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

Abstract

The application discloses a kind of recombinant type I humanized collagen and its preparation method and application, belong to genetic engineering field, by bioinformatics method to the core function fragment of human type I collagen alpha 1 chain as shown in SEQ ID NO.1 is analyzed, design and construct the length of 330 amino acids of recombinant type I humanized collagen rCol1, sequence as shown in SEQ ID NO.3;Recombinant type I humanized collagen rCol1 is hydrophilic protein, with good biocompatibility, and with lower antigenicity, can efficiently promote Schwann cell and neuron neurite regeneration;The recombinant type I humanized collagen rCol1 prepared by the method has high purity, no virus hidden danger, has obvious expression abundance;This protein has important application in biomedical, regenerative medicine, tissue engineering, beauty care and cosmetic preparation and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant type I humanized collagen, its preparation method, and its application. Background Technology

[0002] Collagen, the most abundant protein in mammals, accounts for approximately 30% of the total protein in the human body. It is abundant in animal tissues such as skin, cartilage, and blood vessels, playing a crucial role in growth and development, cell differentiation, and antigen-antibody binding. Currently, 28 types of collagen have been identified in vertebrates. Type I collagen is the most abundant type in vertebrates, interacting with other biomolecules to form basement membranes, ligaments, tendons, skin, and blood vessels, exhibiting high mechanical strength. The most typical characteristic of collagen is its fibrous structure, formed by the helical winding of three peptide chains. It possesses a stable molecular structure, good fiber toughness, and high mechanical strength. The amino acid sequence of the primary structure of the active region of collagen exhibits a periodic pattern of "glycine-XY (where X and Y are any amino acids other than glycine)".

[0003] In recent years, collagen, as a novel medical material, has shown broad application prospects in fields such as cosmetic surgery, burns, trauma, and tissue repair. However, natural collagen has poor water solubility, diverse sources, and is difficult to process deeply. Currently, the main methods for extracting collagen from tissues such as pig skin, cow skin, and fish skin include acid methods, alkaline methods, and various enzymatic methods, but these methods have poor safety and certain toxic side effects on the human body. Furthermore, chemical methods for preparing collagen inevitably result in the loss of its biological activity, and the preparation process is cumbersome, with environmental pollution and high costs associated with the extraction process, significantly limiting its application in medicine and other fields.

[0004] With the rapid development of molecular biology, the preparation of human collagen using insects, mice, and E. coli as carriers through genetic engineering has become a research hotspot. For example, Fan Daidi et al. from Northwest University used E. coli for high-concentration fermentation to produce human collagen, but the protein expression level was less than 30%. This system suffers from biosafety issues such as endotoxins and pyrogens, and the expressed products are mostly inclusion bodies, making isolation and purification difficult, resulting in low product purity and limited clinical application. Currently, research on recombinant human collagen using Pichia pastoris as raw material through fermentation has become a research hotspot both domestically and internationally. Although this fermentation method is pyrogen-free and the product can be secreted extracellularly, it still suffers from many problems such as long fermentation cycles, low yields, and low purity. Therefore, developing a recombinant human collagen raw material with high purity, high safety, high yield, short cycle, strong hydrophilicity, and ease of large-scale production is currently a research focus. Simultaneously, to reduce the human body's rejection of foreign materials, it is necessary to develop a recombinant humanized collagen with lower immunogenicity. Summary of the Invention

[0005] Technical Problem Solved: This invention provides a recombinant type I humanized collagen, its preparation method, and its application. It addresses the core technical problems of existing technologies, such as poor water solubility, strong immunogenicity, poor safety, and certain toxic side effects on the human body from natural collagen; the inevitable loss of biological activity in chemically prepared collagen, cumbersome preparation processes, environmental pollution and high cost during extraction, difficulty in separation and purification, limited clinical application, long fermentation cycles, and low yields. This recombinant humanized collagen has shorter peptide segments, can be directly synthesized or expressed at high throughput, is suitable for large-scale preparation, and is simple to operate with good water solubility.

[0006] To achieve the above objectives, this application provides the following technical solution: A recombinant type I humanized collagen, rCol1 comprising a core amino acid sequence functional fragment as shown in SEQ ID No. 1, SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

[0007] Furthermore, the full-length amino acid sequence of the recombinant type I humanized collagen rCol1 is composed of the functional fragment shown in SEQ ID No. 1 repeated 10 times. The full amino acid sequence of the recombinant type I humanized collagen rCol1 is shown in SEQ ID No. 3: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

[0008] Furthermore, the recombinant type I humanized collagen was efficiently expressed in BL21(DE3) Escherichia coli using the pET30a expression vector, and the purity after purification was >95%.

[0009] Furthermore, the sequence shown in SEQ ID No. 1 exhibits the sequence characteristics of collagen, glycine-XY, where X and Y are any amino acids other than glycine, with a total length of 33 amino acids. The molecular weight of the core functional fragment of the recombinant type I humanized collagen is 3085.31 Da, and the theoretical isoelectric point of the functional fragment is 8.74. The functional fragment of the recombinant type I humanized collagen has transmembrane properties and is a hydrophilic protein. The functional fragment of the recombinant type I humanized collagen has low antigenicity. The functional fragment of the recombinant type I humanized collagen has a secondary structure.

[0010] Furthermore, the recombinant type I humanized collagen rCol1 has a molecular weight of 30690.92 Da and a theoretical isoelectric point of 10.58. The recombinant type I humanized collagen has transmembrane properties and is a hydrophilic protein. The recombinant type I humanized collagen has low antigenicity. The recombinant type I humanized collagen has a peptide chain secondary structure.

[0011] A method for preparing any of the above-mentioned recombinant type I humanized collagen, the method comprising the following steps: Step 1: The human type I collagen alpha 1 chain (Col1α1) was comprehensively analyzed using AlphaFold2 / ColabFold and PEP-FOLD3 methods, and its function was verified. Step 2: Using ExPASy ProtParam and HeliQuest software, the candidate peptides were analyzed for biological characteristics, and the key fragment G677-K709 of the Col1α1 chain was identified as shown in SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK. Step 3: The functional fragment shown in SEQ ID No. 1 was repeated 10 times and its physicochemical properties were determined. Based on the amino acid sequence of the protein, the corresponding base sequence was reversed and the codon was optimized. It was then inserted into the pET30a expression plasmid to construct a recombinant protein. After expression, enzyme digestion and purification, recombinant human collagen type 1 (rCol1) was obtained.

[0012] Preferably, 33 amino acids at the critical segment G677-K709 of the Col1α1 chain were identified; the recombinant type I humanized collagen has a length of 330 amino acids.

[0013] Furthermore, for the potential core region of 33 amino acids at the key segment G677-K709 of the Col1α1 chain, amino acid sequence sequencing, antigenicity analysis, transmembrane analysis, signal peptide analysis, hydrophobicity analysis, hydrophilicity analysis, and secondary structure analysis were performed.

[0014] Furthermore, the functional fragment shown in SEQ ID No. 1 is repeated 10 times to form the complete amino acid sequence of recombinant type I humanized collagen, and amino acid sequence sequencing, antigenicity analysis, transmembrane analysis, signal peptide analysis, hydrophobicity analysis, hydrophilicity analysis, and secondary structure analysis are performed.

[0015] This application also discloses the application of any of the above-mentioned recombinant type I humanized collagen in the preparation of medical biomaterials, beauty and health products or cosmetics.

[0016] The principle of constructing recombinant type I humanized collagen as described in this application is as follows: Searching for "human and collagen type I alpha 1 chain (Col1A1)" in the NCBI database Gene, the alpha1 peptide chain sequence of human type I collagen with 1464 amino acid residues, NP_000079.2, was downloaded; the above-mentioned human collagen type I alpha1 peptide chain with a length of 1464 amino acid residues was then analyzed using the online antigenic epitope prediction tool Predicted. Antigenic Peptides (website: http: / / imed.med.ucm.es / Tools / antigenic.pl) were used to analyze antigenicity. Multiple candidate amino acid fragments were analyzed, and 33 amino acids in the region G677-K709, which had low antigenicity, were selected. The full-length structure of the human type I collagen alpha1 chain was analyzed using relevant software. Multiple candidate amino acid fragments were screened, and these fragments were tandemly repeated. Their biological properties were analyzed in depth, and the resulting structures were further analyzed using relevant software. Finally, recombinant human collagen peptide sequences with different spatial structures and low antigenicity were obtained. From these, the recombinant human collagen sequence with low antigenicity and high hydrophilicity, namely the 10-fold repeat sequence G677-K709, was selected. Using the human type I collagen alpha1 chain as the research object, its structure was analyzed using relevant software, multiple amino acid sequences were determined, and each sequence was tandemly repeated. Their biological properties were compared and studied, and potential polypeptide chains with specific spatial structures and low antigenicity were screened. These were then recombinantly expressed and purified.

[0017] This application provides a recombinant type I humanized collagen, its preparation method, and its application. Compared with the prior art, it has the following advantages: 1. The recombinant type I humanized collagen of this application has a short functional fragment length of 33 amino acids, which has the advantages of high absorption rate, short cycle, and high purity. It has no viral risks and is water-soluble. 2. The functional fragment of the recombinant type I humanized collagen of this invention is short in length, can be directly synthesized, and does not require prokaryotic or eukaryotic expression, thus having the advantages of simple preparation and high yield; 3. This application utilizes a recombinant humanized collagen overexpression plasmid to achieve high-efficiency expression in BL21(DE3). After separation, purification, and identification, the protein purity is greater than 95%. 4. The recombinant humanized collagen of this application has low antigenicity and strong hydrophilicity. Its amino acid composition is basically consistent with the corresponding amino acid sequence of natural collagen. It will not cause immune rejection in vivo, so it can be widely used in clinical practice. Attached Figure Description

[0018] Figure 1 This is a diagram showing the amino acid sequence of the functional fragment of the recombinant type I humanized collagen in this application; Figure 2 This is an amino acid antigenicity analysis diagram of the functional fragment of recombinant type I humanized collagen in this application; Figure 3 This is a transmembrane amino acid analysis diagram of the functional fragment of recombinant type I humanized collagen from this application; Figure 4 This is an analysis diagram of the amino acid signal peptide of the functional fragment of recombinant type I humanized collagen in this application; Figure 5 This is a hydrophobicity analysis diagram of the amino acid functional fragments of recombinant type I humanized collagen in this application; Figure 6 This is a hydrophilicity analysis diagram of the amino acid components of the functional fragment of recombinant type I humanized collagen in this application; Figure 7 This is an amino acid secondary structure analysis diagram of the functional fragment of recombinant type I humanized collagen in this application; Figure 8 This is a diagram of the quality test report for the expression and purification of recombinant type I humanized collagen in this application; Figure 9 This is the complete amino acid sequence of the recombinant type I humanized collagen of this application; Figure 10 This is a full-sequence amino acid antigenicity analysis diagram of the recombinant type I humanized collagen of this application; Figure 11 This is a transmembrane analysis diagram of the full-sequence amino acid composition of the recombinant type I humanized collagen of this application; Figure 12 This is a full-sequence amino acid signal peptide analysis diagram of the recombinant type I humanized collagen of this application; Figure 13 This is a full-sequence amino acid hydrophobicity analysis diagram of the recombinant type I humanized collagen of this application; Figure 14 This is a full-sequence amino acid hydrophilicity analysis diagram of the recombinant type I humanized collagen of this application; Figure 15 This is a full-sequence amino acid secondary structure analysis diagram of the recombinant type I humanized collagen of this application; Figure 16 This is a graph showing the contact angle measurement results of the recombinant type I humanized collagen of this application; Figure 17These are CCK8 cell activity assays of recombinant type I humanized collagen Schwann under different concentrations of rCol1. In this paper, A is the activity assay of Schwann cells after 24 h of growth, B is the activity assay of Schwann cells after 48 h of growth, C is the activity assay of Schwann cells after 72 h of growth, and D is a summary of the Schwann cell activity assays obtained from CCK8 at 24 h, 48 h, and 72 h. Figure 18 These are images showing the CCK8 cell activity of recombinant type I humanized collagen in SD newborn rat cerebral cortex neurons under different concentrations of rCol1. In the images, A is the activity detection image of cortical neurons after 24 hours of growth, B is the activity detection image of cortical neurons after 48 hours of growth, and C is the sum of neuronal activity detected by CCK8 at 24 hours and 48 hours. Figure 19 This is a diagram showing the protrusion growth of Schwann, a recombinant type I humanized collagen, under different concentrations of rCol1. Figure 20 This is a diagram showing the neurite growth of DRG neurons under different concentrations of rCol1 for recombinant type I humanized collagen of this application. In the diagram, A is the neurite growth of DRG neurons, B is the statistical diagram of the longest neurite of DRG neurons, and C is the statistical diagram of the total neurite of DRG neurons. Figure 21 These are diagrams showing the protrusion growth of the recombinant type I humanized collagen DRG explant under different concentrations of rCol1, where A is a diagram showing the protrusion growth of the DRG explant and B is a statistical diagram showing the protrusion length of the DRG explant. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and equivalent alterations or modifications also fall within the scope defined by the claims of this application.

[0020] In this embodiment of the application, type I collagen sequence was selected as the sequence for screening and optimization. The core sequence of the recombinant type I humanized collagen rCol1 is the NCBI reference sequence, as shown in SEQ ID No. 2: MFSFVDLRLLLLLAATALLThGQEEGQVEG QDEDIPPITC VQNGLRYHDR DVWKPEPCRI CVCDNGKVLC DDVICDETKNCPGAEVPEGE CCPVCPDGSE SPTDQETTGV EGPKGDTGPR GPRGPAGPPG RDGIPGQPGL PGPPGPPGPPGPPGLGGNFA PQLSYGYDEK STGGISVPGP MGPSGPRGLP GPPGAPGPQG FQGPPGEPGE PGASGPMGPRGPPGPPGKNG DDGEAGKPGR PGERGPPGPQ GARGLPGTAG LPGMKGHRGF SGLDGAKGDA GPAGPKGEPGSPGENGAPGQ MGPRGLPGER GRPGAPGPAG ARGNDGATGA AGPPGPTGPA GPPGFPGAVG AKGEAGPQGPRGSEGPQGVR GEPGPPGPAG AAGPAGNPGA DGQPGAKGAN GAPGIAGAPG FPGARGPSGP QGPGGPPGPKGNSGEPGAPG SKGDTGAKGE PGPVGVQGPP GPAGEEGKRG ARGEPGPTGL PGPPGERGGP GSRGFPGADGVAGPKGPAGE RGSPGPAGPK GSPGEAGRPG EAGLPGAKGL TGSPGSPGPD GKTGPPGPAG QDGRPGPPGPPGARGQAGVM GFPGPKGAAG EPGKAGERGV PGPPGAVGPA GKDGEAGAQG PPGPAGPAGE RGEQGPAGSPGFQGLPGPAG PPGEAGKPGE QGVPGDLGAP GPSGARGERG FPGERGVQGP PGPAGPRGAN GAPGNDGAKGDAGAPGAPGS QGAPGLQGMP GERGAAGLPGPKGDRGDAGP KGADGPSPGKD GVRGLTGPIG PPGPAGAPGDKGESGPSGPA GPTGARGAPG DRGEPGPPGP AGFAGPPGAD GQPGAKGEPG DAGAKGDAGPPGPAGPAGPPGPIGNVGAPG AKGARGSAGP PGATGFPGAA GRVGPPGPSG NAGPPGPPGP AGKEGGKGPR GETGPAGRPGEVGPPGPPGP AGEKGSPGAD GPAGAPGTPG PQGIAGQRGV VGLPGQRGER GFPGLPGPSG EPGKQGPSGASGERGPPGPM GPPGLAGPPG ESGREGAPGA EGSPGRDGSP GAKGDRGETG PAGPPGAPGA PGAPGPVGPAGKSGDRGETG PAGPAGPVGP VGARGPAGPQ GPRGDKGETG EQGDRGIKGH RGFSGLQGPP GPPGSPGEQGPSGASGPAGP RGPPGSAGAP GKDGLNGLPG PIGPPGPRGR TGDAGPVGPP GPPGPPGPPG PPSAGFDFSFLPQPPQEKAH DGGRYYRADD ANVVRDRDLE VDTTLKSLSQ QIENIRSPEG SRKNPARTCR DLKMCHSDWKSGEYWIDPNQ GCNLDAIKVF CNMETGETCV YPTQPSVAQK NWYISKNPKD KRHVWFGESM TDGFQFEYGGQGSDPADVAI QLTFLRLMST EASQNITYHC KNSVAYMDQQ TGNLKKALLL QGSNEIEIRA EGNSRFTYSVTVDGCTSHTG AWGKTVIEYKTTKTSRLLPII DVAPLDVGAP DQEFGFDVGP VCFL.

[0021] The GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK (SEQ ID No. 1) portion of the above sequence is the amino acid sequence functional fragment selected in this application. Through multiple studies, it has been found that the amino acid sequence functional fragment is superior to commercial human collagen in promoting nerve injury repair. The molecular weight of the recombinant type I humanized collagen peptide functional fragment is 3085.31 Da, and the theoretical isoelectric point of the recombinant type I humanized collagen peptide functional fragment is 8.74. The recombinant type I humanized collagen peptide functional fragment has transmembrane properties and is a hydrophilic protein. The recombinant type I humanized collagen peptide functional fragment has low antigenicity. The recombinant type I humanized collagen peptide functional fragment has a secondary structure.

[0022] The complete amino acid sequence of the recombinant type I humanized collagen polypeptide described in this application consists of the functional fragment shown in SEQ ID No. 1 repeated 10 times. The complete amino acid sequence of the recombinant type I humanized collagen is shown in SEQ ID No. 3: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGP The recombinant type I humanized collagen has a molecular weight of 30690.92 Da and a theoretical isoelectric point of 10.58. It exhibits transmembrane properties, is a hydrophilic protein, and has low antigenicity. Furthermore, it possesses a secondary structural form.

[0023] During the expression of the recombinant type I humanized collagen described in this application, a sequence for affinity chromatography purification and SUMO enzyme digestion needs to be added to its N-terminus, as shown in SEQ ID No. 4: MHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG. This sequence can be removed by protease cleavage, thereby directly obtaining the polypeptide chain with the sequence shown in SEQ ID No. 3. The sequence shown in SEQ ID No. 4 is directly linked to the N-terminus of the first repeat sequence, as shown in SEQ ID No. 5: MHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHRE QIGGGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPG PAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAK GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

[0024] In this invention, recombinant type I humanized collagen rCol1 can be prepared by conventional methods in the art. The recombinant type I humanized collagen is efficiently expressed in BL21 (DE3) Escherichia coli using the pET30a expression vector, and the protein purity after extraction is >95%.

[0025] The preparation method of the recombinant type I humanized collagen specifically includes the following steps: Step 1: The alpha 1 peptide chain of type I collagen was comprehensively analyzed using AlphaFold2 / ColabFold and PEP-FOLD3 methods, and its function was verified. Step 2: Using ExPASy ProtParam and HeliQuest software, the candidate peptides were analyzed for biological characteristics, and the key fragment of the Col1α1 chain Col1α1G677-K709, namely SEQ ID No.1, was identified as GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK. Step 3: Repeat the functional fragment shown in SEQ ID No. 1 10 times and determine its physicochemical properties; based on the protein sequence, reverse the derivation of the corresponding base sequence, optimize its encoding gene, insert it into the expression vector of pET30a, and obtain recombinant type I humanized collagen through transformation, amplification, chromatography, enzyme digestion and repurification.

[0026] The method identified 33 amino acids at the key fragment Col1α1G677-K709 of the Col1α1 chain, and the recombinant type I humanized collagen is 330 amino acids in length. The method then performed amino acid sequence sequencing, antigenicity analysis, transmembrane analysis, signal peptide analysis, hydrophobicity analysis, hydrophilicity analysis, and secondary structure analysis on the potential core region of the identified key fragment Col1α1G677-K709. Finally, the method involved repeating the functional fragment shown in SEQ ID No. 1 10 times to construct the complete amino acid sequence of the recombinant type I humanized collagen, followed by amino acid sequence sequencing, antigenicity analysis, transmembrane analysis, signal peptide analysis, hydrophobicity analysis, hydrophilicity analysis, and secondary structure analysis.

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0028] Example 1: This example provides a method for preparing recombinant type I humanized collagen, specifically including the following steps: 1. Construction of the rCol1 gene expression vector:

[0029] The optimized nucleotide sequence derived from the protein sequence was inserted into the pET30a expression vector to construct an expression plasmid for recombinant type I humanized collagen.

[0030] 2. Transformation of recombinant expression plasmids: The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific procedure is as follows: S1. Take 1 μL of the plasmid and put it into 100 μL of E. coli competent cells BL21(DE3), and incubate on ice for 30 min. S2. Heat shock the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes. S3. Add 700 μL of non-resistant LB (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) to the mixture and incubate at 37°C and 220 rpm for 1 h. S4. Take 200 μL of the bacterial solution and spread it evenly on an LB agar plate containing kanamycin sulfate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 50 μg / mL kanamycin sulfate). S5. Invert the plate and incubate it in a 37°C incubator for about 16 hours until clearly visible colonies grow.

[0031] 3. Induced expression of recombinant type I humanized collagen rCol1: Single colonies were picked from the transformed LB plates and placed in 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate). The culture was incubated at 37°C and 220 rpm until the bacterial OD reached the target value. 600 At a temperature of 0.9-1.1, 2 mL of the bacterial culture was centrifuged to extract the plasmid, which was then digested with enzymes and sequenced for identification. After confirming accurate plasmid construction, another 2 mL of the bacterial culture was added to 1000 mL of LB medium (containing 50 μg / mL kanamycin sulfate) for expansion culture. The culture was allowed to grow until the OD of the bacterial culture was reached. 600 At a concentration of 0.5-0.8, IPTG was added to a final concentration of 0.2 mM to induce expression, and the cells were cultured at 15℃ and 220 rpm for 16 h. Finally, the cells were collected by centrifugation and stored at -20℃ or immediately proceeded to the next purification step.

[0032] 4. Purification of recombinant type I humanized collagen rCol1: The bacterial pellet was lysed by sonication using Tris buffer (50 mM Tris, 300 mM sodium chloride, 20 mM imidazole, 1% Triton X-100, 1 mM MTT, 1 mM PMSF, pH 8.0), and then centrifuged at 17,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0033] The Ni-IDA affinity column was equilibrated with 5 times the column volume of equilibration buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0). Protein supernatant was then added, and the column was placed on a rotary mixer to ensure the target recombinant protein was fully bound to the column. Impurities were then washed with a 20 mM imidazole buffer (20 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0), followed by elution with a 500 mM imidazole solution (500 mM imidazole, 50 mM Tris, 300 mM NaCl, pH 8.0).

[0034] The target protein was collected and dialyzed into 1×PBS (pH 7.4, containing 5% glycerol). Then, an appropriate amount of SUMO protease was added, and the mixture was incubated at 16°C for 3 hours for enzymatic hydrolysis. The digested protein solution was then added to a Ni-IDA affinity column pre-equilibrated with 1×PBS, and the column was vortexed to ensure complete binding of the tag fragment to the column material. Finally, the eluent was collected, which was the target recombinant type I humanized collagen with the tag fragment removed. The protein was filtered through a 0.22 μm filter, and the protein concentration was determined using the Bradford method. The protein was then aliquoted and stored at -80°C or freeze-dried for later use.

[0035] 5. Purity detection of recombinant type I humanized collagen rCol1: The purity of the obtained rCol1 protein was determined by SDS-PAGE. The specific procedure was as follows: 20 μL of purified protein solution was added to 5 μL of 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.05% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), and heated in boiling water at 100℃ for 5 min. The sample mixture was then added to the wells of the SDS-PAGE gel. After electrophoresis, the protein was stained with Coomassie Brilliant Blue (0.1% Coomassie Brilliant Blue R-250, 25% ethanol, 10% glacial acetic acid) for 3 min, followed by destaining with protein destaining solution (10% acetic acid, 5% ethanol). After electrophoresis of the remaining protein, subsequent steps of Western blotting were performed, including incubation with anti-His tag antibody, and the purity of the digested protein was determined. The results are as follows: Figure 8As shown, the apparent molecular weight of the expression product before enzyme digestion was approximately 50 kDa, and the apparent molecular weight of rCol1 after SUMO digestion was approximately 35 kDa, indicating that rCol1 was correctly expressed and purified.

[0036] Example 2, Water solubility test of recombinant humanized collagen: 1.2 mg of recombinant human collagen was dissolved in 1 mL of water. It dissolved directly without stirring, forming a colorless, precipitate-free, and float-free transparent solution. This indicates that the recombinant humanized collagen prepared in this invention has excellent water solubility, verifying its characteristics as a hydrophilic protein.

[0037] The protein solution was then transferred to a 14 mm diameter glass slide using a 200 μL pipette to ensure the slide surface was completely covered with the protein solution. Excess liquid was aspirated from the top of the slide to ensure a uniform liquid layer thickness. Five parallel samples were prepared and frozen overnight at -20°C. The next day, the samples were transferred to a freeze dryer and dried for 24 hours. After drying, the samples were removed and contact angle measurements were performed (e.g., ...). Figure 16 (As shown). This further demonstrates that the recombinant human collagen rCol1 prepared in this invention has excellent water solubility, verifying its characteristics as a hydrophilic protein.

[0038] The recombinant human collagen synthesized using this method is characterized by high purity, absence of viral risks, and high expression levels. This protein has broad application prospects in biomedicine, regenerative medicine, tissue engineering, and related products in the fields of beauty, health care, and cosmetics.

[0039] Example 3, Antigenicity analysis of recombinant human collagen: The recombinant type I humanized collagen comprises a functional fragment of the amino acid sequence shown in SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

[0040] The antigenicity of this protein was analyzed using the online antigenic epitope prediction tool Predicted Antigenic Peptides (website: http: / / imed.med.ucm.es / Tools / antigenic.pl). The analysis revealed that 33 amino acids in the selected region G677-K709 (e.g., ...) were involved. Figure 2 (As shown) has low antigenicity.

[0041] Example 4, Basic Properties Analysis of Recombinant Human Collagen: The recombinant type I humanized collagen comprises a functional fragment of the amino acid sequence shown in SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

[0042] The recombinant human collagen has a full length of 33 amino acids and is a type I peptide of recombinant human collagen.

[0043] The recombinant human collagen has a molecular weight of 3085.31 Da and a theoretical isoelectric point of 8.74.

[0044] like Figure 3 As shown, the recombinant human collagen exhibits transmembrane properties as predicted by TMHMM. Figure 4 As shown, the recombinant human collagen protein, analyzed using the SignalP signal peptide prediction tool, lacks a signal peptide; as... Figure 5 , Figure 6 As shown, ExPASy analysis revealed that this recombinant human collagen is a hydrophilic protein; Figure 7 As shown, the recombinant human collagen was found to have a secondary structure using online SOPMA analysis.

[0045] Example 5, Quality testing of recombinant human collagen: The recombinant type I humanized collagen comprises a functional fragment of the amino acid sequence shown in SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

[0046] The protein sequence (SEQ ID NO.1) is repeated 10 times, which is 330 amino acids in length. Based on the protein sequence, the nucleotide sequence was deduced, codons were optimized, and the sequence was inserted into the pET30a expression vector to construct a high-expression plasmid for recombinant human type I collagen. This plasmid was transformed into BL21(DE3) bacteria for expression. The protein was then isolated, purified, and identified. The purity of the obtained protein is currently ≥95% (e.g., ...). Figure 8 (As shown).

[0047] Example 6, Bioactivity of soluble recombinant type I humanized collagen: Recombinant type I humanized collagen was prepared into protein solutions with concentrations of 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL using sterile water, and then filtered through a 0.22 μm sterile filter for sterilization. 100 μL of the protein solution was added to each well of a 96-well plate and incubated at 37 °C for 2 h. Non-specific binding sites were blocked with 100 μL of 1% bovine serum albumin (BSA), followed by washing twice with sterile water.

[0048] 100 μL of a density of 1×10 5 Schwann cells were added at a concentration of / mL to 96-well plates, with replicates. The control group received only DMEM high-glucose medium. Cells were incubated at 37°C, 5% CO2 for 24h, 48h, and 72h, respectively. The medium was then aspirated from the 96-well plates, and Schwann cell viability was assessed using a CCK-8 assay. Figure 10 As shown.

[0049] The embodiments selected in the above materials are for ease of understanding and not for limiting the process method. Those skilled in the art can easily modify the process flow or transfer it to other cases without inventive change. If these modifications also fall under the category of similar claims or similar technology of this invention, then the intent of this invention also includes these modifications.

Claims

1. A recombinant type I humanized collagen, characterized in that, The recombinant type I humanized collagen rCol1 includes a core amino acid sequence functional fragment as shown in SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

2. The recombinant type I humanized collagen according to claim 1, characterized in that, The full-length amino acid sequence of the recombinant type I humanized collagen rCol1 is composed of 10 repetitions of the functional fragment shown in SEQ ID No.

1. The full amino acid sequence of the recombinant type I humanized collagen rCol1 is shown in SEQ ID No. 3: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGERGFPGERGVQGPPGPAGPRGANGAPGNDGAK.

3. The recombinant type I humanized collagen according to claim 1 or 2, characterized in that, The recombinant type I humanized collagen was efficiently expressed in BL21(DE3) Escherichia coli using the pET30a expression vector, with a protein purity >95%.

4. The recombinant type I humanized collagen according to claim 1, characterized in that, The sequence shown in SEQ ID No. 1 exhibits the sequence characteristics of collagen, glycine-XY, where X and Y are any amino acids other than glycine, with a total length of 33 amino acids. The molecular weight of the core functional fragment of the recombinant type I humanized collagen is 3085.31 Da, and the theoretical isoelectric point of the functional fragment is 8.

74. The functional fragment of the recombinant type I humanized collagen has transmembrane properties and is a hydrophilic protein. The functional fragment of the recombinant type I humanized collagen has low antigenicity and a secondary structure.

5. The recombinant type I humanized collagen according to claim 2, characterized in that, The recombinant type I humanized collagen rCol1 has a molecular weight of 30690.92 Da and a theoretical isoelectric point of 10.

58. The recombinant type I humanized collagen has transmembrane properties and is a hydrophilic protein. The recombinant type I humanized collagen has low antigenicity. The recombinant type I humanized collagen has a peptide chain secondary structure.

6. A method for preparing recombinant type I humanized collagen as described in claim 1 or 2, characterized in that, The preparation method Includes the following steps: Step 1: The human type I collagen alpha 1 chain (Col1α1) was comprehensively analyzed using AlphaFold2 / ColabFold and PEP-FOLD3 methods, and its function was verified. Step 2: Using ExPASy ProtParam and HeliQuest software, the candidate peptides were analyzed for biological characteristics, and the key fragment G677-K709 of the Col1α1 chain was identified as shown in SEQ ID No. 1: GERGFPGERGVQGPPGPAGPRGANGAPGNDGAK. Step 3: The functional fragment shown in SEQ ID No. 1 was repeated 10 times and its physicochemical properties were determined. Based on the amino acid sequence of the protein, the corresponding base sequence was reversed and the codon was optimized. It was then inserted into the pET30a expression plasmid to construct a recombinant protein. After expression, enzyme digestion and purification, recombinant human collagen type 1 (rCol1) was obtained.

7. The method for preparing recombinant type I humanized collagen according to claim 6, characterized in that, The study identified 33 amino acids at the critical segment G677-K709 of the Col1α1 chain; the recombinant type I humanized collagen was 330 amino acids in length.

8. The method for preparing recombinant type I humanized collagen according to claim 7, characterized in that, The process involved amino acid sequence sequencing, antigenicity analysis, transmembrane analysis, signal peptide analysis, hydrophobicity analysis, hydrophilicity analysis, and secondary structure analysis of the potential core region of 33 amino acids at the critical segment G677-K709 of the Col1α1 chain.

9. The method for preparing recombinant type I humanized collagen according to claim 6, characterized in that, The functional fragment shown in SEQ ID No. 1 was repeated 10 times to form the complete amino acid sequence of recombinant type I humanized collagen, and then amino acid sequence sorting, antigenicity analysis, transmembrane analysis, signal peptide analysis, hydrophobicity analysis, hydrophilicity analysis and secondary structure analysis were performed.

10. The use of the recombinant type I humanized collagen as described in claim 1 or 2 in the preparation of medical biomaterials, beauty and health products or cosmetics.

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