A recombinant humanized type IV collagen, expression vector, preparation method and application

Abstract

This invention discloses a recombinant humanized type IV collagen, its expression vector, preparation method, and applications, belonging to the field of collagen. This invention obtains a novel recombinant humanized type IV collagen COL4-HR by truncating and splicing natural human type IV collagen, and optimizes the codons of its encoding gene to obtain a nucleotide sequence with high expression efficiency. A recombinant expression system is constructed using *E. coli* as the host cell to achieve efficient and soluble expression of the target protein. The COL4-HR collagen prepared by this invention exhibits excellent cell adhesion and proliferation activities, as well as good high-temperature and acid / alkali resistance, and can be widely used in skincare products, medical dressings, tissue engineering materials, medical devices, and skin repair drugs.

Description

Technical Field

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

[0002] Type IV collagen is a core structural protein of the human basement membrane. It is a non-fibrous collagen that is widely distributed in the basement membranes of tissues such as skin, blood vessels, and cornea. It plays a key role in maintaining the structural stability of the basement membrane, regulating cell adhesion, migration, proliferation, and tissue repair and regeneration. It is a core functional protein for skin barrier repair, wound healing, and skin stability.

[0003] Traditionally, type IV collagen is extracted from animal placenta and connective tissue through acid hydrolysis, alkaline hydrolysis, or enzymatic hydrolysis. This technology has several inherent drawbacks: First, animal-derived collagen is susceptible to rejection by foreign proteins, easily causing adverse reactions such as allergies and inflammation when applied to humans, exhibiting poor biocompatibility. Second, animal tissues contain extremely low levels of type IV collagen, resulting in low extraction efficiency and significant raw material loss, failing to meet the demands of large-scale industrial production. Third, the extraction process easily damages the triple helix structure of collagen, leading to loss of natural biological activity and posing safety risks due to residual viruses and pathogens. Fourth, traditionally extracted products exhibit poor batch-to-batch stability and uneven content of active ingredients, severely limiting their application in high-end skincare products and medical materials.

[0004] With the development of genetic engineering technology, recombinant humanized collagen is gradually replacing traditional animal-extracted collagen, offering advantages such as no viral risks, low immunogenicity, batch stability, and good water solubility. However, existing recombinant type IV collagen technologies still have significant shortcomings: most technologies use full-length sequence expression, resulting in large protein molecular weights, low expression efficiency, and high purification difficulty; some truncated sequences lack precise screening of core active domains, leading to weak biological activity; simultaneously, the gene codons are not optimized for host cells, resulting in codon bias mismatch, excessively high GC content, and low transcription and translation efficiency, leading to low protein yield, high mass production costs, and difficulty in achieving industrial application. Furthermore, existing products generally suffer from insufficient stability, poor resistance to high temperatures and acids / alkalis, and limitations in storage and application scenarios.

[0005] To address the shortcomings of existing technologies, this invention utilizes bioinformatics to precisely screen the core active functional domains of human type IV collagen, splicing them together to obtain novel short-fragment recombinant collagen sequences. Simultaneously, codon optimization is performed to construct an efficient E. coli expression system, enabling the large-scale preparation of highly active, highly pure, and highly stable recombinant humanized type IV collagen, thus overcoming the deficiencies of existing technologies. 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:

[0007] The present invention provides a recombinant humanized type IV collagen, the amino acid sequence of which is shown in SEQ ID NO:5.

[0008] Furthermore, the recombinant humanized type IV collagen is assembled from the core active functional fragments SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 of the original sequence (SEQ ID NO:1) of human type IV collagen COL4A3.

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

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.7.

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

[0012] Furthermore, the recombinant expression vector is constructed by inserting the nucleotide sequence shown in SEQ ID NO:7 into the vector restriction enzyme site using pET-28a(+) as the backbone vector.

[0013] The present invention also provides a host cell comprising the recombinant expression vector according to the invention.

[0014] 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.

[0015] Furthermore, the host cell is Escherichia coli BL21(DE3) strain.

[0016] The present invention also provides a method for producing the recombinant humanized type IV collagen, characterized by comprising the following steps: (1) culturing the host cells in a culture medium; (2) isolating the recombinant humanized type IV collagen from the host cells.

[0017] The present invention also provides a composition, characterized in that the composition comprises the recombinant humanized type IV collagen, or the recombinant humanized type IV collagen encoded by the nucleic acid molecule, or the recombinant humanized type IV collagen expressed by the recombinant expression vector, or the recombinant humanized type IV collagen produced by the host cell, or the recombinant humanized type IV collagen prepared by the production method.

[0018] The present invention also provides the application of the recombinant humanized type IV collagen or the composition thereof in the preparation of skin repair and anti-aging products.

[0019] Furthermore, the product is a pharmaceutical composition, a medical device, or a cosmetic.

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

[0022] (1) This invention precisely screens and splices the three core active functional domains of human type IV collagen to prepare a novel COL4-HR recombinant protein without redundant sequences, effectively solving the industry pain points of low expression efficiency of traditional full-length proteins and weak biological activity of ordinary truncated proteins. This protein precisely retains the core functions of cell adhesion, proliferation and skin repair. Experimental verification shows that its cell adhesion activity and skin repair performance are significantly better than those of commercial type IV collagen.

[0023] (2) To improve the performance of protein mass production, this invention performs host-specific codon optimization for E. coli, reducing the GC content of the gene from 75.78% to 67.52%, significantly improving the codon fitness index and completely eliminating the transcriptional-translational arrest problem. Experimental data show that the expression level of the target protein after optimization is increased by 85.8% compared with the original sequence, and the proportion of soluble protein is significantly increased, effectively improving mass production capacity and reducing industrial production costs.

[0024] (3) The COL4-HR protein of the present invention has excellent environmental stability and can tolerate a wide range of temperature and acid-base environments. It can maintain high biological activity for a long time in a processing temperature of 40-50℃ and an acid-base system of pH 4.0-9.0 without denaturation or precipitation. It is perfectly suited to the conventional process conditions for skin care product production and storage and medical dressing processing, and solves the defects of poor stability and limited application scenarios of similar proteins.

[0025] (4) The COL4-HR protein prepared by this technology has multiple core advantages, including high activity, high purity, high stability, easy mass production and high safety. It has extremely high industrial application value in the fields of high-end skin repair and skin care products, medical repair materials and tissue engineering scaffolds. Attached Figure Description

[0026] Figure 1 The protein activity retention rate of recombinant humanized type IV collagen COL4-HR under different temperatures and incubation times.

[0027] Figure 2 : Protein activity retention rate of recombinant humanized type IV collagen COL4-HR under different pH conditions. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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 expressed by the symbol "%".

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Example 1: Design of the amino acid sequence of recombinant humanized type IV collagen

[0035] Based on the sequence of human type IV collagen (https: / / www.uniprot.org / uniprotkb / Q28084 / entry; CO4A3, amino acid sequence as shown in SEQ ID NO:1, 471AAs) 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 three highly conserved and highly active core functional fragments (SEQ ID NO:2-4) were selected. Details are shown below:

[0036] GLPGRKGPVGDAGPPGQLGVTGPQGAPGFPGVTIP GQKGDRGPPGSRGNPGMPGPPGPPGSP VEGIKGDKGLMGEPGQRGPPGAI GDMGSPGHPGAPGVPGQPGARGDPGFYGFPGMKGKKGNSGFPGPPGPPGQS GPKGPPGVRGEPGTVKIISLPGSPGSAGEPGMQGEPGPPGPPGDPGPCGPK GKPGEDGPPGTPGPTGEKGNKGCKGEQGPP GSDGLPGLK GKPGDTGPPAAGAVMRGFVFTRHSQTTAIPSCPEGTEPLYSGFSLLFVQGNEQAHGQDLGTLGSCLQRFTTMPFLFCNINDVCNFASRNDYSYWLSTPAMIPMDMAPITGRALEPYISRCTVC EGPAIAIAVHSQTTDIPPCPAGWISLWKGFSFIMFTSAGSEGAGQALASPGSCLEEFRASPFIECHGRGTCNYYSNSYSFWLASLDPKRMFRKPIPSTVKAGELENIISRCQVCMKMRP (SEQ ID NO:1).

[0037] Fragment 1 (SEQ ID NO:2): GQKGDRGPPGSRGNPGMPGPPGPPGSP, amino acids 36–62, contain cell-specific recognition adhesion sites, mediating skin cell basement membrane adhesion;

[0038] Fragment 2 (SEQ ID NO:3): GDMGSPGHPGAPGVPGQPGARGDPGFYGFPGMKGKKGNSGFPGPPGPPGQS, amino acids 86–136, contain core sites for cell proliferation and wound repair, and play a leading role in epidermal cell regeneration;

[0039] Fragment 3 (SEQ ID NO:4): GKPGEDGPPGTPGPTGEKGNKGCKGEQGPPGSDGLPGLK, amino acids 190–228, contain anti-inflammatory microenvironment regulatory sites, which can balance the levels of skin inflammatory factors.

[0040] The fragments shown in SEQ ID NO:2-4 were spliced ​​without gaps to obtain a novel recombinant humanized type IV collagen COL4-HR, with a 117 amino acid sequence as shown in SEQ ID NO:5: GQKGDRGPPGSRGNPGMPGPPGPPGSPGDMGSPGHPGAPGVPGQPGARGDPGFYGFPGMKGKKGNSGFPGPPGPPGQSGKPGEDGPPGTPGPTGEKGNKGCKGEQGPPGSDGLPGLK (SEQ ID NO:5).

[0041] Based on the amino acid sequence shown in SEQ ID NO:5, its coding nucleic acid sequence was reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ), as shown in SEQ ID NO:6, with a full length of 351 bp.

[0042] GGCCAGAAAGGCGATCGCGGCCCGCCCGGGCAGCCGCGGCAACCCGGGCATGCCGGGCCCGCCGGGCCCGCCGGGCAGCCCGGGCGATATGGGGCAGCCCGGGCCATCCGGGCGCCGGGCGTGCCGGGCCAGCCGGGCGCGCGCGGCGATCCGGGCTTTTATGGCTTTCCGGGCATG AAAGGCAAAAAAGGCACAGCGGCTTCCGGGCCCGCCGGGCCCGCCGGGCCAGAGCGGCAAACCGGGCGAAGATGGCCCGCCGGGCACCCCGGGCCCGACCGGCGAAAAAGGCAACAAAGGCTGCAAAGGCGAACAGGGCCCGCCGGGCAGCGATGGCCTGCCGGGCCTGAAA (SEQ ID NO:6).

[0043] 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:7. Compared with the original gene sequence, the GC content decreased from 75.78% to 67.52%, which is more conducive to the efficient expression of the gene by the host bacterium E. coli.

[0044] GGCCAGAAAGGTGATCGTGGTCCGCCGGGCTCTCGTGGTAACCCAGGTATGCCGGGCCCTCCGGGTCCGCCGGGTTCCAGGTGATATGGGCAGCCCTGGTCACCCGGGCGCTCCGGGCGTACCGGGTCAGCCAGGTGCGCGCGGCGATCCGGGTTTCTACGGTTTCCCGGGCATG AAAGGCAAGAAAGGCAACAGCGGCTTCCCGGGTCCGCCGGGTCCGCCTGGTCAGTCCGGCAAACCGGGCGAAGACGGTCCGCCGGGCACCCCGGGCCCGACTGGCGAGAAAGGTAACAAAGGCTGCAAGGGTGAACAGGGCCCGCCGGGCTCTGACGGCCTGCCTGGCCTGAAG (SEQ ID NO:7).

[0045] The sequences SEQ ID NO:6 and SEQ ID NO:7 were synthesized by Shanghai Sangon Biotech. Specific restriction enzyme sites NdeI (5' end) and XhoI (3' end) 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 IV collagen expression vector

[0047] The experimental vector used was the pET-28a(+) prokaryotic expression vector, and the host cell was Escherichia coli BL21(DE3) competent cells. The core reagents included NdeI, XhoI restriction endonuclease, T4 DNA ligase, DNA gel extraction kit, BCA protein quantification kit, kanamycin antibiotic, and IPTG inducer. The culture media included LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and LB solid medium (with 1.5% agar powder added).

[0048] (1) Construction of recombinant expression vector

[0049] Two vectors were constructed: the control group contained the unoptimized gene SEQ ID NO:6+pET-28a(+), and the experimental group contained the codon-optimized gene SEQ ID NO:7+pET-28a(+). The two vectors were constructed using the same method.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] (2) Double verification of positive engineered strains

[0056] 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.

[0057] (3) Parallel induction expression experiment

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] (4) Protein extraction and quantitative detection

[0063] 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.

[0064] Table 1. Expression levels of recombinant type IV collagen before and after codon optimization (n=3, x±SD)

[0065]

[0066] Experimental conclusion: Under completely identical culture, induction, and extraction conditions, the effective expression level of the target type IV collagen in the codon-optimized experimental group (SEQ ID NO:7) increased from 85.12 mg / L to 158.26 mg / L, with an overall expression level increase of 85.8%. This demonstrates that the codon optimization scheme of the present invention can effectively adapt to the E. coli expression system, solve the problem of transcriptional translation arrest of the original gene, significantly improve the mass production capacity of recombinant type IV collagen, and reduce the cost of industrial production.

[0067] Example 3: Purification and biological activity evaluation of recombinant humanized type IV collagen

[0068] In this embodiment, the COL4-HR protein expressed in the experimental group after codon optimization was standardized and purified. The purity was verified by SDS-PAGE. At the same time, HACAT cells and NIH / 3T3 cells were used to quantitatively detect cell proliferation and cell adhesion activity.

[0069] (1) Purification of the target protein

[0070] 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.

[0071] 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;

[0072] 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 fractions.

[0073] 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;

[0074] 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;

[0075] 6) Concentration and preservation: After ultrafiltration concentration, high-purity COL4-HR protein is obtained and aliquoted and stored at -80℃ for later use.

[0076] (2) Protein purity test results

[0077] 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.

[0078] (3) Cell proliferation activity detection (HACAT cells, CCK8 assay)

[0079] Experimental groups: blank control group (pure culture medium), recombinant humanized type IV collagen COL4-HR experimental groups 1-4 (0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL), with 6 replicates per group; HACAT cells were seeded at a density of 5 × 10³ cells / well, cultured at 37℃ and 5% CO2 for 24 h, and the absorbance at 450 nm was measured to calculate the relative cell viability. The results are shown in Table 2.

[0080] Table 2. Effects of different concentrations of COL4-HR on the proliferation of HACAT cells (n=6, x±SD)

[0081]

[0082] (4) Cell adhesion activity assay (BALB / 3T3 cells)

[0083] Recombinant humanized type IV collagen COL4-HR was dissolved in PBS and added to each well of a 96-well cell culture plate at final concentrations of 0.5 mg / ml and 1.0 mg / ml, respectively. The plates were incubated at 37°C for 2 hours, with PBS 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. The results are shown in Table 3. In this example, the positive control group was a commercially available recombinant type IV collagen sample solution, added to the cell culture medium at a concentration of 1.0 mg / ml.

[0084] 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%.

[0085] Table 3 Comparison of COL4-HR collagen cell adhesion activity among different groups (n=6, x±SD)

[0086]

[0087] Experimental conclusion: The present invention COL4-HR is non-cytotoxic, has good cell adhesion and proliferation properties, can significantly promote skin keratinization, and its cell adhesion activity is significantly better than that of commercially available type IV collagen. It has outstanding skin barrier repair and wound adhesion repair capabilities and can be applied in a variety of fields, such as cosmetics or pharmaceutical products.

[0088] Example 4: Stability test of recombinant humanized type IV collagen

[0089] To verify the processing and storage suitability of COL4-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.

[0090] (1) High temperature stability test

[0091] A 1 mg / mL standard COL4-HR protein solution was prepared and divided into three groups. These groups were incubated at constant temperatures of 40℃, 50℃, and 60℃, respectively. Samples were taken at 4 h, 8 h, and 12 h, and cell proliferation activity was assessed after rewarming to room temperature. The activity of fresh, untreated protein was used as a 100% baseline. Results are as follows: Figure 1 As shown.

[0092] 4.2 Acid and alkali stability test

[0093] The pH of 1 mg / mL COL4-HR protein solution was adjusted to 4.0, 5.0, 8.0, and 9.0, respectively, and incubated at room temperature in a sealed environment for 12 h and 24 h. The pH was then adjusted back to 7.4 before cell proliferation activity was assessed. Fresh protein at pH 7.4 was used as a blank control. Protein status was also observed. Results are as follows: Figure 2 As shown.

[0094] Experimental conclusion: COL4-HR protein can remain stable for a long time in conventional processing temperatures of 50℃ and below and in acid-base systems of pH 4.0-9.0. It has a high activity retention rate and no denaturation or precipitation, making it perfectly suitable for the production, processing and room temperature storage conditions of skin care products and medical materials. Its stability is significantly better than that of existing conventional recombinant type IV collagen.

[0095] 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 IV collagen, characterized in that, The amino acid sequence of the recombinant humanized type IV collagen is shown in SEQ ID NO:

5.

2. A nucleic acid molecule encoding the recombinant humanized type IV 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.

7.

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:7 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 IV 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 IV collagen according to claim 1 from the host cells.

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

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

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