WO2024119724A1 - 胶原蛋白肽及其制备方法和用途 - Google Patents

胶原蛋白肽及其制备方法和用途 Download PDF

Info

Publication number
WO2024119724A1
WO2024119724A1 PCT/CN2023/095616 CN2023095616W WO2024119724A1 WO 2024119724 A1 WO2024119724 A1 WO 2024119724A1 CN 2023095616 W CN2023095616 W CN 2023095616W WO 2024119724 A1 WO2024119724 A1 WO 2024119724A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
collagen
collagen peptide
sequence
expression
Prior art date
Application number
PCT/CN2023/095616
Other languages
English (en)
French (fr)
Inventor
杜刚
符德保
王欣
熊雅琴
董越
Original Assignee
武汉佳惟达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉佳惟达生物科技有限公司 filed Critical 武汉佳惟达生物科技有限公司
Publication of WO2024119724A1 publication Critical patent/WO2024119724A1/zh

Links

Definitions

  • the present invention belongs to the field of genetic engineering technology. Specifically, the present application provides a collagen peptide and a preparation method and use thereof.
  • Collagen is a type of structural protein that is abundant in all animals. In the human body, it accounts for 30% of the total protein and three-quarters of the dry weight of the skin. Collagen is the main component of the extracellular matrix, and together with elastin, fibronectin, etc., it forms a network structure for cell growth. Collagen is a key substance that creates the appearance of the skin and provides strength and elasticity for the skin. 80-90% of the collagen in the human body is composed of types I, II and III, and the three different collagens are distributed in different tissues and play different roles. Type I collagen is the main structural protein of bones, skin, tendons, ligaments and dentin. It is also a structural protein of blood vessels, heart valves and aortas. Type II collagen is not as dense as type I and is the main collagen in cartilage. The function of muscles, skin and arteries depends on type III collagen, which usually appears together with type I collagen and plays a key role in promoting elasticity.
  • collagen In addition to serving as the body's structural support function, collagen also has excellent biological functions and provides a good microenvironment for cell growth.
  • Collagen has cell surface receptor recognition sites and cell integrin binding motifs, which can interact with cells to promote cell adhesion, proliferation, growth and differentiation. Due to its good tissue compatibility, collagen is used as a coating material for cardiovascular stents, surface materials for artificial bones and joints, and artificial corneas in medicine. Collagen can tightly bind to the wound to prevent blood from flowing out, and at the same time penetrate into the new tissue as a scaffold for cell growth; in the process of tissue repair and reconstruction, collagen is also conducive to cell recognition and migration. Therefore, collagen is widely used as a wound dressing, which can promote wound repair and wound healing while stopping bleeding.
  • collagen as a signal substance for cell activity, can promote autologous collagen regeneration and is widely used in the field of regenerative medicine, such as mucosal repair, prevention and treatment of hair loss, improvement of eczema, anti-aging and anti-wrinkle, etc.
  • Type III collagen is closely related to skin aging. How to supplement type III collagen has become the pursuit of many beauty seekers.
  • collagen fillers can also promote autologous collagen regeneration, which has unique advantages over traditional passive filling materials. With the in-depth research and industrialization, the application field of collagen has been greatly expanded, and has deeply covered The fields of medical health, beauty and skin care, and food.
  • Natural collagen has a large molecular weight, and the length of a single chain of mature protein is more than 1,000 amino acids. Different chains are cross-linked to form large fibrous structures or mesh structures. Therefore, this type of protein has poor water solubility and needs to be dissolved in acidic conditions, which greatly limits its application.
  • commercially available collagen is still mainly derived from animals, extracted from raw materials such as cowhide, cow Achilles tendon, pig skin, and fish skin.
  • the collagen obtained by this method has a low cost, but its safety cannot be guaranteed and may carry animal viruses.
  • the sequence differences of animal collagen and animal-derived impurities can easily cause human immune responses. Therefore, the application of this type of collagen in medical health and medical beauty skin care is limited.
  • the applicant screened and designed based on the active motifs in the human collagen type III sequence and the hydrophilicity and charge properties of the peptides, and used the E. coli expression system for expression verification, and obtained a collagen peptide with high expression and good water solubility.
  • the expression strain constructed based on the sequence, after induced fermentation, the collagen peptide obtained was 100% consistent with the human collagen sequence after the fusion tag was removed by a specific protease.
  • the collagen peptide can be further developed into a humanized collagen product.
  • the present application provides a collagen peptide, which comprises n amino acid sequences shown in SEQ ID NO.1 and/or SEQ ID NO.2; or the collagen peptide comprises n amino acid sequences that are more than 95% identical to the amino acid sequences shown in SEQ ID NO.1 and/or SEQ ID NO.2, wherein n is 2-50, preferably, n is 8-28, and more preferably, n is 10-26.
  • the collagen peptide comprises n1 tandemly repeated amino acid sequences shown in SEQ ID NO.1, wherein n1 is 2-40, preferably 2-30; or the collagen peptide comprises n2 tandemly repeated amino acid sequences shown in SEQ ID NO.1.
  • the amino acid sequence of SEQ ID NO. 2 is repeated, wherein n2 is 2-50, preferably, 2-30.
  • the N-terminus of the collagen peptide further contains a recognition motif for a protease for tag removal.
  • the recognition motif of the protease is the recognition motif of TEV protease.
  • the recognition motif of the protease contains the sequence shown in SEQ ID NO.3.
  • the collagen peptide has a structure shown in Formula I from N-terminus to C-terminus: Z0-L-Z1 (I)
  • L is none or a linker peptide
  • Z1 is n amino acid sequences represented by SEQ ID NO.1 and/or SEQ ID NO.2, wherein n is 2-50, preferably, n is 8-28, and more preferably, n is 10-26;
  • the collagen peptide may be modified or unmodified.
  • the collagen peptide has an amino acid sequence selected from the following group:
  • amino acid sequence of the collagen peptide is as shown in any one of SEQ ID NO: 4, 6, 8, 10, and 12.
  • amino acid sequence of the collagen peptide is as shown in any one of SEQ ID NO: 4, 6, 8, and 12.
  • the second aspect of the present invention provides a collagen polypeptide, which contains a sequence shown in any one of SEQ ID NO.14 and SEQ ID NO.16.
  • an isolated polynucleotide is provided, wherein the polynucleotide encodes the collagen polypeptide as described in the first aspect or the second aspect of the present invention.
  • the polynucleotide is a codon-optimized polynucleotide.
  • nucleotide sequence is selected from the following group:
  • nucleotide sequence is any one of SEQ ID NO.: 5, 7, 9, 11, 13, 15, 17;
  • nucleotide sequence has ⁇ 95% identity with the nucleotide sequence shown in any one of SEQ ID NO.: 5, 7, 9, 11, 13, 15, 17, preferably ⁇ 98%, more preferably ⁇ 99% identity;
  • the nucleotide sequence includes a DNA sequence, a cDNA sequence, or an mRNA sequence.
  • the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.
  • a vector comprising the polynucleotide as described in the third aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • a host cell contains the vector as described in the fourth aspect of the present invention, or an exogenous polynucleotide as described in the third aspect of the present invention is integrated into the chromosome.
  • a method for preparing the collagen peptide as described in the first aspect of the present invention or the second aspect of the present invention comprising the steps of:
  • step (ii) optionally enzymatically cleaving, purifying and/or separating the collagen peptides obtained in step (i).
  • composition comprising:
  • the composition is used to promote cell proliferation activity and/or increase cell adhesion.
  • the cells include epidermal cells, mucosal epithelial cells, dermal cells, and skin mesenchymal cells.
  • the composition further contains other substances that can promote cell proliferation activity and/or increase cell adhesion.
  • the cosmetically acceptable excipient is selected from the following group: moisturizers, skin conditioners, thickeners, emollients, emulsifiers, antioxidants, preservatives, anti-ultraviolet agents, film formers, oil-soluble gelling agents, organic 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.
  • 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.
  • the composition is a facial cream, lotion, essence, cleanser or essence lotion.
  • the eighth aspect of the present invention there is provided a use of the collagen peptide described in the first aspect of the present invention or the second aspect of the present invention for preparing a composition or product, wherein the composition or product is used for (i) promoting cell proliferation activity and/or (ii) increasing cell adhesion.
  • the composition or product includes a tissue engineering product, a cosmetic, a health product or a medicine.
  • a ninth aspect of the present invention there is provided a method for (i) promoting cell proliferation activity, and/or (ii) increasing cell adhesion, comprising the steps of:
  • An effective amount of the collagen peptide described in the first aspect or the second aspect of the present invention or the composition described in the seventh aspect of the present invention is administered to a subject in need thereof.
  • the application includes external application to skin, external application to mucous membranes, and wound application.
  • 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.
  • the subject is a mammal, such as a human, a pet dog, a cat, a rat or a mouse.
  • the present application provides a method for preparing the above collagen peptide, which comprises gene sequence synthesis; construction of an expression plasmid; expression strain transformation; culture and expression; lysis and extraction steps.
  • the gene sequence contains the coding sequence of the above-mentioned collagen peptide.
  • the gene sequence also contains a recognition motif for a protease for tag removal.
  • the recognition motif of the protease is the recognition motif of TEV protease SEQ ID NO.3.
  • gene sequence is selected from SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, and SEQ ID NO.17.
  • collagen peptide sequence is selected from SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.13.
  • the expression strain is Escherichia coli.
  • the vector used in constructing the expression plasmid is selected from pET28a+ and pET32a+.
  • the present application provides a composition comprising the above-mentioned collagen peptide.
  • composition is a tissue engineering product, a cosmetic, a health product, a food or a pharmaceutical composition.
  • composition also contains auxiliary materials.
  • the present application provides the above-mentioned collagen peptide in the preparation of tissue engineering products, cosmetics, Application in health products, foods or pharmaceutical compositions.
  • sequence identity in this application refers to the identity calculated by algorithms known in the art, including but not limited to blast and GenPast.
  • 95% or more identity includes but is not limited to integer percentages, such as 95%, 96%, 97%, 98%, 99% identity, and 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.
  • the amino acid sequence having more than 95% identity with the amino acid sequence shown in SEQ ID NO.1/SEQ ID NO.2 has the same or similar function and/or higher-order structure as SEQ ID NO.1/SEQ ID NO.2, including but not limited to hydrophilicity, adhesion properties, and binding performance with certain components such as integrins.
  • Such sequences can be designed and manufactured by those skilled in the art through 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.
  • 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.
  • 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.
  • 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.
  • 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 during the expression process.
  • 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.
  • the recognition motifs of the protease used for tag removal in this application include but are not limited to TEV protease, Recognition motifs of 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.
  • 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 may also contain other functional ingredients, such as Chinese and Western medicine ingredients, chemical ingredients, and cytokines for moisturizing, whitening, and tissue repair.
  • the health care products and pharmaceutical compositions in this application can 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 can be products for beauty and tissue repair.
  • Foods and cosmetics can be any form known in the food and cosmetics field.
  • FIG. 1 Electrophoresis of Co8 expression
  • FIG. 4 Electrophoresis of the expression of Co10 (pET-28a(+) expression vector);
  • FIG. 5 Electrophoresis of the expression of Co10 (pET-32a(+) expression vector);
  • FIG. 8 Electrophoresis of Co11 expression
  • FIG. 10 Electrophoresis of Co7 expression
  • FIG. 1 Electrophoresis of Co1F expression
  • FIG. 12 Electrophoresis of T16C expression
  • FIG. 13 Electrophoresis of Co4 expression
  • Figure 14 Test results of the preliminary purified samples placed under different temperature conditions for 12 days;
  • Figure 15 Test results of the preliminary purified samples after 1, 3, and 5 freeze-thaw cycles
  • Figure 17 Test results of Jinbo Bio's samples after being placed at 37°C for 7 days;
  • Figure 20 Comparison of the proliferation-promoting activity of Co8, Jinbo recombinant collagen, and Sigma natural collagen;
  • Figure 21 Comparison of the microscopic observation results of the proliferation-promoting activity of Co8 at different dosages
  • FIG22 Comparison of the cell proliferation activities of Co15 and Co8;
  • the inventors have conducted extensive and in-depth research and have developed a collagen peptide for the first time after a large number of screenings.
  • the collagen peptide comprises n amino acid sequences shown in SEQ ID NO.1 and/or SEQ ID NO.2, wherein n is 2-50, preferably n is 8-28, and more preferably n is 10-26.
  • the inventors have found for the first time that the collagen peptide of the present invention can achieve stable high expression, has good hydrophilicity, stability, purity and biological activity, and can be further developed into tissue engineering products, cosmetics, health products, food or The present invention is completed on this basis.
  • collagen peptide having good hydrophilicity, stability, purity and bioactivity of the present invention are used interchangeably and refer to a collagen peptide comprising n amino acid sequences shown in SEQ ID NO.1 and/or SEQ ID NO.2; or the collagen peptide comprises n amino acid sequences that are more than 95% identical to the amino acid sequences shown in SEQ ID NO.1 and/or SEQ ID NO.2, wherein n is 2-50, preferably, n is 8-28, and more preferably, n is 10-26.
  • the collagen peptide of the present invention has a structure shown in Formula I from N-terminus to C-terminus: Z0-L-Z1 (I)
  • L is none or a linker peptide
  • Z1 is n amino acid sequences represented by SEQ ID NO.1 and/or SEQ ID NO.2, wherein n is 2-50, preferably, n is 8-28, and more preferably, n is 10-26;
  • connecting 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).
  • the collagen peptide of the present invention has an amino acid sequence selected from the group consisting of:
  • the collagen peptide of the present invention contains a sequence shown in any one of SEQ ID NO.14 and SEQ ID NO.16.
  • the term "collagen peptide” 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, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. 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 of the protein. In addition, the term also includes monomeric and polymeric forms of the polypeptides of the present invention. The term also includes linear as well as non-linear polypeptides (such as cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above-mentioned collagen peptides.
  • fragment refers to polypeptides that substantially maintain the function or activity of the fusion protein of the present invention.
  • 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 6His). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.
  • 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.
  • the present invention also provides analogs of the collagen peptides of the present invention.
  • the difference between these analogs and the polypeptides of the present invention may be a difference in amino acid sequence, or a difference in modification that does not affect the sequence, or both.
  • Analogs also include those having residues different from natural L-amino acids (such as D-amino acids).
  • Analogs, and analogs with non-naturally occurring or synthetic amino acids such as ⁇ , ⁇ -amino acids. It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • the collagen peptides of the present invention can also be modified.
  • Modifications 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).
  • polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.
  • polynucleotide of the present invention may include a polynucleotide encoding the collagen peptide of the present invention, or may include additional coding and/or non-coding sequences.
  • 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.
  • 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 peptide encoded by it.
  • 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).
  • 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%.
  • the collagen peptides and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • 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 the relevant sequences.
  • cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences.
  • the relevant sequence 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.
  • artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Typically, very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • 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.
  • the method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention.
  • the RACE method RACE-cDNA terminal rapid amplification method
  • 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.
  • 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.
  • 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:
  • the polynucleotide sequence encoding the collagen peptide can be inserted into a recombinant expression vector.
  • 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.
  • 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.
  • 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.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, resistance and green fluorescent protein (GFP) for Escherichia coli, or tetracycline or ampicillin resistance for Escherichia coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, resistance and green fluorescent protein (GFP) for Escherichia coli, or tetracycline or ampicillin resistance for Escherichia coli.
  • 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.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).
  • 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.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • 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 by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • 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.
  • 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 protein precipitants (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.
  • the present invention first discovered that the collagen peptide of the present invention can achieve stable high expression, has good hydrophilicity, stability, purity and biological activity, and can be further developed into tissue engineering products, cosmetics, health products, foods or pharmaceutical compositions and other products containing collagen.
  • the collagen peptides of the present invention can effectively promote cell proliferation activity and/or increase cell adhesion.
  • the amino acid sequence of the 270-311th position of the human type III collagen ⁇ 1 chain sequence is: GRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAP GERGRP (SEQ ID NO.1).
  • This sequence consists of 42 amino acids and contains the cell binding motifs GER, GE K and the integrin binding motif GLKGEN, which makes it have a good cell adhesion function; this region is rich in charged amino acids (arginine R, lysine K, glutamic acid E), which is conducive to binding water molecules and increasing the hydrophilicity of the protein.
  • the online software predicts that the hydrophobicity value of this peptide segment is far less than 0 (the software prediction results are shown in Figure 1); the rich charged amino acids are also conducive to the formation of a hydrogen bond network between peptide chains, thereby stabilizing the triple helix structure of the collagen peptide, which is closer to the natural state of collagen.
  • the amino acid sequence has not been modified in any way and is 100% humanized.
  • the collagen peptide can be expressed using an existing mature protein expression system, and during expression design, the sequence can be repeated 2 to 40 times in series to obtain collagen peptides of different lengths.
  • the amino acid sequence of the ⁇ 1 chain of human type III collagen (sequence collected by the National Center for Biotechnology Information, NP_000081.2) at positions 1080-1109 is: GSRGAPGPQGPRGDKGETGERGAAGIKGHR (SE Q ID NO.2).
  • This sequence consists of 30 amino acids and contains the cell recognition motif RGD and the binding motif GER, which makes it have good biological activity; this region is rich in charged amino acids (arginine R, lysine K, aspartic acid D, glutamic acid E), which is conducive to binding water molecules and increasing the hydrophilicity of the protein.
  • the online software predicts that the hydrophobic value of this peptide segment is less than 0 (the software prediction results are shown in Figure 2); the rich charged amino acids are also conducive to the formation of a hydrogen bond network between peptide chains, thereby stabilizing the triple helix structure of the collagen peptide, which is closer to the natural state of collagen.
  • This amino acid sequence has not been modified in any way and is 100% humanized.
  • the collagen peptide can be expressed using an existing mature protein expression system, and during expression design, the sequence can be repeated 2 to 50 times in series to obtain collagen peptides of different lengths.
  • SEQ ID NO.1 was repeated 10 times to obtain the sequence of Co8, which has 420 amino acids in total:
  • the p28Co8 plasmid was transformed into E. coli competent cells BL21 (DE3) (Merck, catalog number: CMC0014).
  • the specific operation process is as follows: 1 Take 1 ⁇ L (about 10 ng) of the plasmid in 100 ⁇ L 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 500 ⁇ L of non-resistant LB medium (10 g/L peptone, 5 g/L yeast extract, 10 g/L sodium chloride) to the mixture, and culture at 37°C, 220 rpm for 40 minutes; 4 Take 200 ⁇ L of the bacterial solution and evenly spread it on an LB plate containing kanamycin (10 g/L peptone, 5 g/L yeast extract, 10 g/L sodium chloride, 15 g/L agar, 50 ⁇
  • Culture and induction expression Pick a monoclonal colony from the above LB plate and place it in 2 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin), culture it overnight at 37°C and 220 rpm, transfer it to 200 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin), culture it at 37°C and 220 rpm until the OD600 of the bacterial solution is 0.6-1.0 (culture time 1.5-2 h), 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 h. Collect the bacteria by centrifugation and store them at -20°C or immediately enter the lysis and purification process.
  • 2 LB liquid medium containing 50 ⁇ g/mL kanamycin
  • Bacterial lysis Resuspend the above bacterial precipitate with lysis buffer (20mM PB buffer, 500mM sodium chloride, pH 7.4), with the resuspension ratio of 1g bacterial sludge to 20mL lysis buffer. After fully resuspended, 30mL portions were dispensed into 50mL centrifuge tubes and the bacteria were crushed using an ultrasonic cell crusher (Ningbo Xinzhi, SCIENTZ-II D). The sample was placed in an ice water bath, and the power was set to 250W using a ⁇ 6 amplitude. 3s, stop 3s, total working time 25min. Ultrasonic disruption was used to obtain bacterial lysate, which was then centrifuged at 12000rpm for 20min to fully separate soluble proteins from inclusion bodies.
  • lysis buffer 20mM PB buffer, 500mM sodium chloride, pH 7.4
  • 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 40 ⁇ L, add 10 ⁇ L 5 ⁇ reduced protein loading buffer (Biosharp, catalog number: BL502A) and mix well, place in a metal bath at 98°C for 8min for thermal denaturation, then centrifuge at 12000rpm for 10min, take an appropriate amount of supernatant and add it to the SDS-PAGE protein gel well (GenScript Bio, catalog number: M00930), electrophoresis at 150V for 1h, use Coomassie Brilliant Blue staining solution (Biosharp, catalog number: BL605A) for protein staining for 1h, then decolorize with protein decolorizing solution (10% acetic acid, 5% methanol), change the solution several times until the background is clean, and take pictures on a white light board.
  • the apparent molecular weight of the recombinant collagen peptide Co8 obtained by electrophoresis is about 50 kDa, which is close to the theoretical molecular weight of Co8, 44.6 kDa, indicating that the recombinant collagen peptide Co8 is correctly expressed.
  • the collagen peptide is expressed in a high amount and exists in the form of soluble protein, which is convenient for further purification and scale-up production.
  • SEQ ID NO.1 was repeated three times to obtain the sequence of Co10, which has 126 amino acids in total:
  • a short peptide of 6 amino acids ENLYFQ (SEQ ID NO.3) was also added to its N-terminus to form a recognition motif for TEV protease.
  • the nucleic acid sequence encoding SEQ ID NO.3+SEQ ID NO.6 was calculated according to the central dogma, and codon optimization was performed for E. coli preference, and a stop codon TAA was added to the 3' end to obtain the nucleic acid sequence SEQ ID NO.7.
  • GenScript Biotech Co., Ltd. was commissioned to synthesize the nucleic acid sequence of SEQ ID NO.7, and the expression plasmid p28Co10 of Co10 was obtained by inserting it into the pET-28a(+) expression vector using BamH I-HF and Xho I restriction endonucleases.
  • p28Co10 was transformed, cultured, induced for expression, lysed and detected in the same manner as in Example 2, and the results are shown in Figure 4.
  • the apparent molecular weight of the recombinant collagen peptide expressed by p28Co10 was about 18 kDa by electrophoresis, which was close to its theoretical molecular weight of 16.4 kDa, indicating that the recombinant collagen peptide Co10 was correctly expressed.
  • the recombinant collagen peptide Co10 has good water solubility, but the expression level on p28Co10 is moderate, so the pET-32a(+) expression vector was tried again.
  • the methods for constructing, transforming, culturing, and inducing expression of the Co10 sequence on the pET-32a(+) expression vector are as follows.
  • the bacterial lysis and analysis and detection methods are exactly the same as those in Example 2.
  • the p32Co10 plasmid was transformed into E. coli competent cells BL21 (DE3) (Merck, catalog number: CMC0014).
  • the specific operation process is as follows: 1 Take 1 ⁇ L (about 10 ng) of the plasmid in 100 ⁇ L of Escherichia coli competent cells BL21 (DE3), mix gently and let stand on ice for 30 min; 2 Heat shock the mixture in a 42°C water bath for 45 s, and then quickly put it on ice for 2 min; 3 Add 500 ⁇ L 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, 220 rpm for 40 min; 4 Take 200 ⁇ L 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, 100 ⁇ g
  • Culture and induction of expression Pick a single clone from the above LB plate and place it in 2 mL LB liquid medium (containing 100 ⁇ g/mL ampicillin), culture it overnight at 37°C and 220 rpm, transfer it to 200 mL LB liquid medium (containing 100 ⁇ g/mL ampicillin), and culture it at 37°C and 220 rpm until the OD600 of the bacterial solution is 0.6-1.0. (Cultivation time 1.5-2h), add IPTG (Biosharp, catalog number: BS119) with a final concentration of 0.2mM to induce expression, and the induction conditions are 18°C or 37°C, 220rpm and culture for 16-20h. Collect the cells by centrifugation and store at -20°C or immediately enter the lysis purification process.
  • IPTG Biosharp, catalog number: BS119
  • the expression of p32C10 is shown in Figure 5.
  • the apparent molecular weight of the recombinant collagen peptide expressed by the plasmid is about 33 kDa by SDS-PAGE electrophoresis, which is close to its theoretical molecular weight of 30.58 kDa, indicating that the recombinant collagen peptide Co10 is correctly expressed.
  • the collagen peptide is expressed in a high amount and exists in the form of soluble protein, which is convenient for further purification and scale-up production.
  • SEQ ID NO.1 was repeated 26 times to obtain the sequence of Co15, with a total of 1092 amino acids:
  • sequence length exceeds the natural length of the mature peptide of the ⁇ 1 chain of human type III collagen (1073 amino acids), and is closer to natural type III collagen in terms of structure and function.
  • the expression design of the Col5 sequence was performed to obtain a nucleic acid sequence SEQ ID NO.9 for the codon preference of Escherichia coli, which can encode a polypeptide fused with the TEV protease recognition motif ENLYFQ (SEQ ID NO.3) and Col5 (SEQ ID NO.8).
  • GenScript was commissioned to synthesize the nucleic acid sequence SEQ ID NO.9 and constructed into the pET28a(+) vector according to the method of Example 2, and then transformed into the BL21 host bacteria for screening, culture and induced expression to verify the expression of the designed sequence.
  • the results of SDS-PAGE analysis are shown in Figure 6.
  • the strain can express The molecular weight of the exogenous protein is about 120kDa, which is close to the theoretical molecular weight of Co15 fusion polypeptide 108.86kDa, indicating that the recombinant collagen peptide Co15 is correctly expressed.
  • the collagen peptide has a high expression level and good solubility, and can be further purified and scaled up for production.
  • SEQ ID NO.2 was repeated 12 times to obtain the sequence of Co9, which has 360 amino acids in total:
  • a short peptide of 6 amino acids ENLYFQ (SEQ ID NO.3) was added to the N-terminus of Co9 to form a recognition motif for TEV protease.
  • the nucleic acid sequence encoding SEQ ID NO.3+SEQ ID NO.10 was calculated according to the central dogma, and codon optimization was performed for E. coli preference.
  • the stop codon TAG was added to the 3' end to obtain the nucleic acid sequence SEQ ID NO.11.
  • the SEQ ID NO.11 nucleic acid sequence was inserted into the pET-28a(+) and pET-32a(+) expression vectors, and then transformed, cultured, induced for expression, harvested for lysis, and analyzed for detection.
  • the specific operations are shown in Examples 2 and 3, respectively. Different induction temperatures, culture times, and concentrations of inducer IPTG were tried for the expression strain of Co9, but the ideal expression level was not obtained.
  • Co9 was not expressed on the pET-28a(+) vector (theoretical molecular weight was 39.1 kDa); it was lowly expressed and soluble on the pET-32a(+) vector, with an apparent molecular weight of around 62 kDa and a theoretical molecular weight of 53.3 kDa.
  • the expression level of Co9 is low and cannot be industrialized.
  • SEQ ID NO.2 was repeated three times to obtain the sequence of Co11, which has 90 amino acids in total:
  • a short peptide of 6 amino acids ENLYFQ (SEQ ID NO.3) was added to the N-terminus of Co11 to form a recognition motif for TEV protease.
  • the nucleic acid sequence encoding SEQ ID NO.3+SEQ ID NO.12 was calculated according to the central dogma, and codon optimization was performed for E. coli preference, and a stop codon TAA was added to the 3' end to obtain the nucleic acid sequence SEQ ID NO.13.
  • the SEQ ID NO.13 nucleic acid sequence was inserted into the pET-28a(+) and pET-32a(+) expression vectors successively, and the two expression plasmids were transformed, cultured, induced to express, harvested, lysed, and analyzed and detected according to the specific operations of Examples 2 and 3, respectively. The results are shown in Figure 8.
  • Co11 was lowly expressed on the pET-28a(+) vector, with an apparent molecular weight of around 17 kDa, which is close to the theoretical molecular weight of 13.1 kDa; Co11 was highly expressed on the pET-32a(+) vector, with an apparent molecular weight of around 30 kDa on SDS-PAGE, which is close to the theoretical molecular weight of 27.2 kDa, indicating that Co11 was correctly expressed on both expression vectors.
  • the collagen peptide Co11 was highly expressed on the pET-32a(+) vector, and all existed in the form of soluble protein, which was convenient for further purification and scale-up production.
  • the Co6 sequence is the amino acid sequence of the 480-731th position of the human type III collagen ⁇ 1 chain sequence (sequence included in the National Center for Biotechnology Information, NP_000081.2):
  • the sequence has 252 amino acids, including the sequence fragments in Juyuan Bio's patent 202010631961.8 and Qizhuang Bio's patent 202010717892.2, and contains surrounding sequence fragments.
  • the sequence of Co6 contains more cell binding motifs GER and GEK, and is predicted to have good biological activity. The sequence has not been modified in any way and is 100% humanized.
  • a short peptide of 6 amino acids ENLYFQ (SEQ ID NO.3) was added to the N-terminus of Co6 to form a recognition motif for TEV protease.
  • the nucleic acid sequence encoding SEQ ID NO.3+SEQ ID NO.14 was calculated according to the central dogma, and codon optimization was performed for E. coli preference, and a stop codon TAA was added to the 3' end to obtain the nucleic acid sequence SEQ ID NO.15.
  • the SEQ ID NO.15 nucleic acid sequence was inserted into the pET-28a(+) and pET-32a(+) expression vectors, and the two expression plasmids were transformed, cultured, induced to express, harvested, lysed, and analyzed and tested according to the specific operations of Examples 2 and 3, respectively.
  • the results are shown in Figure 9.
  • Co6 was not expressed on the pET-28a(+) vector (theoretical molecular weight was 26.7 kDa); it was lowly expressed and soluble on the pET-32a(+) vector, with an apparent molecular weight of about 50 kDa and a theoretical molecular weight of 40.8 kDa.
  • the expression level of Co6 is too low to be industrialized.
  • Co7 was designed by directly splicing five segments rich in active motifs such as cell binding motifs and integrin binding motifs from the human type III collagen ⁇ 1 chain sequence (sequence included in the National Center for Biotechnology Information, NP_000081.2) and relatively hydrophilic segments, containing 423 amino acids:
  • the Co7 sequence has many active motifs and is predicted to have good biological functions; it also contains two cysteines at the C-terminus, which helps to form interchain crosslinks and stabilize the triple helix structure. 100% humanized.
  • a short peptide of 6 amino acids ENLYFQ (SEQ ID NO.3) was added to the N-terminus of Co7 to form a recognition motif for TEV protease.
  • the nucleic acid sequence encoding SEQ ID NO.3+SEQ ID NO.16 was calculated according to the central dogma, and codon optimization was performed for E. coli preference, and a stop codon TAG was added to the 3' end to obtain the nucleic acid sequence SEQ ID NO.17.
  • the SEQ ID NO.17 nucleic acid sequence was inserted into the pET-28a(+) and pET-32a(+) expression vectors, and the two expression plasmids were transformed, cultured, induced to express, harvested, lysed, and analyzed and tested according to the specific operations of Examples 2 and 3, respectively.
  • the results are shown in Figure 10.
  • Co7 was not expressed in the pET-28a(+) vector (theoretical The theoretical molecular weight is 43kDa); it is lowly expressed and soluble on the pET-32a(+) vector, with an apparent molecular weight of around 70kDa and a theoretical molecular weight of 57.2kDa.
  • the expression level of Co7 is too low to be industrialized.
  • Co1F is a recombinant collagen sequence reported by Japanese Sri Fine University Press Juming Yao et al. in a 2004 paper. It is composed of 8 repetitions of the sequence fragment GERGDLGPQGIAGQRGVVGERGERGERGAS (SEQ ID NO.18) of natural type I collagen, and the hinge region peptide GPPGPCCGGG (SEQ ID NO.19) of natural type III collagen is added to the C-terminus to promote the formation of triple helix.
  • the full-length amino acid sequence of Co1F is shown in SEQ ID NO.20, which consists of 250 amino acids. This patent expresses this sequence for reference.
  • the nucleic acid sequence encoding SEQ ID NO.20 was calculated based on the central dogma and optimized based on the codon preference of E. coli to improve translation efficiency and avoid high repetition of nucleic acid sequences.
  • the termination codon TGA was added to the 3' end to obtain the nucleic acid sequence SEQ ID NO.21.
  • GenScript Biotech Inc. was commissioned to synthesize the nucleic acid sequence of SEQ ID NO.21, and inserted into the pET-28a(+) expression vector (Novagen, Catalog No.: 70777) using restriction endonucleases Nde I (NEB, Catalog No.: R0111S) and BamH I-HF (NEB, Catalog No.: R3136S) to obtain the expression plasmid p28Co1F of Co1F.
  • p28Co1F was transformed, cultured, induced to express, harvested, lysed, and analyzed and tested according to the specific operations of Example 2. As shown in Figure 11, Co1F was moderately expressed on the pET-28a(+) vector and had good solubility. Its apparent molecular weight was around 30 kDa, which was close to its theoretical molecular weight of 27.0 kDa, indicating that Co1F was expressed correctly. However, for industrialization, the expression level of Co1F was not ideal.
  • T16C is a recombinant collagen sequence protected in Jinbo Patent 201811438582.6. It is composed of 16 repeats of the amino acid sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID NO.22) at positions 483-512 of the ⁇ 1 chain of human type III collagen (sequence included in the National Center for Biotechnology Information of the United States, NP_000081.2) (see SEQ ID NO.23), and a short peptide ENLYFQ (SEQ ID NO.3) is added to the N-terminus to form a recognition motif for TEV protease.
  • the nucleic acid sequence corresponding to T16C directly adopts the sequence SEQ ID NO.7 in patent 201811438582.6 (see SEQ ID NO.24), and entrusts GenScript Biotech Co., Ltd. to perform
  • the protein was synthesized and inserted into the pET-28a(+) expression vector (Novagen, Catalog No.: 70777) and the pET-32a(+) expression vector (Novagen, Catalog No.: 69015) using BamH I-HF (NEB Company, Catalog No.: R3136S) and Xho I (NEB Company, Catalog No.: R0146S) restriction endonucleases, respectively, to obtain expression plasmids p28T16C and p32T16C, respectively.
  • the two expression plasmids were transformed, cultured, induced to express, The cells were harvested, lysed, analyzed and tested, and the results are shown in FIG12 .
  • the electrophoresis band of the p28T16C expression protein was around 55kDa, close to the theoretical molecular weight of 49.1kDa; the electrophoresis band of the p32T16C expression protein was around 68kDa, close to the theoretical molecular weight of 63.3kDa, indicating that the recombinant protein T16C was expressed correctly.
  • T16C was expressed on the pET-28a(+) vector, it existed in the form of inclusion bodies, and there was almost no soluble T16C protein in the supernatant of the lysate, so further purification and renaturation would be very difficult; the protein expressed by T16C on the pET-32a(+) vector had good water solubility, which could be further separated and purified, and could be scaled up to industrialization.
  • Jinbo Bio has successfully industrialized the T16C protein and successfully applied it to skin care products and medical device products.
  • there are still some limiting factors in the capacity expansion of this protein such as the possibility of gelatinous precipitation during the process and the generation of degradation bands during storage.
  • Co4 is a recombinant collagen sequence designed by Guangzhou Qizhuang Biotechnology Co., Ltd., and is authorized for protection in patent 202010717892.2.
  • the Co4 sequence is a direct combination of the amino acid sequence of positions 950-1075 in the ⁇ 1 chain of human type I collagen (sequence included in the National Center for Biotechnology Information, NP_000079.2) and the amino acid sequence of positions 594-728 in the ⁇ 1 chain of human type III collagen (sequence included in the National Center for Biotechnology Information, NP_000081.2), and the hinge region peptide segment GPPGPCCGGG (SEQ ID NO.25) of human type III collagen is added to the C-terminus, with a total length of 271 amino acids, see sequence SEQ ID NO.26.
  • Co4 selects a specific (Gly-XY) repeat sequence on collagen, has an integrin binding domain and a cell proliferation-promoting domain, and the hinge region peptide segment at the C-terminus can promote the formation
  • Co4 has no expression on the pET-28a(+) vector (theoretical molecular weight is 27.3kDa); it has low expression and is soluble on the pET-32a(+) vector, with an apparent molecular weight of around 52kDa and a theoretical molecular weight of 41.5kDa.
  • the experiment showed that the expression performance of the nucleotide sequence optimized by Qizhuang Biotechnology was not good, and the expression level of the sequence was low, making it unsuitable for industrialization.
  • Co8 Co10, Co11, and Co15 have achieved better expression performance, stable high expression, and good hydrophilicity. Their expression levels have reached the level of T16C, far exceeding the levels of Co1F and Co4, and have great industrial development value.
  • the present invention also intercepts and designs some predicted collagen domain sequence fragments on the ⁇ 1 chain of human type III collagen, and constructs strains for expression verification.
  • the fragment lengths range from 60 to 81 amino acids, and after 8, 7 or 6 repetitions, the core sequences of F6, F12, F13, F14 and F15 are formed.
  • Comparative Example According to the method of Example 3, the corresponding nucleic acid sequence was synthesized and constructed into the pET32a (+) vector. The expression of each sequence was verified by SDS-PAGE analysis. The results are shown in Table 2.
  • the stability of Co8, Co10, and Co11 was investigated, including the stability of bacterial lysate and preliminary purified samples under different temperature conditions and after multiple freeze-thaw cycles.
  • the specific implementation is as follows:
  • IPTG Biosharp, catalog number: BS119
  • lysis buffer (20mM PB buffer, 500mM sodium chloride, pH 7.4) at a rate of 20mL buffer per 1g of bacteria, and divide into 50mL centrifuge tubes at 30mL each.
  • the tube was placed in an ice-water bath and the cells were broken using an ultrasonic cell crusher (SCIENTZ-II D, Ningbo Xinzhi) with a ⁇ 6 amplitude, a power of 250 W, ultrasound on for 3 seconds, off for 3 seconds, and a total working time of 25 minutes.
  • the bacterial lysate was obtained by ultrasonic crushing, and the bacterial fragments were removed by centrifugation at 12000 rpm for 20 minutes.
  • the bacterial lysate after centrifugation was purified by a gravity column filled with Ni-TED filler (Hui Yan, product number: HQ060313).
  • the column bed was equilibrated with 5 column volumes of lysis buffer (20mM PB buffer, 500mM sodium chloride, pH 7.4), and the bacterial lysate after centrifugation was added and allowed to flow slowly through the column bed by gravity to allow the recombinant collagen to fully bind to the filler; impurities were rinsed with 10 column volumes of washing buffer (20mM PB buffer, 500mM sodium chloride, 20mM imidazole, pH 7.4), and the target protein was washed with elution buffer (20mM PB buffer, 500mM sodium chloride, 200mM imidazole, pH 7.4), and the eluted samples were collected, desalted with Seplife RG-25 (Lan Xiao, product number: D1007211M) molecular sieves, and equilibrated and rinsed
  • the bacterial lysate after centrifugation and the preliminary purified recombinant collagen sample were sampled, and dispensed into several sterile PCR tubes at 80 ⁇ L/tube.
  • the tubes were marked and placed under different temperature conditions: -80°C, -20°C, 2-8°C, room temperature, and 37°C, with at least 6 tubes in each group.
  • 20 ⁇ L 5 ⁇ reduced protein loading buffer Biosharp, catalog number: BL502A
  • the above-packaged samples were subjected to repeated freeze-thaw treatment. After being frozen at -80°C overnight, they were taken out and placed at room temperature for 4 hours, and then taken back to -80°C refrigerator for overnight. This process was repeated 1, 3, and 5 times, and samples were taken for electrophoresis analysis.
  • FIG 14 is the test results of the preliminary purified samples placed under different temperature conditions for 12 days; the supernatant of the bacterial lysate and the preliminary purified samples of Co8, Co10, and Co11 were subjected to 1, 3, and 5 freeze-thaw cycles, and the target protein and impurity protein increased. There was no significant change in the proteins.
  • Figure 15 shows the test results of the initially purified samples after 1, 3, and 5 freeze-thaw cycles.
  • Figure 16 shows that the initially purified Co15 sample was placed at room temperature or 2-8°C for 15 days without obvious degradation. The experimental results also show that the degradation bands of the Jinbo sample increased significantly after being placed at 37°C for 7 days, as shown in Figure 17.
  • the supernatant of the bacterial lysate was obtained according to the method in Example 11, and then the recombinant collagen was separated and purified using a fully automatic protein purification system (Insect, Unique Auto Pure 100) and affinity filler Ni Focurose FF (IMAC) (Hui Yan, product number HQ060312, 5 mL pre-packed column).
  • a fully automatic protein purification system Insect, Unique Auto Pure 100
  • affinity filler Ni Focurose FF IMAC
  • 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; the column was eluted with 50 mL of wash buffer (20 mM PB buffer, 500 mM sodium chloride, 30 mM imidazole, pH 7.4) 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 absorption 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)
  • the eluted samples were desalted with a molecular sieve filled with Seplife R G-25 filler (Lanxiao, product number: D1007211M).
  • the column model was 16mm/200mm and balanced and flushed with desalting buffer (20mM PB buffer, pH 7.4) at a flow rate of 5mL/min.
  • the target protein was collected according to the A215 absorption value.
  • the regenerated chromatography column was rinsed with 25mL of pure water at a flow rate of 2mL/min; the column bed was equilibrated with 30mL of 20mM PB buffer (pH 7.4) at a flow rate of 2mL/min; the target protein solution containing His-rTEV protease was loaded at a flow rate of 1mL/min and then rinsed with 20mM PB buffer (pH 7.4).
  • the target protein that flowed through was collected according to the A215 absorption value.
  • the sample was a recombinant collagen solution with high purity and without any label.
  • Figure 18 is the purification result of recombinant collagen Co8, and its purity reached more than 99%.
  • the pre-treatment steps for Co15 purification are different: 15 mL of buffer is used for 1 g of the harvested cells. Resuspend in 20mM PB (pH 7.4), crush 3 times at 600bar high pressure, place overnight at 2-8°C to precipitate the target protein, centrifuge and discard the supernatant, resuspend the precipitate in 20mM PB (pH 7.4) containing 0.5M NaCl to dissolve the target protein, centrifuge again to collect the supernatant, and then perform chromatography purification and tag removal according to the method of this embodiment. SDS-PAGE detection was performed on the key step samples, and the results are shown in Figure 19. The purity of Co15 recombinant collagen peptide after nickel column purification reached more than 98%, and high-purity tag-free protein can be obtained by enzyme digestion.
  • Example 13 Cell proliferation promoting activity detection
  • the collagen solution obtained in Example 12 was used to detect the cell proliferation activity, and the CCK8 method was used to test NIH/3T3 cells (Procell, Catalog No. CL-0171).
  • the dehydrogenase in the mitochondria of living cells can reduce the water-soluble tetrazolium salt WST-8 to a yellow formazan substance, which has an absorption peak at a wavelength of 450nm. The higher the absorbance value, the more formazan dye is generated, thereby reflecting the greater number of living cells.
  • microscopic observation is used as an auxiliary means to evaluate the cell proliferation activity.
  • NIH/3T3 cells were seeded into 96-well plates at 5 ⁇ 10 3 cells/well.
  • the culture medium was 200 ⁇ L of DMEM containing 10% fetal bovine serum.
  • the culture conditions were 37°C and 5% CO 2 .
  • the original culture medium was aspirated, and 200 ⁇ L of DMEM culture medium containing 2% fetal bovine serum and 1 ⁇ g/mL, 10 ⁇ g/mL, and 100 ⁇ g/mL collagen was added.
  • the culture was continued for 48 hours. 20 ⁇ L/well of CCK8 reaction reagent was added, and the plates were incubated at 37°C for 1 hour.
  • the absorbance at 450 nm was detected using an enzyme reader.
  • the cell adhesion was tested using the collagen solution obtained in Example 12, and the detection was performed using a 96-well cell culture plate that was not treated with TC.
  • the culture plate was coated with 200 ⁇ L/well of DMEM medium containing 0, 0.5 or 1.0 mg/mL collagen, incubated at 37°C for 1 hour, the coating solution was aspirated, and the culture wells were washed twice with PBS, 200 ⁇ L/well; then NIH/3T3 cells were inoculated at 5 ⁇ 10 4 cells/well, the culture medium was 100 ⁇ L of DMEM containing 10% fetal bovine serum, and cultured at 37°C and 5% CO 2 for 1 hour, the culture was aspirated and the culture wells were washed twice with PBS, 200 ⁇ L/well, and finally 100 ⁇ L of DMEM medium was added again, and the number of adherent cells was observed under a microscope.
  • NIH/3T3 cells have poor adhesion to plates that have not been treated with TC.
  • Collagen can increase the adhesion of cells, thereby increasing the number of adherent cells.
  • This example compares the cell adhesion of recombinant type III collagen from Gavida and type I collagen extracted from human placenta (Sigma#C7774). The former clearly shows better cell adhesion.
  • Figure 23 shows that the number of NIH/3T3 cells that adhere to the plate is very small on the culture plate coated with Sigma#C7774 collagen or without collagen coating, but the culture plate coated with Co8 can significantly increase the number of adherent cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dermatology (AREA)
  • Peptides Or Proteins (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)

Abstract

本发明提供了一种胶原蛋白肽，其包含一个或多个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列，或者一个或多个与SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列有95%以上同一性的氨基酸序列，本发明还提供了该胶原蛋白肽的制备方法和用途。

Description

胶原蛋白肽及其制备方法和用途 发明领域：
本发明属于基因工程技术领域，具体地，本申请提供了一种胶原蛋白肽及其制备方法和用途。
背景技术：
胶原蛋白是所有动物体内含量丰富的一类结构蛋白质。在人体中，占蛋白质总量的30％，占皮肤干重的四分之三。胶原蛋白是细胞外基质的主要成分，与弹性蛋白、纤连蛋白等共同组成细胞生长的网络构架。胶原蛋白是造就皮肤外观的关键物质，为皮肤提供强度与弹性。人体内80-90％的胶原蛋白由I，II和III型组成，而三种不同的胶原蛋白分布在不同的组织中发挥着不同的作用，I型胶原蛋白是骨骼、皮肤、肌腱、韧带和牙本质的主要结构蛋白，它还是血管，心脏瓣膜和主动脉的结构蛋白。II型胶原蛋白的密度不如I型密实，是软骨中的主要胶原蛋白。肌肉、皮肤和动脉的功能实现依赖于III型胶原蛋白，它通常与I型胶原一起出现，在促进弹性方面起着关键作用。
除担任机体的结构支撑功能外，胶原蛋白也具备着卓越的生物学功能，为细胞生长提供良好的微环境。胶原蛋白上有细胞表面受体识别位点，有细胞整合素结合基序，可与细胞相互作用，促进细胞黏附、增殖生长和分化；因其良好的组织相容性，胶原蛋白在医疗上被用作心血管支架的涂层材料、人造骨骼和关节的表面材料以及人造角膜。胶原蛋白能与伤口紧密结合，阻止血液流出，同时渗入新生组织中作为细胞生长的支架；在组织修复和重建过程中，胶原蛋白也有利于细胞的识别和移行。因此胶原蛋白被广泛用作创口敷料，在止血的同时可促进创伤修复、伤口愈合。此外，胶原蛋白作为细胞活动的信号物质可促进自体胶原再生，被广泛应用到再生医学领域，如黏膜修复、防治脱发、改善湿疹、抗衰抗皱等。III型胶原蛋白与皮肤衰老紧密相关，如何补充III型胶原蛋白已成为广大求美者追求的方向；胶原蛋白填充剂，除直接填充作用外，还能促进自体胶原再生，相对于传统的被动填充材料有着独到的优势。随着研究深入和产业化的开展，胶原蛋白的应用领域得到了极大的拓展，已深度覆盖 了医疗健康、美容护肤和食品领域。
天然胶原蛋白分子量很大，成熟蛋白的单链长度都在1000个氨基酸以上，不同链之间相互交联形成大型的纤维状结构或网状结构，因此这类蛋白质的水溶性都较差，需要在酸性条件溶解，这极大限制了其应用。目前市售的胶原蛋白还是以动物来源为主，从牛皮、牛跟腱、猪皮、鱼皮等原材料中提取得到，该方法得到的胶原蛋白成本较低，但安全性不能保障，可能携带动物病毒，另外动物胶原蛋白的序列差异性及动物源杂质很容易引起人的免疫反应，因此这类胶原蛋白在医疗健康和医美护肤方面的应用受到了限制。采用基因工程手段对人胶原蛋白进行重组表达是一个很好的方向，可以从根源上解决安全性和免疫原性的问题，但是胶原蛋白本身的性质，如高分子量、低亲水性，决定这类蛋白很难进行全长链的重组表达。目前的研究主要是对人胶原蛋白的局部片段进行表达，或对多个片段的组合、单个片段的重复进行表达。
在庞大的胶原蛋白序列中如何选取有效的功能片段以及功能片段如何进行组合设计，以表达出稳定、高产、亲水性好的重组蛋白，仍然是胶原蛋白肽产业化的难点。
发明内容
申请人基于人胶原蛋白III型序列中的活性基序以及肽段的亲疏水性、电荷性质进行筛选、设计，并利用大肠杆菌表达体系进行表达验证，得到了高表达、水溶性好的胶原蛋白肽。基于该序列构建的表达菌株，经诱导发酵后得到的胶原蛋白肽经特异性蛋白酶切除融合标签后，与人胶原蛋白序列100％一致。该胶原蛋白肽可进一步开发成为人源化的胶原蛋白产品。
本发明第一方面，本申请提供了一种胶原蛋白肽，所述胶原蛋白肽包含n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列；或者所述胶原蛋白肽包含n个与SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列有95％以上同一性的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26。
进一步地，所述胶原蛋白肽包含n1个串联重复的SEQ ID NO.1所示的氨基酸序列，其中n1为2-40，较佳地，2-30；或者所述胶原蛋白肽包含n2个串联重 复的SEQ ID NO.2所示的氨基酸序列，其中n2位2-50，较佳地，2-30。
在另一优选例中，所述胶原蛋白肽的N端还含有标签去除用蛋白酶的识别基序。
在另一优选例中，所述蛋白酶的识别基序为TEV蛋白酶的识别基序。
在另一优选例中，所述蛋白酶的识别基序含有SEQ ID NO.3所示的序列。
在另一优选例中，所述胶原蛋白肽从N端到C端具有式I所示的结构：
Z0-L-Z1 (I)
式中，
Z0任选的标签去除用蛋白酶的识别基序；
L为无或连接肽；
Z1为n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26；
“-”为键。
在另一优选例中，所述胶原蛋白肽可以是被修饰的或未被修饰的。
在另一优选例中，所述胶原蛋白肽具有选自下组的氨基酸序列：
(a)如SEQ ID NO:4、6、8、10、12中任一所示的序列；
(b)与SEQ ID NO:4、6、8、10、12中任一所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，更佳地至少为98％，更佳地至少为99％的氨基酸序列。
在另一优选例中，所述胶原蛋白肽的氨基酸序列如SEQ ID NO:4、6、8、10、12中任一所示。
在另一优选例中，所述胶原蛋白肽的氨基酸序列如SEQ ID NO:4、6、8、12中任一所示。
本发明第二方面提供了一种胶原蛋白多肽，所述胶原蛋白多肽含有SEQ ID NO.14、SEQ ID NO.16中任一所示的序列。
在本发明的第三方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第一方面或本发明第二方面所述的胶原蛋白多肽。
在另一优选例中，所述多核苷酸为经密码子优化的多核苷酸。
在另一优选例中，所述核苷酸序列选自下组：
(a)所述核苷酸序列如SEQ ID NO.:5、7、9、11、13、15、17中任一所示；
(b)所述核苷酸序列与SEQ ID NO.:5、7、9、11、13、15、17中任一所示的核苷酸序列有≥95％的同一性，优选地≥98％，更优选地≥99％；和
(c)与(a)或(b)所述的核苷酸序列互补的核苷酸序列。
在另一优选例中，所述核苷酸序列包括DNA序列、cDNA序列、或mRNA序列。
在另一优选例中，所述核苷酸序列包括单链序列和双链序列。
在本发明的第四方面，提供了一种载体，所述载体包括如本发明的第三方面所述的多核苷酸。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、或其组合。
在本发明的第五方面，提供了一种宿主细胞，所述的宿主细胞含有如本发明的第四方面所述的载体，或染色体中整合有外源的如本发明的第三方面所述的多核苷酸。
在本发明的第六方面，提供了一种制备如本发明第一方面或本发明第二方面所述的胶原蛋白肽的方法，包括步骤：
(i)在合适的条件下，培养如本发明的第五方面所述的宿主细胞，获得本发明第一方面或本发明第二方面所述的胶原蛋白肽；和
(ii)任选的对步骤(i)中得到的所述胶原蛋白肽进行酶切、纯化和/或分离。
在本发明的第七方面，提供了一种组合物，所述组合物含有：
(I)本发明第一方面或本发明第二方面所述的胶原蛋白肽；和
(II)药学上可接受的载体或化妆品上可接受的赋形剂。
在另一优选例中，所述组合物用于促进细胞增殖活性，和/或增加细胞黏附性。
在另一优选例中，所述细胞包括表皮细胞、粘膜上皮细胞、真皮细胞、皮肤间充质细胞。
在另一优选例中，所述组合物中还含有其他可促进细胞增殖活性，和/或增加细胞黏附性的物质。在另一优选例中，所述化妆品上可接受的赋形剂选自下组：保湿剂、肌肤调理剂、增稠剂、柔润剂、乳化剂、抗氧化剂、防腐剂、抗紫外线剂、成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、香精、盐类、pH调节剂、调理剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分、维生素、氨基酸、核酸、包合化合物，溶剂(如水)，或其组合。
在另一优选例中，所述组合物的剂型选自下组：液体剂型、半固体剂型或固体剂型，更优选地为膏剂、霜剂、乳剂、油剂、粉剂、溶液剂、凝胶剂、喷雾剂、泡沫剂、混悬剂、洗剂或贴剂(sticks)。
在另一优选例中，所述组合物为面霜、乳液、精华液、洁面乳或肌底液。
在本发明的第八方面，提供了本发明第一方面或本发明第二方面所述的胶原蛋白肽的用途，用于制备一组合物或产品，所述组合物或产品用于(i)促进细胞增殖活性，和/或(ii)增加细胞黏附性。在另一优选例中，所述组合物或产品包括组织工程产品、化妆品、保健品或药物。
在本发明的第九方面，提供了一种(i)促进细胞增殖活性，和/或(ii)增加细胞黏附性的方法，包括步骤：
向有所需要的对象施用有效量的本发明第一方面或本发明第二方面所述的胶原蛋白肽或本发明第七方面所述的组合物。
在另一优选例中，所述施用包括皮肤外用、粘膜外用、创口敷贴。
在另一优选例中，所述施用有助于完善皮肤屏障，抚平细纹，补水保湿、美白淡斑，收缩毛孔，淡化黑眼圈；修复鼻腔、口腔、阴道、肛肠等粘膜屏障；止血和促进伤口愈合，预防疤痕形成。
在另一优选例中，所述对象为哺乳动物，如人、宠物狗、猫、大鼠或小鼠。另一方面，本申请提供了上述胶原蛋白肽的制备方法，所述制备方法包括基因序列合成；构建表达质粒；表达菌株转化；培养和表达；裂解和提取步骤。
进一步地，所述基因序列中包含上述胶原蛋白肽的编码序列。
进一步地，所述基因序列中还包含标签去除用蛋白酶的识别基序。
进一步地，所述蛋白酶的识别基序为TEV蛋白酶的识别基序SEQ ID NO.3。
进一步地，所述基因序列选自SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17。
进一步地，所述胶原蛋白肽序列选自SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.13。
进一步地，所述表达菌株为大肠杆菌。
进一步地，所述构建表达质粒时使用的载体选自pET28a+、pET32a+。
另一方面，本申请提供了包含上述胶原蛋白肽的组合物。
进一步地，所述组合物为组织工程产品、化妆品、保健品、食品或者药物组合物。
进一步地，所述组合物中还包含辅料。
另一方面，本申请提供了上述胶原蛋白肽在制备组织工程产品、化妆品、 保健品、食品或者药物组合物中的应用。
本申请中的序列同一性是指通过本领域中已知的算法，包括但不限于blast、GenPast计算所得的同一性。
本申请中95％以上同一性包括但不限于整数百分数，如95％、96％、97％、98％、99％的同一性，以及根据变化位点数量或算法可能得到的带有小数位的同一性，包括但不限于99.9％、99.8％、99.7％、99.6％、99.5％、99.4％、99.3％、99.2％、99.1％等。
本申请中与SEQ ID NO.1/SEQ ID NO.2所示的氨基酸序列有95％以上同一性的氨基酸序列与SEQ ID NO.1/SEQ ID NO.2具有相同或类似的功能和/或高级结构，包括但不限于亲水性、黏附性质、与某些成分如整合素的结合性能等。这样的序列本领域技术人员可以通过蛋白领域和基因领域的现有工具，如蛋白结构预测工具、疏水性预测工具、密码子设计工具、对相应基因序列进行取代、缺失和添加的试剂/试剂盒进行设计和制造。
胶原蛋白肽的编码序列本领域技术人员可以使用现有的氨基酸编码规则知识和设计工具，根据宿主的需要等因素设计获得。
蛋白表达过程中可用的标签，如His、Flag、MBP、GST、HA等本领域技术人员均可以根据需要和相关文献、说明常规选用。
去除标签可以使用选自化学方法、蛋白酶法、内含肽等本领域已知的方法，本领域技术人员均可以根据需要和相关文献、说明常规选用。
本申请制备方法中表达菌株转化以及培养和表达步骤的常规策略和工具本领域技术人员可以常规选用，适合的参数可以通过查阅资料和实验验证确定。例如转化可以使用氯化钙、原生质体、电穿孔等转化方法以及抗性标记筛选、重组子直接鉴定等鉴定方法；表达菌株可以为大肠杆菌、枯草芽孢杆菌、酵母、昆虫细胞、哺乳动物细胞、植物细胞等；表达过程中可使用IPTG、糖诱导、乳链球菌素、pH诱导等诱导系统。
本申请中的表达载体可选用各种市售/记载/研究中的载体，包括但不限于Pllp、pMBP、pET、pBAD、pCAI、pPOW系列载体。
本申请中的标签去除用蛋白酶的识别基序包括但不限于TEV蛋白酶、 Thrombin、Factor Xa、Enterokinase、PreScission、HRV 3C蛋白酶、SUMO蛋白酶等的识别基序，这些识别基序及其对应的核酸序列本领域技术人员可以参考现有文献、说明书或者分子生物学和蛋白领域的一般常识获得。
本申请组合物中的辅料包括但不限于药学、化妆品、保健品、食品中可接受的辅料，包括但不限于填充剂、溶剂、助溶剂、分散剂、粘度调节剂、抗氧化剂、甜味剂、粘合剂、pH调节剂等；本领域技术人员可以根据本领域的常规做法来选用这些辅料。本申请组合物，特别是中还可以包含其他功效成分，如保湿、美白、组织修复用的中西药成分、化学成分和细胞因子等。
本申请中的保健品、药物组合物可以为：注射剂，如注射液、粉针剂等；口服制剂，如片剂、胶囊剂、口服液等；外用制剂，如软膏剂、喷剂、贴剂等。组织工程产品可以为美容用、组织修复用产品。食品和化妆品可选食品和化妆品领域已知可用的形式。
本申请中涉及的现有技术如下：
1 Juming Yao,Satoshi Yanagisawa and Tetsuo Asakura，2004，Design,Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens，J.Biochem.136,643–649
2 ZL201811438582.6，杨霞；多肽、其生产方法和用途
3 ZL201811590564.X，赵健烽，高力虎，冯丽萍，黄建民；重组人源Ⅲ型胶原蛋白、表达菌株及其构建方法
4 ZL202010631961.8，赵健烽，徐鹏程，余继刚，高力虎，冯丽萍，黄建民；重组人源IxIII胶原蛋白、表达菌株及其应用
5 ZL202010717892.2，陈伟，熊盛，孙云起，郭朝万，柳耀平，苏志旭，熊灿，王一婷；一种重组人胶原蛋白肽及其应用
附图说明
图1：SEQ ID NO.1的疏水性预测；
图2：SEQ ID NO.2的疏水性预测；
图3：Co8表达情况电泳图；
图4：Co10(pET-28a(+)表达载体)表达情况电泳图；
图5：Co10(pET-32a(+)表达载体)表达情况电泳图；
图6：Co15表达情况电泳图；
图7：Co9表达情况电泳图；
图8：Co11表达情况电泳图；
图9：Co6表达情况电泳图；
图10：Co7表达情况电泳图；
图11：Co1F表达情况电泳图；
图12：T16C表达情况电泳图；
图13：Co4表达情况电泳图；
图14：初步纯化样品在不同温度条件下放置12天的检测结果；
图15：初步纯化样品经历1次、3次、5次冻融后的检测结果；
图16：初步纯化的Co15在室温或2-8℃放置15天的检测结果
图17：锦波生物的样品在37℃放置7天后的检测结果；
图18：重组胶原蛋白Co8的纯化结果；
图19：重组胶原蛋白Co15的纯化结果；
图20：Co8与锦波重组胶原蛋白、Sigma天然胶原蛋白的促增殖活性比较结果；
图21：不同用量的Co8的促增殖活性显微观察比较结果；
图22：Co15与Co8的促细胞增殖活性比较结果；
图23：Co8与Sigma#C7774对细胞黏附性能的影响。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，首次开发了一种胶原蛋白肽，所述胶原蛋白肽包含n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26，本发明首次发现，本发明的胶原蛋白肽能实现稳定高表达，具备良好的亲水性、稳定性、纯度和生物活性性能，可进一步开发成为组织工程产品、化妆品、保健品、食品或者 药物组合物等含胶原蛋白的产品。在此基础上完成了本发明。
本发明的胶原蛋白肽
如本文所用，术语“本发明具有良好的亲水性、稳定性、纯度和生物活性性能的胶原蛋白肽”、“本发明的胶原蛋白肽”、可互换使用，是指包含n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列；或者所述胶原蛋白肽包含n个与SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列有95％以上同一性的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26的胶原蛋白肽。
在一优选实施方式中，本发明的胶原蛋白肽从N端到C端具有如下式I所示的结构：
Z0-L-Z1 (I)
式中，
Z0任选的标签去除用蛋白酶的识别基序；
L为无或连接肽；
Z1为n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26；
“-”为键。
如本文所用，术语“连接肽”是指具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能的连接肽，优选为柔性连接子(或柔性接头)。
在一优选实施方式中，本发明的胶原蛋白肽具有选自下组的氨基酸序列：
(a)如SEQ ID NO:4、6、8、10、12中任一所示的序列；
(b)与SEQ ID NO:4、6、8、10、12中任一所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，更佳地至少为98％，更佳地至少为99％的氨基酸序列。
在一优选实施方式中，本发明的胶原蛋白肽含有SEQ ID NO.14、SEQ ID NO.16中任一所示的序列。
如本文所用，术语“胶原蛋白肽”还包括具有上述活性的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构 和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述胶原蛋白肽的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与本发明的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
本发明还提供本发明胶原蛋白肽的类似物。这些类似物与本发明的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的 类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
此外，还可以对本发明胶原蛋白肽进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
术语“本发明的多核苷酸”可以是包括编码本发明胶原蛋白肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或胶原蛋白肽的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的胶原蛋白肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的胶原蛋白肽和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。 通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明胶原蛋白肽编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的胶原蛋白肽。一般来说有以下步骤：
(1).用本发明的编码本发明胶原蛋白肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码胶原蛋白肽的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明胶原蛋白肽编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗 性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如人参细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的主要优点包括：
(1)本发明首次发现，本发明的胶原蛋白肽能实现稳定高表达，具备良好的亲水性、稳定性、纯度和生物活性性能，可进一步开发成为组织工程产品、化妆品、保健品、食品或者药物组合物等含胶原蛋白的产品。
(2)本发明的胶原蛋白肽可有效促进细胞增殖活性，和/或增加细胞黏附性。
具体实施方式
下面结合具体实施例详述本发明，但本发明的保护范围不局限于以下实施例。
实施例1基础序列的设计和性质
人III型胶原蛋白α1链序列(美国国立生物技术信息中心收录序列，NP_000081.2)第270-311位氨基酸序列：GRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAP GERGRP(SEQ ID NO.1)，该序列由42个氨基酸组成，含有细胞结合基序GER、GE K和整合素结合基序GLKGEN，使其具有很好的促细胞黏附功能；该区域富含带电氨基酸(精氨酸R，赖氨酸K，谷氨酸E)，有利于结合水分子，增加蛋白的亲水性，在线软件预测该肽段的疏水值均远小于0(软件预测结果如图1所示)；丰富的带电氨基酸也有利于形成肽链间的氢键网络，从而稳定胶原蛋白肽的三股螺旋结构，更接近胶原蛋白的天然状态。该氨基酸序列未做任何修饰，100％人源化。
该胶原蛋白肽可使用现有的成熟的蛋白表达系统进行表达，表达设计时可对该序列进行2-40次的串联重复，从而得到不同长度的胶原蛋白肽。
人III型胶原蛋白α1链序列(美国国立生物技术信息中心收录序列，NP_000081.2)第1080-1109位氨基酸序列：GSRGAPGPQGPRGDKGETGERGAAGIKGHR(SE Q ID NO.2)，该序列由30个氨基酸组成，含有细胞识别基序RGD和结合基序GER，使其具有很好的生物学活性；该区域富含带电氨基酸(精氨酸R，赖氨酸K，天冬氨酸D、谷氨酸E)，有利于结合水分子，增加蛋白的亲水性，在线软件预测该肽段的疏水值均小于0(软件预测结果如图2所示)；丰富的带电氨基酸也有利于形成肽链间的氢键网络，从而稳定胶原蛋白肽的三股螺旋结构，更接近胶原蛋白的天然状态。该氨基酸序列未做任何修饰，100％人源化。
该胶原蛋白肽可使用现有的成熟的蛋白表达系统进行表达，表达设计时可对该序列进行2-50次的串联重复，从而得到不同长度的胶原蛋白肽。
以下进行详细的序列设计和筛选。
实施例2Co8的设计与表达
对SEQ ID NO.1进行10次重复，得到Co8的序列，共420个氨基酸：
对Co8进行重组表达时，在其N端添加6个氨基酸的短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。根据中心法则计算出可以编码出SEQ ID NO.3+SEQ ID NO.4的核酸序列，并在3’端添加终止密码子TAA。对该核酸序列进行针对大肠杆菌偏好性的密码子优化，得到核酸序列SEQ ID NO.5:

表达质粒构建：委托金斯瑞生物科技股份有限公司进行SEQ ID NO.5核酸序列的合成，并通过BamH I-HF(NEB公司货号：R3136S)和Xho I(NEB公司，货号：R0146S)限制性内切酶插入至pET-28a(+)表达载体(Novagen公司，货号：70777)上得到Co8的表达质粒p28Co8。
表达菌株的转化：将p28Co8质粒转化至大肠杆菌感受态细胞BL21(DE3)(Merck公司，货号：CMC0014)。具体操作过程为：①取1μL(约10ng)的该质粒于100μL的大肠杆菌感受态细胞BL21(DE3)中，轻轻混匀后冰上静置30min；②将该混合物于42℃水浴锅中热激45s，然后迅速置冰上静置2min；③向该混合物中加入500μL无抗性的LB培养基(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠)，37℃，220rpm条件下培养40min；④取200μL该菌液均匀的涂布在含有卡那霉素的LB平板上(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠，15g/L琼脂，50μg/mL卡那霉素)；⑤将平板倒置于37℃恒温箱中过夜培养，待长出清晰可见的菌落。
培养和诱导表达：从上述LB平板上挑取单克隆菌落于2mL LB液体培养基(含50μg/mL卡那抗生素)中，37℃、220rpm过夜培养，转接至200mL LB液体培养基(含50μg/mL卡那抗生素)中，37℃、220rpm培养至菌液OD600在0.6-1.0时(培养时间1.5-2h)，加入终浓度为0.2mM的IPTG(Biosharp公司，货号：BS119)进行诱导表达，诱导条件为18℃、220rpm培养16-20h。离心收集菌体，保存于-20℃或立即进入裂解纯化流程。
菌体裂解：用裂解缓冲液(20mM PB缓冲液，500mM氯化钠，pH 7.4)重悬上述菌体沉淀，重悬比例为1g菌泥对应20mL裂解缓冲液。充分重悬均匀后，按30mL每份分装至50mL离心管中，利用超声波细胞粉碎机(宁波新芝，SCIENTZ-II D)进行菌体破碎。样品置冰水浴中，采用Φ6变幅杆，功率设定为250W，超声开 3s，停3s，总工作时长25min。超声破碎得到菌体裂解液，经12000rpm离心20min使可溶性蛋白与包涵体充分分离。
分析检测：采用SDS-PAGE对目的蛋白的表达情况进行监测分析。具体过程为：对离心前的菌体裂解液和离心后的菌体裂解液上清进行取样，取40μL，加入10μL 5×的还原型蛋白上样缓冲液(Biosharp，货号：BL502A)混匀，置金属浴中98℃控温8min进行热变性，然后经12000rpm、10min离心，取适量的上清液加入SDS-PAGE蛋白胶孔中(金斯瑞生物，货号：M00930)，以150V电压电泳1h后，用考马斯亮蓝染色液(Biosharp，货号：BL605A)进行蛋白染色1h，再以蛋白脱色液(10％醋酸，5％甲醇)进行脱色，多次换液至背景干净，在白光板上拍照。
结果如图3所示，重组胶原蛋白肽Co8通过电泳所得的表观分子量在50kDa左右，与Co8的理论分子量44.6kDa相近，表明重组胶原蛋白肽Co8得到正确的表达。该胶原蛋白肽表达量很高，并且都以可溶性蛋白的形式存在，方便进行进一步纯化以及放大生产。
实施例3Co10的设计与表达
对SEQ ID NO.1进行3次重复，得到Co10的序列，共126个氨基酸：
对Co10进行重组表达时，同样在其N端添加6个氨基酸的短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。根据中心法则计算出可编码SEQ ID NO.3+SEQ ID NO.6的核酸序列，并进行针对大肠杆菌偏好性的密码子优化，在3’端添加终止密码子TAA，得到核酸序列SEQ ID NO.7.

委托金斯瑞生物科技股份有限公司进行SEQ ID NO.7核酸序列的合成，同样通过BamH I-HF和Xho I限制性内切酶插入至pET-28a(+)表达载体上得到Co10的表达质粒p28Co10。p28Co10按照与实施例2中相同的方法进行转化、培养、诱导表达、裂解和检测分析，结果如图4所示。p28Co10表达的重组胶原蛋白肽通过电泳所得的表观分子量在18kDa左右，与其理论分子量16.4kDa相近，表明重组胶原蛋白肽Co10得到正确的表达。重组胶原蛋白肽Co10的水溶性很好，但在p28Co10上的表达量中等，因此又尝试了pET-32a(+)表达载体。
Co10序列在pET-32a(+)表达载体上的构建、转化、培养、诱导表达方法如下，菌体裂解和分析检测方法与实施例2完全相同。
表达质粒构建：将金斯瑞生物科技股份有限公司合成的SEQ ID NO.7核酸序列通过BamH I-HF(NEB公司货号：R3136S)和Xho I(NEB公司，货号：R0146S)限制性内切酶插入至pET-32a(+)表达载体(Novagen公司，货号：69015)上得到Co10的表达质粒p32Co10。
表达菌株的转化：将p32Co10质粒转化至大肠杆菌感受态细胞BL21(DE3)(Merck公司，货号：CMC0014)。具体操作过程为：①取1μL(约10ng)的该质粒于100μL的大肠杆菌感受态细胞BL21(DE3)中，轻轻混匀后冰上静置30min；②将该混合物于42℃水浴锅中热激45s，然后迅速置冰上静置2min；③向该混合物中加入500μL无抗性的LB培养基(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠)，37℃，220rpm条件下培养40min；④取200μL该菌液均匀的涂布在含有氨苄青霉素的LB平板上(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠，15g/L琼脂，100μg/mL氨苄青霉素)；⑤将平板倒置于37℃恒温箱中过夜培养，待长出清晰可见的菌落。
培养和诱导表达：从上述LB平板上挑取单克隆菌落于2mL LB液体培养基(含100μg/mL氨苄青霉素)中，37℃、220rpm过夜培养，转接至200mL LB液体培养基(含100μg/mL氨苄青霉素)中，37℃、220rpm培养至菌液OD600在0.6-1.0时 (培养时间1.5-2h)，加入终浓度为0.2mM的IPTG(Biosharp公司，货号：BS119)进行诱导表达，诱导条件为18℃或37℃、220rpm培养16-20h。离心收集菌体，保存于-20℃或立即进入裂解纯化流程。
p32C10的表达情况如图5所示，该质粒表达的重组胶原蛋白肽通过SDS-PAGE电泳得到的表观分子量在33kDa左右，与其理论分子量30.58kDa相近，表明重组胶原蛋白肽Co10得到正确的表达。该胶原蛋白肽表达量很高，并且都以可溶性蛋白的形式存在，方便进行进一步纯化以及放大生产。
实施例4Co15的设计与表达
对SEQ ID NO.1进行26次重复，得到Co15的序列，共1092个氨基酸：
该序列长度超过人III型胶原蛋白α1链成熟肽的天然长度(1073个氨基酸)。在结构和功能方面更接近天然的人III型胶原蛋白。
按照实施例2的方法，对Co15序列进行表达设计，得到针对大肠杆菌密码子偏好性的核酸序列SEQ ID NO.9，该序列可编码出融合TEV蛋白酶识别基序ENLYFQ(SEQ ID NO.3)和Co15(SEQ ID NO.8)的多肽。

委托金斯瑞合成核酸序列SEQ ID NO.9并按照实施例2的方法构建到pET28a(+)载体上，转化至BL21宿主菌中进行筛选培养和诱导表达，验证设计序列的表达情况。经SDS-PAGE分析检测，结果如图6所示，该菌株可表达出表 观分子量在120kDa左右外源蛋白，与Co15融合多肽的理论分子量108.86kDa相近，表明重组胶原蛋白肽Co15得到正确表达。该胶原蛋白肽表达量高且可溶性好，可进一步纯化以及放大生产。
实施例5Co9的设计与表达
对SEQ ID NO.2进行12次重复，得到Co9的序列，共360个氨基酸：
在Co9的N端添加6个氨基酸的短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。根据中心法则计算出可编码SEQ ID NO.3+SEQ ID NO.10的核酸序列，并进行针对大肠杆菌偏好性的密码子优化，在3’端添加终止密码子TAG，得到核酸序列SEQ ID NO.11.

将SEQ ID NO.11核酸序列先后插入到pET-28a(+)和pET-32a(+)表达载体上，进行转化、培养、诱导表达、收获裂解、分析检测，具体操作分别见实施例2和3。对Co9的表达菌株先后尝试了不同的诱导温度、培养时间、诱导剂IPTG浓度等条件，均未获得理想的表达量。表达情况如图7所示，Co9在pET-28a(+)载体上无表达(理论分子量为39.1kDa)；在pET-32a(+)载体上低表达并可溶，表观分子量在62kDa附近，理论分子量为53.3kDa。Co9表达量低，无法进行产业化。
实施例6Co11的设计与表达
对SEQ ID NO.2进行3次重复，得到Co11的序列，共90个氨基酸：
在Co11的N端添加6个氨基酸的短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。根据中心法则计算出可编码SEQ ID NO.3+SEQ ID NO.12的核酸序列，并进行针对大肠杆菌偏好性的密码子优化，在3’端添加终止密码子TAA，得到核酸序列SEQ ID NO.13。
将SEQ ID NO.13核酸序列先后插入到pET-28a(+)和pET-32a(+)表达载体上，分别按照实施例2和3的具体操作对两个表达质粒进行转化、培养、诱导表达、收获裂解、分析检测。结果如图8所示，Co11在pET-28a(+)载体上低表达，表观分子量在17kDa附近，和理论分子量13.1kDa相近；Co11在pET-32a(+)载体上得到了高表达，在SDS-PAGE上的表观分子量在30kDa附近，与理论分子量27.2kDa相近，说明Co11在两个表达载体上均正确表达。胶原蛋白肽Co11在pET-32a(+)载体上表达量高，且都以可溶性蛋白的形式存在，方便进行进一步纯化以及放大生产。
实施例7 Co6的设计与表达
Co6序列选取的是人III型胶原蛋白α1链序列(美国国立生物技术信息中心收录序列，NP_000081.2)第480-731位氨基酸序列：
该序列有252个氨基酸，包含了聚源生物专利202010631961.8和启妆生物专利202010717892.2中的序列片段，并含有周边的序列片段。Co6的序列中含有较多的细胞结合基序GER、GEK，预测具有较好的生物学活性。该序列未做任何修饰，100％人源化。
在Co6的N端添加6个氨基酸的短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。根据中心法则计算出可编码SEQ ID NO.3+SEQ ID NO.14的核酸序列，并进行针对大肠杆菌偏好性的密码子优化，在3’端添加终止密码子TAA，得到核酸序列SEQ ID NO.15.

将SEQ ID NO.15核酸序列先后插入到pET-28a(+)和pET-32a(+)表达载体上，分别按照实施例2和3的具体操作对两个表达质粒进行转化、培养、诱导表达、收获裂解、分析检测。结果如图9所示，Co6在pET-28a(+)载体上无表达(理论分子量为26.7kDa)；在pET-32a(+)载体上有低表达并可溶，表观分子量在50kDa附近，理论分子量为40.8kDa。Co6表达量过低，无法进行产业化。
实施例8 Co7的设计与表达
Co7的设计是从人III型胶原蛋白α1链序列(美国国立生物技术信息中心收录序列，NP_000081.2)中精选了5个富含细胞结合基序、整合素结合基序等活性基序，并且亲水性相对较好的区段，直接拼接而成，含有423个氨基酸：
Co7序列具有较多的活性基序，预测会有较好的生物学功能；并且在C端含有两个半胱氨酸，有助于形成链间交联，稳定三螺旋结构。该未做任何修饰， 100％人源化。
在Co7的N端添加6个氨基酸的短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。根据中心法则计算出可编码SEQ ID NO.3+SEQ ID NO.16的核酸序列，并进行针对大肠杆菌偏好性的密码子优化，在3’端添加终止密码子TAG，得到核酸序列SEQ ID NO.17.
将SEQ ID NO.17核酸序列先后插入到pET-28a(+)和pET-32a(+)表达载体上，分别按照实施例2和3的具体操作对两个表达质粒进行转化、培养、诱导表达、收获裂解、分析检测。结果如图10所示，Co7在pET-28a(+)载体上无表达(理 论分子量为43kDa)；在pET-32a(+)载体上有低表达并可溶，表观分子量在70kDa附近，理论分子量为57.2kDa。Co7表达量过低，无法进行产业化。
实施例9对照序列的设计和表达
对日本学者Juming Yao等设计的序列(命名Co1F)进行表达验证
Co1F是日本学者Juming Yao等在2004年的文献中报道的重组胶原蛋白序列，它是由天然I型胶原蛋白的序列片段GERGDLGPQGIAGQRGVVGERGERGERGAS(SEQ ID NO.18)的8次重复组成，并在C端加上天然III型胶原蛋白的铰链区肽段GPPGPCCGGG(SEQ ID NO.19)来促进三股螺旋的形成。Co1F的全长氨基酸序列如SEQ ID NO.20所示，有250个氨基酸组成。本专利表达该序列用作对照。
根据中心法则计算出可编码SEQ ID NO.20的核酸序列，并根据大肠杆菌的密码子偏好性对其进行优化，以提高翻译效率并避免核酸序列的高度重复。在3’端添加终止密码子TGA，得到核酸序列SEQ ID NO.21.

委托金斯瑞生物科技股份有限公司进行SEQ ID NO.21核酸序列的合成，并通过Nde I(NEB公司货号：R0111S)和BamH I-HF(NEB公司，货号：R3136S)限制性内切酶插入至pET-28a(+)表达载体(Novagen公司，货号：70777)上得到Co1F的表达质粒p28Co1F。
p28Co1F按照实施列2的具体操作进行转化、培养、诱导表达、收获裂解、分析检测。结果如图11所示，Co1F在pET-28a(+)载体上获得中度表达，并具有较好的可溶性。其表观分子量在30kDa附近，与其理论分子量为27.0kDa接近，说明Co1F表达正确。然而，对于产业化而言，Co1F的表达量并不理想。
对锦波专利201811438582.6中的序列(T16C)进行表达验证
T16C是锦波专利201811438582.6中保护的重组胶原蛋白序列，它是由人III型胶原蛋白α1链(美国国立生物技术信息中心收录序列，NP_000081.2)第483-512位氨基酸序列GERGAPGFRGPAGPNGIPGEKGPAGERGAP(SEQ ID NO.22)的16次重复组成(见SEQ ID NO.23)，并在N端添加短肽ENLYFQ(SEQ ID NO.3)以形成TEV蛋白酶的识别基序。
T16C对应的核酸序列直接采用专利201811438582.6中的序列SEQ ID NO.7(见SEQ ID NO.24)，委托金斯瑞生物科技股份有限公司对SEQ ID NO.24进行 合成，并通过BamH I-HF(NEB公司货号：R3136S)和Xho I(NEB公司，货号：R0146S)限制性内切酶先后插入至pET-28a(+)表达载体(Novagen公司，货号：70777)和pET-32a(+)表达载体(Novagen公司，货号：69015)上，分别得到表达质粒p28T16C和p32T16C。
按照实施列2和3的具体操作对两个表达质粒进行转化、培养、诱导表达、 收获裂解、分析检测，结果如图12所示。
T16C在两种表达载体上都表达出了重组蛋白，并且表达量较高。p28T16C表达蛋白的电泳条带在55kDa附近，与理论分子量49.1kDa相近；p32T16C表达蛋白的电泳条带在68kDa附近，与理论分子量63.3kDa相近，说明重组蛋白T16C表达正确。然而T16C在pET-28a(+)载体上表达后都是以包含体的形式存在的，在裂解液上清中几乎没有可溶性的T16C蛋白，因此进一步纯化、复性难度将会很大；T16C在pET-32a(+)载体上表达的蛋白则具有很好的水溶解性，可进一步分离纯化，可放大至产业化。目前锦波生物已经将T16C蛋白成功产业化，并成功应用到护肤品及医疗器械产品中。然而该蛋白在产能放大方面仍存在一些限制因素，比如在工艺过程中可能产生胶状沉淀，在放置过程中会产生降解条带。
对启妆专利202010717892.2中的序列(命名Co4)进行表达验证
Co4是广州启妆生物科技有限公司设计的重组胶原蛋白序列，在专利202010717892.2中授权保护。Co4序列是人I型胶原蛋白α1链(美国国立生物技术信息中心收录序列，NP_000079.2)中第950-1075位的氨基酸序列与人III型胶原蛋白α1链(美国国立生物技术信息中心收录序列，NP_000081.2)中第594-728位氨基酸序列的直接拼合，并在C端添加上人III型胶原蛋白的铰链区肽段GPPGPCCGGG(SEQ ID NO.25)，全长共271个氨基酸，见序列SEQ ID NO.26。Co4选取的是胶原蛋白上特定的(Gly-X-Y)重复序列，具有整合素结合结构域、促细胞增殖结构域，C端的铰链区肽段能促进三股螺旋结构的形成。
启妆生物根据选择的氨基酸序列重新设计和优化了重组人胶原蛋白肽的核苷酸序列，并通过翻译暂停理论，对胶原蛋白的核苷酸序列进行优化，使目的蛋白以可溶的方式表达，并且有充足的时间进行折叠得到正确构象。启妆生 物优化后的核苷酸序列如SEQ ID NO.27.
本研究委托金斯瑞生物科技股份有限公司对SEQ ID NO.27进行合成，并通过BamH I-HF(NEB公司货号：R3136S)和Xho I(NEB公司，货号：R0146S)限制性内切酶先后插入至pET-28a(+)表达载体(Novagen公司，货号：70777)和pET-32a(+)表达载体(Novagen公司，货号：69015)上，分别得到表达质粒p28Co4和p32Co4。按照实施列2和3的具体操作对两个表达质粒进行转化、培养、诱导表达、收获裂解、分析检测，结果如图13所示。
Co4在pET-28a(+)载体上无表达(理论分子量为27.3kDa)；在pET-32a(+)载体上有低表达并可溶，表观分子量在52kDa附近，理论分子量为41.5kDa。实验表明启妆生物优化后的核苷酸序列的表达性能也并不佳，该序列表达量低，不适合产业化。
在启妆生物的专利202010717892.2的实施例中，采用pET-28a(+)/BL21(DE3)表达系统对SEQ ID NO.27进行重组表达并获得成功；但从其SDS-PAGE图可以看到，表达量并不高。尽管如此，该专利的重组胶原蛋白也已经被应用到“丸美”旗下的功效性护肤品中。
实施例10各序列的表达情况汇总
对实施例2-9中的各序列的表达情况进行汇总，如表1所示。
对现有报道的重组胶原蛋白肽序列进行表达，仅锦波生物的T16C可获得高表达，并且可溶性较好，目前该重组胶原蛋白肽已进入产业化，被广泛应用于医疗健康和医美护肤产品中，并获得了中国药监局批准的III类医疗器械证书。
本专利设计的序列中，有Co8、Co10、Co11、Co15获得了较好的表达性能，稳定高表达且亲水性好，其表达量达到了T16C的水平，远远超过了Co1F、Co4的水平，具备很好的产业化开发价值。
表1.各胶原蛋白肽序列的表达情况汇总
注：“+”表示有表达或可溶性表达，“*”表示微弱表达，“-”表示无明显表达，“/”表示不适用。
对比例
除上述实施例外，本发明也对人III型胶原蛋白α1链上一些预测的胶原蛋白结构域序列片段进行截取设计，并构建菌株进行表达验证。片段长度在60-81个氨基酸不等，经8次、7次或6次重复，形成F6、F12、F13、F14、F15的核心序列。对比例按照实施例3的方法，合成相应的核酸序列并构建到pET32a(+)载 体上，转化至BL21(DE3)宿主菌中进行筛选培养和诱导表达，验证各序列的表达情况。经SDS-PAGE分析，结果如表2所示。
表2.对比例序列的表达情况
“-”代表无明显表达。
结果显示，基于预测片段进行重复得到的序列F6、F12、F13、F14、F15，按照与实施例3相同的方式进行菌株构建和诱导表达后，均未检测到明显目的蛋白。这表明即使经过软件预测的胶原蛋白片段，也有很大概率是不能表达的。如何设计和筛选出高表达、可溶性好且具有活性的片段是有较大挑战的。
实施例11稳定性考察
对Co8、Co10、Co11进行稳定性考察，包含菌体裂解液和初步纯化样品在不同温度条件下的放置稳定性和多次冻融后的稳定性。具体实施如下：
分别接种p28Co8-BL21(DE3)、p32Co10-BL21(DE3)、p32Co11-BL21(DE3)甘油菌于含相应抗生素的LB液体培养基中，37℃、220rpm过夜培养；转接至200mL含相应抗生素的LB液体培养基中，37℃、220rpm培养2h，加入终浓度为0.2mM的IPTG(Biosharp公司，货号：BS119)进行诱导表达，诱导条件为18℃、220rpm、20h，离心收集菌体，并洗涤1次。用裂解缓冲液(20mM PB缓冲液，500mM氯化钠，pH 7.4)按1g菌体对应20mL缓冲液重悬菌体，按30mL每份分装至50mL离心 管中，置冰水浴中，利用超声波细胞粉碎机(宁波新芝，SCIENTZ-II D)进行菌体破碎，采用Φ6变幅杆，功率250W，超声开3s，停3s，总工作时长25min。超声破碎得到菌体裂解液，经12000rpm 20min离心去除菌体碎片。
离心后的菌体裂解液经装有Ni-TED填料(汇研，货号：HQ060313)的重力柱进行纯化。以5倍柱体积的裂解缓冲液(20mM PB缓冲液，500mM氯化钠，pH 7.4)平衡柱床，加入离心后的菌体裂解液，靠重力缓慢流过柱床，使重组胶原蛋白充分结合到填料上；用10倍柱体积的洗涤缓冲液(20mM PB缓冲液，500mM氯化钠，20mM咪唑，pH 7.4)漂洗杂蛋白，最后以洗脱缓冲液(20mM PB缓冲液，500mM氯化钠，200mM咪唑，pH 7.4)洗下目的蛋白，收集洗脱样品，经Seplife R G-25(蓝晓，货号：D1007211M)分子筛脱盐，使用脱盐缓冲液(20mM PB缓冲液，pH 7.4)进行平衡和冲洗，得到初步纯化的重组胶原蛋白样品。
对离心后的菌体裂解液和初步纯化的重组胶原蛋白样品进行取样，按80μL/支分装至若干个无菌PCR管中，做好标记，在不同温度条件下放样：-80℃、-20℃、2-8℃、室温、37℃，每组至少6支，一段时间后取样加入20μL 5×还原型蛋白上样缓冲液(Biosharp，货号：BL502A)，混匀，热变性后进行SDS-PAGE检测，具体操作见实施例2。
取上述分装的样品进行反复冻融处理，-80℃冻存过夜后取出至室温放置4h，然后重新收回-80℃冰箱冻存过夜，重复1次、3次、5次，取样进行电泳分析。
取初步纯化的Co15样品按80μL/支分装至若干个无菌PCR管中，在室温和2-8℃条件下分别放置若干支，每隔一段时间取样进行电泳检测。
取锦波生物的“重组III型人源化胶原蛋白冻干纤维”分别以无菌的20mM PB缓冲液(pH 7.4)和生理盐水复溶成1mg/mL，分装至PCR管中，在-80℃、37℃条件下分别放置若干支，一段时间后取样进行电泳检测。
结果显示，Co8、Co10、Co11的菌体裂解液上清和初步纯化样品在不同温度条件下放置1天、5天、12天，目的蛋白无明显降解，杂质蛋白无明显增加，图14是初步纯化样品在不同温度条件下放置12天的检测结果；Co8、Co10、Co11的菌体裂解液上清和初步纯化样品经历1次、3次、5次冻融，目的蛋白和杂质 蛋白均无明显变化，图15是初步纯化样品经历1次、3次、5次冻融后的检测结果。图16显示，经初步纯化的Co15样品在室温或2-8℃放置15天，没有出现明显的降解。实验结果还表明锦波样品在37℃放置7天，其降解条带明显增加，如图17所示。
实施例12高纯度、无标签重组胶原蛋白的制备
按照实施例11中的方法获取菌体裂解液上清，然后采用全自动蛋白纯化系统(英赛思，Unique Auto Pure 100)和亲和填料Ni Focurose FF(IMAC)(汇研，货号HQ060312，5mL预装柱)进行重组胶原蛋白的分离纯化。以30mL的平衡缓冲液(20mM PB缓冲液，500mM氯化钠，10mM咪唑，pH 7.4)平衡柱床，流速2mL/min；以1mL/min流速上样裂解液上清至填料载量的80％；以50mL的洗涤缓冲液(20mM PB缓冲液，500mM氯化钠，30mM咪唑，pH 7.4)淋洗，流速2mL/min；以洗脱缓冲液(20mM PB缓冲液，500mM氯化钠，300mM咪唑，pH 7.4)洗下目的蛋白，根据A215吸收值收集洗脱峰；最后以25mL的再生缓冲液(20mM PB缓冲液，500mM氯化钠，500mM咪唑，pH 7.4)来清洗填料，流速2mL/min。
上述洗脱样品经装有Seplife R G-25填料的(蓝晓，货号：D1007211M)分子筛脱盐，柱型号为16mm/200mm，使用脱盐缓冲液(20mM PB缓冲液，pH 7.4)进行平衡和冲洗，流速5mL/min，根据A215吸收值来收集目的蛋白。
往上述目的蛋白溶液中加入适量的His-rTEV蛋白酶(Solarbio，P2060)，比例约为50EU/mL，于16℃条件下孵育4h后，再次经上述Ni Focurose FF(IMAC)预装柱层析分离。将上述再生后的层析柱用25mL纯水冲洗，流速2mL/min；以30mL 20mM PB缓冲液(pH 7.4)平衡柱床，流速2mL/min；以1mL/min流速上样含有His-rTEV蛋白酶的目的蛋白溶液并继以20mM PB缓冲液(pH 7.4)冲洗，根据A215吸收值来收集穿流的目的蛋白，该样品为高纯度且不带任何标签的重组胶原蛋白溶液。
对个关键步骤的样品进行取样，做SDS-PAGE检测，并分析纯度。图18是重组胶原蛋白Co8的纯化结果，其纯度达到99％以上。
Co15纯化的前处理步骤有所不同：将收获的菌体按1g菌体对应15mL缓冲液 重悬于20mM PB(pH 7.4)中，经600bar高压破碎3次后，置2-8℃条件下放置过夜使目的蛋白析出沉淀，离心弃去上清后以含有0.5M NaCl的20mM PB(pH 7.4)重悬沉淀，使目的蛋白溶解，再次离心收集上清，然后按照本实施例的方法对其进行层析纯化以及标签切除。对关键步骤样品进行SDS-PAGE检测，结果如图19所示，Co15重组胶原蛋白肽经镍柱纯化后纯度达到98％以上，并且可通过酶切获得高纯度的无标签蛋白。
实施例13促细胞增殖活性检测
使用实施例12中得到的胶原蛋白溶液进行促细胞增殖活性的检测，采用CCK8法，使用NIH/3T3细胞(普洛赛，货号CL-0171)进行测试。活细胞线粒体中的脱氢酶能将水溶性四唑盐WST-8还原成黄色甲臜类物质，该物质在450nm波长处有吸收峰，吸光值越高代表生成的甲臜染料越多，从而反映活细胞数量越多。在此实施例中，显微观察作为辅助手段来评估促细胞增殖活性。
将NIH/3T3细胞按5×103cells/孔接种到96孔板中，培养基为含10％胎牛血清的DMEM 200μL，培养条件为37℃，5％CO2，12h后吸出原有培养基，加入200μL含有2％胎牛血清和1μg/mL、10μg/mL、100μg/mL胶原蛋白的DMEM培养基，继续培养48h；加入CCK8反应试剂20μL/孔，37℃孵育1h，使用酶标仪检测450nm的吸光度。
实验比较了佳惟达的重组III型胶原蛋白、锦波的“重组III型人源化胶原蛋白冻干纤维”和人胎盘提取的I型胶原蛋白(Sigma#C7774)，CCK8法结果表明佳惟达的重组III型胶原蛋白的促细胞增殖活性明显高于人胎盘提取的I型胶原蛋白(Sigma#C7774)，也高于锦波的“重组III型人源化胶原蛋白冻干纤维”，图20展示了Co8与锦波重组胶原蛋白、Sigma天然胶原蛋白的比较结果，其中Sigma天然胶原蛋白因需要使用0.5M醋酸溶解，在100μg/mL浓度下可能因为醋酸的影响抑制了细胞的生长。
显微观察结果也同样可以表明佳惟达的重组III型胶原蛋白能够促进NIH/3T3细胞的贴壁生长，图21显示随着Co8添加量的增加，细胞贴壁生长的汇合度明显提高。图22显示，经0.5mg/mL Co15或Co8样品处理后，贴壁生长的细胞密度 明显高于阴性对照组，表明Co15和Co8均具有优异的促细胞增殖效果，Co15效果更佳。
实施例14细胞黏附性检测
使用实施例12中得到的胶原蛋白溶液测试细胞黏附性，采用未经TC处理的96孔细胞培养板进行检测。使用含有0、0.5或1.0mg/mL胶原蛋白的DMEM培养基200μL/孔包被培养板，37℃孵育1h，吸出包被液，以PBS洗涤培养孔两次，200μL/孔；然后按5×104cells/孔接入NIH/3T3细胞，培养基为含10％胎牛血清的DMEM 100μL，37℃、5％CO2条件下培养1h，吸出培养物并以PBS洗涤培养孔两次，200μL/孔，最后重新加入100μL DMEM培养基，在显微镜下观察贴壁细胞的数量。
NIH/3T3细胞在未经TC处理的平板上贴壁能力很差，胶原蛋白可增加细胞的黏附性，从而增加贴壁细胞的数量。该实施例比较了佳惟达的重组III型胶原蛋白和人胎盘提取的I型胶原蛋白(Sigma#C7774)的细胞黏附性，前者明显表现出更好的细胞黏附效果。图23显示NIH/3T3细胞在经Sigma#C7774胶原蛋白包被或无胶原蛋白包被的培养板上贴壁数量很少，但经Co8包被的培养板能显著增加贴壁细胞的数量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种胶原蛋白肽，其特征在于，所述胶原蛋白肽包含n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列；或者所述胶原蛋白肽包含n个与SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列有95％以上同一性的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26。
  2. 根据权利要求1所述的胶原蛋白肽，其特征在于，所述胶原蛋白肽包含n1个串联重复的SEQ ID NO.1所示的氨基酸序列，其中n1为2-40，较佳地，2-30；或者所述胶原蛋白肽包含n2个串联重复的SEQ ID NO.2所示的氨基酸序列，其中n2位2-50，较佳地，2-30。
  3. 如权利要求1所述的胶原蛋白肽，其特征在于，所述胶原蛋白肽的N端还含有标签去除用蛋白酶的识别基序。
  4. 如权利要求3所述的胶原蛋白肽，其特征在于，所述蛋白酶的识别基序含有SEQ ID NO.3所示的序列。
  5. 如权利要求1所述的胶原蛋白肽，其特征在于，所述胶原蛋白肽从N端到C端具有式I所示的结构：
    Z0-L-Z1  (I)
    式中，
    Z0任选的标签去除用蛋白酶的识别基序；
    L为无或连接肽；
    Z1为n个SEQ ID NO.1和/或SEQ ID NO.2所示的氨基酸序列，其中n为2-50，较佳地，n为8-28，更佳地，n为10-26；
    “-”为键。
  6. 如权利要求1所述的胶原蛋白肽，其特征在于，所述胶原蛋白肽具有选自下组的氨基酸序列：
    (a)如SEQ ID NO:4、6、8、10、12中任一所示的序列；
    (b)与SEQ ID NO:4、6、8、10、12中任一所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，更佳地至少为98％，更佳地至少为99％的氨基酸序列。
  7. 一种胶原蛋白多肽，其特征在于，所述胶原蛋白多肽含有SEQ ID NO.14、SEQ ID NO.16中任一所示的序列。
  8. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1或7所述的胶原蛋白多肽。
  9. 一种载体，其特征在于，所述载体包括权利要求8所述的多核苷酸。
  10. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求9所述的载体，或染色体中整合有外源的如权利要求8所述的多核苷酸。
  11. 一种制备如权利要求1或7所述的胶原蛋白肽的方法，其特征在于，包括步骤：
    (i)在合适的条件下，培养如权利要求10所述的宿主细胞，获得权利要求1或7所述的胶原蛋白肽；和
    (ii)任选的对步骤(i)中得到的所述胶原蛋白肽进行酶切、纯化和/或分离。
  12. 一种组合物，其特征在于，所述组合物含有：
    (I)权利要求1或7所述的胶原蛋白肽；和
    (II)药学上可接受的载体或化妆品上可接受的赋形剂。
  13. 权利要求1或7所述的胶原蛋白肽的用途，其特征在于，用于制备一组合物或产品，所述组合物或产品用于(i)促进细胞增殖活性，和/或(ii)增加细胞黏附性。
  14. 一种(i)促进细胞增殖活性，和/或(ii)增加细胞黏附性的方法，其特征在于，包括步骤：
    向有所需要的对象施用有效量的权利要求1或7所述的胶原蛋白肽或权利要求12所述的组合物。
PCT/CN2023/095616 2022-12-06 2023-05-22 胶原蛋白肽及其制备方法和用途 WO2024119724A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211556168.1 2022-12-06
CN202211556168.1A CN118146352A (zh) 2022-12-06 2022-12-06 胶原蛋白肽及其制备方法和用途

Publications (1)

Publication Number Publication Date
WO2024119724A1 true WO2024119724A1 (zh) 2024-06-13

Family

ID=91287543

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/095616 WO2024119724A1 (zh) 2022-12-06 2023-05-22 胶原蛋白肽及其制备方法和用途

Country Status (2)

Country Link
CN (1) CN118146352A (zh)
WO (1) WO2024119724A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103122027A (zh) * 2012-11-26 2013-05-29 杨霞 一种重组人源胶原蛋白及其生产方法
CN103725623A (zh) * 2013-12-19 2014-04-16 西安巨子生物基因技术股份有限公司 一种分泌表达人Ⅲ型胶原α链蛋白的毕赤酵母工程菌及其构建方法与应用
CN109593126A (zh) * 2018-11-28 2019-04-09 山西锦波生物医药股份有限公司 多肽、其生产方法和用途
CN109988243A (zh) * 2019-01-29 2019-07-09 江苏悦智生物医药有限公司 重组人源Ⅲ型胶原蛋白α1链及其应用
CN110194795A (zh) * 2019-06-13 2019-09-03 郭伟 一种重组人源胶原蛋白及其应用
CN111944057A (zh) * 2020-07-23 2020-11-17 广州启妆生物科技有限公司 一种重组人胶原蛋白肽及其应用
CN113880941A (zh) * 2020-07-03 2022-01-04 江苏江山聚源生物技术有限公司 重组人源IxIII胶原蛋白、表达菌株及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103122027A (zh) * 2012-11-26 2013-05-29 杨霞 一种重组人源胶原蛋白及其生产方法
CN103725623A (zh) * 2013-12-19 2014-04-16 西安巨子生物基因技术股份有限公司 一种分泌表达人Ⅲ型胶原α链蛋白的毕赤酵母工程菌及其构建方法与应用
CN109593126A (zh) * 2018-11-28 2019-04-09 山西锦波生物医药股份有限公司 多肽、其生产方法和用途
CN109988243A (zh) * 2019-01-29 2019-07-09 江苏悦智生物医药有限公司 重组人源Ⅲ型胶原蛋白α1链及其应用
CN110194795A (zh) * 2019-06-13 2019-09-03 郭伟 一种重组人源胶原蛋白及其应用
CN113880941A (zh) * 2020-07-03 2022-01-04 江苏江山聚源生物技术有限公司 重组人源IxIII胶原蛋白、表达菌株及其应用
CN111944057A (zh) * 2020-07-23 2020-11-17 广州启妆生物科技有限公司 一种重组人胶原蛋白肽及其应用

Also Published As

Publication number Publication date
CN118146352A (zh) 2024-06-07

Similar Documents

Publication Publication Date Title
CN111944057B (zh) 一种重组人胶原蛋白肽及其应用
CN110606896B (zh) 重组人源III型胶原蛋白α1链及其应用
CN110845603B (zh) 人胶原蛋白17型多肽、其生产方法和用途
AU640115B2 (en) Cloning and expression of transforming growth factor beta-2
JPH04505705A (ja) 血管内皮細胞成長因子の生産およびそれをコードするｄｎａ
CN113683679B (zh) 一种重组i型人源化胶原蛋白c1l6t及其制备方法和用途
US20130237486A1 (en) Collagen
JPH04502916A (ja) 精製した毛様体神経栄養因子
JPH10502360A (ja) ヒトインターロイキン４のアンタゴニストもしくは部分的アゴニストとして用いられる新規ｈＩＬ−４突然変異蛋白質
CN113637068B (zh) 一种重组i型人源化胶原蛋白c1l5t及其制备方法和用途
ES2686968T3 (es) Proteínas de fusión bifuncionales para inhibir la angiogénesis en el microambiente tumoral y para activar las respuestas inmunitarias adaptativas y los genes y usos de las mismas
CN112745394B (zh) 一种重组类人胶原蛋白及其制备方法和应用
CN112941081B (zh) 一种纤连蛋白突变体及其制备方法与应用
CN113683680A (zh) 一种重组ⅰ型人源化胶原蛋白c1l1t及其制备方法和用途
US9102755B2 (en) Highly stabilized epidermal growth factor mutants
JP7459362B1 (ja) 組換えヒト型化コラーゲンおよびその応用
Kim et al. Construction of chimeric human epidermal growth factor containing short collagen-binding domain moieties for use as a wound tissue healing agent
CN111333715B (zh) 一种类i型胶原蛋白纤维的制备方法
JPH08503858A (ja) 生物活性ポリペプチド融合ダイマー
CN118047857A (zh) 一种生物合成人体结构性材料的制备方法
CN102671185A (zh) 少棘蜈蚣多肽毒素omega-SLPTX-Ssm1a的应用
CN116874590B (zh) 一种重组iii型胶原蛋白及其制备方法
CN111620953B (zh) 类胶原融合蛋白组合物及制备方法
WO2024119724A1 (zh) 胶原蛋白肽及其制备方法和用途
CN113683681B (zh) 一种重组i型人源化胶原蛋白c1l3t及其制备方法和用途