Collagen domain, collagen, recombinant collagen expression bacterium and use thereof

Abstract

The present application provides a collagen domain, collagen, recombinant collagen expression bacterium, and use thereof, and belongs to the field of genetic engineering. Stability prediction and sequence design are carried out on human collagen to obtain a collagen structural domain with high native human collagen homology, and recombinant human collagen having a triple helix structure is directly expressed in Escherichia coli. The designed high-thermal-stability collagen fragment can be correctly folded to form a triple helix structure, whereas low-thermal-stability fragments cannot be correctly folded. Further, the designed high-thermal-stability recombinant human collagen type I can self-assemble to form periodic bright and dark stripes similar to native collagen type I. The described collagen domain and collagen can further be used in the fields of biomedicine and tissue engineering for bionic recombinant collagen having structural functions, and can be used for tissue culture, dental tissue repair, and so on.

Description

Collagen domain, collagen, recombinant collagen expression bacteria and their applications Technical Field

[0001] The present invention relates to collagen domains, collagen, recombinant collagen expression bacteria and applications, and specifically to a method for directly expressing recombinant collagen with a triple helix structure in Escherichia coli. The recombinantly expressed human type I collagen can self-assemble into regular bionic fibers, belonging to the field of genetic engineering technology. Background Art

[0002] Collagen is the most abundant structural protein in the human body, accounting for approximately 30% of the total protein in the body. It is widely distributed in tissues such as bone, tendon, cartilage, and skin. Collagen is composed of three polypeptide chains arranged around a central axis to form a right-handed triple helix. This triple helix can further assemble into higher-order collagen fibrils, which perform their functions in the body. Therefore, the triple helix structure of collagen is the foundation of its biological functions. Types I, II, and III collagens account for 80-90% of the total collagen content in the human body. Type I collagen is the most abundant functional protein in animals. The self-assembled collagen fibrils, characterized by overlapping and interstitial regions, exhibit a band-like morphology, commonly known as the D-cycle, under transmission electron microscopy. The D-cycle is considered a key structural element that confers collagen's various functions, and is involved in the load-bearing properties of tissues, bone mineralization, and the regulation of cell differentiation and adhesion during tissue development. Type II collagen is found in the cartilage of the ribs, nose, larynx, and trachea, and can control the symptoms of joint-related diseases such as osteoarthritis. Type III collagen, along with type I, plays a role in the skin, ligaments, blood vessels, and joints, and is closely linked to the repair process and quality of skin injuries. In recent years, with the advancement of intelligent manufacturing in bioengineering, the demand for high-performance biomimetic biomaterials has also increased. Collagen materials, due to their excellent biocompatibility and low immunogenicity, have enormous potential for application in skin injury treatment, vascular stent engineering, cartilage and bone defect repair, skin care, hemostatic sponges, and drug delivery, including coatings and medical nanoparticles.

[0003] Currently, the main source of collagen is animal extraction, but its potential immunogenicity limits its application in the field of biomedical materials. Polypeptide chains with characteristic collagen sequences can also be obtained through chemical synthesis, but this is costly and the length of the synthesized polypeptide chains is limited, making them unsuitable for large-scale production. Genetically engineering human collagen to express natural or optimized sequences in microorganisms to produce recombinant collagen is gaining increasing attention and becoming a research hotspot. This not only addresses the potential viral risks associated with traditional extraction methods, but also allows for sequence modification to increase the hydrophilicity of collagen according to actual needs, resulting in samples of stable quality and high safety.

[0004] Microbial expression systems have the advantages of a clear genetic background, ease of genetic manipulation, short fermentation cycles, and high expression levels, and are widely used for heterologous protein expression. However, due to the current difficulty in achieving humanized post-translational modifications in microbial expression systems, the expressed human collagen cannot be modified to fold into a triple helical structure or self-assemble into a higher-order structure. In addition, due to the particularity of collagen structure, the current research on the forces acting on collagen folding is not yet completely clear, which also makes the sequence design of heterologous expression of human collagen lack sufficient theoretical support. Therefore, the problem of recombinantly expressing human collagen to fold into a triple helical structure and further assemble into regular collagen higher-order structures remains difficult to solve.

[0005] Currently, there are some reports showing that human collagen can be heterologously expressed by microorganisms such as Escherichia coli, but there is at least one problem:

[0006] (1) The sequence has low homology with the natural collagen. The sequence is obtained by random truncation, modification and repetition / splicing based on experience. It has a triple helix structure verified by circular dichroism. For example, the literature "The self-assembly of a mini-fibril with axial periodicity from a designed collagen-mimetic triple helix" and "To achieve self-assembled collagen mimetic fibrils using designed The 108-amino acid collagen domain Col108 reported in the "Preparation, Structural Characterization and Performance Analysis of Recombinant Human-like Collagen" is a splicing of 4 short sequence fragments in the collagen domain of human type I collagen, and its homology with the natural sequence is only 45.61%; CN115521373A discloses a triple-helical recombinant humanized type I collagen, a preparation method and its application. The expressed recombinant humanized type I collagen has a triple-helical structure and can self-assemble to form collagen fibers. The collagen domain fragment of the above patent is a functional motif inserted into the Col108 fragment reported in the above literature, and has a low homology with the natural sequence; the degree thesis "Preparation, Structural Characterization and Performance Analysis of Recombinant Human-like Collagen" designed a recombinant human-like collagen single fragment with a 38-amino acid sequence and analyzed The sequence is repeated 4 or 8 times and expressed in Escherichia coli. The synthesized human-like collagen has a triple helix structure, but a sequence search of the single-fragment collagen sequence cannot match human collagen; CN115819557A discloses a triple helix recombinant humanized type II collagen, a preparation method and an application. The expressed recombinant humanized type II collagen has a triple helix structure and can self-assemble to form collagen fibers. The sequence matches the human collagen sequence for up to 7 consecutive amino acid residues; and CN115521372A discloses a triple helix recombinant humanized type III collagen, a preparation method and an application. The sequence matches the natural sequence for up to 9 consecutive amino acids, and a sequence search cannot match human collagen.

[0007] (2) The expressed collagen has low stability and no triple helical structure at room temperature. For example, the paper “Recombinant expression of hydroxylated human collagen in Escherichia coli” promotes its folding into a triple helical structure by co-expressing the prolyl and lysyl hydroxylases of mimic viruses and human type III collagen fragments, but T m The value is only 24.3°C. Collagen with low stability is prone to lose its triple helix structure during in vivo and in vitro applications, and thus cannot function.

[0008] (3) The expressed collagen has not been characterized by standardized triple helices, and the triple helical structure cannot be determined. According to the "Guidelines for the Evaluation of Recombinant Humanized Collagen Raw Materials" and Nature Protocols, 2006: VOL.1, NO.6, 2527, the paper "Selective expression of nonsecreted triple-helical and secret single-chain recombinant collagen fragments in the yeast Pichia pastoris" recombinantly expressed human type III collagen fragments in Pichia pastoris, and its subsequent study "Expression of recombinant human type I-III collagens in the yeast Pichia pastoris" co-expressed proline hydroxylase with human type I, II, and III collagens in Pichia pastoris, but neither study characterized the triple helical structure; "Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers" promoted the folding of human type I collagen into a triple helical structure by co-expressing chicken proline hydroxylase and human type I collagen, but the article only measured T by thermal curve at 197nm. mThe value is 30℃. The absorption peak at this wavelength is often similar to the protein spectrum in the unfolded state, and it cannot be used as a standard collagen triple helix characterization method, so the triple helix structure cannot be determined; CN114276435A discloses a recombinant human type III collagen and its application, which selects a segment containing 123 amino acid sequences, and directionally replaces the tripeptide sequence in the segment sequence, repeats it, and connects a specific sequence at the -C terminus. It is expressed using Pichia pastoris, and the triple helix structure is not characterized; CN114774460A discloses yeast recombinant human type I triple helix collagen and its preparation method, which selects human type I collagen α1 chain sequence and hydroxylase for co-expression, and patent CN114480471A discloses yeast recombinant human type III triple helix collagen and its preparation method, which selects human type III collagen α1 chain sequence and hydroxylase for co-expression; CN11 Patents 1087464B disclose a recombinant human type III collagen with a functional structure and its expression method, which selects a partial sequence fragment of human type III collagen and co-expresses it with a hydroxylase; CN112851797B discloses a recombinant human type III collagen and its preparation method and use, which splices fragments of human type III collagen with cell binding ability and co-expresses them with a hydroxylase; CN116555320A discloses a recombinant human type III triple-helical collagen engineered bacteria and its construction method and use, which selects a human type III collagen α1 chain sequence and co-expresses it with a hydroxylase; and CN116082494A discloses a recombinant human type III collagen polypeptide, expression vector, expression strain, and construction method, which selects a 54-amino acid polypeptide fragment with strong hydrophilicity and stability from the human type III collagen sequence and expresses it in Pichia pastoris. None of the above seven patents characterize the triple helical structure, and whether it can actually form a triple helical structure remains unknown.

[0009] In addition, in the early research process, the inventor team's CN111333715B (a method for preparing type I collagen fibers) disclosed a method for preparing type I collagen fibers with N and C termini (GPP) n Sequence is based on the collagen sequence with continuous Gly-Xaa-Yaa triplets inserted in the middle to form a ribbon fiber with periodic light and dark stripes, and CN111499729B (A method for regulating the periodic length of type I collagen fiber stripes), with N and C termini (PPG) nThe human collagen sequence was used as the basis, and continuous collagen sequences with different numbers of Gly-Xaa-Yaa triplets were inserted in the middle to form ribbon fibers with periodic light and dark stripes with different dark stripe lengths. None of the above methods systematically designed the human collagen sequence; the inventor team's master's thesis "Hierarchical self-assembly of collagen polypeptides induced by multiple non-covalent forces" selected human type I collagen sequence fragments for expression in Escherichia coli, which can form a triple helical structure, but did not assemble to form a fiber structure similar to natural human collagen.

[0010] Therefore, it is necessary to develop a collagen sequence that has high homology with natural human collagen and can achieve exogenous expression of a triple helical structure based on systematic thermal stability analysis.

[0011] Summary of the Invention

[0012] In order to solve at least one problem of the above-mentioned recombinant human collagen, such as low homology with natural human collagen, difficulty in heterologous expression to form a triple helical structure, or difficulty in further self-assembly to form a higher-order structure, the present invention intercepts the collagen fragments of human type I, II, and III collagen for sequence splicing and design through systematic thermal stability prediction analysis, and obtains a collagen domain (also known as a collagen domain or collagen domain) with high homology to natural collagen; further, a repeat sequence module is introduced at both ends of the collagen domain, and the designed collagen sequence is expressed in Escherichia coli. It is found that the designed high thermal stability collagen fragments can all be correctly folded to form a triple helical structure, while the low thermal stability fragments cannot be correctly folded. In addition, the designed high thermal stability recombinant human type I collagen can self-assemble to form periodic light and dark stripes similar to natural type I collagen. The present invention has developed a sequence of collagen with high homology to natural human collagen that can achieve exogenous expression of a triple helical structure, and has achieved expression, which can meet the needs of the biomedical and tissue engineering fields for recombinant collagen with structural functions.

[0013] The first object of the present invention is to provide an amino acid sequence encoding a collagen domain, wherein the amino acid sequence has:

[0014] (1) an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7; or

[0015] (2) an amino acid sequence obtained by combining two sequences as shown in any one of SEQ ID NOs. 1 to 3; or

[0016] (3) An amino acid sequence obtained by repeating 2 to 3 times the sequence shown in any one of SEQ ID NOs. 1 to 7.

[0017] In one embodiment, the amino acid sequences shown in SEQ ID NOs. 1 to 7 are obtained by sequence truncation or further sequence splicing and design from collagen fragments of natural type I, II, and III human collagen.

[0018] In one embodiment, the amino acid sequences shown in SEQ ID NO. 1 to 7 are obtained by predicting the thermal stability of natural human collagen types I, II, and III, and selecting the amino acid sequences with high predicted T m The amino acid sequence is used as the prediction of the collagen triple helix structure of the collagen domain. m The value is 38-39℃.

[0019] The amino acid sequence is used as a prediction of the collagen triple helical structure of the collagen domain m The specific prediction method is as follows: take the first triplet unit (XYG) of the triple helix structure as the starting point of the continuous numbering, calculate the average relative stability for each XYG triplet, and obtain the thermal stability value of each triplet; then take n consecutive triplets, calculate the average thermal stability value of these n consecutive triplets, which is the thermal stability prediction value of the collagen domain sequence; among them, the thermal stability value of a single triplet i refers to the thermal stability value of a window composed of 10 consecutive triplets in the interval [i-5,i+5]; the thermal stability value of the window T windows By the window main chain tendency value T bb and window side chain interaction value T side Decide,

[0020] The second object of the present invention is to provide a single protein chain for expressing collagen, wherein the single protein chain contains the above-mentioned amino acid sequence encoding the collagen domain.

[0021] In one embodiment, the structure of the protein single chain includes: a folding domain, a repeat sequence module, and a collagen domain.

[0022] In one embodiment, the structure of the protein single chain includes, from N-terminus to C-terminus: a folding domain, a {repeating sequence module, a collagen domain}m, a repeating sequence module, where m is an integer and m≥1.

[0023] Optionally, m is 1 or 2.

[0024] In one embodiment, a glycine can be added to the end of the amino acid sequence of the collagen single chain.

[0025] In one embodiment, the introduction of the folding domain assists collagen in folding to form a triple helical structure.

[0026] Optionally, the folding domain is a V-domain, and its amino acid sequence is as shown in SEQ ID NO.13; optionally, the folding domain is a coiled-coil domain, and its amino acid sequence is as shown in SEQ ID NO.14.

[0027] In one embodiment, the introduction of the repeat sequence module can assist in the folding of the collagen triple helix and improve its thermal stability. Optionally, there are multiple repeat sequence modules, located at both ends of the collagen domain or at both ends of multiple collagen domains; for example, when expressing type II collagen, there can be multiple collagen domains, and the multiple collagen domains are connected by repeat sequence modules. Optionally, the sequences of the repeat sequence modules can be the same or different.

[0028] In one embodiment, the repeat sequence module adopts (GPP) n Optionally, when multiple repeat sequence modules are included, each repeat sequence module (GPP) n The values ​​of n can be the same or different. n By adjusting the number of n, the molecules can further assemble to form a fiber structure. n Collagen(GPP) n Mode, two n are equal, (GPP) n The value of n satisfies 5<n≤30, and the value of n disclosed in CN 111333715 B disclosed in the previous research of the inventor team can be referred to. n Collagen(GPP) n Collagen(GPP) n Mode, the three n's may not be equal.

[0029] In one embodiment, the folding domain and the repeat sequence module are connected by an enzyme cleavage site, such as LVPRGSP (sequence such as SEQ ID NO.21). Alternatively, the folding domain V-domain and the repeat sequence module (GPP) n They are connected by LVPRGS (sequence shown in SEQ ID NO.22).

[0030] In one embodiment, the structure of the protein single chain for expressing collagen comprises, from N-terminus to C-terminus, the following sequence: a folding domain, an enzyme cleavage site, {repeating sequence module, collagen domain}m, and a repeating sequence module; wherein m is greater than or equal to 1. Optionally, m is 1 or 2.

[0031] In one embodiment, the front end (N-terminus) of the folding domain has a 6×His tag.

[0032] In one embodiment, the protein single chain for expressing collagen has a structure as shown in FIG10 ; or a structure as shown in FIG11 .

[0033] In one embodiment, the structure of the protein single chain for expressing collagen is: a first repeating sequence module-collagen domain-second repeating sequence module; at least one of the first repeating sequence module and the second repeating sequence module has an amino acid sequence as shown in SEQ ID NO. 27 (abbreviated as KD2) or SEQ ID NO. 28 (abbreviated as KD3), and the other has an amino acid sequence as shown in SEQ ID NO. 23, i.e. (GPP) 10 (abbreviated as P10);

[0034] Alternatively, the amino acid sequences of the first repeating sequence module and the second repeating sequence module are both as shown in SEQ ID NO.23.

[0035] In one embodiment, the structure of the single protein chain of the collagen is from N-terminus to C-terminus: {KD2, collagen domain}m, KD2; or {P10, collagen domain}m, KD2; or {KD3, collagen domain}m, P10; or {P10, collagen domain}m, KD3; or {P10, collagen domain}m, P10; wherein m=1.

[0036] The third object of the present invention is to provide a nucleotide sequence encoding the collagen domain, or a nucleotide sequence encoding the protein single chain for expressing collagen, or a gene encoding the protein single chain for expressing collagen, as well as a plasmid or cell expressing the gene.

[0037] Optionally, the plasmid may be a pColdIII series or a pET series plasmid.

[0038] Optionally, the cells are Escherichia coli cells, including E. coli BL21, E. coli BL21 (DE3), E. coli Rosetta (DE3), E. coli BL21 (DE3) pLysS / pLysE or E. coli Origami2 (DE3).

[0039] Optionally, the cell is E. coli BL21 (DE3).

[0040] The fourth object of the present invention is to provide a collagen protein, which is composed of three of the above-mentioned protein single chains coiled around a common central axis to form a triple helical structure.

[0041] In one embodiment, the structure of the collagen protein single chain from segment N to segment C is: {repeating sequence module, collagen domain}m, repeating sequence module, where m is an integer and m≥1.

[0042] In one embodiment, the repeat sequence module and the repeat sequence module are (GPP) n ; Optionally, (GPP) n The value of n satisfies 5<n≤30.

[0043] In one embodiment, the structure of the protein single chain for expressing collagen is: a first repeating sequence module-collagen domain-second repeating sequence module; at least one of the first repeating sequence module and the second repeating sequence module has an amino acid sequence as shown in SEQ ID NO. 27 (abbreviated as KD2) or SEQ ID NO. 28 (abbreviated as KD3), and the other has an amino acid sequence as shown in SEQ ID NO. 23, i.e. (GPP) 10 (abbreviated as P10);

[0044] Alternatively, the amino acid sequences of the first repeating sequence module and the second repeating sequence module are both as shown in SEQ ID NO.23.

[0045] In one embodiment, the structure of the single protein chain of the collagen is from N-terminus to C-terminus: {KD2, collagen domain}m, KD2; or {P10, collagen domain}m, KD2; or {KD3, collagen domain}m, P10; or {P10, collagen domain}m, KD3; or {P10, collagen domain}m, P10; wherein m=1.

[0046] The fifth object of the present invention is to provide collagen fibers formed by the self-assembly of the collagen polymer.

[0047] In one embodiment, the collagen is collagen type I. Optionally, the collagen fibers have periodic light and dark stripes; and optionally, the collagen fibers exhibit bright stripes when negatively stained under TEM.

[0048] In one embodiment, the collagen fibers can be prepared by (GPP) n The regulation of the number is obtained; optionally, the repetitive sequence module in the regulation is (GPP) 10 , corresponding to the length of the bright stripe is 10nm.

[0049] In one embodiment, the amino acid sequence of the collagen domain of the present invention is introduced into the collagen domain region of type I collagen, so that the dark stripes in the collagen fibers reach: (number of amino acids in the collagen domain region ÷ 3 × 0.9) ± 1 nm.

[0050] The sixth object of the present invention is to provide a product containing the collagen of the present invention, which is a collagen containing a triple helical structure; the triple helical structure collagen has the collagen domain of the present invention, or is composed of the protein single chain of the present invention coiled around a common central axis.

[0051] The products are products in the fields of beauty, chemical industry, food and health products, medical / biomedical fields, cosmetics, and feed, such as beauty cosmetics (facial masks, essences, creams, facial masks, etc.), artificial collagen casings, nutritional health products (collagen powder, oral liquid), medical dressings, hemostatic materials, artificial bone scaffolds, injectable fillers, artificial blood vessels, eye drops, drug sustained-release carriers, etc.

[0052] In one embodiment, the product is a dressing, comprising a collagen film layer containing the triple helix structure collagen of the present invention; optionally, further comprising a drug-loaded cellulose film layer; optionally, the number of layers of the collagen film layer is between 2 and 4, and the number of layers of the drug-loaded cellulose film layer is between 1 and 3, and one layer of drug-loaded cellulose film layer is correspondingly arranged between two collagen film layers.

[0053] In one embodiment, the product is an injectable filler, comprising: dissolving the collagen solution of the present invention with a mass fraction of 1.0% to 5.0% in water for injection, dissolving the medical sodium hyaluronate solution with a mass fraction of 0.5% to 2.0% in water for injection, mixing them in a certain proportion, adding a certain amount of medical-grade hydroxyapatite, mixing and stirring, adding an appropriate amount of medical-grade glycerin during the stirring process, mixing evenly, and vacuum degassing to obtain the finished product.

[0054] In one embodiment, the product is an artificial bone scaffold material, which is prepared by preparing the collagen of the present invention into a collagen solution of a certain concentration, then adjusting the pH to 6.5-7.5, adding glutaminase and nano-hydroxyapatite, transferring the solution to a mold to react for a period of time, cooling, inactivating the enzyme, and freeze-drying to obtain the artificial bone scaffold material.

[0055] In one embodiment, the product contains the above-mentioned protein single chain or the above-mentioned collagen or the above-mentioned collagen fiber.

[0056] In one embodiment, the product further contains one or more of vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenols, and natural plant extracts.

[0057] The seventh object of the present invention is to provide an application in the preparation of collagen-containing products in the fields of biology, chemical industry, food, medicine, biomaterials, tissue engineering or cosmetics, wherein the application includes utilizing the amino acid sequence encoding the collagen domain, protein single chain, collagen, collagen fiber of the present invention, or the nucleotide sequence encoding the collagen domain, the nucleotide sequence encoding the protein single chain for expressing collagen, the gene encoding the protein single chain for expressing collagen, or the plasmid or cell expressing the gene.

[0058] The present invention also provides a method for expressing a collagen having a triple helical structure, wherein the method comprises constructing a collagen using a collagen domain of the present invention having a specific amino acid sequence; wherein the amino acid sequence encoding the collagen domain has:

[0059] (1) an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7; or

[0060] (2) an amino acid sequence obtained by combining two sequences as shown in any one of SEQ ID NOs. 1 to 3; or

[0061] (3) An amino acid sequence obtained by repeating 2 to 3 times the sequence shown in any one of SEQ ID NOs. 1 to 7.

[0062] In one embodiment, the method comprises: constructing a single protein chain for expressing collagen, and expressing it in a host to obtain a recombinant collagen with a triple helical structure; wherein the structure of the single protein chain for expressing collagen comprises, from N-terminus to C-terminus, the following sequence: a folding domain, a restriction enzyme cleavage site, {repeating sequence module, collagen domain}m, and a repeating sequence module; wherein m is greater than or equal to 1. Optionally, m is 1 or 2.

[0063] In one embodiment, the method further comprises: performing enzymatic digestion to remove the folding domain of the recombinant collagen to obtain collagen that still maintains a triple helical structure.

[0064] The present invention also provides a method for improving the stability, solubility or yield of collagen, comprising connecting a first repeating sequence module to the N-terminus of a collagen domain and a second repeating sequence module to the C-terminus, constructing a single collagen chain having a structure of the first repeating sequence module-collagen domain-second repeating sequence module, and expressing the first repeating sequence module-collagen domain-second repeating sequence module through a host cell;

[0065] At least one of the first and second repeating sequence modules has an amino acid sequence as shown in SEQ ID NO. 27 (abbreviated as KD2) or SEQ ID NO. 28 (abbreviated as KD3), and the other has an amino acid sequence as shown in SEQ ID NO. 23, i.e. (GPP)10 (abbreviated as P10).

[0066] In one embodiment, the amino acid sequence of the collagen domain has:

[0067] (1) an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7; or

[0068] (2) an amino acid sequence obtained by combining two sequences as shown in any one of SEQ ID NOs. 1 to 3; or

[0069] (3) An amino acid sequence obtained by repeating 2 to 3 times the sequence shown in any one of SEQ ID NOs. 1 to 7.

[0070] The recombinant human collagen provided by the present invention can fold into a triple helical structure and can be controlled to self-assemble into a regular high-order biomimetic fiber structure. The present invention predicts the thermal stability of natural type I, II, and III collagen and selects high / low predicted T m The sequence of the value is used to form the collagen domain. Optionally, different types of designed collagen sequences are introduced into the collagen domain with the structure shown in Figure 10 or Figure 11; the introduction of the folding domain (such as the V-domain of SEQ ID NO.13 or the coiled-coil domain of SEQ ID NO.14) assists the collagen in folding to form a triple helical structure; the repeated sequence module (such as (GPP) n ) can assist the folding of collagen triple helix and improve its thermal stability; through (GPP) n By adjusting the number of molecules, the molecules can further assemble to form a fiber structure, which exhibits the morphological characteristics of bright stripes under negative staining in TEM.

[0071] The present invention also conducted a thermal stability analysis on the obtained recombinant collagen, performed TEM characterization, and determined the high thermal stability fragments in the design of type I, II, and III collagen. m Value and prediction T m The values ​​of the two fragments (38-39°C) deviate, but they can all fold correctly to form a triple helical structure, while the fragments with low thermal stability are predicted to be unable to fold to form a triple helical structure. Beneficial effects

[0072] 1. The collagen domain of the present invention is obtained by sequence splicing and designing collagen fragments of natural human type I, II, and III collagen, and has high homology with natural human collagen; among them, the fragments directly cut from natural human collagen have 100% homology with the natural sequence, and the collagen domain sequence obtained by splicing has more than 57% homology with the natural sequence.

[0073] 2. Based on the prediction of the thermal stability of human collagen sequences, the present invention conducted sequence screening and design, and successfully achieved heterologous expression of different types of highly thermally stable human collagen fragments in Escherichia coli.

[0074] 3. The source collagen sequence of the present invention, predicted T m The thermal transition temperature T of the collagen domain was determined by circular dichroism spectroscopy. m The value is also closer to human body temperature.

[0075] 4. This invention utilizes sequences with high homology to human collagen to achieve expression in Escherichia coli of human type I collagen with a triple helical structure that self-assembles into regular, high-order biomimetic fibers, resolving the current difficulties in expressing recombinant human collagen. Furthermore, the resulting human type I collagen self-assembles into fibers with periodic light and dark stripes, similar to the morphological characteristics of type I collagen. This approach addresses the demand for structurally functional recombinant collagen in the biomedical and tissue engineering fields.

[0076] 5. This invention incorporates integrin binding sites into a designed, highly stable human type I collagen sequence, enabling it to fold into a stable triple helical structure and self-assemble into a fibrous morphology. This invention enables the application of recombinant collagen in tissue culture and dental tissue repair, and also provides a reference for the introduction of other functional motifs into collagen sequences.

[0077] 6. The present invention is to repeat sequence (GPP) 10 Through transformation, we obtained collagen composed of multiple different repeating sequences, which is composed of (GPP) 10 Compared with the collagen composed of proteins (V-P10BP10), the yield of the modified collagen (V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BKD3) was increased by more than 23.4%, the heat resistance temperature was increased by 2°C, the solubility was improved, and good salt tolerance was maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is a schematic diagram of the axial and lateral side chain interactions in the triple helix structure of collagen.

[0079] FIG2 is a graph showing the relative stability of type I collagen.

[0080] FIG3 is a schematic diagram of the sequence design of collagen.

[0081] Figure 4 is an SDS-PAGE identification diagram after collagen purification; lanes 1-7 are respectively purified V-HC1-1, V-HC1-2, V-HC1-3, V-HC1-12, V-HC1-22, V-HC1F and V-HC1E, and arrows represent target bands; M: protein marker.

[0082] Figure 5 is an SDS-PAGE identification diagram after collagen purification; lanes 1-2 and 4-6 are the purified V-HC2A, V-HC2B, V-HC3A, V-HC3B and V-HC3C, respectively, and arrows represent the target bands; M: protein marker.

[0083] FIG6 is a circular dichroism spectrum identification diagram of the designed type I collagen; (a) is the full wavelength spectrum; (b) is the thermal curve.

[0084] Figure 7 is the circular dichroism spectrum identification diagram of the designed type II and III collagen; (a) is the full wavelength spectrum and thermal curve of type II collagen; (b) is the full wavelength spectrum and thermal curve of type III collagen.

[0085] Figure 8 shows the fiber morphology of the designed collagen self-assembled; (a)-(b) are the TEM characterization and bandwidth statistics of bright and dark stripes of HC1-1, HC1-2 and HC1-3.

[0086] Figure 9 shows the fiber morphology of the designed collagen self-assembled; (c)-(d) are the TEM characterization and bandwidth statistics of bright and dark stripes of HC1-12 and HC1-22.

[0087] FIG10 is a single protein chain for expressing collagen, the structure of which is folded domain-repeating sequence-collagen domain-repeating sequence.

[0088] FIG11 is a single protein chain for expressing collagen, the structure of which is folded domain-repeating sequence-collagen domain-repeating sequence-collagen domain-repeating sequence.

[0089] Figure 12 is an SDS-PAGE image of collagen with V-domain after trypsin digestion. In the figure, 1 to 5 correspond to sequences P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3, respectively. Arrows represent target bands; M: protein marker.

[0090] FIG13 is a graph showing the production of collagen with different sequences.

[0091] FIG14 is a full-wavelength spectrum, thermal curve, and first-order derivative of the thermal curve of collagen.

[0092] FIG15 shows the full wavelength spectrum, thermal shift curve and first derivative of the thermal shift curve of collagen at 0 mM, 10 mM and 100 mM, wherein A to D represent KD3BP10, P10BKD2, KD2BKD2 and P10BKD3, respectively.

[0093] FIG16 shows the preparation of different collagen samples with a concentration of 2 mmol / L, dissolved in 10 mM sodium phosphate buffer, and placed at 4° C. for 1 day and then photographed and recorded. DETAILED DESCRIPTION

[0094] Culture medium:

[0095] LB medium (g / L): tryptone 10, yeast powder 5, NaCl 10, agar powder 15 (solid);

[0096] TB medium (g / L): tryptone 12, yeast powder 24, glycerol 4 mL, KH2PO4 2.31, K2HPO4 12.54;

[0097] Culture Method: Pipette 50 μL of the bacterial suspension from the glycerol tube containing the target gene into 20 mL of LB (Amp-resistant) and culture overnight at 37°C, 200 rpm. Transfer 1% of the suspension to 100 mL of TB (Amp-resistant) fermentation broth and culture at 37°C, 200 rpm, for 24 hours. Add IPTG to a final concentration of 1 mmol / L and incubate at 25°C, 200 rpm, for 10 hours. Then transfer the suspension to 15°C for 14 hours.

[0098] Protein purification method: After the fermentation is completed, the cells are collected, crushed and centrifuged, and the supernatant is collected and filtered through a 0.45 μm water filter membrane. TM For HP 5mL affinity purification, equilibrate with 5 column volumes of binding buffer A (20 mmol / L Na2HPO4, 20 mmol / L NaH2PO4, 500 mmol / L NaCl, 10 mmol / L imiazole, pH 7.4) before loading the sample at a flow rate of 5 mL / min. After loading, gradient elution with elution buffer B (20 mmol / L Na2HPO4, 20 mmol / L NaH2PO4, 500 mmol / L NaCl, 500 mmol / L imiazole, pH 7.4) was performed to obtain the target protein, and the purification was analyzed by SDS-PAGE.

[0099] Trypsin removal salt: Purified collagen was dissolved in water to a concentration of 4 mg / mL. 200 μL of each sample was added with 2.5 g / L of trypsin at molar ratios of 20:1, 200:1, and 2000:1. Digestion was performed in a 16°C waterbath, with samples taken every 3 hours. Finally, the digestion was continued in an incubator for 12 hours. Purity was verified by SDS-PAGE analysis. After digestion under optimal conditions, desalting was performed using HiTrap Desalting. Peak samples were collected and freeze-dried under vacuum.

[0100] Sample stability identification: The desalted samples were vacuum freeze-dried and circular dichroism was used to identify the full wavelength and thermal stability. The specific steps are: the freeze-dried sample was dissolved in 10mmol / L, pH 7.0 sodium phosphate buffer to a 1mg / mL solution, and then equilibrated at 4°C for 48h before circular dichroism identification. The full wavelength is the CD spectrum of 190-250nm measured at 4°C with an interval of 1nm, and the average scanning time is 5s. The thermal curve is obtained by monitoring the CD signal at 225nm, increasing the temperature from 4°C to 70°C at a rate of 10°C / h, equilibrating at each temperature for 8s, and the melting temperature (T m ) is obtained by taking the median of the absorbance values ​​corresponding to the fitted thermal curve at 4°C and 70°C. This data represents the stability of the sample.

[0101] Transmission electron microscopy characterization: The lyophilized collagen sample was dissolved in 10 mmol / L sodium phosphate buffer (pH 7.0) to a final concentration of 0.5 mmol / L. The solution was then self-assembled at 4°C for 4 days. A 5 μL drop of the assembled sample was placed on a copper grid and adsorbed for 30 seconds. Excess liquid was removed with filter paper. Then, 5 μL of 0.75% phosphotungstic acid was added for negative staining. The stain was then aspirated and the sample was air-dried. The sample was observed and imaged on a Hitachi H-7650 transmission electron microscope at 80 kV. At least five TEM images with clear fields of view were selected, and the bandwidths of the bright and dark fringes were measured using ImageJ. At least 200 measurements were taken for each sample and averaged.

[0102] Thermal stability analysis method:

[0103] The steps include:

[0104] (1) expressing a gene encoding a protein having a structure as shown in FIG10 or a structure as shown in FIG11 in E. coli BL21 (DE3);

[0105] (2) Purify the intracellular expressed product to obtain the purified protein, and perform SDS-PAGE identification;

[0106] (3) The purified sample was digested with trypsin, and after SDS-PAGE confirmation of complete removal of the V-domain, it was desalted and lyophilized;

[0107] (4) The lyophilized collagen sample was prepared into a solution with a final concentration of 1 mg / mL using 10 mmol / L sodium phosphate buffer, and then balanced at 4°C for 48 h. The identification was performed by full wavelength circular dichroism and thermal temperature scanning.

[0108] (5) The lyophilized type I collagen sample was prepared into a collagen solution with a final concentration of 0.5 mmol / L using 10 mmol / L sodium phosphate buffer, and the solution was equilibrated at 4°C for 4 days before TEM characterization.

[0109] Example 1: Design of collagen domain sequences

[0110] The full-length sequence of natural human collagen is subjected to protein computational analysis and thermal stability prediction to obtain a sequence fragment with high thermal stability. The fragment is directly intercepted or further spliced ​​to obtain the collagen domain sequence. The target collagen domain sequence is obtained and used as the thermal stability prediction value T of the collagen triple helix structure of the collagen domain. m The value is between 38 and 39 degrees Celsius.

[0111] Among them, the thermal stability prediction value (T m The prediction method of the collagen domain sequence is as follows: taking the first triplet unit (XYG) of the triple helix structure as the starting point of the consecutive numbering, the average relative stability of each XYG triplet is calculated to obtain the thermal stability value of each triplet; then, n consecutive triplets are taken and the average thermal stability value of these n consecutive triplets is calculated, which is the thermal stability prediction value of the collagen domain sequence. The target collagen domain sequence of the present invention ensures that the thermal stability prediction value T of the collagen domain sequence is as large as possible when n is as large as possible. m The value is between 38 and 39 degrees Celsius.

[0112] The thermal stability value of a single triplet i refers to the thermal stability value of a window consisting of 10 consecutive triplets in the interval [i-5, i+5].

[0113] The thermal stability value T of the window windows By the window main chain tendency value T bb and window side chain interaction value T side Decision, among others,

[0114] The T bb Obtained as follows:

[0115] (1) Based on the host-guest system, the most stable triplet Pro-Hyp-Gly was used as the host, and only 19 non-Pro residues were single-point mutated to the Pro at position X to construct the guest. The thermal stability value T of each guest was measured. m , which is the main chain tendency value at different X positions; similarly, objects were constructed by single-point mutation of 20 natural amino acids of Hyp at position Y in the Pro-Hyp-Gly triplet, and the thermal stability value T of each object was measured. m , which is the main chain tendency value at different Y positions;

[0116] (2) The calculation of the main chain tendency value of any triplet XYG is based on the types of residues at positions X and Y in the triplet, and the main chain tendency value of position X and position Y in (1) are found. The main chain tendency value T of the corresponding position X is used to calculate the main chain tendency value of the triplet XYG. X and the main chain tendency value T at position Y Y Add them together to get T X +T Y For example, the main chain tendency value of Ala-Ala-Gly triplet is T X +T Y , T X (X=Ala) represents the T of Ala-Hyp-Gly determination m Value, T Y (Y=Ala) represents the T of Pro-Ala-Gly determination m value;

[0117] (3) Window main chain tendency value T bb , is the calculation method of the main chain tendency value of any triplet XYG in (2), and the main chain tendency values ​​of all triplets in the window are summed up; where the window includes 3 chains, each chain has 10 triplets (i.e., 60 triplets), that is, T bb =∑(T X +T Y ).

[0118] The T side Produced by all side chain interactions within the window, T side =∑ΔT Lat +∑ΔT Axi .

[0119] Where, ΔT Axi represents the axial interaction value between two chains of adjacent triplets, ΔT Lat represents the lateral interaction value between adjacent triplets on two chains.

[0120] The triple helix fold constrains interactions between adjacent chains to two geometric structures: axial and lateral (Figure 1). Axial interactions occur when the Y- and X-positions of two adjacent chains interact parallel to the helical axis, while lateral interactions occur when the Y- and X-positions of two adjacent chains interact perpendicular to the helical axis.

[0121] ΔT Axi and ΔT Lat , which is determined and calculated by double mutation experiments, represents the difference between the thermal stability of the double mutation at position Y and position X and the sum of the stability of the single mutation at position Y or position X in the axial or transverse geometry, respectively. The formula is as follows: ΔT = T YX -(T YP +T OX )-T OP ;

[0122] Among them, T OP The experimentally measured T when the Y position is Hyp and the X position is Pro m Value; T OX represents the experimentally measured T when the single point mutation X position is present and Y position is still Hyp m Value; T YP represents the experimentally measured T when the single point mutation Y position is present and the X position is still Pro. m Value; T YX Indicates that when the Y and X positions are double mutated, that is, the Y position is not Hyp and the X position is not Pro, the experimentally measured T m value.

[0123] For example, calculate the lateral action value (ΔT Lat ), the thermal stability value of the double mutation was T YX (Y=Lys, X=Asp), and the corresponding X-position single point mutation determination T m The value is T OX (X=Asp,Y=Hyp), the Tm value of single point mutation at position Y is T YP (Y=Lys,X=Pro) Main thermal stability value T OP In the lateral interaction, position Y can be mutated into 20 other natural amino acids, and position X can be mutated into 19 other natural amino acids. There are 20×19=380 combinations of different Y and X positions, corresponding to 380 lateral interaction values ​​(ΔT Lat ). Axial interaction (ΔT Axi ) was measured using a similar method, and 380 axial interaction values ​​were also obtained. For details, please refer to the master's thesis of the inventor team Liu Han "The Effect of Amino Acid Components on the Thermal Stability of Collagen-like Polypeptides".

[0124] The window unit contains three chains (chains a, b, and c), arranged with a one-residue offset, and each chain has 10 triplets (as shown in Figure 1). Chains a and b, and between chain b and c, each contain 10 lateral and 9 axial interaction pairs; chain c and chain a contain 9 lateral and 8 axial interaction pairs. Therefore, in the 10-triplet window, there are 29 lateral interaction values ​​and 26 axial interaction pairs between the three chains, which are summed to obtain ∑ΔT. Lat and ∑ΔT Axi The sum of the contributions of all axial and lateral side chain interaction pairs within the window is T side .

[0125] The above method involves experimentally determining T m Thermal stability was determined using circular dichroism spectroscopy. Specifically, lyophilized pure host or guest collagen peptide powder was weighed and dissolved in 10 mM phosphate buffer (pH 7.0) to prepare a high-concentration (1 mM) stock solution. The host and guest peptide stock solutions were further diluted to a final concentration of 0.2 mM and mixed in a 1:1:1 ratio of chain a, chain b, and chain c. The mixture was heated at 80°C for 10 minutes to unfold the folded triple helix into a single-stranded disordered state. The mixture was then incubated at 4°C for at least 24 hours to allow for full self-assembly into a well-folded collagen triple helix. Circular dichroism (CD) spectroscopy was performed on a Chirascan instrument (Applied Photophysics Ltd, England). Wavelength scans were performed at 4°C from 190 nm to 260 nm with a step size of 1 nm. Thermal shift experiments were performed at 225 nm, with a gradient heating rate of 1°C / 6 min from 4°C to 80°C. T was calculated by fitting the first-order derivative of the thermal shift curve. m value.

[0126] Figure 2 shows the relative stability profile of type I collagen, obtained by calculating the thermal stability values ​​of each triplet in the native human type I collagen sequence and plotting the average relative stability curve according to the above method. Consecutive triplets with high thermal stability values ​​can be intercepted from Figure 2 to obtain sequence fragments with higher thermal stability, or these intercepted sequences with higher thermal stability can be further spliced ​​to obtain collagen domain sequences.

[0127] Referring to the above method, sequence fragments with higher thermal stability were extracted from natural human type I collagen α1 chain (NCBI accession number NP_000079.2), human type II collagen α1 chain (NCBI accession number NP_001835.3), and human type III collagen α1 chain (NCBI accession number NP_000081.2), or the extracted sequences with higher thermal stability were further spliced ​​to obtain collagen domain sequences. m The collagen domain sequence with a T value of 38-39°C and a high triple helix tendency was used as the target sequence. m Sequences with lower values ​​and low triple helix propensity were used as controls.

[0128] Among them, different types of collagen predict T m Several sequences with values ​​between 38 and 39°C are as follows:

[0129] (1) Amino acid sequences shown in SEQ ID NOs. 1 to 7; (wherein, SEQ ID NOs. 1 to 3 are fragments or multiple fragments selected from natural type I human collagen, named HC1-1, HC1-2, and HC1-3 of type I collagen, and predicted T m 38.4℃, 38.5℃, 38.2℃ respectively; SEQ ID NO.4 is obtained by splicing a fragment or multiple fragments cut from natural type II human collagen, named HC2A of type II collagen, and predicted T m 38.3℃; SEQ ID NO.5~7 are fragments or multiple fragments obtained by splicing from natural type III human collagen, named HC3A, HC3B, HC3C of type III collagen, predicted T m 38.8℃, 38.8℃, 39.0℃ respectively)

[0130] (2) The amino acid sequence obtained by combining any two sequences of SEQ ID NO.1 to 3, such as SEQ ID NO.8 obtained by combining SEQ ID NO.1 and SEQ ID NO.2 (named HC1-12, predicted T m is 38.4℃);

[0131] (3) The amino acid sequence obtained by repeating 2 to 3 times any of the sequences shown in SEQ ID NO.1 to 7, for example, SEQ ID NO.9 obtained by repeating 2 times (named HC1-22, predicted T m is 38.4℃).

[0132] Predicted T mSeveral sequences with lower values ​​(36-37°C) are as follows: SEQ ID NO.10-12 (named HC1E, HC1F, HC2B, predicted T m 37.1℃, 36.3℃, and 36.5℃ respectively).

[0133] As shown in Table 1, in SEQ ID NO. 1-12, all directly selected sequence fragments have not been sequence modified (100% homology with natural human collagen sequence), and all spliced ​​collagen domain sequences have a homology greater than 57% with natural human collagen sequence.

[0134] Table 1

[0135] Example 2: Design of collagen sequence

[0136] A protein single chain containing the collagen domain of Example 1 was designed, wherein the structure of the protein single chain includes: a folded domain, a repeat sequence module, and a collagen domain.

[0137] The introduction of the folding domain assists collagen in folding to form a triple helical structure. Optionally, the folding domain is a V-domain or a coiled-coil domain; optionally, the amino acid sequence of the V-domain is as shown in SEQ ID NO. 13; optionally, the amino acid sequence of the coiled-coil domain is as shown in SEQ ID NO. 14.

[0138] The introduction of the repeat sequence module can assist the folding of the collagen triple helix and improve its thermal stability. Optionally, there are multiple repeat sequence modules, located at both ends of the collagen domain or at both ends of multiple collagen domains; for example, when expressing type II collagen, there can be multiple collagen domains, and the multiple collagen domains are connected by repeat sequence modules. Optionally, the sequences of the repeat sequence modules can be the same or different. Optionally, the repeat sequence module adopts (GPP) n Optionally, when multiple repeat sequence modules are included, each repeat sequence module (GPP) n The value of n can be the same or different; considering that proline at the end of the sequence may be detrimental to protein expression, an additional glycine is added to the end of the collagen amino acid sequence.

[0139] As an example, this embodiment designs the amino acid sequence of a single-chain collagen protein structure as shown in Figure 10. The amino acid sequence of the V-domain is shown in SEQ ID NO. 13, and the collagen domain sequence uses SEQ ID NOs. 1 to 12 of Example 1.

[0140] As one example, as shown in FIG3 , for a sequence derived from type I collagen, a repeating sequence module (Gly-Pro-Pro) is inserted at both ends of the sequences HC1-1, HC1-2, HC1-3, HC1-12, HC1-22, HC1E, and HC1F in Example 1. 10 Short peptide, abbreviated as (GPP) 10 (SEQ ID NO. 23), and after inserting the folded V-domain at the N-terminus, the sequences were named V-HC1-1, V-HC1-2, V-HC1-3, V-HC1-12, V-HC1-22, V-HC1E, and V-HC1F, respectively. The HC1-12 sequence is a spliced ​​combination of the HC1-1 and HC1-2 sequences, and the HC1-22 sequence is a spliced ​​combination of two HC1-2 sequences. The amino acid sequence of V-HC1-1 is shown in SEQ ID NO. 15, and the nucleotide sequence encoding V-HC1-1 is shown in SEQ ID NO. 16. The amino acid sequences of V-HC1-2, V-HC1-3, V-HC-12, V-HC1-22, V-HC1E, and V-HC1F were obtained by replacing the corresponding collagen domain sequences in Example 1 based on the amino acid sequence of V-HC1-1.

[0141] For sequences derived from type II and III collagen, and considering the morphology of native collagen, short peptides (Gly-Pro-Pro)5, (Gly-Pro-Pro)4, and (Gly-Pro-Pro)6 were inserted into the N-terminus, middle, and C-terminus of the collagen fragments in the sequences HC2A, HC2B, HC3A, HC3B, and HC3C, abbreviated as (GPP)5 (SEQ ID NO. 24), (GPP)4 (SEQ ID NO. 25), and (GPP)6 (SEQ ID NO. 26). The sequences were named V-HC2A, V-HC2B, V-HC3A, V-HC3B, and V-HC3C, respectively. The sequence design is shown in Figure 3. The amino acid sequence of V-HC2A is shown in SEQ ID NO. 17, and the nucleotide sequence encoding V-HC2A is shown in SEQ ID NO. 18. The amino acid sequence of V-HC2B was obtained by replacing the corresponding collagen domain sequence in Example 1 based on the amino acid sequence of V-HC2A. The amino acid sequence of V-HC3A is shown in SEQ ID NO. 19, and the nucleotide sequence encoding V-HC3A is shown in SEQ ID NO. 20. The amino acid sequences of V-HC3B and V-HC3C are obtained by replacing the corresponding collagen domain sequences in Example 1 based on the amino acid sequence of V-HC3A.

[0142] Example 3: Construction of recombinant plasmids and recombinant bacteria

[0143] When synthesizing the nucleotide sequence of a single protein chain (such as the single protein chain of Example 2), the base GC is introduced at the 5' flanking end, and Nco I and Bam HI restriction sites are introduced at the 5' and 3' ends, respectively. Subsequently, the synthesized genes are respectively inserted between Nco I and Bam HI of the pColdIII-M plasmid to obtain the corresponding recombinant collagen plasmid. The pColdIII-M plasmid is a plasmid in which the Nde I restriction site on the pColdIII plasmid is mutated to an Nco I restriction site. The correctly sequenced recombinant plasmids are respectively transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing ampicillin, cultured and screened, and glycerol-protected to obtain recombinant bacteria containing recombinant collagen.

[0144] Example 4: Expression, purification and enzyme cleavage optimization of collagen sequences

[0145] The recombinant bacteria obtained in Example 3 were cultured in shake flasks, the cells were collected, crushed and centrifuged, and the supernatant was collected and filtered using His Trap. TM HP 5mL was used for affinity purification, and samples at imidazole concentrations of 175mmol / L and 400mmol / L were collected. The SDS-PAGE identification of the samples is shown in Figures 4 and 5. The theoretical molecular weights of V-HC1-1, V-HC1-2, V-HC1-3, V-HC1-12, V-HC1-22, V-HC1E and V-HC1F are 25.13kDa, 24.81kDa, 28.15kDa, 34.38kDa, 34.33kDa, 25.15kDa and 26.09kDa, respectively. Their apparent molecular weights on SDS-PAGE are around 36kDa, 35kDa, 40kDa, 48kDa, 48kDa, 37kDa and 38kDa, respectively (as shown in Figure 4); V-HC2A, V-HC2B, V-HC3A The theoretical molecular weights of V-HC3B, V-HC3C are 34.32 kDa, 37.15 kDa, 34.70 kDa, 34.28 kDa and 32.61 kDa, respectively. Their apparent molecular weights on SDS-PAGE are approximately 37 kDa, 44 kDa, 43 kDa, 43 kDa and 38 kDa, respectively (as shown in Figure 5), which are approximately 1.4 times the theoretical molecular weight. This may be due to the presence of more proline in the collagen sequence, resulting in the target protein migrating slower on SDS-PAGE than proteins of the same molecular weight, which is consistent with literature reports.

[0146] Removal of the folding domain is a prerequisite for the self-assembly of collagen molecules through a transverse, head-to-tail staggered arrangement, ultimately promoting the formation of striated fibrils. Therefore, when designing the sequence, a trypsin cleavage site, LVPRGS, was introduced between the collagen domain and the folding domain. This allows the folding domain to be removed by digestion with an appropriate amount of trypsin, resulting in a pure collagen domain structure. Under the action of trypsin, the V-domain will be digested into multiple short peptides containing 2-20 amino acid residues. If the collagen domain is correctly folded into a rigid triple helical structure under the action of the V-domain, it will not be digested by trypsin in a short period of time.

[0147] V-HC1-2 was selected as a model protein for optimization of trypsin digestion conditions. Results showed that at a molar ratio of 20:1 and digestion time of 3 hours, the V-domain and contaminants were essentially completely digested, leaving only a single band with a molecular weight of approximately 25 kDa, corresponding to 1.4 times the molecular weight of the HC1-2 collagen domain. The band faded after 12 hours, likely due to prolonged digestion in a high-concentration trypsin solution, resulting in the cleavage of a small portion of the triple helix. At a molar ratio of 200:1, a slight band remained at 3 hours, but disappeared after 6 hours, indicating that the V-domain was essentially excised, with no significant signs of band fading within 12 hours. At a molar ratio of 2000:1, the V-domain was not completely excised after 9 hours of digestion, and the band gradually disappeared around 12 hours before digestion. Based on the digestion results, a molar ratio of 200:1 was selected for digestion, with a digestion time of 6-12 hours.

[0148] Example 5: SDS-PAGE analysis after collagenase cleavage

[0149] Under the optimal enzymatic digestion conditions of Example 4, five types of collagen were digested. The results showed that collagens V-HC1-1, V-HC1-2, V-HC1-3, and V-HC1-22 were all single bands after trypsin digestion, and the purity reached electrophoretic purity. The apparent molecular weight corresponded to 1.4 times the theoretical molecular weight after enzymatic digestion.

[0150] Example 6: Collagen forming a triple helical structure and circular dichroism characterization of the sequence

[0151] To confirm the secondary structure of the collagen domain, the enzymatically desalted lyophilized collagen sample from Example 5 was prepared into a 1 mg / mL solution in 10 mmol / L sodium phosphate buffer and equilibrated at 4°C for 48 hours. After equilibration, a full-wavelength scan was performed using circular dichroism spectroscopy.

[0152] For the design of type I human collagen, as shown in Figure 6(a), HC1-1, HC1-2, and HC1-3 all showed characteristic positive absorption peaks at 225nm, indicating that the three collagens were correctly folded to form a triple helical structure with the assistance of the V-domain. As shown in Figure 6, the low-prediction fragments HC1E and HC1F used as controls did not have characteristic positive absorption peaks at 225nm, indicating that they could not correctly fold to form a triple helical structure. Circular dichroism was further used to determine the thermal transition temperature of the collagen domain. The predicted T values ​​of HC1-1, HC1-2, and HC1-3 were 0. m 38.4℃, 38.5℃ and 38.2℃ respectively, as shown in Figure 6(b). The thermal curves from 4℃ to 70℃ at 225nm were detected by circular dichroism spectroscopy, and the thermal curves were fitted (see Table 1). The results showed that the T m 37.2℃, 38.7℃ and 32.4℃ respectively, while the predicted T values ​​of the low-prediction fragments HC1E and HC1F as controls were 0. m The thermal transitions of HC1E and HC1F were 37.1°C and 36.3°C, respectively. The thermal transition curves from 4°C to 70°C at 225 nm were detected by circular dichroism spectroscopy, as shown in Figure 6. No thermal transitions of HC1E and HC1F could be measured, indicating that HC1E and HC1F were not correctly folded to form a triple helical structure.

[0153] In addition, the collagen HC1-12 and HC1-22, which were formed by combining the thermally stable fragments 1 and 2, can also fold correctly to form a triple helical structure; among them, the predicted T m 38.4℃ and 38.4℃ respectively. The T values ​​of HC1-12 and HC1-22 were detected by circular dichroism. m The values ​​were 33.0°C and 33.6°C, respectively, indicating that the extension of the collagen domain leads to a certain degree of decreased thermal stability. Analysis suggests that the reason may be that the growth of the collagen sequence and the splicing of the two sequences prevent the V-domain from transmitting the force of triple helix folding from the N-terminus to the more distal C-terminus. As a result, the triple helix formed in some regions is less rigid, more loose, and unfolds more rapidly, resulting in decreased thermal stability.

[0154] For the design of type II and III human collagen, in order to confirm the secondary structure of the collagen domain, full wavelength scanning was performed using circular dichroism. As shown in Figure 7, HC2A, HC3A, HC3B and HC3C all showed characteristic positive absorption peaks at 225nm, indicating that the four collagens were correctly folded to form a triple helical structure with the assistance of the V-domain. However, the low-prediction fragment HC2B used as a control had no characteristic positive absorption peak at 225nm, indicating that it could not correctly fold to form a triple helical structure. Circular dichroism was further used to determine the thermal transition temperature of the collagen domain. The predicted T values ​​of HC2A, HC3A, HC3B and HC3C were 0. m 38.3℃, 38.8℃, 38.8℃ and 39.0℃ respectively, as the predicted T of HC2B with low stability prediction. m As shown in Figure 7, the thermal curves from 4°C to 70°C at 225nm were detected by circular dichroism spectroscopy, and the thermal curves were fitted (see Table 2). The T values ​​of HC2A, HC3A, HC3B, and HC3C were m The thermal transitions of the low-prediction fragment HC2B were 28.2°C, 25.1°C, 28.2°C and 30.3°C, respectively. The thermal transition of the low-prediction fragment HC2B could not be measured, indicating that HC2B was not correctly folded to form a triple helical structure.

[0155] The above results show that the prediction T m At 38-39℃, the high thermal stability collagen fragments can all fold correctly to form triple helical structures, and the predicted T m Fragments with low thermal stability below 38°C cannot fold correctly, indicating that by computationally predicting the thermal stability of human collagen, collagen fragments with different thermal stabilities can be effectively designed and heterologously expressed in Escherichia coli.

[0156] Table 2 Prediction and fitting T of human collagen m

[0157] Example 7: Collagen Fibers Formed by Collagen Polymer Self-Assembly (Characterization of Collagen Sequence Self-Assembly Morphology)

[0158] To observe whether collagen domains can self-assemble into higher-order structures in a high-concentration solution, the lyophilized type I collagen HC1-1, HC1-2, HC1-3, HC1-12, and HC1-22 of Example 1 were dissolved in 10 mmol / L sodium phosphate buffer to prepare a 0.5 mmol / L solution. After assembly at 4°C for 4 days, the solution was negatively stained and its morphological characteristics were characterized by TEM.

[0159] As shown in Figures 8 and 9, ribbon-like fibers with periodic light and dark stripes can be observed in the field of view, which are similar to the fiber morphology of natural type I collagen, indicating that the collagen domains of the designed collagen can self-assemble to form biomimetic microfiber structures. The literature reports that the length of each Gly-Pro-Pro triplet is 1.0nm, and the length of each XYGs triplet is 0.9nm. The length of the light and dark stripes was measured using ImageJ (see Table 3). The results showed that the lengths of the light stripes of HC1-1, HC1-2, HC1-3, HC1-12 and HC1-22 were approximately 10.6nm, 10.3nm, 11.7nm, 10.2nm and 9.9nm, respectively, which are consistent with (GPP) 10 The theoretical length of the repeating sequence module is 10 nm; the lengths of the dark stripes are approximately 32.2 nm, 32.3 nm, 42.8 nm, 63.8 nm and 64.5 nm, respectively, which are consistent with the theoretical values.

[0160] Furthermore, Figures 8 and 9 show that HC1-22 forms more ribbon-like fibers than HC1-12, indicating that HC1-22 self-assembles better than HC1-12. This may be due to the higher thermal stability of HC1-2 compared to HC1-1, which affects the assembly efficiency. Furthermore, the results also reveal that HC1-12 and HC1-22 form fewer ribbon-like fibers, and their self-assembly performance is inferior to that of the shorter HC1-1 and HC1-2, both in terms of fiber length and fiber aggregation morphology.

[0161] Table 3 Statistics of collagen fiber bandwidth

[0162] Example 8: Construction of collagen with different repeat sequences and performance testing

[0163] 1. Constructing collagen single chains with different repeating sequences

[0164] Based on Example 2, the amino acid sequences of the first repeat sequence module and the second repeat sequence module in the collagen single chain shown in Figure 10 were changed, and collagen domain HC1-2 (amino acid sequence as shown in SEQ ID NO.2), folding domain V-domain (amino acid sequence as shown in SEQ ID NO.13), and enzyme cleavage site LVPRGSP (amino acid sequence as shown in SEQ ID NO.21) were used as examples to construct collagens with different repeat sequences.

[0165] The specific structure is as follows (m=1):

[0166] (1) The first repeating sequence module was changed to KD3 (amino acid sequence shown in SEQ ID NO. 28), and the amino acid sequence of the second repeating sequence module was changed to P10 (amino acid sequence shown in SEQ ID NO. 23). The collagen structure was: folded domain, {KD3, HC1-2}m, P10, and the complete amino acid sequence was shown in SEQ ID NO. 29;

[0167] (2) The first repeating sequence module is changed to P10, and the amino acid sequence of the second repeating sequence module is changed to KD3. The collagen structure is: folding domain, {P10, HC1-2}m, KD3, and the complete amino acid sequence is shown in SEQ ID NO. 30;

[0168] (3) The first repeating sequence module is changed to P10, and the amino acid sequence of the second repeating sequence module is changed to KD2 (the amino acid sequence is shown in SEQ ID NO. 27). The collagen structure is: folded domain, {P10, HC1-2}m, KD2, and the complete amino acid sequence is shown in SEQ ID NO. 31;

[0169] (4) Changing the first repeating sequence module to KD2, the amino acid sequence of the second repeating sequence module to KD2, and the collagen structure thereof is: folding domain, {KD2, HC1-2}m, KD2, and the complete amino acid sequence is shown in SEQ ID NO. 33;

[0170] (5) The first repeating sequence module is changed to P10, and the amino acid sequence of the second repeating sequence module is P10. The collagen structure is: folding domain, {P10, HC1-2}m, P10, and the complete amino acid sequence is shown in SEQ ID NO.32.

[0171] Collagen proteins were prepared according to the method of Example 3 and named V-KD3BP10, V-P10BKD3, V-P10BKD2, V-KD2BKD2, and V-P10BP10 according to the amino acid sequences of the first and second repeating sequence modules, respectively, where "B" represents the collagen domain (the collagen domain in this example is HC1-2), "V" represents the folding domain V-domain, and "P10" represents (GPP). 10 The amino acid sequence is shown in SEQ ID NO.23, the amino acid sequence of "KD2" is shown in SEQ ID NO.27, and the amino acid sequence of "KD3" is shown in SEQ ID NO.28. The amino acid sequence of the prepared collagen is shown in SEQ ID NO.29 to SEQ ID NO.33.

[0172] The collagen was expressed, purified, and enzyme digestion optimized according to the method of Example 4 to prepare enzyme-digested collagen KD3BP10, P10BKD3, P10BKD2, KD2BKD2, and P10BP10. SDS-PAGE was used according to the method of Example 5 to verify the results, as shown in FIG12 .

[0173] The verified proteins were desalted, lyophilized, and weighed to obtain their yields, as shown in Figure 13. The yields of recombinant collagens V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3 were 55.02 mg / L, 83.25 mg / L, 68.02 mg / L, 73.51 mg / L, and 91.98 mg / L, respectively. The yields of the repetitive sequence-modified collagens V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3 increased by 23.6% to 67.18% compared to V-P10BP10, with V-P10BKD3 having the highest yield.

[0174] 2. Circular dichroism characterization of collagen and sequence structure

[0175] The secondary structures of the collagen domains of KD3BP10, P10BKD3, P10BKD2, KD2BKD2, and P10BP10 prepared above were determined. The collagen was lyophilized and prepared to a 1 mg / mL solution in 10 mmol / L sodium phosphate buffer. The solution was equilibrated at 4°C for 24 hours. After equilibration, full-wavelength circular dichroism spectroscopy was performed.

[0176] The results, as shown in Figure 14, show that P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 all exhibit characteristic positive absorption peaks at 225 nm, indicating that all six collagens correctly fold into triple helical structures with the assistance of the V-domain. Circular dichroism spectroscopy was used to monitor the thermal shift curves at 225 nm from 4°C to 80°C. The Tm values ​​of the collagens were determined by taking the first derivative of the thermal shift curves. The Tm value for P10BP10 was 35°C, while the Tm values ​​for the remaining sequences were all 37°C.

[0177] The above results show that the collagen sequences designed in the present invention can all be correctly folded to form a triple helical structure, and the Tm value is 2°C higher than that of the P10BP10 protein sequence. The designed sequences can all improve the thermal stability of proteins.

[0178] 3. Salt tolerance test of collagen with different repeat sequences

[0179] The collagen proteins KD3BP10, P10BKD3, P10BKD2, KD2BKD2, and P10BP10 prepared above were tested for their stability in high-concentration salt solutions. The collagen was lyophilized and dissolved in 10 mmol / L sodium phosphate buffer to prepare a 1 mg / mL solution. On this basis, 100 mmol and 200 mmol of sodium chloride were added to the solution. The results are shown in Figure 15. Circular dichroism spectroscopy was used to monitor the thermal curve from 4°C to 80°C at 225 nm. The Tm value of the collagen was obtained by taking the first-order derivative of the thermal curve. The results showed that the Tm value of each sequence was still 37°C in a high-salt environment. This shows that the modified collagen still has good salt tolerance.

[0180] 4. Solubility test of collagen with different repeat sequences

[0181] The solubility of the collagen proteins KD3BP10, P10BKD3, P10BKD2, KD2BKD2, and P10BP10 prepared above was tested. The collagen proteins were lyophilized and dissolved in 10 mmol / L sodium phosphate buffer to a sample concentration of 2 mmol / L. The solution was then incubated at 4°C for one day. As shown in Figure 16, P10BP10 precipitated, while the other proteins did not. This demonstrates that, at the same concentration, the modified collagen proteins exhibited reduced turbidity and improved molecular solubility compared to the original P10BP10.

[0182] 5. Performance testing of single-chain collagen prepared from collagen domains with different amino acid sequences

[0183] Based on 1 "Constructing collagen single chains with different repeat sequences", the collagen domain HC1-2 (amino acid sequence as shown in SEQ ID NO.2) was further changed to HC1-1 (amino acid sequence as shown in SEQ ID NO.1), HC1-3 (amino acid sequence as shown in SEQ ID NO.3), HC2A (amino acid sequence as shown in SEQ ID NO.4), HC3A (amino acid sequence as shown in SEQ ID NO.5), HC3B (amino acid sequence as shown in SEQ ID NO.6), HC3C (amino acid sequence as shown in SEQ ID NO.7), HC1-12 (amino acid sequence as shown in SEQ ID NO.8), and HC1-22 (amino acid sequence as shown in SEQ ID NO.9) to obtain multiple collagen single chains, which were expressed and purified to analyze the versatility of the repeat sequence modification method of the present invention for improving collagen production, heat resistance, and salt resistance.

[0184] The results showed that the yield of the above collagen single chain was increased by more than 15% compared with (GPP)10; the heat resistance temperature Tm of the purified collagen was increased by about 2°C or maintained at about 37-38°C, and it also had good salt tolerance.

[0185] Example 9: Products containing collagen

[0186] A product containing collagen can be a product in the fields of beauty, chemical industry, food and health products, medical / biomedical fields, cosmetics, and feed; for example, beauty cosmetics (facial masks, essences, creams, facial masks, etc.), artificial collagen casings, nutritional health products (collagen powder, oral liquid), medical dressings, hemostatic materials, artificial bone scaffolds, injectable fillers, artificial blood vessels, eye drops, drug sustained-release carriers, etc.

[0187] In the product containing collagen, the collagen has the collagen domain sequence of Example 1 of the present invention, or has the collagen sequence prepared in Example 2, or the enzymatically cleaved collagen obtained in Example 4, or the enzymatically cleaved collagen obtained in Example 8.

[0188] Furthermore, the collagen is a collagen expressing a triple helical structure.

[0189] Furthermore, the collagen is type I, type II or type III collagen.

[0190] Furthermore, for the above-mentioned collagen-containing product, other components, formulas, and preparation processes, those skilled in the art can adopt any existing method.

[0191] Optionally, the product is a dressing, comprising a collagen film layer containing the triple helix structure collagen of the present invention; optionally, further comprising a drug-loaded cellulose film layer; optionally, the number of layers of the collagen film layer is between 2 and 4, and the number of layers of the drug-loaded cellulose film layer is between 1 and 3, and one layer of drug-loaded cellulose film layer is correspondingly arranged between two collagen film layers.

[0192] Optionally, the product is an injectable filler, comprising: dissolving the collagen solution of the present invention with a mass fraction of 1.0% to 5.0% in water for injection, dissolving the medical sodium hyaluronate solution with a mass fraction of 0.5% to 2.0% in water for injection, mixing them in a certain proportion, then adding a certain amount of medical-grade hydroxyapatite, mixing and stirring, adding an appropriate amount of medical-grade glycerin during the stirring process, mixing evenly, and vacuum degassing to obtain the finished product.

[0193] Optionally, the product is an artificial bone scaffold material, which is prepared by preparing the collagen of the present invention into a collagen solution of a certain concentration, adjusting the pH to 6.5-7.5, adding glutaminase and nano-hydroxyapatite, transferring the solution to a mold to react for a period of time, cooling, inactivating the enzyme, and freeze-drying the solution to obtain the artificial bone scaffold material.

[0194] The present invention relates to the sequence:

[0195] SEQ ID NO.1: Amino acid sequence of HC1-1

[0196] SEQ ID NO.2: Amino acid sequence of HC1-2

[0197] SEQ ID NO.3: Amino acid sequence of HC1-3

[0198] SEQ ID NO.4: Amino acid sequence of HC2A

[0199] SEQ ID NO.5: Amino acid sequence of HC3A

[0200] SEQ ID NO.6: Amino acid sequence of HC3B

[0201] SEQ ID NO.7: Amino acid sequence of HC3C

[0202] SEQ ID NO.8: Amino acid sequence of HC1-12

[0203] SEQ ID NO.9: Amino acid sequence of HC1-22

[0204] SEQ ID NO.10: Amino acid sequence of HC1E

[0205] SEQ ID NO.11: Amino acid sequence of HC1F

[0206] SEQ ID NO.12: Amino acid sequence of HC2B

[0207] SEQ ID NO.13: Amino acid sequence of V-domain

[0208] SEQ ID NO.14: Amino acid sequence of coiled-coil domain

[0209] SEQ ID NO.15: Amino acid sequence of V-HC1-1

[0210] SEQ ID NO.16: Nucleotide sequence of V-HC1-1

[0211] SEQ ID NO.17: Amino acid sequence of V-HC2A

[0212] SEQ ID NO.18: Nucleotide sequence of V-HC2A

[0213] SEQ ID NO.19: Amino acid sequence of V-HC3A

[0214] SEQ ID NO.20: Nucleotide sequence of V-HC3A

[0215] SEQ ID NO.21: LVPRGSP

[0216] SEQ ID NO. 22: LVPRGS

[0217] SEQ ID NO.23: GPPGPPGPPGPPGPPGPPGPPGPPGPPGPP

[0218] SEQ ID NO.24: GPPPGPPGPPGPP

[0219] SEQ ID NO.25: GPPGPPGPPGPP

[0220] SEQ ID NO.26:GPPGPPGPPGPPGPPGPP

[0221] SEQ ID NO.27 (amino acid sequence of KD2): GPPGPPGPKGDPGPPGPPGPKGDPGPPGPP

[0222] SEQ ID NO.28 (amino acid sequence of KD3): GPPGPKGDPGPPGPKGDPGPPGPKGDPGPP SEQ ID NO.29 (amino acid sequence of V-KD3BP10):

[0223] SEQ ID NO.30 (amino acid sequence of V-P10BKD3):

[0224] SEQ ID NO.31 (amino acid sequence of V-P10BKD2):

[0225] SEQ ID NO.32 (amino acid sequence of V-P10BP10):

[0226] SEQ ID NO.33 (amino acid sequence of V-KD2BKD2):

[0227] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An amino acid sequence encoding a collagen domain, characterized in that The amino acid sequence has: (1) an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7; or (2) an amino acid sequence obtained by combining two sequences as shown in any one of SEQ ID NOs. 1 to 3; or (3) An amino acid sequence obtained by repeating 2 to 3 times a sequence as shown in any one of SEQ ID NOs. 1 to 7.

2. A single protein chain for expressing collagen, characterized in that: The protein single chain contains the amino acid sequence encoding the collagen domain of claim 1.

3. The protein single chain according to claim 2, characterized in that The structure of the protein single chain includes, from the N-terminus to the C-terminus: a folding domain, {a repeating sequence module, a collagen domain}m, a repeating sequence module, where m is an integer and m≥1; Optionally, m is 1 or 2.

4. The protein single chain according to claim 3, characterized in that There is also a histidine tag at the front end of the folding domain.

5. The protein single chain according to claim 3, characterized in that The folding domain can assist collagen in folding to form a triple helical structure; Optionally, the folded domain is a V-domain or a coiled-coil domain; Optionally, the folding domain is a V-domain, and its amino acid sequence is as shown in SEQ ID NO.13; Optionally, the folded domain is a coiled-coil domain, and its amino acid sequence is as shown in SEQ ID NO.

14.

6. The protein single chain according to claim 3, characterized in that There are multiple repeat sequence modules, which are located at both ends of the collagen domain or at both ends of multiple collagen domains; Optionally, the sequences of the repeat modules may be the same or different; Optionally, the repetitive sequence module adopts (GPP) n ; Optionally, (GPP) n The value of n satisfies 5<n≤30.

7. The protein single chain according to claim 2, characterized in that The structure of the protein single chain used to express collagen is: first repeating sequence module-collagen domain-second repeating sequence module; Wherein, the amino acid sequence of at least one of the first repeating sequence module or the second repeating sequence module is shown as SEQ ID NO.27 or SEQ ID NO.28, and the amino acid sequence of the other is shown as SEQ ID NO.23; Alternatively, the amino acid sequences of the first repeating sequence module and the second repeating sequence module are both as shown in SEQ ID Shown in NO.23; Optionally, the amino acid sequence of the first repeating sequence module of the collagen single chain is shown as SEQ ID NO.23, and the amino acid sequence of the second repeating sequence module is shown as SEQ ID NO.27; Optionally, the amino acid sequence of the first repeating sequence module of the collagen single chain is shown as SEQ ID NO.28, and the amino acid sequence of the second repeating sequence module is shown as SEQ ID NO.23; Optionally, the amino acid sequence of the first repeating sequence module of the collagen single chain is shown as SEQ ID NO.23, and the amino acid sequence of the second repeating sequence module is shown as SEQ ID NO.28; Optionally, the first repeating sequence module and the second repeating sequence module of the collagen single chain are both as shown in SEQ ID NO.27; Optionally, the first repeating sequence module and the second repeating sequence module of the collagen single chain are both as shown in SEQ ID NO.

23.

8. A nucleotide sequence encoding the collagen domain of claim 1, or a nucleotide sequence encoding a single-chain protein for expressing collagen as described in any one of claims 2-6, or a gene encoding a single-chain protein for expressing collagen as described in any one of claims 2-6, and a plasmid or cell expressing the gene.

9. A gene encoding the single-chain protein of claim 7, or a plasmid or cell carrying the gene encoding the single-chain protein of claim 7.

10. A collagen, characterized in that: The protein single chains described in any one of claims 2 to 6 are coiled around a common central axis to form a triple helical structure.

11. A collagen, characterized in that: Composed of the single protein chain described in claim 7.

12. Collagen fibers formed by the self-assembly of the collagen polymer according to claim 10 or 11.

13. A product containing triple helical collagen, characterized in that: The triple helical collagen has a collagen domain with the amino acid sequence of claim 1, or is formed by the protein single chain of any one of claims 2 to 6 coiled around a common central axis.

14. The product containing triple helix collagen according to claim 13, characterized in that: The products mentioned are those in the fields of beauty, chemical industry, food and health products, medical / biomedical field, cosmetics and feed, such as beauty cosmetics (facial masks, essences, creams, facial masks, etc.), artificial collagen casings, nutritional health products (collagen powder, oral liquid), medical dressings, hemostatic materials, artificial bone scaffolds, injectable fillers, artificial blood vessels, eye drops, drug sustained-release carriers, etc.

15. A collagen product, characterized in that: It has the protein single chain according to claim 7, the collagen according to claim 11, or the collagen fiber according to claim 12.

16. The collagen product according to claim 15, characterized in that: The product also contains one or more of vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenols, and natural plant extracts.

17. A method for expressing collagen having a triple helical structure, characterized in that: The method is to construct collagen with a specific collagen domain; wherein the amino acid sequence encoding the collagen domain has: (1) an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7; or (2) an amino acid sequence obtained by combining two sequences as shown in any one of SEQ ID NOs. 1 to 3; or (3) An amino acid sequence obtained by repeating 2 to 3 times a sequence as shown in any one of SEQ ID NOs. 1 to 7.

18. A method for improving the stability, solubility or yield of collagen, characterized in that: The first repeating sequence module is connected to the N-terminus of the collagen domain, and the second repeating sequence module is connected to the C-terminus, to construct a collagen single chain with a structure of the first repeating sequence module-collagen domain-second repeating sequence module, and expressed by a host cell; wherein the amino acid sequence of at least one of the first repeating sequence module or the second repeating sequence module is as shown in SEQ ID NO.27 or SEQ ID NO.28; The amino acid sequence of the collagen domain has: (1) an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7; or (2) an amino acid sequence obtained by combining two sequences as shown in any one of SEQ ID NOs. 1 to 3; or (3) An amino acid sequence obtained by repeating 2 to 3 times a sequence as shown in any one of SEQ ID NOs. 1 to 7.

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