11 or SEQ ID NO: 12.

[0025] In a second aspect, the present invention provides a polynucleotide encoding the above-mentioned recombinant humanized collagen polypeptide.

[0026] In a third aspect, the present invention provides an expression vector containing the above-mentioned polynucleotide.

[0027] In a fourth aspect, the present invention provides a host cell containing the above-mentioned expression vector, and the host cell is selected from eukaryotic cells or prokaryotic cells.

[0028] The eukaryotic cell is preferably a CHO cell, yeast cell, HEK293, BHK-21, C127, MDCK, NAMALWA, VERO, SP2 / 0, COS, and the yeast cell is more preferably Pichia pastoris or Saccharomyces cerevisiae.

[0029] The prokaryotic cell is preferably an Escherichia coli cell, and the Escherichia coli cell is more preferably BL21(DE3), BL21(DE3)pLysS, BL21 Star(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta 2(DE3) or Rosetta2(DE3)pLysS.

[0030] In a fifth aspect, the present invention provides a method for preparing a recombinant humanized collagen:

[0031] (1) Optimize the codons of the amino acid sequence of the recombinant humanized collagen according to the codon preference of the expression system;

[0032] (2) Connect the optimized nucleotide sequences into an expression vector respectively to construct an expression recombinant plasmid vector;

[0033] (3) Transform or transfect the host cell;

[0034] (4) Use the host cell to express the recombinant protein and purify the recombinant protein;

[0035] The host cell is selected from eukaryotic cells or prokaryotic cells. The eukaryotic cells are preferably CHO cells, yeast cells, HEK293, BHK-21, C127, MDCK, NAMALWA, VERO, SP2 / 0, COS. The yeast cells are more preferably Pichia pastoris or Saccharomyces cerevisiae. The prokaryotic cells are preferably Escherichia coli cells, and the Escherichia coli cells are more preferably BL21(DE3), BL21(DE3)pLysS, BL21 Star(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta 2(DE3) or Rosetta2(DE3)pLysS.

[0036] In a sixth aspect, the present invention provides the use of the above-mentioned recombinant humanized collagen polypeptide in the preparation of a product, wherein the product is preferably a biomedical material, a tissue engineering product, a cosmetic, a food, a health product or a drug, a medical device and medical beauty.

[0037] In a seventh aspect, the present invention provides the use of the above-mentioned recombinant humanized collagen polypeptide in the preparation of a product having the function of promoting cell adhesion or serving as a filler.

[0038] As used herein, the term "variant" refers to a peptide or polypeptide whose amino acid sequence has been changed by the insertion, deletion, or substitution of one or more amino acids compared to a reference sequence, but which retains at least one biological activity. Variants described in any embodiment herein include amino acid sequences having at least 90%, preferably at least 92%; more preferably at least 94%; more preferably at least 95%, such as at least 96%, 98%, 99%, or 99.5% sequence identity with a reference sequence (such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 as described herein) and retaining the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI. Mutants also include amino acid sequences having one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of the reference sequence while still retaining the biological activity of the reference sequence. The one or more generally refers to 1 - 10, preferably 9, more preferably 8, more preferably 7, more preferably 6, more preferably 5, more preferably 4, more preferably 3, more preferably 2, or 1. The substitutions are preferably conservative substitutions. For example, in the art, when conservative substitutions are made with amino acids having similar or closely related properties, the function of a protein or polypeptide is generally not changed. "Amino acids having similar or closely related properties" include, for example, families of amino acid residues having similar side chains, which families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β - branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, replacing one or several positions with another amino acid residue from the same side chain class in the polypeptides of the present invention will not substantially affect their activity.

[0039] All reagents used in the present invention can be commercially purchased.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] (1) The recombinant human type III collagen designed in the present invention can be highly expressed in three expression systems: yeast, Escherichia coli, and CHO cells. By tandem repeating or specifically combining the specific functional core sequences selected in the present invention, it can have biological properties superior to those of control molecules and commercially available collagen products when expressed in Escherichia coli and other expression systems.

[0042] (2) By using the CHO cell expression system to express the humanized type III collagen designed in the present invention, the obtained collagen is more in line with the structural characteristics of natural collagen and has a high content of hydroxyproline. It overcomes the problem that Escherichia coli and yeast lack prolyl hydroxylase by themselves, and cannot obtain hydroxylated collagen when expressing collagen alone, and thus cannot effectively form a triple helix structure.

[0043] (2) Compared with commercially available collagen products, the recombinant humanized type III collagen of the present invention has much better cell adhesion activity and proliferation activity than commercially available collagen products, and thus has better biological functional activity.

[0044] It can be seen that the present invention provides a recombinant humanized type III collagen in the technical field with structural characteristics and functional activities similar to natural collagen, solves the problems existing in existing products, and thus the recombinant humanized type III collagen has more excellent practical application value compared with the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings used in the description of the specific embodiments or examples.

[0046] Figure 1 A - C show the electrophoresis detection results of the recombinant humanized type III collagen molecules YM - 3, 4, 5, 6, 7, 8, 9 constructed by the present invention in CHO cells and the control molecule 1;

[0047] Figure 2 Show the electrophoresis detection results of the recombinant humanized type III collagen molecules YM - 12, 16, 17 constructed by the present invention in Escherichia coli and the control molecule 2;

[0048] Figure 3 A - H show the circular dichroism spectrometer detection results of the recombinant humanized type III collagen molecules YM - 3, 4, 5, 6, 7, 8, 9 constructed by the present invention and the control molecule 1;

[0049] Figure 4 A - B show the circular dichroism spectrometer detection results of the recombinant humanized type III collagen molecule YM - 17 constructed by the present invention and the marketed product Weiyimei of Shanxi Jinbo Biotech;

[0050] Figure 5 Show the cell adhesion activity detection results of the recombinant humanized type III collagen molecules YM - 3, 4, 7, 8, 9, the control molecule 1 and the marketed product Weiyimei of Shanxi Jinbo Biotech constructed by the present invention;

[0051] Figure 6A - G show the cell adhesion activity of the recombinant humanized collagen molecules YM - 3, 4, 7, 8, 9, control molecule 1 and the listed product Weiyimei of Shanxi Jinbo Biotech before detecting cell adhesion activity;

[0052] Figure 7 Show the cell adhesion activity test results of the recombinant humanized type III collagen molecules YM - 3, 7, 8, 12, 16, 17, control molecule 1, control molecule 2 and the listed product Weiyimei of Shanxi Jinbo Biotech at different concentrations;

[0053] Figure 8 Show the cell proliferation activity test results of the recombinant humanized type III collagen molecule YM8, the listed product Weiyimei of Shanxi Jinbo Biotech and PBS negative control. Detailed implementation manners

[0054] The principles and features of the present invention will be described below in conjunction with examples. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific implementation manners and not for limiting the protection scope of the present invention. The test methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer. When the examples give a numerical range, it should be understood that unless otherwise stated in the present invention, any value between the two endpoints of each numerical range and either of the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field of the present invention. In addition to the specific methods, equipment, and materials used in the examples, according to the knowledge of those skilled in the technical field of the present invention and the records of the present invention, any method, equipment, and material similar to or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0055] Example 1 Design of recombinant human collagen molecule

[0056] According to the human type III collagen sequence, a protein structure sequence containing the core sequences 1-4 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) and / or SEQ ID NO: 5 is designed, as shown in Table 1. At the N-terminus, the signal peptide sequence shown in SEQ ID NO: 13 or methionine is selected as the N-terminal structural protein according to the expression system, and at the C-terminus, the histidine-tag amino acid sequence shown in SEQ ID NO: 14 for protein detection is included, obtaining the recombinant human type III collagen with molecule numbers YM3-YM9, YM12, YM16, YM17 and the amino acid sequences of control molecule 1 and control molecule 2 proteins designed according to the listed product Vivym of Shanxi Jinbo Biotech Co., Ltd., as shown in Table 1.

[0057] According to the codon preferences of the CHO cell and Escherichia coli expression systems, the amino acid sequences of the above-mentioned YM-numbered molecular proteins are codon-optimized to obtain the corresponding specific nucleotide sequences, as shown in Table 1.

[0058] Table 1 Design of YM-numbered molecular proteins

[0059]

[0060] Example 2: Expression and purification of recombinant human collagen molecules

[0061] 2-1 Protein expression in the CHO cell system

[0062] Specific implementation method:

[0063] (1) GenScript Corporation in Suzhou synthesized the sequences according to the nucleotide sequences of the above-mentioned molecules, and cloned the nucleotide sequences encoding the above-mentioned proteins into the expression vector pcDNA3.4 (GenScript), then transfected into CHO cells for expression to obtain the above-mentioned molecular proteins.

[0064] (2) For the constructed pcDNA3.4 plasmid, according to the standard plasmid construction protocol of Lonza, the target gene was cloned into the SPB-007 (Lonza) expression vector, and the nucleotide sequence was verified by sequencing.

[0065] (3) Construct a stable cell pool according to the standard manual of Lonza. After electroporation, place it in CD CHO Medium (Gibco, catalog number: 12490003). After 24 hours, change to the pressurized culture medium, and culture it in a shaking incubator at 37 °C, 8% CO2, 80% humidity, and 220 rpm; when the cell viability reaches more than 80% and the cell density reaches 1*10 6 / mL or more, expand the culture and transfer it to a 125 mL shake flask for culture.

[0066] (4) Transfer the recovered cells to a 1L shake flask for culture, with an initial density of 5*10 5 / mL, a volume of 200 mL, and the medium being Dynamis Medium (Gibco, catalog number: 2439302). Incubate in a shaker at 37°C, 8% CO2, 80% humidity, and 120 rpm; on the 5th day, transfer the cells to a shaker at 33°C, 8% CO2, 80% humidity, and 120 rpm; starting from the 3rd day, add Cell Boost 7a (Cytiva, catalog number: SH31026.01) and Cell Boost 7B (Cytiva, catalog number: SH31027.02CN) daily; starting from the 7th day, add glucose (Sigma, catalog number: G5388) daily; harvest the supernatant on the 12th day for protein purification.

[0067] Purification of the protein expressed by the 2-2 CHO cell system

[0068] Purify the recombinant protein components obtained from 2-1 expression through nickel column affinity chromatography and molecular sieve chromatography to obtain high-purity protein.

[0069] Specific implementation method:

[0070] (1) Sample treatment: Centrifuge the CHO cell culture solution expressing the recombinant protein molecule at 12000g for 30 min at high speed to harvest the cell supernatant; then use a Cobetter capsule filter 0.45 + 0.2μm filter (purchased from Cobetter, model: Bricap C01:180cm 2 ) and a membrane package (purchased from Millipore, model: 10 kDa) for clarification filtration.

[0071] (2) Nickel ion affinity chromatography: Use a Histrap excel-5ml (purchased from Cytiva) nickel column for purification. First, equilibrate the nickel column with the equilibration buffer TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4); load the sample; after loading, wash the nickel column with the TBS buffer; then wash away the impurity proteins with 20 mM imidazole + TBS buffer; finally, elute the target protein with 500 mM imidazole + TBS buffer; after elution, detect the purity of the target protein by SDS-PAGE.

[0072] (3) Molecular sieve purification: Collect the target protein purified by the nickel column, concentrate it to 1 ml with a concentrator tube, and then perform separation and purification on a molecular sieve (purchased from Cytiva, model: Superdex 200pg 10 / 300GL). The elution buffer is TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). Collect the eluted protein components, and after elution, detect the protein purity by SDS-PAGE.

[0073] Figure 1 A - C show the expression and purification results of the purified recombinant humanized type III collagen molecule and control molecule 1. Among them, Figure 1 A is the SDS - PAGE detection result of YM - 5 and 6, Figure 1 B is the SDS - PAGE detection result of YM - 3, 4, 7, 8, and 9, Figure 1 C is the SDS - PAGE detection result of control molecule 1.

[0074] 2 - 3 Expression of proteins in E. coli system

[0075] Specific implementation method:

[0076] (1) According to the codon preference of the E. coli expression system, the amino acid sequences of the above - mentioned molecules were codon - optimized, synthesized by GenScript Corporation in Suzhou, and GenScript Corporation in Suzhou was entrusted to clone the nucleotide sequences encoding the above - mentioned proteins into the expression vector pET21a (GenScript).

[0077] (2) Induction expression conditions: Inoculate the BL21(DE3) positive monoclonal strain into 800 mL LB (Amp+) medium, culture at 37°C and 220 rpm for 4 - 5 hours. When the OD600 of the culture medium is about 0.6 - 0.8, transfer the bacterial solution to 18°C, add IPTG to a final concentration of 0.5 mM, and induce protein expression at 200 rpm for 16 hours.

[0078] (3) Centrifuge to collect bacteria: Centrifuge the cultured bacterial solution at 7000g at room temperature to collect the bacterial cells, discard the culture medium, and resuspend the bacterial cells with 80 mL of 150 mM NaCl, 20 mM Tris 7.4 solution.

[0079] (4) Ultrasonic disruption: Place the resuspended bacterial solution in an ice - water bath for ultrasonic disruption. At 50% power, ultrasonic for 3 seconds, stop for 7 seconds, and the total ultrasonic time is 12 minutes.

[0080] (5) Centrifuge to collect the target protein: Centrifuge at 13000g at 4°C for 30 minutes, collect the supernatant of the cell lysate for protein purification.

[0081] 2 - 4 Purification of proteins expressed in E. coli system

[0082] The recombinant protein components obtained in 2 - 3 were purified by nickel - column affinity chromatography and molecular sieve chromatography to obtain high - purity proteins.

[0083] Specific implementation method:

[0084] (1) Purification of the target protein by Histrap: The washing solution is 20 mM Tris-HCl, 150 mM NaCl, pH 7.4; the elution solution is 20 mM Tris-HCl, 150 mM NaCl, 500 mM Imidazole, pH 7.4; use a Histrap excel-5ml nickel column for purification; after equilibrating the Histrap excel-5ml with the washing solution for 10 CV, load the sample; after loading, wash the chromatography column with the washing solution for 10 CV; wash with 2% elution solution for 10 CV to remove impurities; linearly elute the target protein with 2%-100% elution solution for 15 CV; after elution, detect the protein purity by SDS-PAGE.

[0085] (2) Purification of the target protein by cation exchange column: The washing solution is 50 mM sodium phosphate, pH 6.0; the elution solution is 50 mM sodium phosphate, 1 M NaCl, pH 6.0. Use HiTrap SP FF for purification; after equilibrating with the washing solution for 10 CV, dilute the target protein purified by the nickel column with the washing solution and then load the sample; after loading, wash with the washing solution for 10 CV; linearly elute the target protein with 0%-100% elution solution for 15 CV; after elution, detect the protein purity by SDS-PAGE.

[0086] Figure 2 The expression and purification results of the purified recombinant humanized type III collagen molecules YM-12, 16, 17 and the control molecule 2 are shown.

[0087] Example 3 Circular Dichroism Detection of Recombinant Humanized Type III Collagen Molecules

[0088] Circular dichroism (CD) spectroscopy is a common method for characterizing the secondary and tertiary structures of biological macromolecules such as proteins. The CD spectrum of collagen has a negative peak at a wavelength near 195 nm and a positive peak at a wavelength near 221 nm, indicating that collagen has a triple helix structure.

[0089] The recombinant humanized type III collagen molecules prepared in the present invention were identified by circular dichroism spectroscopy (Applied Photophysics, model: Chirascan V100).

[0090] As Figure 3 As shown in A-H, the recombinant humanized collagen molecules YM-3, 4, 7, 8, 9 prepared in the present invention and the control molecule 1 have typical negative peaks near 195 nm and typical positive peaks near 221 nm, which conform to the characteristic triple helix structure of natural collagen.

[0091] As Figure 4As shown in A-B, the recombinant humanized type III collagen molecule YM-17 expressed by Escherichia coli prepared in the present invention and the marketed product of Shanxi Jinbo Biotech Co., Ltd., Weiyimei, show typical negative peaks near 195 nm, but the positive peak absorption value near 221 nm is weak.

[0092] Example 4: Detection of cell adhesion activity of recombinant humanized type III collagen molecule

[0093] The concentrations of the recombinant humanized collagen molecules YM-3, 4, 7, 8, 9, 12, 16, 17, control molecule 1, control molecule 2 provided by the present invention and the marketed product of Shanxi Jinbo Biotech Co., Ltd. (recombinant type III humanized collagen freeze-dried fiber - Weiyimei, production batch number: 07230502) were detected by ultraviolet absorption method. The ultraviolet light absorption of the samples was measured at 215 nm and 225 nm respectively, and the protein concentration was calculated using the formula C (mg / mL) = 0.144 × (A215 - A225), where A215 < 1.5. The concentrations of the proteins to be tested were adjusted to 0.5 mg / mL, 0.25 mg / mL, 0.1 mg / mL, 0.01 mg / mL, 0.001 mg / mL, and 0.0001 mg / mL.

[0094] Specific implementation method:

[0095] (1) NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNM6, and the culture and subculture methods were carried out according to the cell instruction manual) were cultured normally.

[0096] (2) 100 μL of various protein solutions and blank PBS solution as a control were added to the 96-well plate and placed overnight at 4°C.

[0097] (3) Block with inactivated 1% BSA at 37°C for 1 hour, wash twice with PBS, add 5 x 104 well-grown NIH / 3T3 cells to each well, and incubate at 37°C for 3 hours. Observe the cell adhesion status and take pictures with an inverted microscope (Leica, Germany, model: DMi8).

[0098] (4) Wash each well 4 times with PBS.

[0099] (5) Detect the absorbance value at OD490nm using an LDH detection kit (Beyotime, C0017). Cell relative adhesion rate = (experimental group absorbance value at 490nm - negative control group absorbance value at 490nm) / (whole cell group absorbance value at 490nm - negative control group absorbance value at 490nm) × 100%.

[0100] Cell adhesion activity of recombinant humanized collagen molecule in CHO expression system

[0101] The results are as Figure 5 、Figure 6 As shown, at protein concentrations of 0.1 mg / mL and 0.01 mg / mL, the cell adhesion activities of the recombinant humanized collagen YM-3, 4, 7, 8, 9 prepared by mammalian cell expression of the present invention and the control molecule 1 are superior to the marketed product Wei Yimei of Shanxi Jinbo Biological. In addition, the cell adhesion activities of the recombinant humanized collagen YM-3, 4, 7, 8, 9 prepared by the present invention are significantly higher than those of the control molecule 1.

[0102] like Figure 6 As shown, it is the cell adhesion state at a protein concentration of 0.01 mg / mL. Compared with the control molecule 1 and the marketed product Wei Yimei of Shanxi Jinbo Biological, the recombinant humanized collagen YM-3, 4, 7, 8, 9 prepared by the present invention can make the cells adhere to the wall very well and fully stretch into the fibroblast morphology, thereby showing significantly better cell adhesion activity, and YM-3 and 4 also show cell adhesion activity comparable to that of the control molecule 1.

[0103] 4-2 Effects of the concentration of recombinant humanized collagen molecules and expression of recombinant proteins through different expression systems on cell adhesion activity

[0104] The results are as follows Figure 7 As shown, the cell adhesion activity of the recombinant humanized type III collagen prepared by the present invention exhibits a concentration-dependent relationship, and as the concentration of the recombinant humanized type III collagen decreases, its cell adhesion activity also decreases accordingly.

[0105] The cell adhesion activity of YM-3, 7, and 8 is better than that of YM-12, 16, and 17, which means that the cell adhesion activity of humanized type III collagen expressed by CHO is better than that of the molecule expressed by E. coli. However, the cell adhesion activity of humanized type III collagen expressed by CHO or E. coli is better than that of control molecule 1, control molecule 2, and the marketed product Wei Yi Mei.

[0106] Example 5: Detection of cell proliferation activity of recombinant humanized type III collagen molecule

[0107] The protein concentration detection method in Example 4 was used to detect the protein concentrations of control molecule 1, control molecule 2, the recombinant type III humanized collagen molecules YM-7, 8, 16, 17 provided by the present invention, and the purchased Shanxi Jinbo Biological marketed product Wei Yimei, and the concentrations of all proteins to be tested were adjusted to 0.1 mg / mL.

[0108] Specific implementation method:

[0109] (1) Normally culture NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNM6, culture and passaging methods refer to the cell instructions).

[0110] (2) Add 100 μL of various protein solutions and a blank PBS solution control to a 96-well plate, and place it at 4 °C overnight.

[0111] (3) Block with inactivated 1% BSA at 37 °C for 1 hour, wash twice with PBS, add 200 μL of 0.5 × 10 4 well-grown NIH / 3T3 cells and culture medium to each well as a blank control, and culture at 37 °C for 48 hours.

[0112] (4) Add 20 μL of CCK-8 solution (Beyotime, C0038) to each well, and measure the absorbance at OD450nm after incubation at 37 °C for 1 hour. Cell relative proliferation = (experimental group 450nm absorbance - blank control group 450nm absorbance) / (PBS group 450nm absorbance - blank control group 450nm absorbance) × 100%.

[0113] The results are as Figure 8 shown. The cell proliferation activities of the recombinant humanized type III collagen YM-7, 8, 16, and 17 prepared by mammalian cell expression in the present invention are all superior to the marketed product Weiyimei of Shanxi Jinbo Biotech Co., Ltd., and the cell proliferation activities of YM-7 and 8 are superior to those of YM-16 and 17. It is worth noting that the recombinant humanized type III collagen YM-8 prepared in the present invention shows excellent cell proliferation activity, almost twice that of the marketed product Weiyimei.

[0114] Example 6: Detection of hydroxyproline content in recombinant humanized type III collagen molecules

[0115] Use a hydroxyproline (HYP) content detection reagent (Solarbio, BC0255) to detect the hydroxyproline content of the recombinant type III humanized collagen molecules YM-3, 7, and 8 expressed by CHO cells provided in the present invention and the marketed product Weiyimei of Shanxi Jinbo Biotech Co., Ltd.

[0116] Specific implementation method:

[0117] (1) Dilute the hydroxyproline standard at 0.5 mg / ml with ultrapure water to prepare standard curve solutions containing 30 μg / ml, 20 μg / ml, 15 μg / ml, 10 μg / ml, and 2 μg / ml of hydroxyproline.

[0118] (2) Take samples of about 1 mg of total protein of YM-3, 7, 8 and Weiyimei and place them in a 10-ml glass digestion tube. Add concentrated hydrochloric acid with the same volume as the sample. After screwing on the lid, place it in an oven at 110 °C for high-temperature digestion for 6 h. Then take it out and let it cool to room temperature. Add 2 ml of ultrapure water. Adjust the pH to 6-8 with 5 M sodium hydroxide under a pH meter. Transfer the solution to a 5-ml volumetric flask, rinse the digestion tube 2 times with 500 μl of water, and transfer the rinsed water to the 5-ml volumetric flask as well. Finally, make up the volume to the scale with water.

[0119] (3) Pipette 60 μl of the blank solution, linear solutions of each concentration of hydroxyproline standard, and test solutions of YM-3, 7, 8 and Weiyimei into different 2-ml EP tubes respectively. Add 60 μl of chloramine T solution to each tube, mix well and let stand for 20 minutes. Then add 60 μl of p-dimethylbenzaldehyde solution and 120 μl of water. After mixing, heat at 60 °C for 15 minutes. Take it out and cool for 15 minutes. Take 200 μl and place it in a 96-well plate, and measure its absorbance at 560 nm with an ultraviolet spectrophotometer. Calculate the concentration of hydroxyproline according to the standard curve.

[0120] The results are shown in Table 2. The marketed product Weiyimei of Shanxi Jinbo Biotech does not contain hydroxyproline, while the recombinant humanized type III collagen YM-3, 7, 8 prepared by expressing mammalian cells CHO cells contains hydroxyproline. Therefore, the recombinant humanized type III collagen prepared by the present invention has structural characteristics and functional activities similar to those of natural collagen, and thus has better applicability of natural collagen.

[0121] Table 2 shows the detection results of the hydroxyproline content of the recombinant humanized type III collagen molecules YM-3, 7, 8 constructed by the present invention and the marketed product Weiyimei of Shanxi Jinbo Biotech

[0122] Sample Name Determined HYP Concentration (μg / ml) HYP Content (%) YM-3 5.18 2.61 YM-7 5.17 2.55 YM-8 5.15 2.55 Shanxi Jinbo Biotechnology - Weiyimei 0 0

[0123] In summary, the above-mentioned embodiments and the accompanying drawings are only the preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant humanized collagen, characterized in that, The recombinant humanized collagen sequence comprises: (1) Core sequence 1, where the core sequence 1 is the reference sequence shown as GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID NO: 1) or its variant sequence; and (2) A sequence selected from core sequence 2, 3, 4 or a combination thereof, where the core sequence 2 is the reference sequence shown as GPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPK (SEQ ID NO: 2) or its variant sequence, the core sequence 3 is the reference sequence shown as GPPGIKGPAGIPGFPGMKGHRGFDGRNGEK (SEQ ID NO: 3) or its variant sequence, and the core sequence 4 is the reference sequence shown as GAPGPQGPRGDKGETGERGAAGIKGHRGFP (SEQ ID NO: 4) or its variant sequence; The variant sequence is a sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations or deletions at the N-terminus or C-terminus of the corresponding reference sequence.

2. The recombinant humanized collagen according to claim 1, wherein The C-terminus further contains the amino acid sequence shown as GAPGPCCGG (SEQ ID NO: 5).

3. The recombinant humanized collagen according to claim 1 or 2, characterized in that, The recombinant humanized collagen comprises the following sequences: (1) Core sequence 1 and core sequence 2; (2) Core sequence 1, core sequence 3, and core sequence 4; or (3) Core sequence 1, core sequence 2, core sequence 3, and core sequence 4.

4. The recombinant humanized collagen according to claim 3, wherein The recombinant humanized collagen comprises multiple repeating units 1 or repeating units 2. The repeating unit 1 is a sequence group composed of core sequence 1 and core sequence 2, and the repeating unit 2 is a sequence group composed of core sequence 1, core sequence 3, and core sequence 4. Preferably, the recombinant humanized collagen comprises 2 - 20 repeating units 1 or repeating units 2, and multiple repeating units are connected in series directly or indirectly. The indirect series connection is preferably through a linker.

5. The recombinant humanized collagen according to claim 3 or 4, characterized in that, The recombinant humanized collagen sequence is selected from: (1) The sequence shown as SEQ ID NO: 6 or 7, or a sequence having 95%, 96%, 97%, 98%, 99%, or 100% identity with the said sequence; (2) The recombinant humanized collagen sequences containing 2, 4, or 6 of the repeating units 1 are the sequences shown as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or a sequence having 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; (3) The recombinant humanized collagen sequences containing 3 or 6 of the repeating units 2 are the sequences shown as SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence having 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 or SEQ ID NO:

12.

6. A polynucleotide encoding the recombinant humanized collagen polypeptide according to any one of claims 1 to 5.

7. An expression vector comprising the polynucleotide according to claim 6.

8. A host cell comprising the expression vector according to claim 7, wherein the host cell is selected from eukaryotic cells or prokaryotic cells, the eukaryotic cells are preferably CHO cells, yeast cells, HEK293, BHK-21, C127, MDCK, NAMALWA, VERO, SP2 / 0, COS, the yeast cells are more preferably Pichia pastoris and Saccharomyces cerevisiae, the prokaryotic cells are preferably Escherichia coli cells, and the Escherichia coli cells are more preferably BL21(DE3), BL21(DE3)pLysS, BL21 Star(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta 2(DE3) and Rosetta 2(DE3)pLysS.

9. The preparation method of the recombinant humanized collagen polypeptide according to any one of claims 1 to 5, characterized in that, Comprising: (1) Codon-optimizing the amino acid sequence of the recombinant humanized collagen according to the codon preference of the expression system; (2) Separately ligating the optimized nucleotide sequence into an expression vector to construct an expression recombinant plasmid vector; (3) Transforming or transfecting a host cell; (4) Using the host cell to express a recombinant protein and purifying the recombinant protein; The host cell is selected from eukaryotic cells or prokaryotic cells, the eukaryotic cells are preferably CHO cells, yeast cells, HEK293, BHK-21, C127, MDCK, NAMALWA, VERO, SP2 / 0, COS, the yeast cells are more preferably Pichia pastoris and Saccharomyces cerevisiae, the prokaryotic cells are preferably Escherichia coli cells, and the Escherichia coli cells are more preferably BL21(DE3), BL21(DE3)pLysS, BL21 Star(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta 2(DE3) and Rosetta 2(DE3)pLysS.

10. Use of the recombinant humanized collagen according to any one of claims 1-5 in the preparation of a product, wherein the product is preferably a biomedical material, a tissue engineering product, a cosmetic, a food, a health product or a drug, a medical device and a medical beauty product, and more preferably, the product has the functions of medical beauty, cosmetics, medical devices and biomedical materials.

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