Recombinant IV type collagen as well as preparation method and application thereof

Abstract

The invention provides recombinant IV-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The recombinant IV type collagen with the whitening effect is designed on the basis of a part of active amino acid sequences in a natural human IV type collagen alpha1 chain, and the amino acid sequence of the recombinant IV type collagen with the whitening effect is shown as SEQ ID NO: 1; the recombinant IV-type collagen has the activity of promoting cell adhesion and migration, can inhibit synthesis of melanin, has a whitening effect, does not generate immune response when being applied to a human body, and has the value of being applied to the fields of cosmetics, medicines, health care products, medical instruments and biological materials.

Description

Technical Field

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

[0002] The pigmentation of human skin determines the color of skin, eyes and hair, and can also protect the skin from damage by ultraviolet radiation. If there is a disorder in melanin metabolism, it will cause pigmentation diseases such as spots, freckles, chloasma and melanoma. The metabolic process of melanin includes the production of melanin, the transport of melanin from melanocytes to keratinocytes, and the distribution and degradation of melanin in keratinocytes.

[0003] In recent years, regulating melanin has gradually become a research hotspot. The existing whitening methods mainly include inhibiting melanin production, inhibiting melanin transport, and promoting melanin metabolism. The synthesis of melanin mainly involves three enzymes in the tyrosinase gene family, namely tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2). Among them, tyrosinase is the key rate-limiting enzyme in the melanin synthesis pathway, catalyzing tyrosine to generate dopa and further generate dopaquinone, or catalyzing tyrosine direct oxidation to generate dopaquinone, which is the substrate for melanin synthesis. Therefore, inhibiting the activity of tyrosinase can block the biosynthesis reaction chain of melanin, reduce the production of melanin, and achieve a whitening effect. At present, the ingredients that inhibit tyrosinase activity mainly include tranexamic acid, azelaic acid, phenethyl resorcinol, potassium methoxysalicylate (4MSK), tranexamic acid, etc.; the ingredients that inhibit melanin transport include niacinamide, soy protein, 1-methylhydantoin-2-imide, diglucosyl gallic acid, etc.; to promote melanin metabolism, acid raw materials are usually used to exfoliate old keratin, such as glycolic acid and β-hydroxy acids such as salicylic acid. Although the above whitening ingredients can reduce pigmentation, some substances are cytotoxic, and acid raw materials may bring the risk of irritation to the skin. As for other whitening agents such as ascorbic acid, retinoids and nonapeptide-1, ascorbic acid is unstable and easily oxidized, retinoids may cause irritant dermatitis, and nonapeptide-1 has a relatively weak effect, and its whitening effect may depend on the concentration and formula used and the reaction of individual skin. Therefore, it is necessary to find a natural, non-toxic, stable, efficient and easily absorbed melanin regulator.

[0004] In the prior art, a novel tyrosinase inhibitory peptide, namely Asp-Gly-Leu (DGL), Gly-Ala-Arg (GAR) and Ser-Asp-Trp (SDW), was identified from donkey collagen by combining computer screening with in vitro activity verification. All three short peptides have potent tyrosinase inhibitory activity, among which His85, His244, His259 and Asn260 are key residues driving the interaction between the peptide and tyrosinase. According to the results of the tyrosinase inhibition experiment, it was found that the DGL tripeptide was the most effective among the three short peptides, because different types of interactions greatly affect the structural stability and binding affinity between the peptide and tyrosinase. A total of 9 hydrogen bonds (7 carbon-hydrogen bonds and 2 conventional hydrogen bonds) are formed between the DGL short peptide and tyrosinase, including 6 amino acid residues in tyrosinase, namely 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, and it can directly interact with tyrosinase to inhibit the formation of dopaquinone.

[0005] The basement membrane has functions such as signal conduction, permeation, and barrier, which can prevent harmful substances from entering the dermis. When the main component of the basement membrane, type IV collagen, is reduced, the continuity of the basement membrane will be destroyed, causing the epidermal cells to lose contact with the basement membrane and directly connect to the exposed dermis. The damaged basement membrane allows melanocytes and melanin to enter the dermis, and melanin cannot be excreted with the normal metabolism of keratinocytes, resulting in severe pigmentation. Therefore, the stability of the basement membrane can not only improve the metabolism of melanin, but also prevent melanin from entering the dermis from the epidermis, and can protect the basement membrane of the skin for whitening and freckle removal. The increase of type IV collagen can improve the stability of the basement membrane of the skin, and type IV collagen can promote cell regeneration, and by promoting the metabolism of skin cells, it helps reduce pigmentation, thereby achieving the effect of whitening. At present, there is no relevant research record of type IV collagen with whitening effect, and it is necessary to develop a recombinant type IV collagen with whitening effect and high transdermal absorption rate. Summary of the invention

[0006] In view of some deficiencies in the prior art, the present invention provides recombinant type IV collagen and 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 will 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 shown in SEQ ID NO: 1, or an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with 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, wherein 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 above-mentioned tandem recombinant type IV collagen, and the nucleic acid sequence is as shown in SEQ ID No:4.

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

[0019] The present invention also provides a recombinant engineered bacterium, comprising the nucleic acid molecule as described above, or comprising the recombinant expression vector as described above, or expressing the recombinant type IV collagen as 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 the deposit number being CGMCC No.25815.

[0022] Preferably, the recombinant engineered bacteria is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being 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 using the recombinant type IV collagen as a basic unit for tandem repetition to construct a tandem recombinant type IV collagen;

[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, and obtaining recombinant engineered bacteria expressing high-copy collagen;

[0027] (4) Fermentation-induced expression of the recombinant engineered bacteria expressing high-copy collagen 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 with an amino acid 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 a nucleotide sequence as 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 Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number of CGMCC NO.32113.

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

[0039] The present invention also provides a composition, which comprises 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 theoretical molecular weight of the recombinant type IV collagen is 3.3 kDa. It has a typical GXY triplet structure, which is 100% consistent with the sequence of natural human type IV collagen, and 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, and will not cause skin intolerance. Therefore, it is expected to be applied to the fields of cosmetics, medicines, 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 promotion activity or cell migration promotion activity.

[0044] (2) When constructing a method for preparing recombinant type IV collagen, the recombinant type IV collagen is repeatedly expressed in series, which can indirectly increase the copy number and increase the expression amount of the recombinant type IV collagen. The preparation method of the present invention obtains a recombinant collagen that is 100% consistent with the natural human type IV collagen sequence and will not produce an immune response when applied to the human body.

[0045] In addition, the linker used in the sequence design of the present invention will be cut and removed during the process of collagen secretion in cells, thereby obtaining a recombinant collagen with 100% homology to the corresponding region of natural collagen. The present invention uses strain HCPB-PPKEX2 to design and express recombinant type IV collagen, and directly obtains the target protein without introducing exogenous proteins or using any proteases that cut in vitro, which not only avoids the risk of exogenous protein residues, but also shortens the time and cost of subsequent purification process.

[0046] (3) The host bacteria of the recombinant type IV collagen of the present invention is Pichia pastoris, which can secrete the protein for extracellular expression, and can effectively avoid problems such as impurities caused by lysing the bacteria when extracting the product. As a eukaryotic organism, it can perform post-translational modifications on the secreted recombinant protein, such as glycosylation and phosphorylation, and the produced recombinant protein does not contain pathogens, viral inclusion bodies or heat sources, has high safety, and low fermentation cost. 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 alignment result of the NC terminal sequence of recombinant type IV collagen 1CB1K.

[0050] Figure 4 Results of cell adhesion assay.

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

[0052] Figure 6 Calculate the cell migration rate.

[0053] Figure 7 This is a graph showing 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 is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. In the embodiments of the present invention, all that is not described in detail is accomplished by conventional experimental methods, and all that is not described in detail in the processes involved in the embodiments is understandable and easily implemented by those skilled in the art based on the product manual 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 the tandem sequence contains recombinant type IV collagen has a recognition and cutting site for Kex2 enzyme, CPB enzyme, and STE13 enzyme between each adjacent two basic units, and these enzymes can cut and remove the non-natural collagen sequence (i.e., Linker) between the two tandem sequences; in addition, the design of repeated tandem expression of amino acid sequences can achieve the purpose of indirectly increasing the copy number, so as to achieve the purpose of increasing the expression amount of exogenous protein. After transcription and translation, the sequence in the present invention retains a protein sequence that is 100% consistent with the natural human type IV collagen sequence. The reason why this effect can be achieved is that the sequence of Linker is determined as LKKREA in the present invention, wherein L comes from the type IV collagen sequence itself, and KKREA is an additional non-natural collagen sequence. Linker can be completely cut and removed by Kex2 enzyme, CPB enzyme, and STE13 enzyme in the secretion pathway of the protein.

[0057] Kex2 enzyme is a calcium ion-dependent serine protease expressed by yeast microorganisms (naturally including Pichia pastoris) itself, which 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, so in the present invention, when the amino acid KKREA is added at the end of 1CB1K to form the sequence LKKREA as Linker for repeated tandem expression, KKREA can be completely cut and removed, and non-natural collagen sequences will not be introduced in the final recombinant protein product.

[0058] Yeast microorganisms have the STE 13 gene, which expresses the Ste 13 protease (strictly speaking, it belongs to the dipeptidyl aminopeptidase) in the cell, which can cut the amino acid sequence of EA and EAEA at the amino terminal of the protein. Recombinant carboxypeptidase B (CPB enzyme) can specifically cut the basic amino acids (especially K and R) at the carboxyl terminal of the protein until the basic amino acids at the C terminal are cut off, and other non-basic amino acids are exposed at the C terminal of the protein. The three proteases work together to remove non-collagenous amino acids such as KR at the C terminal and EA at the N terminal, respectively, to maintain the sequence homology between the recombinant collagen and natural human collagen.

[0059] The host bacteria of the recombinant type IV collagen of the present invention include one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula. In the following examples, HCPB-PPKEX2 with a deposit number of CGMCC No. 25815 in Pichia pastoris is used for specific description. The HCPB-PPKEX2 expresses human CPB enzyme, and the recombinant human carboxypeptidase HCPB can specifically cleave the basic amino acids (especially K and R) at the carboxyl terminal of the protein until the basic amino acids at the C-terminus are cleaved, and other non-basic amino acids are exposed at the C-terminus of the protein. That is, the non-human KKR sequence introduced by the Linker can be removed.

[0060] Embodiment 1:

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

[0062] The 379-412AA portion of the natural human type IV collagen α1 chain (Uniprot database P02462, https: / / www.uniprot.org / Uniprotkb / P02462 / entry#sequences) was named 1CB1K. The 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 with 1CB1K as the basic unit, and the basic unit was repeated 12 times in series. An amino acid sequence KKREA that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste13 enzyme was added between every two basic units to form an amino acid sequence as shown in SEQ ID No: 2, i.e., the recombinant type IV collagen after series connection, 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 composed of the endoplasmic reticulum and the Golgi apparatus through transcription and translation, the Kex2 enzyme will split it between KKR and EA in each KKREA, and then the two amino acids of EA at the amino terminal will be cut and removed by Ste 13 protease, and its sequence is shown in SEQ ID NO: 3. Then, the three amino acids of KKR at the carboxyl terminal of the sequence described in SEQ ID NO: 3 will be cut and removed by CPB enzyme, and finally a recombinant type IV collagen with 34 amino acids is secreted, that is, 1CB1K with an amino acid sequence as 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 termination codon TGATAA was added to the 3' end of the sequence. Then, Nanjing GenScript Biotech Co., Ltd. was 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 the 1210-1248 position 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, and 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, and then a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used to recover the linearized plasmid, and the DNA concentration in the resulting solution was controlled to be above 100 ng / μL as much as possible.

[0078] The linearized plasmid was electroporated into the competent cells of the host strain Pichia pastoris HCPB-PPKEX2 (from the strain number: CGMCC No. 25815), and the electroporated bacterial solution was spread on the MD plate, with 100 μL to 200 μL spread on one plate, and allowed to stand at room temperature for 10 minutes, and inverted 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 use a sterile triangular applicator to gently scrape off the His+ transformants on the surface of the plate 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 a single colony appeared, thereby obtaining a recombinant engineered bacterium containing pPIC9K-1CB1K-12.

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

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

[0082] The above-mentioned recombinant engineered bacteria containing pPIC9K-1CB1K-12 were placed in a 100mL Erlenmeyer flask containing 15mL BMGY medium and cultured at 28-30°C and 220rpm until OD600 was about 10 (24h). Centrifuge at 1500-3000×g for 5min at room temperature, collect the bacteria, resuspend the bacteria with BSM medium to make OD600 about 10, and continue to grow for 2 days on a shaker at 28-30°C and 220rpm. Add 100% methanol to the culture medium every 24h to a final concentration of 1.0% (v / v) for induction. After 16h of induction, the bacterial liquid sample can be collected. When the induction time is 48h, the induction effect is better. After the induction is completed, take an appropriate amount of sample and place it in a centrifuge tube, centrifuge it at 12000×g for 5min at 4°C, collect the supernatant, and test it immediately or store it 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 min, and then subjected to SDS-PAGE detection. The SDS-PAGE 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 48-hour induced expression supernatant of recombinant type IV collagen 1CB1K with whitening effect was subjected to N-terminal and C-terminal sequence alignment (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 by the present invention is consistent with the theoretical sequence, which means 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 "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 in proportion 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 number C7774) was weighed in proportion, dissolved in ultrapure water, and acetic acid was added to a pH value of 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 the 96-well plate. Coat each sample in 4 wells, incubate in a 37°C, 5% CO2 (v / v) incubator for 1 hour, remove excess coating solution from the wells, add 10 μL of 1% BSA-PBS solution, incubate in a 37°C, 5% CO2 incubator for 1 hour, remove the liquid in the wells after incubation, wash three times with D-PBS, discard the washing solution, seal with sealing film and place at 4°C 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 under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. The cells were diluted to 5×10 using complete culture medium premixed 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 the incubation, three replicate samples were measured. The fourth well was used to adjust the microscope parameters and its measured value was not used.

[0098] The specific detection steps are:

[0099] At least 4 × 4 digital tile images (fluorescence) of each of the 3 wells were captured using an inverted microscope. Each well was filled with D-PBS to form a "reverse meniscus", purged of air bubbles and covered with a sealing film. The plate was centrifuged (inverted) at a relative centrifugal force (RCF) of 300g at 22°C for 5 min. After centrifugation, the sealing film was discarded, the supernatant was removed from the well, washed once with D-PBS, and 100 μL of D-PBS was added. For each of the 3 wells, a total of 25 fluorescent tile digital images were taken (at least 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 numbers before and after centrifugation were measured, 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 of each experimental group were analyzed by one-way ANOVA (Dunnett method). The measurement results are shown in 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, NIH / 3T3 cells consistent with those 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 the 6-well plate. The interval between each two lines is 0.5 cm to 1 cm. The lines are drawn horizontally through the holes, ensuring that there are 3 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). Put it in a 37℃, 5% CO2 incubator for culture. Take samples at 0, 6h, and 24h, take pictures, and the test results are as follows: Figure 5 shown.

[0106] (3) Data processing: The scratch area of ​​each image 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 shown in Figure 2. Figure 6 shown.

[0107] Combination 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 a more excellent 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 cell B16 (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 1×10 4 / well density plated 96-well cell culture plates, washed with PBS after 24 hours and replaced with 1640 medium diluted with 0.01%, 0.03%, 0.1%, 0.3%, 0.5%, 1% recombinant type IV collagen solution, each concentration set 3 replicates. After 72 hours of cell culture incubator, CCK-8 was added, and cytotoxicity was detected at 450nm wavelength using an enzyme reader. The test results are shown as follows Figure 7 shown.

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

[0114] (2) Testing of the whitening effect of recombinant collagen:

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

[0116] Blank control: cell culture medium (Gibco, catalog number 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 1×10 4 / well density plate 6-well cell culture plate, culture for 24h, remove the culture medium, rinse with PBS, replace with 1640 culture medium (containing 2% serum) diluted with different concentrations of samples. Set up 3 replicates for each concentration. After 72h of culture in the cell culture incubator, discard the supernatant, add 500μL of trypsin to each well for 1-2min digestion, add complete culture medium to terminate digestion, blow and mix the cells, collect the cell suspension in a centrifuge tube, and take a small amount for cell counting.

[0120] Take 1 mL of cells collected after trypsin digestion and centrifuge at 3000r / min for 10 minutes, discard the supernatant, add 1 mL of 1 mol / L NaOH aqueous solution containing 10% (volume fraction) DMSO, seal the centrifuge tube with a sealing film and heat it in an 80°C water bath for 30 minutes, and measure the absorbance at 405nm. Among them, 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. Inhibition rate of melanin synthesis by recombinant type IV collagen 1CB1K at different concentrations in B16 (mouse melanoma cells)

[0123]

[0124] Combining Table 1 and Figure 8It can be seen that the melanin synthesis inhibition rate of the positive control (100 μg / mL α-arbutin) is 17.92% ± 10.37%, which is significantly different from the blank control (P value 0.04022), indicating that the test system is effective. Type IV collagen 1CB1K has no inhibition on B16 melanin synthesis at a test concentration of 0.01%, while the melanin synthesis inhibition rate at a test concentration of 0.03% is 21.51% ± 8.98%, and the melanin synthesis inhibition rate at a test concentration of 0.1% is 17.20% ± 5.69%, both higher than the blank control. This shows that recombinant type IV collagen 1CB1K has an inhibitory effect on melanin synthesis at concentrations of 0.03% and 0.1%, and has a whitening effect.

[0125] In summary, the present invention designs a recombinant type IV collagen with whitening effect 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 effect 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 effect, and will 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.

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

Claims

1. A recombinant type IV collagen, characterized in that: The sequence of the recombinant type IV collagen is shown in i or ii: i. an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 1; 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.

2. The recombinant type IV collagen according to claim 1, characterized in that The recombinant type IV collagen also includes: 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.

3. The recombinant type IV collagen according to claim 2, characterized in that 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.

4. The recombinant type IV collagen according to claim 3, characterized in that The amino acids that can be recognized and cleaved by Kex2 enzyme, CPB enzyme, and Ste13 enzyme include KKREA.

5. The recombinant type IV collagen according to any one of claims 2 to 4, characterized in that The sequence of the recombinant type IV collagen is shown in i or ii: i. The amino acid sequence is shown in SEQ ID No: 2; 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.

6. A nucleic acid encoding the tandem recombinant type IV collagen according to any one of claims 2 to 5.

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

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

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

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

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

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

13. The recombinant engineered bacterium according to any one of claims 9 to 12, characterized in that: The recombinant engineered bacteria are deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC NO.32113.

14. 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 using the recombinant type IV collagen as a basic unit for tandem repetition to construct a tandem recombinant type IV collagen; (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, and obtaining recombinant engineered bacteria expressing high-copy collagen; (4) Fermentation-induced expression of the recombinant engineered bacteria expressing high-copy collagen to obtain recombinant type IV collagen.

15. The preparation method according to claim 14, characterized in that: The sequence of the recombinant type IV collagen is shown in i or ii: i. an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO: 1; 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.

16. The preparation method according to claim 14, characterized in that: In step (1), the series-connected recombinant type IV collagen contains 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, and Ste13 enzyme as linkers.

17. The preparation method according to claim 16, characterized in that: The amino acids that can be recognized and cleaved by Kex2 enzyme, CPB enzyme, and Ste13 enzyme include KKREA.

18. The preparation method according to claim 14, 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.

19. The preparation method according to claim 14, characterized in that: In step (2), the vector of the recombinant plasmid includes pPICZαB, pFLDα, and pPIC9K.

20. The preparation method according to claim 19, characterized in that: The vector is pPIC9K.

21. The preparation method according to claim 14, characterized in that: In step (3), the host bacteria includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula.

22. The preparation method according to claim 14, characterized in that: In step (3), the recombinant engineered bacteria are deposited in the General Microbiological Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC NO.32113.

23. Recombinant type IV collagen prepared by the method according to any one of claims 14 to 22.

24. A composition, characterized in that The composition comprises the recombinant type IV collagen described in claim 1, or the recombinant type IV collagen described in any one of claims 2 to 5, or the tandem recombinant type IV collagen encoded by the nucleic acid described in any one of claims 6 to 7, or the recombinant expression vector described in claim 8, or the recombinant engineered bacteria described in any one of claims 9 to 13, or the recombinant type IV collagen obtained according to the method described in any one of claims 14 to 22.

25. A product, characterized in that The product comprises the recombinant type IV collagen described in claim 1, or the recombinant type IV collagen described in any one of claims 2 to 5, or the recombinant type IV collagen encoded by the nucleic acid described in any one of claims 6 to 7, or the recombinant expression vector described in claim 8, or the recombinant engineered bacteria described in any one of claims 9 to 13, or the recombinant type IV collagen obtained by the method described in any one of claims 14 to 22, or the composition described in claim 24; 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.

26. Use of the recombinant type IV collagen according to claim 1, or the recombinant type IV collagen according to any one of claims 2 to 5, or the recombinant type IV collagen encoded by the nucleic acid according to any one of claims 6 to 7, or the recombinant expression vector according to claim 8, or the recombinant engineered bacteria according to any one of claims 9 to 13, or the recombinant type IV collagen obtained according to the method according to any one of claims 14 to 22, or the composition according to claim 24, or the product according to claim 25 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.

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