A recombinant human type III collagen and its preparation method

Abstract

This invention relates to the field of genetic engineering technology, and in particular to a recombinant humanized collagen and its preparation method. The recombinant humanized type III collagen of this invention can be expressed efficiently and stably in three expression systems: yeast, *E. coli*, and CHO cells, and exhibits superior biological activity compared to commercially available collagen products. The recombinant humanized type III collagen of this invention possesses structural characteristics and functional activities similar to natural collagen, and therefore has broad application prospects in biomedical materials, tissue engineering products, cosmetics, food, health products or pharmaceuticals, medical devices, and medical aesthetics.

Description

Recombinant human type III collagen and preparation method thereof Technical Field

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

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

[0003] Collagen can be divided into several types, including type I, type II, type III, and type IV. Type I and type III collagen are primarily found in connective tissues such as the skin, tendons, ligaments, and joints, and are the two most abundant types of collagen in the skin. Type I collagen is the mainstay of the skin, accounting for 80-85% of the total collagen content. It provides strong structural support and strength, and loss of type I collagen can lead to facial wrinkles and depressions. Type III collagen, which is a loose, mesh-like structure and relatively fine, accounts for 10-15% of the total collagen content in the skin. It provides elasticity and stress resistance to the skin, and promotes elasticity repair.

[0004] At present, the collagen used in commercial applications mainly includes type I and type III. Type I is mainly used in the field of facial masks, while type III is used in the field of medical beauty fillers and medical devices. The raw materials of collagen products on the market are mainly extracted from the connective tissues of animals such as pigs, cattle, and fish through acid, alkali, and enzyme methods. However, collagen extracted from animals has certain risks of viral infection, human rejection, and the destruction of collagen structure during the extraction process, resulting in reduced efficacy and poor stability (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 risks, excellent biological compatibility and efficacy, and low immunogenicity. It effectively avoids the viral risks and rejection reactions of traditional animal-derived collagen, and overcomes the disadvantages of traditional animal collagen, such as uncertain clinical efficacy and unstable quality of the end product due to differences in animal age and species.

[0005] Currently, due to the low cost, short cycle, and easy cultivation of microbial fermentation systems, recombinant collagen is mainly produced and expressed in Escherichia coli and Pichia pastoris. However, E. coli and yeast lack proline hydroxylase, so when expressing collagen alone, hydroxylated collagen cannot be obtained, and the triple helical structure cannot be effectively formed, which in turn inhibits 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, in order to solve the current difficulties in recombinant collagen production, there is an urgent need for a simple and efficient method for preparing recombinant collagen and obtaining recombinant human-like collagen with better functional properties and a conformation closer to nature. Summary of the Invention

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

[0008] In a first aspect, the present invention provides a recombinant humanized collagen protein, wherein the recombinant humanized collagen protein sequence comprises:

[0009] (1) core sequence 1, wherein the core sequence 1 is the sequence shown in SEQ ID NO: 1 (reference sequence) or a variant sequence thereof, and

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

[0011] Preferably, the variant sequence of SEQ ID NO: 1 is a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are mutated or deleted at the N-terminus or C-terminus of 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 a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are mutated or deleted at the N-terminus or C-terminus of 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 a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are mutated or deleted at the N-terminus or C-terminus of 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 a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are mutated or deleted 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 comprises a signal peptide at the N-terminus and an amino acid sequence shown in SEQ ID NO: 5 and / or a histidine tag at the C-terminus, wherein 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] Furthermore, the recombinant humanized collagen comprises the following sequence combination:

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

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

[0022] In one embodiment, the recombinant humanized collagen comprises a repeating unit 1 consisting of core sequence 1 and core sequence 2, or a repeating unit 2 consisting of core sequence 1, core sequence 3 and core sequence 4, and the number of repetitions of the repeating unit 1 or the 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 that is 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0024] Specifically, the recombinant humanized collagen with repeating unit 2 contains 3 or 6 repeating units 2, and the specific sequence is preferably SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to 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 comprising the above-mentioned polynucleotide.

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

[0028] The eukaryotic cells are preferably CHO cells, yeast cells, HEK293, BHK-21, C127, MDCK, NAMALWA, VERO, SP2 / 0, COS, and the yeast cells are 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, Rosetta2(DE3) or Rosetta 2(DE3)pLysS.

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

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

[0032] (2) connecting the optimized nucleotide sequences into expression vectors to construct expression recombinant plasmid vectors;

[0033] (3) transforming or transfecting host cells;

[0034] (4) using the host cell to express the recombinant protein and purifying 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, Rosetta2 (DE3) or Rosetta 2 (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 products, wherein the products are preferably biomedical materials, tissue engineering products, cosmetics, foods, health products or medicines, medical devices and medical cosmetology.

[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 is altered by insertion, deletion or substitution of one or more amino acids compared to a reference sequence, but which retains at least one biological activity. The variants described in any embodiment herein include amino acid sequences that have 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 described herein) and retain the biological activity of the reference sequence. The sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Mutants also include amino acid sequences that have 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 term "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 substitution is preferably a conservative substitution. For example, in the art, conservative substitution with amino acids having similar or similar properties generally does not alter the function of the protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in a polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

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

[0040] Compared with the prior art, the present invention has the following beneficial effects:

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

[0042] (2) The present invention uses a CHO cell expression system to express the humanized type III collagen designed in the present invention, so that the obtained collagen more closely matches the structural characteristics of natural collagen and has a higher content of hydroxyproline. This overcomes the problem that E. coli and yeast, due to their own lack of proline hydroxylase, cannot produce hydroxylated collagen and cannot effectively form a triple helical structure when expressing collagen alone.

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

[0044] It can be seen that the present invention provides a recombinant humanized type III collagen with structural characteristics and functional activities similar to natural collagen in the field of this technology, solving the problems of existing products. Therefore, the recombinant humanized type III collagen has better practical application value than the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0047] FIG2 shows the electrophoresis detection results of the recombinant humanized type III collagen molecules YM-12, 16, and 17 expressed in E. coli constructed by the present invention and the control molecule 2;

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

[0049] 4A-B show the dichroic spectrometer test results of the recombinant humanized type III collagen molecule YM-17 constructed by the present invention and the marketed product Wei Yi Mei Yuan of Shanxi Jinbo Biological;

[0050] FIG5 shows the cell adhesion activity test results of the recombinant humanized type III collagen molecules YM-3, 4, 7, 8, 9 constructed by the present invention, the control molecule 1, and Wei Yimei, a marketed product of Shanxi Jinbo Biological;

[0051] Figures 6A-G show images of the cell adhesion state detection before detecting cell adhesion activity of the recombinant humanized collagen molecules YM-3, 4, 7, 8, 9 constructed by the present invention, the control molecule 1, and the marketed product Wei Yimei of Shanxi Jinbo Biological;

[0052] FIG7 shows the results of cell adhesion activity tests of the recombinant humanized type III collagen molecules YM-3, 7, 8, 12, 16, 17, control molecule 1, control molecule 2, and Shanxi Jinbo Biological's marketed product Wei Yimei at different concentrations;

[0053] FIG8 shows the cell proliferation activity test results of the recombinant humanized type III collagen molecule YM8 constructed by the present invention, Weiyimei, a marketed product of Shanxi Jinbo Biological, and a PBS negative control. DETAILED DESCRIPTION

[0054] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for the purpose of describing specific specific embodiments and are not intended to limit the scope of protection of the present invention. The test methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the respective manufacturers. When the examples give a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, according to the prior art knowledge of those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the examples of the present invention can also be used to implement the present invention.

[0055] Example 1 Recombinant human collagen molecule design

[0056] Based on the human type III collagen sequence, a protein structure sequence comprising 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 was designed, as shown in Table 1, and a signal peptide sequence represented by SEQ ID NO: 13 or methionine was selected as the N-terminal structural protein at the N-terminus according to the expression system, and a histidine tag amino acid sequence represented by SEQ ID NO: 14 for protein detection was included at the C-terminus, resulting in recombinant human type III collagen proteins numbered YM3-YM9, YM 12, YM 16, and YM 17, as well as amino acid sequences of control molecules 1 and 2 designed based on Wei Yimei, a marketed product of Shanxi Jinbo Biological, as shown in Table 1.

[0057] According to the codon preference of CHO cells and E. coli expression systems, the amino acid sequence of the above-mentioned YM numbered molecular protein was codon optimized to obtain the corresponding specific nucleotide sequence, as shown in Table 1.

[0058] Table 1 YM numbered molecular protein design

[0059] Example 2: Expression and purification of recombinant human collagen molecules

[0060] 2-1 Protein expression in CHO cell system

[0061] Specific implementation methods:

[0062] (1) Suzhou GenScript Co., Ltd. designed and synthesized the nucleotide sequence of the above-mentioned molecule, cloned the nucleotide sequence encoding the above-mentioned protein into the expression vector pcDNA3.4 (GenScript), and then transfected it into CHO cells for expression to obtain the above-mentioned molecular protein.

[0063] (2) The constructed pcDNA3.4 plasmid was cloned into the SPB-007 (Lonza) expression vector according to the Lonza standard plasmid construction protocol, and the nucleotide sequence was verified by sequencing.

[0064] (3) Stably transfected cell pools were constructed according to the Lonza standard manual. After electroporation, cells were placed in CD CHO Medium (Gibco, Cat. No. 12490003). After 24 hours, the cells were replaced with pressurized culture medium and cultured in a shaker at 37°C, 8% CO2, 80% humidity, and 220 rpm. The cells were cultured until the cell viability reached above 80% and the cell density reached 1 × 10 6 / mL or above to expand the culture and transfer to 125mL shake flask culture.

[0065] (4) The recovered cells were transferred to a 1 L shake flask for culture at an initial density of 5*10 5 / mL, volume 200mL, culture medium is Dynamis Medium (Gibco, catalog number: 2439302), placed in 37 ° C, 8% CO2, 80% humidity, 120 rpm shaker culture; on the 5th day, the cells were transferred to 33 ° C, 8% CO2, 80% humidity, 120 rpm shaker culture; starting from the 3rd day, Cell Boost 7a (Cytiva, catalog number: SH31026.01) and Cell Boost 7B (Cytiva, catalog number: SH31027.02CN) were added daily, starting from the 7th day, glucose (Sigma, catalog number: G5388) was added daily; on the 12th day, the supernatant was harvested for protein purification.

[0066] Purification of proteins expressed in 2-2CHO cell system

[0067] The recombinant protein component obtained by expression of 2-1 was purified by nickel column affinity chromatography and molecular sieve chromatography to obtain high-purity protein.

[0068] Specific implementation methods:

[0069] (1) Sample processing: The CHO cell culture medium expressing the recombinant protein molecule was centrifuged at 12000g for 30 min, and the cell supernatant was harvested; the supernatant was then filtered using a Cobetter capsule filter 0.45+0.2μm (purchased from Cobetter, model: Bricap C01: 180cm 2 ) and membrane package (purchased from Millipore, model: 10kDa) for clarification and filtration.

[0070] (2) Nickel ion affinity chromatography: Purification was performed using a Histrap excel-5ml (purchased from Cytiva) nickel column. The nickel column was first equilibrated with equilibration buffer TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). Samples were loaded. After loading, the nickel column was rinsed with TBS buffer. Contaminated proteins were then washed with 20 mM imidazole + TBS buffer. Finally, the target protein was eluted with 500 mM imidazole + TBS buffer. The purity of the target protein was detected by SDS-PAGE after elution.

[0071] (3) Molecular sieve purification: The target protein after nickel column purification was collected and concentrated to 1 ml using a concentrator tube, and then separated and purified using molecular sieves (purchased from Cytiva, model: Superdex 200pg 10 / 300GL). The elution buffer was TBS (20mM Tris-HCl, 150mM NaCl, pH7.4). The eluted protein fractions were collected and the protein purity was detected by SDS-PAGE after elution.

[0072] Figures 1A-C show the expression and purification results of the purified recombinant humanized type III collagen molecules and control molecule 1. Figure 1A shows the SDS-PAGE detection results of YM-5 and 6, Figure 1B shows the SDS-PAGE detection results of YM-3, 4, 7, 8, and 9, and Figure 1C shows the SDS-PAGE detection results of control molecule 1.

[0073] 2-3 Protein expression in E. coli system

[0074] Specific implementation methods:

[0075] (1) The amino acid sequence of the above-mentioned molecule was codon-optimized according to the codon preference of the E. coli expression system, and the sequence was synthesized by Suzhou GenScript Co., Ltd., which was then commissioned to clone the nucleotide sequence encoding the above-mentioned protein into the expression vector pET21a (GenScript).

[0076] (2) Induction expression conditions: Inoculate BL21 (DE3) positive monoclonal bacteria into 800 mL LB (Amp+) medium and culture at 37°C and 220 rpm for 4-5 hours. When the OD600 of the culture solution is about 0.6-0.8, transfer the culture solution to 18°C, add IPTG to a final concentration of 0.5 mM, and induce protein expression at 200 rpm for 16 hours.

[0077] (3) Harvesting bacteria by centrifugation: The cultured bacterial solution was centrifuged at 7000 g at room temperature to collect the bacterial cells. The culture medium was discarded and the bacterial cells were resuspended in 80 mL of 150 mM NaCl, 20 mM Tris 7.4 solution.

[0078] (4) Ultrasonic disruption: Place the resuspended bacterial solution in an ice-water bath for ultrasonic disruption. Ultrasonication at 50% power for 3 seconds, then 7 seconds on, for a total of 12 minutes.

[0079] (5) Collect the target protein by centrifugation: 13000g, 4℃, 30 minutes, collect the cell disruption supernatant for protein purification.

[0080] 2-4 Purification of proteins expressed in E. coli system

[0081] The recombinant protein component obtained by expression in 2-3 was purified by nickel column affinity chromatography and molecular sieve chromatography to obtain high-purity protein.

[0082] Specific implementation methods:

[0083] (1) Purify the target protein using Hi strap: the washing solution is 20mM Tris-HCl, 150mM NaCl, pH 7.4; the elution solution is 20mM Tris-HCl, 150mM NaCl, 500mM Imidazole, pH 7.4; use Hi strap excel-5ml nickel column for purification; after equilibrating the Hi strap excel-5ml with the washing solution for 10CV, load the sample; after loading the sample, rinse the column with the washing solution for 10CV; rinse with 2% elution solution for 10CV to wash away impurities; linearly elute the target protein with 2%-100% elution solution for 15CV; after elution, detect the protein purity by SDS-PAGE.

[0084] (2) Purification of the target protein using a cation exchange column: the wash solution is 50 mM sodium phosphate, pH 6.0; the elution solution is 50 mM sodium phosphate, 1 M NaCl, pH 6.0. Purification is performed using HiTrap SP FF. After 10 CV of equilibration with the wash solution, the target protein purified on the nickel column is diluted with the wash solution and loaded onto the sample. After loading, the protein is washed with the wash solution for 10 CV. The target protein is linearly eluted with 0%-100% elution solution for 15 CV. After elution, the protein purity is determined by SDS-PAGE.

[0085] FIG2 shows the expression and purification results of the purified recombinant humanized type III collagen molecules YM-12, 16, 17 and the control molecule 2.

[0086] Example 3 Circular dichroism detection of recombinant humanized type III collagen molecules

[0087] Circular dichroism (CD) spectrum is a common method used to characterize the secondary and tertiary structures of biological macromolecules such as proteins. The CD spectrum of collagen has a negative peak at a wavelength near 195nm and a positive peak at a wavelength near 221nm, indicating that collagen has a triple helical structure.

[0088] The recombinant humanized type III collagen molecules prepared in the present invention were identified by circular dichroism using a circular dichroism spectrometer (Applied Photophysics, model: Chirascan V100).

[0089] As shown in Figures 3A-H, the recombinant humanized collagen molecules YM-3, 4, 7, 8, 9 and the control molecule 1 prepared by the present invention have typical negative peaks near 195 nm and typical positive peaks near 221 nm, which are consistent with the characteristic triple helical structure of natural collagen.

[0090] As shown in FIG4A-B , the recombinant humanized type III collagen molecule YM-17 expressed by E. coli prepared by the present invention and the marketed product Wei Yimei of Shanxi Jinbo Biological have a typical negative peak near 195 nm, but the positive peak absorption value near 221 nm is relatively weak.

[0091] Example 4: Detection of cell adhesion activity of recombinant humanized type III collagen molecules

[0092] The concentrations of the recombinant humanized collagen molecules YM-3, 4, 7, 8, 9, 12, 16, and 17 provided by the present invention, control molecule 1, control molecule 2, and the purchased Shanxi Jinbo Biological marketed product (recombinant type III humanized collagen freeze-dried fiber - Wei Yimei, production batch number: 07230502) were detected by ultraviolet absorption. The ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated using the formula C (mg / mL) = 0.144 × (A215-A225), 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.

[0093] Specific implementation methods:

[0094] (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).

[0095] (2) Add 100 μL of various protein solutions and a blank PBS solution as a control to a 96-well plate and incubate at 4°C overnight.

[0096] (3) Block the wells with inactivated 1% BSA at 37°C for 1 hour, wash twice with PBS, add 5 × 104 well-cultured NIH / 3T3 cells to each well, and incubate at 37°C for 3 hours. Observe the cell adhesion and take photos using an inverted microscope (Leica, Germany, model: DMi8).

[0097] (4) Wash each well 4 times with PBS.

[0098] (5) Detect the absorbance at OD490nm using the LDH detection kit (Biyuntian, C0017). Relative cell adhesion rate = (absorbance at 490nm of the experimental group - absorbance at 490nm of the negative control group) / absorbance at 490nm of the whole cell group - absorbance at 490nm of the negative control group × 100%.

[0099] Cell adhesion activity of recombinant humanized collagen expressed in the 4-1CHO expression system

[0100] The results, as shown in Figures 5 and 6, demonstrate that at protein concentrations of 0.1 mg / mL and 0.01 mg / mL, the cell adhesion activities of the recombinant humanized collagens YM-3, 4, 7, 8, and 9 expressed in mammalian cells according to the present invention, as well as those of the control molecule 1, were superior to those of Wei Yi Mei, a marketed product from Shanxi Jinbo Biological. Furthermore, the cell adhesion activities of the recombinant humanized collagens YM-3, 4, 7, 8, and 9 produced according to the present invention were significantly higher than those of the control molecule 1.

[0101] As shown in Figure 6, the cell adhesion state at a protein concentration of 0.01 mg / mL is shown. Compared with control molecule 1 and Shanxi Jinbo Biological's marketed product Weiyimei, the recombinant humanized collagen proteins YM-3, 4, 7, 8, and 9 prepared by the present invention can enable cells to adhere well and fully expand into fibroblast morphology, thereby exhibiting significantly better cell adhesion activity. YM-3 and 4 also showed cell adhesion activity comparable to control molecule 1.

[0102] 4-2 Effects of Recombinant Humanized Collagen Molecular Concentration and Expression of Recombinant Proteins in Different Expression Systems on Cell Adhesion Activity

[0103] The results are shown in FIG7 . The cell adhesion activity of the recombinant humanized type III collagen prepared by the present invention showed a concentration-dependent relationship. As the concentration of the recombinant humanized type III collagen decreased, the cell adhesion activity also decreased.

[0104] The cell adhesion activity of YM-3, 7, and 8 was superior to that of YM-12, 16, and 17, indicating that the cell adhesion activity of humanized type III collagen expressed in CHO cells was superior to that of the molecule expressed in E. coli. However, the cell adhesion activity of humanized type III collagen expressed in both CHO cells and E. coli cells was superior to that of control molecules 1 and 2, as well as the marketed product Weiyimei.

[0105] Example 5: Detection of cell proliferation activity of recombinant humanized type III collagen molecules

[0106] 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, and 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.

[0107] Specific implementation methods:

[0108] (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).

[0109] (2) Add 100 μL of various protein solutions and a blank PBS solution as a control to a 96-well plate and incubate at 4°C overnight.

[0110] (3) Block with inactivated 1% BSA at 37°C for 1 hour, wash twice with PBS, and add 200 μL of 0.5×10 4 NIH / 3T3 cells in good culture status and culture medium were used as blank controls and cultured at 37°C for 48 hours.

[0111] (4) Add 20 μL of CCK-8 solution (Biyuntian, C0038) to each well and incubate at 37°C for 1 hour. Measure the absorbance at OD450 nm. Relative cell proliferation = (absorbance at 450 nm of the experimental group - absorbance at 450 nm of the blank control group) / absorbance at 450 nm of the PBS group - absorbance at 450 nm of the blank control group × 100%.

[0112] The results, as shown in Figure 8, show that the cell proliferation activities of the recombinant humanized type III collagen proteins YM-7, 8, 16, and 17 produced by mammalian cell expression in the present invention were superior to those of Wei Yi Mei, a marketed product from Shanxi Jinbo Biological. The cell proliferation activities of YM-7 and 8 were superior to those of YM-16 and 17. Notably, the recombinant humanized type III collagen protein YM-8 produced by the present invention exhibited excellent cell proliferation activity, nearly twice that of the marketed product Wei Yi Mei.

[0113] Example 6: Detection of Hydroxyproline Content in Recombinant Humanized Type III Collagen Molecules

[0114] The hydroxyproline (HYP) content detection reagent (Solebo, BC0255) was used to detect the hydroxyproline content of the recombinant type III humanized collagen molecules YM-3, 7, and 8 expressed by CHO cells provided by the present invention and the purchased Shanxi Jinbo Biological marketed product Wei Yimei.

[0115] Specific implementation methods:

[0116] (1) Dilute 0.5 mg / ml of hydroxyproline standard 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.

[0117] (2) Take a sample of about 1 mg of YM-3, 7, 8 and Weiyimei protein in a 10 ml glass digestion tube, add concentrated hydrochloric acid of equal volume to the sample, tighten the lid and place it in a 110 ° C oven for high temperature digestion for 6 hours, then remove it and cool it to room temperature, add 2 ml of ultrapure water. Adjust the pH to 6-8 with 5M sodium hydroxide under a pH meter, transfer the solution to a 5 ml volumetric flask, rinse the digestion tube twice with 500 μl of water, and transfer the rinse water to a 5 ml volumetric flask. Finally, dilute to the mark with water.

[0118] (3) Pipette 60 μl of each of the blank solution, linear solutions of hydroxyproline standards at various concentrations, and YM-3, 7, 8, and Weiyimei test solutions into separate 2 ml EP tubes. 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, mix well, and heat at 60°C for 15 minutes. Remove and cool for 15 minutes. Take 200 μl of the solution and place it in a 96-well plate. Measure its absorbance at 560 nm using a UV spectrophotometer and calculate the hydroxyproline concentration based on the standard curve.

[0119] The results, as shown in Table 2, show that Shanxi Jinbo Biological's marketed product, Wei Yimei, does not contain hydroxyproline, while the recombinant humanized type III collagens YM-3, 7, and 8 produced by the present invention through mammalian CHO cell expression do contain hydroxyproline. Therefore, the recombinant humanized type III collagen produced by the present invention possesses structural characteristics and functional activities similar to those of natural collagen, and thus possesses the applicable effects of natural collagen.

[0120] Table 2 shows the results of hydroxyproline content test of recombinant humanized type III collagen molecules YM-3, 7, 8 constructed by the present invention and Weiyimei, a marketed product of Shanxi Jinbo Biological.

[0121] In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant humanized collagen, characterized in that, The recombinant humanized collagen sequence comprises: (1) Core sequence 1, which 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, wherein core sequence 2 is the reference sequence shown as GPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPK (SEQ ID NO: 2) or its variant sequence, core sequence 3 is the reference sequence shown as GPPGIKGPAGIPGFPGMKGHRGFDGRNGEK (SEQ ID NO: 3) or its variant sequence, and 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 acids mutated or deleted at the N-terminus or C-terminus of the corresponding reference sequence.

2. The recombinant humanized collagen according to claim 1, characterized in that, The C-terminus also 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 tandem 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, 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) Optimizing the codons of the recombinant humanized collagen amino acid sequence 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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