A stable recombinant collagen type iii and its use

Abstract

The application provides a kind of stable recombinant collagen type III and its application, the recombinant collagen type III includes the amino acid sequence as shown in SEQ ID NO.1.The application efficiently prepares the recombinant human collagen type III by genetic engineering technology, and high purity product is obtained by combining purification process.The recombinant human collagen type III has excellent water solubility and stability, and can be stored at room temperature for more than one year.In addition, the completely humanized sequence eliminates the immunogenicity and pathogen contamination risk of animal-derived collagen, ensuring the safety of application, and has excellent effects of inhibiting hyaluronidase, cell adhesion, repair, soothing and the like.The application provides a new collagen raw material for food, medicine, health care products, cosmetics, medical device materials and scientific research fields, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a stable recombinant type III collagen and its applications. Background Technology

[0002] Currently, artificially prepared recombinant collagen has poor stability because the stability of the natural triple helix structure of collagen is highly dependent on the hydroxylation of proline and lysine. This modification is completely absent in prokaryotic expression systems (such as E. coli), and its expression efficiency in eukaryotic microbial systems such as yeast is much lower than that in human cells. This results in poor thermal stability of recombinant products, making them more prone to denaturation and degradation. Summary of the Invention

[0003] To address the aforementioned problems in the prior art, this invention provides a stable recombinant type III collagen and its applications.

[0004] In a first aspect, the present invention provides a recombinant type III collagen, the recombinant type III collagen comprising the amino acid sequence shown in SEQ ID NO.1.

[0005] According to the present invention, a recombinant type III collagen is preferably further comprising a tag, wherein the tag is selected from at least one of His tag, GST tag, MBP tag, Strep tag and FLAG tag.

[0006] More preferably, the tag is a His tag, and the recombinant type III collagen includes the amino acid sequence shown in SEQ ID NO.3.

[0007] More preferably, the amino acid sequence of the recombinant type III collagen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO.1 and has the same function.

[0008] More preferably, the amino acid sequence of the recombinant type III collagen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO.3 and has the same function.

[0009] In some specific embodiments, the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.1 or SEQ ID NO.3.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding recombinant type III collagen, wherein the recombinant type III collagen comprises the amino acid sequence shown in SEQ ID NO.1.

[0011] According to the present invention, a nucleic acid molecule is preferably provided, wherein the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0012] According to a nucleic acid molecule provided by the present invention, preferably, the recombinant type III collagen further includes a tag, the tag being selected from at least one of His tag, GST tag, MBP tag, Strep tag and FLAG tag.

[0013] More preferably, the tag is a His tag, the recombinant type III collagen includes the amino acid sequence shown in SEQ ID NO.3, and the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.4.

[0014] More preferably, the nucleotide sequence of the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.2 and encodes a protein with the same function.

[0015] More preferably, the nucleotide sequence of the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.4 and encodes a protein with the same function.

[0016] In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO.4.

[0017] Thirdly, the present invention provides a biological material comprising a nucleic acid molecule encoding recombinant type III collagen, wherein the recombinant type III collagen comprises an amino acid sequence as shown in SEQ ID NO.1; the biological material is recombinant DNA, an expression cassette, a transposon, a vector, a cell, or a microorganism.

[0018] According to a biological material provided by the present invention, preferably, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0019] According to a biomaterial provided by the present invention, preferably, the recombinant type III collagen further includes a tag, the tag being selected from at least one of His tag, GST tag, MBP tag, Strep tag and FLAG tag.

[0020] More preferably, the tag is a His tag, the recombinant type III collagen includes the amino acid sequence shown in SEQ ID NO.3, and the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.4.

[0021] More preferably, the nucleotide sequence of the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.2 and encodes a protein with the same function.

[0022] More preferably, the nucleotide sequence of the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.4 and encodes a protein with the same function.

[0023] In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO.4.

[0024] According to a biomaterial provided by the present invention, preferably, the carrier uses a yeast expression vector as a backbone.

[0025] More preferably, the carrier uses the pPICZαA carrier as its framework.

[0026] More preferably, the nucleic acid molecule is located between the EcoR I site and the Not I site of the pPICZαA vector.

[0027] According to the present invention, the microorganisms preferably include yeast.

[0028] More preferably, the yeast is Pichia pastoris.

[0029] More preferably, the Pichia pastoris is Pichia pastoris strain X33.

[0030] Fourthly, the present invention provides a conjugate comprising the recombinant type III collagen and a chemical moiety conjugated thereto.

[0031] Fifthly, the present invention provides a composition comprising the recombinant type III collagen.

[0032] This invention provides a product containing the recombinant type III collagen, or the nucleic acid molecule, or the biological material, or the conjugate, or the composition thereof; the product is food, medicine, health product, cosmetic, medical biomaterial, or medical device.

[0033] According to a product provided by the present invention, preferably, the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 20% (m / v).

[0034] More preferably, if the product has repair and / or soothing functions, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 5% (m / v).

[0035] More preferably, if the product has repair and / or soothing functions, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 2.5% (m / v).

[0036] Most preferably, if the product has a repair function, then the effective concentration of the recombinant type III collagen is 0.5% (m / v).

[0037] Most preferably, if the product has a soothing function, then the effective concentration of the recombinant type III collagen is 2.5% (m / v).

[0038] Most preferably, if the product has repair and soothing functions, then the effective concentration of the recombinant type III collagen is 1% (m / v).

[0039] More preferably, if the product has a moisturizing function, then the effective concentration of the recombinant type III collagen is 1% (m / v) to 20% (m / v).

[0040] More preferably, if the product has a moisturizing function, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0041] Most preferably, if the product has a moisturizing function, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0042] More preferably, if the product has the function of inhibiting hyaluronidase activity, then the effective concentration of the recombinant type III collagen is 1% (m / v) to 20% (m / v).

[0043] More preferably, if the product has the function of inhibiting hyaluronidase activity, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0044] Most preferably, the product has the function of inhibiting hyaluronidase activity, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0045] More preferably, if the product has the function of promoting cell migration and / or reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 5% (m / v).

[0046] More preferably, if the product has the function of promoting cell migration and / or reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 2.5% (m / v).

[0047] Most preferably, if the product has the function of promoting cell migration, then the effective concentration of the recombinant type III collagen is 0.5% (m / v).

[0048] Most preferably, if the product has the function of reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 2.5% (m / v).

[0049] Most preferably, if the product has the function of promoting cell migration and reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 1% (m / v).

[0050] More preferably, if the product has the function of promoting cell adhesion, then the effective concentration of the recombinant type III collagen is 5% (m / v) to 20% (m / v).

[0051] More preferably, if the product has the function of promoting cell adhesion, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0052] Most preferably, if the product has the function of promoting cell adhesion, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0053] More preferably, the inflammatory factor includes nitric oxide.

[0054] In a sixth aspect, the present invention provides the application of the recombinant type III collagen, the nucleic acid molecule, the biomaterial, the conjugate, or the composition thereof in the preparation of food, pharmaceuticals, health products, cosmetics, medical biomaterials, or medical devices.

[0055] According to the present invention, the recombinant type III collagen, the nucleic acid molecule, the biomaterial, the conjugate, or the composition thereof are used in the preparation of food, pharmaceuticals, health products, cosmetics, medical biomaterials, or medical devices. Preferably, in the food, pharmaceuticals, health products, cosmetics, medical biomaterials, or medical devices, the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 20% (m / v).

[0056] More preferably, the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 5% (m / v).

[0057] More preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has repair and / or soothing functions, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 5% (m / v).

[0058] More preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has repair and / or soothing functions, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 2.5% (m / v).

[0059] Most preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has a repair function, then the effective concentration of the recombinant type III collagen is 0.5% (m / v).

[0060] Most preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has a soothing function, then the effective concentration of the recombinant type III collagen is 2.5% (m / v).

[0061] Most preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has repair and soothing functions, then the effective concentration of the recombinant type III collagen is 1% (m / v).

[0062] More preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has a moisturizing function, then the effective concentration of the recombinant type III collagen is 1% (m / v) to 20% (m / v).

[0063] More preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has a moisturizing function, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0064] Most preferably, if the food, drug, health product, cosmetic, medical biomaterial or medical device has a moisturizing function, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0065] The application of the recombinant type III collagen, the nucleic acid molecules, the biomaterials, the conjugates, or the compositions described herein in the preparation of products with moisturizing, repairing, and / or soothing functions should also be within the scope of protection of this invention. Preferably, the products are food, pharmaceuticals, health products, cosmetics, medical biomaterials, or medical devices.

[0066] Preferably, the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 20% (m / v).

[0067] More preferably, if the product has repair and / or soothing functions, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 5% (m / v).

[0068] More preferably, if the product has repair and / or soothing functions, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 2.5% (m / v).

[0069] Most preferably, if the product has a repair function, then the effective concentration of the recombinant type III collagen is 0.5% (m / v).

[0070] Most preferably, if the product has a soothing function, then the effective concentration of the recombinant type III collagen is 2.5% (m / v).

[0071] Most preferably, if the product has repair and soothing functions, then the effective concentration of the recombinant type III collagen is 1% (m / v).

[0072] More preferably, if the product has a moisturizing function, then the effective concentration of the recombinant type III collagen is 1% (m / v) to 20% (m / v).

[0073] More preferably, if the product has a moisturizing function, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0074] Most preferably, if the product has a moisturizing function, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0075] The application of the recombinant type III collagen, the nucleic acid molecules, the biomaterials, the conjugates, or the compositions described herein in the preparation of products that inhibit hyaluronidase activity, promote cell migration, promote cell adhesion, and / or reduce the content of inflammatory factors should also be within the scope of protection of this invention. Preferably, the products are food, pharmaceuticals, health products, cosmetics, medical biomaterials, or medical devices.

[0076] Preferably, the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 20% (m / v).

[0077] More preferably, if the product has the function of inhibiting hyaluronidase activity, then the effective concentration of the recombinant type III collagen is 1% (m / v) to 20% (m / v).

[0078] More preferably, if the product has the function of inhibiting hyaluronidase activity, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0079] Most preferably, the product has the function of inhibiting hyaluronidase activity, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0080] More preferably, if the product has the function of promoting cell migration and / or reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 5% (m / v).

[0081] More preferably, if the product has the function of promoting cell migration and / or reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 0.5% (m / v) to 2.5% (m / v).

[0082] Most preferably, if the product has the function of promoting cell migration, then the effective concentration of the recombinant type III collagen is 0.5% (m / v).

[0083] Most preferably, if the product has the function of reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 2.5% (m / v).

[0084] Most preferably, if the product has the function of promoting cell migration and reducing the content of inflammatory factors, then the effective concentration of the recombinant type III collagen is 1% (m / v).

[0085] More preferably, if the product has the function of promoting cell adhesion, then the effective concentration of the recombinant type III collagen is 5% (m / v) to 20% (m / v).

[0086] More preferably, if the product has the function of promoting cell adhesion, then the effective concentration of the recombinant type III collagen is 10% (m / v) to 20% (m / v).

[0087] Most preferably, if the product has the function of promoting cell adhesion, then the effective concentration of the recombinant type III collagen is 20% (m / v).

[0088] Preferably, the inflammatory factor includes nitric oxide.

[0089] In a seventh aspect, the present invention provides a method for preparing recombinant type III collagen, wherein, under the condition that the recombinant type III collagen is allowed to be expressed, microorganisms in the biological material are cultured, and the recombinant type III collagen is recovered from the culture product.

[0090] According to the present invention, a method for preparing recombinant type III collagen preferably includes yeast as the microorganism.

[0091] More preferably, the yeast is Pichia pastoris.

[0092] More preferably, the Pichia pastoris is Pichia pastoris strain X33.

[0093] According to the method for preparing recombinant type III collagen provided by the present invention, preferably, the culture temperature is 25℃~35℃.

[0094] More preferably, the culture temperature is 30°C.

[0095] According to a method for preparing recombinant type III collagen provided by the present invention, preferably, the preparation method further includes: purifying the recovered recombinant type III collagen.

[0096] More preferably, the purification method is selected from any one or more of affinity chromatography, ion exchange, hydrophobic chromatography, chromatographic chromatography, and salting out.

[0097] More preferably, the affinity chromatography uses a nickel column.

[0098] More preferably, the recombinant type III collagen is eluted from the nickel column using an eluent containing 110 mM to 130 mM imidazole.

[0099] More preferably, the eluent contains 120 mM imidazole.

[0100] More preferably, the eluent further contains 10mM to 30mM Tris and 90mM to 110mM NaCl.

[0101] More preferably, the eluent also contains 20 mM Tris and 100 mM NaCl.

[0102] More preferably, the pH of the eluent is 7.8 to 8.2.

[0103] More preferably, the pH of the eluent is 8.0.

[0104] More preferably, the preparation method further includes: purifying and dialyzing the recovered recombinant type III collagen sequentially.

[0105] More preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa.

[0106] More preferably, the buffer solution used for dialysis is 8mM to 12mM PB containing 40mM to 60mM NaCl, with a pH of 7.4.

[0107] More preferably, the buffer solution used for dialysis is 10mM PB containing 50mM NaCl, with a pH of 7.4.

[0108] The present invention has the following beneficial effects:

[0109] This invention utilizes genetic engineering technology to efficiently prepare recombinant human type III collagen, combined with purification processes to obtain a high-purity product. This recombinant human type III collagen exhibits excellent water solubility and stability, and can be stored at room temperature for over one year. Furthermore, its fully humanized sequence eliminates the immunogenicity and pathogen contamination risks associated with animal-derived collagen, ensuring application safety. It also possesses excellent effects in inhibiting hyaluronidase, promoting cell adhesion, repair, and soothing. This invention provides a new collagen raw material for the food, pharmaceutical, health product, cosmetic, medical device materials, and scientific research fields, and has broad application prospects. Attached Figure Description

[0110] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0111] Figure 1 This is a spectrum of the recombinant plasmid pPICZαA-CollagenⅢ provided in Example 1 of this invention.

[0112] Figure 2 This is the result of double enzyme digestion identification of the recombinant plasmid pPICZαA-CollagenⅢ provided in Example 1 of this invention.

[0113] Figure 3 The results of SDS-PAGE electrophoresis of the recombinant strain expressing recombinant type III collagen provided in Example 2 of this invention are shown; M is the protein molecular weight standard, and the bands from largest to smallest are 140kDa, 115kDa, 80kDa, 65kDa, 50kDa, 40kDa, 30kDa, 25kDa, 15kDa and 10kDa; negative is the negative control pPICZαA / X33; lanes 1 to 10 correspond to 10 single colonies.

[0114] Figure 4 This is the hydrophilicity prediction result of recombinant type III collagen provided in Example 4 of the present invention.

[0115] Figure 5 This is the stability evaluation result of the recombinant type III collagen provided in Example 4 of the present invention; A to D represent storage days of 0 days, 90 days, 180 days, and 365 days, respectively; M is the protein molecular weight standard, with the bands decreasing in size as follows: 140kDa, 115kDa, 80kDa, 65kDa, 50kDa, 40kDa, 30kDa, 25kDa, 15kDa, and 10kDa; lanes 1 to 4 correspond to storage temperatures of 37℃, 25℃, 4℃, and -20℃, respectively.

[0116] Figure 6 This is the statistical result of the percentage of cell adhesion under different concentrations of recombinant type III collagen in the cell adhesion effect evaluation provided in Example 4 of the present invention; BC is the blank group. p<0.01.

[0117] Figure 7 This is a cell nuclear fluorescence staining image under different concentrations of recombinant type III collagen treatment in the cell adhesion effect evaluation provided in Example 4 of the present invention.

[0118] Figure 8 This is the statistical result of cell viability rate under different concentrations of recombinant type III collagen treatment in the evaluation of repair effect provided in Example 4 of the present invention; BC is the blank control group.

[0119] Figure 9 This is the statistical result of cell migration rate under different concentrations of recombinant type III collagen treatment in the evaluation of repair effect provided in Example 4 of the present invention; BC is the blank control group. p<0.05, p<0.01.

[0120] Figure 10 These are photographs of cell scratch experiments performed under different concentrations of recombinant type III collagen in the evaluation of repair effects provided in Example 4 of this invention.

[0121] Figure 11 This is the statistical result of cell viability rate under different concentrations of recombinant type III collagen treatment in the evaluation of the soothing effect provided in Example 4 of the present invention; BC is the blank group.

[0122] Figure 12 This is the statistical result of the relative NO content in cells under different concentrations of recombinant type III collagen treatment in the evaluation of the soothing effect provided in Example 4 of the present invention; BC is the blank group, M is the model group, and PC is the positive control group. This indicates that the difference is statistically significant compared to the model group. P<0.01. Detailed Implementation

[0123] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0124] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0125] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available. The Pichia pastoris X33 strain used in the following examples, i.e., the X33 engineered strain, was preserved by Jinpu Nuoan Protein Engineering Technology (Beijing) Co., Ltd.; the restriction endonucleases EcoRI and Not I were purchased from NEB (Beijing) Co., Ltd.; the tryptone, yeast extract, bleomycin, and BCA protein quantification kit were purchased from Thermo Fisher Scientific; the protein electrophoresis buffer, staining solution, and destaining solution were purchased from Genscript Biotech Co., Ltd.; and all other reagents were domestically produced analytical grade.

[0126] The formulations of the culture media used in the examples are as follows: (1) YPDS solid medium (100ml): Dissolve 1g yeast extract and 2g peptone in 90ml water; autoclave for 20min; add 10mL 20% glucose, 18.22g sorbitol and 2g agar powder, and dissolve thoroughly; (2) BMGY liquid medium (1L): Dissolve 10g yeast powder and 20g peptone in 700ml water, autoclave for 20min; cool to room temperature, add 100ml 1M potassium phosphate buffer pH6.0 (room temperature), 100ml 13.4% yeast nitrogen base (YNB), 100ml 10% glycerol (GyY) and 2ml 0.02% Biotin, mix thoroughly; (3) BMMY liquid medium (1L): Dissolve 10g yeast powder and 20g peptone in 700ml water, sterilize for 20min; cool to room temperature, add 100ml 1M potassium phosphate buffer pH6.0 (room temperature), 100ml 13.4% YNB, 100ml 5% methanol (Methanol, M) and 2ml 0.02% Biotin, mix thoroughly; (4) YPD solid medium (100ml): Dissolve 1g yeast extract and 2g peptone in 90ml water; autoclave for 20min; add 10mL 20% glucose and 2g agar powder, dissolve thoroughly.

[0127] Example 1: Construction and identification of recombinant plasmid expressing recombinant type III collagen

[0128] 1. Construction of recombinant plasmids

[0129] This invention targets the amino acid sequence characteristics of human type III collagen, selects specific functional fragments for sequence optimization, and modifies them in combination with the codon bias of Pichia pastoris. Finally, the amino acid sequence of the recombinant type III collagen (Collagen III) used for expression is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0130] After adding a His tag to the C-terminus of the Collagen III protein, the amino acid sequence shown in SEQ ID NO.3 was obtained. The corresponding coding gene sequence was then synthesized by Qingke Biotechnology Co., Ltd. to obtain the Collagen III gene (nucleotide sequence shown in SEQ ID NO.4), which was then ligated into the Pichia pastoris expression vector pPICZαA, ultimately yielding the desired result. Figure 1 The recombinant plasmid shown is named pPICZαA-CollagenⅢ.

[0131] 2. Identification of recombinant plasmids

[0132] 2 µl of the recombinant plasmid pPICZαA-CollagenⅢ was identified by double digestion with EcoRI and NotI, and the digestion products were collected for 1% agarose gel electrophoresis.

[0133] like Figure 2 As shown, two fragments of 3594 bp and 1803 bp are visible, respectively. This indicates that the CollagenⅢ gene has been successfully ligated into the pPICZαA vector, and the recombinant plasmid has been successfully constructed.

[0134] Example 2 Construction and identification of recombinant strains expressing recombinant type III collagen

[0135] 1. Transformation of recombinant plasmids and screening of high-copy transformants

[0136] pPICZαA-CollagenⅢ was transformed into competent cells of X33 engineered bacteria, plated onto YPDS solid medium containing 200 μg / ml bleomycin, incubated at 30°C for 20 min, then inverted for 2–3 days. Single colonies grown on the plates were transferred to YPD solid medium containing 200 μg / ml bleomycin for high-copy transformant screening.

[0137] The method for screening high-copy transformants is as follows:

[0138] Colonies from YPDS solid medium containing 200 μg / ml bleomycin were transferred to YPD solid medium containing 200 μg / ml bleomycin and numbered. The plates were then incubated upside down at 30°C for 16–18 h. Ten single colonies were then picked from each YPD solid medium plate and inoculated into 10 ml of BMGY liquid medium. After incubation at 30°C and 280 rpm for 24 h, samples were taken and examined under a microscope to confirm the absence of contamination.

[0139] 2. Identification of high-copy transformants

[0140] The bacterial culture of each single colony was transferred to 10 ml of BMMY liquid medium and cultured at 30℃ with shaking at 280 rpm for 120 h. 200 μl of the bacterial culture was taken, centrifuged at 6000 rpm for 5 min, the precipitate was discarded, and 50 μl of the supernatant was added to 10 μl of 6× protein loading buffer. The mixture was boiled at 100℃ for 10 min, and the sample was taken for SDS-PAGE electrophoresis. X33 engineered bacteria transformed with the pPICZαA vector (i.e., pPICZαA / X33) was used as a negative control.

[0141] like Figure 3As shown, compared with the supernatant of the negative control, all 10 single colonies exhibited a distinct protein band at a position greater than 140 kDa, consistent with the expected size of Collagen III protein, indicating that all 10 single colonies successfully expressed and secreted Collagen III protein into the culture medium. The sixth single colony showed the highest Collagen III protein expression level, and was named pPICZαA-CollagenⅢ / X33 strain for subsequent fermentation and purification of Collagen III protein.

[0142] After purification and culture, 24 tubes of pPICZαA-CollagenⅢ / X33 were preserved and designated as the F0 generation. A series of tests were performed on the F0 generation pPICZαA-CollagenⅢ / X33. The results showed that when the recombinant strain was streaked onto YPD solid medium containing 200 μg / ml bleomycin, the colonies were large, thick, smooth, moist, mostly milky white, and had an alcoholic aroma, consistent with yeast culture characteristics. Under a standard optical microscope, the recombinant strain appeared round, oval, or sausage-shaped. PCR identification confirmed that the recombinant strain could amplify the target fragment of 1803 bp.

[0143] Example 3: Fermentation and purification of recombinant type III collagen

[0144] 1. pPICZαA-CollagenⅢ / X33 fermentation

[0145] pPICZαA-CollagenⅢ / X33 was inoculated at a rate of 0.1% v / v into a 500ml reaction tube containing 100ml of BMGY liquid medium and amplified at 30℃ and 280rpm.

[0146] OD of the bacterial culture 600 When the OD value reaches 5.0, change the solution to remove the OD. 600 The bacterial culture with a concentration of 1.0 was inoculated into 100 ml of BMMY liquid medium and induced to express at 30°C and 280 rpm for 72 h before fermentation was terminated.

[0147] 2. Protein purification

[0148] Collect the fermentation broth, centrifuge at 6000 rpm for 5 min, remove the precipitate, and collect the supernatant. Adjust the pH of the supernatant to 8.0, then filter through a 0.22 μm membrane, collect the filtrate, and purify it using a Ni column, as detailed below:

[0149] First, rinse the column with 5 column volumes of water; then, equilibrate the column with 5 column volumes of Ni-A buffer (20 mM Tris, 100 mM NaCl, pH 8.0); load the sample, which is the filtrate described above; after loading, rinse the column with 10 column volumes of Ni-Abuffer to remove any sample or contaminating proteins that did not adhere to the column; finally, rinse the column with Ni-B buffer (20 mM Tris, 100 mM NaCl, 120 mM imidazole, pH 8.0), collect the eluent in a sterile bottle, and monitor the UV absorbance of the eluent at 280 nm in real time. Retain the eluent corresponding to the first protein peak to obtain the purified Collagen III protein.

[0150] SDS-PAGE electrophoresis showed that the purified Collagen III protein exhibited a target protein band at a position greater than 140 kDa, indicating successful purification.

[0151] 3. Dialysis and lyophilization

[0152] The purified Collagen III protein was dialyzed using a dialysis bag containing buffer (10 mM PB, 50 mM NaCl, pH 7.4) and a MW3500 (i.e., a molecular weight cutoff of 3.5 kDa). After dialysis for 24 hours, the dialysate was collected and lyophilized to obtain a lyophilized sample of recombinant type III collagen, which appeared as a white solid powder.

[0153] Example 4 Performance evaluation of recombinant type III collagen

[0154] 1. Hydrophilic

[0155] (1) Evaluation method

[0156] The hydrophilicity of recombinant type III collagen (amino acid sequence as shown in SEQ ID NO.3) was predicted using the website https: / / web.expasy.org / protscale / .

[0157] (2) Evaluation results

[0158] like Figure 4 As shown, the recombinant type III collagen of the present invention (amino acid sequence as shown in SEQ ID NO.3) has no hydrophobic regions, indicating that it has extremely strong hydrophilicity and extremely high solubility.

[0159] 2. Stability

[0160] (1) Evaluation method

[0161] The lyophilized samples of recombinant type III collagen prepared in Example 3 were placed in sealed bags and stored at 37°C, 25°C, 4°C and -20°C, respectively. SDS-PAGE electrophoresis was performed on samples taken at 0 days, 90 days, 180 days and 365 days of storage to analyze the degradation of recombinant type III collagen.

[0162] (2) Evaluation results

[0163] like Figure 5 As shown in A to D, the recombinant type III collagen of the present invention did not degrade during storage at -20℃ to 37℃ for one year, demonstrating excellent stability.

[0164] 3. Hyaluronidase inhibition effect

[0165] Hyaluronic acid, also known as hyaluronic acid, is a naturally occurring glycosaminoglycan found in living organisms. It has high hydrophilicity and is a major component of the extracellular matrix. Its main functions include maintaining skin moisture and elasticity, promoting wound healing, and regulating angiogenesis. HA also participates in regulating inflammation and allergic reactions. Therefore, HA is closely related to skin sensitivity, skin hydration, and anti-aging. Hyaluronidase (HAase) is a specific cleavage enzyme of HA. Excessive HAase activity leads to HA degradation; therefore, inhibiting HAase can maintain HA function, achieving soothing, moisturizing, and anti-aging effects. The nitrogen atom in cetyltrimethylammonium bromide (CTAB) can pair with the oxygen atom in the carboxyl group of HA to form a water-insoluble HA-CTAB complex. The amount of HA in the solution can be determined by detecting the content of the HA-CTAB complex. Hyaluronidase hydrolyzes HA, reducing its content in the solution. By detecting changes in the amount of HA in the solution, the inhibitory effect of the sample on hyaluronidase can be indirectly determined.

[0166] (1) Evaluation method

[0167] 1) Instruments

[0168] Microplate reader, analytical balance, and electric thermostatic incubator.

[0169] 2) Experimental materials

[0170] Preparation of positive control: Epigallocatechin gallate (EGCG, CAS No.: 989-51-5) was dissolved in water to prepare an EGCG solution with a working concentration of 0.5 mg / mL.

[0171] Preparation of test samples: The lyophilized sample of recombinant type III collagen prepared in Example 3 was dissolved in water and prepared into recombinant type III collagen solutions with working concentrations of 20% (m / v), 2% (m / v) and 1% (m / v) respectively, which were used as test samples for testing.

[0172] Preparation of hyaluronic acid solution: Dissolve hyaluronic acid powder (CAS No.: 9004-61-9) in water to prepare a hyaluronic acid solution with a concentration of 0.04% (m / v).

[0173] 3) Experimental Procedure

[0174] Set up sample wells (T), sample background wells (T0), enzyme reaction wells (C), solvent background wells (C0), positive control wells (P), and positive control background wells (P0) in a 96-well microplate. Add samples according to the information shown in Table 1, with three parallel replicates per well.

[0175] Table 1. Sample loading details for each well of the 96-well microplate.

[0176]

[0177] The sample well corresponding to the test sample with a recombinant type III collagen concentration of 20% (m / v) is designated as T-20, the sample well corresponding to the test sample with a recombinant type III collagen concentration of 2% (m / v) is designated as T-2, and the sample well corresponding to the test sample with a recombinant type III collagen concentration of 1% (m / v) is designated as T-1.

[0178] After adding the reagents shown in Table 1 to each well, the 96-well microplate was incubated at 37°C for 10 min. Then, 40 μl of hyaluronic acid solution was added to the corresponding well, and the plate was incubated at 37°C for 45 min. Afterward, 80 μl of CTAB solution was added to each well, and the plate was precipitated at 37°C for 5 min. The microplate was then transferred to a microplate reader, and the absorbance of each well was measured at 400 nm.

[0179] 4) Criteria for determining test validity

[0180] The test is valid if the test results meet the following conditions:

[0181] a) The inhibition rate of the positive control EGCG should be above 50%;

[0182] b) If the inhibition rate of the sample wells is significantly higher than that of the enzyme reaction wells (P<0.05), then the test sample has an inhibitory effect on hyaluronidase activity.

[0183] (2) Evaluation results

[0184] Table 2. Average absorbance and average suppression rate of each well (Meant±SD)

[0185]

[0186] Note: This indicates that the difference between the sample wells and the enzyme reaction wells is statistically significant (P < 0.05).

[0187] As shown in Table 2, under the conditions of this test, the inhibition rate of the positive control well (EGCG) was 52.53% (≥50%), indicating that the experimental system was effective. Different concentrations of recombinant type III collagen all showed some inhibitory effect on hyaluronidase activity. When the concentration of recombinant type III collagen was 20% (m / v), the inhibitory effect on hyaluronidase activity was the strongest and significantly better than EGCG, with an inhibition rate of 66.08%. Using the same method, the inhibition rate of recombinant type III collagen at a concentration of 0.5% (m / v) was also significantly higher than that of the enzyme reaction wells, indicating that it also inhibited hyaluronidase activity. This demonstrates that the recombinant type III collagen provided by this invention can inhibit hyaluronidase activity and has excellent moisturizing effects.

[0188] 4. Cell adhesion effect

[0189] (1) Evaluation method

[0190] 1) Instruments

[0191] Inverted microscope, carbon dioxide incubator, centrifuge, cell counter, biosafety cabinet and fluorescence microscope.

[0192] 2) Experimental materials

[0193] Sample solution preparation: The lyophilized sample of recombinant type III collagen prepared in Example 3 was dissolved in DPBS to prepare recombinant type III collagen solutions with working concentrations of 20% (m / v), 10% (m / v) and 5% (m / v).

[0194] Experimental cells: Human immortalized keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences. The HaCaT cells used in this experiment were passaged to the 9th generation.

[0195] 3) Experimental Procedure

[0196] Sample groups and blank groups were set up in 96-well cell culture plates. 100 μL of sample solutions of different concentrations (20% (m / v), 10% (m / v) and 5% (m / v)) were added to the cell wells of the sample groups, and were labeled as 20%-sample group, 10%-sample group and 5%-sample group, respectively. Three parallel replicates were set up for each concentration. 100 μL of DPBS was added to the cell wells of the blank groups.

[0197] Incubate the cell culture plate in a 37°C, 5% CO2 incubator for 1-4 hours, then discard the solution in each well to obtain a coated 96-well plate for later use.

[0198] HaCaT cells cultured to the logarithmic growth phase were trypsinized, and the cells were collected, prepared into a cell suspension, and counted. HaCaT cells were resuspended in cell culture medium containing Hoechst 33342 live cell staining solution, and then seeded at 100 μL per well into coated 96-well plates. After seeding, the plates were incubated in a carbon dioxide (CO2) incubator.

[0199] After culture, discard the culture medium in the cell culture plate, add 100 μL of DPBS to each well, and gently shake to wash three times. Photograph the cells in each well using a fluorescence microscope, count the number of cell nuclei, and calculate the cell adhesion percentage using the formula: Cell adhesion percentage (%) = (Vt / Vc) × 100%; Vt: number of cell nuclei in the sample group, Vc: number of cell nuclei in the BC group.

[0200] (2) Evaluation results

[0201] Table 3. Cell adhesion percentage results for different groups (Meant±SD)

[0202]

[0203] Note: This indicates that the difference is statistically significant compared to the control group (BC). P<0.01.

[0204] As shown in Table 3 and Figures 6-7 As shown, when the concentrations of recombinant type III collagen were 20% (m / v), 10% (m / v), and 5% (m / v), the cell adhesion percentage was significantly higher than that of the control group (P<0.01). Using the same method, the cell adhesion percentages at concentrations of 0.5% (m / v) and 2.5% (m / v) of recombinant type III collagen were also significantly higher than those of the control group. This indicates that the recombinant type III collagen provided by this invention has excellent cell adhesion-promoting ability.

[0205] 5. Repair effect

[0206] (1) Evaluation method

[0207] 1) Instruments

[0208] Inverted microscope, cell counter, ELISA reader, CO2 incubator, centrifuge and biosafety cabinet.

[0209] 2) Experimental materials

[0210] Preparation of MTT working solution: Dilute with complete culture medium for HaCaT cells to a working concentration of 1 mg / mL MTT working solution and use immediately.

[0211] Sample solution preparation: The lyophilized sample of recombinant type III collagen prepared in Example 3 was dissolved in complete culture medium to prepare recombinant type III collagen solutions with working concentrations of 10% (m / v), 5% (m / v), 2.5% (m / v), 1% (m / v) and 0.5% (m / v).

[0212] Experimental cells: Immortalized human keratinocytes (HaCaT) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. HaCaT cells passaged to the 9th generation were used for cytotoxicity testing, and HaCaT cells passaged to the 8th generation were used for cell scratch testing. Cells used in both tests were different cryopreserved cells from the same cell line.

[0213] 3) Experimental Procedure

[0214] Cytotoxicity assay: Cytotoxicity was tested using the CCK8 method, and a suitable concentration of sample solution was selected for cell scratch testing based on the cytotoxicity results.

[0215] Cell seeding: HaCaT cells cultured to the logarithmic growth phase were digested, centrifuged, and counted. The HaCaT cells were resuspended in complete culture medium and seeded into 6-well plates at 2 mL per well according to the corresponding groups. The plates were then incubated at 37°C and 5% CO2 for 24 ± 2 h. Before seeding, five parallel straight lines were drawn on the back of the 6-well plate with a marker, with a spacing of approximately 0.5 cm.

[0216] Cell scratching: When the cell confluence reaches 98% or more, use a sterile pipette tip to draw a straight line in each well of the cell, perpendicular to the bottom of the 6-well plate. The direction of the scratching should be perpendicular to the straight line drawn with the marker pen. After scratching, discard the original culture medium and wash the cells 3 times with DPBS.

[0217] Sample addition and culture: Add 2 mL of sample solution of different concentrations to each well of the 6-well plate, and add 2 mL of complete culture medium to the blank control group (BC). Continue to culture in a 37℃, 5% CO2 incubator.

[0218] Photography and detection: At 0h, 24h and 48h of culture, the scratched areas in the cell wells were photographed under an inverted microscope to record cell migration and calculate the cell migration rate. The formula is: Cell migration rate (%) = (0h scratch area - scratch area at each time point) / 0h scratch area × 100%.

[0219] (2) Evaluation results

[0220] 1) Cytotoxicity test results

[0221] Table 4. HaCaT cell viability results after treatment with different concentrations of recombinant type III collagen (Meant±SD)

[0222]

[0223] As shown in Table 4 and Figure 8 As shown, when the concentration of recombinant type III collagen is 0.5% (m / v) to 2.5% (m / v), the cell viability of HaCaT cells is >85%, indicating that recombinant type III collagen has no obvious toxicity to HaCaT cells within this concentration range. Cell scratch assays were subsequently performed using this concentration range.

[0224] 2) Cell scratch test results

[0225] Table 5. Cell migration rate results under different concentrations of recombinant type III collagen treatment (Meant±SD)

[0226]

[0227] Note: This indicates that the difference was statistically significant compared to the blank control group (BC). P<0.05, P<0.01.

[0228] As shown in Table 5 and Figures 9-10 As shown, at 24h and 48h of culture, the cell migration rate was significantly higher than that of the blank control group when the concentration of recombinant type III collagen was 1% (m / v) and 0.5% (m / v) (P<0.05), indicating that the recombinant type III collagen provided by the present invention significantly promotes cell migration at concentrations of 0.5% (m / v) to 1% (m / v) and has excellent repair effects.

[0229] 6. Soothing effect

[0230] (1) Evaluation method

[0231] 1) Instruments

[0232] Inverted microscope, ELISA reader, CO2 incubator, centrifuge, cell counter, biosafety cabinet.

[0233] 2) Experimental materials

[0234] Preparation of MTT working solution: Dilute with complete culture medium for RAW264.7 cells to a working concentration of 1 mg / mL MTT working solution and use immediately.

[0235] Sample solution preparation: The lyophilized sample of recombinant type III collagen prepared in Example 3 was dissolved in complete culture medium to prepare recombinant type III collagen solutions with working concentrations of 10% (m / v), 5% (m / v), 2.5% (m / v), 1% (m / v) and 0.5% (m / v).

[0236] Preparation of positive control solution: Dexamethasone (DXM) was used as a positive control. DXM was prepared into a working concentration of 100 μg / ml using complete culture medium and used immediately.

[0237] Preparation of modeling solution: Prepare a working concentration of 1 μg / ml LPS solution using complete culture medium and use immediately.

[0238] Experimental cells: Mouse monocytic leukemia cells (RAW264.7) were purchased from the Kunming Cell Bank, Chinese Academy of Sciences. RAW264.7 cells passaged to the 10th generation were used for the cytotoxicity test, and RAW264.7 cells passaged to the 6th generation were used for the NO inhibition test. Cells used in both tests were different cryopreserved tubes of the same cell line.

[0239] 3) Test Grouping

[0240] Set up the test groups according to the information shown in Table 6.

[0241] Table 6 Grouping of NO Inhibition Test

[0242]

[0243] 4) Experimental Procedure

[0244] Cytotoxicity assay: The CCK8 assay was used for cytotoxicity experiments. Based on the cytotoxicity results, an appropriate sample concentration was selected for subsequent NO inhibition tests.

[0245] Cell seeding: RAW264.7 cells cultured to the logarithmic growth phase were trypsinized, and the cells were collected, prepared into a cell suspension, and counted. RAW264.7 cells were resuspended in complete culture medium and seeded into 96-well plates at 100 μL per well. After seeding, the plates were incubated in a CO2 incubator for 24 h ± 2 h.

[0246] Induction and drug administration: Discard the culture medium in the 96-well plate, and add the corresponding solutions to the corresponding wells according to the grouping shown in Table 6, with a total solution volume of 200 μL per well. Incubate the 96-well plate in a CO2 incubator for 24 h ± 2 h.

[0247] Cell supernatant collection: After the culture is completed, the cell culture medium is collected in a sterile centrifuge tube, centrifuged, and the supernatant is collected and stored in an ultra-low temperature freezer at -80℃ for later use.

[0248] NO content detection: The NO content in the cell supernatant of each group was detected using an in vitro inflammatory factor nitric oxide (NO) content assay kit. The relative NO content and NO inhibition rate were calculated using the following formulas: relative NO content (%) = (T / C) × 100%, NO inhibition rate (%) = (1-T / C) × 100%; T is the average NO content of each test sample group; C is the average NO content of the model group.

[0249] (2) Evaluation results

[0250] 1) Cytotoxicity test results

[0251] Table 7. RAW264.7 cell viability results after treatment with different concentrations of recombinant type III collagen (Meant±SD)

[0252]

[0253] As shown in Table 7 and Figure 11 As shown, when the concentration of recombinant type III collagen was 0.5% (m / v) to 5% (m / v), the cell viability of RAW264.7 cells was >85%, indicating that recombinant type III collagen had no significant toxicity to RAW264.7 cells within this concentration range. Based on this result and experimental requirements, NO inhibition tests were subsequently conducted using a concentration range of 0.5% (m / v) to 2.5% (m / v).

[0254] 2) Results of in vitro inflammatory factor NO content measurement

[0255] Table 8. Statistical results of NO content in RAW264.7 cells treated with different concentrations of recombinant type III collagen (Meant±SD)

[0256]

[0257] Note: This indicates that the difference is statistically significant compared to the model group. P<0.01; NA indicates that the calculation formula is not applicable.

[0258] As shown in Table 8 and Figure 12 As shown, the relative NO content in the model group was significantly higher than that in the blank control group, while the relative NO content in the positive control group was significantly lower than that in the model group. When the concentration of recombinant type III collagen was 0.5% (m / v) to 2.5% (m / v), the relative NO content in the sample groups was significantly lower than that in the model group, showing a concentration-dependent effect. Among them, the relative NO content was lowest and the NO inhibition rate was highest when the concentration of recombinant type III collagen was 2.5% (m / v), which was basically equivalent to that in the positive control group. This indicates that the recombinant type III collagen provided by this invention can significantly inhibit the release of inflammatory factors in vitro and has excellent soothing effects.

[0259] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

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

1.

2. The recombinant type III collagen according to claim 1, characterized in that, The recombinant type III collagen also includes a tag, which is selected from at least one of the following: His tag, GST tag, MBP tag, Strep tag, and FLAG tag.

3. The recombinant type III collagen according to claim 2, characterized in that, The tag is a His tag, and the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.

3.

4. A nucleic acid molecule, characterized in that, The recombinant type III collagen as described in any one of claims 1 to 3 is encoded.

5. A biomaterial, characterized in that, It includes the nucleic acid molecule as described in claim 4; The biological material is recombinant DNA, expression cassette, transposon, vector, cell or microorganism.

6. A composition, characterized in that, It includes the recombinant type III collagen as described in any one of claims 1 to 3.

7. A method for preparing recombinant type III collagen, characterized in that, If the expression of the recombinant type III collagen as described in any one of claims 1 to 3 is permitted, the microorganism described in claim 5 is cultured, and the recombinant type III collagen is recovered from the culture product.

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