Preparation method and application of recombinant humanized III-type collagen F3

Abstract

The invention provides a preparation method and application of recombinant humanized III-type collagen F3. The collagen peptide comprises n amino acid sequences as shown in SEQ ID NO. 1; or the collagen peptide comprises n amino acid sequences which have more than 95% of identity with the amino acid sequence as shown in SEQ ID NO.1, n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10. The invention also provides a preparation method and application of the collagen peptide. The collagen peptide has stable high expression, good hydrophilicity, stability, purity and biological activity, and can be further developed into collagen-containing products such as tissue engineering products, cosmetics, health care products, food or pharmaceutical compositions.

Description

Field of the Invention

[0001] The present invention belongs to the technical field of genetic engineering. Specifically, the present application provides a preparation method and use of recombinant humanized type III collagen F3. Background Art

[0002] Collagen is a general term for a class of biological polymer proteins. It is not only one of the main components of animal connective tissue, but also the most abundant and widely distributed functional protein in mammals, accounting for about 25% to 30% of the total protein content. At the tissue and organ level, collagen is an important structural protein in skin, bones, teeth, cornea, tendons and ligaments, which plays a role in supporting and protecting body tissues. At the cellular level, collagen is the main component of the extracellular matrix, which can promote cell adhesion, growth and differentiation, and maintain the normal function of cells or tissues and organs. So far, 29 types of collagen have been found in vertebrates. According to whether the spatial structure of collagen is fibrous, it can be divided into two categories: fibrous and non-fibrous. Type I and type III collagen are the two types of fibrous collagen with the highest content, with a total content of >90%, mainly present in the dermis; among them, type I collagen is thick and solid, with the function of supporting skin hardness and maintaining skin firmness, and type III collagen is small and has good elasticity, which has the function of maintaining skin elasticity and promoting scar healing. A decrease in type III collagen content can lead to skin aging. Supplementing type III collagen through external application and implantation can alleviate skin aging problems. Therefore, type III collagen is widely used in cosmetics, medical materials, medical beauty industry and other fields.

[0003] Natural type III collagen has a triple helix structure in terms of spatial structure. The triple helix region (also called the collagen region) has regular characteristics in the primary structure, that is, Gly-XY (X and Y represent any amino acid residue other than Gly, usually proline and hydroxyproline) is repeated in the amino acid residues of the α1 peptide chain. Under the action of the N-terminal and C-terminal domains of the collagen region, the three collagen peptide chains form a stable triple helix structure in a right-handed superhelical manner with the same axis as the center. At this time, the N-terminal and C-terminal domains are removed by proteases. In a higher-level spatial structure, the triple helix collagen will form fibrils through intermolecular cross-linking, and the fibrils will further form collagen fibers to exert biological functions.

[0004] There are currently two main methods for obtaining collagen. The first method uses animal tissues or organs to extract collagen, but animal-derived collagen has the following risks in the industrialization process: 1) The final product is complex in composition and the structure of the active ingredient is unclear; 2) Non-human protein is immunogenic and may cause allergic or immune rejection reactions; 3) There may be a risk of animal-derived virus infection. The second method uses genetic engineering technology to express and purify recombinant collagen through biological fermentation. It has the advantages of a single final product component, few impurities, no animal viruses, and no immune risks. Therefore, the use of genetic engineering combined with fermentation engineering to recombinantly express collagen has become the mainstream trend for obtaining collagen. Due to the complex structure and large molecular weight of natural collagen, as well as the inherent technical limitations of the microbial fermentation expression system, it is difficult to express the full length of collagen. The current research mainly focuses on expressing a certain segment of collagen, expressing multiple segments spliced ​​together, or expressing multiple repetitions of a single small segment. However, how to select functional sequence segments from the collagen sequence is a key and difficult point affecting the basic research and industrialization of collagen.

[0005] Therefore, there is an urgent need in this field to develop collagen peptides with high expression, good water solubility and better activity. Summary of the invention

[0006] The purpose of the present invention is to provide a collagen peptide with high expression, good water solubility and better activity.

[0007] In view of the shortcomings of the above-mentioned prior art, the applicant used bioinformatics to analyze the collagen functional domains present in the mature segment of type III collagen, and predicted a total of 11. Subsequently, the sequence of the collagen functional domain was obtained, and it was repeated many times. After codon optimization and gene synthesis, the expression was verified in Escherichia coli, and a high-expression collagen was screened. After fermentation expression, enzyme tag cleavage and affinity purification, a high-purity recombinant humanized collagen with no exogenous amino acid residues was obtained, and its sequence was 100% consistent with the human collagen sequence. Further experiments showed that the recombinant humanized type III collagen of the present invention has the characteristics of strong stability and not easy to degrade. Cell activity detection shows that the recombinant humanized type III collagen of the present invention has obvious activity in promoting cell adhesion.

[0008] In a first aspect of the present invention, the present application provides a collagen protein, wherein the collagen protein comprises n amino acid sequences shown in SEQ ID NO.1; or the collagen protein comprises n amino acid sequences having more than 95% identity with the amino acid sequence shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10.

[0009] In another preferred embodiment, the N-terminus of the collagen further contains a recognition motif for a protease for tag removal.

[0010] In another preferred embodiment, the recognition motif of the protease is the recognition motif of TEV protease.

[0011] In another preferred embodiment, the recognition motif of the protease contains the sequence shown in SEQ ID NO.3.

[0012] In another preferred embodiment, the collagen has a structure shown in Formula I from N-terminus to C-terminus:

[0013] Z0-L-Z1(I)

[0014] In the formula,

[0015] Z0 is the recognition motif of an optional protease for tag removal;

[0016] L is none or a linker peptide;

[0017] Z1 is n amino acid sequences shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10;

[0018] “-” is a key.

[0019] In another preferred embodiment, the collagen may be modified or unmodified.

[0020] In another preferred embodiment, the collagen has an amino acid sequence selected from the following group:

[0021] (A) having the sequence shown in SEQ ID NO. 4 or 8;

[0022] (B) a polypeptide having ≥80% homology (preferably, ≥90% homology; more preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the amino acid sequence shown in SEQ ID NO. 4 or 8, and the polypeptide has the activity of promoting cell adhesion;

[0023] (C) A derivative polypeptide formed by substituting, deleting or adding 1 to 5 amino acid residues of the amino acid sequence shown in SEQ ID NO. 4 or 8 and retaining the activity of promoting cell adhesion.

[0024] The second aspect of the present invention provides an isolated polynucleotide encoding the collagen as described in the first aspect of the present invention.

[0025] In another preferred embodiment, the polynucleotide is a codon-optimized polynucleotide.

[0026] In another preferred embodiment, the nucleotide sequence is selected from the following group:

[0027] (a) the nucleotide sequence is as shown in SEQ ID NO.5;

[0028] (b) the nucleotide sequence has ≥95% identity with the nucleotide sequence shown in any one of SEQ ID NO.5, preferably ≥98%, more preferably ≥99% identity; and

[0029] (c) a nucleotide sequence complementary to the nucleotide sequence described in (a) or (b).

[0030] In another preferred embodiment, the nucleotide sequence includes a DNA sequence, a cDNA sequence, or an mRNA sequence.

[0031] In another preferred embodiment, the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.

[0032] The third aspect of the present invention provides a vector, which comprises the polynucleotide as described in the second aspect of the present invention.

[0033] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, viral vector, transposon, or a combination thereof.

[0034] In another preferred embodiment, the vector comprises one or more promoters, which are operably connected to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selective marker, nucleic acid restriction site, and / or homologous recombination site.

[0035] In another preferred embodiment, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0036] In another preferred embodiment, the vector includes an expression vector, a shuttle vector, and an integration vector.

[0037] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector as described in the third aspect of the present invention.

[0038] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell.

[0039] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0040] The fifth aspect of the present invention provides a method for preparing the collagen as described in the first aspect of the present invention, comprising the steps of:

[0041] (i) culturing the host cell according to the fourth aspect of the present invention under appropriate conditions to obtain the collagen according to the first aspect of the present invention; and

[0042] (ii) optionally enzymatically cleaving, purifying and / or separating the collagen obtained in step (i).

[0043] The sixth aspect of the present invention provides a composition comprising:

[0044] (I) the collagen described in the first aspect of the present invention; and

[0045] (II) a pharmaceutically acceptable carrier or a cosmetically acceptable excipient.

[0046] In another preferred embodiment, the composition is used to increase cell adhesion.

[0047] In another preferred embodiment, the cells include epidermal cells, mucosal epithelial cells, dermal cells, and skin mesenchymal cells.

[0048] In another preferred embodiment, the composition further contains other substances that can increase cell adhesion.

[0049] In another preferred embodiment, the cosmetically acceptable excipient is selected from the following group: moisturizers, skin conditioners, thickeners, emollients, emulsifiers, antioxidants, preservatives, UV inhibitors, film formers, oil-soluble gelling agents, organically modified clay minerals, resins, flavors, salts, pH adjusters, conditioners, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients, vitamins, amino acids, nucleic acids, inclusion compounds, solvents (such as water), or combinations thereof.

[0050] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, semisolid dosage form or solid dosage form, more preferably ointment, cream, emulsion, oil, powder, solution, gel, spray, foam, suspension, lotion or sticks.

[0051] In another preferred embodiment, the composition is a facial cream, lotion, essence, cleanser or essence lotion.

[0052] In another preferred embodiment, the composition is a pharmaceutical composition.

[0053] In another preferred embodiment, the composition further comprises auxiliary materials.

[0054] The seventh aspect of the present invention provides the use of the collagen described in the first aspect of the present invention for preparing a composition or product, wherein the composition or product is used to increase cell adhesion.

[0055] In another preferred embodiment, the composition or product includes a tissue engineering product, a cosmetic, a health product or a medicine.

[0056] The eighth aspect of the present invention provides a method for increasing cell adhesion, comprising the steps of:

[0057] An effective amount of the collagen described in the first aspect of the present invention or the composition described in the sixth aspect of the present invention is administered to a subject in need thereof.

[0058] In another preferred embodiment, the application includes external application to skin, external application to mucous membranes, and wound application.

[0059] In another preferred embodiment, the application helps to improve the skin barrier, smooth fine lines, moisturize, whiten and lighten spots, shrink pores, and lighten dark circles; repair the mucosal barriers of the nasal cavity, oral cavity, vagina, anorectum, etc.; stop bleeding and promote wound healing, and prevent scar formation.

[0060] In another preferred embodiment, the subject is a human or non-human mammal.

[0061] In another preferred embodiment, the non-human mammal includes a pet dog, cat, monkey, rabbit, rat or mouse.

[0062] A ninth aspect of the present invention provides a method for increasing cell adhesion, comprising the steps of:

[0063] Cells are cultured in the presence of the collagen described in the first aspect of the present invention or the composition described in the sixth aspect of the present invention, thereby increasing cell adhesion.

[0064] In another preferred embodiment, the cells include epidermal cells, mucosal epithelial cells, dermal cells, and skin mesenchymal cells.

[0065] In another preferred embodiment, the cells are cells cultured in vitro.

[0066] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0067] On the other hand, the present application provides the use of the above collagen in the preparation of tissue engineering products, cosmetics, health products, foods or pharmaceutical compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 .SDS-PAGE was used to detect the induced expression of p32F3 and the control p32F12.

[0069] Figure 2 .The stability results of p32F3 after being placed at different temperatures for 30 days and 53 days.

[0070] Figure 3.The stability results of p32F3 after freezing and thawing once, three times and five times.

[0071] Figure 4 .Affinity chromatography purification of p32F3.

[0072] Figure 5 .Affinity chromatography purification after TEV digestion of p32F3.

[0073] Figure 6 .p32F3 enhances cell adhesion. DETAILED DESCRIPTION

[0074] The inventors have conducted extensive and in-depth research and a large number of screenings, and for the first time developed a collagen protein, wherein the collagen protein comprises n amino acid sequences shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10. The inventors have discovered for the first time that the collagen protein of the present invention has the characteristics of strong stability and not easy to degrade, and cell activity detection shows that the collagen protein has obvious activity of promoting cell adhesion, and the collagen protein of the present invention has the characteristics of high expression amount, and can be further developed into products containing collagen protein such as tissue engineering products, cosmetics, health products, foods or pharmaceutical compositions. The present invention has been completed on this basis.

[0075] The sequence identity in this application refers to the identity calculated by algorithms known in the art, including but not limited to blast and GenPast.

[0076] In the present application, 95% or more identity includes but is not limited to integer percentages, such as 95%, 96%, 97%, 98%, 99% identity, as well as decimal identities that may be obtained based on the number of variation sites or algorithms, including but not limited to 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, etc.

[0077] The amino acid sequences in the present application that have more than 95% identity with the amino acid sequence shown in SEQ ID NO.1 have the same or similar functions and / or higher-order structures as SEQ ID NO.1, including but not limited to hydrophilicity, adhesion properties, binding properties with certain components such as integrins, etc. Such sequences can be designed and manufactured by those skilled in the art using existing tools in the protein and gene fields, such as protein structure prediction tools, hydrophobicity prediction tools, codon design tools, and reagents / kits for substitution, deletion and addition of corresponding gene sequences.

[0078] The coding sequence of collagen peptide can be designed by those skilled in the art using the existing knowledge of amino acid coding rules and design tools according to factors such as the needs of the host.

[0079] Tags available in the protein expression process, such as His, Flag, MBP, GST, HA, etc., can be routinely selected by those skilled in the art according to needs and relevant literature and instructions.

[0080] The tag can be removed by a method known in the art such as chemical method, protease method, intein method, etc., and those skilled in the art can select them according to the needs and relevant literature and instructions.

[0081] Conventional strategies and tools for the transformation of expression strains and the steps of culture and expression in the preparation method of the present application can be routinely selected by those skilled in the art, and suitable parameters can be determined by consulting materials and experimental verification. For example, transformation methods such as calcium chloride, protoplasts, electroporation, and identification methods such as resistance marker screening and direct identification of recombinants can be used for transformation; the expression strain can be Escherichia coli, Bacillus subtilis, yeast, insect cells, mammalian cells, plant cells, etc.; induction systems such as IPTG, sugar induction, nisin, and pH induction can be used in the expression process.

[0082] The expression vector in this application can be selected from various commercially available / recorded / researched vectors, including but not limited to Pllp, pMBP, pET, pBAD, pCAI, and pPOW series vectors.

[0083] The recognition motifs of the protease for tag removal in the present application include but are not limited to the recognition motifs of TEV protease, Thrombin, Factor Xa, Enterokinase, PreScission, HRV 3C protease, SUMO protease, etc. These recognition motifs and their corresponding nucleic acid sequences can be obtained by those skilled in the art by referring to existing literature, instructions or general knowledge in the field of molecular biology and protein.

[0084] The excipients in the composition of the present application include, but are not limited to, excipients acceptable in pharmacy, cosmetics, health products, and food, including, but not limited to, fillers, solvents, cosolvents, dispersants, viscosity modifiers, antioxidants, sweeteners, adhesives, pH modifiers, etc. Those skilled in the art can select these excipients according to conventional practices in the art. The composition of the present application, in particular, may also contain other functional ingredients, such as Chinese and Western medicine ingredients, chemical ingredients, and cytokines for moisturizing, whitening, and tissue repair.

[0085] The health care products and pharmaceutical compositions in this application may be: injections, such as injections, powder injections, etc.; oral preparations, such as tablets, capsules, oral liquids, etc.; external preparations, such as ointments, sprays, patches, etc. Tissue engineering products may be cosmetic or tissue repair products. Foods and cosmetics may be in any form known in the food and cosmetics field.

[0086] Collagen of the present invention

[0087] As used herein, the terms "recombinant humanized type III collagen of the present invention" and "collagen of the present invention" are used interchangeably and refer to a collagen peptide comprising n amino acid sequences shown in SEQ ID NO.1; or the collagen peptide comprises n amino acid sequences that are more than 95% identical to the amino acid sequence shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10.

[0088] In a preferred embodiment, the collagen peptide of the present invention has a structure shown in Formula I from N-terminus to C-terminus:

[0089] Z0-L-Z1 (I)

[0090] In the formula,

[0091] Z0 is the recognition motif of an optional protease for tag removal;

[0092] L is none or a linker peptide;

[0093] Z1 is n amino acid sequences shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10;

[0094] “-” is a key.

[0095] As used herein, the term "connector peptide" refers to a connecting peptide having sufficient length and flexibility to ensure that the two connected proteins have sufficient spatial freedom to perform their functions, preferably a flexible linker (or flexible linker).

[0096] In a preferred embodiment, the collagen peptide of the present invention has an amino acid sequence selected from the group consisting of:

[0097] (A) having the sequence shown in SEQ ID NO. 4 or 8;

[0098] (B) a polypeptide having ≥80% homology (preferably, ≥90% homology; more preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the amino acid sequence shown in SEQ ID NO. 4 or 8, and the polypeptide has the activity of increasing cell adhesion;

[0099] (C) A derivative polypeptide formed by substituting, deleting or adding 1 to 5 amino acid residues of the amino acid sequence shown in SEQ ID NO. 4 or 8 and retaining the activity of promoting cell adhesion.

[0100] As used herein, the term "collagen" also includes variant forms having the above-mentioned activities. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, preferably 1) amino acids, and addition or deletion of one or several (usually within 3, preferably within 2, and more preferably within 1) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. In addition, the term also includes the polypeptides of the present invention in monomeric and polymeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0101] The present invention also includes active fragments, derivatives and analogs of the above-mentioned collagen peptides. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention can be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6XHis). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0102] A preferred class of active derivatives refers to polypeptides formed by replacing at most 3, preferably at most 2, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0103] Table A

[0104] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0105] The present invention also provides analogs of the collagen of the present invention. The difference between these analogs and the polypeptide of the present invention may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

[0106] In addition, the collagen of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0107] The term "polynucleotide of the present invention" may include a polynucleotide encoding the collagen of the present invention, or may include additional coding and / or non-coding sequences.

[0108] The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or collagen peptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the collagen protein encoded by it.

[0109] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.

[0110] The collagen and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0111] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0112] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0113] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0114] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0115] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0116] Expression vector

[0117] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the collagen peptide coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0118] The polynucleotide sequence of the present invention can be used to express or produce recombinant collagen peptides by conventional recombinant DNA technology. Generally speaking, the following steps are involved:

[0119] (1) Transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the collagen of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0120] (2) Host cells cultured in a suitable culture medium;

[0121] (3) Isolate and purify proteins from culture medium or cells.

[0122] In the present invention, the polynucleotide sequence encoding the collagen peptide can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0123] Methods well known to those skilled in the art can be used to construct expression vectors containing the collagen peptide encoding DNA sequence of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0124] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0125] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0126] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast, plant cells (such as ginseng cells).

[0127] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0128] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0129] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0130] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0131] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0132] The main advantages of the present invention include:

[0133] (1) The present invention first discovered that the collagen of the present invention has the characteristics of strong stability and non-degradability. Cell activity detection shows that collagen has the activity of increasing cell adhesion. In addition, the collagen of the present invention has the characteristic of high expression level. It can be further developed into tissue engineering products, cosmetics, health products, foods or pharmaceutical compositions and other products containing collagen.

[0134] (2) The collagen of the present invention can effectively increase the adhesion of cells.

[0135] (3) The present invention uses bioinformatics to analyze the collagen functional domains present in the mature segment of type III collagen, and a total of 11 are predicted. Subsequently, the sequence of the collagen functional domain was obtained, and it was repeated many times. After codon optimization and gene synthesis, it was expressed and verified in Escherichia coli, and 1 highly expressed collagen was screened. After fermentation expression, enzyme tag cleavage and affinity purification, a high-purity recombinant humanized collagen with no exogenous amino acid residues was obtained, and its sequence was 100% consistent with the human collagen sequence. Further experiments showed that the recombinant humanized type III collagen of the present invention has the characteristics of strong stability and not easy to degrade. Cell activity detection shows that the recombinant humanized type III collagen of the present invention has obvious activity in promoting cell adhesion.

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually based on conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise stated, the reagents and materials in the embodiments of the present invention are all commercially available products.

[0137] Example 1 Bioinformatics prediction of collagen functional domains

[0138] The amino acid sequence of human type III collagen α1 chain is from the National Center for Biotechnology Information, with the accession number NP_0 00081.2 (https: / / www.ncbi.nlm.nih.gov / protein / NP_000081.2), and the total length is 1466aa. SMART (https: / / smart.embl.de / ) and CD-search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) online bioinformatics prediction software were further used to predict the collagen functional domain of type III collagen α1 chain, and the results of repeated predictions of the two software were combined to obtain a total of 11 functional domain segments. The collagen functional domain sequence between positions 237 and 293: GRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGET GAPGLKGENGLPGEN (SEQ ID NO.1) was named F3, and the collagen functional domain sequence between positions 168 and 233: GLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGES (SEQ ID NO.2) was named F12 and used as control sequences.

[0139] Example 2 Construction of expression vectors for F3 and F12 sequences and induction of expression

[0140] Sequence design and codon optimization: In order to analyze the expression of F3 and F12, we repeated them for different times according to the length of the functional domain sequence. The F3 sequence contains 57 amino acids, which are used as the basic unit and repeated 8 times. To achieve 100% humanization, the TEV protease recognition cleavage motif ENLYFQ (SEQ ID NO.3) was added to the N-terminus of the F3 sequence repeated 8 times. The amino acid sequence of the fusion protein is shown in SEQ ID NO.4 or 8:

[0141] ENLYFQGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGEN*(SEQ ID NO.4)

[0142] GRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGEN*(SEQ ID NO.8)

[0143] According to the central dogma, the amino acid sequence of the F3 sequence repeated 8 times was reverse translated to obtain the encoded RNA sequence, and the termination codon TAG was added to the 3' end. The RNA sequence was further optimized for the codon preference of Escherichia coli. The optimized RNA sequence is shown in SEQ ID NO.5

[0144]

[0145]

[0146] The control sequence F12 contains 66 amino acids, which are used as the basic unit and repeated 7 times. Similarly, in order to achieve 100% humanization, the TEV protease recognition cleavage motif ENLYFQ (SEQ ID NO.3) is added to the N-terminus of the F12 sequence repeated 7 times, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.6: ENLYFQ GLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPG PPGAIGPSGPAGKDGESGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQ GPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGLAGYPGPAGPPGPPGP PGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGES*

[0147] According to the central dogma, the amino acid sequence of the F12 sequence repeated 7 times was reverse translated to obtain the encoded RNA sequence, and the termination codon TAG was added at the 3' end. The RNA sequence was further optimized for the codon preference of Escherichia coli. The optimized RNA sequence is shown in SEQ ID NO.7

[0148]

[0149] Expression vector construction: GenScript Biotech Co., Ltd. was commissioned to synthesize the nucleic acid sequence, and inserted into the pET-32a(+) expression vector (Novagen, Catalog No.: 69015) using BamHI-HF (NEB Company, Catalog No.: R3136S) and XhoI (NEB Company, Catalog No.: R0146S) restriction endonucleases to obtain expression plasmids, which were named p32F3 and p32F12, respectively.

[0150] Transformation of expression strains: The p32F3 and p32F12 expression plasmids were transformed into Escherichia coli competent cells BL21 (DE3) (Merck, catalog number: CMC0014), respectively. The specific operation process is as follows: ① Take 5 μL (about 500 ng) of the plasmid into about 30 μL of Escherichia coli competent cells BL21 (DE3), mix gently and let stand on ice for 30 minutes; ② Heat shock the mixture in a 42°C water bath for 45 seconds, and then quickly put it on ice for 2 minutes; ③ Add 200 μL of non-resistant LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) to the mixture, and culture it at 37°C, 220rpm for 40 minutes; ④ Take 200 μL of the bacterial solution and evenly spread it on an LB plate containing ampicillin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 50 μg / mL ampicillin); ⑤ Invert the plate in a 37°C incubator for overnight culture until clearly visible colonies grow.

[0151] Induced expression: Pick a single clone from the above LB plate and place it in 2 mL LB liquid medium (containing 50 μg / mL ampicillin), culture it at 37°C and 220 rpm overnight, transfer it to 200 mL LB liquid medium (containing 50 μg / mL ampicillin), culture it at 37°C and 220 rpm until the bacterial solution OD600 is 0.6-1.0, add IPTG (Biosharp, catalog number: BS119) with a final concentration of 0.2 mM to induce expression, and the induction conditions are 18°C ​​and 220 rpm for 16-20 hours. Collect the bacteria by centrifugation and store them at -20°C or immediately enter the lysis purification process.

[0152] Bacterial lysis: Resuspend the above bacterial pellet with lysis buffer (20mM PB buffer, 500mM sodium chloride, pH 7.4), and the resuspension ratio is 1g bacterial mud corresponding to 20mL lysis buffer. After fully resuspended and evenly distributed, 30mL each portion is dispensed into 50mL centrifuge tubes, and the bacteria are crushed using an ultrasonic cell crusher (Ningbo Xinzhi, SCIENTZ-IID). The sample is placed in an ice water bath, using a Φ6 amplitude rod, the power is set to 250W, the ultrasound is turned on for 3sec, stopped for 3sec, and the total working time is 25min. The bacterial lysate is obtained by ultrasonic crushing, and the soluble protein and inclusion body are fully separated by centrifugation at 12000rpm for 20min.

[0153] Analysis and detection: SDS-PAGE was used to monitor and analyze the expression of the target protein. The specific process is: sample the bacterial lysate before centrifugation and the supernatant of the bacterial lysate after centrifugation, take 40μL, add 10μL 5× reduced protein loading buffer (Biosharp, catalog number: BL502A) and mix well, place in a metal bath at 95℃ for 5min for thermal denaturation, then centrifuge at 12000rpm for 10min, take 5μL of supernatant and add it to the SDS-PAGE protein gel well (GenScript Biotech, catalog number: M00930), electrophoresis at 160V for 1h, then use a decolorizer (GenScript Biotech, catalog number: M00930) for staining and decolorization, and take pictures on a white light plate. The theoretical size of the p32F3 recombinant protein is 63.08kDa, and the theoretical size of the control plasmid p32F12 recombinant protein is 52.85kDa. The test results are as follows Figure 1 As shown in A, the target band was observed at the 70 kDa position of p32F3, and the expression level was high. The detection results of the control plasmid p32F12 were as follows Figure 1 As shown in B, the control plasmid was found to have no expression. Comparative analysis of the brightness of the target protein in the whole p32F3 bacteria and the supernatant showed that the p32F3 protein was mainly in the supernatant, indicating that it existed in a soluble form.

[0154] Example 3: Stability Analysis of p32F3 Recombinant Collagen

[0155] The p32F3 stability treatment sample was obtained from the supernatant of the bacterial lysate after centrifugation in Example 2. The bacterial lysate after centrifugation was sampled, and 40 μL was dispensed into several EP tubes and marked, and placed at -80°C, -20°C, 2-8°C, 25°C, and 37°C respectively. After treatment for 30 days and 53 days, 10 μL of 5× reduced protein loading buffer (Biosharp, item number: BL502A) was added to the samples, mixed well, and denatured at 95°C for 5 minutes before SDS-PAGE detection. The specific operations of SDS-PAGE detection and staining and photography are shown in Example 2. Figure 2The results showed that the supernatant of p32F3 bacterial lysate was placed at -80℃, -20℃, 2-8℃, and 25℃ for 30 days ( Figure 2 A) and 53d( Figure 2 B) After storage at 37°C for 53 days, the target protein was slightly degraded.

[0156] The repeated freeze-thaw samples were obtained from the supernatant of the bacterial lysate after centrifugation in Example 2. 40 μL was taken and distributed into several EP tubes for repeated freeze-thaw treatment (frozen overnight at -80°C and then taken out and placed at room temperature for 4 hours, which was recorded as one freeze-thaw). After repeating this process 1, 3, and 5 times, samples were taken for SDS-PAGE detection. The specific operations of SDS-PAGE detection and staining photography are shown in Example 2. Figure 3 The results showed that the p32F3 protein underwent 1 ( Figure 3 A), 3 times ( Figure 3 B), 5 times ( Figure 3 C) After freezing and thawing, the target protein did not degrade. The results of the different temperature treatment experiments and repeated freezing and thawing experiments showed that the p32F3 protein has strong stability.

[0157] Example 4: Purification and tag removal of p32F3 recombinant collagen

[0158] The supernatant of the bacterial lysate was obtained according to the method in Example 2, and then the recombinant collagen was separated and purified using a fully automatic protein purification system (Insert, Unique Auto Pure 100) and affinity filler Ni Focurose FF (IMAC) (Hui Yan, product number HQ060312, 5 mL pre-packed column). The column bed was equilibrated with 30 mL of equilibration buffer (20 mM PB buffer, 500 mM sodium chloride, 10 mM imidazole, pH 7.4) at a flow rate of 2 mL / min; the lysate supernatant was loaded at a flow rate of 1 mL / min to 80% of the filler loading; 50 mL of wash buffer (20 mM PB buffer, 500 mM sodium chloride, 30 mM imidazole, pH 7.4) was used for elution at a flow rate of 2 mL / min; the target protein was washed with elution buffer (20 mM PB buffer, 500 mM sodium chloride, 300 mM imidazole, pH 7.4), and the elution peak was collected according to the A215 absorbance value; finally, the filler was cleaned with 25 mL of regeneration buffer (20 mM PB buffer, 500 mM sodium chloride, 500 mM imidazole, pH 7.4) at a flow rate of 2 mL / min. The eluted sample was desalted by molecular sieves filled with Seplife RG-25 filler (Lanxiao, catalog number: D1007211M), the column model was 16mm / 200mm, and the desalting buffer (20mM PB buffer, pH 7.4) was used for balancing and washing, the flow rate was 5mL / min, and the target protein was collected according to the A215 absorption value. The target protein was detected by SDS-PAGE. Figure 4 It can be seen that the target protein does not exist in the flow-through and the rinse solution, and the target protein is enriched in the eluate, indicating that p32F3 can enrich the target protein in large quantities through affinity chromatography on a nickel column. The protein purity reaches about 80% through grayscale analysis.

[0159] Take the purified p32F3 protein solution, add an appropriate amount of His-rTEV protease (Solarbio, P2060), the ratio is about 50EU / mL, incubate at 16°C for 4h, and then separate again by the above-mentioned Ni Focurose FF (IMAC) pre-loaded column chromatography. Rinse the above-mentioned regenerated chromatography column with 25mL of pure water at a flow rate of 2mL / min; balance the column bed with 30mL of 20mM PB buffer (pH7.4) at a flow rate of 2mL / min; load the target protein solution containing His-rTEV protease at a flow rate of 1mL / min and then rinse with 20mM PB buffer (pH 7.4), collect the target protein that flows through according to the A215 absorption value, and the sample is a recombinant collagen solution with high purity and without any label. Take a sample and perform SDS-PAGE detection on the target protein, Figure 5The results showed that the tag of p32F3 protein had been completely removed, and the untagged p32F3 protein was obtained by flow-through, with a single protein band and a purity of about 95%.

[0160] Example 5 Activity Detection of p32F3 Recombinant Collagen

[0161] The high-purity collagen solution without any label obtained in Example 4 was used to test the cell adhesion, and the 96-well cell culture plate without TC treatment was used for detection. The culture plates were coated with 200 μL / well of DMEM medium containing p32F3 collagen (concentration of 1.0 mg / mL), incubated at 37°C for 1 hour, the coating solution was aspirated, and the culture wells were washed twice with PBS, 200 μL / well; then 5×10 4 NIH / 3T3 cells were inoculated into 100 μL DMEM containing 10% fetal bovine serum at 37°C and 5% CO2 for 1 hour. The culture was aspirated and the culture wells were washed twice with PBS at 200 μL / well. Finally, 100 μL DMEM culture medium was added again and the number of adherent cells was observed under a microscope. NIH / 3T3 cells have poor adhesion ability on plates not treated with TC. Collagen can increase the adhesion of cells, thereby increasing the number of adherent cells. Figure 6 The results showed that compared with the negative control, i.e., the culture plate without collagen coating ( Figure 6 A) Compared with NIH / 3T3 cells cultured on p32F3 recombinant collagen coated plates ( Figure 6 B) can significantly increase the number of adherent cells.

[0162] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A collagen protein, characterized in that The collagen contains n amino acid sequences shown in SEQ ID NO.1; or the collagen contains n amino acid sequences that are more than 95% identical to the amino acid sequence shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10.

2. The collagen according to claim 1, characterized in that The collagen has a structure shown in Formula I from the N-terminus to the C-terminus: Z0-L-Z1(I) In the formula, Z0 is the recognition motif of an optional protease for tag removal; L is none or a linker peptide; Z1 is n amino acid sequences shown in SEQ ID NO.1, wherein n is 2-20, preferably, n is 4-15, more preferably, n is 5-15, and more preferably, n is 6-10; "-" is a key.

3. The collagen according to claim 1, characterized in that The collagen has an amino acid sequence selected from the group consisting of: (A) having the sequence shown in SEQ ID NO. 4 or 8; (B) a polypeptide having ≥80% homology (preferably, ≥90% homology; more preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the amino acid sequence shown in SEQ ID NO. 4 or 8, and the polypeptide has the activity of promoting cell adhesion; (C) A derivative polypeptide formed by substituting, deleting or adding 1 to 5 amino acid residues of the amino acid sequence shown in SEQ ID NO. 4 or 8 and retaining the activity of promoting cell adhesion.

4. An isolated polynucleotide encoding the collagen according to claim 1.

5. A carrier, characterized in that The vector comprises the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the vector as claimed in claim 5.

7. A method for preparing the collagen according to claim 1, characterized in that: Includes steps: (i) culturing the host cell according to claim 6 under appropriate conditions to obtain the collagen according to claim 1; and (ii) optionally enzymatically cleaving, purifying and / or separating the collagen obtained in step (i).

8. A composition, characterized in that The composition contains: (I) the collagen according to claim 1; and (II) a pharmaceutically acceptable carrier or a cosmetically acceptable excipient.

9. The use of collagen according to claim 1, characterized in that: Used to prepare a composition or product for increasing cell adhesion.

10. A method for increasing cell adhesion, characterized in that: Includes steps: Cells are cultured in the presence of the collagen of claim 1 or the composition of claim 8, thereby increasing cell adhesion.

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