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

Abstract

The invention belongs to the technical field of protein engineering and genetic engineering, and particularly relates to recombinant IV-type humanized collagen as well as a preparation method and application thereof. A section of brand new recombinant collagen is designed and obtained through methods such as screening, replacement and splicing, the nucleotide sequence of the recombinant collagen is optimized through pichia pastoris codon preference, then a corresponding genetic engineering yeast strain is constructed and obtained, and the recombinant collagen is successfully prepared through fermentation. Experiments prove that the recombinant IV-type humanized collagen prepared by the invention is large in molecular weight and high in yield, and compared with commercially available recombinant collagen, the recombinant IV-type humanized collagen has better free radical scavenging performance and cell proliferation and migration promoting performance, and has good antioxidant and skin barrier repair functions, so that the recombinant IV-type humanized collagen has good practical application value.

Description

Technical Field

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

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Collagen is the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. At present, about 29 different types of collagen have been identified. According to the order of discovery of collagen, it can be divided into type I, type II, type III, type IV, etc. Among them, type IV collagen mainly exists in the basement membrane of cells 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, making up for the function of the basement membrane of the skin, and helping the combination of the epidermis and the dermis; making the skin structure complete and firm, preventing aging and wrinkles; playing an important role in cell signal transduction, angiogenesis inhibition and other processes, and has excellent potential to become a raw material for medical cosmetics. In recent years, with the gradual deepening of functional activity research, type IV collagen has gradually been used 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-derived viruses, and product quality differences caused by individual differences in animals, which limits the application and development of new products. Recombinant collagen is synthesized by genetic engineering technology and has the characteristics of higher biological activity and biocompatibility, lower immunogenicity, and better water solubility. Therefore, its application range is wider and has received strong attention from the market. The inventors found that the most studied recombinant collagens that have been commercialized are type I, type III, and type XVII, most of which come from microbial expression systems, while there are fewer related studies on recombinant type IV collagen. The reported type IV recombinant collagens are basically small molecular weight collagens, which are difficult to meet the market demand for different molecular weight ranges. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a macromolecular recombinant type IV humanized collagen, a preparation method and application thereof. Experiments have shown that the macromolecular recombinant type IV humanized collagen produced by the present invention has excellent free radical scavenging performance and cell proliferation and adhesion performance, and its product performance is better than the existing commercially available recombinant collagen, and is very suitable for industrial large-scale production. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] The first aspect of the present invention provides a recombinant humanized type IV collagen, wherein the recombinant humanized type IV collagen has:

[0008] a1) the amino acid sequence 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] The second aspect of the present invention provides a nucleic acid molecule, which can encode the above-mentioned recombinant humanized type IV collagen.

[0011] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule.

[0012] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the recombinant expression vector or the nucleic acid molecule, or is capable of expressing the recombinant humanized type IV collagen.

[0013] The fifth aspect of the present invention provides a method for preparing the recombinant type IV humanized collagen, comprising: culturing the host cells to express the recombinant type IV humanized collagen; and isolating and purifying the recombinant type IV humanized collagen.

[0014] The sixth aspect of the present invention provides the use of the above-mentioned recombinant humanized type IV collagen in the preparation of health products, cosmetics, medical beauty products or medicines.

[0015] In the present invention, the cosmetics are skin cosmetics, and further, the cosmetics can be skin care products.

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

[0017] A seventh aspect of the present invention provides a cosmetic or medical beauty product, wherein the cosmetic or medical beauty product at least comprises the recombinant type IV humanized collagen.

[0018] Beneficial technical effects of one or more of the above technical solutions:

[0019] The above technical scheme designs a new recombinant collagen protein through screening, replacement, splicing and other methods. The nucleotide sequence is optimized by Pichia pastoris codon preference, and then the corresponding genetically engineered yeast strain is constructed, and the recombinant collagen protein is successfully prepared by fermentation.

[0020] Experiments have proved that the recombinant type IV humanized collagen prepared by the above technical scheme has a large molecular weight and high yield. Compared with commercially available recombinant collagen, it has better free radical scavenging performance and cell proliferation and migration promoting performance, and has good antioxidant and skin barrier repair functions, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

[0023] Figure 2 It is a bar graph of DPPH free radical scavenging rate of three different collagens in the present invention; wherein, comparative example 1 represents a comparative experimental group of commercially available recombinant collagen; C4B represents an experimental group of recombinant humanized collagen prepared using Example 5 of the present invention.

[0024] Figure 3 It is a bar graph of the relative mRNA expression of FLG in HaCaT cells under the action of different collagens in the present invention; wherein, control group 1 represents a blank control group; control group 2 represents a commercially available recombinant collagen control group; C4B represents the recombinant humanized collagen C4B group prepared in Example 5 of the present invention; in the figure, compared with control group 1, P<0.01(**), P<0.001(***), which is considered to have significant differences. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0027] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0028] In a typical embodiment of the present invention, a recombinant humanized type IV collagen is provided, wherein the recombinant humanized type IV collagen has:

[0029] a1) the amino acid sequence 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 specific embodiment of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule is capable of encoding the above-mentioned type IV humanized collagen.

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

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

[0034] b2) a nucleotide sequence that can hybridize with the complementary sequence of the nucleotide sequence shown in SEQ ID NO.2 under stringent conditions and encodes a protein that is substantially equivalent to the protein encoded by the nucleotide sequence shown in SEQ ID NO.2;

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

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

[0037] In the present invention, the term "substantially equivalent protein" refers to an amino acid sequence that does not affect the activity of the protein (polypeptide) despite the fact that one or more amino acids are changed by substitution, deletion, replacement, addition or insertion.

[0038] In the present invention, examples of changes that do not affect activity include conservative substitutions. The term "conservative substitution" refers to replacing 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 substantially unchanged. Examples thereof include situations where a hydrophobic amino acid residue is replaced with another hydrophobic amino acid residue and situations where a polar amino acid residue is replaced with another polar amino acid residue with the same charge. Functionally similar amino acids capable of such substitutions are each amino acid known in the art.

[0039] In the present invention, the term "stringent conditions" refers to the conditions for washing the membrane in a solution with a low salt concentration at a high temperature after hybridization. Those skilled in the art can appropriately determine the conditions. For example, the conditions include washing in a solution with 2×SSC (1×SSC: 15 mM sodium citrate, 150 mM sodium chloride) and 0.5% SDS at 60°C for 20 minutes, and washing in a solution with 0.2×SSC (1×SSC: 15 mM sodium citrate, 150 mM sodium chloride) and 0.1% SDS at 60°C for 15 minutes.

[0040] In the present invention, for nucleotide sequences, the term "identity" (homology) refers to the degree of base pairing that constitutes each sequence in the sequence to be compared. At this time, the presence of gaps and the characteristics of amino acids are taken into account. Any value of "identity" shown in this specification can be a value calculated using a homology search program known to those skilled in the art. For example, this value can be easily calculated in FASTA, BLAST, etc. by using default (initial setting) parameters.

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

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

[0043] In the present invention, the term "a nucleotide sequence encoding a protein substantially equivalent to the protein encoded by the (each) nucleotide sequence" refers to a nucleotide sequence, the protein encoded by which has the same activity as "the protein encoded by the (each) nucleotide sequence".

[0044] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the nucleic acid molecule.

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

[0046] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell comprises the recombinant expression vector or the nucleic acid molecule, or is capable of expressing the recombinant humanized type IV collagen.

[0047] The host cell may be a cell of bacteria, fungi, or actinomycetes. Furthermore, the host fungus may be a yeast among fungi (specifically, it may be Pichia pastoris).

[0048] In another specific embodiment of the present invention, a method for preparing the recombinant humanized type IV collagen is provided, comprising: culturing the host cells to express the recombinant humanized type IV collagen; and isolating and purifying the recombinant humanized type IV collagen.

[0049] In another specific embodiment of the present invention, there is provided use of the above-mentioned recombinant humanized type IV collagen in the preparation of health products, cosmetics, medical beauty products or medicines.

[0050] In the present invention, the cosmetics are skin cosmetics, and further, the cosmetics can be skin care products.

[0051] It should be noted that the medical beauty products in the present invention refer to products used in the field of medical beauty, which are of a different level from ordinary daily skin care products and beauty salon skin care products. Some commonly used medical beauty products include medical beauty masks, dressings, etc. The use of these products needs to be carried out by professional medical staff, or relevant guidance is required. Medical beauty products, such as dressings, can be used for dermatitis, eczema, sensitive skin, and those with impaired skin barrier function after photorejuvenation, laser beauty, fruit acid rejuvenation, etc.

[0052] In the present invention, the drug may be an antioxidant drug.

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

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

[0055] Furthermore, the cosmetics are specifically skin care products.

[0056] Of course, the cosmetics may also contain any other raw material ingredients allowed to be added in the cosmetics field, including but not limited to solvents, solubilizers, suspending agents, stabilizers, emulsifiers, thickeners, adhesives, preservatives, pH adjusters, wetting agents, moisturizers, fragrances, pearlescent agents, surfactants, compounding agents, colorants, flavor enhancers and / or combinations thereof, etc. Those skilled in the art may select and add according to actual conditions, and no specific limitation is made here.

[0057] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

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

[0059] In this embodiment, the amino acid sequence of human type IV collagen α2 chain protein (GenBank: KAI4063798) was obtained through the NCBI database. Through in-depth mining and analysis of the sequence information, and based on the characteristics of the natural collagen amino acid sequence and the reported active sites, an amino acid sequence C4B was finally obtained through screening and splicing. The recombinant collagen of the present invention has a total of 748 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.1, which is obtained by splicing 34 amino acids repeated 22 times. The sequence protein has a high molecular weight, contains multiple biological active sites of human type IV collagen, has high biocompatibility, and has low use risks, which ensures the realization of the biological function of the recombinant collagen, and is a recombinant humanized collagen.

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

[0061] According to the codon preference of Pichia pastoris, the sequence of recombinant collagen C4B was optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2. Subsequently, the nucleotide sequence of the codon-optimized recombinant collagen C4B was entrusted to GenScript Biotech Co., Ltd. for full gene synthesis, and cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-C4B. After DNA sequencing and comparison, the recombinant sequence was correct. After linearization of the recombinant expression plasmid pPIC9K-C4B using SalI fast-cutting enzyme, it was electrotransferred into the P. pastoris GS115 expression host cell, and the obtained recombinant transformant was screened by geneticin G418 to obtain high-copy recombinant Pichia pastoris P. pastoris GS115 / pPIC9K-C4B.

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

[0063] The obtained genetically recombinant engineered bacteria P. pastoris GS115 / pPIC9K-C4B was fermented in a shake flask. The specific fermentation steps are as follows: Pick a single clone and inoculate it in 40mL of YPD medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L), and culture it at 30°C and 200rpm for 24h. Transfer it to 40mL of the initial expression medium BMGY at a 10% inoculation rate and culture it at 30°C and 200rpm for 24h. Collect the bacteria by centrifugation, wash the bacteria with physiological saline, and then replace it with 40mL of induced expression medium BMMY, culture it at 30°C and 200rpm, add pure methanol to the culture medium every 24h to a final concentration of 1.0% (v / v), and induce expression for 96h.

[0064] The supernatant of the fermentation of the recombinant engineering bacteria and the supernatant of the fermentation of the control bacteria were subjected to SDS-PAGE protein electrophoresis analysis. The electrophoresis analysis results were as follows: Figure 2 As shown in A. At around 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).

[0065] Example 4 Preparation of recombinant collagen C4B

[0066] The recombinant bacteria P. pastoris GS115 / pPIC9K-C4B were cultured in shake flasks, and the fermentation supernatant was sequentially purified by hollow fiber clarification, ultrafiltration membrane concentration and replacement, ion exchange chromatography, ultrafiltration membrane desalination, and freeze-drying (① hollow fiber clarification: the fermentation supernatant was filtered through a 500 kDa hollow fiber membrane to sterilize and remove macromolecular impurities; ② ultrafiltration membrane concentration and replacement: the feed liquid was concentrated using a 10 kDa ultrafiltration membrane package (manufacturer: Sartorius) and ion A solution (20 mM Tris, pH7.5) for replacement until the conductivity at the permeate end is equivalent to that of ion A solution; ③ ion exchange chromatography: anion exchange chromatography is used, the recombinant protein C4B is negatively charged, eluted with 150mM sodium chloride, and the elution peak is collected; ④ ultrafiltration membrane desalination: the feed liquid is desalted with a 10kDa ultrafiltration membrane bag until the conductivity at the permeate end is reduced to 70μS / cm; ⑤ freeze drying: the ultrafiltration concentrate obtained above is filtered with a 0.45μm microfiltration membrane and then placed in a freeze drying tray for freeze drying) to obtain pure recombinant collagen. The purity of the protein purified by the above steps is greater than 95%, and the yield is greater than 50%.

[0067] Example 5: Effects of recombinant humanized collagen on cell proliferation and migration

[0068] The effect of the recombinant type IV collagen C4B of the present application in promoting cell proliferation: The experimental results of the CCK-8 method showed that the cell proliferation rates of ESF cells at final concentrations of 1.0 mg / mL and 0.5 mg / mL were 131.52% and 119.65%, which were significantly higher than those of the negative control group (1× maintenance culture medium), and it is believed that it has a certain effect in promoting ESF cell proliferation.

[0069] The recombinant type IV collagen C4B of the present application promotes cell migration: the results of the cell scratch experiment show that the 24h wound healing percentage of the negative control group (NC) is 57.65%, and the 24h wound healing percentages (Wound Closure%) of the 0.2mg / mL and 0.1mg / mL recombinant type IV collagen sample groups are significantly greater than that of the negative control group (NC), which are approximately 70.03% and 79.36%, respectively. It is believed that the 0.2mg / mL and 0.1mg / mL recombinant type IV collagen sample groups both have a significant effect on promoting HaCaT wound healing.

[0070] Example 6 Antioxidant Experiment of Recombinant Collagen

[0071] Take 1 mg / mL, 2.5 mL of the purified stock solution of recombinant humanized collagen in Example 4 and place it in a test tube, add 2.5 mL of DPPH free radical solution (0.1 mmoL / L) dissolved in 95% ethanol, shake the mixture vigorously for 10 seconds, and then place it at room temperature for reaction for 30 minutes. 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 was purchased from Hebei Naco Biotechnology Co., Ltd., model 3CH-FD05). DPPH free radical scavenging activity = (OD blank-OD sample) / OD blank, where 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 as follows Figure 2 As shown, the recombinant humanized collagen C4B of the present invention has obvious DPPH free radical scavenging ability compared with the blank control, and its DPPH free radical scavenging rate is higher than the DPPH free radical scavenging rate of commercially available recombinant collagen. Therefore, 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 cornified envelope, ensuring the integrity of the skin barrier. FLG deficiency can lead to disordered lipid bilayer structure and delayed maturation, while also causing reduced compactness of stratum corneum cells, increased intercellular permeability and decreased photoprotection, ultimately leading to damaged skin barrier.

[0075] Human immortalized keratinocytes (HaCaT cells) were collected and cell suspension was prepared with high-glucose DMEM cell culture medium. 2 mL of cell suspension was added to each well of a 6-well plate. The cell number was 2.6 × 10 5 / well. The experiment set up a blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), and a recombinant humanized collagen C4B group prepared in Example 5 of the present invention (experimental group), and each group was set up with 3 replicate wells. The 6-well plate was placed in a cell culture incubator (37°C, 5% CO2) and incubated for 24 hours. When the cell fusion rate reached 50% to 60%, the culture medium was discarded, and 2 mL of high-glucose DMEM cell culture medium was added to each well of the blank control group; 2 mL of high-glucose DMEM cell culture medium containing the corresponding collagen was added to the other two groups, respectively, wherein the concentration of collagen in the high-glucose DMEM cell culture medium was 1 mg / mL. After the 6-well plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours, the cells were washed twice with 2 mL of PBS buffer in each well, and 1 mL of RNAiso Plus was added according to the Total RNA extraction kit. After blowing and lysing the cells, the samples were collected. According to the instructions of the kit, RNA extraction, reverse transcription and fluorescence quantitative PCR detection experiments were carried out to detect the relative expression of mRNA of the barrier-related protein FLG. 2 -△△CT Method to calculate.

[0076] The ability of different collagens to promote the mRNA expression of FLG in HaCaT cells Figure 3 As shown, compared with the blank control group (control group 1), the commercially available recombinant collagen (control group 2) and the recombinant humanized collagen C4B prepared in Example 4 of the present invention (experimental group) can significantly increase the relative expression of FLG mRNA, among which the recombinant humanized collagen prepared by the present invention has a stronger ability to promote the mRNA expression of FLG than the commercially available recombinant collagen. Therefore, the recombinant humanized collagen of the present invention has obvious skin barrier repair function.

[0077] Matters not covered by the present invention are known technologies.

[0078] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant type IV humanized collagen, characterized in that: The recombinant humanized type IV collagen has: a1) the amino acid sequence shown in SEQ ID NO.1; a2) an amino acid sequence having at least 90% sequence homology with SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule can encode the recombinant humanized type IV collagen according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule contains all or part of at least one nucleotide sequence selected from the following b1) to b4): b1) the nucleotide sequence shown in SEQ ID NO.2; b2) a nucleotide sequence that can hybridize with the complementary sequence of the nucleotide sequence shown in SEQ ID NO.2 under stringent conditions and encodes a protein that is substantially identical to the protein encoded by the nucleotide sequence shown in SEQ ID NO.2; b3) the nucleotide sequence shown in SEQ ID NO.2, wherein one or more nucleotides are deleted, substituted, inserted or added, and the nucleotide sequence encodes a protein substantially equivalent to the protein encoded by the nucleotide sequence shown in SEQ ID NO.2; b4) A nucleotide sequence having at least 90% identity with the polynucleotide of the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence encodes a protein substantially identical to the protein encoded by the nucleotide sequence shown in SEQ ID NO.

2.

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

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

6. The host cell according to claim 5, characterized in that The host cell is a cell of bacteria, fungi, or actinomycetes; further, the host fungus is a yeast among fungi; and further, it is Pichia pastoris.

7. A method for preparing the recombinant humanized type IV collagen according to claim 1, characterized in that: include: Cultivating the host cell according to any one of claims 5 to 6, thereby expressing the recombinant humanized type IV collagen; And, separating and purifying the recombinant humanized type IV collagen.

8. Use of the recombinant humanized type IV collagen according to claim 1 in the preparation of health products, cosmetics, medical beauty products or medicines.

9. A cosmetic or medical beauty product, characterized in that: The cosmetic or medical beauty product at least comprises the recombinant humanized type IV collagen according to claim 1.

10. The cosmetic or medical beauty product according to claim 9, characterized in that: The cosmetics are skin care products.

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