A recombinant collagen type VII and a preparation method and application thereof

Abstract

The application discloses a kind of recombinant VII type collagen and its preparation method and application, belong to the field of bioengineering technology.The amino acid sequence of the recombinant VII type collagen of the present application is as shown in SEQ ID NO.1, the recombinant collagen has good effect of promoting aquaporin 3, cysteine-aspartic acid protease 14 and endosperm protein expression, has moisturizing repair efficacy to skin;And the recombinant protein is derived from human natural protein, application in human body will not produce immune response, can be applied as safe and effective cosmetic material.In addition, the fermentation purification process of the recombinant human VII type collagen of the present application is simple to operate, with the condition of expanding production, can be mass industrialized production.Therefore, the recombinant protein provided in the present application has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a recombinant type VII collagen, its preparation method, and its applications. Background Technology

[0002] Collagen is a biological macromolecule and a major component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein, and even more than 80% in some organisms. There are many types of collagen. Currently, at least 30 genes encoding collagen chains have been discovered, which can form more than 16 types of collagen molecules. Based on their structure, they can be classified into fibrous collagen, basement membrane collagen, microfibrillary collagen, anchored collagen, hexagonal reticular collagen, non-fibrous collagen, transmembrane collagen, etc.

[0003] Type VII collagen is a major component of anchoring fibers in the skin's basement membrane and is crucial for maintaining the integrity of the dermal-epidermal junction. Currently, the main preparation methods are: animal tissue extraction, cell culture, and chemical synthesis. While animal tissue extraction can yield high levels of bioactive type VII collagen, the tissue content is low, and there are issues such as viral risks, immune rejection, and low yield. Cell culture yields fully human proteins with lower immunogenicity risks; however, even using well-established expression systems, expressing and secreting full-length collagen with a specific structure is extremely difficult, if not impossible. Chemical synthesis, while producing extremely high purity and precise sequences, is limited to short peptides and lacks spatial structure. These methods are insufficient to meet the large-scale demands of cosmetics and medical devices. To commercialize type VII collagen products, a new production process is needed to safely and effectively express recombinant type VII collagen while retaining its relevant efficacy.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant type VII collagen, its preparation method and application, which has good moisturizing and repairing effects.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a recombinant type VII collagen, the amino acid sequence of which is any one of the following (A1) to (A3): (A1) The amino acid sequence of recombinant type VII collagen is shown in SEQ ID NO.1; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type VII collagen described in (A1); (A3) has at least 90% identity with the recombinant type VII collagen described in (A1) or the fusion protein described in (A2) and has a moisturizing and repairing amino acid sequence.

[0007] Secondly, the present invention provides a biomaterial related to the above-mentioned recombinant type VII collagen, which is any one of items (B1) to (B4): (B1) The nucleic acid molecule encoding the above-mentioned recombinant type VII collagen; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A host cell containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

[0008] Thirdly, the present invention provides a method for preparing the above-mentioned recombinant type VII collagen, which includes: introducing a nucleic acid molecule encoding the above-mentioned recombinant type VII collagen into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then separating and purifying the recombinant type VII collagen after fermentation culture.

[0009] Fourthly, the present invention provides a composition comprising the above-described recombinant type VII collagen.

[0010] Fifthly, the present invention provides the application of the above-mentioned recombinant type VII collagen in the preparation of moisturizing products.

[0011] Sixthly, the present invention provides the application of the above-mentioned recombinant type VII collagen in the preparation of barrier repair products.

[0012] The present invention has the following beneficial effects: The recombinant type VII collagen of this invention effectively promotes the expression of aquaporin 3, cysteine-aspartic protease 14, and endoplasmin, thus providing moisturizing and repairing effects on the skin. Furthermore, this recombinant protein is derived from natural human protein and will not induce an immune response in the human body, making it a safe and effective cosmetic material. In addition, the fermentation and purification process of the recombinant human type VII collagen of this invention is simple to operate and allows for large-scale industrial production. Therefore, the recombinant protein provided by this invention has promising application prospects. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 The electrophoresis results are for the recombinant type VII collagen purified in Example 1; Figure 2 The results of aquaporin expression level detection in Example 2; Figure 3 The results of the expression level detection of cysteine-aspartic protease 14 in Example 3; Figure 4 The results are the expression levels of the inner lining protein in Example 4. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0016] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0017] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0018] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0019] As used in this invention, the term "nucleic acid molecule" is a collective term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), a biological macromolecular compound composed of many nucleotide monomers, and one of the most fundamental substances of life. A nucleotide sequence refers to the arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA, and in some cases, nucleic acid molecules can be modified for use in the vectors of this invention, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids from one or more given cells or isolated from said one or more given cells.

[0020] As used herein, the term "expression cassette" refers to a nucleic acid construct comprising coding and regulatory sequences operable upon introduction into a host cell, resulting in transcription and / or translation of RNA or polypeptide, respectively. An expression cassette should be understood to include a promoter that allows transcription to begin, an open reading frame of the target gene, and a transcription terminator. Typically, the promoter sequence is positioned upstream of the target gene at a distance compatible with expression control.

[0021] As used herein, the term "expression vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) into a recipient cell. Besides the commonly used *E. coli* plasmid vector, there are many other artificially constructed plasmid vectors suitable for microorganisms, yeast, plants, etc. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. The most commonly used vector type is the "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques. Recombinant expression vectors may contain a form suitable for expressing nucleic acids in host cells, meaning that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0022] As used in this invention, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., of a nucleic acid construct or expression vector containing the polynucleotides of this invention. The host cell can be any cell useful in the production of the relevant protein. To produce the relevant protein, the nucleic acid encoding the protein can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. Methods for introducing vectors into host cells are well known, such as using electroporation, and can also include transfection, microinjection, gene gun technology, liposome-mediated methods, etc.

[0023] The following is provided merely to aid in understanding the invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made by any person within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0025] This invention provides a recombinant type VII collagen, which is extracted from human type VII collagen, and its specific amino acid sequence is as follows: GQKGEPGEMGLRGQVGPPGDPGLPGRTGAPGPQGPPGSATAKGERGFPGADGRPGSPGRAGNPGTPGAPGLKGSPGLPGPRGDPGERGPRGPKGEPGAPGQVIGGEGPGLPGRKGDPGPSGPPGPRGPLGDPGPRGPPGLPGTAMKGDKGDRGEERGPPGPGEGGIAPGEPGLPGLPGSPGPQGPVGPPGKKG EKGDSEDGAPGLPGQPGSPGEQGPRGPPGAIGPKGDRGFPGPLGEAGEKGERGPPGPAGSRGLPGVAGRPGAKGPEGPPGPTGRQGEKGEPGRPGDPAVVGPAVAGPKGEKGDVGPAGPRGATGVQGERGPPGLVLPGDPGPKGDPGDRGPIGLTGRAGPPGDSGPPGEKGDPGRPGPPGPVGPRGRD (SEQ ID NO.1).

[0026] The present invention selected the above fragment because, on the one hand, the fragment is derived from the α-1 chain of human type VII collagen, which does not produce an immune response and is therefore safe; on the other hand, the fragment is smaller in size and can be expressed efficiently; moreover, the fragment retains and enhances the skin moisturizing and repairing effects of type VII collagen.

[0027] To facilitate subsequent separation and purification, the recombinant type VII collagen can also be tagged at the N-terminus and / or C-terminus to obtain a fusion protein. Protein tags can be broadly categorized into three types based on their function: detection tags, expression and purification tags, and tracer tags. Commonly used expression and purification tags include: His, GST, MBP, CBD, Strep-tag, Halo-Tag, SNAP-tag, SUMO, NusA, TrxA, DsbA, Flag, and c-Myc. This invention does not limit the type of tag; those skilled in the art can adjust it as needed.

[0028] In some embodiments, the amino acid sequence of the above-mentioned recombinant type VII collagen may also be an amino acid sequence that has at least 90% identity with the above-mentioned recombinant type VII collagen or fusion protein and has moisturizing and repairing effects.

[0029] The percentage of sequence identity is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with a reference polypeptide sequence, introducing vacancies where necessary to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0030] In some embodiments, having at least 90% sequence identity can specifically be a range of any one or any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0031] Based on the above-mentioned recombinant type VII collagen, the present invention may also provide a nucleic acid molecule that encodes the above-mentioned recombinant type VII collagen.

[0032] In specific implementation schemes, the aforementioned nucleic acid molecules can be optimized according to the codon preferences of the host cell before being used to artificially synthesize gene fragments. It should be understood that all nucleic acid molecules capable of being translated into the aforementioned amino acid sequences are within the scope of protection of this invention.

[0033] In some embodiments, the nucleotide sequence of the above-mentioned nucleic acid molecule is shown in SEQ ID NO.1.

[0034] Furthermore, the present invention can also provide an expression cassette or expression vector comprising the aforementioned nucleic acid molecule. The expression cassette further contains regulatory sequences, such as a promoter and a terminator; the expression vector comprises the aforementioned nucleic acid molecule or expression cassette.

[0035] In some embodiments, the expression vectors described above include, but are not limited to, pPICZαA.

[0036] The present invention may also provide a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned expression cassette or the above-mentioned expression vector, and the host cell is capable of expressing the above-mentioned recombinant type VII collagen.

[0037] In a specific implementation plan, the host cell can be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis; eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells. Preferably, the host cell is Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, or Bacillus subtilis. More preferably, the host cell is Pichia pastoris X-33.

[0038] To obtain recombinant type VII collagen with the required purity and stability, this invention provides a corresponding preparation method, which includes: introducing the nucleic acid molecule of the above-mentioned recombinant type VII collagen into an expression vector, then introducing the obtained recombinant plasmid into a host cell, followed by fermentation culture and then separation and purification to obtain the above-mentioned recombinant type VII collagen.

[0039] In the specific implementation plan, the preparation of recombinant type VII collagen using host cells refers to fermenting and culturing host cells, and the culture medium and culture conditions are well known to those skilled in the art.

[0040] In some embodiments, the culture medium described above may be YPD culture medium.

[0041] The present invention does not impose any restrictions on the expression method, which can be selected and adjusted as needed. For example, the expression can be one or a combination of two of the following: compositional expression and induced expression. The inducer for induced expression can be IPTG, β-galactoside, methanol, ethanol, etc.

[0042] In some embodiments, recombinant type VII collagen is obtained by inducing expression in the host cells described above, specifically by adding methanol to induce expression when the bacterial cell density reaches a certain value.

[0043] As for the methods of separation and purification, they include one or a combination of several of the following: filtration, salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, membrane separation, etc.

[0044] In some embodiments, the separation and purification process involves: after fermentation, centrifuging to collect the supernatant, and then performing membrane separation, chromatography, or other steps on the supernatant. Preferably, the chromatography method is chromatographic chromatography or ion exchange chromatography.

[0045] This invention, through screening the aforementioned protein fragments and expressing them using a Pichia pastoris expression system, followed by specific fermentation and purification processes, yields recombinant type VII collagen with high yield and purity. This recombinant protein also promotes the expression of aquaporin 3 (AQP3), cysteine-aspartic protease 14 (Caspase-14), and inner lining protein (IVL). Therefore, the recombinant type VII collagen of this invention can be industrially produced, particularly for applications in cosmetics and other fields.

[0046] In some embodiments, the recombinant type VII collagen described above can be used to prepare moisturizing cosmetics.

[0047] In some embodiments, the recombinant type VII collagen described above can be used to prepare barrier repair cosmetics.

[0048] In some embodiments, the cosmetics mentioned above include: aqueous cosmetics, emulsion cosmetics, gel cosmetics, and sheet cosmetics. Specifically, cosmetics can be skin care water, serum, lotion, cream, mask, sunscreen, BB cream, and makeup base, etc.

[0049] The recombinant type VII collagen of the present invention can be prepared into cosmetics with moisturizing and repairing effects. The main active ingredient in the cosmetic is the aforementioned recombinant type VII collagen, and it may also include conventional cosmetic excipients. These cosmetic excipients include, but are not limited to, at least one of water, moisturizers, pH adjusters, neutralizers, skin conditioning agents, pH buffers, emulsifiers, thickeners, solubilizers, preservatives, preservative synergists, or fragrances.

[0050] This specification is intended to enable those skilled in the art to practice the invention. Various modifications to the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0051] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0052] Example 1 This embodiment describes recombinant collagen and its preparation. The preparation steps are as follows: S1. The codons were obtained from the amino acid sequence of recombinant type VII collagen (SEQ ID NO: 1). Without changing the amino acid sequence, the codons of the gene encoding the recombinant type VII collagen were optimized using codon optimization software. The optimized gene sequence (SEQ ID NO: 2) was synthesized into a whole gene, and XhoI and XbaI recognition sites and signal peptide recognition sites were added at the 5' and 3' ends, respectively. The gene was then ligated into the expression vector pPICZαA to obtain the recombinant plasmid pPICZαA-Ⅶ-0380. S2. The plasmid obtained in step S1 was linearized with restriction endonuclease Sac I and transformed into Pichia pastoris strain X-33 by electroporation. The strain was cultured at 30°C for 48 h to obtain Pichia pastoris genetically engineered strain. S3. The expression strain obtained in step S2 is cultured to express the target protein, yielding crude recombinant type VII collagen; specific steps include: 1): The expression strain obtained in step S2 is inoculated into YPD medium and cultured to obtain seed culture; 2): Take a portion of the seed culture and inoculate it into liquid culture medium. When the OD600 reaches 15, inoculate it into a BSM fermenter with an initial volume of 5L at a 10% inoculation rate. The culture temperature is 30℃, pH = 5.5, and dissolved oxygen is controlled at 20%~30%. 3): Once the glycerol is depleted, start glycerol feeding culture. Stop glycerol feeding when the strain grows to OD600 of 200. Continue culture until dissolved oxygen rises significantly again. After confirming that the glycerol is completely depleted, start adding methanol for induction culture. The induction temperature is 28℃ and the pH is 5.0. Induce for 48 hours, then remove from the tank and collect the supernatant by centrifugation. 4): The target protein obtained in step S3 was separated and purified using a combination of ion exchange chromatography and membrane separation to obtain recombinant type VII collagen. The purified recombinant type VII collagen solution and the marker SDS-PAGE were then analyzed. PAGE electrophoresis results are as follows Figure 1 As shown. The first column on the left shows the purified recombinant type VII collagen sample, and the second column shows the markers. SDS The results of electrophoresis using the PAGE method showed that the purity of the target protein of the recombinant type VII collagen after separation and purification was approximately 95%.

[0053] Example 2 This embodiment utilizes the recombinant type VII collagen obtained in Example 1 above, and performs real-time quantitative PCR (RT-PCR). The expression level of aquaporin 3 (AQP3) was detected by qPCR to investigate the moisturizing effect of recombinant VII collagen.

[0054] Harvesting healthy HSF cells, resuspending them in DMEM cell culture medium and adjusting the cell concentration to 1.2 × 10⁻⁶ cells / year. 5 Cells / mL. 2 mL of cell suspension was evenly seeded into each well of a six-well plate and incubated at 37°C in a 5% CO2 incubator.

[0055] The experiment included a blank group, a control group, and a sample group. The control group contained full-length type VII collagen, while the sample group contained recombinant type VII collagen with different concentrations. After incubation at 37°C and 5% CO2 for 24 h, total mRNA was extracted.

[0056] RNA concentration was measured using an ultra-micro spectrophotometer. A concentration greater than 20 ng / μL and an A260 / A280 value greater than 1.8 were considered acceptable samples. RevertAid was used. TM First-strand cDNA synthesis kit was used to synthesize cDNA. SYBR Green qPCR Mix, cDNA template, primers (sequences shown in Table 3), and RNase were used. Dissolve free ddH2O at 4℃ and prepare the Real-Time PCR reaction system on ice. The qPCR reaction system is shown in Table 2.

[0057] Table 1 Primer sequence list

[0058] Table 2 q-PCR reaction system

[0059] A two-step PCR reaction was performed. The reaction program was as follows: Step 1: 95℃ pre-denaturation for 300 s; Step 2: 95℃ denaturation for 20 s, annealing at 55℃ for 20 s, and extension at 72℃ for 20 s, for 40 reaction cycles. 2 -ΔΔCt The method determines relative to β The mRNA expression level of each target of Actin mRNA.

[0060] The results of AQP3 expression levels are as follows: Figure 2 As shown. By Figure 2 It can be seen that recombinant type VII collagen can promote the expression of AQP3, and the promotion rate of type VII collagen is stronger than that of the full-length fragment.

[0061] Example 3 This embodiment uses HSF cells and the recombinant type VII collagen obtained in Example 1 above, and performs real-time quantitative PCR (RT-PCR). The expression level of cysteine-aspartic protease 14 (Caspase-14) was detected by qPCR to investigate the moisturizing and repairing effects of recombinant VII collagen.

[0062] Harvesting healthy HSF cells, resuspending them in DMEM cell culture medium and adjusting the cell concentration to 1.2 × 10⁻⁶ cells / year. 5 Cells / mL. 2 mL of cell suspension was evenly seeded into each well of a six-well plate and incubated at 37°C in a 5% CO2 incubator.

[0063] The experiment included a blank group, a control group, and a sample group. The control group contained full-length type VII collagen, while the sample group contained recombinant type VII collagen with different concentrations. After incubation at 37°C and 5% CO2 for 24 h, total mRNA was extracted.

[0064] RNA concentration was measured using an ultra-micro spectrophotometer. A concentration greater than 20 ng / μL and an A260 / A280 value greater than 1.8 were considered acceptable samples. RevertAid was used. TM First-strand cDNA synthesis kit was used to synthesize cDNA. SYBR Green qPCR Mix, cDNA template, primers (sequences shown in Table 3), and RNase were used. Free ddH2O was dissolved at 4°C, and Real-time reaction was carried out on ice. The preparation of the Time PCR reaction system and the qPCR reaction system are shown in Table 4.

[0065] Table 3 Primer sequence list

[0066] Table 4 q-PCR reaction system

[0067] A two-step PCR reaction was performed. The reaction program was as follows: Step 1: 95℃ pre-denaturation for 300 s; Step 2: 95℃ denaturation for 20 s, annealing at 55℃ for 20 s, and extension at 72℃ for 20 s, for 40 reaction cycles. 2 -ΔΔCt The method determines relative to β The mRNA expression level of each target of Actin mRNA.

[0068] The results of Caspase-14 expression levels are as follows: Figure 3 As shown. By Figure 3 It can be seen that recombinant type VII collagen can promote the expression of Caspase-14, and the promotion rate of type VII collagen is stronger than that of the full-length fragment.

[0069] Example 4 In this embodiment, the recombinant type VII collagen obtained in Example 1 was used to detect the expression level of inner lining protein (IVL) by real-time quantitative PCR (RT-qPCR) using HSF cells, and the effect of recombinant type VII collagen on barrier repair was investigated.

[0070] Harvesting healthy HSF cells, resuspending them in DMEM cell culture medium and adjusting the cell concentration to 1.2 × 10⁻⁶ cells / year. 5 Cells / mL. 2 mL of cell suspension was evenly seeded into each well of a six-well plate and incubated at 37°C in a 5% CO2 incubator.

[0071] The experiment included a blank group, a model group, a control group, and a sample group. The model was established by treating with 30 μg / mL SLS. The control group contained full-length type VII collagen, while the sample group contained recombinant type VII collagen with different concentrations. After incubation at 37℃ in a 5% CO2 incubator for 24 h, total mRNA was extracted.

[0072] RNA concentration was measured using an ultra-micro spectrophotometer. A concentration greater than 20 ng / μL and an A260 / A280 value greater than 1.8 were considered acceptable samples. RevertAid was used. TM First-strand cDNA synthesis kit was used to synthesize cDNA. SYBR Green qPCR Mix, cDNA template, primers (sequences shown in Table 5), and RNase were used. Dissolve free ddH2O at 4℃ and prepare the Real-Time PCR reaction system on ice. The qPCR reaction system is shown in Table 6.

[0073] Table 5 Primer sequence list

[0074] Table 6 q-PCR reaction system

[0075] A two-step PCR reaction was performed. The reaction program was as follows: Step 1: 95℃ pre-denaturation for 300 s; Step 2: 95℃ denaturation for 20 s, annealing at 55℃ for 20 s, and extension at 72℃ for 20 s, for 40 reaction cycles. 2 -ΔΔCt The method determines relative to β The mRNA expression level of each target of Actin mRNA.

[0076] The results of IVL expression levels are as follows: Figure 4 As shown. By Figure 4 It can be seen that recombinant type VII collagen can promote the expression level of IVL, and the promotion rate of type VII collagen is stronger than that of the full-length fragment.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant type VII collagen, characterized in that, The amino acid sequence of the recombinant type VII collagen is any one of the following (A1) to (A3): (A1) The amino acid sequence of the recombinant type VII collagen is shown in SEQ ID NO.1; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type VII collagen described in (A1); (A3) has an amino acid sequence that is at least 90% identical to the recombinant type VII collagen described in (A1) or the fusion protein described in (A2) and has moisturizing and repairing effects.

2. The biomaterial of recombinant type VII collagen according to claim 1, characterized in that, It can be any one of items (B1) to (B4): (B1) A nucleic acid molecule encoding the recombinant type VII collagen of claim 1; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A host cell containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. The method for preparing recombinant type VII collagen as described in claim 1, characterized in that, include: The nucleic acid molecule encoding the recombinant type VII collagen as described in claim 1 is introduced into an expression vector, and then the obtained recombinant plasmid is introduced into a host cell. After fermentation culture, the recombinant type VII collagen is separated and purified to obtain the recombinant type VII collagen.

5. The preparation method according to claim 4, characterized in that, The expression vector includes pPICZα.

6. The preparation method according to claim 4, characterized in that, The host cell includes Pichia pastoris.

7. A composition, characterized in that, Includes the recombinant type VII collagen as described in claim 1.

8. The use of the recombinant type VII collagen according to claim 1 in the preparation of moisturizing products.

9. The use of the recombinant type VII collagen according to claim 1 in the preparation of barrier repair products.

10. The application according to claim 9, characterized in that, The product is used to promote the expression of aquaporin 3, cysteine-aspartic protease 14, and inner lining protein.

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