WO2023284900A2 - 重组人胶原蛋白多肽及其应用 - Google Patents

重组人胶原蛋白多肽及其应用 Download PDF

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WO2023284900A2
WO2023284900A2 PCT/CN2022/118748 CN2022118748W WO2023284900A2 WO 2023284900 A2 WO2023284900 A2 WO 2023284900A2 CN 2022118748 W CN2022118748 W CN 2022118748W WO 2023284900 A2 WO2023284900 A2 WO 2023284900A2
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recombinant human
human collagen
collagen polypeptide
nucleic acid
host cell
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WO2023284900A3 (zh
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范代娣
范翠英
段志广
贺婧
严建亚
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陕西巨子生物技术有限公司
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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
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the technical field of bioengineering, in particular to a recombinant human collagen polypeptide and its application.
  • Collagen is one of the most abundant proteins in animals. It is widely distributed in mammalian skin, bones, tendons, ligaments, cartilage and blood vessels. It is the main protein component of connective tissue and an important component of extracellular matrix. Element. The content of collagen in the human body accounts for 25% to 30% of the total protein, and it is rich in amino acids such as glycine, proline, and hydroxyproline that the human body needs. Collagen, a structural protein, plays an important role in protecting the body and supporting organs. This makes collagen widely used in medicine, food, beauty and biomaterials, etc. It has been a hot research topic in recent years.
  • Collagen exists widely in the extracellular matrix, and its structure is a triple helix formed by the winding of three polypeptide chains. Collagen types vary by their combination of three alpha chains. So far, 26 genetically distinct collagen types have been identified. It can be roughly divided into the following categories: fiber-forming collagen, basement membrane collagen, microfibrillar collagen, anchor fibrils, transmembrane collagen, and incompletely characterized collagen.
  • type I collagen whose triple helix composition is mainly ⁇ 1 and ⁇ 2 chains, which are the most abundant in bones, tendons, skin, cornea and ligaments
  • type II collagen whose triple helix composition is [ ⁇ 1(II )] 2 , present in tissues such as hyaline cartilage, vitreous body, and intervertebral disc
  • type III collagen whose triple helix composition is [ ⁇ 1(III)] 3 , mainly distributed in embryos, gastrointestinal tract, and periodontal ligament
  • type IV collagen Protein whose triple-helix composition form is [ ⁇ 1(IV)] 3 , [ ⁇ 1(IV)] 2 ⁇ 2(VI), [ ⁇ 2(I)] 3 , is a component of basement membrane, and contains a lot of hydroxylysine.
  • the sugar content is relatively high, containing both 4-hydroxyproline and 3-hydroxyproline.
  • Collagen is the scaffold of basement membrane, bone, cartilage and other structures, and can be used in the treatment of burns, trauma and corneal diseases. Collagen plays an important role in wound healing, organ development and tissue repair. Clinical applications include: medical collagen injections for cosmetic plastic surgery, collagen membranes for burns and trauma treatment, collagen hemostatic sponges for wound hemostasis, etc. Collagen is an important component of the extracellular matrix of the skin, and has the effects of moisturizing, replenishing skin collagen, and anti-aging. Because collagen has the above biological functions, collagen has a wide range of applications in the field of medicine and health (such as hemostasis, beauty, drug delivery, wound repair, drug sustained release, etc.).
  • the main source of collagen is extracted from animal skin, bone, and other tissues by acid, alkaline hydrolysis and enzymatic hydrolysis. Since it is derived from animal tissues, there is a risk of cross-infection for animal-derived diseases or human-infected diseases. With the increasing maturity of genetic engineering technology, the preparation of recombinant collagen by molecular biological methods has gradually replaced the trend of extracting collagen from animal tissues. Recombinant collagen produced in prokaryotic or eukaryotic expression systems has better cell adhesion, tissue compatibility, skin permeability, stability, and can promote cell formation and growth, compared with traditional animal collagen Its unique advantages (no virus hidden danger, water solubility, low rejection, etc.).
  • the purpose of the present invention is to provide a recombinant human collagen polypeptide, which can be used for inhibiting angiogenesis factors, regulating endothelial cell plasticity, preparing cell growth promoting agents, and preparing cell adhesion promoting agents. It can be widely used in the fields of biochemical industry and cosmetics.
  • a recombinant human collagen polypeptide the amino acid sequence of the recombinant human collagen polypeptide is as shown in SEQ ID NO: 2 or an amino acid sequence having at least 90% identity with SEQ ID NO: 2.
  • nucleic acid molecule wherein the nucleic acid molecule encodes the recombinant human collagen polypeptide described in item 1, preferably the nucleic acid molecule is labeled with a label, more preferably, the label is 6*His, GST, MBP or SUMO .
  • a vector wherein the vector comprises the nucleic acid molecule described in item 2, preferably, the vector is pPIC9k, pPICZ ⁇ A, pGAPZA, pHIL-S1 or pYAM75P.
  • a host cell comprising the vector according to item 3.
  • the host cell according to item 4 wherein the host cell is a yeast host cell, preferably Pichia pastoris, more preferably Pichia pastoris.
  • a method for preparing the recombinant human collagen polypeptide described in item 1, comprising the steps of:
  • An active agent that inhibits the growth of vascular endothelial cells or a cell proliferation-promoting agent or a cell adhesion-promoting agent comprising the recombinant human collagen polypeptide described in Item 1 or the recombinant human collagen encoded by the nucleic acid molecule described in Item 2
  • the recombinant human collagen polypeptide described in item 1 or the recombinant human collagen polypeptide encoded by the nucleic acid molecule described in item 2 or the recombinant human collagen polypeptide expressed by the vector described in item 3 or expressed by the vector described in item 4 or 5 The application of the recombinant human collagen polypeptide produced by the host cell in the preparation of an active agent for inhibiting the growth of vascular endothelial cells, an agent for promoting cell proliferation or an agent for promoting cell adhesion.
  • the recombinant human collagen polypeptide of the present invention can be used to inhibit the growth of vascular endothelial cells, and can promote cell proliferation and cell adhesion.
  • Figure 1 is a schematic diagram of the Pichia pastoris expression vector and the recombinant human collagen polypeptide expression vector.
  • Figure 2 is a schematic diagram of the obtained recombinant collagen expression and purification, wherein, M is protein marker, 1 is recombinant protein expression, 2 is empty vector expression, 3 is flow-through, 4 is NTA-10mM washing, 5 is NTA- 40mM elution, 6 is NTA-200mM elution, 7 is the protein concentrated by ultrafiltration.
  • Fig. 3A is a schematic diagram of the inhibitory effect of the recombinant human collagen polypeptide of the present invention and commercially available collagen on vascular endothelial cells.
  • Fig. 3B is a schematic diagram of the inhibition rate of the recombinant human collagen polypeptide of the present invention on vascular endothelial cells.
  • Fig. 4A is a schematic diagram of the cell proliferation promoting effect of the recombinant human collagen polypeptide of the present invention and commercially available collagen.
  • Fig. 4B is a schematic diagram showing that the recombinant human collagen polypeptide of the present invention promotes cell proliferation.
  • Fig. 5A is a schematic diagram of the effect of the recombinant human collagen polypeptide of the present invention on promoting cell adhesion with commercially available collagen.
  • Fig. 5B is a schematic diagram of the recombinant human collagen polypeptide of the present invention promoting cell adhesion.
  • the present invention provides a recombinant human collagen polypeptide, the amino acid sequence of the recombinant human collagen polypeptide is as shown in SEQ ID NO: 2 or at least 90%, 91%, 92%, 93% with SEQ ID NO: 2 Amino acid sequences that are %, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the recombinant collagen polypeptide is creatively designed by the inventors on the basis of collagen, and the recombinant collagen polypeptide can be used to inhibit the growth of vascular endothelial cells and promote cell proliferation and adhesion, wherein,
  • amino acid sequence shown in SEQ ID NO:2 is as follows:
  • the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned recombinant human collagen polypeptide.
  • nucleotide sequence of described nucleic acid molecule is as shown in SEQ ID NO:1, and its nucleotide sequence is as follows:
  • the nucleic acid molecules may include those comprising naturally and/or non-naturally occurring nucleotides and bases, for example including those with backbone modifications, and the nucleic acid molecules refer to polymers of nucleotides, nucleotides Such polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA and PNA.
  • Nucleotide sequence refers to the linear sequence that makes up a nucleic acid molecule.
  • the nucleic acid molecule comprises cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs described herein, such as for codon optimization.
  • the sequence may be designed to contain terminal restriction site sequences for the purpose of cloning into a vector.
  • the nucleic acid molecule encoding the recombinant human collagen polypeptide can be obtained from a variety of sources, such as by means of DNA in or isolated from one or more given cells. Obtained by polymerase chain reaction (PCR) amplification of the encoding nucleic acid.
  • PCR polymerase chain reaction
  • the nucleotide sequence encoding said recombinant human collagen polypeptide is codon optimized.
  • codon optimization involves balancing the percentage of selected codons with the abundance of published human transfer RNAs such that neither is overloaded or constrained. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between the transfected gene and the host cell may affect protein expression and immunogenicity of the nucleic acid construct.
  • codons are chosen to select those codons that are in balance with the frequency of human usage. Often, the redundancy of amino acid codons is such that different codons encode one amino acid.
  • the resulting mutation in selecting codons for substitution, it may be desirable that the resulting mutation be a silent mutation such that the codon change does not affect the amino acid sequence.
  • the last nucleotide of a codon can be left unchanged without affecting the amino acid sequence.
  • the nucleic acid molecule is labeled with a tag, preferably, the tag is 6*His, GST (glutathione sulfhydryl transferase), MBP (maltose binding protein) or SUMO (small ubiquitin-related modification things).
  • the present invention provides a vector comprising the above-mentioned nucleic acid molecule.
  • the vector is pPIC9k, pPICZ ⁇ A, pGAPZA, pHIL-S1 or pYAM75P.
  • one or more nucleic acids encoding the above-mentioned recombinant human collagen polypeptides are cloned into one or more suitable expression vectors
  • the expression vectors can be any suitable recombinant expression vectors, and can be used for transformation or Transfect any suitable host.
  • Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
  • the vector may contain regulatory sequences (such as transcriptional and translational initiation and termination codons) specific for the type of host (e.g., bacteria, fungi, plant or animal) into which the vector is to be introduced, as appropriate and considering that the vector is DNA-based Still based on RNA.
  • regulatory sequences such as transcriptional and translational initiation and termination codons
  • the present invention provides a host cell comprising the above-mentioned vector.
  • nucleic acids encoding recombinant human collagen polypeptides can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be readily isolated and sequenced using conventional techniques (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding recombinant collagen).
  • 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 the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations.
  • the method for introducing the vector into the host cell is well known, such as using electric shock to introduce the vector into the host cell or using the CaCl method to introduce the foreign DNA into the prokaryotic cell or adopting the liposome - mediated transfection method to introduce the foreign DNA Introduce into eukaryotic cells or use virus vector infection method to transfer gene into host cells.
  • the host cell is a yeast host cell, preferably Pichia pastoris, more preferably Pichia pastoris, such as GS115 and KM71 strains.
  • the present invention provides a method for preparing the above-mentioned recombinant human collagen polypeptide, which comprises the following steps:
  • the expression of the host cell refers to culturing the host cell, and the culture medium and culture conditions are well known to those skilled in the art, for example, YPD, BMGY, BMMY, YNB and other media are used for culturing.
  • the present invention does not make any limitation on the expression method, which can be confirmed as needed, for example, the expression is induced expression, and for the induced expression, the inducer can be IPTG, ⁇ -galactoside, methanol, ethanol, etc., preferably methanol.
  • the present invention does not make any limitations, which can be determined according to, for example, Ni-nickel affinity chromatography, reduced glutathione affinity chromatography, maltose affinity chromatography or SUMO affinity chromatography.
  • the recombinant human collagen polypeptide provided by the present invention can be used to inhibit the growth of vascular endothelial cells, and can promote cell proliferation and adhesion.
  • the recombinant collagen polypeptide provided by the present invention is an amino acid sequence designed on the basis of collagen, and the recombinant collagen polypeptide having the amino acid sequence shown in SEQ ID NO: 2 can be used to inhibit the growth of vascular endothelial cells, And it can promote the proliferation and adhesion of cells. Compared with commercially available collagen, it can significantly inhibit the growth of vascular endothelial cells, and significantly promote the proliferation and adhesion of cells.
  • the present invention provides the application of the above-mentioned recombinant human collagen polypeptide in biochemical industry or cosmetics.
  • the present invention provides an active agent for inhibiting the growth of vascular endothelial cells, which comprises the above-mentioned recombinant human collagen polypeptide or the above-mentioned recombinant human collagen polypeptide encoded by the nucleic acid molecule or expressed by the above-mentioned vector
  • the present invention provides a cell growth-promoting agent, which comprises the above-mentioned recombinant human collagen polypeptide or the above-mentioned recombinant human collagen polypeptide encoded by the nucleic acid molecule or the recombinant human collagen expressed by the above-mentioned vector Protein polypeptide or recombinant human collagen polypeptide produced by the above-mentioned host cells.
  • the present invention provides a cell adhesion-promoting agent, which comprises the above-mentioned recombinant human collagen polypeptide or the above-mentioned recombinant human collagen polypeptide encoded by the nucleic acid molecule or the recombinant human collagen polypeptide expressed by the above-mentioned vector.
  • a cell adhesion-promoting agent which comprises the above-mentioned recombinant human collagen polypeptide or the above-mentioned recombinant human collagen polypeptide encoded by the nucleic acid molecule or the recombinant human collagen polypeptide expressed by the above-mentioned vector.
  • Collagen polypeptide or recombinant human collagen polypeptide produced by the above-mentioned host cells.
  • the recombinant human collagen polypeptide described in the present invention or the recombinant human collagen polypeptide encoded by the above-mentioned nucleic acid molecule or the recombinant human collagen polypeptide expressed by the above-mentioned vector or produced by the above-mentioned host cell Application of the recombinant human collagen polypeptide in the preparation of an active agent for inhibiting the growth of vascular endothelial cells, an agent for promoting cell proliferation or an agent for promoting cell adhesion.
  • the present invention generally and/or specifically describes the materials and test methods used in the test.
  • % means wt%, ie weight percentage.
  • the reagents or instruments used, whose manufacturers are not indicated, are all commercially available conventional reagent products.
  • Embodiment 1 Preparation of recombinant human collagen polypeptide
  • the sequence of the upstream primer P1 is shown in SEQ ID NO: 3, and its nucleotide sequence is: 5'-CGGAATTCATCAAGGGTGATCG-3', which contains an EcoRI restriction endonuclease site;
  • the sequence of the downstream primer P2 is shown in SEQ ID NO: 4, and its nucleotide sequence is: 5'-TTGCGGCCGCTTTGTCACCT-3', which contains a NotI restriction endonuclease site.
  • the reaction conditions of PCT are as follows: pre-denaturation at 98°C for 1 min, denaturation at 98°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, and extension at 72°C for 10 min after 30 cycles.
  • the reaction system is shown in Table 1:
  • the recovered PCR product and the expression vector pPIC9K (purchased from Invitrogen) were digested with restriction endonucleases EcoRI and Not I.
  • the enzyme digestion reaction system is shown in Table 2:
  • the digested product was recovered by the gel recovery kit and ligated with T4 DNA ligase (purchased from Thermo Fisher Scientific (China) Co., Ltd.).
  • the ligation temperature was 16°C and the ligation time was 30 min.
  • the ligation reaction was shown in Table 3. Show:
  • the ligation product was transformed into Escherichia coli DH5 ⁇ competent cells (purchased from Thermo Fisher Scientific (China) Co., Ltd.), mixed evenly, ice-bathed for 30 minutes, and water-bathed at 42°C for 60s (do not shake), then ice-bathed for 1- 2min; add 500 ⁇ L LB medium, incubate on a shaking table at 37°C for 1h; shake up the bacterial solution, spread 100 ⁇ L on an ampicillin agar plate (LB/AP+), and incubate at 37°C for 12-16 hours.
  • Escherichia coli DH5 ⁇ competent cells purchased from Thermo Fisher Scientific (China) Co., Ltd.
  • the sequencing primers are general sequencing primers for the plasmid pPIC9K. If the DNA sequencing results are consistent with the designed gene sequence, it is determined that the expression vector has been successfully constructed. At the same time, a recombinant E. coli transformed with an empty vector pPIC9K is constructed as a control.
  • Transformants with correct DNA sequencing were inoculated in LB liquid medium containing 100mg/mL Amp, cultured overnight at 37°C with shaking at 220rpm, and a large number of pPIC9k-COL-S plasmids were extracted (using a large plasmid extraction kit, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), take an appropriate amount for serial dilution, and then detect the plasmid concentration by agarose gel electrophoresis. Digest pPIC9k-COL-S with Sacl I and linearize the vector. The enzyme digestion system is shown in Table 4:
  • the fermentation supernatant was subjected to SDS-PAGE electrophoresis detection, wherein the concentration of the stacking gel was 5%, the concentration of the separating gel was 15%, and the loading volume of the supernatant was 15 ⁇ L.
  • the results showed that the empty vector pPIC9k was not expressed in the yeast strain protein, while the pPIC9k-COL-S collagen polypeptide was expressed in the yeast strain GS115, as shown in FIG. 2 .
  • the recombinant protein uses Ni-nickel affinity chromatography, and the fermentation supernatant is added to the Ni-nickel affinity chromatography medium and mixed to fully bind the target protein to the Ni-nickel affinity chromatography gel, and the binding flow rate is 0.6mL/min .
  • the recombinant human collagen polypeptide described in Example 1 was used to act on VEC (human vascular endothelial cells) cells to carry out cell inhibition experiments.
  • VEC human vascular endothelial cells
  • the specific steps are as follows: Inoculate human HUVEC cells (purchased from ATCC cell bank, catalog number: CRL-1730) in a sterile 96-well plate, 200 ⁇ l medium per well, and use M199 complete culture medium for cell culture, each well Add 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL recombinant human (COL-S) collagen polypeptide respectively.
  • Inhibition rate (blank group OD-experimental group OD) ⁇ 100%/blank group.
  • Commercially available collagen 1mg/mL 0.607 0.615 0.649
  • the inhibition rate of recombinant collagen of the present invention to vascular endothelium is more than 60%, while the inhibition rate of commercially available collagen is below 40%, which shows that the inhibition rate of the recombinant collagen of the present invention is below 40%.
  • the recombinant collagen has a strong inhibitory effect on vascular endothelial cells.
  • HSF human skin fibroblast
  • the specific steps are as follows: Inoculate human HSF cells (purchased from ATCC cell bank, product catalog number is HTX2132) in a sterile 96-well plate with 200 ⁇ L of medium per well, and use DMEM/F12 complete culture medium for cell culture, and each well is separately Add 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL recombinant human (COL-S) collagen polypeptide.
  • Proliferation rate (experimental group OD-blank group OD) ⁇ 100%/blank group.
  • the cell proliferation rate of the recombinant collagen of the present invention is close to 80%, while the cell proliferation rate of commercially available collagen is lower than 40%, indicating that the cell proliferation rate of the present invention Recombinant collagen can significantly promote cell proliferation.
  • HSF human skin fibroblast
  • Promoting cell adhesion rate (experimental group OD-blank group OD) ⁇ 100%/blank group.
  • the cell adhesion rate of the recombinant collagen of the present invention is 80%, while the cell adhesion rate of commercially available collagen is lower than 60%.
  • the recombinant collagen can significantly promote cell adhesion.
  • the recombinant human collagen polypeptide provided by the present invention can inhibit the growth of vascular endothelial cells, and can promote cell proliferation and adhesion, so that the recombinant human collagen polypeptide can be applied to the fields of biochemical industry or cosmetics .

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Abstract

本发明公开了一种重组人胶原蛋白多肽及其应用。本发明所述的重组人胶原蛋白多肽的氨基酸序列如SEQ ID NO:2所示或者与SEQ ID NO:2具有至少90％同一性的氨基酸序列。本发明所述的重组人胶原蛋白多肽可以用于抑制血管内皮细胞的生长，可以促进细胞增殖和促进细胞粘附。

Description

重组人胶原蛋白多肽及其应用 技术领域
本发明涉及生物工程技术领域，尤其涉及一种重组人胶原蛋白多肽及其应用。
背景技术
胶原蛋白是动物体内含量最为丰富的蛋白质之一，广泛地分布在哺乳动物的皮肤、骨骼、肌腱、韧带、软骨和血管之中，它是结缔组织的主要蛋白成分，也是细胞外基质的重要组成成分。人体中胶原蛋白含量占总蛋白的25％～30％，并且富含人体需要的甘氨酸、脯氨酸、羟脯氨酸等氨基酸。胶原蛋白这种结构性蛋白起着保护机体、支撑器官的重要作用。这使得胶原蛋白在医药、食品、美容以及生物材料等方面有很广泛的应用，近年来一直是大家研究的热点。
胶原蛋白广泛存在于细胞外基质，其结构为三股多肽链缠绕形成的三螺旋。胶原蛋白的类型因其三条α链的组合不同而各不相同。到目前为止，已经发现了遗传学上截然不同的胶原蛋白类型有26种。大致可以分为以下几类：纤维形成胶原、基底膜胶原、微纤维胶原、锚原纤维、跨膜区胶原、未完全定性胶原。其中主要的类型有：I型胶原蛋白，其三螺旋组成主要是α1、α2链，在骨骼、肌腱、皮肤、角膜和韧带中含量最高；II型胶原蛋白，其三螺旋组成是[α1(II)] 2，存在于透明软骨、玻璃体和椎间盘等组织中；III型胶原蛋白，其三螺旋组成是[α1(III)] 3，主要分布在胚胎、胃肠道、牙周膜；IV型胶原蛋白，其三螺旋组成形式是[α1(IV)] 3，[α1(IV)] 2α2(VI)，[α2(I)] 3，是基底膜组成成分，羟赖氨酸含量特别多，含糖量比较高，既含有4-羟脯氨酸又含有3-羟脯氨酸。
胶原蛋白是基底膜、骨骼、软骨等结构的支架，可以用于烧伤、创伤和眼角膜疾病的治疗。胶原蛋白在伤口愈合、器官发育和组织修复等方面扮演着重要角色，临床上的应用包括：美容整形用的医用胶原注射剂，烧伤、创伤治疗的胶原膜，创伤止血的胶原蛋白止血海绵等。胶原蛋白是皮肤细胞外 基质的重要组成成分，具有保湿、补充皮肤胶原、抗衰老等方面的功效。由于胶原蛋白具有以上生物学功能，所以胶原蛋白在医药卫生领域(如止血、美容、药物传递、创伤修复、药物缓释等)有着广泛的应用。
目前胶原蛋白主要来源方式是从动物皮肤、骨头、等组织中采用酸、碱性水解和酶水解的方法提取得到。由于来源于动物组织，对于动物源疾病或者人传染疾病有交叉感染的风险。随着基因工程技术的日益成熟，采用分子生物学方法制备重组胶原蛋白逐渐替代了从动物组织中提取胶原蛋白的趋势。在原核或真核表达系统中生产的重组胶原蛋白具有比较好的细胞粘附性、组织相容性、皮肤透过性、稳定性、可促细胞形成和生长，与传统动物胶原蛋白相比较有其独特的优越性(无病毒隐患、水溶性、低排异反应等)。
发明内容
本发明的目的在于提供了一种重组人胶原蛋白多肽，所述的重组人胶原蛋白多肽可用于抑制血管生长因子，调节内皮细胞可塑性、制备促细胞生长剂、制备促细胞粘附剂中。可广泛应用于生物化工以及化妆品领域。
本发明具体技术方案如下：
1.一种重组人胶原蛋白多肽，所述重组人胶原蛋白多肽的氨基酸序列如SEQ ID NO:2所示或者是与SEQ ID NO:2具有至少90％同一性的氨基酸序列。
2.一种核酸分子，其中，所述核酸分子编码项1所述的重组人胶原蛋白多肽，优选所述核酸分子用标签标记，进一步优选，所述标签为6*His、GST、MBP或SUMO。
3.一种载体，其中，所述载体包含项2所述的核酸分子，优选所述载体为pPIC9k、pPICZαA、pGAPZA、pHIL-S1或pYAM75P。
4.一种宿主细胞，其包含项3所述的载体。
5.根据项4所述的宿主细胞，其中，所述宿主细胞为酵母宿主细胞，优选为毕赤酵母，进一步优选为巴斯德毕赤酵母。
6.一种制备项1所述重组人胶原蛋白多肽的方法，其包含下述步骤：
将项4或5所述的宿主细胞进行表达，然后进行分离纯化得到。
7.根据项6所述的方法，其中，所述分离纯化的方法为Ni-镍亲和层析法、还原型谷胱甘肽亲和层析法、麦芽糖亲和层析法或SUMO亲和层析法
8.项1所述的重组人胶原蛋白多肽或项2所述的核酸分子所编码的重组 人胶原蛋白多肽或由项3所述的载体所表达的重组人胶原蛋白多肽或由项4或5所述的宿主细胞生产的重组人胶原蛋白多肽在生物化工或化妆品中的应用。
9.一种抑制血管内皮细胞生长的活性剂或促细胞增殖剂或促细胞粘附剂，其包含项1所述的重组人胶原蛋白多肽或项2所述的核酸分子所编码的重组人胶原蛋白多肽或由项3所述的载体所表达的重组人胶原蛋白多肽或由项4或5所述的宿主细胞生产的重组人胶原蛋白多肽。
10.项1所述的重组人胶原蛋白多肽或项2所述的核酸分子所编码的重组人胶原蛋白多肽或由项3所述的载体所表达的重组人胶原蛋白多肽或由项4或5所述的宿主细胞生产的重组人胶原蛋白多肽在制备抑制血管内皮细胞生长的活性剂、促细胞增殖剂或促细胞粘附剂中的应用。
发明的效果
本发明所述的重组人胶原蛋白多肽，可以用于抑制血管内皮细胞的生长，可以促进细胞增殖和促进细胞粘附。
附图说明
图1是毕赤酵母表达载体和重组人胶原蛋白多肽表达载体示意图。
图2是所得到的重组胶原蛋白表达以及纯化示意图，其中，M是蛋白Marker，1为重组蛋白表达，2为空载体表达，3为流穿，4为NTA-10mM洗杂，5为NTA-40mM洗杂，6为NTA-200mM洗脱，7为超滤浓缩后蛋白。
图3A是本发明所述的重组人胶原蛋白多肽与市售胶原蛋白对血管内皮细胞抑制作用的示意图。
图3B是本发明所述的重组人胶原蛋白多肽对血管内皮细胞抑制率的示意图。
图4A是本发明所述的重组人胶原蛋白多肽与市售胶原蛋白促细胞增殖作用的示意图。
图4B是本发明所述的重组人胶原蛋白多肽促细胞增殖的示意图。
图5A是本发明所述的重组人胶原蛋白多肽与市售胶原蛋白促细胞粘附作用的示意图。
图5B是本发明所述的重组人胶原蛋白多肽促细胞粘附的示意图。
具体实施方式
下面结合附图所描述的实施方式对本发明做以详细说明，其中所有附图中相同的数字表示相同的特征。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式，而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然而所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
本发明提供了一种重组人胶原蛋白多肽，所述重组人胶原蛋白多肽的氨基酸序列如SEQ ID NO:2所示或者是与SEQ ID NO:2具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
所述重组胶原蛋白多肽是发明人在胶原蛋白的基础上创造性地设计得到，所述的重组胶原蛋白多肽可以用于抑制血管内皮细胞的生长以及能够促进细胞的增殖和粘附，其中，
SEQ ID NO:2所示的氨基酸序列如下：
Figure PCTCN2022118748-appb-000001
本发明提供了一种核酸分子，其中，所述核酸分子编码上述所述的重组人胶原蛋白多肽。
所述核酸分子的核苷酸序列如SEQ ID NO:1所示，其核苷酸序列如下：
Figure PCTCN2022118748-appb-000002
Figure PCTCN2022118748-appb-000003
所述核酸分子可以包括包含天然和/或非天然存在的核苷酸和碱基的那些，例如包括具有骨架修饰的那些，所述核酸分子指的是核苷酸的聚合物，核苷酸的此类聚合物可以含有天然和/或非天然核苷酸，并且包括但不限于DNA、RNA和PNA。核苷酸序列指的是构成核酸分子的线性序列。
在一些情况下，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发明所述的构建体中，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。
在一些情况下，编码所述重组人胶原蛋白多肽的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。
在一个实施方案中，编码所述重组人胶原蛋白多肽的核苷酸序列是经密码子优化的。通常，密码子优化涉及使所选择的密码子的百分比与已公开的人类转移RNA的丰度平衡，使得没有一者过载或受限。在一些情况下，这可能是必要的，因为大多数氨基酸由超过一种密码子编码，并且密码子使用因生物而异。经转染的基因与宿主细胞之间的密码子使用差异可能会影响核酸构建体的蛋白质表达和免疫原性。通常，对于密码子优化，选择密码子以选择与人类使用频率平衡的那些密码子。通常，氨基酸密码子的冗余度使得不同的密码子编码一种氨基酸。在一些实施方案中，在选择用于置换的密码子时，可能需要所得突变是沉默突变，使得密码子改变不影响氨基酸序列。通常，密码子的最后一个核苷酸可以保持不变而不会影响氨基酸序列。
在一个实施方案中，所述核酸分子用标签标记，优选的，所述标签为6*His、GST(谷胱甘肽巯基转移酶)、MBP(麦芽糖结合蛋白)或SUMO(小泛素相关修饰物)。
本发明提供了一种载体，所述载体包含上述所述的核酸分子。
在一个实施方案中，所述载体为pPIC9k、pPICZαA、pGAPZA、pHIL-S1或pYAM75P。
例如，将编码上述所述的重组人胶原蛋白多肽的一种或多种核酸克隆到合适的一种或多种表达载体中，表达载体可以是任何合适的重组表达载体，并且可以用于转化或转染任何合适的宿主。合适的载体包括设计用于繁殖和扩增或用于表达或用于两者的那些，如质粒和病毒。
所述载体可以含有调节序列(如转录和翻译起始和终止密码子)，其对待引入载体的宿主的类型(例如，细菌、真菌、植物或动物)具有特异性，酌情并考虑载体是基于DNA还是基于RNA。
本发明提供了一种宿主细胞，其包含上述所述的载体。
为了产生重组人胶原蛋白多肽，可以将编码重组人胶原蛋白多肽的核酸分离，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，例如使用电击将载体导入宿主细胞中或者使用CaCl 2法将外源DNA导入到原核细胞中或者采用脂质体介导的转染法将外源DNA导入到真核细胞中或者采用病毒载体感染法将基因转入到宿主细胞中。
在一个实施方案中，所述宿主细胞为酵母宿主细胞，优选为毕赤酵母，进一步优选为巴斯德毕赤酵母，例如为GS115、KM71菌株。
本发明提供了制备上述所述重组人胶原蛋白多肽的方法，其包括下述步骤：
将上述所述的宿主细胞进行表达，然后进行分离纯化得到。
所述将宿主细胞进行表达指的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，例如使用YPD、BMGY、BMMY、YNB等培养基进行培养。
对于表达方式，本发明不作任何限制，其可以根据需要进行确认，例如表达为诱导表达，对于诱导表达，其诱导剂可以为IPTG、β-半乳糖苷、甲 醇、乙醇等，优选为甲醇。
对于分离纯化的方法，本发明不作任何限制，其可以根据进行确定，例如可以使用Ni-镍亲和层析法、还原型谷胱甘肽亲和层析法、麦芽糖亲和层析法或SUMO亲和层析法。
本发明所提供的重组人胶原蛋白多肽，其可以用于抑制血管内皮细胞生长，并且能够促进细胞的增殖和粘附。
本发明所提供的重组胶原蛋白多肽，其是在胶原蛋白的基础上经过设计所得到的氨基酸序列，具有SEQ ID NO:2所示氨基酸序列的重组胶原蛋白多肽可以用于抑制血管内皮细胞生长，并且能够促进细胞的增殖和粘附，其与市售的胶原蛋白相比，能够显著抑制血管内皮细胞生长，以及显著促进细胞的增殖和粘附。
本发明提供了上述所述的重组人胶原蛋白多肽在生物化工或化妆品中的应用。
本发明提供了一种抑制血管内皮细胞生长的活性剂，其包含上述所述的重组人胶原蛋白多肽或上述所述的核酸分子所编码的重组人胶原蛋白多肽或由上述所述的载体所表达的重组人胶原蛋白多肽或由上述所述的宿主细胞生产的重组人胶原蛋白多肽。
本发明提供了一种促细胞生长剂，其包含上述所述的重组人胶原蛋白多肽或上述所述的核酸分子所编码的重组人胶原蛋白多肽或由上述所述的载体所表达的重组人胶原蛋白多肽或由上述所述的宿主细胞生产的重组人胶原蛋白多肽。
本发明提供了一种促细胞粘附剂，其包含上述所述的重组人胶原蛋白多肽或上述所述的核酸分子所编码的重组人胶原蛋白多肽或由上述所述的载体所表达的重组人胶原蛋白多肽或由上述所述的宿主细胞生产的重组人胶原蛋白多肽。
本发明所述的重组人胶原蛋白多肽或上述所述的核酸分子所编码的重组人胶原蛋白多肽或由上述所述的载体所表达的重组人胶原蛋白多肽或由上述所述的宿主细胞生产的重组人胶原蛋白多肽在制备制备抑制血管内皮细胞生长的活性剂、促细胞增殖剂或促细胞粘附剂中的应用。
实施例
本发明对试验中所用到的材料以及试验方法进行一般性和/或具体的描述，在下面的实施例中，如果无其他特别的说明，％表示wt％，即重量百分数。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1重组人胶原蛋白多肽的制备
1.重组人胶原蛋白多肽表达载体的构建
委托生工生物工程(上海)股份有限公司合成重组胶原蛋白肽的核苷酸序列(核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2)。以该核苷酸序列片段为模板，设计含有酶切位点的上下游引物进行PCR反应。其中，上游引物加入6*His标签方便后续的纯化。
上游引物P1的序列如SEQ ID NO:3所示，其核苷酸序列为：5'-CGGAATTCATCAAGGGTGATCG-3'，其含有EcoRI限制性内切酶酶切位点；
下游引物P2的序列如SEQ ID NO:4所示，其核苷酸序列为：5'-TTGCGGCCGCTTTGTCACCT-3'，其含有NotI限制性内切酶酶切位点。
PCT的反应条件如下：98℃预变性1min，98℃变性10s，60℃退火10s，72℃延伸10s，进行30循环后72℃延伸10min，反应体系如表1所示：
表1 PCR反应体系
试剂 用量
模板DNA 1μL
PrimeSTAR Max DNA Polymerase 12.5μL
引物P1 0.5μL
引物P2 0.5μL
ddH 2O 0.5μL
用限制性内切酶EcoR Ⅰ和Not I对回收的PCR产物和表达载体pPIC9K(购自于Invitrogen公司)进行酶切。酶切反应体系如表2所示：
表2表达载体以及胶原蛋白多肽PCR产物的酶切反应
试剂 用量
pPIC9K载体或PCR产物 10μL(≦1μg)
限制性内切酶 0.5μL
10×buffer 2μL
ddH 2O 7.5μL
酶切产物经胶回收试剂盒回收后用T4 DNA连接酶(购自于赛默飞世尔科技(中国)有限公司)连接，连接温度为16℃，连接时间为30min，连接反应如表3所示：
表3表达载体与胶原蛋白多肽的连接反应
试剂 用量
酶切后的PCR产物片段(50ng/μL) 2μL
酶切后的pPIC9K载体(25ng/μL) 2μL
10×Ligation Buffer 1μL
T4 DNA Ligase 0.5μL
ddH 2O 4.5μL
将连接产物转化入大肠杆菌DH5α感受态细胞(购自于赛默飞世尔科技(中国)有限公司)中，混匀，冰浴30min，42℃水浴60s(不能摇动)，再冰浴1-2min；加入500μL LB培养基，37℃摇床孵育1h；摇匀菌液，取100μL涂布于氨苄青霉素琼脂平板(LB/AP+)上，倒置于37℃培养12～16小时。琼脂平板上挑取单菌落作为模板，然后以上游引物P1(SEQ ID NO:3)和下游引物P2(SEQ ID NO:4)进行PCR。PCR筛选的阳性克隆子进一步使用限制性酶EcoRI、NotI进行酶切反应鉴定。两者都为阳性结果的克隆子进行测序分析。测序引物是针对质粒pPIC9K的通用测序引物，若DNA测序结果与设计的基因序列相符则确定此表达载体构建成功，同时构建转化空载体pPIC9K的重组大肠杆菌做对照。
2.重组人胶原蛋白多肽的表达和纯化
接种DNA测序正确的转化子于含100mg/mL Amp的LB液体培养基中，37℃，220rpm振荡培养过夜，进行pPIC9k-COL-S质粒大量提取(采用质粒大提试剂盒，购自于天根生化科技(北京)有限公司)，取适量进行梯度稀释后琼脂糖凝胶电泳检测质粒浓度。用Sacl I单酶切pPIC9k-COL-S，线性化载体，其酶切体系如表4所示：
表4重组胶原蛋白多肽的线性化酶切反应
试剂 用量
pPIC9k-COL-S 20μL
Sal I 1μL
100×BSA 0.5μL
10×buffer H 5μL
ddH 2O 5μL
在上述酶切后的DNA进行纯化后，取10μL线性化DNA pPIC9k-COL-S加入到80μL GS115酵母感受态细胞中，其操作示意图如图1所示，并转移到预冷的0.2cm电极杯中，冰浴5min。将电极杯上的冰仔细擦干后放到电极槽中拧紧，放下玻璃罩，调节电压1500V，电阻200Ω，电容50μF进行转化。电击完后马上打开盖子加入1mL冰上预冷的1M山梨醇，轻轻混合后将液体全部转移到1.5mL灭菌离心管中，涂MD板。将平板在30℃生化培养箱中倒置培养2～5天，直到出现单菌落。以COL-S引物(上游引物P1和下游引物P2)进行菌落PCR阳性筛选，在超净工作台中将长出目的条带的菌落依次点到含0.25mg/mL～4.0mg/mL G418的YPD平板上，进行高拷贝筛选。
取高拷贝单菌落接种于YPD培养基中30℃，220rpm振荡培养过夜，取适量培养液转接BMGY培养基30℃，220rpm振荡过夜至OD600达到2～8，取适量培养物中转移至50mL灭菌离心管中，室温4000rpm离心3min后弃上清，用BMMY培养基将菌体重新悬浮至OD600等于1以后，30℃，250rpm诱导培养，酵母菌株GS115以及转化了空载体pPIC9k的酵母菌株GS115用同样方法诱导培养。每24h加250μL甲醇。每24h取1mL样，12000rpm离心1min后将上清和菌体分别保存于-20℃中以便后续的检测。诱导5天后停止培养，将发酵液离心，分别保存上清液和菌体。
将发酵上清液进行SDS-PAGE电泳检测，其中浓缩胶浓度为5％，分离胶浓度为15％，上清液上样量为15μL。60V电压下跑胶30min后将其调至100V继续电泳1～2h左右直到溴酚蓝达到胶板底部停止电泳，考马斯亮蓝R-250染色30min后用脱色液脱色1h左右，更换几次脱色液，直至蛋白条带清晰显现。结果表明，空载体pPIC9k在酵母菌株蛋白没有表达，而pPIC9k-COL-S胶原蛋白多肽在酵母菌株GS115中表达，如图2所示。
重组蛋白利用Ni-镍亲和层析，将发酵上清液加入到Ni-镍亲和层析介质中混合使目的蛋白与Ni-镍亲和层析胶充分结合，结合流速为0.6mL/min。用NTA-10mM咪唑缓冲液清洗基线，流速为1mL/min；之后用NTA-40mM咪唑缓冲液洗杂蛋白，流速为1mL/min，NTA-200mM咪唑缓冲液洗脱目的蛋白，流速为1mL/min；在上清液以及NTA-200mM洗脱后的回收液中发现目标蛋白，大小约为14KD，与预期结果吻合。纯化后的蛋白经超滤离心后，上样20μg，基本上无杂带，蛋白纯度超过95％，如图2所示。
实验例1重组人胶原蛋白多肽在抑制血管内皮细胞中的应用
将实施例1所述的重组人胶原蛋白多肽作用于VEC(人血管内皮细胞)细胞进行细胞抑制实验。具体步骤如下：将人HUVEC细胞(购自于ATCC细胞库，产品目录号为：CRL-1730)接种于无菌96孔板中，每孔200μl培养基，细胞培养使用M199完全培养液，每孔分别加入0.25mg/mL、0.5mg/mL、1mg/mL、2mg/mL重组人(COL-S)胶原蛋白多肽。每个组浓度设置三个复孔，空白对照组加入等体积的培养液，置于37℃培养箱培48h。弃上清，每孔加入100μL培养液和50μL MTT溶液，摇匀，放进培养箱中孵育4h，4h后吸出混合液，加入150μL DMSO来溶解紫色晶体甲瓒，震荡10min，放进酶标仪中，检测波长570nm，测定每孔的吸光度值，重组人胶原蛋白多肽抑制HUVEC内皮细胞数据如表5所示，其与市售胶原蛋白(购自于Abcam公司)对血管内皮细胞的抑制作用示意图如图3A所示，抑制生长曲线如图3B所示，其中，
抑制率＝(空白组OD-实验组OD)×100％/空白组。
表5不同浓度的重组人胶原蛋白多肽以及市售胶原蛋白对血管内皮细胞的抑制作用的吸光度值
  复孔1 复孔2 复孔3
对照 1.036 0.912 0.986
0.25mg/mL 0.735 0.769 0.786
0.5mg/mL 0.617 0.623 0.653
1mg/mL 0.396 0.312 0.306
2mg/mL 0.469 0.475 0.482
市售胶原蛋白1mg/mL 0.607 0.615 0.649
从表5及图3A至图3B可以看出，本发明所述的重组胶原蛋白对血管内皮的抑制率在60％以上，而市售胶原蛋白的抑制率在40％以下，说明本发明所述的重组胶原蛋白对血管内皮细胞具有很强的抑制作用。
实验例2重组人胶原蛋白多肽在促细胞增殖中的应用
使用实施例1所述的重组人胶原蛋白多肽作用于HSF(人皮肤成纤维细胞)细胞进行细胞增殖实验。具体步骤如下：将人HSF细胞(购自于ATCC 细胞库，产品目录号为HTX2132)接种于无菌96孔板中，每孔200μL培养基，细胞培养使用DMEM/F12完全培养液，每孔分别加入0.25mg/mL、0.5mg/mL、1mg/mL、2mg/mL重组人(COL-S)胶原蛋白多肽。每个组浓度设置三个复孔，空白对照组加入等体积的培养液，置于37℃培养箱培养48h。弃上清，每孔加入100μL培养液和50μL MTT溶液，摇匀，放进培养箱中孵育4h，4h后吸出混合液，加入150μL DMSO来溶解紫色晶体甲瓒，震荡10min，放进酶标仪中，检测波长570nm，测定每孔的吸光度值，重组胶原蛋白多肽促HSF(人皮肤成纤维细胞)细胞增殖数据如表6所示，其与市售胶原蛋白(购自于Abcam公司)对人皮肤成纤维细胞增殖作用示意图如图4A所示，增殖率曲线如图4B所示，其中，
增殖率＝(实验组OD-空白组OD)×100％/空白组。
表6不同浓度的重组胶原蛋白多肽以及市售胶原蛋白对皮肤成纤维细胞的增殖作用的吸光度值
  复孔1 复孔2 复孔3
对照 0.523 0.562 0.539
0.25mg/mL 0.625 0.692 0.612
0.5mg/mL 0.723 0.713 0.701
1mg/mL 0.953 0.975 0.964
2mg/mL 0.946 0.956 0.976
市售胶原蛋白(1mg/mL) 0.715 0.736 0.729
从表6及图4A至4B可以看出，本发明所述的重组胶原蛋白促细胞增值率接近于80％，而市售胶原蛋白的促细胞增值率低于40％，说明本发明所述的重组胶原蛋白能够显著促进细胞的增殖。
实验例3重组人胶原蛋白多肽在促细胞粘附中的应用
使用实施例1所述的重组人胶原蛋白多肽作用于HSF(人皮肤成纤维细胞)细胞进行促细胞粘附实验。具体步骤如下：将人HSF细胞接种于无菌96孔板中，每孔200μL培养基，空白对照组只加PBS，37℃放置2h后用PBS清洗各组3遍，洗去未粘附的细胞，每孔加入100μL培养液和50μL MTT溶液，摇匀，放进培养箱中孵育4h，后吸出混合液，加入150μL DMSO来溶解紫色晶体甲瓒，震荡10min，放进酶标仪中，检测波长570nm，测定每孔 的吸光度值。
胶原蛋白多肽促HSF(人皮肤成纤维细胞)细胞粘附数据如表7所示，其与市售胶原蛋白(购自于Abcam公司)对人皮肤成纤维细胞的粘附作用示意图如图5A所示，粘附率曲线如图5B所示，其中，
促细胞粘附率＝(实验组OD-空白组OD)×100％/空白组。
表7不同浓度的重组胶原蛋白多肽以及市售胶原蛋白对皮肤成纤维细胞的促粘附作用的吸光度值
  复孔1 复孔2 复孔3
对照 0.326 0.352 0.348
0.25mg/mL 0.432 0.415 0.426
0.5mg/mL 0.510 0.521 0.513
1mg/mL 0.629 0.611 0.634
2mg/mL 0.613 0.602 0.615
市售胶原蛋白(1mg/mL) 0.523 0.551 0.534
从表7以及图5A至图5B可以看出，本发明所述的重组胶原蛋白的促细胞粘附率为80％，而市售胶原蛋白的促细胞粘附率低于60％，说明本发明所述的重组胶原蛋白能够显著促进细胞的粘附。
综上所述，本发明所提供的重组人胶原蛋白多肽能够抑制血管内皮细胞的生长，能够促进细胞的增殖和粘附，从而可以将所述的重组人胶原蛋白多肽应用于生物化工或者化妆品领域。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (10)

  1. 一种重组人胶原蛋白多肽，所述重组人胶原蛋白多肽的氨基酸序列如SEQ ID NO:2所示或者是与SEQ ID NO:2具有至少90％同一性的氨基酸序列。
  2. 一种核酸分子，其中，所述核酸分子编码权利要求1所述的重组人胶原蛋白多肽，优选所述核酸分子用标签标记，进一步优选，所述标签为6*His、GST、MBP或SUMO。
  3. 一种载体，其中，所述载体包含权利要求2所述的核酸分子，优选所述载体为pPIC9k、pPICZαA、pGAPZA、pHIL-S1或pYAM75P。
  4. 一种宿主细胞，其包含权利要求3所述的载体。
  5. 根据权利要求4所述的宿主细胞，其中，所述宿主细胞为酵母宿主细胞，优选为毕赤酵母，进一步优选为巴斯德毕赤酵母。
  6. 一种制备权利要求1所述重组人胶原蛋白多肽的方法，其包含下述步骤：
    将权利要求4或5所述的宿主细胞进行表达，然后进行分离纯化得到。
  7. 根据权利要求6所述的方法，其中，所述分离纯化的方法为Ni-镍亲和层析法、还原型谷胱甘肽亲和层析法、麦芽糖亲和层析法或SUMO亲和层析法
  8. 权利要求1所述的重组人胶原蛋白多肽或权利要求2所述的核酸分子所编码的重组人胶原蛋白多肽或由权利要求3所述的载体所表达的重组人胶原蛋白多肽或由权利要求4或5所述的宿主细胞生产的重组人胶原蛋白多肽在生物化工或化妆品中的应用。
  9. 一种抑制血管内皮细胞生长的活性剂或促细胞增殖剂或促细胞粘附剂，其包含权利要求1所述的重组人胶原蛋白多肽或权利要求2所述的核酸分子所编码的重组人胶原蛋白多肽或由权利要求3所述的载体所表达的重组人胶原蛋白多肽或由权利要求4或5所述的宿主细胞生产的重组人胶原蛋白多肽。
  10. 权利要求1所述的重组人胶原蛋白多肽或权利要求2所述的核酸分子所编码的重组人胶原蛋白多肽或由权利要求3所述的载体所表达的重组人胶原蛋白多肽或由权利要求4或5所述的宿主细胞生产的重组人胶原蛋白多 肽在制备抑制血管内皮细胞生长的活性剂、促细胞增殖剂或促细胞粘附剂中的应用。
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CN117003857B (zh) * 2023-09-28 2024-01-05 英特菲尔(成都)生物制品有限责任公司 一种具有透皮吸收性能的胶原蛋白及其制备方法与应用
CN117304306B (zh) * 2023-09-28 2024-06-04 广东普言生物科技有限公司 一种重组Ⅲ型胶原蛋白Pro.C3及其制备方法和应用
CN117285616A (zh) * 2023-10-07 2023-12-26 广东省卓肽医药有限公司 一种重组人源化i+iii型胶原蛋白及应用
CN117285616B (zh) * 2023-10-07 2024-02-27 广东省卓肽医药有限公司 一种重组人源化i+iii型胶原蛋白及应用
CN117603343A (zh) * 2024-01-19 2024-02-27 四川大学 阻断bFGF的新型胶原来源天然短肽及应用
CN117603343B (zh) * 2024-01-19 2024-04-26 四川大学 阻断bFGF的胶原来源天然短肽及应用

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