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

Abstract

The application belongs to the technical field of protein engineering and genetic engineering, and particularly relates to a recombinant type IV humanized collagen, a preparation method and application thereof. A novel recombinant collagen-like protein is obtained through screening, replacement and splicing, and the nucleotide sequence thereof is optimized according to the codon bias of Pichia pastoris, and then a corresponding genetic engineering yeast strain is constructed and obtained, and the recombinant collagen protein is successfully prepared through fermentation. It is proved through tests that the recombinant type IV humanized collagen prepared by the application has large molecular weight and high yield, has better free radical scavenging performance, cell proliferation and migration promotion performance compared with the commercially available recombinant collagen, and has good antioxidant and skin barrier repair functions, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein engineering and genetic engineering, and particularly relates to a recombinant humanized type IV collagen, a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application, and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those of ordinary skill in the art.

[0003] Collagen is the most abundant and widely distributed functional protein in mammals, accounting for 25-30% of the total protein. At present, about 29 different collagen types have been identified, which can be divided into types I, II, III, IV, etc. according to the order of discovery. Among them, type IV collagen mainly exists in the cell basement membrane and is an important component of the basement membrane. Its functions include: increasing the stability and elasticity of the basement membrane, maintaining the connection between cells and matrix, compensating for the function of the skin basement membrane, helping the combination of the epidermis and the dermis; making the skin structure complete and compact, preventing aging and wrinkles; playing an important role in cell signal transduction, angiogenesis inhibition, etc., and having excellent potential as a raw material for medical and cosmetic products. In recent years, with the gradual deepening of functional activity research, type IV collagen has been gradually applied as a raw material in the fields of beauty and skin care, medical devices, etc.

[0004] Different collagens can be divided into animal-derived collagen and recombinant collagen according to their sources. Collagen obtained from animal connective tissue has problems such as harsh extraction and purification conditions, animal source viruses, and product quality differences caused by animal individual differences, which limit the application and development of new products. Recombinant collagen is synthesized by genetic engineering technology, and has higher biological activity and biocompatibility, lower immunogenicity, better water solubility, etc. Therefore, its application range is more extensive, and it has attracted strong attention from the market. The inventors found that among the currently commercialized recombinant collagens, the most studied are types I, III and XVII, most of which come from microbial expression systems, while there are fewer studies on recombinant type IV collagen. The reported recombinant type IV collagen is basically a small molecular weight collagen, which is difficult to meet the market demand for different molecular weight ranges. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a macromolecular recombinant type IV humanized collagen, a preparation method and application thereof.

[0006] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

[0007] In a first aspect, the present application provides a recombinant type IV humanized collagen, which has:

[0008] a1) an amino acid sequence as shown in SEQ ID NO. 1; or,

[0009] a2) an amino acid sequence having at least 90% sequence homology with SEQ ID NO. 1.

[0010] In a second aspect, the present application provides a nucleic acid molecule capable of encoding the above-mentioned recombinant type IV humanized collagen.

[0011] In a third aspect, the present application provides a recombinant expression vector comprising the nucleic acid molecule.

[0012] In a fourth aspect, the present application provides a host cell comprising the recombinant expression vector or the nucleic acid molecule, or capable of expressing the recombinant type IV humanized collagen.

[0013] In a fifth aspect, the present application provides a preparation method of the recombinant type IV humanized collagen, comprising: culturing the host cell to express the recombinant type IV humanized collagen; and isolating and purifying the recombinant type IV humanized collagen.

[0014] In a sixth aspect, the present application provides the use of the above-mentioned recombinant type IV humanized collagen in the preparation of health products, cosmetics, medical aesthetic products or pharmaceuticals.

[0015] In the present application, the cosmetic is a skin cosmetic, and further, the cosmetic can be a skin care product.

[0016] Cell proliferation and migration are the basis of physiological processes such as anti-inflammatory, coagulation, wound healing, and angiogenesis. Based on the cell proliferation and migration activity, antioxidant and skin barrier repair function of the recombinant type IV humanized collagen of the present application, the recombinant type IV humanized collagen of the present application can be used in the fields of cosmetics, health products, medical aesthetic products and pharmaceuticals.

[0017] In a seventh aspect of the present application, a cosmetic or medical product is provided, which comprises at least the recombinant humanized collagen IV.

[0018] The above one or more technical solutions have the beneficial technical effects of:

[0019] The above technical solution obtains a new recombinant collagen protein through screening, replacement, splicing and other methods, and the nucleotide sequence is optimized according to the codon preference of Pichia pastoris, and then a corresponding genetically engineered yeast strain is constructed and successfully fermented to prepare the recombinant collagen protein.

[0020] The recombinant humanized collagen IV prepared by the above technical solution has a large molecular weight and a high yield, and has better free radical scavenging performance and cell proliferation and migration promotion performance compared with commercially available recombinant collagen proteins, and has good antioxidant and skin barrier repair functions, thus having good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0022] Figure 1 FIG. 1 is a SDS-PAGE protein electrophoresis result diagram of fermentation supernatant of recombinant collagen C4B in the present application; in the diagram, lane M represents standard protein with a molecular weight of 180 kDa; lane 1 represents fermentation supernatant of control bacteria P. pastoris GS115 / pPIC9K-C4B; and lane 2 represents fermentation supernatant of recombinant bacteria P. pastoris GS115 / pPIC9K.

[0023] Figure 2 FIG. 3 is a DPPH free radical scavenging rate column diagram of three different collagens in the present application; wherein, comparative example 1 represents a commercially available recombinant collagen comparative experiment group; and C4B represents a recombinant humanized collagen experiment group prepared by the present application example 5.

[0024] Figure 3 FIG. 4 is a mRNA relative expression amount column diagram of FLG in HaCaT cells under the action of different collagens in the present application; wherein, control group 1 represents a blank control group; control group 2 represents a commercially available recombinant collagen control group; and C4B represents a recombinant humanized collagen C4B group prepared by the present application example 5; in the diagram, P<0.01(**), P<0.001(***) compared with the control group 1 are considered to have significant differences. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed description is illustrative only, and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0026] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0027] The application will be further described with reference to the following examples, but it will be appreciated that the examples are illustrative of the application and are not intended to limit its scope. If the specific conditions of the experiment are not mentioned in the examples, the experiment is usually carried out under the conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels if not otherwise specified.

[0028] In one exemplary embodiment of the application, a recombinant humanized collagen type IV is provided, the recombinant humanized collagen type IV has:

[0029] a1) an amino acid sequence as shown in SEQ ID NO. 1; or,

[0030] a2) an amino acid sequence having at least 90% sequence homology with SEQ ID NO. 1.

[0031] In another exemplary embodiment of the application, a nucleic acid molecule is provided, the nucleic acid molecule is capable of encoding the humanized collagen type IV described above.

[0032] The nucleic acid molecule contains all or part of at least one nucleotide sequence selected from b1) to b4) below:

[0033] b1) a nucleotide sequence as shown in SEQ ID NO. 2;

[0034] b2) a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence as shown in SEQ ID NO. 2 under stringent conditions, and encoding a protein substantially identical to the protein encoded by the nucleotide sequence as shown in SEQ ID NO. 2;

[0035] b3) a nucleotide sequence represented by SEQ ID NO. 2 in which one or more nucleotides are deleted, substituted, inserted or added, and the nucleotide sequence encodes a protein substantially identical to the protein encoded by the nucleotide sequence represented by SEQ ID NO. 2;

[0036] b4) a nucleotide sequence having at least 90% identity to the polynucleotide of the nucleotide sequence represented by SEQ ID NO. 2, and the nucleotide sequence encodes a protein substantially identical to the protein encoded by the nucleotide sequence represented by SEQ ID NO. 2.

[0037] In the present application, the term "substantially identical protein" means an amino acid sequence which does not affect the activity of the protein (polypeptide), although one or more amino acids are actually changed by substitution, deletion, substitution, addition or insertion.

[0038] In the present application, examples of changes which do not affect the activity include conservative substitutions. The term "conservative substitution" means substitution of a preferably 1 to 40, more preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 8, and most preferably 1 to 4 amino acid residues with other chemically similar amino acid residues, while the activity of the polypeptide is not substantially changed. Examples thereof include cases where a certain hydrophobic amino acid residue is substituted with another hydrophobic amino acid residue and cases where a certain polar amino acid residue is substituted with another polar amino acid residue having the same charge. Functionally similar amino acids which can make such substitutions are known for each amino acid in the art.

[0039] In the present application, the term "stringent conditions" in the present application means conditions under which washing of the membrane is performed at a high temperature in a solution having a low salt concentration after hybridization, which can be appropriately determined by those skilled in the art, for example, the conditions include washing for 20 minutes at 60°C in a solution having 2xSSC (lxSSC: 15mM sodium citrate, 150mM sodium chloride) and 0.5% SDS, and include washing for 15 minutes at 60°C in a solution having 0.2xSSC (lxSSC: 15mM sodium citrate, 150mM sodium chloride) and 0.1% SDS.

[0040] In the present application, the term "identity" (homology) with respect to nucleotide sequences means the degree of base pairing constituting each sequence in the sequences to be compared. At this time, the presence of a gap and the characteristics of the amino acid are taken into consideration. Any value of "identity" shown in the present specification can be a value calculated using a homology search program known to those skilled in the art. For example, the value can be easily calculated by using default (initial setting) parameters in FASTA, BLAST, etc.

[0041] In the present application, "identity" of a nucleotide sequence is 90% or more, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more.

[0042] In the present application, the term "deletion, substitution, insertion or addition of one or more nucleotides in a polynucleotide" means that the change is made by known methods such as site-specific mutagenesis, or substitution of various nucleotides to the extent that can occur naturally. The number of changed nucleotides is one or more nucleotides. For example, one to several nucleotides, or 1, 2, 3, or 4 nucleotides.

[0043] In the present application, the term "a nucleotide sequence encoding a protein substantially identical to the protein encoded by the (each) nucleotide sequence" means a nucleotide sequence encoding a protein having an identical activity to the protein encoded by "the (each) nucleotide sequence".

[0044] In still another embodiment of the present application, there is provided a recombinant expression vector comprising the nucleic acid molecule.

[0045] The recombinant expression vector is obtained by effectively linking the above-mentioned nucleic acid molecule to an expression vector, which is a plasmid, and further can be a pPIC9K series plasmid.

[0046] In still another embodiment of the present application, there is provided a host cell comprising the recombinant expression vector or the nucleic acid molecule, or capable of expressing the recombinant type IV humanized collagen.

[0047] The host cell can be a cell of bacteria, fungi, actinomycetes, and further, the host cell can be a yeast (particularly, Pichia) among fungi.

[0048] In still another embodiment of the present application, there is provided a method for preparing the recombinant type IV humanized collagen, comprising: culturing the host cell, thereby expressing the recombinant type IV humanized collagen; and isolating and purifying the recombinant type IV humanized collagen.

[0049] In still another embodiment of the present application, there is provided a use of the above-mentioned recombinant type IV humanized collagen in the preparation of health care products, cosmetics, medical beauty products, or pharmaceutical products.

[0050] In the present application, the cosmetic is a skin cosmetic, and further, the cosmetic can be a skin care product.

[0051] It should be noted that the medical and beauty product in the present application refers to a product used in the field of medical and beauty, which is different from ordinary daily skin care products and beauty parlor skin care products in level. Some commonly used medical and beauty products include medical and beauty masks, dressings, etc. The use of these products is required to be performed by professional medical personnel or relevant guidance. The medical and beauty products, such as dressings, can be applied to persons with impaired skin barrier function after treatment of dermatitis, eczema, sensitive skin, and photorejuvenation, laser beauty, fruit acid skin activation, etc.

[0052] In the present application, the drug can be an antioxidant drug.

[0053] Cell proliferation and migration are the basis of physiological processes such as anti-inflammatory, coagulation, wound healing, and angiogenesis. Based on the cell proliferation and migration activity of the recombinant IV type humanized collagen, as well as the antioxidant and skin barrier repair function, the recombinant IV type humanized collagen of the present application can be used in the fields of cosmetics, health products, medical and beauty products, and drugs.

[0054] In another specific embodiment of the present application, a cosmetic or medical and beauty product is provided, which at least comprises the recombinant IV type humanized collagen.

[0055] Further, the cosmetic is specifically a skin care product.

[0056] Of course, the cosmetic can also comprise other raw material components allowed to be added in the field of cosmetics, including but not limited to solvents, solubilizers, suspending agents, stabilizers, emulsifiers, thickening agents, binders, preservatives, pH regulators, wetting agents, humectants, fragrances, pearl agents, surfactants, complexing agents, coloring agents, flavor enhancers, and / or combinations thereof, etc. A person skilled in the art can select and add according to the actual situation, which is not specifically limited here.

[0057] The present application is further explained and described by the following examples, but does not constitute a limitation on the present application. It should be understood that these examples are only used to illustrate the present application and do not limit the scope of the present application. In the following examples, the test methods for indicating specific conditions are generally performed under conventional conditions.

[0058] Example 1 Sequence selection of recombinant humanized IV type collagen

[0059] The amino acid sequence of human collagen type IV alpha 2 chain protein (GenBank: KAI4063798) is obtained from the NCBI database, and a segment of amino acid sequence C4B is finally obtained through deep mining analysis of the sequence information, screening and splicing according to the characteristics of the natural collagen amino acid sequence and the reported active site. The recombinant collagen protein has 748 amino acids, and the specific amino acid sequence is shown as SEQ ID NO. 1, which is obtained by splicing 34 amino acids for 22 times. The sequence protein has high molecular weight, contains multiple biological active sites of human collagen type IV, has high biocompatibility, low use risk, and ensures the realization of the biological function of the recombinant collagen protein, thereby realizing the recombinant humanized collagen protein.

[0060] Example 2 Construction of humanized collagen type IV gene expression system

[0061] According to the codon bias of Pichia pastoris, the sequence of the recombinant collagen C4B is optimized, and the optimized nucleotide sequence is shown as SEQ ID NO. 2. Subsequently, the nucleotide sequence of the codon-optimized recombinant collagen C4B is entrusted to Jinshui Biotechnology Co., Ltd. for whole gene synthesis, and is cloned into the EcoRI and NotI enzyme cutting sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-C4B. The recombinant sequence is correct through DNA sequencing comparison. After linearization treatment of the recombinant expression plasmid pPIC9K-C4B using SalI fast cutting enzyme, it is electroporated into P.pastoris GS115 expression host cells to obtain the recombinant transformant, and high copy recombinant P.pastoris GS115 / pPIC9K-C4B is obtained through geneticin G418 screening.

[0062] Example 3 Shake flask fermentation of recombinant humanized collagen type IV engineering yeast strain

[0063] The obtained gene recombinant engineering bacteria P.pastoris GS115 / pPIC9K-C4B are subjected to shake flask fermentation culture. The specific fermentation steps are as follows: a single colony is inoculated in 40 mL of YPD culture medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L), and is cultured at 30℃, 200 rpm for 24 h. According to the inoculation amount of 10%, it is transferred to 40 mL of initial expression culture medium BMGY, and is cultured at 30℃, 200 rpm for 24 h. The bacterial cells are collected by centrifugation, washed with normal saline, and then replaced into 40 mL of induction expression culture medium BMMY, and are cultured at 30℃, 200 rpm. Pure methanol is added to the culture medium every 24 h to a final concentration of 1.0% (v / v) to induce expression for 96 h.

[0064] SDS-PAGE protein electrophoresis analysis was performed on the fermentation supernatant of the recombinant engineering bacteria and the fermentation supernatant of the control bacteria, and the electrophoresis analysis results are shown in Figure 1. Figure 2 As shown in Figure 1, near the protein molecular weight of 125 kDa, the fermentation supernatant of the recombinant engineering bacteria P. pastoris GS115 / pPIC9K-C4B (lane 2) has an additional protein band (indicated by the arrow position).

[0065] Example 4 Preparation of recombinant collagen C4B

[0066] The recombinant bacteria P. pastoris GS115 / pPIC9K-C4B were subjected to shake flask culture, and the fermentation supernatant was sequentially subjected to the purification steps of hollow fiber clarification, ultrafiltration membrane concentration displacement, ion exchange chromatography, ultrafiltration membrane desalting, and freeze-drying (① Hollow fiber clarification: the fermentation supernatant was filtered through a 500 kDa hollow fiber membrane to remove bacteria and large molecular impurities; ② Ultrafiltration membrane concentration displacement: a 10 kDa ultrafiltration membrane package (manufacturer: Sartorius) was used to concentrate the feed liquid while using ion A liquid (20 mM Tris, pH 7.5) for displacement, until the conductivity of the permeate end was equivalent to that of ion A liquid; ③ Ion exchange chromatography: anion exchange chromatography was used, and the recombinant protein C4B was negatively charged and eluted with 150 mM sodium chloride, and the elution peak was collected; ④ Ultrafiltration membrane desalting: a 10 kDa ultrafiltration membrane package was used to desalt the feed liquid until the conductivity of the permeate end was reduced to 70 μS / cm; ⑤ Freeze-drying: the ultrafiltration concentrated liquid obtained above was filtered through a 0.45 μm microfiltration membrane and then placed in a freeze-drying tray for freeze-drying) to obtain the recombinant collagen protein pure product. The protein purified through the above steps has a purity of greater than 95% and a yield of greater than 50%.

[0067] Example 5 Cell proliferation and migration promoting effect of recombinant humanized collagen

[0068] The recombinant type IV collagen C4B of the present application promotes cell proliferation: the CCK-8 method experiment results show that the cell proliferation rate of ESF cells at a final concentration of 1.0 mg / mL and 0.5 mg / mL is 131.52% and 119.65%, which is significantly higher than that of the negative control group (1x maintenance medium), indicating that it has a certain effect on promoting ESF cell proliferation.

[0069] The recombinant collagen type IV C4B of the present application promotes cell migration: the results of the cell scratch test show that the wound healing percentage of the negative control group (NC) is 57.65% after 24 hours, and the wound healing percentage of the 0.2 mg / mL and 0.1 mg / mL recombinant collagen type IV sample groups is significantly higher than that of the negative control group (NC), which is about 70.03% and 79.36% respectively, so it is considered that the 0.2 mg / mL and 0.1 mg / mL recombinant collagen type IV sample groups have obvious wound healing promotion effect on HaCaT.

[0070] Example 6 Antioxidant experiment of recombinant collagen

[0071] Take 1 mg / mL, 2.5 mL of the recombinant humanized collagen stock solution in Example 4 and add 2.5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol into a test tube. Vigorously shake the mixture for 10 s, then place it at room temperature for 30 min. After the reaction is completed, measure the absorbance of the reaction mixture at 517 nm. Use distilled water instead of the sample solution as a blank control, and use commercially available and self-developed recombinant collagen for comparative experiments (Comparative Example 1, the recombinant collagen is purchased from Hebei Nakobio Technology Co., Ltd., model number 3CH-FD05). DPPH free radical scavenging activity = (OD blank - OD sample) / OD blank, wherein OD blank is the absorbance value of the blank control group, and OD sample is the absorbance value of the recombinant humanized collagen or commercially available recombinant collagen group.

[0072] The results are shown in Table 1. Figure 2 As shown in Table 1, the recombinant humanized collagen C4B of the present application has obvious DPPH free radical scavenging ability compared with the blank control, and its DPPH free radical scavenging rate is higher than that of the commercially available recombinant collagen, so the recombinant collagen provided in this example has obvious antioxidant effect.

[0073] Example 7 Barrier repair experiment

[0074] Filaggrin (FLG) is one of the important components of the keratin envelope, which ensures the integrity of the skin barrier. FLG deficiency can cause disorder of the lipid bilayer structure, delay of maturation, and at the same time, cause decrease of the tightness of the keratin layer cells, increase of the permeability between cells and decrease of the light protection, ultimately leading to damage of the skin barrier.

[0075] Collect human immortal keratinocytes (HaCaT cells), and prepare a cell suspension with high glucose DMEM cell culture solution. Add 2 mL of the cell suspension to each well of a 6-well plate, and the number of cells is 2.6 x 10 5 / well. The experimental blank control group (control group 1), the commercial recombinant collagen control group (control group 2), and the recombinant humanized collagen C4B group (experimental group) prepared in Example 5 of the application were set up, with 3 replicate wells in each group. The 6-well plate was incubated in a cell incubator (37℃, 5% CO2) for 24 h. When the cell confluence rate reached 50%-60%, the culture solution was discarded, 2 mL of high-sugar DMEM cell culture solution was added to each well of the blank control group, and 2 mL of high-sugar DMEM cell culture solution containing the corresponding collagen was added to each well of the other two groups, wherein the concentration of the collagen in the high-sugar DMEM cell culture solution was 1 mg / mL. After the 6-well plate was incubated in the incubator (37℃, 5% CO2) for 24 h, the cells in each well were washed twice with 2 mL of PBS buffer. According to the Total RNA extraction kit, 1 mL of RNAiso Plus was added, the cells were lysed by blowing, and the sample was collected. According to the kit instructions, the RNA extraction, reverse transcription, and fluorescence quantitative PCR detection experiments were carried out to detect the mRNA relative expression of the barrier-related protein FLG. The 2 -△△CT method was used for calculation.

[0076] The mRNA expression promoting ability of different collagens in HaCaT cells is shown in Figure 3 Table 2. Compared with the blank control group (control group 1), the commercial recombinant collagen (control group 2) and the recombinant humanized collagen C4B prepared in Example 4 of the application (experimental group) can significantly increase the mRNA relative expression of FLG. The recombinant humanized collagen prepared in the application has a stronger mRNA expression promoting ability than the commercial recombinant collagen, so the recombinant humanized collagen of the application has obvious skin barrier repair function.

[0077] The remaining matters of the application are known technologies.

[0078] The above examples are only for illustrating the technical concept and characteristics of the application, and the purpose is to enable those skilled in the art to understand the content of the application and implement it, and cannot limit the protection scope of the application. Any equivalent changes or modifications made according to the spirit and essence of the application should be covered within the protection scope of the application.

Claims

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

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is capable of encoding the recombinant humanized collagen type IV of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 2 or 3.

5. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 4 or the nucleic acid molecule of any one of claims 2-3, or is capable of expressing the recombinant humanized collagen type IV of claim 1.

6. The host cell of claim 5, wherein, The host cell is a cell of bacteria or fungi.

7. The host cell of claim 6, wherein The host cell is a cell of yeast in fungi.

8. The host cell of claim 7, wherein, The yeast is Pichia pastoris.

9. A method of preparing the recombinant humanized collagen type IV according to claim 1, wherein, Comprising: culturing the host cell of any one of claims 5-8, thereby expressing the recombinant humanized collagen type IV; and purifying the recombinant humanized collagen type IV.

10. Use of the recombinant humanized collagen type IV of claim 1 in the preparation of a health product, a cosmetic product, or a medical aesthetic product.

11. A cosmetic or medical aesthetic product, characterized in that, The cosmetic product or the medical aesthetic product comprises at least the recombinant humanized collagen type IV of claim 1.

12. The cosmetic or medical product according to claim 11, wherein The cosmetic product is a skin care product.

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