Recombinant type IV collagen and its preparation method and application

Abstract

The present invention provides recombinant type IV collagen, a preparation method and application thereof, and belongs to the field of bioengineering technology. The present invention designs a recombinant type IV collagen with whitening efficacy based on a partial active amino acid sequence in the α1 chain of natural human type IV collagen. The amino acid sequence of the recombinant type IV collagen with whitening efficacy is shown in SEQ ID NO: 1. The recombinant type IV collagen has the activity of promoting cell adhesion and migration, can inhibit the synthesis of melanin, has whitening efficacy, and does not produce an immune response when applied to the human body. The recombinant type IV collagen has value in the fields of cosmetics, medicines, health products, medical devices and biomaterials.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to recombinant type IV collagen and a preparation method and application thereof. Background Art

[0002] Human skin pigmentation determines the color of the skin, eyes, and hair, and also protects the skin from damage by ultraviolet radiation. Disorders in melanin metabolism can cause pigmentation disorders such as spots, freckles, chloasma, and melanoma. Melanin metabolism involves melanin production, its transport from melanocytes to keratinocytes, and its distribution and degradation within keratinocytes.

[0003] In recent years, regulating melanin has become a research hotspot. Existing whitening methods mainly include inhibiting melanin production, inhibiting melanin transport, and promoting melanin metabolism. Melanin synthesis primarily involves three enzymes in the tyrosinase gene family: tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). Tyrosinase is a key rate-limiting enzyme in the melanin synthesis pathway, catalyzing the conversion of tyrosine to dopa and further to dopaquinone, or catalyzing the direct oxidation of tyrosine to dopaquinone, a substrate for melanin synthesis. Therefore, inhibiting tyrosinase activity can block the melanin biosynthesis reaction chain, reduce melanin production, and achieve a whitening effect. Currently, ingredients that inhibit tyrosinase activity include tranexamic acid, azelaic acid, phenylethyl resorcinol, potassium methoxysalicylate (4MSK), and tranexamic acid. Ingredients that inhibit melanin transport include niacinamide, soy protein, 1-methylhydantoin-2-imide, and diglucosyl gallic acid. To promote melanin metabolism, acids such as glycolic acid and β-hydroxy acids like salicylic acid are often used to exfoliate old keratin. While these whitening ingredients can reduce pigmentation, some are cytotoxic, and acids may pose a risk of skin irritation. Other whitening agents, such as ascorbic acid, retinoids, and nonapeptide-1, are unstable and easily oxidized. Retinoids may cause irritant dermatitis, while nonapeptide-1 is relatively weak. Their whitening effectiveness may depend on the concentration and formulation used, as well as individual skin reactions. Therefore, there is a need for a natural, non-toxic, stable, highly effective, and easily absorbed melanin regulator.

[0004] Prior art has identified novel tyrosinase inhibitory peptides from donkey collagen, namely Asp-Gly-Leu (DGL), Gly-Ala-Arg (GAR), and Ser-Asp-Trp (SDW), through a combination of computer screening and in vitro activity validation. All three peptides exhibit potent tyrosinase inhibitory activity, with His85, His244, His259, and Asn260 being key residues driving the interaction between the peptides and tyrosinase. Tyrosinase inhibition experiments revealed that the DGL tripeptide was the most potent of the three peptides, as different interaction types significantly influence the structural stability and binding affinity between the peptide and tyrosinase. The DGL tripeptide forms a total of nine hydrogen bonds (seven carbon-hydrogen bonds and two conventional hydrogen bonds) with tyrosinase, involving six amino acid residues in tyrosinase: His61, His85, His259, His244, Asn260, and Glu256. The C-terminal leucine of the DGL short peptide formed five hydrophobic interactions (one alkyl interaction and four π-alkyl interactions) with three residues of tyrosinase Val283, His263 and Phe264. The hydrophobic aliphatic residue leucine is very important for the inhibition of tyrosinase. It can directly interact with tyrosinase to inhibit the formation of dopaquinone.

[0005] The basement membrane performs signal transduction, permeability, and barrier functions, preventing harmful substances from entering the dermis. A decrease in type IV collagen, a key component of the basement membrane, disrupts its continuity, causing epidermal cells to lose contact with the basement membrane and connect directly to the exposed dermis. This damaged basement membrane allows melanocytes and melanin to enter the dermis, preventing melanin from being metabolized and excreted by keratinocytes, leading to severe hyperpigmentation. Therefore, a stable basement membrane not only improves melanin metabolism but also prevents melanin from entering the dermis from the epidermis, potentially promoting whitening and freckle removal by protecting the skin's basement membrane. Increasing type IV collagen improves the stability of the skin's basement membrane and promotes cell regeneration. By boosting skin cell metabolism, it helps reduce hyperpigmentation, thereby achieving a whitening effect. Currently, there is no documented research on type IV collagen with whitening properties. There is a need to develop a recombinant type IV collagen with whitening properties and high transdermal absorption. Summary of the Invention

[0006] In response to some deficiencies in the prior art, the present invention provides recombinant type IV collagen, a preparation method and application thereof; the present invention designs a recombinant type IV collagen based on a partial active amino acid sequence in the α1 chain of natural human type IV collagen, and the amino acid sequence of the recombinant type IV collagen is shown in SEQ ID NO: 1 or SEQ ID NO: 2; the recombinant type IV collagen has the activity of promoting cell adhesion and migration, can inhibit the synthesis of melanin, has a whitening effect, and does not produce an immune response when applied to the human body, and has value in the fields of cosmetics, medicines, health products, medical devices and biomaterials.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0008] The present invention first provides a recombinant type IV collagen, the sequence of which is shown in i or ii:

[0009] i. an amino acid sequence as set forth in SEQ ID No: 1, or an amino acid sequence having 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to SEQ ID NO: 1;

[0010] ii. A protein derived from i, wherein the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity.

[0011] The present invention also provides a recombinant type IV collagen, which further comprises: a collagen obtained by taking the recombinant type IV collagen according to claim 1 as a basic unit and repeating it in series n times, where n is an integer greater than or equal to 3 and less than or equal to 20.

[0012] Preferably, two adjacent basic units are connected with an amino acid that can be recognized and cleaved by Kex2 enzyme, CPB enzyme, or Ste13 enzyme as a linker.

[0013] Preferably, the amino acid that can be recognized and cleaved by Kex2 enzyme, CPB enzyme, or Ste13 enzyme includes KKREA.

[0014] Preferably, the sequence of the tandem recombinant type IV collagen is as shown in i or ii:

[0015] i. The amino acid sequence is shown in SEQ ID No: 2;

[0016] ii. A protein derived from i, wherein the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity.

[0017] The present invention also provides a nucleic acid encoding the aforementioned tandem recombinant type IV collagen, wherein the nucleic acid sequence is shown as SEQ ID No: 4.

[0018] The present invention also provides a recombinant expression vector comprising the above nucleic acid.

[0019] The present invention also provides a recombinant engineered bacterium comprising the nucleic acid molecule described above, or comprising the recombinant expression vector described above, or expressing the recombinant type IV collagen described above.

[0020] Preferably, the host bacteria of the recombinant engineered bacteria include Pichia pastoris, Saccharomyces cerevisiae or Hansenula.

[0021] Preferably, the host cell is Pichia pastoris, more preferably HCPB-PPKEX2, with a deposit number of CGMCC No. 25815.

[0022] Preferably, the recombinant engineered bacteria is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC NO.32113.

[0023] The present invention also provides a method for preparing the above-mentioned recombinant type IV collagen, the preparation method comprising:

[0024] (1) selecting and designing the sequence of recombinant type IV collagen, and then constructing a tandem recombinant type IV collagen using the recombinant type IV collagen as a basic unit by tandem repetition;

[0025] (2) constructing a recombinant expression vector expressing the tandem recombinant type IV collagen;

[0026] (3) electroporating the constructed recombinant expression vector into the host bacteria, screening and verifying to obtain recombinant engineered bacteria expressing high-copy collagen;

[0027] (4) Fermentation-induced expression of recombinant engineered bacteria expressing high-copy collagen was performed to obtain recombinant type IV collagen.

[0028] Preferably, the sequence of the recombinant type IV collagen is as shown in i or ii:

[0029] i. The amino acid sequence is shown in SEQ ID No: 1;

[0030] ii. A protein derived from i, wherein the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity.

[0031] Preferably, in step (1), the series-connected recombinant type IV collagen contains 3 to 20 basic unit recombinant type IV collagens; and two adjacent basic units are connected by amino acids that can be recognized and cleaved by Kex2 enzyme, CPB enzyme, or Ste13 enzyme as a linker.

[0032] Preferably, the amino acid that can be recognized and cleaved by Kex2 enzyme, CPB enzyme, or Ste13 enzyme includes KKREA.

[0033] Preferably, in step (1), the nucleic acid of the tandem recombinant type IV collagen comprises the nucleotide sequence shown in SEQ ID No: 4, or a degenerate sequence thereof.

[0034] Preferably, in step (2), the vector of the recombinant plasmid includes pPICZαB, pFLDα, and pPIC9K.

[0035] Preferably, the vector is pPIC9K.

[0036] Preferably, in step (3), the host bacteria includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula.

[0037] Preferably, in step (3), the recombinant engineered bacteria is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC NO.32113.

[0038] The present invention also provides recombinant type IV collagen prepared according to the above method.

[0039] The present invention also provides a composition comprising the above-mentioned recombinant type IV collagen, or the above-mentioned tandem recombinant type IV collagen, or the tandem recombinant type IV collagen encoded by the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria, or the recombinant type IV collagen obtained according to the above-mentioned method.

[0040] The present invention also provides a product, which comprises the above-mentioned recombinant type IV collagen, or the recombinant type IV collagen encoded by the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria, or the recombinant type IV collagen obtained according to the above-mentioned method, or the above-mentioned composition; the product includes drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products with whitening, cell adhesion promoting activity or cell migration promoting activity.

[0041] The present invention also provides the use of the above-mentioned recombinant type IV collagen, or the recombinant type IV collagen encoded by the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria, or the recombinant type IV collagen obtained according to the above-mentioned method, or the above-mentioned composition, or the above-mentioned product in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products with whitening, cell adhesion promoting activity or cell migration promoting activity.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The present invention designs a recombinant type IV collagen 1CB1K with whitening effect based on the partial active amino acid sequence in the α1 chain of natural human type IV collagen. The recombinant type IV collagen has a theoretical molecular weight of 3.3 kDa and has a typical GXY triplet structure, which is 100% consistent with the sequence of natural human type IV collagen. It will not produce an immune response when applied to the human body. The recombinant type IV collagen of the present invention has a good activity in promoting cell adhesion and migration, and can also inhibit the synthesis of melanin. The recombinant type IV collagen can achieve whitening effect by inhibiting melanin synthesis without causing skin intolerance. Therefore, it is expected to be applied to the fields of cosmetics, pharmaceuticals, health products, medical devices and biomaterials, and can be used to prepare drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products with whitening, cell adhesion or cell migration promoting activities.

[0044] (2) In constructing a method for preparing recombinant type IV collagen, the present invention employs repeated tandem expression of the recombinant type IV collagen to indirectly increase the copy number, thereby increasing the expression level of the recombinant type IV collagen. The preparation method of the present invention yields a recombinant collagen that is 100% identical in sequence to natural human type IV collagen and does not produce an immune response when applied to the human body.

[0045] Furthermore, the linker used in the sequence design of this invention is cleaved and removed during the collagen's secretion process, resulting in a recombinant collagen protein with 100% homology to the corresponding region of native collagen. This invention utilizes the strain HCPB-PPKEX2 to engineer and express recombinant type IV collagen, directly obtaining the target protein without the introduction of exogenous proteins or the use of any in vitro cleavage proteases. This eliminates the risk of exogenous protein residues and reduces the time and cost of subsequent purification processes.

[0046] (3) The host bacteria for the recombinant type IV collagen of the present invention is Pichia pastoris, which can secrete the protein for extracellular expression, effectively avoiding problems such as impurities introduced by bacterial lysis during product extraction. As a eukaryotic organism, it can perform post-translational modifications on the secreted recombinant protein, such as glycosylation and phosphorylation. The resulting recombinant protein is free of pathogens, viral inclusion bodies, or pyrogens, thus having high safety and low fermentation costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the map of the pPIC9K-1CB1K vector.

[0048] Figure 2 This is the SDS-PAGE electrophoresis detection result of the recombinant type IV collagen 1CB1K expression supernatant (induced for 48 hours).

[0049] Figure 3 This is the result of sequence alignment of the NC terminus of recombinant type IV collagen 1CB1K.

[0050] Figure 4 The results of cell adhesion test.

[0051] Figure 5 These are photos of cell migration.

[0052] Figure 6 Calculation results for cell migration rate.

[0053] Figure 7 The figure shows the cytotoxicity assay of recombinant type IV collagen 1CB1K at different concentrations.

[0054] Figure 8 The inhibition rate of recombinant type IV collagen 1CB1K on melanin synthesis in B16 (mouse melanoma cells) at different concentrations. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, all that is not explained in detail is accomplished using conventional experimental methods, and the processes involved in the embodiments are understood and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the art, and therefore will not be described in detail.

[0056] In the following examples, the recombinant type IV collagen constructed after tandem is provided with recognition and cleavage sites for the enzymes Kex2, CPB, and STE13 between each two adjacent basic units. These enzymes can cleave and remove the non-native collagen sequence (i.e., linker) between the two tandem sequences. In addition, the design of repeated tandem expression of amino acid sequences can indirectly increase the copy number, thereby achieving the purpose of increasing the expression level of the exogenous protein. After transcription and translation, the sequence of the present invention retains a protein sequence that is 100% identical to the natural human type IV collagen sequence. This effect is achieved because the linker sequence is determined as LKKREA in the present invention, where L comes from the type IV collagen sequence itself and KKREA is an additional non-native collagen sequence. The linker can be completely cleaved and removed by the enzymes Kex2, CPB, and STE13 in the secretory pathway of the protein.

[0057] The Kex2 enzyme is a calcium-dependent serine protease expressed by yeast microorganisms (naturally including Pichia pastoris) that can specifically recognize and cut the carboxyl-terminal peptide bonds of dibasic amino acids such as RR and KR in the amino acid sequence. Taking KR as an example, the position of R is set to p1, the position of K is set to p2, and so on. In order to improve the cutting efficiency, amino acid sequences such as EA and EAEA are added after p1. At the same time, the amino acids of p3 and p4 also have different acidity and alkalinity and charge, etc., so that the Kex2 enzyme exhibits different cutting efficiencies. It has been verified that in Pichia pastoris, when p3 is K, Kex2 has the expected cutting efficiency, and K can be cut by CPB enzyme. Therefore, in the present invention, when the amino acid KKREA is added at the end of 1CB1K to form the sequence LKKREA as a linker for repeated tandem expression, KKREA can be completely cut and removed, and non-native collagen sequences will not be introduced into the final recombinant protein product.

[0058] Yeast microorganisms harbor the STE13 gene, which expresses the Ste13 protease (strictly speaking, a dipeptidyl aminopeptidase) within their cells. This protease cleaves the amino acid sequences EA and EAEA at the amino termini of proteins. Recombinant carboxypeptidase B (CPB) specifically cleaves basic amino acids (especially K and R) at the carboxyl terminus of proteins, until all the basic amino acids at the C-terminus are cleaved, leaving the remaining non-basic amino acids exposed at the C-terminus. These three proteases work together to remove non-collagenous amino acids, such as KR at the C-terminus and EA at the N-terminus, respectively, maintaining the sequence homology between the recombinant collagen and native human collagen.

[0059] The host bacteria for the recombinant type IV collagen described herein include one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula. The following examples illustrate this using HCPB-PPKEX2 from Pichia pastoris, with accession number CGMCC No. 25815. HCPB-PPKEX2 expresses human CPB enzyme. The recombinant human carboxypeptidase HCPB specifically cleaves basic amino acids (particularly K and R) at the carboxyl terminus of proteins until all basic amino acids are cleaved, leaving the remaining non-basic amino acids exposed at the C-terminus. This allows for the removal of non-human KKR sequences introduced by the linker.

[0060] Example 1:

[0061] S1. Design of amino acid sequence of recombinant type IV collagen:

[0062] The 379-412AA portion of the native human type IV collagen α1 chain (Uniprot database P02462, https: / / www.uniprot.org / Uniprotkb / P02462 / entry#sequences) was named 1CB1K. 1CB1K has a total of 34 amino acids, a theoretical molecular weight of 3.3 kDa, and its amino acid sequence is shown in SEQ ID NO: 1.

[0063] A repeating tandem sequence was designed using 1CB1K as the basic unit. The basic unit was repeated 12 times in series, and an amino acid sequence KKREA, which can be cut and removed by Kex2 enzyme, CPB enzyme and Ste13 enzyme, was added between every two basic units to form the amino acid sequence shown in SEQ ID No: 2, i.e., the recombinant type IV collagen after tandem, recorded as 1CB1K-12, with a total of 463 sequences.

[0064] SEQ ID NO: 1:

[0065] GQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGL;

[0066] SEQ ID NO: 2:

[0067] GQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKRE AGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKR EAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKK REAGQAGAPGFPGERGEKGDRGFPGTSLPGPSRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSRDGLKKREAGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGL.

[0068] When 1CB1K-12 enters the protein secretion pathway, which is comprised of the endoplasmic reticulum and Golgi apparatus, after transcription and translation, the Kex2 enzyme cleaves it between the KKR and EA in each KKREA. The two amino acids of EA at the amino terminus are then cleaved and removed by the Ste 13 protease, resulting in the sequence shown in SEQ ID NO: 3. Subsequently, the three amino acids of KKR at the carboxyl terminus of the sequence described in SEQ ID NO: 3 are cleaved and removed by the CPB enzyme, ultimately secreting the 34-amino acid recombinant type IV collagen protein, 1CB1K, whose amino acid sequence is shown in SEQ ID NO: 1.

[0069] SEQ ID NO: 3:

[0070] GQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLKKR.

[0071] S2. Synthesis of DNA sequences and construction of recombinant plasmids:

[0072] The DNA sequence encoding the tandem recombinant type IV collagen 1CB1K-12 was designed and optimized, and a double stop codon TGATAA was added to the 3' end of the sequence. Nanjing GenScript Biotech Co., Ltd. was then commissioned to synthesize the optimized 1CB1K-12 nucleic acid sequence SEQ ID NO: 4.

[0073] The synthesized gene fragment as shown in SEQ ID No: 5 was cloned into the pPIC9K empty vector (purchased from Thermo Fisher Scientific). After cloning, the 39 bp sequence at sites 1210-1248 was replaced so that the target fragment was accurately inserted into the secretory vector reading frame containing the secretion signal α-factor to obtain a recombinant expression vector expressing 1CB1K, named pPIC9K-1CB1K-12. Its map is shown in FIG. Figure 1 shown.

[0074] SEQ ID NO: 4:

[0075]

[0076] S3. Construction of recombinant engineering strains and screening of strains:

[0077] 10 μg of pPIC9K-1CB1K-12 obtained in step S2 was digested with Sal I (Dalian TaKaRa Company, specific operations were carried out according to the kit instructions) at 37°C for 2 h to linearize it. Then, a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used to recover the linearized plasmid. The DNA concentration in the resulting solution was controlled as much as possible to be above 100 ng / μL.

[0078] The linearized plasmid was electroporated into the competent cells of the host strain Pichia pastoris HCPB-PPKEX2 (from strain number: CGMCC No. 25815). The electroporated bacterial solution was spread on MD plates, with 100 μL to 200 μL spread on one plate. The plate was allowed to stand at room temperature for 10 minutes and then inverted and cultured at 30°C for 2-5 days until a single colony (positive transformant) appeared.

[0079] Add 2 mL of sterile double-distilled water to the surface of the MD plate, then gently scrape the His+ transformants on the surface of the plate with a sterile triangular applicator and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double-distilled water and take 10 6 The cells were spread on a YPD plate containing 0.5 mg / mL G418, inverted, and cultured at 30°C for 3 to 4 days until single colonies appeared, thereby obtaining recombinant engineered bacteria containing pPIC9K-1CB1K-12.

[0080] The recombinant engineered bacteria containing pPIC9K-1CB1K-12 were sent to the General Microbiology Center of the China Culture Collection Administration, with the culture collection number: CGMCC No. 32113. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is: September 29, 2024; the taxonomic name is: Pichia pastoris Komagataella phaffii.

[0081] S4. Induced expression and identification of recombinant collagen:

[0082] The recombinant engineered bacteria containing pPIC9K-1CB1K-12 were placed in a 100 mL Erlenmeyer flask containing 15 mL of BMGY medium and cultured at 28-30°C and 220 rpm to an OD600 of approximately 10 (24 h). The cells were centrifuged at 1500-3000 × g for 5 min at room temperature, harvested, and resuspended in BSM medium to an OD600 of approximately 10. The cells were then grown on a shaker at 28-30°C and 220 rpm for 2 days. 100% methanol was added to the culture medium every 24 h to a final concentration of 1.0% (v / v) for induction. After 16 h of induction, a sample of the bacterial culture was collected. An induction time of 48 h was found to be more effective. After induction, an appropriate amount of sample was placed in a centrifuge tube and centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was collected and tested immediately or stored at -80°C for later use.

[0083] The collected expression supernatant was added with 2× loading buffer, heated in a 100°C metal bath for 10 minutes, and then subjected to SDS-PAGE analysis. The SDS-PAGE analysis of the expression supernatant is as follows: Figure 2 As shown in the figure, it can be seen that the recombinant type IV collagen 1CB1K with whitening effect is effectively secreted and expressed in the supernatant, and the electrophoresis band is single (collagen will have a certain electrophoretic migration delay during electrophoresis, so its apparent molecular weight during electrophoresis will be higher than the theoretical value).

[0084] The N-terminal and C-terminal sequence alignment of the 48-hour induced expression supernatant of recombinant type IV collagen 1CB1K with whitening effect was performed (commissioned by Beijing Biotech Biotechnology Co., Ltd.). The data comparison results are as follows: Figure 3 As shown, the results show that the sequence alignment result of the recombinant type IV collagen 1CB1K with whitening effect obtained in the present invention is consistent with the theoretical sequence, which indicates that the recombinant type IV collagen 1CB1K with whitening effect is successfully obtained.

[0085] Example 2: Recombinant collagen cell adhesion activity experiment

[0086] In this example, natural human collagen (Sigma, catalog number C7774) was used as a positive control, and recombinant type IV collagen 1CB1K was used as an experimental sample. The adhesion activity of the recombinant type IV collagen described in the present invention was investigated using the "YY / T 1849-2022 Recombinant Collagen Appendix B Cell Adhesion Assay - Centrifugation Method".

[0087] The specific steps are as follows:

[0088] (1) Sample preparation:

[0089] Experimental samples: Under sterile conditions, recombinant type IV collagen 1CB1K sample was weighed proportionally and dissolved in ultrapure water to obtain a recombinant type IV collagen solution with a concentration of 5 mg / mL;

[0090] Positive control sample: Natural human collagen (Sigma, Catalog No. C7774) was weighed in proportion, dissolved in ultrapure water, and acetic acid was added to pH 3.0 to obtain a natural human collagen solution with a concentration of 5 mg / mL.

[0091] When ready for use, both the experimental sample and the positive control sample were diluted to 0.5 mg / mL using serum-free DMEM medium and sterilized by filtration through a 0.22 μm sterile filter.

[0092] Blank sample: D-PBS phosphate buffer.

[0093] (2) Coating preparation:

[0094] Add 100 μL of experimental sample, positive control sample, and blank sample to a 96-well plate. Coat four wells with each sample and incubate in a 37°C, 5% CO2 (v / v) incubator for 1 hour. Remove excess coating solution from the wells and add 10 μL of 1% BSA-PBS solution. Incubate in a 37°C, 5% CO2 incubator for 1 hour. After incubation, remove the liquid from the wells and wash three times with D-PBS. Discard the wash solution, seal with sealing film, and store at 4°C until ready for use.

[0095] (3) Cell preparation:

[0096] NIH / 3T3 cells (ATCC CRL-1658 mouse embryonic fibroblasts) were cultured in a 37°C, 5% CO2 cell culture incubator. The cell density and status were observed daily under an inverted microscope. When the cells grew to 80% to 90% of the culture flask, the cells were passaged or seeded. The cells were diluted to 5×10 using complete culture medium pre-mixed with Hoechst-33342 fluorescent dye (10%). 4 pieces / mL.

[0097] 100 μL of cells were added to the wells, covered with aluminum foil, and incubated at 37°C, 5% CO2 for 1 h. After incubation, three replicate samples were measured. The fourth well was used to adjust the microscope parameters, and its measurement value was not used.

[0098] The specific detection steps are:

[0099] At least 4 × 4 digital tile images (fluorescence) were captured from each of the three wells using an inverted microscope. Each well was filled with D-PBS to form a "reverse meniscus," purged of air bubbles, and covered with Parafilm. The plate was centrifuged (inverted) at 300 g relative centrifugal force (RCF) at 22°C for 5 min. After centrifugation, the Parafilm was discarded, and the supernatant was removed from the wells. After washing once with D-PBS, 100 μL of D-PBS was added. A total of 25 digital fluorescent tile images were captured for each of the three wells (a minimum of a 4 × 4 matrix with 10% tile overlap is recommended), and approximately 2400 to 3600 cells were counted for each sample (800–1200 cells / well × 3 wells).

[0100] The cell number before and after centrifugation was determined, and the percentage of adhesion was calculated according to the formula V = Nt / Nc (Nt: cell number after centrifugation, Nc: cell number before centrifugation). The data differences among the experimental groups were analyzed using one-way variance analysis (Dunnett method). The measurement results were as follows: Figure 4 shown.

[0101] from Figure 4 It can be seen that compared with the blank group and the positive control group of natural human collagen, the recombinant type IV collagen of the present invention has better bioadhesion activity.

[0102] Example 3: Recombinant collagen cell migration experiment

[0103] In this example, the same NIH / 3T3 cells as in Example 2 were used to examine the cell migration ability of recombinant type IV collagen and positive control natural collagen (Sigma, catalog number C7774).

[0104] (1) Experimental preparation: First, use a marker to draw straight and even lines on the back of a 6-well plate. The interval between each line is 0.5 cm to 1 cm. The lines are drawn horizontally through the holes, ensuring that there are 3 straight lines passing through each hole. Add about 5×10 5 cells were cultured.

[0105] (2) Scratch test: On the second day of cell culture, use the tip of the gun to measure the ruler and scratch the horizontal line on the back as vertically as possible. The tip of the gun should be vertical and not tilted. Then rinse the cells with PBS three times, remove the scratched cells, and add serum-free culture medium containing the test sample as the experimental group. The concentration is 0.05% (mass volume ratio). Place in a 37℃, 5% CO2 incubator for culture. Take samples at 0, 6, and 24 hours, take pictures, and the test results are as follows: Figure 5 shown.

[0106] (3) Data processing: The scratch area of each picture was calculated using ImageJ image processing software, and the migration rate of each group of cells was calculated by dividing the total area of the migrating cells in the fixed scratch area by the initial area of the fixed scratch area. The time was used as the horizontal axis and the migration area ratio was used as the vertical axis (unit %). The photos of the experimental group and the control group at the initial time 0 and the end of the experiment were compared. The chi-square test was used to analyze the differences in the data of each experimental group. The test results are as follows: Figure 6 shown.

[0107] Combine Figure 5 and Figure 6 It can be seen that compared with the blank group and the natural human collagen control group, the recombinant type IV collagen of the present invention has better cell migration promoting activity.

[0108] Example 4: Whitening efficacy test of recombinant collagen

[0109] In this example, the recombinant type IV collagen was first tested for cytotoxicity, and then the whitening efficacy was tested based on the results of the cytotoxicity test. The test results are as follows:

[0110] (1) Recombinant collagen cytotoxicity test:

[0111] Under the experimental conditions of this example, CCK-8 cell counting kit (Adamas Life, catalog number C8022) and mouse melanoma B16 cells (Xinrun Bio, catalog number CM3076) were used to investigate the cytotoxicity of the recombinant type IV collagen of the present invention. The specific investigation steps are as follows:

[0112] Cytotoxicity assay: B16 cells were cultured at a rate of 1×10 4 / well density plated 96-well cell culture plates, after 24 hours of PBS washing and replacement with 1640 medium diluted with mass volume concentration of 0.01%, 0.03%, 0.1%, 0.3%, 0.5%, 1% recombinant type IV collagen solution, each concentration of three replicates. After 72 hours of cell culture incubator incubation, CCK-8 was added, and cytotoxicity was detected using a microplate reader at a wavelength of 450nm. The test results are as follows Figure 7 shown.

[0113] As can be seen from the figure, when the concentration of recombinant type IV collagen solution is 0.01%, 0.03% and 0.1%, the survival rate of mouse melanoma cell B16 is above 90%, and it has no toxicity to cells. Therefore, these three concentrations were selected for subsequent whitening efficacy testing.

[0114] (2) Whitening efficacy test of recombinant collagen:

[0115] In this example, mouse melanoma cells B16 were used as the research subjects to determine the inhibitory effect of the recombinant type IV collagen of the present invention on melanin synthesis, thereby evaluating the whitening efficacy of the recombinant type IV collagen of the present invention.

[0116] Blank control: cell culture medium (Gibco, cat. no. 61870036);

[0117] Positive control: 100 μg / mL α-arbutin;

[0118] Experimental groups: 0.01%, 0.03% and 0.1% recombinant type IV collagen.

[0119] B16 cells were cultured at a rate of 1 × 10 4 Plate cells at a density of 100 μL / well in a 6-well cell culture plate and incubate for 24 hours. After removing the culture medium, rinse with PBS and replace with samples diluted with 1640 medium (containing 2% serum) at different concentrations. Set up three replicates for each concentration. After 72 hours of incubation in a cell culture incubator, discard the supernatant and add 500 μL of trypsin to each well for 1-2 minutes. After that, add complete culture medium to terminate the digestion. Mix the cells by pipetting. Collect the cell suspension into a centrifuge tube and take a small amount for cell counting.

[0120] Centrifuge 1 mL of cells collected after trypsin digestion at 3000 rpm for 10 minutes, discard the supernatant, add 1 mL of 1 mol / L NaOH aqueous solution containing 10% (volume fraction) DMSO, seal the tube with parafilm, heat in an 80°C water bath for 30 minutes, and measure absorbance at 405 nm. Melanin synthesis inhibition rate = (1 - absorbance of sample group / absorbance of control group) * 100%.

[0121] The significant difference P value was analyzed by GraphPad Prism software. The melanin synthesis inhibition rate of blank control, positive control and test sample was analyzed by t test method using GraphPad Prism software. The significant differences are shown in Table 1 and Figure 6 shown.

[0122] Table 1. Inhibitory effect of recombinant type IV collagen 1CB1K on melanin synthesis in B16 (mouse melanoma cells) at different concentrations

[0123]

[0124] Combined with Table 1 and Figure 8As can be seen, the positive control (100 μg / mL α-arbutin) showed a melanin synthesis inhibition rate of 17.92% ± 10.37%, which was significantly different from the blank control (P value 0.04022), indicating that the experimental system is effective. At a test concentration of 0.01%, type IV collagen 1CB1K did not inhibit B16 melanin synthesis. However, at a test concentration of 0.03%, the melanin synthesis inhibition rate was 21.51% ± 8.98%, and at a test concentration of 0.1%, the melanin synthesis inhibition rate was 17.20% ± 5.69%, both higher than the blank control. This indicates that recombinant type IV collagen 1CB1K has an inhibitory effect on melanin synthesis at concentrations of 0.03% and 0.1%, demonstrating its whitening efficacy.

[0125] In summary, the present invention designs a recombinant type IV collagen with whitening efficacy based on a partial active amino acid sequence in the α1 chain of natural human type IV collagen. The amino acid sequence of the recombinant type IV collagen with whitening efficacy is shown in SEQ ID NO: 1; the recombinant type IV collagen has the activity of promoting cell adhesion and migration, can inhibit the synthesis of melanin, has whitening efficacy, and does not produce an immune response when applied to the human body. It has value in the fields of cosmetics, medicines, health products, medical devices and biomaterials.

[0126] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A recombinant type IV collagen, characterized in that The amino acid sequence of the recombinant type IV collagen is shown in SEQ ID No:

1.

2. A tandem recombinant type IV collagen, characterized in that: The tandem recombinant type IV collagen is a collagen obtained by repeating the recombinant type IV collagen according to claim 1 as a basic unit n times in series, where n is an integer greater than or equal to 3 and less than or equal to 20; Two adjacent basic units are connected by amino acids that can be recognized and cut by Kex2 enzyme, CPB enzyme, and Ste13 enzyme as a linker.

3. The recombinant type IV collagen according to claim 2, characterized in that The amino acid sequence that can be recognized and cut by Kex2 enzyme, CPB enzyme and Ste13 enzyme is KKREA.

4. The tandem recombinant type IV collagen according to claim 2, characterized in that The amino acid sequence of the recombinant type IV collagen is shown in SEQ ID No:

2.

5. The nucleic acid encoding the tandem recombinant type IV collagen according to claim 2.

6. The nucleic acid according to claim 5, characterized in that The nucleic acid sequence includes SEQ ID No: 4, or a degenerate sequence thereof.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 5 or 6.

8. A recombinant engineered bacterium, characterized in that The method comprises the nucleic acid according to claim 5 or 6, or comprises the recombinant expression vector according to claim 7, or expresses the recombinant type IV collagen according to claim 1.

9. The recombinant engineered bacterium according to claim 6, characterized in that The host bacteria of the recombinant engineering bacteria include Pichia pastoris, Saccharomyces cerevisiae or Hansenula.

10. The recombinant engineered bacterium according to claim 9, characterized in that The host bacteria is Pichia pastoris.

11. The recombinant engineered bacterium according to claim 10, characterized in that The host bacteria is HCPB-PPKEX2, and its deposit number is CGMCC No.25815.

12. The recombinant engineered bacterium according to any one of claims 8 to 11, characterized in that The recombinant engineered bacteria are deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC NO.32113.

13. The method for preparing recombinant type IV collagen according to claim 1, characterized in that: The preparation method comprises: (1) selecting and designing the sequence of recombinant type IV collagen, and then constructing a tandem recombinant type IV collagen using the recombinant type IV collagen as a basic unit by tandem repetition; The tandem recombinant type IV collagen comprises 3 to 20 basic unit recombinant type IV collagens; two adjacent basic units are connected by amino acids that can be recognized and cut by Kex2 enzyme, CPB enzyme, or Ste13 enzyme as a linker; (2) constructing a recombinant expression vector expressing the tandem recombinant type IV collagen; (3) electroporating the constructed recombinant expression vector into the host bacteria, screening and verifying to obtain recombinant engineered bacteria expressing high-copy collagen; (4) Fermentation-induced expression of recombinant engineered bacteria expressing high-copy collagen was performed to obtain recombinant type IV collagen.

14. The preparation method according to claim 13, characterized in that The amino acid sequence that can be recognized and cut by Kex2 enzyme, CPB enzyme and Ste13 enzyme is KKREA.

15. The preparation method according to claim 13, characterized in that In step (1), the nucleic acid of the tandem recombinant type IV collagen comprises the nucleotide sequence shown in SEQ ID No: 4, or a degenerate sequence thereof.

16. The preparation method according to claim 13, characterized in that In step (2), the recombinant plasmid vector includes pPICZαB, pFLDα, and pPIC9K.

17. The preparation method according to claim 16, characterized in that The vector is pPIC9K.

18. The preparation method according to claim 13, characterized in that In step (3), the host bacteria includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula.

19. The preparation method according to claim 13, characterized in that In step (3), the recombinant engineered bacteria are deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC NO.32113.

20. Recombinant type IV collagen prepared by the method according to any one of claims 13 to 19.

21. A composition characterized in that The composition comprises the recombinant type IV collagen according to claim 1, or the tandem recombinant type IV collagen according to claim 2, or the tandem recombinant type IV collagen encoded by the nucleic acid according to any one of claims 5 to 6, or the recombinant expression vector according to claim 7, or the recombinant engineered bacteria according to any one of claims 8 to 12, or the recombinant type IV collagen obtained according to the method according to any one of claims 13 to 19.

22. The product, characterized in that The product comprises the recombinant type IV collagen according to claim 1, or the tandem recombinant type IV collagen according to claim 2, or the recombinant type IV collagen encoded by the nucleic acid according to any one of claims 5 to 6, or the recombinant expression vector according to claim 7, or the recombinant engineered bacteria according to any one of claims 8 to 12, or the recombinant type IV collagen obtained by the method according to any one of claims 13 to 19, or the composition according to claim 21; the product includes a drug, medical device, biomaterial, tissue engineering product or cosmetic with a whitening effect.

23. Use of the recombinant type IV collagen according to claim 1, or the tandem recombinant type IV collagen according to claim 2, or the recombinant type IV collagen encoded by the nucleic acid according to any one of claims 5 to 6, or the recombinant expression vector according to claim 7, or the recombinant engineered bacteria according to any one of claims 8 to 12, or the recombinant type IV collagen obtained by the method according to any one of claims 13 to 19, or the composition according to claim 21, or the product according to claim 22 in the preparation of drugs, medical devices, biomaterials, tissue engineering products or cosmetics with whitening effects.

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