Method for fermenting recombinant humanized collagen by using escherichia coli and application of recombinant humanized collagen

Abstract

The invention discloses a method for fermenting recombinant humanized collagen by using escherichia coli and application of the recombinant humanized collagen, and belongs to the field of collagen. The amino acid sequence of the prepared recombinant humanized III-type collagen is shown as SEQ ID NO.4-7, the recombinant humanized III-type collagen is formed by connecting a plurality of repetitive units of natural collagen in series and has the advantages of being good in biocompatibility and high in safety, in addition, the nucleotide sequence for coding the recombinant collagen is designed according to the codon optimization theory, and the recombinant humanized III-type collagen has the advantages of being good in biocompatibility and high in safety. And carrying out fermentation expression on escherichia coli to obtain the recombinant humanized III-type collagen. The humanized III-type collagen provided by the invention can significantly improve the cell adhesion activity and proliferation activity, and can be widely applied to the fields of cosmetics, medical instruments and the like.

Description

[0001] The parent application for this divisional application is: application date January 13, 2025, application number 202510050269.9, invention title "A method for fermenting recombinant humanized collagen using Escherichia coli and its application". Based on the lack of unity of invention in the first examination opinion issued on February 25, 2025, for the parent application, this divisional application is hereby made. Technical Field

[0002] This invention relates to the field of collagen, specifically to a method for fermenting recombinant humanized collagen using Escherichia coli and its application. Background Technology

[0003] Collagen is one of the most important and abundant proteins in mammals. It is generally white, transparent, and unbranched fibrils, serving as the basic structural support for skin and bones. It can account for 25% to 35% of total protein and is mainly distributed in human skin, blood vessels, bones, tendons, teeth, and cartilage. It acts as the main matrix and scaffold of these tissues, protecting and connecting them, and playing a vital physiological role in the body. Therefore, collagen has wide applications in the pharmaceutical and cosmetic industries.

[0004] Human type III collagen is a transmembrane non-fibroblast collagen, a homogeneous trimer composed of three identical α1(XVII) chains, divided into three major structural domains: intracellular, transmembrane, and extracellular. Human type III collagen is a component of hemidesmosomes in cells and plays a crucial role in the epithelial cell-basement membrane process, regulating epithelial cell adhesion, separation, and developmental differentiation, and is essential for keratinocyte differentiation and regeneration. Human type III collagen is extremely rare in humans, and similarly scarce in animals, making extraction very difficult and preventing mass production. Furthermore, animal-derived collagen products inevitably pose immunogenicity and potential biosafety risks such as viral and disease transmission, limiting their widespread application. Due to legal and ethical constraints, human type III collagen can only be used in research, resulting in the current lack of commercially available type III collagen products derived from humans or other animals, further restricting its research and application. Currently, the main approach to addressing these issues is through biotechnology such as genetic engineering to produce collagen.

[0005] The application of recombinant collagen requires large-scale, high-density, and high-expression fermentation production and purification, with *E. coli* being an ideal expression host. However, as a eukaryote, *E. coli* inevitably regulates the expression of heterologous proteins when large-scale expression and secretion of these proteins occur, meaning that intracellular biological resources are heavily utilized. This regulation is primarily manifested in the degradation of the expressed recombinant protein. Recombinant collagen is produced by constructing engineered bacteria containing the full-length or partial gene fragments of collagen using genetic engineering techniques. These engineered bacteria then synthesize collagen, and finally, high-purity recombinant collagen is obtained through specific purification processes. Constructing genetically engineered strains using genetic engineering techniques is of great significance for the preparation of high-quality human-like collagen. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to recombinantly express a new recombinant humanized type III collagen using genetic engineering technology, specifically a method and application of recombinant humanized collagen fermented with Escherichia coli.

[0007] Based on the above, the technical solution of the present invention is implemented as follows:

[0008] The present invention provides a recombinant humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO.4-7.

[0009] The present invention also provides a nucleic acid molecule encoding the recombinant humanized type III collagen.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.8-11.

[0011] The present invention also provides a recombinant expression vector containing the nucleic acid molecule.

[0012] The present invention also provides a host cell comprising the recombinant expression vector or expressing the collagen.

[0013] Furthermore, the host cell is Escherichia coli.

[0014] It should be noted that the host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc., of nucleic acid constructs or expression vectors containing the polynucleotides of this application. The host cell can be any cell useful in the production of recombinant humanized collagen according to this application. To produce recombinant collagen, the nucleic acid encoding the recombinant collagen can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. This nucleic acid can be easily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding recombinant collagen). The host cell refers to a cell in which exogenous nucleic acid has been introduced, including the progeny of such cells. The host cell includes transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. The progeny may not be completely identical to the parent cells in terms of nucleic acid content, but may contain mutations. Methods for introducing vectors into host cells are well known, such as using electroporation to introduce vectors into host cells; other methods include transfection, microinjection, gene gun technology, liposome-mediated methods, etc. The host cell can be a prokaryotic or eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli; preferably, the eukaryotic cell is Pichia pastoris.

[0015] This invention also provides a method for producing the recombinant humanized type III collagen, characterized by comprising the following steps:

[0016] (1) The host cells are cultured in a culture medium;

[0017] (2) The recombinant humanized type III collagen was isolated from the host cell.

[0018] The present invention also provides a composition comprising the recombinant humanized type III collagen, or the recombinant humanized type III collagen encoded by the nucleic acid molecule, or the recombinant humanized type III collagen expressed by the recombinant expression vector, or the recombinant humanized type III collagen produced by the host cell, or the recombinant humanized type III collagen prepared by the production method.

[0019] The present invention also provides articles comprising the composition, wherein the articles are pharmaceutical compositions, medical devices or cosmetics.

[0020] Furthermore, the product is one or more of the following: biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, and pharmaceutical excipients.

[0021] Furthermore, the application area of ​​the cosmetic is not particularly limited and can be the face, hands, legs, torso, etc. The form of the composition is not particularly limited, as long as it achieves the intended function. For example, the composition can be a solid, liquid, or gel composition.

[0022] The present invention also provides the use of the composition in the preparation of pharmaceutical compositions, medical devices or cosmetics.

[0023] This invention provides the use of recombinant humanized type III collagen in the preparation of medicaments for promoting cell adhesion, cell proliferation and / or wound healing.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The recombinant humanized type III collagen of this invention is formed by multiple repeating units tandemly, and these repeating units are derived from the amino acid sequence of natural type III collagen, thus possessing advantages such as good biocompatibility and high safety. The recombinant humanized type III collagen of this invention exhibits excellent effects in promoting cell adhesion, cell proliferation, and / or wound healing, and can be widely used in cosmetics and pharmaceutical products. The preparation method of the recombinant humanized type III collagen of this invention is simple and easy to operate, suitable for large-scale industrial production, and therefore has broad application prospects. Attached Figure Description

[0026] Figure 1 Cytotoxicity evaluation of recombinant humanized type III collagen.

[0027] Figure 2 Cell proliferation activity of recombinant humanized type III collagen.

[0028] Figure 3 Cell adhesion-promoting activity of recombinant humanized type III collagen. Detailed Implementation

[0029] The present invention will be illustrated below with examples to make the technical solution of the present invention easier to understand and master, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the medicinal materials and reagents can be obtained commercially; and the performance of products from different sources does not have a significant impact.

[0030] The features, beneficial effects, and advantages of this invention will become apparent to those skilled in the art upon reading the contents of this specification.

[0031] Unless otherwise specified, all percentages, fractions, and ratios are calculated based on the total mass of the compositions of the present invention. The term "mass content" may be represented by the symbol "%".

[0032] As used in this article, "medical device" refers to instruments, equipment, appliances, in vitro diagnostic reagents and calibrators, materials and other similar or related items that are used directly or indirectly on the human body.

[0033] As used herein, “isolation” refers to the separation of the target peptide from cultured host cells, for example, by disrupting the host cells and purifying the target peptide. In cases where the purified target peptide carries a purification tag, such as a Trx or His tag, “isolation” also includes the enzymatic removal of the Trx or His tag.

[0034] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and components may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention may comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0035] Example 1: Design of the amino acid sequence of recombinant humanized type III collagen

[0036] In this invention, the human natural type III collagen COL-III sequence was selected for screening and optimization. The human type III collagen sequence is the NCBI reference sequence: KAI4037253.1 (SEQ ID No. 12), see https: / / www.ncbi.nlm.nih.gov / protein / KAI4037253.1. Details are as follows:

[0037] MMSFVQKGSWLLLALLHPTIILAQQEAVEGGCSLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPN GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGIC ESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSP GSPGYQGPPGEPGQAGPSGPPGPPGA IGPPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR GFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMG ARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQ GESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPG GPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGD AGAPGER GPGPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETG PPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFK INTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDV LDVQLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL(SEQ IDNo.12).

[0038] The bold underlined portion of the above sequence is the amino acid sequence selected in this invention. Through extensive research, the applicant has discovered that the selected sequence exhibits high water solubility, high recombinant expression yield, simple purification process, and better bioactivity than commercially available collagen or other sequences in SEQ ID No. 12.

[0039] This invention is partly based on the finding that peptides containing at least 41 consecutive amino acid residues as in SEQ ID No. 12 exhibit better biomaterial properties than commercially available human collagen; therefore, the sequences of several specific amino acid regions were tested.

[0040] COL-HR-1: GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGIC (SEQ ID NO. 1).

[0041] COL-HR-2: GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR (SEQ ID NO. 2).

[0042] COL-HR-3: GESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERG GPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGP PGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGER (SEQ ID NO. 3).

[0043] The recombinant humanized type III collagen COL-HR-111 in this invention is a triplicate repeat sequence of COL-HR-1 (SEQ ID No. 1), comprising 123 amino acids. The amino acid sequence of COL-HR-111 is as follows:

[0044] GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICGQGPQGPKG DPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICGQGPQGPKGDPGPPGIPGRNGD PGIPGQPGSPGSPGPPGIC (SEQ ID NO. 4).

[0045] The DNA sequence of COL-HR-111 is as follows: GGCCAGGGCCCGCAGGGCCCGAAAG GCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCATTCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGCGGCCAGGGCCCGCAGGGCCCGAAAGGCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCAT TCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGCGGCCAGGGCCCGCAGGGCCCGAAAGGCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCATTCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGC.

[0046] After codon optimization of the above DNA sequence, the GC content changed from 80.49% before optimization to 69.38% after optimization. The sequence is as follows: GGTCAGGGTCCACAAGGCCCGAAAGGCGATCCTGG TCCGCCAGGCATTCCGGGCCGTAACGGTGATCCGGGCATCCCTGCCAGCCGGGCTCTCCGGGTTCCCCTGGTCCGCCGGGTATTTGTGGCCAGGGTCCTCAGGGCCCAAAAGGTGATCCTGGTCCGCCTGGTATCCCGGGCCGCAACGGTGATCCTGGCATCCCGGGC CAGCCGGGTTCCCCGGGTTCCCCGGGCCCGCCAGGCATCTGCGGTCAGGGTCCACAGGGCCCAAAGGGTGACCCGGGTCCACCGGGTATTCCGGGTCGCAATGGTGATCCGGGTATTCCGGGCCAGCCAGGTTCTCCTGGCTCTCCGGGTCCGCCGGGTATCTGC(SEQ ID NO.8).

[0047] The recombinant humanized type III collagen COL-HR-222 in this invention is a triplicate repeat sequence of COL-HR-2 (SEQ ID No. 2), comprising 316 amino acids. The amino acid sequence of COL-HR-222 is as follows:

[0048] GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR(SEQ ID NO.5).

[0049] The DNA sequence of COL-HR-222 is as follows: GGCAGCCCGGGCTATCAGGGCCCGC CGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGC.

[0050] After codon optimization, the GC content of the above DNA sequence changed from 80.09% to 69.91%, and its sequence is as follows: GGCTCCCCAGGTTACCAGGGTCCGCCGGGCGAACC (SEQ ID NO.9).

[0051] The recombinant humanized type III collagen COL-HR-333 in this invention is a triplicate repeat sequence of COL-HR-3 (SEQ ID No. 3), comprising 378 amino acids. The amino acid sequence of COL-HR-333 is as follows:

[0052] GESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGER(SEQ ID NO.6)。

[0053]

[0054]

[0055] The recombinant humanized type III collagen COL-HR-123 in this invention is a linker sequence of COL-HR-1, COL-HR-2, and COL-HR-3, comprising 239 amino acids. The amino acid sequence of COL-HR-123 is as follows:

[0056] GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGESGRPGP PGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGER(SEQ ID NO.7).

[0057] The DNA sequence of COL-HR-123 is as follows: GGCCAGGGCCCGCAGGGCCCGAAAG GCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCATTCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGCGGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGAGCGGCCCGCGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGCCGGGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGGGCAAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCGATAAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCGGCGGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCGGGCGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGC。

[0058] After codon optimization, the GC content of the above DNA sequence was changed from 80.89% to 69.87%, and the sequence is as follows: GGTCAGGGTCCGCAGGGTCCTAAGGGCGATCCAGG (SEQ ID NO.11).

[0059] The DNA sequences of the collagen mentioned above, SEQ ID NO. 8-11, are all codons of the recombinant gene optimized based on codon preference and expression capacity in the E. coli expression host using the Exp Optimizer codon optimization tool.

[0060] Table 1 Sequence List of Recombinant Humanized Type III Collagen

[0061] Recombinant humanized type III collagen amino acid sequence DNA sequence COL-HR-111 SEQ ID NO.4 SEQ ID NO.8 COL-HR-222 SEQ ID NO.5 SEQ ID NO.9 COL-HR-333 SEQ ID NO.6 SEQ ID NO.10 COL-HR-123 SEQ ID NO.7 SEQ ID NO.11

[0062] Example 2: Construction and expression of recombinant humanized type III collagen expression vector

[0063] The collagen gene sequence is shown in SEQ ID NO.8-11 and was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd.

[0064] Vectors were constructed and the target collagen proteins COL-HR-111, COL-HR-222, COL-HR-333, and COL-HR-123 were expressed and purified using the same method as in Example 2 of the applicant's authorized patent CN118324899B. The specific steps are as follows:

[0065] (1) The DNA fragments of SEQ ID NO.8-11 above were cloned and amplified by PCR, and the target gene was recovered by agarose gel electrophoresis. Using the EcoRI and NotI restriction sites added to the primers, the gene fragments were cut into sticky ends by adding the corresponding restriction endonucleases. The resulting fragments were then co-incubated with the corresponding enzyme-digested pPIC9K expression vector (Thermo Fisher Scientific), and T4 DNA ligase (TaKaRa, catalog number 2011A) was added. Ligation was performed at 4°C for 12 hours, followed by transformation into *E. coli* DH5α (purchased from Sangon Biotech (Shanghai) Co., Ltd.). White spot clones were selected using ampicillin sodium-LB plates. Plasmids were extracted using the alkaline lysis method of molecular cloning, and positive clones were identified by restriction enzyme digestion and PCR. Sequencing confirmed that the recombinant plasmids contained the correct gene sequence reading frame. The recombinant expression vectors pPIC9K-COL-HR-111, pPIC9K-COL-HR-222, pPIC9K-COL-HR-333, and pPIC9K-COL-HR-123 were successfully constructed and sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0066] (2) The correct pPIC9K-COL-HR-111, pPIC9K-COL-HR-222, pPIC9K-COL-HR-333 and pPIC9K-COL-HR-123 expression vectors were transformed into Escherichia coli BL21(DE3) (purchased from Sangon Biotech (Shanghai) Co., Ltd.). Single-spot clones were screened using ampicillin sodium-LB plates and cultured overnight in 5 ml LB medium. The cells were then transferred at a ratio of 1:100 and cultured in shake flasks at 37°C until the OD 600 reached 0.5. IPTG stock solution was added at a ratio of 1:5000 and cultured at 20°C for 8 hours. The cells were collected by centrifugation and stored at -20°C or immediately proceeded to the next purification step.

[0067] (3) Wash the mixed bacterial pellet with PBS buffer, resuspend the pellet in approximately 500 ml of bacterial solution with 20-40 ml of PBS buffer, lyse the bacteria with lysozyme and Triton X-100, sonicate for 60 min in an ice-water mixture, centrifuge at 12000 rpm / min for 20 min, and collect the supernatant. At this point, the solution contains large amounts of the target recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333, and COL-HR-123, respectively.

[0068] (4) Ammonium sulfate was selected for preliminary purification by precipitation and fractional salting out. After exploring the saturation of ammonium sulfate, the optimal saturation of ammonium sulfate salting out was found to be 20%. The Ni-NTA His-Bind Resin column was washed with PBS buffer. The column was then mixed with the desalted and purified solution and incubated for 30 min at room temperature or on ice. The column was then loaded with the solution, and impurities were washed with PBS containing 15 mM imidazole, leaving only pure type III collagen on the column. An appropriate amount of His-tagged Prescission Protein was added to the column, and the column was incubated for 2 hours at room temperature or on ice to release the type III collagen. The collagen was then eluted with PBS to obtain high-purity recombinant humanized type III collagen COL-HR-111 (SEQ ID NO.4), COL-HR-222 (SEQ ID NO.5), COL-HR-333 (SEQ ID NO.6), and COL-HR-123 (SEQ ID NO.8), which were then lyophilized for later use.

[0069] Example 3: Evaluation of the cytotoxicity of recombinant humanized type III collagen

[0070] HeLa cells that have grown to 80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin and cultured in complete medium to a cell density of 1×10⁻⁶ cells / year. 5Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C with 5% CO2 saturated humidity. After 24 h of cell culture, the complete culture medium was aspirated. Recombinant humanized type III collagen solution diluted to 0.05 mg / mL in high-glucose DMEM medium was added to the experimental groups (three replicates per concentration). The control group consisted of cells cultured in DMEM medium, and the blank group consisted of cells-free DMEM medium. Both groups were cultured for another 24 h at 37°C with 5% CO2 saturated humidity. 10 μL of CCK-8 reagent was added to each group, and the cells were incubated for 2 h. The absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. Cell viability was calculated based on the mean absorbance of each group using the following formula:

[0071]

[0072] In this embodiment, the positive control group was a commercially available recombinant type III collagen sample solution, which was added to the cell culture medium at a concentration of 0.05 mg / ml, with 3 replicates per group.

[0073] Experimental results are as follows Figure 1 As shown, the cell viability of each experimental group was above 100%, indicating that the recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333 and COL-HR-123 prepared in this invention are all non-cytotoxic and have good safety.

[0074] Example 4: Evaluation of the cell proliferation-promoting activity of recombinant humanized type III collagen

[0075] BALB / c 3T3 cells (mouse embryonic fibroblasts) in good logarithmic growth phase (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were selected and seeded in 96-well cell culture plates. After digestion with 0.25% trypsin, centrifugation, and resuspending in DMEM medium containing 10% fetal bovine serum, the cells were counted. BALB / c 3T3 cells were seeded in 96-well culture plates at approximately 5 × 10⁶ cells per well. 3 A blank control without cells was set up, and the cells were cultured overnight at 37°C and 5% CO2. Recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333, and COL-HR-123 prepared in Example 2 were added to final concentrations of 0.05 mg / ml and 0.1 mg / ml, respectively, with three replicates per group, and cultured at 37°C and 5% CO2 for 48 h. Four h before the end of the culture, 5 g / L MTT solution was added, and the culture was continued for another 4 h before termination. The liquid in the wells was aspirated, the cells were washed once with PBS, dissolved and crystallized in DMSO, and the absorbance of each well was measured at 570 nm.

[0076] Cell proliferation rate = (absorbance value of measurement well - absorbance value of blank control group) / (absorbance value of cell control group - absorbance value of blank control group) × 100%.

[0077] In this embodiment, the positive control group was a commercially available recombinant type III collagen sample solution, with 3 parallel samples in each group.

[0078] like Figure 2 As shown, the experimental results indicate that, under the conditions of concentrations of 0.05 mg / ml and 0.1 mg / ml, both the collagen in the control group and the recombinant humanized type III collagen of this application promoted the proliferation of BALB / c 3T3 cells, and the effect was better than that of commercially available recombinant type III collagen.

[0079] Example 5: Evaluation of the cell adhesion-promoting activity of recombinant humanized type III collagen

[0080] In Example 2, recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333, and COL-HR-123 were dissolved in PBS and added to final concentrations of 0.05 mg / ml and 0.1 mg / ml, respectively. 50 μL of the recombinant humanized type III collagen solution was added to each well of a 96-well cell culture plate and incubated at 37°C for 2 hours. PBS was added to the control wells. BALB / 3T3 cells were digested with trypsin, counted, and 5 × 10⁵ mg / ml solution was added to each well. 4 Cells were cultured at 37°C and 5% CO2 for 2 hours. The cells were washed three times with PBS to remove unadhered cells, and then 200 μL of LMEM medium was added. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C and 5% CO2 for 2 hours. The absorbance of the 96-well plate was read at 450 nm and 630 nm using a microplate reader. Using 630 nm as the reference wavelength, the absorbance was measured at 450 nm, and the results were recorded.

[0081] Cell adhesion promotion rate = (absorbance value of experimental group at 450nm - absorbance value of negative control group at 450nm) / absorbance value of negative control group at 450nm × 100%.

[0082] In this embodiment, the positive control group was a commercially available recombinant type III collagen sample solution, which was added to the cell culture medium at a concentration of 0.10 mg / ml, with 3 replicates per group.

[0083] Experimental results are as follows Figure 3As shown, the experimental results indicate that, under the conditions of concentrations of 0.05 mg / ml and 0.1 mg / ml, both the collagen in the control group and the recombinant humanized type III collagen of this application promoted the adhesion of BALB / c 3T3 cells, and the effect was better than that of commercially available recombinant type III collagen.

[0084] Based on the above embodiments, the recombinant humanized type III collagen expressed in this invention has a uniform molecular weight, good solubility, and excellent cell adhesion and proliferation properties, and can be applied in a variety of fields, such as cosmetics or pharmaceutical products.

[0085] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A recombinant humanized type III collagen, characterized in that, The amino acid sequences of the collagen are shown in SEQ ID NO.6-7.

2. A nucleic acid molecule encoding the recombinant humanized type III collagen as described in any one of claims 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO.10-11, respectively.

4. A recombinant expression vector comprising the nucleic acid molecule according to any one of claims 2-3.

5. A host cell comprising the recombinant expression vector according to claim 4 or expressing the collagen according to claim 1.

6. The host cell according to claim 5, characterized in that, The host cell is Escherichia coli.

7. The method for producing recombinant humanized type III collagen according to claim 1, characterized in that, The procedure includes the following steps: (1) culturing the host cells according to claim 5 in a culture medium; and (2) isolating the recombinant humanized type III collagen according to any one of claims 1 from the host cells.

8. A composition, characterized in that, The composition comprises recombinant humanized type III collagen according to any one of claims 1, or recombinant humanized type III collagen encoded by a nucleic acid molecule according to any one of claims 2-3, or recombinant humanized type III collagen expressed by a recombinant expression vector according to claim 4, or recombinant humanized type III collagen produced by a host cell according to any one of claims 5-6, or recombinant humanized type III collagen prepared by the production method according to claim 7.

9. An article comprising the composition according to claim 8, characterized in that, The product is a pharmaceutical composition, a medical device, or a cosmetic.

10. Use of the composition of claim 8 or the article of claim 9 in the preparation of a medicament for promoting wound healing.

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