A recombinant humanized type XVII collagen, its preparation method and application

Abstract

This invention discloses a recombinant humanized type XVII collagen, its preparation method, and its applications, belonging to the field of collagen. Based on the natural human type XVII collagen sequence, this invention screens five core active polypeptide fragments and splices them together to obtain a novel recombinant humanized collagen COL17-HR. Codon optimization addresses the defects of high GC content and low expression efficiency in *E. coli* of the original human gene. Using the pET-28a(+) / BL21(DE3) prokaryotic expression system, the optimized gene expression level significantly increased by 82.6%. The collagen of this invention is free of animal-derived contamination and has extremely low immunogenicity, possessing high industrial application value and market prospects in the fields of skin barrier repair, wound healing, anti-aging skincare, hair follicle care, and medical repair materials.

Description

Technical Field

[0001] This invention relates to the field of collagen, specifically to a recombinant humanized type XVII collagen, its preparation method, and its application. Background Technology

[0002] Type XVII collagen (COL17A1) is a key transmembrane functional protein of the epidermal-dermal basement membrane in human skin. It is mainly expressed in keratinocytes and is a core component of the hemidesmosome structure. It plays a core physiological role in maintaining epidermal adhesion stability, repairing damaged basement membranes, regulating skin cell proliferation and differentiation, delaying skin photoaging, and nourishing hair follicle stem cells. It has an irreplaceable role in sensitive skin repair, wound healing, epidermal barrier reconstruction, and maintaining healthy hair follicles.

[0003] Natural human-derived type XVII collagen has a large molecular weight and complex sequence structure, containing transmembrane hydrophobic domains and a large number of disordered redundant sequences. In its natural state, it is an insoluble fibrous protein, and its expression level in human tissues is extremely low, making it impossible to prepare on a large scale using animal tissue extraction methods. Traditional animal-derived collagen extraction processes suffer from drawbacks such as immune rejection of foreign proteins, residual viral pathogens, damage to the protein triple helix structure, and severe loss of activity, making them unsuitable for high-end skincare and medical repair applications.

[0004] Current technologies for the industrialization of recombinant type XVII collagen still face numerous insurmountable technical bottlenecks, severely hindering its large-scale application and market penetration. Firstly, most existing technologies directly clone and express the full-length human type XVII collagen sequence. This sequence contains numerous hydrophobic transmembrane domains and disordered redundant regions, making it highly susceptible to protein misfolding and the formation of numerous inactive inclusion bodies during heterologous expression. This results in extremely low soluble protein yields, failing to meet the demands of industrial-scale production. Secondly, some truncated modification technologies lack precise bioinformatics functional domain screening support, relying mostly on empirical random truncation. This fails to accurately preserve core active sites, leaving behind a large number of invalid sequences, leading to weak bioactivity, poor skin repair effects, and insufficient product competitiveness in the prepared collagen. Thirdly, the base structure of the human natural type XVII collagen encoding gene is unique, with an extremely high GC content and enrichment of numerous rare E. coli codons. This easily leads to the formation of stable mRNA secondary structures during transcription, severely hindering transcription and translation processes. This results in low prokaryotic expression efficiency, high fermentation production costs, and difficulty in achieving large-scale mass production. Fourth, the currently used purification processes in the industry are simple and have low refining levels, resulting in products with many residual proteins and substandard purity. Furthermore, the recombinant collagen produced has poor temperature and acid / alkali resistance, making it highly susceptible to inactivation and denaturation during high-temperature processing in skincare products, wide-pH formulation adjustments, and long-term storage at room temperature, thus limiting its applicability. Fifth, existing research largely focuses on basic protein expression, lacking systematic parallel controlled experiments, quantitative activity verification, and stability data. This results in low technological maturity, poor industrialization feasibility, and an inability to meet the stringent quality standards of high-end cosmetics and medical repair materials.

[0005] To address the shortcomings of existing technologies, this invention employs precise bioinformatics functional domain screening to splice together highly active short-fragment recombinant type XVII collagen. Simultaneously, it conducts targeted codon balance optimization and establishes standardized fermentation and high-precision purification processes to obtain highly expressed, highly pure, highly active, and highly stable humanized type XVII collagen, thus completely solving the industry pain points of existing technologies, such as difficulty in mass production, low activity, poor stability, and limited applications. Summary of the Invention

[0006] Based on the technical problems existing in the prior art, the technical solution of the present invention is implemented as follows: The present invention provides a recombinant humanized type XVII collagen, the amino acid sequence of which is shown in SEQ ID NO:7.

[0007] Furthermore, the recombinant humanized type XVII collagen is assembled from the core active functional fragments SEQ ID NO:2-6 of the original sequence (SEQ ID NO:1) of human type XVII collagen COL17A1.

[0008] The present invention also provides a nucleic acid molecule encoding the recombinant humanized type XVII collagen described above.

[0009] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.9.

[0010] The present invention also provides a recombinant expression vector comprising the aforementioned nucleic acid molecule.

[0011] Furthermore, the recombinant expression vector is constructed by inserting the nucleotide sequence shown in SEQ ID NO:9 into the vector restriction enzyme site using pET-28a(+) as the backbone vector.

[0012] The present invention also provides a host cell comprising the recombinant expression vector according to the invention.

[0013] It should be noted that "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., by nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. "Host cell" encompasses any progeny of the parent cell that is not entirely identical to the parent cell due to mutations during replication. The host cell can be any cell useful in the production of recombinant humanized collagen according to the present invention. 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 the recombinant collagen). The host cell refers to a cell in which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations. Methods for introducing vectors into host cells are well-known, such as electroporation, transfection, microinjection, gene gun technology, and liposome-mediated methods. The host cell is 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.

[0014] Furthermore, the host cell is Escherichia coli BL21(DE3) strain.

[0015] The present invention also provides a method for producing the recombinant humanized type XVII collagen, characterized by comprising the following steps: (1) culturing the host cells in a culture medium; (2) isolating the recombinant humanized type XVII collagen from the host cells.

[0016] The present invention also provides a composition, characterized in that the composition comprises the recombinant humanized type XVII collagen, or the recombinant humanized type XVII collagen encoded by the nucleic acid molecule, or the recombinant humanized type XVII collagen expressed by the recombinant expression vector, or the recombinant humanized type XVII collagen produced by the host cell, or the recombinant humanized type XVII collagen prepared by the production method.

[0017] The present invention also provides the application of the recombinant humanized type XVII collagen or the composition thereof in the preparation of skin repair, anti-aging and hair follicle care products.

[0018] Furthermore, the product is a pharmaceutical composition, a medical device, or a cosmetic.

[0019] 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.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention precisely screens and splices the five core active functional domains of human type XVII collagen to prepare COL17-HR recombinant protein, which fully retains the core biological functions of natural type XVII collagen in maintaining epidermal-dermal connection, repairing damaged basement membrane and rebuilding skin barrier, and avoids the defects of full-length protein being difficult to dissolve, easy to form inclusion bodies and lack of activity.

[0021] (2) To improve protein mass production performance, this invention performs host-specific codon optimization for E. coli, reducing the GC content of the gene from 79.81% to 68.74%, significantly reducing the proportion of rare codons and unfolding the overfolded structure of mRNA. Experimental data show that the expression level of the target protein after optimization is increased by 82.6% compared with the original sequence, and the proportion of soluble protein is significantly increased, effectively improving mass production capacity and reducing industrial production costs.

[0022] (3) The COL17-HR protein of this invention has excellent environmental stability and can tolerate a wide range of temperature and acid and alkaline environments. It is perfectly suited to the conventional process conditions for skin care product production and storage and medical dressing processing. It has extremely high industrial application value and market prospects in the fields of sensitive skin barrier repair, superficial wound healing and repair, skin basement membrane regeneration and repair, high-end anti-aging skin care and medical repair materials. Attached Figure Description

[0023] Figure 1Translation curve of recombinant humanized type XVII collagen described in this invention (the wavy line is the blue line, and the horizontal line in the middle is the red line); wherein, A is SEQ ID NO:8 before optimization, and B is SEQ ID NO:9 after optimization.

[0024] Figure 2 : Comparison of Western blot expression of recombinant type XVII collagen in this invention.

[0025] Figure 3 The protein activity retention rate of recombinant humanized type XVII collagen COL17-HR under different temperatures and incubation times.

[0026] Figure 4 The protein activity retention rate of recombinant humanized type XVII collagen COL17-HR under different pH conditions. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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 "%".

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Example 1: Design of the amino acid sequence of recombinant humanized type XVII collagen Based on the sequence of human type XVII collagen (https: / / www.uniprot.org / uniprotkb / Q9UMD9 / entry; Collagen alpha-1(XVII) chain, amino acid sequence as shown in SEQ ID NO:1, 1497AAs) obtained from the uniport database, and taking advantage of the highly repetitive amino acid sequences of natural collagen, SMART functional domain prediction and NCBI Conserved Domain analysis were used to remove non-biologically active redundant sequences, and five highly conserved and highly active core functional fragments (SEQ ID NO:2-6) were screened. Details are shown below: MDVTKKNKRDGTEVTERIVTETVTTRLTSLPPKGGTSNGYAKTASLGGGSRLEKQSLTHGSSGYINSTGSTRGHASTSSYRRAHSPASTLPNSPGSTFERKTHVTRHAYEGSSSGNSSPEYPRKEFASSSTRGRSQTRESEIRVRLQSASPSTRWTELDDVKRLLKGSRSASVSPTRNSSNTLPIPKKGTVETKIVTASSQSVSGTYDATILDANLPSHVWSSTLPAGSSMGTYHNNMTTQSSSLLNTNAYSAGSVFGVPNNMASCSPTLHPGLSTSSSVFGMQNNLAPSLTTLSHGTTTTSTAYGVKKNMPQSPAAVNTGVSTSAACTTSVQSDDLLHKDCKFLILEKDNTPAKKEMELLIMTKDSGKVFTASPASIAATSFSEDTLKKEKQAAYNADSGLKAEANGDLKTVSTKGKTTTADIHSYGSSGGGGSGGGGGVGGAGGGPWGPAPAWCPCGSCCSWWKWLLGLLLTWLLLLGLLFGLIALAEEVRKLKARVDELERIRRSILPYGDSMDRIEKDRLQGMAPAAGADLDKIGLHSDSQEELWMFVRKKLMMEQENGNLRGSP GPKGDMGSPGPKGDRGFPGTPGIPGPLGHPG PQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAA GEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPM GPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQG PRGEQ GLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPAGLPGHQEVLNLQ GPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSF LSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQ GPPGPPGPQGPKGDKGDPGVPGALGIPSGP SEGGSSSTMYVSGPPGPPGPPGPPGSISSSGQEIQQYISEYMQSDSIRSYLSGVQGPPGPPGPPGPVTTITGETFDYSELASHVVSYLRTSGYGVSLFSSSISSEDILAVLQRDDV RQYLRQYLMMGPRGPPGPPGASGDGSLLSLDYAELSSRILSYMSSSGISIGLPGPPGPPGLPGTSYEELLSLLRGSEFRGIVGPPGPPGPPGIPGNVWSSISVEDLSSYLHTAGLSF IPGPPGPPGPPGPRGPPGVSGALATYAAENSDSFRSELISYLTSPDVRSFIVGPPGPPGPQGPPGDSRLLSTDASHSRGSSSSSHSSSVRRGSSYSSSMSTGGGGAGSLGAGGAF GEAAGDRGPYGTDIGPGGGYGAAAEGGMYAGNGGLLGADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQGPPGQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQK GEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDK GDQVYAGRRRRRSIAVKP (SEQ ID NO: 1).

[0034] Fragment 1 (SEQ ID NO:2): GPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAA, amino acids 570-649, the core segment of cell basement membrane specific adhesion, mediating the adhesion of skin cells to the basement membrane.

[0035] Fragment 2 (SEQ ID NO:3): GPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQ, amino acids 704-757, promotes keratinocyte migration and proliferation, and accelerates epidermal wound healing and regeneration.

[0036] Fragment 3 (SEQ ID NO:4): GPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSF, amino acids 857-896, strengthens the stability of epithelial cell junctions and effectively repairs defects in the epidermal barrier structure.

[0037] Fragment 4 (SEQ ID NO:5): GPPGPPGPQGPKGDKGDPGVPGALGIPSGPS, amino acids 951-981, stabilize the hemidesmosome structure, anchor the epidermal-dermal junction, and maintain the integrity of the skin basement membrane.

[0038] Fragment 5 (SEQ ID NO:6): GEMGTGPKGDRGPAGPPGHPGPPGPRGHKGEKGDK, amino acids 1444-1479, regulates the orderly regeneration of epidermal cells, repairs the photodamage barrier, and improves the state of skin aging and damage.

[0039] The fragments shown in SEQ ID NO:2-6 were spliced ​​without gaps to obtain a novel recombinant humanized type XVII collagen COL17-HR, with a 241-amino acid sequence as shown in SEQ ID NO:7: GPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA GPDGHQGPRGEQGPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSFGPPGPPGPQGPKGDKGDPGVPGALGIPSGPSGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDK (SEQ ID NO:7).

[0040] Based on the amino acid sequence shown in SEQ ID NO:7, its coding nucleic acid sequence was reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ), as shown in SEQ ID NO:8, with a full length of 723bp.

[0041] GGCCCGAAAGGCGATATGGGCAGCCCGGGCCCGAAAGGCGATCGCGGCTTTCCGGGCACCCCGGGCATTCCGGGCCCGCTGGGCCATCCGGGCCCGCAGGGCCCGAAAGGCCAGAAAGGCAGCGTGGGCGATCCGGGCATGGAAGGCCCGATGGGCCAGCGCGGCCGCGAAGGCCCGATGGGCCCGCGCGGCGAAGCGGGCCCGCCGGGCAGCGGCGAAAAAGGCGAACGCGGCGCGGCGGGCCCGCCGGGCCCGAAAGGCGATCAGGGCGAAAAAGGCCCGCGCGGCCTGACCGGCGAACCGGGCATGCGCGGCCTGCCGGGCGCGGTGGGCGAACCGGGCGCGAAAGGCGCGATGGGCCCGGCGGGCCCGGATGGCCATCAGGGCCCGCGCGGCGAACAGGGCCCGCCGGGCCCGCCGGGCCCGCCCGGCCCGCCGGGCCCGAGCATTCCGGGCCCGCCGGGCCCGCCCGGCCCGCCGGGCGAAGGCCTGCCGGGCCCGCCGGGCCCGCCGGGCAGCTTTGGCCCGCCGGGCCCGCCGGGCCCGCAGGGCCCGAAAGGCGATAAAGGCGATCCGGGCGTGCCGGGCGCGCTGGGCATTCCGAGCGGCCCGAGCGGCGAAATGGGCACCCCGGGCCCGAAAGGCGATCGCGGCCCGGCGGGCCCGCCGGGCCATCCGGGCCCGCCGGGCCCGCGCGGCCATAAAGGCGAAAAAGGCGATAAA(SEQ ID NO:8)。

[0042] The nucleotide sequence shown in SEQ ID NO:8 has a GC content as high as 79.81%, indicating a severe imbalance in base ratios. It also exhibits a high proportion of high-frequency rare codons in *E. coli*, resulting in extremely high mRNA secondary structure free energy and a tendency to form stable hairpin folds. This significantly hinders transcription and translation in *E. coli*, leading to extremely low protein expression levels and poor translation efficiency, failing to meet mass production requirements. Using the online codon optimization tool (ExpOptimizer) (https: / / www.novopro.cn / tools / codon-optimization.html) targeting the codon preference library for *E. coli* BL21(DE3), the optimized nucleotide sequence is shown in SEQ ID NO:9. Compared to the original gene sequence, this sequence reduces the GC content from 79.81% to 68.74%, making it more conducive to efficient gene expression in the host bacterium *E. coli*.

[0043] GGTCCGAAAGGCGATATGGGTTCTCCTGGCCCTAAAGGTGATCGTGGTTTCCCAGGTACCCCAGGCATTCCGGGTCCGCTGGGCCACCCGGGCCCGCAGGGTCCGAAAGGTCAGAAAGGTTCTGTTGGTGACCCAGGTATGGAAGGTCCGATGGGTCAGCGTGGCCGTGAAGGTCCGATGGGTCCGCGTGGCGAAGCAGGTCCGCCTGGTTCTGGCGAAAAAGGTGAACGCGGTGCGGCAGGTCCGCCGGGTCCGAAAGGCGACCAGGGTGAAAAAGGTCCGCGTGGTCTGACCGGTGAGCCGGGTATGCGTGGTCTGCCGGGCGCGGTAGGCGAACCGGGCGCTAAAGGCGCAATGGGTCCGGCGGGTCCGGATGGCCACCAGGGTCCGCGTGGTGAACAGGGCCCGCCGGGTCCACCTGGTCCGCGTGGTCCGCCGGGTCCGTCCATCCCTGGCCCGCCGGGTCCGCGTGGTCCTCCGGGTGAGGGCCTGCCGGGTCCGCCTGGCCCTCCGGGTTCTTTTGGCCCGCCAGGTCCGCCGGGTCCGCAGGGCCCGAAAGGTGATAAAGGTGATCCGGGTGTTCCGGGCGCGCTGGGTATTCCGAGCGGCCCGTCCGGCGAGATGGGCACCCCAGGCCCGAAAGGCGATCGTGGTCCAGCAGGTCCACCGGGTCACCCGGGCCCGCCGGGCCCACGTGGTCATAAGGGTGAAAAAGGTGACAAA (SEQ ID NO:9).

[0044] The translation pause curves calculated by the RiboTempo software for type XVII collagen proteins before and after codon optimization are shown respectively as Figure 1As shown in A (before optimization) and B (after optimization), the red line of the translation pausing curve forms translation pausing sites in the predetermined region (the red line is lower than the blue line). The translation rate of the unoptimized nucleotide bases in SEQ ID NO: 8 on the ribosome is relatively constant, without any decrease in translation rate. However, the optimized SEQ ID NO: 9 shows a significant decrease in translation rate on the ribosome throughout the translation process, with more translation pausing sites appearing (the red line is lower than the blue line). This allows the translated collagen sufficient time to fold, thus yielding a collagen product with higher biological activity and expression levels.

[0045] Shanghai Sangon Biotech Co., Ltd. synthesized SEQ ID NO:8 and SEQ ID NO:9 sequences. NdeI (5' end) and XhoI (3' end) specific restriction enzyme sites were introduced at both ends of the gene, respectively. Sequencing after synthesis verified that the sequences were 100% correct, with no base mutations, deletions, or insertions, and can be directly used for vector construction and expression.

[0046] Example 2 Construction and expression of recombinant humanized type XVII collagen expression vector The experimental vector used was the pET-28a(+) prokaryotic expression vector, and the host cell was Escherichia coli BL21(DE3) competent cells.

[0047] (1) Construction of recombinant expression vector Two vectors were constructed: the control group contained the unoptimized gene SEQ ID NO:8+pET-28a(+), and the experimental group contained the codon-optimized gene SEQ ID NO:9+pET-28a(+). The two vectors were constructed using the same method.

[0048] 1) Double enzyme digestion reaction: Prepare a 50 μL standard enzyme digestion system containing 1 μg of target gene / vector plasmid, 1 μL of NdeI, 1 μL of XhoI, 5 μL of 10×QuickCut Buffer, and sterile deionized water to make up the system; after mixing, digest at 37℃ for 4 h to ensure that the target fragment and vector are completely linearized.

[0049] 2) Enzyme digestion product recovery: After enzyme digestion, the target nucleic acid fragments were separated by 1% agarose gel electrophoresis. The products were purified using a commercial DNA gel recovery kit, and the nucleic acid purity was tested and OD was controlled. 260 The value is between 1.8 and 2.0.

[0050] 3) Ligation of target fragment with vector: Prepare a 10 μL ligation system according to the vector:target fragment molar ratio of 1:3, including linearized vector, target gene, 0.5 μL T4 DNA ligase, 1 μL 10×T4 Buffer, and sterile water to make up the system; after mixing by low speed centrifugation, ligate overnight in a 16℃ metal bath for 12 h.

[0051] 4) Transformation of competent cells: Add 5 μL of ligation product to 100 μL of BL21(DE3) competent cells, mix gently, and incubate on ice for 30 min; perform precise heat shock at 42℃ for 90 s, quickly remove and cool on ice for 2 min to complete plasmid transformation; add 900 μL of antibiotic-free LB liquid medium to the system, and incubate at 37℃ and 180 rpm for 1 h.

[0052] 5) Initial screening of positive strains: Take 200 μL of the revived bacterial solution and spread it evenly on LB solid plates containing 100 μg / mL kanamycin. Incubate at 37°C inverted for 12 h until single colonies grow evenly. Select round, plump, and uniformly sized single colonies for later use.

[0053] (2) Double verification of positive engineered strains To eliminate false-positive strains containing empty vectors or missing inserted fragments, both groups of single colonies underwent dual verification using colony PCR and plasmid double enzyme digestion. First, colony PCR amplification was performed; strains showing specific bands of corresponding size on electrophoresis were initially screened as positive. Subsequently, plasmids from positive strains were extracted and again verified by NdeI and XhoI double enzyme digestion. Electrophoresis detected both the vector band and the target gene band simultaneously, with fragment sizes completely consistent with theoretical values, proving successful vector construction, correct insertion of the target gene, and absence of mutations. Finally, qualified positive engineered strains for the control and experimental groups were obtained and stored at 4°C for later use.

[0054] (3) Parallel induction expression experiment 1) Seed culture: Two groups of verified positive single colonies were picked and inoculated into 20 mL of LB liquid medium containing 100 μg / mL kanamycin. The culture was carried out overnight at 37℃ with shaking at 180 rpm for 12 h to obtain a stable seed culture.

[0055] 2) Expanded fermentation culture: Following a uniform inoculation ratio of 1%, the two groups of seed cultures were transferred to 500 mL of LB liquid medium with equivalent resistance. The cultures were incubated at 37°C with continuous shaking at 180 rpm, and OD was monitored in real time. 600 Stop culturing when the value stabilizes at 0.6 to ensure that the growth status of the two groups of bacteria is completely consistent.

[0056] 3) Low-temperature induction of expression: IPTG inducer was added to both groups of bacterial cultures, with a final concentration of 0.25 mmol / L. The culture parameters were adjusted to 30℃ and 90 rpm low-temperature and low-speed shaking, and expression was induced for 6 h.

[0057] 4) Cell collection: After the induction culture is completed, the two groups of bacterial solutions are separately dispensed into centrifuge tubes, centrifuged at 5000 rpm and 4℃ for 20 min to completely remove the supernatant culture medium, collect the pure bacterial precipitate, and store it at -20℃ for short-term use in subsequent protein extraction and quantification.

[0058] (4) Protein extraction and quantitative detection Equal volumes of Binding Buffer (20 mM imidazole, 12 mM sodium phosphate, 500 mM NaCl, pH 7.4) were added to both groups of bacterial pellets to fully resuspend the cells. The cells were then disrupted using an ultrasonic cell disruptor with uniform parameters: 300 W power, 2 s operation, 3 s pause, total disruption time 4 min, under ice bath conditions to avoid high-temperature protein denaturation. After disruption, the cells were centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant soluble protein solution was collected. The BCA protein quantification kit was used, with three replicates per group, to measure absorbance at 450 nm. The mean and standard deviation of the sample protein concentration were calculated, and the final results are shown in Table 1.

[0059] Table 1. Expression levels of recombinant type XVII collagen before and after codon optimization (n=3, x±SD)

[0060] Table 1 shows that, under completely identical culture, induction, and extraction conditions, the effective expression level of the target type XVII collagen in the codon-optimized experimental group (SEQ ID NO:9) increased from 79.65 mg / L to 145.42 mg / L, representing an overall increase of 82.6%. This demonstrates that the codon optimization strategy of this invention completely solves the defects of excessively high GC content and transcriptional translation arrest in the human COL17A1 gene, significantly improving the soluble expression capacity of recombinant type XVII collagen. This provides reliable process support for the large-scale industrial production of highly active barrier repair and wound repair functional proteins, and effectively reduces fermentation preparation costs.

[0061] (6) Western Blot protein expression experiment To further verify the expression differences and protein specificity of recombinant type XVII collagen between the two groups before and after codon optimization, this experiment used Western blotting for quantitative analysis. The WB results are as follows: Figure 2 As shown, both groups of samples exhibited a single, specific target band at the target molecular weight, demonstrating that the recombinant protein of this invention has strong expression specificity and is free from interference from other proteins. Furthermore, the grayscale of the band in the codon-optimized experimental group was significantly higher than that in the unoptimized control group, proving that codon optimization can significantly improve the overall expression level of recombinant type XVII collagen, which is consistent with the trend of BCA quantitative detection results.

[0062] Example 3: Purification and biological activity evaluation of recombinant humanized type XVII collagen In this embodiment, the COL17-HR protein expressed in the codon-optimized experimental group was purified, and the biological activity of collagen was quantitatively detected by constructing an epidermal wound scratch model and a Transwell epidermal barrier model using HaCaT keratinocytes.

[0063] (1) Purification of the target protein 1) Cell disruption: Take the cell pellet from the codon-optimized experimental group, add Binding Buffer (20mM imidazole, 12mM sodium phosphate, 500mM NaCl, pH 7.4) at a ratio of 1:20, resuspend thoroughly, and sonicate on ice bath with the same parameters as in Example 2. 2) Coarse separation: Centrifuge at 12000 rpm and 4℃ for 15 min, collect the supernatant crude protein solution, and filter through a 0.45 μm filter membrane to remove impurities; 3) Nickel column affinity chromatography: After equilibration of the Ni-NTA column, load the sample, elute with 10 column volumes of elution buffer to remove contaminating proteins, elute with gradient elution buffer to remove the target protein, and collect the elution peaks in segments. 4) Acid-adjusting purification: Add 0.25M NaCl to the eluent, adjust the pH to 4.0 with phosphoric acid, incubate at 30℃ for 60 min to remove impurities and denatured proteins, and centrifuge to collect the supernatant; 5) Buffer replacement: Dialyze with 100-fold volume of PBS buffer (100mM NaCl, pH 7.4) at 4℃ for 18 hours, changing the dialysate every 6 hours; 6) Concentration and preservation: After ultrafiltration concentration, high-purity COL17-HR protein is obtained and aliquoted and stored at -80℃ for later use.

[0064] (2) Protein purity test results The purified protein was detected by 12% SDS-PAGE electrophoresis. A single, clear band was observed, with no impurities or degradation bands. The protein purity was ≥98% according to grayscale scanning analysis.

[0065] Cellular scratch wound migration and healing activity assay Human immortalized keratinocytes (HaCaT) were used to construct an in vitro cell scratch wound model to simulate the epidermal wound healing process and verify the activity of recombinant collagen in promoting wound repair and accelerating epidermal migration and regeneration. The experiment included a blank control group (pure culture medium) and experimental groups 1-3 with 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL recombinant collagen COL17-HR, with 6 replicates per group. HaCaT cells were seeded in 6-well plates and cultured routinely until complete cell confluence to form a monolayer. A sterile 200 μL pipette tip was used to make vertical and uniform scratches. The cells were gently washed with PBS to remove detached cells and eliminate any residual suspended cells at the scratch edges. Culture medium containing different concentrations of recombinant collagen COL17-HR was added, and the plates were incubated at 37°C in a 5% CO2 incubator in the dark. Microscopic images of the healed area were taken at 0 h, 24 h, and 48 h. The remaining scratch area was statistically analyzed using ImageJ software, and the wound healing rate was calculated. The results are shown in Table 2.

[0066] Table 2. Effects of different concentrations of recombinant collagen on wound healing rate of HaCaT cells (n=6, x±SD)

[0067] (4) Epidermal barrier function repair experiment Using a HaCaT cell monolayer barrier model, the transepithelial electrical resistance (TEER) was measured to evaluate the repair and enhancement capabilities of recombinant collagen on the skin's epidermal barrier. The experiment included a blank control group, a positive control group using commercially available type XVII collagen, and an experimental group using 1.0 mg / mL of the recombinant type XVII collagen COL17-HR from this invention, with six replicates per group. HaCaT cells were seeded in Transwell chambers and cultured until a dense monolayer barrier structure was formed. Initial TEER values ​​were measured. Corresponding collagen samples were added to each group and cultured for 48 hours. Transepithelial electrical resistance values ​​were measured at 24 and 48 hours of culture. Higher resistance values ​​indicated better epidermal barrier integrity and density. The results are shown in Table 3.

[0068] Table 3. Effects of each experimental group on the TEER value of the epidermal barrier (n=6, x±SD)

[0069] Experimental Conclusions: The recombinant humanized type XVII collagen COL17-HR prepared in this invention exhibits good biocompatibility and no cytotoxicity, possessing excellent dual repair functions for epidermal wounds and skin barrier. Concentration gradient experiments show that this protein can promote HaCaT keratinocyte migration and accelerate epidermal wound closure in a concentration-dependent manner, with 1.0 mg / mL being the optimal concentration for best wound healing and repair. TEER barrier assay results confirm that the recombinant collagen of this invention can significantly increase the transepithelial electrical resistance of the epidermal monolayer barrier, effectively enhancing the density and structural integrity of the skin barrier, and its repair activity is significantly superior to commercially available type XVII collagen. This invention retains the core functions of natural type XVII collagen in stabilizing the basement membrane, rebuilding the epidermal barrier, and promoting wound regeneration, demonstrating clear technical advantages and application value for repairing damaged skin barriers, healing superficial wounds, and repairing sensitive skin.

[0070] Example 4: Stability test of recombinant humanized type XVII collagen To verify the processing and storage suitability of COL17-HR protein, this embodiment conducted temperature and pH tolerance experiments, using protein bioactivity retention rate as the core evaluation index to quantitatively detect protein stability. (1) High temperature stability test A 1 mg / mL standard COL17-HR protein solution was prepared and divided into three groups. The groups were incubated at constant temperatures of 40℃, 50℃, and 60℃, respectively. Samples were taken at 4 h, 8 h, and 12 h. After rewarming to room temperature, the barrier repair function activity of the protein after heat stress was quantitatively evaluated using the HaCaT cell TEER epidermal barrier resistance assay. The activity of fresh, untreated protein was used as a 100% baseline, and the protein activity retention rate of each group was calculated. The results are as follows: Figure 3 As shown.

[0071] (2) Acid and alkali resistance test The pH of 1 mg / mL COL17-HR protein solution was adjusted to 4.0, 5.0, 8.0, and 9.0, respectively, and incubated at room temperature in a sealed container for 12 h and 24 h. After adjusting the pH back to 7.4, the barrier repair activity retention capacity of the protein under different acid-base stresses was evaluated using the TEER epidermal barrier function assay. The clarity of the protein solution was observed visually simultaneously, with fresh protein at pH 7.4 as a blank control. The protein state was also observed. The results are as follows: Figure 4 As shown.

[0072] Experimental Conclusion: This experiment used TEER (Tissue-Assisted Evolution) epidermal barrier repair activity as the core quantitative evaluation index to systematically verify the environmental tolerance and stability of recombinant humanized type XVII collagen COL17-HR. The experiment showed that this protein possesses excellent thermal stability and acid-base tolerance. Under conventional skincare processing temperatures of 40–50℃, the barrier repair activity retention rate can reach over 95% after long-term incubation. It can withstand high-temperature sterilization of raw materials and heat treatment processes. Simultaneously, it is compatible with pH 4.0–9.0 acid-base formulation systems, showing no turbidity or denaturation after 24 hours of standing at room temperature. It exhibits high retention rate of core activities for wound healing and skin barrier repair with extremely low functional attenuation. This invention, recombinant humanized type XVII collagen COL17-HR, solves the industry pain points of traditional type XVII collagen, such as poor structural stability, easy loss of repair activity, weak formulation compatibility, and limited processing and storage. It is compatible with various skincare formulations and medical dressing preparation processes, including acidic soothing and alkaline effects, resulting in a longer product shelf life and strong industrial adaptability and practicality.

[0073] 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 XVII collagen, characterized in that, The amino acid sequence of the recombinant humanized type XVII collagen is shown in SEQ ID NO:

7.

2. A nucleic acid molecule encoding the recombinant humanized type XVII collagen of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

9.

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

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector was constructed by inserting the nucleotide sequence shown in SEQ ID NO:9 into the vector restriction enzyme site using pET-28a(+) as the backbone vector.

6. A host cell comprising the recombinant expression vector according to any one of claims 4-5.

7. The host cell according to claim 6, characterized in that, The host cell is Escherichia coli BL21(DE3) strain.

8. The method for producing recombinant humanized type XVII collagen according to claim 1, characterized in that, The method includes the following steps: (1) culturing the host cells according to any one of claims 6-7 in a culture medium; (2) isolating the recombinant humanized type XVII collagen according to claim 1 from the host cells.

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

10. The use of the recombinant humanized type XVII collagen of claim 1 or the composition of claim 9 in the preparation of skin repair, anti-aging and hair follicle care products.

Images