Collagen fusion polypeptide as well as application and preparation method thereof

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to collagen fusion polypeptide as well as application and a preparation method thereof. The collagen fusion polypeptide provided by the invention comprises: (a) a first polypeptide of recombinant III-type collagen; and (b) a second polypeptide comprising a trimerized folding domain. The collagen fusion polypeptide provided by the invention has the advantages of low immunogenicity, good biocompatibility, high biological activity and the like, can significantly promote proliferation and migration of human skin fibroblasts, and has stronger ability of promoting regeneration of endogenous collagen. The collagen fusion polypeptide has a triple helix structure similar to natural collagen, and can be self-assembled to form collagenous fibers which are good in morphology and similar to natural collagen. The method can be widely applied to the fields of skin repair dressings, biological materials, artificial implants, medical instruments, cosmetics, health care products, food and the like.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and specifically relates to a collagen fusion polypeptide and its application and preparation method. Background Art

[0002] Collagen is a biological macromolecular structural protein and the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of the total protein. It is closely related to maintaining the morphology and structure of tissues and organs, maintaining skin elasticity, wound healing, calcification, blood coagulation and aging, and is widely used in medical materials, cosmetics, and food industries.

[0003] Natural collagen is composed of three helical polypeptide chains. The amino acid sequence of the peptide chain follows the Gly-XY principle. Gly is glycine, X is usually proline, and Y is other amino acids. The three polypeptide chains in an alpha helical structure are intertwined to form a right-handed composite helical structure. Since the position of glycine on each chain is fixed, the three chains can be tightly intertwined. At a higher structural level, the collagen triple helix will further associate into collagen fibrils and collagen fibers. At present, the industry has a relatively mature technology for extracting collagen from different tissues such as skin, tendons, and bones of animals such as cattle and pigs using acid, alkali or enzyme treatment methods and processes. However, the animal extraction method easily destroys the triple helical structure and biological activity of collagen, and the molecular weight of the obtained collagen and its peptides is uneven, the batch quality is uneven, and there are risks such as hidden dangers of virus transmission. At the same time, large-scale preparation will cause excessive pressure on animal demand.

[0004] Recombinant collagen prepared by genetic engineering technology can overcome the risks brought by the above-mentioned animal extraction method. Based on the advantages of easy operation of chassis cells, clear genetic background, high safety, high cost-effectiveness, controllable expression, and easy large-scale production, it can express collagen of specific molecular weight from pigs, cattle, fish and humans, and has obvious application advantages in the fields of cosmetics, biomaterials, etc.

[0005] Regarding collagen, many studies have been done in this field. Currently, most recombinantly expressed collagen is a certain fragment or spliced ​​combination of a single peptide chain, or a forward tandem repetitive expression of the sequence. Recombinant collagens of different molecular weights have different solubility, different expression levels in host cells, and the degree of absorption of the product by the skin and the biological activity may also be different. CN112851797B discloses a recombinant human type III collagen and its preparation method and use, splicing fragments of human type III collagen with cell binding ability and co-expressing with hydroxylase. CN111363029B discloses a recombinant human type III collagen, an expression strain and a construction method thereof, and a polypeptide fragment of 498 amino acids with strong hydrophilicity in the human type III collagen sequence is selected for expression in Pichia pastoris. CN103122027B is expressed in Escherichia coli by connecting the amino acids GPPGPCCGGG in the hinge region of the type II collagen peptide to the end of the polypeptide. CN113185612B discloses a long-acting recombinant type III collagen, comprising a basic repeating unit and a terminal amino acid sequence, and expressing the target polypeptide in Saccharomyces cerevisiae. CN111087463B discloses a long-fragment recombinant type III collagen, and obtains efficient expression in Escherichia coli. The recombinant collagen polypeptide prepared by the above patents lacks the characteristic structural spectrum and fiber morphology of collagen, and it is not yet known whether it forms a triple helical structure.

[0006] CN109593126B adds the hinge region sequence to the C-terminus of the polypeptide to help gelation, but it will cause the polypeptide to form a colloidal precipitate in advance during the fermentation culture process, making it difficult to purify the product and resulting in a reduced yield. The post-translational modification processing system using prokaryotic expression systems such as Escherichia coli is imperfect, lacking some necessary modification enzymes, which affects the biological activity of the expression product; during the production process, the target protein is often expressed in the form of inclusion bodies, and the expression vector contains unnecessary tags, and the bacteria itself contains endotoxin risks, which increases the separation and purification costs of large-scale preparation of products. Pichia pastoris is a recognized safe organism (GRAS), which has a natural secretion mechanism and efficient expression ability, and can perform post-translational modifications unique to eukaryotic cells, which is crucial for the functions of certain proteins. The synthesis of procollagen to mature collagen in vivo undergoes special post-translational modifications, glycosylation, hydroxylation, cross-linking and other processes, and this process depends on multiple collagen-specific partners and modifying enzymes, such as prolyl 3-hydroxylase (P3H) and lysine hydroxylase (P4H) involved in the hydroxylation of proline and lysine residues, and protein partners that stabilize the correct folding of the triple helix structure of collagen in cells, including heat shock protein 47 (HSP47), heat shock protein-related lumen-binding protein (BiP) and P4H β subunit (PDI, also known as disulfide isomerase), etc., making the production of recombinant collagen with correct triple helix conformation and high biological activity challenging. Domestic and foreign researchers mainly promote the formation and stability of the triple helix structure of recombinant collagen from the structural design of collagen polypeptides. CN117186210A discloses a recombinant human collagen and its application. Through the systematic prediction and analysis of thermal stability, the collagen fragments of human type I collagen are intercepted for sequence splicing and design, and the repetitive sequence modules (GPP) n are introduced at both ends of the collagen domain. After expression in Escherichia coli, it is found that the designed high thermal stability collagen fragments can be correctly folded to form a triple helix structure, while the low thermal stability fragments cannot be correctly folded. CN116082493B discloses a method for constructing a variant that improves the stability of collagen and its application, including avoiding unstable GXY triplet, removing potential MMP enzyme cleavage sites, eliminating chemical unstable factors, and increasing the RGD content in the sequence on the basis of the original collagen sequence. Collagen with low stability is prone to lose the triple helix structure in in vitro and in vivo applications, so that it cannot exert biological activity. Therefore, further obtaining a recombinant human collagen with stable triple helix structure, good fiber appearance, and better biological activity is still a goal that is constantly pursued in this field. Summary of the invention

[0007] Technical Problems to be Solved by the Invention

[0008] In view of the shortcomings of the above-mentioned existing recombinant collagen preparation technology, the present invention provides a Pichia pastoris engineered bacterium containing a recombinant type III collagen fusion polypeptide, and also provides a construction method, fermentation culture and separation and purification method of the Pichia pastoris engineered bacterium of the recombinant type III collagen fusion polypeptide. The recombinant type III collagen fusion polypeptide provided by the present invention has a triple helix structure similar to that of natural collagen and a good fiber morphology, and can be prepared on a large scale by a biosynthetic method.

[0009] In view of this, the present invention proposes the following technical solutions:

[0010] In a first aspect, the present invention provides a collagen fusion polypeptide comprising: (a) a basic repeating unit of recombinant type III collagen; and (b) a trimerization folding domain.

[0011] In some embodiments, the recombinant type III collagen is human recombinant type III collagen.

[0012] In some embodiments, the trimerization foldon domain is derived from a human type III collagen peptide or a T4 phage fibrin foldon domain.

[0013] In some embodiments, the collagen fusion polypeptide further has at least one of the following characteristics:

[0014] (1) Has a triple helix structure;

[0015] (2) Self-assembly to form collagen fibers;

[0016] (3) Promote the proliferation and migration of human skin fibroblasts;

[0017] (4) Promote the regeneration of endogenous collagen.

[0018] In some preferred embodiments, the collagen fusion polypeptide has a flexible triple helical structure.

[0019] In some preferred embodiments, the collagen fusion polypeptide is a trimer.

[0020] In some embodiments, the trimerization fold domain is located at the N-terminus, the C-terminus, or between the N-terminus and the C-terminus of the basic repeating unit.

[0021] In some preferred embodiments, the trimerization fold domain is located at the N-terminus, the C-terminus, or between the N-terminus and the C-terminus of the basic repeating unit.

[0022] In some more preferred embodiments, the trimerization fold domain is located at the C-terminus of the basic repeating unit.

[0023] In some embodiments, the number of the basic repeating unit is one or more.

[0024] In some embodiments, the number of basic repeating units is 1-30, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30.

[0025] In some preferred embodiments, the number of the basic repeating units is 1-20, preferably 12.

[0026] In some embodiments, the basic repeating unit has an amino acid sequence as shown in SEQ ID NO:1, or has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0027] SEQ ID NO: 1:

[0028] GAPGFRGPAGPNGIPGEKGPAGERGAPGPA.

[0029] In some embodiments, the trimerization folding domain comprises a repetitive sequence having an amino acid sequence as shown in any one of SEQ ID NOs: 2-4, or the repetitive sequence having an amino acid sequence that differs from the amino acid sequence shown in any one of SEQ ID NOs: 2-4 by at least one amino acid.

[0030] In some embodiments, the amino acid difference includes amino acid substitution, addition, deletion, modification, replacement, etc., preferably amino acid substitution, more preferably conservative amino acid substitution.

[0031] In some embodiments, the trimerization fold domain comprises 1-10 repeat sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0032] In some preferred embodiments, the trimerization fold domain comprises 1-5 repeat sequences.

[0033] In some more preferred embodiments, the trimerization fold domain comprises three repeat sequences.

[0034] SEQ ID NO:2:GAPCCGGG.

[0035] SEQ ID NO:3:GPCCGG.

[0036] SEQ ID NO:4:GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0037] In some embodiments, the collagen fusion polypeptide further comprises a polypeptide for enhancing stability, extending half-life and / or separating and purifying, such as serum albumin, Fc protein, His-tagged protein, GST-tagged protein, MBP-tagged protein, SUMO-tagged protein, Strep-tagged protein, NusA-tagged protein, FLAG-tagged protein.

[0038] In some embodiments, the collagen fusion polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 5-9.

[0039] SEQ ID NO:5:

[0040] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGF RGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPCCGGG*.

[0041] SEQ ID NO:6:

[0042] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPCCGG*。

[0043] SEQ ID NO:7:

[0044] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPCCGGGGAPCCGGGGAPCCGGG*。

[0045] SEQ ID NO:8:

[0046] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAG PNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPCCGGGPCCGGGPCCGG*.

[0047] SEQ ID NO:9:

[0048] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGI PGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGYIPEAPRDGQAYVRKDGEWVLLSTFL*.

[0049] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding any of the aforementioned collagen fusion polypeptides.

[0050] In some embodiments, the isolated nucleic acid molecule comprises a codon-optimized nucleic acid molecule as set forth in any one of SEQ ID NOs: 10-14.

[0051] SEQ ID NO:10:

[0052]

[0053] SEQ ID NO:11:

[0054]

[0055] SEQ ID NO:12:

[0056]

[0057] SEQ ID NO:13:

[0058]

[0059] SEQ ID NO:14:

[0060]

[0061] In some preferred embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule that is codon-optimized for a eukaryotic cell, wherein the eukaryotic cell is preferably a yeast cell, more preferably Pichia pastoris.

[0062] In some embodiments, the isolated nucleic acid molecule further comprises a nucleic acid molecule encoding protein disulfide isomerase (PDI) and / or endoplasmic reticulum oxidoreductase (ERO1).

[0063] In some preferred embodiments, the nucleic acid molecule encoding protein disulfide isomerase has a nucleotide sequence as shown in SEQ ID NO:15.

[0064] In some preferred embodiments, the nucleic acid molecule encoding the endoplasmic reticulum oxidoreductase has a nucleotide sequence as shown in SEQ ID NO:16.

[0065] In a third aspect, the present invention provides a recombinant vector comprising any of the aforementioned isolated nucleic acid molecules.

[0066] In some embodiments, the recombinant vector is a recombinant expression vector suitable for eukaryotic cells.

[0067] In some preferred embodiments, the recombinant vector is a recombinant expression vector suitable for yeast cells.

[0068] In some more preferred embodiments, the recombinant expression vector is a recombinant expression vector suitable for Pichia pastoris, such as pPICZαA, pPIC3.5K, pPIC9K, pHIL-S1, or pYAM75P vector.

[0069] In some embodiments, the recombinant vector further comprises a regulatory element, which includes a cis-expression element or a trans-acting factor, such as a promoter, a terminator, an enhancer, a ribosome binding site, and the like.

[0070] In a fourth aspect, the present invention provides a host cell comprising any of the aforementioned isolated nucleic acid molecules or recombinant vectors.

[0071] In some embodiments, the host cell comprises a eukaryotic cell or a prokaryotic cell.

[0072] In some preferred embodiments, the host cell is a yeast cell.

[0073] In some more preferred embodiments, the host cell is Pichia pastoris, preferably Pichia pastoris, more preferably Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strain.

[0074] In some embodiments, the host cell is used to express the isolated nucleic acid molecule or recombinant vector contained therein, so as to translate and express any of the aforementioned collagen fusion polypeptides having biological activity or biological function.

[0075] In a fifth aspect, the present invention provides a composition comprising any of the aforementioned collagen fusion polypeptides and at least one physiologically acceptable auxiliary material or excipient.

[0076] In some embodiments, the physiologically acceptable excipients and excipients include, but are not limited to: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavors, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0077] In a sixth aspect, the present invention provides use of any of the aforementioned collagen fusion polypeptides, isolated nucleic acid molecules, recombinant vectors, host cells or compositions in the preparation of products for wound repair, tissue repair and / or medical cosmetology.

[0078] In some embodiments, the dosage form of the product includes a solid dosage form, a liquid dosage form, or a semisolid dosage form.

[0079] In some preferred embodiments, the dosage form of the product includes a repair solution, a gargle, a cream (such as an ointment), a patch (such as an application), a gel (including a gel dressing), a filler, an injection, an implant, a freeze-dried fiber or a freeze-dried powder.

[0080] In a seventh aspect, the present invention provides a method for preparing any of the aforementioned collagen fusion polypeptides, the preparation method comprising:

[0081] (i) culturing any of the aforementioned host cells under appropriate culture conditions so that the host cell expresses the collagen fusion polypeptide;

[0082] (ii) harvesting the host cells or culture containing the collagen fusion polypeptide;

[0083] (iii) isolating and / or purifying the collagen fusion polypeptide from the host cell or culture of step (ii).

[0084] Preferably, the step (i) includes a bacterial enrichment stage and an induction expression stage, by adding ethanol to the culture medium.

[0085] Preferably, in step (iii), the collagen fusion polypeptide is purified by salting out and / or cation exchange chromatography.

[0086] In some embodiments, the suitable culture conditions in step (i) include:

[0087] 1) a medium containing a carbon source and / or a nitrogen source, preferably a medium suitable for Pichia pastoris, more preferably a BSM medium;

[0088] 2) Suitable temperature, preferably 25°C-30°C, for example, 30°C for fermentation and 26°C for induction expression;

[0089] 3) Induction method, preferably ethanol-induced expression.

[0090] In some embodiments, the purification in step (iii) comprises at least one of the following:

[0091] Salting out, dialysis, ultrafiltration and ion chromatography.

[0092] In some preferred embodiments, the salting out uses a saturated ammonium sulfate solution.

[0093] In some preferred embodiments, the dialysis uses a 1-10KD dialysis bag, preferably a 3KD dialysis bag.

[0094] In some preferred embodiments, the ultrafiltration uses a 1-10KD ultrafiltration tube, preferably a 3KD ultrafiltration tube.

[0095] In some preferred embodiments, the ion chromatography is cationic chromatography, preferably a PrePack SP Purose6XL strong cationic chromatography column.

[0096] In an eighth aspect, a trimerization folding domain is used in the preparation of collagen polypeptides.

[0097] In some embodiments, the trimerization foldon domain is derived from a type III collagen peptide or a foldon domain of T4 bacteriophage fibrin.

[0098] In some embodiments, the collagen polypeptide is human recombinant type III collagen.

[0099] In some embodiments, the trimerization folding domain is used to promote the collagen polypeptide to form a triple helical structure, self-assemble into collagen fibers and / or improve stability.

[0100] In some embodiments, the trimerization folding domain comprises a repetitive sequence having an amino acid sequence as shown in any one of SEQ ID NOs: 2-4, or the repetitive sequence having an amino acid sequence that differs from the amino acid sequence shown in any one of SEQ ID NOs: 2-4 by at least one amino acid.

[0101] In some embodiments, the collagen polypeptide comprises a basic repeating unit, which has an amino acid sequence as shown in SEQ ID NO:1, or has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0102] In some preferred embodiments, the number of the basic repeating units is 1-30, preferably 12.

[0103] In some preferred embodiments, the trimerization folding domain is located at any position of the collagen polypeptide, including the N-terminus, the C-terminus, or between the N-terminus and the C-terminus.

[0104] In some preferred embodiments, the collagen fusion polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 5-9.

[0105] In a ninth aspect, the present invention provides a method of skin care, comprising administering any of the aforementioned collagen fusion polypeptides and / or compositions to a subject in need thereof, preferably contacting the composition with the skin tissue of the subject.

[0106] In some preferred embodiments, the skin care comprises skin anti-wrinkle, skin oil control, skin repair and / or skin soothing.

[0107] In a tenth aspect, the present invention provides a method for promoting fibroblast proliferation, comprising administering any of the aforementioned collagen fusion polypeptides and / or compositions to fibroblasts, preferably contacting the collagen fusion polypeptide and / or composition with fibroblasts.

[0108] In some preferred embodiments, the fibroblasts are skin fibroblasts.

[0109] In an eleventh aspect, the present invention provides a method for promoting fibroblast migration, comprising administering any of the aforementioned collagen fusion polypeptides and / or compositions to fibroblasts, preferably contacting the collagen fusion polypeptide and / or composition with fibroblasts.

[0110] In some preferred embodiments, the fibroblasts are skin fibroblasts.

[0111] In a twelfth aspect, the present invention provides a method for promoting the regeneration of endogenous collagen in fibroblasts, comprising administering any of the aforementioned collagen fusion polypeptides and / or compositions to the fibroblasts, preferably contacting the collagen fusion polypeptide and / or composition with the fibroblasts.

[0112] In some preferred embodiments, the fibroblasts are skin fibroblasts.

[0113] Compared with the prior art, the present invention has at least the following beneficial effects:

[0114] (1) The present invention provides a recombinant type III collagen fusion polypeptide, wherein the amino acid sequence of the collagen is derived from the α1 chain of human type III collagen. Compared with collagen derived from animals, the collagen has the advantages of no hidden danger of virus transmission and low immunogenicity.

[0115] (2) The recombinant type III collagen fusion polypeptide provided by the present invention has a triple helical structure similar to natural collagen and can self-assemble to form collagen fibers with good morphology similar to natural collagen.

[0116] (3) The recombinant type III collagen fusion polypeptide provided by the present invention has important functional sites of human type III collagen, good biocompatibility and high biological activity, can significantly promote the proliferation and migration of human skin fibroblasts, and has a stronger ability to promote the regeneration of endogenous collagen.

[0117] (4) The genetically engineered bacterial expression system of the recombinant type III collagen fusion polypeptide provided by the present invention has good solubility in extracellular secretion, high yield, simple and convenient preparation process, avoids impurities caused by bacterial lysis, does not need to remove labels and endotoxins, and is easy to achieve large-scale production.

[0118] (5) The highly biologically active recombinant type III collagen fusion polypeptide prepared by the present invention can be widely used in the fields of skin repair dressings, biomaterials, artificial implants, medical devices, cosmetics, health products, food, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 This is the map of the recombinant type III collagen fusion polypeptide expression plasmid pPIC9K-PLA-C1. Compared with pPIC9K-PLA-C1, the maps of pPIC9K-PLA-C2, pPIC9K-PLA-C3, pPIC9K-PLA-C4, and pPIC9K-PLA-C5 are exactly the same except for the target gene fragment, so only the plasmid map of pPIC9K-PLA-C1 is shown here.

[0120] Figure 2 The results of nucleic acid electrophoresis for PCR identification of yeast strain expressing recombinant type III collagen fusion polypeptide.

[0121] Figure 3 This is the SDS-PAGE electrophoresis result of the shake flask fermentation sample of recombinant type III collagen fusion polypeptide.

[0122] Figure 4 The SDS-PAGE electrophoresis results show the effects of overexpression of Pdi gene, Ero1 gene and co-expression of Pdi / Ero1 on the expression level of col-P4 protein.

[0123] Figure 5 This is a scanning electron microscope (SEM) image of recombinant type III collagen fusion polypeptide.

[0124] Figure 6 The circular dichroism characterization results of recombinant type III collagen fusion polypeptide.

[0125] Figure 7 The effect of different concentrations of recombinant type III collagen fusion polypeptide on the proliferation of skin fibroblasts. The samples were compared with the competitor control by t-test statistical analysis. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0126] Figure 8 The effect of different concentrations of recombinant type III collagen fusion polypeptide on the migration of skin fibroblasts. The samples were compared with the competitor control by t-test statistical analysis. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0127] Fig. 9 The effect of 0.001% recombinant type III collagen fusion polypeptide on endogenous collagen regeneration, samples were compared with competitor controls for t-test statistical analysis, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. DETAILED DESCRIPTION

[0128] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0129] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0130] All publications and patents mentioned in this disclosure are hereby incorporated by reference into this disclosure in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0131] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0132] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0133] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0134] As shown in the present disclosure, the term "about" means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0135] The numerical ranges used in the present disclosure should be understood to have enumerated all the numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0136] As used in this disclosure, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the disclosure, especially in the claims, may be replaced by the term "consisting of".

[0137] As used herein, the terms "optional", "either", "any", or "any" mean that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. As used herein, "a" and "an" are used in the present invention to refer to one or more than one of the grammatical objects.

[0138] As shown in the present disclosure, “and / or” should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0139] As shown in the present disclosure, the terms "polypeptide", "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. A polypeptide can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0140] As shown in the present disclosure, the term "amino acid" refers to the 20 naturally occurring amino acids that can be translated from the gene code and constitute the protein chain, including L-amino acids and D-amino acids, unless otherwise specified, and also refers to chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are usually not bound to proteins such as norleucine, and chemically synthesized compounds with amino acid properties known in the art. For example, analogs or mimetics of Phe or Pro that can make the conformational constraints of peptide compounds the same as those of natural phenylalanine or proline are included in the definition of "amino acid" and are known to those skilled in the art. The analogs and mimetics mentioned in this article refer to "functional equivalents" of amino acids. Other examples of amino acids and amino acid analogs have been given by Roberts and Vellaccio (The Peptides: Andlysis, Synthesis, Biology, Eds. Gross and Meienhofer, Vol. 5, P. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference).

[0141] As shown in the present disclosure, the term "amino acid substitution" refers to replacing an amino acid residue present in a parent sequence with another amino acid residue. The amino acid in the parent sequence can be substituted, for example, via chemical peptide synthesis or by recombinant methods known in the art. Therefore, reference to "substitution at position X" refers to replacing the amino acid present in position X with an alternative amino acid residue. In some embodiments, the substitution type can be described according to the pattern AXY, where A is a single letter code corresponding to an amino acid naturally present in position X, and Y is a substituted amino acid residue. In other aspects, the substitution type can be described according to the pattern XY, where Y is a single letter code corresponding to replacing an amino acid residue naturally present in position X.

[0142] As indicated in the present disclosure, the term "conservative amino acid substitution" or "conservative substitution of amino acids" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the prior art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative.

[0143] As shown in the present disclosure, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0144] As shown in the present disclosure, the term "nucleic acid molecule" or "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA, cDNA or RNA fragments. When applied to nucleic acids or polynucleotides, the term "isolated" refers to nucleic acid molecules (DNA or RNA) that have been removed from their natural environment, such as for the purposes of the present disclosure, it is believed that the recombinant polynucleotides encoding the antigen-binding proteins contained in the vector are isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or recombinant polynucleotides purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides disclosed herein. According to the isolated polynucleotides or nucleic acids of the present disclosure, further include such molecules produced by synthesis. In addition, polynucleotides or nucleic acids may include regulatory elements, such as promoters, enhancers, ribosome binding sites or transcription termination signals.

[0145] As used in this disclosure, the term "polynucleotide" or "nucleotide" is intended to encompass a single nucleic acid as well as a plurality of nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA).

[0146] As shown in the present disclosure, the term "vector" refers to a vector that can replicate autonomously in a host cell, preferably a multi-copy vector. As used herein, the term "vector" is capable of transporting a nucleic acid molecule of another nucleic acid to which it is connected. In addition, the vector generally has markers such as antibiotic resistance genes for selecting transformants. In addition, the vector may have a promoter and / or terminator for expressing the introduced gene. The vector may be, for example, a vector derived from a bacterial plasmid, a viral vector, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a clay mid, a phage mid, etc. The term "recombinant vector" refers to a vector modified as needed for carrying an exogenous DNA fragment (a DNA fragment of a gene of interest) into a host cell for replication and expression, including a recombinant cloning vector and a recombinant expression vector, preferably a recombinant expression vector. In some instances, the recombinant vector may contain a variety of elements for controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site. The vector may contain a nucleic acid of the present invention to facilitate introduction into a cell for expression. The vector may contain expression control elements operably connected to the nucleic acid, such as a promoter, a terminator, and / or an enhancer.

[0147] As used herein, the term "human recombinant type III collagen" refers to a recombinant protein consisting of or substantially consisting of a sequence derived from human type III collagen. In the present disclosure, human recombinant type III collagen may consist of or substantially consist of a fragment or multiple repeats of a fragment derived from human type III collagen.

[0148] As shown in the present disclosure, the term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plants, or animal cells. In some examples, the host cell is a yeast cell. "Yeast cell" means any yeast host cell that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or a recombinant expression vector comprising a polynucleotide of the present disclosure. The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In the present disclosure, the preferred yeast cell is Pichia pastoris. More preferably, Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strain.

[0149] As used in this disclosure, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0150] As used herein, the term "codon optimized" means that a nucleotide sequence encoding a polypeptide has been configured to contain codons preferred by a host cell or organism to improve gene expression and translation efficiency in the host cell or organism.

[0151] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. If the specific conditions are not specified in the embodiment, it is carried out according to the conditions of normal conditions or manufacturer's suggestion. If the manufacturer is not specified in all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in the publication of many publications.

[0152] Example 1: Construction of a recombinant type III collagen fusion polypeptide expression system

[0153] (1) Synthesis of gene sequences and construction of recombinant expression vectors

[0154] The present invention optimizes type III collagen fragments and folding domains according to the codon preference of Pichia pastoris. The sequences are commissioned to Nanjing GenScript Biotech Co., Ltd. for synthesis. The five gene fragments are respectively designated as pUC-19-C1, pUC19-C2, pUC19-C3, pUC19-C4, and pUC-19-C5. The nucleotide sequences are shown in SEQ ID NOs: 10-14. The synthesized gene fragment and the outer loop of the vector pPIC9K-PLA were amplified respectively, and after being cleaned and recovered by a DNA gel recovery kit (Beijing Qingke Biotechnology Co., Ltd., Catalog No. TSP602-200), the five target genes were respectively connected to the vector pPIC9K-PLA by a one-step cloning kit (ClonExpress MultiS One Step Cloning Kit, Catalog No. C113-01) using a homologous recombination ligation kit, and the ligation products were transformed into competent Escherichia coli DH5α (ClonExpress Biotechnology Co., Ltd., Catalog No. C502-02), and positive clones were screened on an LB resistance plate containing ampicillin (Amp), and a primer pair (upstream primer F1 (SEQ ID NO: 17): 5'-ATGAGATTTCCTTCAATTTTTACTG-3', downstream primer R1 (SEQ ID NO: 18): 5'-ATGAGATTTCCTTCAATTTTTACTG-3', downstream primer R2 (SEQ ID NO: 20): 5'-ATGAGATTTCCTTCAATTTTTACTG-3') was used. NO:18): 5'-CTTATCGATAAGCTTGCACAAAC-3') was verified by colony PCR, and the recombinant plasmid was extracted for sequencing identification (commissioned by Beijing Qingke Biotechnology Co., Ltd.), and the recombinant expression vectors pPIC9K-PLA-C1, pPIC9K-PLA-C2, pPIC9K-PLA-C3, pPIC9K-PLA-C4, and pPIC9K-PLA-C5 were obtained. The relevant plasmid maps are shown in Figure 1 shown.

[0155] (2) Transformation, strain screening and PCR identification of recombinant engineering strains

[0156] 10 μg of the recombinant expression vector verified to be correct in step (1) above was digested with SalⅠ (New England Biolabs Biotechnology Co., Ltd., catalog number R0139) at 37°C overnight to linearize it, and then the linearized plasmid was recovered using a PCR product purification kit. The plasmid was mixed with Pichia pastoris GS115 competent cells and placed in an electroporation cup for electroporation transformation. The electroporated bacterial solution was placed in a 30°C shaker at 200 rpm for 2 hours to recover, and centrifuged at 5000 rpm for 10 minutes. The bacterial cells were collected and re-dissolved in 500 μL of sterile water, and 100-200 μL was spread on a YND plate (glucose 20 g / L, YNB 6.7 g / L, agar 15 g / L), and inverted and cultured at 30°C for 2-3 days until a single white colony grew.

[0157] Add 5 mL of sterile water to the YND plate, gently scrape the His-positive transformants on the surface of the plate with a coating stick, transfer the bacterial suspension to a 10 mL centrifuge tube, and dilute to 10 with sterile water. 7 Take 100-200 μL of the bacterial suspension of cells and spread them on YPD plates (yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, agar 15 g / L) containing 1, 2, 3, and 4 mg / mL G418, and incubate them upside down at 30°C for 3-5 days until a single colony grows. The recombinant strain may obtain integrated plasmids with different copy numbers during homologous recombination integration, among which Kan R The gene confers resistance to G418 on yeast cells, and the number of integrated plasmids is positively correlated with the resistance level of G418. Transformants with different copy numbers were obtained by growing them on YPD plates containing different concentrations of G418, and then multi-copy, highly expressed genetically engineered strains were obtained, which were named col-P1, col-P2, col-P3, col-P4, and col-P5, respectively.

[0158] The genomic DNA of the transformants on the plate was extracted using an efficient plant genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number DP350, refer to the instructions for the method), and PCR identification was performed using the primer pair upstream primer F1 (SEQ ID NO: 19): 5'-ATGAGATTTCCTTCAATTTTTACTG-3', downstream primer R1 (SEQ ID NO: 20): 5'-CTTATCGATAAGCTTGCACAAAC-3'), and the reaction system is shown in Table 1:

[0159] Table 1

[0160]

[0161] The PCR reaction parameters are shown in Table 2:

[0162] Table 2

[0163]

[0164] The amplified products were detected by 1% agarose gel electrophoresis. Figure 2 As shown, the recombinant strains col-P1, col-P2, col-P3, col-P4, and col-P5 correspond to the target gene-containing bands of 1721bp, 1715bp, 1769bp, 1748bp, and 1778bp, respectively, proving that the target gene has been integrated into the chromosome of the recombinant engineering strain.

[0165] Example 2: Fermentation of genetically engineered strains and expression of recombinant type III collagen fusion polypeptide

[0166] The glycerol-preserved bacterial suspension of the transformant identified correctly by PCR in Example 1 was transferred to a 100 mL Erlenmeyer flask containing 5 mL YPD medium (10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose) at a 1% inoculum and cultured at 30°C, 200 rpm until OD = 2-6. The bacterial cells were collected by centrifugation at 5000 g for 10 min and suspended in 50 mL BSM basal salt medium (0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 1.47 g / L trisodium citrate, 20 g / L glucose, 10 g / L ammonium sulfate, potassium phosphate buffer (pH 6.0) 0.1mol / L, trace elements 2mL / L. Main trace element components: copper sulfate pentahydrate 6.0g / L, potassium iodide 0.8g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate dihydrate 0.2g / L, boric acid 0.02g / L, calcium sulfate 0.5g / L, cobalt chloride 0.5g / L, zinc chloride 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, sulfuric acid 5.0m / L, biotin 0.2g / L) were placed in a 250mL conical flask, placed in a 30℃ shaker at 200rpm for culture, and induced after 24h. The induction temperature was adjusted to 26℃. During this period, ethanol with a final concentration of 1.0% was added every 24h to induce expression. Every 24h, 200μL was sampled into a 2mL EP tube, centrifuged at 12000g for 5min, and the fermentation supernatant was collected respectively. Take 12 μL of supernatant and add 3 μL of 5× protein loading buffer, shake and mix well, heat in a boiling water bath for 10 min to denature the protein. Use 4-20% Tris-Glycine protein electrophoresis precast gel (Shanghai Bioengineering Co., Ltd., Cat. No. C661104-0001) for SDS-PAGE electrophoresis, load 10 μL per well, and perform electrophoresis at 120V constant voltage for 60 min. After the electrophoresis, use Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) on a 70rpm horizontal shaker for staining for 1-2 h, and use decolorizing solution (10% acetic acid, 5% ethanol) for decolorization for 1-5 h.

[0167] The SDS-PAGE results are as follows Figure 3 As shown, the recombinant collagen fusion polypeptides of col-P1, col-P2, col-P3, col-P4, and col-P5 can be efficiently secreted in the extracellular culture supernatant, all of which are single bands with apparent molecular weights of about 35-40 kD. The theoretically predicted molecular weights are 34173 Da, 34045 Da, 35379 Da, 34994 Da, and 36634 Da, respectively. Due to the special amino acid sequence, structure, and post-translational modification of collagen, its migration rate on SDS-PAGE may be lower than that of globular proteins with the same molecular weight.

[0168] Example 3: Optimization of engineered strains to improve collagen expression

[0169] The cysteine ​​residues in the terminal folding structure sequence designed in the present invention form disulfide bonds in the collagen molecule, which plays a vital role in the activity and stability of collagen. Protein disulfide isomerase (PDI) catalyzes the formation, reduction and isomerization of disulfide bonds, and endoplasmic reticulum oxidoreductase (ERO1) provides the oxidative environment required for the formation of disulfide bonds. The endogenous disulfide isomerase gene of yeast is inserted into the downstream of the GAP promoter of plasmid pGAPZαA to construct plasmids containing different molecular chaperone proteins. Using the yeast genome as a template, the Pdi gene fragment (Gene ID: 8201243, the nucleotide sequence is shown in SEQID NO: 15) and the Ero1 gene fragment (Gene ID: 8197528, the nucleotide sequence is shown in SEQ ID NO: 16) were amplified. Using the vector pGAPZαA as a template, the vector fragment was amplified using a primer pair. The two gene fragments and the vector fragment were assembled into a circular plasmid using a seamless cloning kit, and transformed into Escherichia coli competent cells. The vectors pGAPZαA-OEpdi and pGAPZαA-OEero1 for overexpressing Pdi and Ero1 were obtained by PCR and sequencing verification. On the basis of verifying the correct construction of the pGAPZαA-OEero1 vector, the expression cassette including the GAP promoter, the Ero1 gene fragment and the AOX1 terminator was amplified and connected to the vector pGAPZαA-OEpdi to obtain the co-expression vector pGAPZαA-OEpdi / ero1. 10 μg of the overexpression plasmid was linearized by enzyme digestion with BglⅡ (New England Biolabs Biotechnology Co., Ltd., catalog number R0144V), and electroporation was transformed and integrated into the chromosome of the Pichia pastoris strain col-P4 in Example 1, and the engineered strains overexpressing Pdi and Ero1 alone and co-expressing the two were obtained, respectively, and named col-P45, col-P46, and col-P47. The expression was induced according to the fermentation method in Example 2 and the expression of collagen was detected.

[0170] The results of fermentation for 120 hours are as follows Figure 4 The results showed that the promoting effect on the expression of collagen fusion polypeptide was as follows: the expression levels of col-P47 and col-P46 were equivalent, and col-P47 / col-P46>col-P45>col-P4. Therefore, the overexpression of Pdi and Ero1 alone had a significant promoting effect on the expression of collagen fusion polypeptide, and the co-expression of Pdi and Ero1 was equivalent to the effect of overexpressing Ero1 alone.

[0171] SEQ ID NO:15

[0172]

[0173] SEQ ID NO:16

[0174] ATGAGGATAGTAAGGAGCGTAGCTATCGCAATAGCCTGTCATTGTATAACAGCGTTAG

[0175] CAAACCCTCAAATCCCTTTTGACGGCAACTACACCGAGATCATCGTGCCAGATACCGA

[0176] AGTTAACATCGGACAGATTGTAGATATTAACCACGAAATAAAACCCAAACTGGTGGAA

[0177] CTGGTCAACACAGACTTCTTCAAATATTACAAATTAAACCTATGGAAACCATGTCCGTT

[0178] TTGGAATGGTGATGAGGGATTCTGCAAGTATAAGGATTGCTCTGTCGACTTTATCACTG

[0179] ATTGGTCTCAGGTGCCTGATATCTGGCAACCAGACCAATTGGGTAAGCTTGGAGATAA

[0180] CACGGTACATAAGGATAAGGGCCAAGATGAAAATGAGCTGTCCTCAAATGATTATTGC

[0181] GCTTTGGATAAAGACGACGATGAAGATTTAGTATATGTCAATTTGATTGATAACCCTGA

[0182] AAGATTCACCGGTTATGGTGGTCAGCAATCTGAATCTATTTGGACTGCGGTCTATGATG

[0183] AGAACTGTTTCCAGCCGAATGAAGGATCACAATTGGGTCAAGTTGAAGACCTCTGTTT

[0184] GGAGAAACAGATCTTTTACCGATTGGTTTCTGGTTTGCATTCTAGTATCTCCACCCACC

[0185] TCACAAACGAATATCTGAATTTGAAAATGGAGCATACGAACCAAATTTGAAACAGTT

[0186] CATGATCAAAGTTGGGTATTTTACTGAAAGAATTCAAAACTTACATCTCAATTATGTCC

[0187] TTGTATTGAAGTCACTAATAAAGCTACAAGAATACAATGTTATCGACAATCTACCTCTC

[0188] GATGACTCTTTGAAAGCTGGTCTTAGCGGTTTAATATCTCAAGGAGCACAGGGTATTAA

[0189] CCAGAGCTCTGATGATTATCTATTTAACGAGAAGGTTCTTTTCCAAAATGACCAAAATG

[0190] ATGATTTGAAAAATGAATTCCGTGACAAATTCCGCAACGTGACTAGATTAATGGATTG

[0191] TGTCCATTGCGAGAGATGCAAATTATGGGGAAAATTGCAAACTACAGGGTACGGGACT

[0192] GCATTGAAGATTCTATTTGATTTGAAGAATCCTAATGACTCCATCAATTTAAAGAGAGT

[0193] TGAGTTAGTTGCTCTAGTCAACACATTCCATAGATTGTCCAAATCTGTTGAAAGCATTG

[0194] AAAACTTTGAAAAACTATATAAGATTCAACCGCCAACGCAGGATCGTGCATCAGCGTC

[0195] GTCCGAATCCTTAGGCCTTTTCGATAACGAAGATGAACAAAATCTCCTCAACTCGTTTT

[0196] CGGTTGATCAGGCAGTCATTTCATCGAAAGAGGCACCAGAAGAAATCAAAAGCAAAC

[0197] CTGTTGGAAAAGCCGCATATAAACAAAACAGTTGTCCATCATTGGGTTTCAAAATCTAT

[0198] CAAAGAAGCATTCCATGAAGAACTTCACGCATTTATTGATGCAATTGGATTTATATTGA

[0199] ACTCTTACAGGACTTTGCCCAAGCTGTTGTACACACTTTTCCTCGTTAAATCATCTGAAT

[0200] TATGGGACATTTTCATTGGCACTCAAAGGCACCGAGATACCACATATAGAGTAGACTTGTAA.

[0201] Example 4: Isolation and purification of recombinant type III collagen fusion polypeptide

[0202] The fermented liquid obtained according to the method of Example 2 was centrifuged at 10000 rpm for 10 min to remove the bacterial cells, and the supernatant was collected. A saturated ammonium sulfate solution was added to a saturation of 30%, and the mixture was stirred and mixed thoroughly and allowed to stand at room temperature for 1 hour. The precipitate was collected by centrifugation at 12000 rpm for 20 min. The precipitate was dissolved in ultrapure water and dialyzed overnight with a 3KD dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd., item number SP131204), and concentrated 5-10 times with a 3KD ultrafiltration tube (Merck, item number RT-UFC900396-5) to obtain a chromatography loading solution. The AKTA Pure 150 (Cytiva) machine was loaded with a PrePack SP Purose 6XL strong cation chromatography column, and 5 column volumes were balanced with buffer A (50mM Tris-HCl solution, pH=8), the flow rate was 5mL / min, the loop was cleaned with buffer A before loading, the sample loading volume was 100mL, and buffer B (50mM Tris-HCl solution containing 100-1000mM NaCl, pH=8) was used for elution, and the elution peaks at each stage were collected. The molecular weight and purity of collagen were detected by SDS-PAGE, and recombinant type III collagen fusion polypeptide with a purity of more than 95% could be obtained by elution with 300mM NaCl. After overnight dialysis, it was freeze-dried for subsequent activity detection.

[0203] Example 5: Assembly morphology of recombinant type III collagen fusion polypeptide

[0204] The freeze-dried recombinant type III collagen fusion polypeptide sample prepared in Example 4 was cut and gold-sprayed, and then the morphological structure was observed and evaluated using a Hitachi-Regulus 8100 emission scanning electron microscope (SEM).

[0205] like Figure 5 As shown, the recombinant type III collagen fusion polypeptides of the present invention all have a layered structure, and a fibrous structure is observed. Among them, the fiber structures of the collagen fusion polypeptides col-P3 and col-P4 are relatively regular, and can form collagen fibers with good morphology and similar to natural collagen. The layered and fibrous structures can be used for wound treatment and play an active role in wound healing as a collagen sponge.

[0206] Example 6: Circular dichroism characterization of recombinant type III collagen fusion polypeptide

[0207] The triple helix structure is a typical feature of collagen and is closely related to the thermal stability of collagen. The triple helix structure of collagen has specific spectral characteristics in circular dichroism. The lyophilized collagen sample was prepared into a solution with a final concentration of 1 mg / mL using 10 mmol / L, pH 7.0 sodium phosphate buffer. After equilibration at 4°C for 48 hours using a 1 mm cuvette, the circular dichroism spectrum was scanned at full wavelength (190-260 nm) with a wavelength interval of 1 nm and a data sampling time of 0.5 s at each wavelength.

[0208] like Figure 6 As shown, the recombinant type III collagen fusion polypeptide has a strong negative absorption peak at 198nm and a weak positive absorption peak at about 221nm, which is the characteristic absorption peak of the collagen triple helix structure, indicating that the recombinant collagen fusion polypeptide can spontaneously fold to form a triple helix structure. Different terminal trimerization folding domain sequences promote the formation of a triple helix structure of collagen fusion polypeptides to different levels, among which the collagen fusion polypeptide col-P5 has a more significant positive peak at 221nm, indicating that its triple helix level is higher.

[0209] Example 7: Biological activity of recombinant type III collagen fusion polypeptide

[0210] (1) Cell proliferation assay

[0211] The cells were cultured in 10% serum medium (10 mL fetal bovine serum plus 90 mL DMEM basal medium). Human fibroblasts grown to 80-90% were collected and counted. Based on the counting results, 0.5-1×10 4Each well was inoculated into a 96-well cell culture plate. The recombinant type III collagen fusion polypeptide prepared in Example 4 was prepared into a collagen solution with a final concentration of 0.001% and 0.01% with 10% serum culture medium, and the culture medium in each well was 200 μL. The zeroing group was a cell-free inoculation, and only 200 μL of 10% serum culture medium was added to each well; the blank control group was a well inoculated with cells and 200 μL of 10% serum culture medium was added to each well, and commercially available recombinant type III collagen was used as a competitive control. According to the experimental design, group administration was carried out, and three replicates were designed for each group. After the sample was completed, the 96-well plate was placed in an incubator (37°C, 5% CO2, 95% RH) and cultured for 24 hours. The cell culture medium was discarded, and 200 μL of cytotoxicity detection solution (5mL CCK-8 reagent plus 45mL DMEM basal culture medium) was added to each well, and the incubation continued for 0.5-2 hours in the cell culture incubator, and the absorbance value was measured at 450nm using an enzyme marker. The relative cell viability was calculated according to the formula: relative cell viability (%) = (sample well OD-zero well OD) ÷ (blank control well OD-zero well OD) × 100.

[0212] like Figure 7 As shown, compared with the blank control, the cell viability of the experimental group added with recombinant type III collagen fusion polypeptides col-P1, col-P2, col-P3, and col-P4 was greater than 100%, which promoted the viability of human skin fibroblasts; the cell viability of col-P1, col-P2, col-P3, col-P4, and col-P5 collagen fusion polypeptides at a concentration of 0.01% was significantly higher than that of the competitive product control, and the lowest cell viability of col-P5 at a concentration of 0.001% was 110.47%, and the highest cell viability of col-P2 protein at a concentration of 0.01% could reach 128.63%. The results show that the recombinant type III collagen polypeptide of the present invention has a significant pro-proliferation effect on skin fibroblasts.

[0213] (2) Cell migration assay

[0214] The cell scratch method was used to determine the cell migration promoting effect of the recombinant type III collagen fusion polypeptide prepared in Example 4. The cells were cultured using a 10% serum medium (10 mL of fetal bovine serum plus 90 mL of DMEM basal medium). The medium containing 1% serum was used as a blank control. The collagen in Example 4 was dissolved in a 1% serum medium to prepare migration fluid culture medium with a final concentration of 0.001% and 0.01%. The cultured cells were human dermal fibroblasts. The cells were digested, and the culture medium was discarded by centrifugation after the digestion was terminated, and the cells were washed 1-2 times with PBS. The cells were resuspended in a medium containing 1% serum, and the cell density was adjusted to 5×10 5 / mL. Place the plug-in (ibidi, Culture-Insert 2-well) in a 12-well plate, add 70 μL of cell suspension to each well, and culture for 12-24 hours. Carefully remove the upper chamber with tweezers, add migration culture medium to each well, take pictures under a microscope, and record it as 0 hours. Place the cells in an incubator and culture for 6 hours, take pictures under a microscope, and record it as 6 hours. Measure the scratch area with imageJ. The calculation formula is as follows: Wound healing rate (%) = (scratch area at 6 hours - scratch area at 0 hours) / scratch area at 0 hours × 100%.

[0215] In vitro cell migration experiments directly reflect the interaction between cells and extracellular matrix and between cells under the influence of matrix. Cell migration activity is an effective indicator of the biological activity of collagen. The higher the migration rate and the faster the speed, the better the biological activity of collagen. Figure 8 As shown, the cell migration activity of the purified freeze-dried samples of col-P2, col-P3, col-P4, and col-P5 was significantly better than that of the competitor control, and the collagen samples at a concentration of 0.001% had higher cell migration activity than the corresponding proteins at a concentration of 0.01%. This result shows that the recombinant type III collagen fusion polypeptide of the present invention has a significant effect on promoting migration of skin fibroblasts.

[0216] (3) Collagen regeneration experiment

[0217] The cells were cultured in 10% serum medium (10 mL fetal bovine serum plus 90 mL DMEM basal medium). Human fibroblasts grown to 80-90% were collected and counted. Based on the counting results, 0.5-1×10 4Each well was inoculated into a 96-well cell culture plate. The recombinant type III collagen fusion polypeptide prepared in Example 4 was prepared into a collagen solution with a final concentration of 0.001% using 10% serum culture medium. The culture medium in each well was 200 μL. The zeroing group was a cell-free inoculation, and only 200 μL of 10% serum culture medium was added to each well; the blank control group was a well inoculated with cells and 200 μL of 10% serum culture medium was added to each well. According to the experimental design, group administration was carried out, and three replicates were designed for each group. After the sample was completed, the 96-well plate was placed in an incubator (37°C, 5% CO2, 95% RH) and cultured for 48 hours. The cell culture supernatant medium was collected and type I collagen ELISA kit was used to detect type III collagen (supplier: Elabscience, product number: E-EL-H0869, for detailed operations, please refer to the kit manual), type III collagen ELISA kit was used to detect type III collagen (supplier: Elabscience, product number: E-EL-H6049, for detailed operations, please refer to the kit manual), type IV collagen ELISA kit was used to detect type IV collagen (supplier: Elabscience, product number: E-EL-H0178, for detailed operations, please refer to the kit manual), and type VII collagen ELISA kit was used to detect type VII collagen (supplier: Elabscience, product number: E-EL-H0781, for detailed operations, please refer to the kit manual).

[0218] like Fig. 9 As shown, compared with the competitive product control, the addition of 0.001% recombinant type III collagen fusion polypeptides col-P1, col-P2, col-P3, col-P4 and col-P5 can significantly promote the increase in the content of type III collagen and type VII collagen; col-P2, col-P3, col-P4 and col-P5 can significantly promote the increase in the content of type I collagen and type IV collagen. The results indicate that the recombinant type III collagen fusion polypeptide of the present invention can effectively increase the regeneration synthesis of endogenous collagen in fibroblasts and improve skin elastic support.

[0219] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A collagen fusion polypeptide, characterized in that: It comprises: (a) a basic repeating unit of recombinant type III collagen; (b) a trimerization folding domain; Preferably, the recombinant type III collagen is human recombinant type III collagen; Preferably, the trimerization folding domain is derived from a human type III collagen peptide or a T4 bacteriophage fibrin foldon domain; Preferably, the collagen fusion polypeptide also has at least one of the following characteristics: (1) Has a triple helix structure; (2) Self-assembly to form collagen fibers; (3) Promote the proliferation and migration of human skin fibroblasts; (4) Promote the regeneration of endogenous collagen.

2. The collagen fusion polypeptide according to claim 1, characterized in that: The trimerization folding domain is located at the N-terminus, the C-terminus, or between the N-terminus and the C-terminus of the basic repeating unit, preferably the C-terminus.

3. The collagen fusion polypeptide according to claim 1 or 2, characterized in that: The number of the basic repeating units is 1-30, preferably 1-20, more preferably 12; Preferably, the basic repeating unit has an amino acid sequence as shown in SEQ ID NO:1, or has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

4. The collagen fusion polypeptide according to any one of claims 1 to 3, characterized in that: The trimerization folding domain comprises a repeating sequence, wherein the repeating sequence has an amino acid sequence as shown in any one of SEQ ID NOs: 2-4, or the repeating sequence has an amino acid sequence that differs from the amino acid sequence shown in any one of SEQ ID NOs: 2-4 by at least one amino acid; Preferably, the trimerization folding domain comprises 1-5 repeating sequences; more preferably, the trimerization folding domain comprises 3 repeating sequences; More preferably, the collagen fusion polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 5-9.

5. The collagen fusion polypeptide according to claim 1, characterized in that: The collagen fusion polypeptide also includes polypeptides for enhancing stability, extending half-life and / or separating and purifying, such as serum albumin, Fc protein, His-tagged protein, GST-tagged protein, MBP-tagged protein, SUMO-tagged protein, Strep-tagged protein, NusA-tagged protein, and FLAG-tagged protein.

6. An isolated nucleic acid molecule, characterized in that It encodes the collagen fusion polypeptide described in any one of claims 1 to 5; Preferably, the isolated nucleic acid molecule further comprises a nucleic acid molecule encoding a protein disulfide isomerase and / or an endoplasmic reticulum oxidoreductase.

7. A recombinant vector, characterized in that: It comprises the isolated nucleic acid molecule according to claim 6.

8. A host cell, characterized in that It comprises the isolated nucleic acid molecule of claim 6 or the recombinant vector of claim 7; Preferably, the host cell comprises a eukaryotic cell or a prokaryotic cell; more preferably, the host cell comprises a yeast cell; most preferably, the host cell is Pichia pastoris.

9. A composition, characterized in that The method comprises the collagen fusion polypeptide according to any one of claims 1 to 5 and at least one physiologically acceptable auxiliary material.

10. Use of the collagen fusion polypeptide according to any one of claims 1 to 5, the isolated nucleic acid molecule according to claim 6, the recombinant vector according to claim 7, the host cell according to claim 8 or the composition according to claim 9 in the preparation of a product for wound repair, tissue repair and / or medical cosmetology; Preferably, the dosage form of the product includes a solid dosage form, a liquid dosage form or a semisolid dosage form; Preferably, the product includes a skin repair dressing, a biomaterial, an artificial implant, a medical device, a cosmetic, a health product or a food.

11. A method for preparing the collagen fusion polypeptide according to any one of claims 1 to 5, characterized in that: The preparation method comprises: (i) culturing the host cell according to claim 8 under appropriate culture conditions so that the host cell expresses the induced expression of the collagen fusion polypeptide; (ii) harvesting the host cells or culture containing the collagen fusion polypeptide; (iii) isolating and / or purifying the collagen fusion polypeptide from the host cell or culture of step (ii).

12. Use of a trimerization folding domain in the preparation of collagen polypeptides; Preferably, the trimerization folding domain is derived from the foldon domain of type III collagen peptide or T4 bacteriophage fibrin; Preferably, the collagen polypeptide is human recombinant type III collagen; Preferably, the trimerization folding domain is used to promote the collagen polypeptide to form a triple helical structure, self-assemble to form collagen fibers and / or improve stability; Preferably, the trimerization folding domain comprises a repeat sequence, the repeat sequence having an amino acid sequence as shown in any one of SEQ ID NOs: 2-4, or the repeat sequence having an amino acid sequence that differs from the amino acid sequence shown in any one of SEQ ID NOs: 2-4 by at least one amino acid; Preferably, the collagen polypeptide comprises a basic repeating unit having an amino acid sequence as shown in SEQ ID NO: 1, or having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO: 1; More preferably, the number of the basic repeating units is 1-30, and most preferably, the number of the basic repeating units is 12.

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