WO2024002149A1 - 一种重组iii型胶原蛋白及其制备方法 - Google Patents

一种重组iii型胶原蛋白及其制备方法 Download PDF

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Publication number
WO2024002149A1
WO2024002149A1 PCT/CN2023/103141 CN2023103141W WO2024002149A1 WO 2024002149 A1 WO2024002149 A1 WO 2024002149A1 CN 2023103141 W CN2023103141 W CN 2023103141W WO 2024002149 A1 WO2024002149 A1 WO 2024002149A1
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Prior art keywords
type iii
iii collagen
recombinant type
cell
collagen
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PCT/CN2023/103141
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English (en)
French (fr)
Inventor
周伟
陆雨婷
徐国霞
张晶
刘明明
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Bloomage Biotech Tianjin Co Ltd
Bloomage Biotech Co Ltd
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Bloomage Biotech Tianjin Co Ltd
Bloomage Biotech Co Ltd
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Priority to EP23830322.6A priority Critical patent/EP4549456A4/en
Priority to KR1020257002336A priority patent/KR20250028405A/ko
Publication of WO2024002149A1 publication Critical patent/WO2024002149A1/zh
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • This application belongs to the field of biotechnology. Specifically, this application relates to a recombinant type III collagen and a preparation method thereof.
  • Collagen is a biopolymer that is widely present in animal connective tissues, accounting for approximately 25% to 30% of the total protein in the body. It is the functional protein with the widest distribution and the largest mass fraction in mammals. Because of its good biocompatibility, biodegradability, low immunogenicity and biological activity (for example, it can promote cell proliferation, differentiation, migration, repair and hemostasis), it is widely used in cosmetics, food, medicine , tissue engineering and other fields.
  • the current mainstream collagen preparation methods include animal-derived extraction methods and genetic engineering methods.
  • the extraction of animal-derived collagen is mainly industrialized through acid and enzymatic methods.
  • the cost is relatively lower, but there are risks of animal-derived disease infections, Allogeneic collagen may cause immune rejection or allergic reactions, production capacity limitations and other issues.
  • Genetic engineering technology produces recombinant collagen with single components, high safety, and controllable production process.
  • the use of genetic engineering technology to recombinantly express collagen has become a very significant research direction.
  • Genetic engineering involves different expression systems such as Escherichia coli, yeast, insect cells, mammalian cells, and genetically modified crops.
  • Expression systems such as mammalian cells and insect cells have high costs and long cycles, making it difficult to meet the needs of industrialization.
  • microbial fermentation The cost of producing recombinant collagen is low, the cycle is short, it is easy to culture, and it is easier to produce commercially.
  • this application uses genetic engineering technology to recombinantly express a new type III collagen, which can avoid viral risks and rejection reactions, has a definite molecular weight, and has mild production process conditions, which is conducive to maintaining the biological integrity of the protein. active.
  • this application provides a method for preparing the recombinant type III collagen.
  • this application relates to the following aspects:
  • a recombinant type III collagen characterized in that the recombinant type III collagen is any one of the following A1)-A3):
  • A1 A protein composed of the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3;
  • A2 A protein related to the recombinant type III collagen obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in A1);
  • A3 A protein that has more than 85% identity with A1) or A2) and is related to the recombinant type III collagen.
  • nucleic acid molecule characterized in that the nucleic acid molecule encodes the recombinant type III collagen described in item 1.
  • nucleic acid molecule according to item 2, characterized in that the nucleic acid molecule is any one of the following C1) or C2):
  • nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 is obtained by modifying and/or substituting and/or deleting and/or adding one or several nucleotides.
  • a host cell characterized in that the host cell contains the collagen described in Item 1 or the nucleic acid molecule described in Item 2 or the vector described in Item 4.
  • the host cell according to item 5 characterized in that the host cell is a prokaryotic cell or a eukaryotic cell.
  • the recombinant type III collagen is expressed by using the host cell described in any one of Items 5-7, and is then separated and purified.
  • Recombinant type III collagen according to item 1 or recombinant type III collagen encoded by the nucleic acid molecule according to item 2 or 3, or produced by the host cell according to any one of items 5-7
  • composition characterized in that the composition includes the recombinant type III collagen according to item 1, or the recombinant type III collagen encoded by the nucleic acid molecule according to item 2 or 3, or is composed of the recombinant type III collagen according to item 2 or 3. Recombinant collagen produced by the host cell according to any one of items 5 to 7.
  • composition according to item 10 which is used as food, cosmetics, health care products or pharmaceutical products.
  • An anti-inflammatory method comprising administering the recombinant type III collagen described in item 1 or the composition described in item 10 to a subject in need thereof.
  • a method for promoting cell proliferation, maintaining cell activity, cell differentiation or cell migration which includes administering the recombinant type III collagen described in Item 1 or the composition described in Item 10 to a subject in need.
  • this application adopts the following strategies: 1) Completely optimize the codons encoding various amino acids of recombinant type III collagen to the preferred codons of Pichia pastoris to facilitate recombination. Expression of type III collagen; 2) Optimize high-density fermentation conditions and processes to explore its high expression potential.
  • the active amino acid sequence of recombinant type III collagen is spliced, and the corresponding base sequence is codon-optimized according to the codon preference of Pichia pastoris for expressing heterologous proteins.
  • the Pichia pastoris expression system used in this application does not contain endotoxin, has low production cost and high protein expression.
  • the amino acid composition of the recombinant type III collagen produced by it is consistent with the ⁇ 1 chain of natural collagen. It will not cause immune rejection when applied to the human body. It can be widely used in the fields of food, cosmetics, health products and pharmaceutical products, and can be produced on an industrial scale. Compared with commercially available collagen products, its cell proliferation, migration and other effects are better.
  • the recombinant type III collagen of the present application can also significantly promote cell adhesion.
  • Cell adhesion is a dynamic process and the most basic life activity of cells. Therefore, the recombinant type III collagen of the present application can promote cell proliferation and maintain cell activity. , plays a key role in cell differentiation and cell migration.
  • the recombinant type III collagen of the present application also has anti-inflammatory effects.
  • Figure 1 is a plasmid map of the recombinant expression vector pPIC9K-COL in Example 2.
  • Figure 2 is an SDS-PAGE electrophoresis diagram of the recombinant type III collagen fermentation supernatant in Example 5.
  • Figure 3 is a graph showing the results of collagen promoting cell proliferation in Example 6. Different letters “a”, “b”, “c”, and “d” represent significant differences between data, p ⁇ 0.05.
  • Figure 4 is a diagram showing the results of collagen cell migration in Example 7.
  • Figure 5 is a graph showing the results of collagen cell migration in Example 7. Different letters “a”, “b”, “c”, “d”, and “e” represent significant differences between data, p ⁇ 0.05.
  • Figure 6 shows the adhesion effect of recombinant type III collagen on the HaCaT cell model.
  • Figure 7 shows the anti-inflammatory effect of recombinant type III collagen on the HaCaT cell model.
  • the present application provides a recombinant type III collagen.
  • amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • recombinant protein refers to using recombinant DNA or recombinant RNA technology to obtain a recombinant vector connected with a gene fragment that can be translated into a target protein, and then transforming it into a host cell that can express the target protein to express a specific Recombinant protein molecules are obtained from proteins.
  • In vitro recombinant protein expression systems mainly include the following: 1. Prokaryotic cell expression systems, such as E. coli; eukaryotic cell expression systems, such as yeast; mammalian cell expression systems, such as CHO cells, HEK293; and insect cell expression systems.
  • Amino acid sequence refers to the order in which amino acids are connected to form a peptide chain (or polypeptide).
  • Recombinant collagen refers to collagen prepared through genetic recombination technology, in which the DNA or RNA encoding collagen is usually inserted into a suitable expression vector and transformed into collagen molecules expressed in host cells. DNA or RNA is inserted into the host through homologous recombination or other methods known in the art.
  • Type I collagen is in the form of bundles, supporting the skin structure and maintaining skin toughness; while type III collagen is more like a fine network, scattered around type I, tightly meshing dermal cells and water, determining the elasticity and tenderness of the skin. Slipperiness. Type I and type III collagen are mainly found in connective tissues such as skin, tendons, ligaments, and blood vessels, forming an extracellular matrix network structure that supports organs and protects the body.
  • Another aspect of the application provides a nucleic acid molecule.
  • the nucleic acid molecule encodes recombinant type III collagen.
  • the sequence of the nucleic acid molecule is shown in SEQ ID NO.4.
  • the nucleic acid molecule encodes recombinant type III collagen.
  • the sequence of the nucleic acid molecule is shown in SEQ ID NO.5.
  • the nucleic acid molecule encodes recombinant type III collagen.
  • the sequence of the nucleic acid molecule is shown in SEQ ID NO. 6.
  • nucleic acid molecules are the general terms of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecular compounds polymerized from many nucleotide monomers and are one of the most basic substances of life.
  • Nucleotide sequence refers to the order of bases in DNA or RNA.
  • the nucleic acid molecule contains cDNA.
  • the nucleic acid molecule can be modified for use in the vector of the present application, such as for codon optimization.
  • sequences can be designed to contain terminal restriction site sequences for the purpose of cloning into a vector.
  • Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids within or isolated from one or more given cells. .
  • PCR polymerase chain reaction
  • Another aspect of the present application provides a vector comprising the above-mentioned nucleic acid molecule.
  • vector refers to a vector that is used in genetic engineering recombinant DNA technology.
  • target gene DNA fragment
  • it must include three parts: genetic marker gene, replication region, and target gene.
  • E. coli plasmid vectors many other artificially constructed plasmid vectors suitable for microorganisms, yeasts, plants, etc. have also been developed.
  • Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide species known in the art.
  • plasmid refers to a circular double-stranded DNA circle into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors eg, non-episomal mammalian vectors
  • some vectors can direct the expression of target genes.
  • Such vectors are referred to herein as "expression vectors.”
  • the recombinant expression vector may comprise a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements, which may be based on the nucleic acid sequence for expression and may be operably linked to the nucleic acid sequence to be expressed. host cells to choose.
  • the vectors are recombinant expression vectors pPIC9K-COL1, pPIC9K-COL2, and pPIC9K-COL3.
  • Another aspect of the present application provides a host cell comprising the above-mentioned vector.
  • a host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the present application.
  • "Host cell” encompasses any descendant of a parent cell that is not identical to the parent cell due to mutations caused by the replication process.
  • the host cell can be any cell useful in the production of recombinant humanized collagen of the present application.
  • the nucleic acid encoding the recombinant collagen can be isolated and inserted into one or more vectors for further cloning/or expression in host cells.
  • the host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of passage number.
  • the offspring may not be identical in nucleic acid content to the parent cells, but may contain mutations.
  • Methods for introducing vectors into host cells are well known. For example, electroporation is used to introduce vectors into host cells. The method can also be transfection, microbiology, etc.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.
  • the prokaryotic cell is Escherichia coli; preferably, the eukaryotic cell is Pichia pastoris.
  • Another aspect of the present application provides a method for preparing recombinant type III collagen, which method includes the following steps: using the above-mentioned host cells to express the recombinant type III collagen, and then isolating and purifying the obtained recombinant type III collagen.
  • the use of host cells for expression refers to culturing the host cells, and the culture medium and culture conditions are well known to those skilled in the art. This application does not impose any restrictions on the expression method, which can be confirmed as needed.
  • the expression method is induced expression.
  • the separation and purification methods include salting out method, ultrafiltration method, affinity chromatography method, gel filtration chromatography method, chromatography method, acid-base precipitation method and membrane separation method, preferably ultrafiltration method.
  • the present application provides the above-mentioned recombinant type III collagen, or the recombinant type III collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant type III collagen produced by the above-mentioned host cells in the preparation of food, cosmetics, health care products or pharmaceutical products. application.
  • compositions include the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • the composition is used as a food, cosmetic, nutraceutical or pharmaceutical product.
  • Another aspect of the present application provides the use of the above-mentioned recombinant type III collagen or the above-mentioned composition in promoting cell proliferation, maintaining cell activity, cell differentiation or cell migration.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • the The cells described above are HaCaT cells.
  • the composition includes the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • the use is for non-therapeutic purposes.
  • Another aspect of the present application provides the use of the above recombinant type III collagen or the above composition in anti-inflammation.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • the anti-inflammatory is anti-inflammatory to HaCaT cells.
  • the composition includes the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • the use is for non-therapeutic purposes.
  • Another aspect of the present application provides the use of the above-mentioned recombinant type III collagen or the above-mentioned composition in the preparation of drugs for promoting cell proliferation, maintaining cell activity, cell differentiation or cell migration.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • the cells are HaCaT cells.
  • the composition includes the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • Another aspect of the present application provides the use of the above recombinant type III collagen or the above composition in the preparation of drugs for anti-inflammatory.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • the anti-inflammation is anti-inflammation on HaCaT cells.
  • the composition includes the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • Another aspect of the present application provides a method for promoting cell proliferation, maintaining cell activity, cell differentiation or cell migration, which includes administering the above recombinant type III collagen or the above composition to a subject in need.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • the cells are HaCaT cells.
  • the composition includes the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • Another aspect of the present application provides an anti-inflammatory method, which includes administering the above recombinant type III collagen or the above composition to a subject in need thereof.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 1.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 2.
  • the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO. 3.
  • the cells are HaCaT cells.
  • the composition includes the above-mentioned recombinant type III collagen, or the above-mentioned recombinant type III collagen encoded by the nucleic acid molecule, or the above-mentioned recombinant collagen produced by the host cell.
  • anti-inflammation refers to helping the body resist the occurrence and spread of inflammation.
  • the tissue defends against the stimulation produced by various damage factors.
  • the pathological process produced during defense is an inflammatory reaction.
  • Through recombinant collagen or other The composition fights the inflammation caused by these factors, a process called anti-inflammatory.
  • Cell proliferation is an important life characteristic of organisms, which refers to the proliferation of cells by division. Single-celled organisms produce new individuals by cell division. Multicellular organisms produce new cells through cell division to replenish aging or dead cells in the body.
  • Cell differentiation refers to The process of cells from the same source gradually producing cell groups with different morphological structures and functional characteristics. The result is that the cells are different in space, and the same cell is different from its previous state in time.
  • Cell migration refers to cell crawling, cell movement or cell movement, which refers to the movement of cells after receiving migration signals or sensing the gradient of certain substances.
  • Cell migration is an alternating process in space and time of the extension of pseudopods at the cell head, the establishment of new adhesion, and the contraction of the cell body tail.
  • Cell migration is one of the basic functions of normal cells, a physiological process of normal growth and development of the body, and a common form of movement of living cells. Cell migration is involved in processes such as embryonic development, angiogenesis, wound healing, immune response, inflammatory response, atherosclerosis, and cancer metastasis.
  • Example 1 Sequence design of recombinant type III collagen
  • YPD yeast extract peptone dextrose
  • 1L contains 10g of yeast extract
  • G418 concentrations 0.5g/L, 1g/L, and 2g/L respectively.
  • Tryptone 20g, glucose 20g, agar powder 20g culture medium plate, culture at 30°C, and obtain transformants through high-concentration antibiotic culture screening.
  • the Pichia pastoris transformant can grow on a plate containing high concentration of G418, it means that the transformant contains multiple copies of the target gene, that is, multiple recombinant fragments have entered P. pastoris and been integrated into it through homologous recombination. on chromosomes. After this step, high-copy, highly-expressible recombinant Pichia pastoris engineering strains can be obtained.
  • the selected transformants were inoculated into 50mL YPD medium, 30°C, 200rpm, cultured with shaking for 24h, and used as primary seeds to be transferred into 280mL YPD medium, 30°C, 200rpm, cultured for 24h, and used as secondary seeds for transfer
  • main fermentation base salt (1L contains 40g of glycerin, 18.2g of K 2 SO 4 , 26.7mL of H 3 PO 4 , 0.93g of CaSO 4 2H 2 O, 14.9g of MgSO 4 and 4.13g of KOH). 10L tank fermentation medium. During the fermentation process, the growth temperature is 26°C, the pH value is 5.3, and the dissolved oxygen is controlled at about 20%.
  • the dissolved oxygen rises sharply, it indicates that the glycerol in the basal salt medium has been exhausted, and the starting rate is 15mL/L/h.
  • Add glycerol with a mass percentage concentration of 50%, and the glycerin with a mass percentage concentration of 50% contains 10mL/L trace element PTM1 (1L contains CuSO 4 ⁇ 5H 2 O 6g, NaI 0.08g, MnSO 4 ⁇ H 2 O 3g, Na 2 MoO 4 ⁇ H 2 O 0.2g, H 3 BO 3 0.02g, H 2 SO 4 5mL, CoCl 2 ⁇ 6H 2 O 0.5g, ZnCl 2 20g, FeSO 4 ⁇ 7H 2 O 75g, biotin 0.2 g, mixed), the dissolved oxygen is maintained at 15% to 20%.
  • BALB/C3T3 cells (mouse embryonic fibroblasts) in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were inoculated into a 96-well plate at a density of 1 ⁇ 10 5 cells/mL, with 100 ⁇ L per well, and divided into control groups. and experimental group. Place in a carbon dioxide cell incubator and incubate for 24 hours at 37°C and 5% CO2 .
  • Use serum-free culture medium (purchased from Jiangsu Kaiji Biotechnology Co., Ltd.) to prepare three recombinant type III collagen sample (COL1, COL2, COL3) solutions, fish collagen and commercially available recombinant collagen.
  • each solution is 400 ⁇ g/ mL, and filter the solution with a 0.22 ⁇ m filter to sterilize.
  • BALB/C3T3 cells were routinely cultured for 24 hours, discard the old culture medium and add 100 ⁇ L of serum-free culture medium or 100 ⁇ L of recombinant type III collagen sample solution.
  • the control group added the same amount of serum-free culture medium, and the experimental group added 100 ⁇ L of recombinant type III collagen.
  • Collagen sample solution 3 parallel samples per group.
  • recombinant type III collagen COL1, COL2, COL3, fish collagen and commercially available recombinant collagen acted on BALB/C3T3 cells for 24 hours.
  • collagen concentration was 400 ⁇ g/mL
  • COL1 and COL3 The relative cell proliferation rates of fish collagen, fish collagen and commercially available recombinant collagen were significantly higher than those of the control group, increasing by 20.7%, 24.3%, 11.2% and 19.7% respectively.
  • the relative cell proliferation rate of COL1 was significantly higher than that of fish collagen
  • the relative cell proliferation rate of COL3 was significantly higher than that of fish collagen and commercially available recombinant collagen.
  • the ability of COL2 to promote cell proliferation was not significantly different from that of the control group.
  • Example 7 Detection of cell migration activity of recombinant collagen by scratch method
  • HacaT cells in the logarithmic growth phase (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were taken as samples. After routine digestion, they were seeded into ibidi chambers in a 24-well plate at a density of 3 ⁇ 10 5 cells/mL. Add 70 ⁇ L of cell suspension to each of the left and right wells of the chamber, and add 400 ⁇ L of serum-free culture medium (purchased from Jiangsu Kaiji Biotechnology Co., Ltd.) outside the chamber. Place in a 37°C, 5% CO2 incubator for 24 hours.
  • the cell migration rate of COL3 was 3.6 times that of the control group, COL1 was 3.04 times that of the control group, and COL2 was 2.49 times that of the control group. Therefore, COL1, COL2, and COL3 all have strong cell migration activity. Compared with fish collagen and commercially available recombinant collagen, the relative cell migration rates of COL3 and COL1 were significantly higher than fish collagen and commercially available collagen.
  • Example 8 Evaluation of the adhesion effect of recombinant type III collagen on HaCaT cell model
  • Example 9 Evaluation of the anti-inflammatory effect of recombinant type III collagen on HaCaT cell model
  • inoculate cells Take HaCaT cells and use DMEM medium containing 10% FBS. When the cell density is about 80%, digest with 0.05% trypsin, centrifuge at 1000 rpm for 5 min, resuspend and count, inoculate into a 6-well plate, incubate at 37°C, 5 Culture under % CO 2 conditions. Then perform sample processing, aspirate the medium after 24 hours of seeding the plate, and add samples of corresponding concentrations. After the cells grow and become confluent, use a 200 ⁇ L pipette tip to make scratches in the cells, wash the cells three times with 1 ⁇ PBS, and add serum-free DMEM medium. The supernatant was collected after 6 hours, and the expression levels of inflammatory factors in each group were detected by ELISA.
  • the scratch treatment group can cause a significant increase in the inflammatory factor PGE 2 , which increased by 8.16 times; while the recombinant type III collagen treatment group COL1+scratch group, COL2+scratch group, The COL3+ scratch group could significantly inhibit the scratch-induced increase in PGE 2 expression by 69.2%, 49.5% and 66.1% respectively.
  • the scratch treatment group can cause a significant increase in TNF- ⁇ , an increase of 86.9%; while the recombinant type III collagen treatment group COL1+scratch group, COL2+scratch group, COL3+scratch group can significantly increase Inhibition of scratch-induced increase in TNF- ⁇ expression by 30.3%, 18.9%, and 27.8% respectively. This shows that recombinant type III collagen has good anti-inflammatory effects.

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Abstract

提供一种重组III型胶原蛋白及其制备方法和应用，重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1、2、3所示。

Description

一种重组III型胶原蛋白及其制备方法 技术领域
本申请属于生物技术领域，具体地，本申请涉及一种重组III型胶原蛋白及其制备方法。
背景技术
胶原蛋白是一种生物高分子，它广泛存在于动物结缔组织中，约占体内蛋白总量的25％～30％，是哺乳动物体内分布最广、质量分数最多的功能性蛋白。因其具有良好的生物相容性、可生物降解性、低免疫原性以及生物活性(例如，可促进细胞增殖、分化、迁移，用于修复和止血)，被广泛应用于化妆品、食品、医药、组织工程等领域。
目前主流的胶原蛋白制备方法包括动物源提取法和基因工程法两大类，动物源胶原蛋白的提取主要通过酸法和酶法进行产业化制备，成本相对更低，但存在动物源疾病感染、异源胶原蛋白可能导致免疫排斥或过敏反应、产能限制等问题。基因工程技术生产重组胶原蛋白组分单一、安全性高、生产过程可控。鉴于胶原蛋白具有广阔的应用前景，特别是在高端医用材料、保健品等领域对优质胶原蛋白的大量需求，利用基因工程技术重组表达胶原蛋白成为一个极具意义的研究方向。
基因工程涉及大肠杆菌、酵母、昆虫细胞、哺乳动物细胞、转基因作物等不同表达体系，哺乳动物细胞、昆虫细胞等表达体系成本高、周期长，难以满足产业化需求，相比之下，微生物发酵生产重组胶原蛋白成本低、周期短、培养较容易，更易于商业化生产。
细菌表达系统普遍存在诸如：产生的致热原致使表达产物难以应用于临床；目的蛋白以包涵体形式表达，致使产物纯化困难；原核表达系统的翻译后加工修饰体系不完善，表达产物的生物活性较低等缺点，采用巴氏毕赤酵母(Pichia pastoris)表达系统，可避免这些缺点，并能对翻译的蛋白质进行一定的翻译后修饰(包括二硫键形成和糖基化等)，有力支撑蛋白质生物学功 能的实现。以巴氏毕赤酵母建立表达系统具有微生物表达系统可高密度发酵生产、培养成本低、蛋白表达水平高等规模化工业生产的优点，且其可分泌于胞外，避免了菌体裂解过程带来的杂质蛋白，同时，其细胞壁成分中不含内毒素、肽聚糖。
发明内容
针对现有技术存在的上述问题，本申请利用基因工程技术重组表达一种新的III型胶原蛋白，其可避免病毒隐患以及排异反应，且分子量确定，生产过程条件温和，利于保持蛋白的生物活性。同时，本申请提供了该重组III型胶原蛋白的制备方法。
具体来说，本申请涉及以下方面：
1.一种重组III型胶原蛋白，其特征在于，所述重组III型胶原蛋白为如下A1)-A3)中的任一种：
A1)SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列组成的蛋白质；
A2)将A1)所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与所述重组III型胶原蛋白相关的蛋白质；
A3)与A1)或A2)具有85％以上同一性且与所述重组III型胶原蛋白相关的蛋白质。
2.一种核酸分子，其特征在于，所述核酸分子编码项1所述的重组III型胶原蛋白。
3.根据项2所述的核酸分子，其特征在于，所述核酸分子为如下C1)或C2)中的任一种：
C1)核苷酸序列为SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6的DNA分子；
C2)将SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与C1)所示的DNA分子具有85％以上的同一性，且具有相同功能的DNA分子。
4.一种载体，其中，所述载体包含项3所述的核酸分子。
5.一种宿主细胞，其特征在于，所述宿主细胞包含项1所述的胶原蛋白或项2所述的核酸分子或项4所述的载体。
6.根据项5所述的宿主细胞，其特征在于，所述宿主细胞为原核细胞或真核细胞。
7.根据项6所述的宿主细胞，其特征在于，所述真核细胞为毕赤酵母。
8.一种制备项1所述的重组III型胶原蛋白的方法，其特征在于，所述方法包括以下步骤：
利用项5-7中任一项所述的宿主细胞表达所述重组III型胶原蛋白，然后进行分离纯化得到。
9.根据项1所述的重组III型胶原蛋白，或根据项2或3所述的核酸分子编码的重组III型胶原蛋白，或由根据项5-7中任一项所述的宿主细胞产生的重组III型胶原蛋白在制备食品、化妆品、保健品或药械产品中的应用。
10.一种组合物，其特征在于，所述组合物包括根据项1所述的重组III型胶原蛋白，或根据项2或3所述的核酸分子编码的重组III型胶原蛋白，或由根据项5-7中任一项所述的宿主细胞产生的重组胶原蛋白。
11.根据项10的组合物，其用作食品、化妆品、保健品或药械产品。
12.一种项1所述的重组III型胶原蛋白或项10所述的组合物在促进细胞增殖、维持细胞活性、细胞分化或细胞迁移中的用途。
13.一种项1所述的重组III型胶原蛋白或项10所述的组合物在抗炎中用途。
14.根据项12或13所述的用途，其是用于非治疗目的的。
15.一种项1所述的重组III型胶原蛋白或项10所述的组合物在制备用于促进细胞增殖、维持细胞活性、细胞分化或细胞迁移的产品中的用途。
16.一种项1所述的重组III型胶原蛋白或项10所述的组合物在制备用于抗炎的产品中的用途。
17.一种抗炎方法，其包括向有此需要的受试者施用项1所述的重组III型胶原蛋白或项10所述的组合物。
18.一种促进细胞增殖、维持细胞活性、细胞分化或细胞迁移的方法，其包括向有此需要的受试者施用项1所述的重组III型胶原蛋白或项10所述的组合物。
本申请为尽可能提高巴氏毕赤酵母的表达水平，采用以下策略：1)将编码重组III型胶原蛋白的各种氨基酸的密码子完全优化为巴氏毕赤酵母的偏好密码子以利于重组III型胶原蛋白的表达；2)对高密度发酵条件及工艺进行优化，发掘其高表达潜力。
有益效果
本申请将重组III型胶原蛋白的活性氨基酸序列进行拼接，并根据巴氏毕赤酵母表达异源蛋白的密码子偏好性对相应的碱基序列进行密码子优化。通过菌株筛选及参数优化后，发酵液上清总蛋白浓度较优化前提高50％。
本申请采用的巴氏毕赤酵母表达系统，不含有内毒素，生产成本低，蛋白表达量较高。其生产的重组III型胶原蛋白的氨基酸组成与天然胶原蛋白α1链一致，应用于人体不会产生免疫排斥，可以广泛应用于食品、化妆品、保健品和药械产品领域，并进行工业化大规模生产。与市售的胶原蛋白产品相比，其细胞增值、迁移等功效更好。
本申请的重组III型胶原蛋白还能够显著促进细胞粘附，细胞粘附是一个动态过程，是细胞最基本的生命活动，因此，本申请的重组III型胶原蛋白在促进细胞增殖、维持细胞活性、细胞分化和细胞迁移中起到关键的作用。此外，本申请的重组III型胶原蛋白还具有抗炎的作用。
附图说明
图1是实施例2中重组表达载体pPIC9K-COL的质粒图谱。
图2是实施例5中重组III型胶原蛋白发酵上清液SDS-PAGE电泳图。
图3是实施例6中胶原蛋白促细胞增殖的结果图。不同字母“a”、“b”、“c”、“d”代表数据间具有显著性差异，p<0.05。
图4是实施例7中胶原蛋白细胞迁移的结果图。
图5是实施例7中胶原蛋白细胞迁移率的结果图。不同字母“a”、“b”、“c”、“d”、“e”代表数据间具有显著性差异，p<0.05。
图6是重组Ⅲ型胶原蛋白对HaCaT细胞模型的粘附作用。
图7是重组Ⅲ型胶原蛋白对HaCaT细胞模型的抗炎作用。
具体实施方式
下面结合附图和具体实施例来详细说明本申请。应理解，以下实施例仅用于说明本申请而不用于限制本申请的范围。
尽管本文使用了特定的术语，但它们仅用于一般性和描述性的意义，而不是为了限制的目的。除非另有定义，否则本文使用的所有技术和科学术语具有与公开描述的主题所属领域的普通技术人员通常理解的含义相同。
本文描述或引用的技术和程序是本领域技术人员通常熟知的且经常使用的常规方法。
本申请一方面提供了一种重组III型胶原蛋白。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。
在上述具体实施方案中，重组蛋白是指利用重组DNA或重组RNA技术，获得连接有可以翻译成目的蛋白的基因片段的重组载体，之后将其转入可以表达目的蛋白的宿主细胞从而表达特定的重组蛋白分子，而获得的蛋白质。体外重组蛋白的表达系统主要包括以下几种:1.原核细胞表达系统，例如大肠杆菌；真核细胞表达系统，如酵母；哺乳动物细胞表达系统，例如细胞CHO，HEK293；及昆虫细胞表达系统。氨基酸序列是指氨基酸相互连接形成肽链(或多肽)的顺序。根据氨基酸的结构和它们连接在一起的方式，氨基酸序列只能按照一个方向读取，并且以特定形式形成肽。所有氨基酸都具有一个常规结构，包含一个碳，和分别位于两侧的一个氨基NH3及羧基COOH。一个氨基酸的氨基和另一个氨基酸的羧基之间形成肽键，形成氨基酸序列。重组胶原蛋白是指通过基因重组技术制备的胶原蛋白，其中通常将编码胶原蛋白的DNA或RNA插入合适的表达载体中，转化进入宿主细胞中表达的胶原蛋白分子。将DNA或RNA通过同源重组或本领域公知的其它方式插入宿 主染色体，并因此用于转化宿主细胞以产生蛋白质。人体胶原蛋白根据组织部位、生理功能、分子结构不同而分为28种以上胶原蛋白，研究多的为I型、II型和III型胶原蛋白。其中I型胶原和III型胶原是皮肤中含量最多的2种胶原成分。I型胶原呈条束状，支撑皮肤结构，保持皮肤韧性；而III型胶原更像是细密的网状，散布在I型周围，紧紧网住真皮细胞与水分，决定着皮肤的弹性和嫩滑度。I型和III型胶原蛋白主要存在皮肤、肌腱、韧带、血管等结缔组织，构成细胞外基质网状结构，起到支撑器官、保护机体的作用，还与细胞附着、细胞迁移有关。市售胶原蛋白99％是从猪皮、牛皮、驴皮、鱼皮、鱼鳞等动物组织中提取的动物源胶原蛋白，对比人胶原蛋白在基因序列上存在根本性差异，属于非同源物质。进入人体后会被皮肤的免疫层所阻隔，无法深入到肌底层、真皮层。上述重组III型胶原蛋白的基因序列与人III型胶原蛋白基因序列高度一致，组织相容性好。
本申请另一方面提供了一种核酸分子。
在一个具体的实施方案中，所述核酸分子编码重组III型胶原蛋白。所述核酸分子的序列如SEQ ID NO.4所示。
在一个具体的实施方案中，所述核酸分子编码重组III型胶原蛋白。所述核酸分子的序列如SEQ ID NO.5所示。
在一个具体的实施方案中，所述核酸分子编码重组III型胶原蛋白。所述核酸分子的序列如SEQ ID NO.6所示。
在上述具体实施方案中，核酸分子是脱氧核糖核酸(DNA)和核糖核酸(RNA)的总称，是由许多核苷酸单体聚合成的生物大分子化合物，为生命的最基本物质之一。核苷酸序列指的是DNA或RNA中碱基的排列顺序。核酸分子含有cDNA，在一些情况下，可以修饰核酸分子以用于本申请载体中，如用于密码子优化。在一些情况下，出于克隆到载体的目的，可以将序列设计为含有末端限制性位点序列。核酸分子可以从多种来源获得，如通过一种或多种给定细胞内的或从所述一种或多种给定细胞中分离的编码核酸的聚合酶链式反应(PCR)扩增获得。
本申请另一方面提供了一种载体，所述载体包含上述的核酸分子。
在一个具体的实施方案中，载体是指在基因工程重组DNA技术中将 DNA片段(目的基因)转移至受体细胞的一种能自我复制的DNA分子。在基因工程中作为最常用，最简单的载体，必须包括三部分:遗传标记基因，复制区，目的基因。除常用的大肠杆菌质粒载体外，还发展了许多人工构建的其它适用于微生物、酵母、植物等的质粒载体。载体包括但不限于：单链，双链或部分双链的核酸分子；包含一个或多个游离末端，没有游离末端(例如环状)的核酸分子；包含DNA，RNA或两者的核酸分子；以及本领域已知的其它多核苷酸种类。一种类型的载体是“质粒”，其是指可以插入额外DNA片段的环状双链DNA环，例如通过标准分子克隆技术。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和游离型哺乳动物载体)。其它载体(例如，非游离型哺乳动物载体)在引入宿主细胞后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。此外，某些载体能够指导目的基因的表达。此类载体在本文中称为“表达载体”。重组表达载体可以包含适于在宿主细胞中表达核酸的形式，这意味着重组表达载体包括一种或多种调节元件，其可以基于用于表达的、可以与待表达的核酸序列可操作地连接的宿主细胞来选择。所述载体为重组表达载体pPIC9K-COL1、pPIC9K-COL2、pPIC9K-COL3。
本申请另一方面提供了一种宿主细胞，所述宿主细胞包含上述载体。
在一个具体的实施方案中，宿主细胞是指任何细胞类型，其易受包含本申请的多核苷酸的核酸构建体或表达载体的转化、转染、转导等。“宿主细胞”涵盖亲本细胞的任何后代，其由于复制过程发生突变与亲本细胞不完全相同。宿主细胞可以是在本申请的重组人源化胶原蛋白生产中有用的任何细胞。为了产生重组胶原蛋白，可以将编码重组胶原蛋白的核酸分离，并且将其插入一种或多种载体中，以在宿主细胞中进一步克隆/或表达。可以使用常规技术(例如，通过使用能够与编码重组胶原蛋白的基因特异性结合的寡核苷酸探针)容易地分离和测序这种核酸。所述宿主细胞是指已引入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括转化体和转化细胞，其包括原代转化细胞和源自其的后代，不考虑传代次数。后代在核酸含量上可能与亲代细胞不完全相同，但可能含有突变。对于载体导入宿主细胞中的方法是公知的，例如使用电转化将载体导入宿主细胞中，所述方法还可以是转染、微 注射技术、基因枪技术、脂质体介导法等。所述宿主细胞为原核细胞或真核细胞。所述宿主细胞选自毕赤酵母、酿酒酵母、大肠杆菌、枯草芽孢杆菌中的任意一种。优选地，所述原核细胞为大肠杆菌；优选地，所述真核细胞为巴氏毕赤酵母。
本申请另一方面提供了一种制备重组III型胶原蛋白的方法，所述方法包括以下步骤：利用上述的宿主细胞表达所述重组III型胶原蛋白，然后进行分离纯化得到。
在一个具体的实施方案中，所述利用宿主细胞表达指的是将宿主细胞进行培养，培养基和培养条件对于本领域技术人员来说是公知的。对于表达方式，本申请不作任何限制，其可以根据需要进行确认，例如表达方式为诱导表达。所述分离纯化的方法包括盐析法、超滤法、亲和层析法、凝胶过滤层析法、色谱层析法、酸碱沉淀法和膜分离法，优选为超滤法。
本申请另一方面提供了上述重组III型胶原蛋白，或上述核酸分子编码的重组III型胶原蛋白，或上述宿主细胞产生的重组III型胶原蛋白在制备食品、化妆品、保健品或药械产品中的应用。
本申请另一方面提供了一种组合物。所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。
在一个具体的实施方案中，组合物用作食品、化妆品、保健品或药械产品。
本申请另一方面提供了上述重组III型胶原蛋白或上述组合物在促进细胞增殖、维持细胞活性、细胞分化或细胞迁移中的用途。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。在一个具体的实施方案中，所 述细胞为HaCaT细胞。在一个具体的实施方案中，所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。在一个具体的实施方案中，所述用途是用于非治疗目的的。
本申请另一方面提供了上述重组III型胶原蛋白或上述组合物在抗炎中用途。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。在一个具体的实施方案中，所述抗炎为对HaCaT细胞抗炎。在一个具体的实施方案中，所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。在一个具体的实施方案中，所述用途是用于非治疗目的的。
本申请另一方面提供了上述重组III型胶原蛋白或上述组合物在制备用于促进细胞增殖、维持细胞活性、细胞分化或细胞迁移的药物中的用途。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。在一个具体的实施方案中，所述细胞为HaCaT细胞。在一个具体的实施方案中，所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。
本申请另一方面提供了上述重组III型胶原蛋白或上述组合物在制备用于抗炎的药物中的用途。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。在一个具体的实施方案中， 所述抗炎为对HaCaT细胞抗炎。在一个具体的实施方案中，所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。
本申请另一方面提供了一种促进细胞增殖、维持细胞活性、细胞分化或细胞迁移的方法，其包括向有此需要的受试者施用上述重组III型胶原蛋白或上述组合物。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。在一个具体的实施方案中，所述细胞为HaCaT细胞。在一个具体的实施方案中，所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。
本申请另一方面提供了一种抗炎的方法，其包括向有此需要的受试者施用上述重组III型胶原蛋白或上述组合物。
在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.1所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.2所示。在一个具体的实施方案中，所述重组III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。在一个具体的实施方案中，所述细胞为HaCaT细胞。在一个具体的实施方案中，所述组合物包括上述的重组III型胶原蛋白，或上述的核酸分子编码的重组III型胶原蛋白，或上述的宿主细胞产生的重组胶原蛋白。
在上述具体实施方案中，抗炎是指帮助机体抵抗炎症的发生和蔓延，此时组织对各种损伤因子产生的刺激进行防御，防御时产生的病理过程为炎症反应，通过重组胶原蛋白或其组合物能够抵抗这些因素造成的炎症，此过程称为抗炎。细胞增殖是生物体的重要生命特征，是指细胞以分裂的方式进行增殖。单细胞生物，以细胞分裂的方式产生新的个体。多细胞生物，以细胞分裂的方式产生新的细胞，用来补充体内衰老或死亡的细胞。细胞分化是指 同一来源的细胞逐渐产生出形态结构、功能特征各不相同的细胞类群的过程，其结果是在空间上细胞产生差异，在时间上同一细胞与其从前的状态有所不同。细胞分化的本质是基因组在时间和空间上的选择性表达，通过不同基因表达的开启或关闭，最终产生标志性蛋白质。细胞迁移是指细胞爬行、细胞移动或细胞运动，是指细胞在接收到迁移信号或感受到某些物质的梯度后而产生的移动。细胞迁移为细胞头部伪足的延伸、新的黏附建立、细胞体尾部收缩在时空上的交替过程。细胞迁移是正常细胞的基本功能之一，是机体正常生长发育的生理过程，也是活细胞普遍存在的一种运动形式。胚胎发育、血管生成、伤口愈合、免疫反应、炎症反应、动脉粥样硬化、癌症转移等过程中都涉及细胞迁移。
本说明书被认为足以使本领域技术人员能够实施本发明。除了本文所示和所描述的那些之外，根据前面的描述，本发明的各种修改对于本领域技术人员而言将是显而易见的，并且落入所附权利要求的范围内。
实施例1：重组III型胶原蛋白的序列设计
利用NCBI(https://www.ncbi.nlm.nih.gov/)网站获取天然人III型胶原蛋白α1链蛋白(参考序列号NP_000081)，根据优选人III型胶原蛋白成熟肽的有效序列，进行功能片段组合及等电点，蛋白稳定性等理化性质优化，设计出3条III型胶原蛋白序列COL1、COL2、COL3，具体序列详见序列表中的SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3。
实施例2：重组III型胶原蛋白的基因合成
按照巴氏毕赤酵母表达异源蛋白的密码子偏好性，设计并人工全基因合成3条III型胶原蛋白的核苷酸序列并亚克隆至pPIC9K质粒上，具体序列详见序列表中的SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6。全基因合成由南京金斯瑞生物科技股份有限公司完成，重组质粒载体命名为pPIC9K-COL1、pPIC9K-COL2、pPIC9K-COL3(图1)。
实施例3：巴氏毕赤酵母电转化
首先对pPIC9K-COL1、pPIC9K-COL2、pPIC9K-COL3重组质粒进行Sac I(购于Thermo Fisher公司)线性化，反应体系如下：
上述试剂加好后，混匀，37℃水浴反应2h，终止反应前，先取出5μL，在质量百分浓度为1.0％的琼脂糖凝胶电泳上检测是否酶切完全。之后，用DNA纯化试剂盒对线性化的pPIC9K-COL1、pPIC9K-COL2、pPIC9K-COL3质粒进行回收。
分别将5μg经Sac I内切酶线性化的pPIC9K-COL1、pPIC9K-COL2、pPIC9K-COL3质粒，与100μL巴氏毕赤酵母感受态细胞混匀，转至电极间距0.2cm的冰预冷的电转化杯中，电击4～10毫秒，加入1mL冰预冷的1M的山梨醇溶液将菌体混匀，涂布于MD(Minimal Dextrase；1L MD含有YNB 13.4g，生物素0.2g，葡萄糖20g，琼脂粉20g)培养基平板，30℃倒置培养2～3天，待MD培养基平板上长出菌落。
实施例4：多拷贝插入重组子的筛选
将MD培养基平板上长出的菌落用无菌牙签对应接种到G418浓度分别为0.5g/L、1g/L、2g/L的YPD(酵母浸出粉胨葡萄糖；1L中含有酵母提取物10g，胰蛋白胨20g，葡萄糖20g，琼脂粉20g)培养基平板上，30℃培养，通过高浓度抗生素培养筛选来获得转化子。毕赤酵母转化子若能在含高浓度G418的平板上生长，说明该转化子含有多拷贝的目的基因，即有多个重组片段进入了巴氏毕赤酵母体内并通过同源重组整合至其染色体上。经过这一步筛选可得到的高拷贝、可高效表达的重组巴氏毕赤酵母工程菌种。
实施例5：重组III型胶原蛋白的发酵
将筛选到的转化子接种于50mL YPD培养基中，30℃，200rpm，振荡培养24h，作为一级种子转接于280mL YPD培养基中，30℃，200rpm，培养24h，作为二级种子转接入装有3.5L的主发酵基础盐(1L中含甘油40g、K2SO4 18.2g、H3PO4 26.7mL、CaSO4·2H2O 0.93g、MgSO4 14.9g、KOH 4.13g混合制成)培养基的10L罐发酵。发酵过程中，生长温度26℃，pH值为5.3，溶氧控制在20％左右，当溶氧陡然上升时，表明基础盐培养基中的甘油已耗尽，开始速率为15mL/L/h流加质量百分浓度为50％的甘油，在质量百分浓度为50％甘油中含10mL/L微量元素PTM1(1L中含CuSO4·5H2O 6g、NaI 0.08g、MnSO4·H2O 3g、Na2MoO4·H2O 0.2g、H3BO3 0.02g、H2SO4 5mL、CoCl2·6H2O 0.5g、ZnCl2 20g、FeSO4·7H2O 75g、生物素0.2g，混合制成)，溶氧维持在15％～20％。发酵液的湿菌重达到180-200mg/mL时，停止补料，饥饿1h，甘油耗尽，流加甲醇诱导发酵，甲醇中含10mL/L PTM1微量元素，以速率为2mL/L/h流加2～3h，然后提高速率至10.9mL/L/h流加进行发酵。通过调节转速、罐压和通气量使溶氧大于15％，诱导发酵96h，发酵液上清电泳结果见图2，泳道M为蛋白Marker，其余泳道分别为COL1、COL2和COL3。
实施例6：重组III型胶原蛋白促细胞增殖/细胞毒性实验
取对数生长期BALB/C3T3细胞(小鼠胚胎成纤维细胞)(购自中国科学院上海细胞库)，以1×105个/mL密度接种于96孔板，每孔100μL，分为对照组和实验组。置于二氧化碳细胞培养箱，37℃、5％CO2常规培养24h。用无血清培养液(购自江苏凯基生物技术股份有限公司)配制三种重组III型胶原蛋白样品(COL1、COL2、COL3)溶液、鱼胶原和市售重组胶原，每种溶液浓度为400μg/mL，并将溶液用0.22μm滤膜过滤除菌。BALB/C3T3细胞常规培养24h后，弃去旧培养液，加入100μL无血清培养液或100μL重组III型胶原蛋白样品溶液，对照组为加入等量无血清培养液，实验组为加入100μL重组III型胶原蛋白样品溶液，每组3个平行样。继续培养24h后，弃去培养液，每孔加入100μL用无血清培养液稀释10倍的CCK-8(购自生工生物工程(上海)股份有限公司中)，放入细胞培养箱中继续孵育2h。采用CCK-8法检测细胞 相对增殖率，于450nm波长处用酶标仪测定吸光度。计算细胞相对增殖率(RGR)％＝实验组吸光度值/正常对照组吸光度值×100％。
如图3所示，本实施例中，重组III型胶原蛋白COL1、COL2、COL3、鱼胶原以及市售重组胶原作用于BALB/C3T3细胞24h，在胶原蛋白浓度为400μg/mL时，COL1、COL3、鱼胶原和市售重组胶原的促细胞相对增值率均显著高于对照组，分别提高了20.7％、24.3％、11.2％以及19.7％。此外，COL1的促细胞相对增值率显著高于鱼胶原，COL3的促细胞相对增值率显著高于鱼胶原和市售重组胶原。COL2的促细胞增殖能力与对照组无显著性差异。
实施例7：划痕法检测重组胶原蛋白细胞迁移活性
取处于对数生长期的HacaT细胞(购自中国科学院上海细胞库)作为样品，常规消化后以3×105个/mL的密度接种于24孔板中的ibidi小室。小室的左、右两孔各加入70μL细胞悬液，小室外补充400μL无血清培养液(购自江苏凯基生物技术股份有限公司)。放置于37℃、5％的CO2培养箱培养24h。取出ibidi小室，拍照，弃去培养液，加入1mL配制好的400μg/mL重组III型胶原蛋白COL1、COL2、COL3、鱼胶原和市售重组胶原的溶液，对照组加入1mL的无血清培养液。继续培养24h后观察并拍照。使用Image J软件(https://imagej.nih.gov/ij)对细胞迁移的图片进行处理，分析细胞划痕区域的愈合率。获得初始划痕面积和无细胞空白区域面积数据，计算迁移率，细胞迁移率(％)＝(1-划痕区域面积/初始划痕区域面积)×100％。
体外细胞迁移实验在一定程度上模拟了体内细胞迁移的过程，直接反映了细胞与胞外基质及基质影响下细胞之间的相互作用。细胞迁移活性是更有效表征胶原蛋白生物学活性的指标，迁移率越高，速度越快，说明胶原蛋白的生物学活性越佳。结果如图4所示，24h拍摄的细胞迁移实际对比图(两黑色线内为初始及细胞迁移后划痕伤口区域)及图5所示的计算出的细胞相对迁移率比较可知，COL1、COL2、COL3迁移率均显著高于对照组，其中细胞迁移率COL3为对照组的3.6倍，COL1为对照组的3.04倍，COL2为对照组的2.49倍，因此，COL1、COL2、COL3均具有较强的细胞迁移活性。与鱼胶原和市售重组胶原相比，COL3和COL1的细胞相对迁移率均显著高于鱼胶原和市售胶原。
实施例8：重组III型胶原蛋白对HaCaT细胞模型的粘附作用评估
待测样品及浓度如下表：
首先进行样品处理，用1x PBS配制2mg/ml的重组Ⅲ型胶原蛋白COL1、COL2、CoL3，将配制好的重组Ⅲ型胶原蛋白CoL3加到96孔板，阴性对照C组加入1x PBS，并在37℃培养箱放置2h，然后，轻轻吸除液体，用5％BSA在37℃培养箱封闭1h，用无血清培养基清洗2次。然后接种细胞，取HaCaT细胞用含10％FBS的DMEM培养基，待细胞密度约80％，用0.05％胰酶消化，1000rpm离心5min，用无血清培养基重悬。将无血清培养基重悬的HaCaT细胞以1x105个/孔的密度加入孔中，并在37℃下孵育1h，之后用PBS洗涤4次，加入MTT，3h后检测吸光度。用Excel软件处理分析MTT的吸光值，通过以下公式计算相对粘附率：
用Graphpad Prism绘图软件进行数据处理，结果表示为平均值±SD。组间比较采用t检验，*P<0.05表示有统计学差异，**P<0.01表示有显著性统计学差异，***P<0.001为有极其显著的统计学差异。
结果如图6显示，与阴性对照C组相比，2mg/ml的重组Ⅲ型胶原蛋白COL1、COL2、CoL3对HaCaT细胞相对粘附率均显著高于对照组，分别提高了144％、67％、114％，均有显著促粘附作用。
实施例9：重组Ⅲ型胶原蛋白对HaCaT细胞模型的抗炎作用评估
待测样品及浓度如下表：

首先接种细胞，取HaCaT细胞用含10％FBS的DMEM培养基，待细胞密度约80％，用0.05％胰酶消化，1000rpm离心5min，重悬计数，接种于6孔板，在37℃、5％CO2条件下培养。然后进行样品处理，种板24h后吸去培养基，分别加入对应浓度样本。待细胞生长融合后，用200μL枪头在细胞中制造划痕，用1×PBS洗涤细胞3次，加入无血清DMEM培养基。6h后收集上清，ELISA检测各组炎症因子的表达水平。
结果如图7显示，与阴性对照组C相比，划痕处理组可以引起炎症因子PGE2显著上升，增加了8.16倍；而重组Ⅲ型胶原蛋白处理组COL1+划痕组、COL2+划痕组、COL3+划痕组可以显著抑制划痕引起的PGE2表达增加，分别抑制了69.2％、49.5％和66.1％。与阴性对照组C相比，划痕处理组可以引起TNF-α的显著上升，增加了86.9％；而重组Ⅲ型胶原蛋白处理组COL1+划痕组、COL2+划痕组、COL3+划痕组可以显著抑制划痕引起的TNF-α表达增加，分别抑制了30.3％、18.9％、27.8％。说明重组Ⅲ型胶原蛋白具有良好的抗炎作用。
序列表：


Claims (13)

  1. 一种重组III型胶原蛋白，其特征在于，所述重组III型胶原蛋白为如下A1)-A3)中的任一种：
    A1)SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列组成的蛋白质；
    A2)将A1)所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与所述重组III型胶原蛋白相关的蛋白质；
    A3)与A1)或A2)具有85％以上同一性且与所述重组III型胶原蛋白相关的蛋白质。
  2. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1所述的重组III型胶原蛋白。
  3. 根据项2所述的核酸分子，其特征在于，所述核酸分子为如下C1)或C2)中的任一种：
    C1)核苷酸序列为SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6的DNA分子；
    C2)将SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与C1)所示的DNA分子具有85％以上的同一性，且具有相同功能的DNA分子。
  4. 一种载体，其中，所述载体包含权利要求2所述的核酸分子。
  5. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求1所述的胶原蛋白或权利要求2所述的核酸分子或权利要求4所述的载体。
  6. 根据权利要求5所述的宿主细胞，其特征在于，所述宿主细胞为原核细胞或真核细胞。
  7. 根据权利要求6所述的宿主细胞，其特征在于，所述真核细胞为毕赤酵母。
  8. 一种制备权利要求1所述的重组III型胶原蛋白的方法，其特征在于，所述方法包括以下步骤：
    利用权利要求5-7中任一项所述的宿主细胞表达所述重组III型胶原蛋白，然后进行分离纯化得到。
  9. 根据权利要求1所述的重组III型胶原蛋白，或根据权利要求2或3所述的核酸分子编码的重组III型胶原蛋白，或由根据权利要求5-7中任一项所述的宿主细胞产生的重组III型胶原蛋白在制备食品、化妆品或药械产品中的应用。
  10. 一种组合物，其特征在于，所述组合物包括根据权利要求1所述的重组III型胶原蛋白，或根据权利要求2或3所述的核酸分子编码的重组III型胶原蛋白，或由根据权利要求5-7中任一项所述的宿主细胞产生的重组III型胶原蛋白。
  11. 一种权利要求10的组合物在制备食品、化妆品或药械产品中的应用。
  12. 一种权利要求1所述的重组III型胶原蛋白或权利要求10所述的组合物在制备用于促进细胞增殖、维持细胞活性、细胞分化或细胞迁移的产品中的用途。
  13. 一种权利要求1所述的重组III型胶原蛋白或权利要求10所述的组合物在制备用于抗炎的产品中的用途。
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