Recombinant XVII type collagen with anti-inflammatory effect and preparation method thereof

Abstract

The invention discloses recombinant XVII type collagen with an anti-inflammatory effect and a preparation method of the recombinant XVII type collagen, and belongs to the technical field of protein engineering. The recombinant XVII type collagen provided by the invention comprises a plurality of repetitive units, and the repetitive units are as shown in SEQ ID NO. 1. Compared with natural XVII type collagen, the recombinant XVII type collagen is smaller in molecular weight, more stable and capable of being directly used for product production; meanwhile, the recombinant XVII type collagen disclosed by the invention can be combined with an inflammatory factor receptor to inhibit activation of a signal channel, so that the anti-inflammatory activity is improved, and therefore, the recombinant XVII type collagen disclosed by the invention can be used as a core active component to be applied to preparation of anti-inflammatory related products, and has a relatively high development prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, in particular to a recombinant collagen XVII with anti-inflammatory effect and a preparation method thereof. BACKGROUND

[0002] Collagen is the most abundant protein in the human body and is the main component of the extracellular matrix, which is essential for maintaining the structural integrity and elasticity of the skin. As a member of the transmembrane collagen family, collagen XVII plays a key role in maintaining skin barrier, cell adhesion and wound repair. Based on the above characteristics, collagen XVII is applied to the preparation of functional skincare products, such as eye masks, facial masks, freeze-dried powders, etc. with effects of moisturizing, anti-wrinkle, etc. It is also used to prepare anti-hair loss care products, as well as medical dressings, skin fillers and therapeutic drugs, etc., which has a broad application prospect.

[0003] Currently, the conventional preparation methods are animal tissue extraction and genetic engineering expression. However, the animal tissue extraction method has the risk of immunogenicity and pathogen contamination, and the conventional genetic engineering expression method does not optimize the anti-inflammatory functional sequence, resulting in the product being unable to effectively inhibit the release of inflammatory factors. In addition, the amino acid sequence of the existing recombinant collagen XVII is mostly a natural fragment cut, and the anti-inflammatory active site is not strengthened by repeated sequence design, which is difficult to meet the high efficiency demand of sensitive skin care, wound inflammation repair, etc. Therefore, designing a high anti-inflammatory activity recombinant collagen XVII with specific repeated sequences and establishing a high-efficiency preparation process have become technical problems to be solved in the field.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a recombinant collagen XVII with anti-inflammatory effect and a preparation method thereof, which can be efficiently expressed and has good anti-inflammatory effect.

[0006] In the first aspect, the present application provides a recombinant collagen XVII, whose amino acid sequence is any one of the following (A1)~(A3): (A1) The amino acid sequence of the recombinant collagen XVII comprises a plurality of repeated units, the repeated unit is shown as SEQ ID NO. 1, and the number of repetitions is 1~20; (A2) A fusion protein obtained by connecting a tag to the N terminus and / or C terminus of the recombinant collagen XVII in (A1); (A3) An amino acid sequence having at least 90% identity to the recombinant collagen XVII of (A1) or the fusion protein of (A2) and having anti-inflammatory efficacy.

[0007] In a second aspect, the present application provides a biomaterial related to the recombinant collagen XVII, which is any one of (B1) to (B4): (B1) A nucleic acid molecule encoding the recombinant collagen XVII; (B2) An expression cassette containing the nucleic acid molecule of (B1); (B3) A recombinant vector containing the nucleic acid molecule of (B1) or the expression cassette of (B2); (B4) A host cell containing the nucleic acid molecule of (B1), the expression cassette of (B2), or the recombinant vector of (B3).

[0008] In a third aspect, the present application provides a method for preparing the recombinant collagen XVII, which comprises: introducing the nucleic acid molecule encoding the recombinant collagen XVII into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then performing fermentation culture, separation and purification to obtain the recombinant collagen XVII.

[0009] In a fourth aspect, the present application provides a composition comprising the recombinant collagen XVII.

[0010] In a fifth aspect, the present application provides the use of the recombinant collagen XVII in the preparation of an anti-inflammatory product.

[0011] The present application has the following advantages: The present application obtains a new recombinant collagen XVII by taking a peptide segment composed of 14 amino acid residues as a core sequence and repeating it multiple times. The recombinant collagen XVII has a smaller molecular weight and is more stable than the natural collagen XVII, and can be directly used for product production. Meanwhile, the recombinant collagen XVII of the present application can inhibit signal pathway activation by binding to an inflammatory factor receptor, thereby improving anti-inflammatory activity. Therefore, the recombinant collagen XVII of the present application can be used as a core active ingredient in the preparation of anti-inflammatory related products, and has a high development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0013] Figure 1 SDS-PAGE electrophoresis map of the purified protein in Example 1. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be obtained by market purchase.

[0015] Unless otherwise defined in the following, all technical and scientific terms used in the present application are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Although it is believed that the following terms are well understood by one of ordinary skill in the art, the following definitions are set forth to better clarify the present application.

[0016] As used in the present application, the terms "comprising", "containing", "having" or "including" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting of is considered a preferred embodiment of the term "comprising". If in the following a group is defined to comprise at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists only of these embodiments.

[0017] The indefinite article "a" or "an" or the definite article "the" as used in the specification and in the claims are intended to include both the singular and the plural form unless otherwise indicated.

[0018] The following are provided only to aid in understanding the present application. These definitions should not be construed to have a scope less than that understood by those skilled in the art.

[0019] The technical solutions of the present application will be further described in detail below in combination with specific embodiments, but do not constitute any limitation to the present application. Any limited number of modifications made by anyone within the scope of the claims of the present application are still within the scope of the claims of the present application.

[0020] In order to optimize the anti-inflammatory function sequence of human collagen XVII, the present application finds that the sequence of some segments contains multiple anti-inflammatory active sites (such as Asp, Glu acidic amino acid residues and Gly-Pro structure analog fragments) through bioinformatics analysis software, which can inhibit the activation of signal pathway by combining with inflammatory factor receptors, based on which, the present application selects the polypeptide fragment as shown in SEQ ID NO. 1 as a core sequence, takes the fragment as a repeating unit, and repeatedly obtains a new recombinant collagen XVII, and the recombinant collagen XVII has good anti-inflammatory efficacy.

[0021] The present application provides a recombinant collagen XVII formed by a plurality of repeating units in series, and the amino acid sequence of the repeating unit is GADFAGDLDYNELA (SEQ ID NO. 1).

[0022] The repeating number n of the repeating unit can be 1-20, specifically 2, 4, 6, 8, 10, or other multiples of 2, and preferably the repeating number is 20 times. The specific amino acid sequence of the recombinant collagen protein composed of 20 repeating units in series is as follows: GADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELAGADFAGDLDYNELA (SEQ ID NO. 2).

[0023] In order to facilitate subsequent separation and purification, the recombinant collagen XVII can also be connected with a tag at the N-terminal and / or C-terminal to obtain a fusion protein. According to the function, the protein tag can be roughly divided into three categories of detection tag, expression and purification tag and tracking tag. The commonly used expression and purification tags at present are His, GST, MBP, CBD, Strep-tag, Halo-Tag, SNAP-tag, SUMO, NusA, TrxA, DsbA, Flag and c-Myc. The type of the tag is not limited in the present application, and those skilled in the art can adjust according to the needs.

[0024] In some embodiments, the amino acid sequence of the recombinant collagen XVII can also be an amino acid sequence having at least 90% identity to the recombinant collagen XVII or fusion protein described above and having anti-inflammatory efficacy.

[0025] The percent identity of a sequence is the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, and one skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0026] In some embodiments, the at least 90% sequence identity can be specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any range between any two of them. The identity limitation of the present application is due in part to the fact that the repeat units described above can be distinguished from the sequence protection scope of the present application by adding linkers or regulatory sequences, etc.

[0027] The present application provides a nucleic acid molecule encoding the recombinant collagen XVII described above.

[0028] Nucleic acid molecules are a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecular compounds polymerized by many nucleotide monomers, and are one of the most basic substances of life. The nucleotide sequence refers to the arrangement order of bases in DNA or RNA. The nucleic acid molecule contains cDNA, and in some cases, the nucleic acid molecule can be modified for use in the vectors of the present application, such as for codon optimization. In some cases, the sequence can be designed to contain terminal restriction site sequences for the purpose of cloning into vectors. The nucleic acid molecule can be obtained from various sources, such as by polymerase chain reaction (PCR) amplification of the encoding nucleic acid within or isolated from the given cell or cells.

[0029] In specific embodiments, the nucleic acid molecule described above can be synthesized by artificially synthesizing the gene fragment after optimizing the relevant gene sequence according to the codon bias of the host cell. It should be understood that the nucleic acid molecule capable of being translated into the amino acid sequence described above is within the protection scope of the present application.

[0030] For expression in host cells, the nucleic acid molecule can also be codon-optimized, and a signal peptide cleavage site and a restriction site can be added at both ends to facilitate later genetic manipulation. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 3.

[0031] The present application provides an expression cassette comprising the above-mentioned nucleic acid molecule. Meanwhile, the expression cassette also contains regulatory sequences such as promoters and terminators.

[0032] An expression cassette refers to a nucleic acid construct comprising coding sequences and regulatory sequences operably linked when introduced into a host cell, resulting in transcription and / or translation of RNA or polypeptide, respectively. An expression cassette is understood to include a promoter that allows the initiation of transcription, an open reading frame of the gene of interest, and a transcription terminator. Typically, the promoter sequence is placed upstream of the gene of interest, compatible with expression control.

[0033] The present application provides an expression vector comprising the above-mentioned nucleic acid molecule or expression cassette.

[0034] An expression vector refers to a self-replicating DNA molecule that transfers a DNA fragment (gene of interest) to a recipient cell in genetic engineering recombinant DNA technology. In addition to commonly used E. coli plasmid vectors, many other artificially constructed plasmid vectors suitable for microorganisms, yeast, plants, etc. have been developed. 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, without free ends (e.g. circular); nucleic acid molecules containing DNA, RNA or both; and other polynucleotide species known in the art. The most commonly used vector type is "plasmid", which refers to a circular double-stranded DNA ring that can insert additional DNA fragments, for example by standard molecular cloning techniques. A recombinant expression vector can comprise a form suitable for expression of a nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell used for expression, which can be operably linked to the nucleic acid sequence to be expressed.

[0035] In some embodiments, the above-mentioned expression vector includes but is not limited to pPIC9K and pET-28a.

[0036] The present application provides 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 collagen type XVII.

[0037] The host cell refers to any cell type which is susceptible to transformation, transfection, transduction, etc. of a nucleic acid construct or expression vector comprising the polynucleotide of the present application. The host cell can be any cell which is useful in the production of the recombinant collagen XVII of the present application. In order to produce the recombinant collagen XVII, the nucleic acid encoding the recombinant collagen XVII can be isolated and inserted into one or more vectors for further cloning / expression in host cells. The methods for introducing the vector into the host cell are well known, for example, the vector is introduced into the host cell using electroporation, which can also be transfection, microinjection technique, gene gun technique, liposome-mediated method, etc.

[0038] In specific embodiments, the above-mentioned host cell can be a prokaryotic cell or a eukaryotic cell, including: bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, etc.; eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, plant cells, etc. Preferably, the host cell is Pichia pastoris and Escherichia coli.

[0039] The present application provides a method for preparing a recombinant collagen XVII, which comprises: introducing the above-mentioned nucleic acid molecule of the recombinant collagen XVII into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then performing fermentation culture, followed by separation and purification to obtain the above-mentioned recombinant collagen XVII.

[0040] In specific embodiments, the preparation of the recombinant collagen XVII by the host cell refers to the fermentation culture of the host cell, and the culture medium and culture conditions are well known to those skilled in the art.

[0041] As for the expression mode, the present application does not make any limitation, which can be selected and adjusted as needed, for example, the expression can be one of constitutive expression, induced expression, or a combination of both expressions, wherein the inducer of induced expression can be IPTG, β-galactoside, methanol, ethanol, etc.

[0042] In some embodiments, the recombinant collagen XVII is obtained by induced expression of the above-mentioned host cell, specifically: when the density of the recombinant Pichia pastoris reaches 1.0, methanol is added for induced expression; when the density of the recombinant Escherichia coli reaches 0.5, IPTG is added for induced expression. Among them, the temperature of the induction stage of the recombinant Pichia pastoris is 30°C, and the induction time is 84h; the temperature of the induction stage of the recombinant Escherichia coli is 28°C, and the induction time is 5h.

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

[0044] In some embodiments, the separation and purification is performed by centrifugation to collect the supernatant after fermentation, and the supernatant is filtered and chromatographed. Preferably, the chromatography is anion exchange chromatography and gel filtration chromatography.

[0045] The present application provides use of the recombinant collagen XVII in preparation of anti-inflammatory products, including cosmetics and pharmaceuticals, preferably the pharmaceuticals are external use pharmaceuticals.

[0046] In some embodiments, the cosmetics are skin care products. The cosmetic dosage forms can be aqueous solutions, emulsions, creams, and the like, and other cosmetic products derived from the above basic cosmetic categories, such as facial cleansers, sunscreens, toners, creams, eye creams, serums, hand creams, body lotions, and masks, and the like.

[0047] In some embodiments, the external use pharmaceuticals include, but are not limited to, gels, solutions / sprays, creams, lyophilized powders, medical dressings, and the like.

[0048] The cosmetics and external use pharmaceuticals can further comprise other ingredients allowed to be added in the cosmetic and pharmaceutical fields, including, but not limited to, suspending agents, emulsifying agents, emollients, humectants, pH adjusting agents, thickening agents, fragrances, and the like. Those skilled in the art can select and add them according to the actual situation, which are not specifically limited herein.

[0049] The present specification is considered sufficient to enable one of ordinary skill in the art to practice the present application. Various modifications of the application in accordance with the previous description will be apparent to those skilled in the art from the foregoing description, and fall within the scope of the appended claims.

[0050] The features and properties of the present application are further described in detail below in conjunction with the embodiments.

[0051] Example 1 This example is the construction of recombinant Pichia pastoris GS115 and the expression of recombinant collagen XVII, and the specific methods are as follows: 1. Vector construction: pPIC9K vector (9.3 kb) and synthetic gene were digested with EcoR I and Xho I at 37°C for 2 h; T4 DNA ligase was added and connected at 16°C overnight; the ligation product was transformed into E. coli DH5α, and positive clones were selected for sequencing verification to obtain recombinant vector pPIC9K-Col17.

[0052] 2. Recombinant vector verification: After double enzyme digestion (EcoR I / Xho I), electrophoresis showed that the vector fragment was about 9.3 kb, and the inserted gene fragment was about 840 bp. The sequencing result after ligation was consistent with the designed sequence with 100% consistency, proving that the recombinant vector pPIC9K-Col17 was successfully constructed.

[0053] 3. Pichia pastoris transformation: The recombinant vector was linearized with Sal I, and was introduced into Pichia pastoris GS115 by an electric transformation instrument (1.5 kV, 25 μF, 200 Ω); was coated on an MD plate and was cultured at 30°C for 4 d; a single colony was inoculated on a YPD plate containing 2.0 mg / mL G418, and a high-copy strain was screened.

[0054] 4. Induction of expression: The high-copy strain was inoculated into BMGY medium, was cultured at 30°C and 200 r / min until the OD 600nm =4; the bacterial cells were collected by centrifugation, were resuspended in BMMY medium, and were induced at 30°C for 84 h by adding methanol to a final concentration of 0.8%, and the methanol was added once every 24 h.

[0055] 5. Purification: The fermentation supernatant was subjected to DEAE-Sepharose chromatography (eluted with Tris-HCl buffer containing 0.35 mol / L NaCl pH 8.0) and Sephacryl S-200 chromatography.

[0056] In the DEAE-Sepharose chromatography stage, the target protein was eluted at a NaCl concentration of 0.3-0.4 mol / L; in the Sephacryl S-200 chromatography stage, the elution volume of the target protein corresponded to a molecular weight of about 31 kDa, which was consistent with the theoretical value. SDS-PAGE electrophoresis (as shown in FIG. 2) showed a single band, and the purity reached 97.2%. Figure 1

[0057] The method of the present embodiment can obtain the target protein, and the expression level can reach 280 mg / L, which is significantly higher than the expression level (100-150 mg / L) of a conventional natural sequence fragment.

[0058] Example 2 The present embodiment is the construction of a recombinant Escherichia coli and the expression of a recombinant type XVII collagen, and the specific method is as follows: 1. Vector construction: The synthetic gene (SEQ ID NO. 3) was inserted into the EcoR I / Xho I site of the pET-28a vector to construct a recombinant vector pET-28a-Col17, and DH5α was transformed and verified. ​2. E. coli transformation: the recombinant vector was introduced into BL21 (DE3) by heat shock method (42℃, 90s), and then coated on LB plate containing 50 μg / mL kanamycin and cultured at 37℃ for 12h; 3. Induction of expression: the positive strain was inoculated into LB medium and cultured at 37℃ until OD 600nm = 0.7, and then IPTG was added to a final concentration of 0.3 mmol / L, and induction was performed at 28℃ and 180 r / min for 5h; 4. Purification: the bacterial cells were broken by ultrasonic wave (200W, working for 3s, interval of 5s, 30min), and the supernatant was subjected to Ni-NTA affinity chromatography (eluted with buffer containing 200 mmol / L imidazole) and Sephacryl S-200 chromatography.

[0059] The method of the present embodiment can obtain the target protein, and the expression level can reach 320 mg / L, which is significantly higher than the expression level (100-150 mg / L) of the conventional natural sequence fragment.

[0060] Example 3 Anti-inflammatory performance identification of recombinant collagen XVII and related sequences The present embodiment is an anti-inflammatory performance comparative identification of recombinant collagen XVII, a commercially available sample and a specific recombinant fragment, which is as follows: Inflammation is a protective response of human tissues when stimulated by various damages, which is characterized by redness, swelling, heat, pain and dysfunction, and is involved in foreign body clearance and tissue repair, etc. Excessive inflammatory response can lead to excessive expression of inflammatory mediators, causing damage to the body. When acute inflammation occurs, mononuclear macrophages are activated to induce the release of TNF-α, IL-6 and other inflammatory mediators, and then induce the activation of immune response. As a key structural protein at the true epidermal junction, the effect of different sequence forms of collagen XVII on inflammation regulation has not been fully determined. The present embodiment provides a theoretical support for its anti-inflammatory application through comparative analysis.

[0061] 1. Experimental materials and design 1.1 Preparation of test samples Sample A: recombinant collagen XVII of the present application; Sample B: commercially available sample; Sample C: recombinant collagen XVII fragment C (containing NC16A domain mutant, aa 150-300, which is an unpublished region).

[0062] All samples were prepared by the same purification process (nickel column affinity chromatography + gel filtration chromatography), and the purity was verified by SDS-PAGE to be ≥95%, and the endotoxin content was <0.1 EU / μg, so as to ensure that the experimental interference was controllable.

[0063] 1.2 Cell inflammation model construction Logarithmic growth phase mouse macrophage RAW264.7 cells were inoculated in a 96-well plate at a concentration of 1×10 4 After 24 hours of culture at 37°C and 5% CO2, LPS (final concentration 1 μg / mL) was added to induce inflammation, and the above four samples were added at final concentrations of 10 μg / mL, 20 μg / mL, and 50 μg / mL, respectively.

[0064] Five control groups were set up: control group 1 (no LPS, no sample, i.e. blank control group), control group 2 (LPS, no sample, i.e. model control group), control group 3 (LPS + 10 μg / mL dexamethasone, i.e. positive control group), control group 4 (LPS + 20 μg / mL patent CN116640205A disclosed fragment), and control group 5 (LPS + 20 μg / mL XVII collagen fragment with the amino acid sequence GPPGPSGDPGKPGL-SEQ ID NO. 4). Each group had six replicates, and after 24 hours of culture, the supernatant was collected for detection of inflammatory factors.

[0065] 2. Inflammation factor detection method and results Human TNF-α and IL-6 ELISA detection kits were used, and the operation was strictly in accordance with the kit instructions. The absorbance value at 450 nm was detected by an enzyme-labeled instrument, and the factor content was calculated by a standard curve. The formula for calculating the inhibition rate of inflammatory factors was as follows: Inhibition rate (%) = [(model control group factor content - experimental group factor content) / (model control group factor content - blank control group factor content)] × 100% The detection results are shown in Table 1, and the data are expressed as "mean ± standard deviation". One-way ANOVA was performed on the data, and P<0.05 was considered statistically significant.

[0066] Table 1 Inflammatory factor content and inhibition rate of different XVII collagen and fragments

[0067] In Table 1, the sample concentration unit is μg / mL, the TNF-α content unit is pg / mL, and the IL-6 content unit is pg / mL.

[0068] 3. Results analysis and conclusion 3.1 Concentration-dependent anti-inflammatory characteristics All samples with significant anti-inflammatory activity (samples A, B, C) showed the feature of "low to medium concentration activity rising, high concentration activity slightly falling", with 20 μg / mL as the optimal induction concentration, consistent with the saturation activation effect of inflammatory cell signaling pathways. The activity decline at high concentration (50 μg / mL) may be related to the functional site shielding caused by protein aggregation, suggesting that the dose range needs to be controlled in actual application.

[0069] 3.2 Anti-inflammatory performance comparison of different sequence forms The recombinant collagen of the present application (sample A) showed the best performance at 20 μg / mL concentration, with TNF-α inhibition rate of 49.1% and IL-6 inhibition rate of 46.5%, significantly higher than the prior art control group (inhibition rate of 24.6%-36.5%), confirming the effectiveness of its sequence optimization; The commercially available recombinant collagen (sample B) showed stable anti-inflammatory activity at a concentration of 20 μg / mL or more, with TNF-α inhibition rate of 32.4%, proving that the anti-inflammatory activity of the commercially available sample is relatively weak compared to the present application, but is within the inhibition rate range of the prior art; The anti-inflammatory activity of fragment C (sample C) was lower than that of the recombinant collagen of the present application (sample A) at all concentrations, proving that not all functional fragments have good anti-inflammatory effect.

[0070] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant type XVII collagen, characterized in that, The amino acid sequence of the recombinant type XVII collagen is any one of the following (A1) to (A3): (A1) The amino acid sequence of the recombinant type XVII collagen contains multiple repeating units, as shown in SEQ ID NO.1, with a repeating number of 1 to 20; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type XVII collagen described in (A1); (A3) has an amino acid sequence that is at least 90% identical to the recombinant type XVII collagen described in (A1) or the fusion protein described in (A2) and has anti-inflammatory effects.

2. The recombinant type XVII collagen according to claim 1, characterized in that, The amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO.

2.

3. A biomaterial related to the recombinant type XVII collagen as described in claim 1, characterized in that, It can be any one of items (B1) to (B4): (B1) A nucleic acid molecule encoding the recombinant type XVII collagen as described in 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); Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

3.

4. The method for preparing recombinant type XVII collagen as described in claim 1 or 2, characterized in that, include: The nucleic acid molecule encoding the recombinant type XVII collagen as described in claim 1 or 2 is introduced into an expression vector, and then the obtained recombinant plasmid is introduced into a host cell. After fermentation culture, the recombinant type XVII collagen is separated and purified to obtain the recombinant type XVII collagen.

5. The preparation method according to claim 4, characterized in that, The expression vectors include pPIC9K and pET-28a.

6. The preparation method according to claim 4, characterized in that, The host cells include Pichia pastoris and Escherichia coli.

7. A composition, characterized in that, Includes the recombinant type XVII collagen as described in claim 1 or 2.

8. The use of the recombinant type XVII collagen as described in claim 1 or 2 in the preparation of anti-inflammatory products.

9. The application according to claim 8, characterized in that, The product is used to reduce the inflammatory factors IL-6 and / or TNF-α.

10. The application according to claim 8, characterized in that, The products include cosmetics and topical medicines.

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