WO2024032467A1 - 高稳定性重组胶原蛋白、构建方法及其应用 - Google Patents

高稳定性重组胶原蛋白、构建方法及其应用 Download PDF

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WO2024032467A1
WO2024032467A1 PCT/CN2023/111028 CN2023111028W WO2024032467A1 WO 2024032467 A1 WO2024032467 A1 WO 2024032467A1 CN 2023111028 W CN2023111028 W CN 2023111028W WO 2024032467 A1 WO2024032467 A1 WO 2024032467A1
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seq
collagen
sequence
recombinant collagen
recombinant
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French (fr)
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王丽萍
刘小海
钱晨明
程鹏飞
豆荣昆
凡孝菊
钱松
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江苏创健医疗科技股份有限公司
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Priority to EP23851688.4A priority Critical patent/EP4567044A1/en
Publication of WO2024032467A1 publication Critical patent/WO2024032467A1/zh

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    • 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
    • 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
    • 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]
    • 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
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present invention relates to the field of collagen technology, and in particular to highly stable recombinant collagen, construction methods and applications.
  • Collagen is the most abundant type of protein in the animal body. It is an important extracellular matrix component and plays an important role in cell migration, cell metabolism, cell signaling pathway response, platelet aggregation, and the maintenance and regulation of normal physiological functions of cells, tissues, and organs. It plays an important role in damage repair and other aspects. Collagen has good biocompatibility, bioactivity and degradability, and is now widely used in many fields such as chemical industry, medicine, food, cosmetics and so on.
  • the technology for extracting collagen from animal tissues is relatively mature, and this type of collagen has a long history of application.
  • the acid and alkali and other raw materials used in the traditional collagen production process are not friendly to the environment, and the extracted collagen peptides have uneven properties, large batch-to-batch differences, and potential safety risks of viral infection.
  • recombinant collagen produced by genetic engineering technology is increasingly Be taken seriously.
  • the research and application of recombinant collagen has a history of more than 30 years.
  • the existing literature and patents mainly focus on the expression of human collagen single chains in different hosts and the expression of truncated collagen gene single chains. There is also a small amount of attention.
  • the co-expression of collagen and related post-expression modification enzymes obtains collagen with a triple helical structure.
  • the expression of recombinant collagen involved in the existing technology mostly uses human collagen sequences (CN201010527766.7, CN201510535383.7, CN201911093124.8).
  • the expression products are mostly random coils, less regular secondary structures, and less There are higher-order structures such as tertiary and quaternary structures.
  • water-soluble proteins are easy to process and use, the selection of amino acid sequences tends to be highly water-soluble. This causes such linear recombinant collagen to be easily degraded by proteases in the host.
  • the highly water-soluble protein cannot fold into a stable high-level structure due to the small content of hydrophobic regions, and unstable side chains, Residues and peptide bonds are prone to deamidation, oxidation, and hydrolysis, which may ultimately lead to the production of process-related impurities and degradation products, increasing immunogenicity.
  • the present invention aims to obtain a recombinant collagen sequence with improved stability through mutation of the existing amino acid sequence or selection of the amino acid sequence, simplify the purification process, and improve protein yield and storage stability.
  • the first aspect of the present invention provides a method for constructing a collagen variant with improved stability, which includes selecting from the group consisting of avoiding unstable GXY triplets and removing potential MMP enzyme cleavage sites based on the original collagen sequence. , eliminate chemical instability factors, and increase one or more mutations in the RGD content of the sequence.
  • the original collagen comprises the amino acid sequence shown in SEQ ID NO: 1 or has an amino acid sequence that is more than 80%, more than 85%, more than 90%, more than 95%, or more than 96% identical to SEQ ID NO: 1 Amino acid sequences with above, above 97%, above 98%, and above 99% identity.
  • G in the GXY triplet is glycine
  • X and Y can be any amino acids
  • collagen is composed of GXY motifs.
  • said avoiding destabilizing GXY triplets includes doing so in the initial collagen sequence. Mutations selected from GPL ⁇ GPS, GIA ⁇ GPA, GDR ⁇ GER, GAA ⁇ GPA.
  • the removal of potential MMP cleavage sites includes removing potential MMP2 and/or MMP9 cleavage sites; preferably, mutations of GLA ⁇ GPA and/or GIK ⁇ GEK are performed.
  • the chemical instability factors include sites that are prone to deamidation, hydrolysis, or oxidation; preferably, mutations selected from the following are made: 1) NG ⁇ QG; 2) F ⁇ P; 3 )MPGPR ⁇ PPGPR; 4) While retaining the original RGD sequence, mutate other R to K; 5) While retaining the original RGD sequence, mutate other D to E.
  • a second aspect of the invention provides collagen variants with improved stability obtained according to the method of the first aspect of the invention.
  • the sequence of the collagen variant comprises a sequence selected from the group consisting of more than 80%, more than 85% similarity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6. , amino acid sequences with more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% identity.
  • sequence of the collagen variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6.
  • a third aspect of the present invention provides a highly stable recombinant collagen, the recombinant collagen comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6 or contains a product selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6 that has a value of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97 % or more, 98% or more, or 99% or more identical amino acid sequences.
  • a fourth aspect of the present invention provides an isolated polynucleotide encoding a collagen variant with improved stability according to the second aspect of the present invention or a highly stable recombinant collagen according to the third aspect of the present invention. .
  • the polynucleotide comprises a polynucleotide selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 that is 80%, 85%, 90% More than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identical nucleic acid sequences.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.
  • a fifth aspect of the invention provides a vector comprising the isolated polynucleotide of the fourth aspect of the invention.
  • the vector is a eukaryotic vector.
  • the vector is a prokaryotic vector.
  • a fifth aspect of the present invention provides a host cell comprising the isolated polynucleotide of the fourth aspect of the present invention or the vector of the fifth aspect of the present invention.
  • the host cell is a eukaryotic cell.
  • the host cell is Pichia pastoris. More preferably, the Pichia pastoris is deposited in the General Microorganism Center of China Microbial Culture Collection Committee, and the deposit numbers are CGMCC No. 24687, CGMCC No. 24688, CGMCC No. 24689, CGMCC No. 24690.
  • the host cell is a prokaryotic cell.
  • the prokaryotic cells are genetically engineered bacteria.
  • a sixth aspect of the present invention provides a composition comprising the collagen variant with improved stability according to the second aspect of the present invention or the highly stable recombinant collagen according to the third aspect of the present invention.
  • the seventh aspect of the present invention provides products, which products include the collagen variant with improved stability according to the second aspect of the present invention, the highly stable recombinant collagen according to the third aspect of the present invention, and the sixth aspect of the present invention. provided group compound.
  • the eighth aspect of the present invention provides the collagen variant with improved stability described in the second aspect of the present invention, the highly stable recombinant collagen described in the third aspect of the present invention, the composition provided by the sixth aspect of the present invention or the present invention.
  • the present invention provides a method for constructing highly stable collagen variants, which includes avoiding unstable GXY triplets, removing potential MMP enzyme cleavage sites, eliminating chemical instability factors, and increasing the RGD content in the sequence. This method can be extended to expression systems other than Pichia pastoris.
  • the recombinant protein involved in the present invention has similar physical and chemical properties and biological functions to the original sequence. When used as a biological material, it has higher stability and high batch-to-batch consistency, which facilitates quality control and is beneficial to product stability.
  • Figure 1 shows the results of recombinant collagen protein spectrum detection.
  • Figure 2 shows SDS-PAGE of recombinant collagen shake flask expression supernatant (induced 48h).
  • Figure 3 shows the SDS-PAGE image of each recombinant collagen 5L tank fermentation supernatant.
  • Figure 4 shows SDS-PAGE of recombinant collagen sponge.
  • Figure 5 shows the M4 reversed-phase chromatography results of recombinant collagen sponge.
  • Figure 6 shows the stability of recombinant collagen liquid at different temperatures.
  • Figure 7 shows the stability of recombinant collagen liquid at different pH.
  • Figure 8 shows cell adhesion activity assay.
  • the invention provides a method for preparing a highly stable collagen variant, performance identification of the collagen variant obtained by the method, and its application. Includes the following method steps:
  • Point mutations made the total sequence contain 8 RGD motifs, MPGPR ⁇ PPGPR , with a homology of 92% to the original sequence; select a stable sequence that does not contain the above-mentioned unstable GXY triplet, potential MMP enzyme cleavage sites, and chemical instability factors in the original sequence and splice it into a monomer, and repeat it 5 times to form M6.
  • DNA sequences containing M2, M4, M5, and M6 were synthesized, and the exogenous DNA was connected into the expression vector pPIC9K to construct recombinant expression vectors expressing M2, M4, M5, and M6 respectively.
  • Preliminary identification of the expressed proteins by SDS-PAGE electrophoresis showed that: M2, M4, M5, and M6 can be efficiently secreted and expressed extracellularly.
  • the target band accounts for more than 90%, there are few degradation fragments, and the size is consistent with the appearance of collagen. Migration characteristics.
  • the liquid phase analysis results were consistent with SDS-PAGE, and the target band content reached more than 90%.
  • a fermentation tank was used for high-density fermentation experiments, and SDS-PAGE verified that the main bands accounted for more than 85%; the target bands obtained after one-step cation exchange purification accounted for 95%.
  • NIH/3T3 cells cultured in vitro were used to conduct cell adhesion between M2, M4, M5, and M6 and the original sequence.
  • the experiment showed that there was no significant difference in the adhesion activity of M2, M4, M5, and M6 to the original sequence, and was different from that of commercial human Collagen is basically the same.
  • the recombinant proteins M2, M4, M5, and M6 were lyophilized into sponges and subjected to different temperatures (-20°C, 4°C, 25°C, 40°C, 60°C) and different pH (4, 7, 9) conditions. Under the stability test, SDS-PAGE and liquid phase methods were used to monitor the changes in the content of the target band. The stability of M2, M4, M5, and M6 was significantly higher than that of the original sequence.
  • CCIC Huatongwei International Inspection (Suzhou) Co., Ltd. was entrusted to use in vitro cultured mammalian L-929 cells to test the potential cytotoxic effect of recombinant collagen M4 in accordance with the method requirements of GB/T16886.5-2017.
  • New Zealand rabbits were used to observe the potential intradermal reaction test of recombinant collagen M4.
  • the guinea pig maximum dose test method was used to observe the potential skin sensitization effect of recombinant collagen M4. The results showed no cytotoxicity, intradermal reactions and skin sensitization.
  • the original sequence is the sequence in CN201310033299.6. This sequence is concatenated with monomers 908-1136AA in the sequence of type 3 collagen (https://www.uniprot.org/uniprot/P02461), and the C-terminal is fused with a 6His tag. Its sequence Such as SEQ ID NO: 1:
  • M2 changes the triplet with a stability score of 0.0 to a triplet with a score of 2.0 or 1.0.
  • the specific change sequence is: GPLGIA (38-43, 270-275) ⁇ GPSGPA, GDR (125-127, 155-157, 357- 359, 387-389) ⁇ GER, GAA (194-196, 426-428) ⁇ GPA.
  • GLA potential MMP2, MMP9 recognition site, 49-51, 94-96, 277-279, 322-324
  • GPA potential MMP2 recognition site
  • GIK potential MMP2 recognition site, 196-198, 424-426
  • GEK potential MMP2 recognition site, 196-198, 424-42
  • NG sites susceptible to deamidation, 78-79, 129-130, 306-307, 357-358
  • QG sensitive to light-induced oxidation, 203, 431) ⁇ P. Its sequence is as SEQ ID NO: 2:
  • M4 is based on M2, retains the original RGD sequence, and mutates other R to K (Various endogenous proteases of Pichia pastoris recognize basic amino acids and cleave them at the C-terminal. The most common basic amino acids at the enzyme cleavage site are arginine). Its sequence is as SEQ ID NO: 3:
  • M5 is based on M2, retaining the original RGD sequence, and mutating other Ds to E (glutamine is prone to hydrolysis). Point mutations make the total sequence contain 8 RGD motifs, MPGPR ⁇ PPGPR (M is located at the Kex2 recognition site On the -5 position, the -4 position is P that hinders enzyme digestion, and M is prone to oxidation, so it is mutated). Its sequence is as SEQ ID NO: 4:
  • sequence is as SEQ ID NO: 5 :GNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGET
  • M6 is SEQ ID NO.5 repeated 5 times, and the sequence is as SEQ ID NO:6:
  • polynucleotide sequences encoding M2, M4, M5, and M6 are shown in SEQ ID NO: 7-10 respectively.
  • Nanjing GenScript Biotechnology Co., Ltd. was entrusted to complete: synthesize DNA fragments expressing M2, M4, M5, and M6, and clone the synthesized gene fragments into the pPIC9K empty vector (purchased from Thermo Fisher Scientific).
  • the target fragment is accurately inserted into the reading frame of the secretion vector containing the secretion signal ⁇ -factor, and a recombinant plasmid expressing M2, M4, M5, and M6 is obtained.
  • Example 3 Construction of recombinant engineering strains and strain screening
  • the linearized plasmid was electrotransformed into the host strain Pichia pastoris SMD1168 (purchased from Thermo Fisher Scientific) competent cells, and the electrotransduced bacterial solution was spread on the MD plate, and each 100 ⁇ L to 200 ⁇ L was applied to one plate. , let stand at room temperature for 10 minutes, and incubate upside down at 30°C for 2-5 days until a single colony (positive transformant) appears.
  • 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 the yeast and been integrated into the yeast chromosome through homologous recombination. After this step, high-copy, highly-expressible recombinant yeast engineered strains can be screened.
  • the constructed samples of the four engineered bacteria were sent to the General Microbiology Center of the China Microbial Culture Collection Committee.
  • the bacterial culture collection numbers are: No. 24687, No. 24688, No. 24689, and No. 24690.
  • the bacterial liquid sample can be collected.
  • the sampling volume is 1 mL, placed in a 1.5 mL EP tube, centrifuged at 12000g for 5 min at 4°C, and the expression supernatant is collected.
  • the sample to be tested is stored at -80°C for later use.
  • the expected bands of M4, M5, and M6 on SDS-PAGE were cut out, enzymatically digested with trypsin, and the tryptic peptides of recombinant collagen were detected by Nano-HPLC-MS/MS mass spectrometry (submitted to Suzhou Putai Biotechnology Co., Ltd.) and compare the detected peptides with the theoretical sequences.
  • Figure 1 The peptides detected after M4, M5, and M6 were enzymatically digested all belong to the theoretical sequences corresponding to the collagen variants, indicating that each collagen variant was successfully expressed.
  • Seed medium YPG (yeast powder 10g/L, peptone 20g/L, glycerol 10g/L); fermentation medium (NH 4 H 2 PO 4 190.4g/L, KH 2 PO 4 10.06g/L, CaSO 4 ⁇ 2H 2 O1.18g/L, K 2 SO 4 18.2g/L, MgSO 4 ⁇ 7H 2 O14.9g/L, glycerol 40g/L); feed medium (50% W/V glycerol, add a trace amount of 12mL PTM1 per liter elements); induction medium (100% methanol, add 12 mL PTM1 trace elements per liter); PTM1: filter sterilize with a 0.22 ⁇ m filter, and store at 4°C. After high-temperature sterilization of the fermentation medium, add PTM1 when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.
  • fermentation medium NH 4 H 2 PO 4 190.4g/L, KH 2 PO 4 10.06g/L, CaSO 4
  • the batch culture conditions and induced expression conditions of the engineering strain are as follows: the fed-batch culture method is used, and the culture temperature is 30°C.
  • Buffer A 20mMKH 2 PO4, pH 4.0;
  • Buffer B 20mMKH 2 PO4, 1M NaCl, pH 4.0.
  • Recombinant proteins M2, M4, M5 and M6 were mixed into a 1 mg/ml solution by adding UP water to a freeze-dried sponge. Filter it with a 0.22 ⁇ m filter membrane and distribute it into sterile centrifuge tubes. Place them at 60°C, 40°C, 25°C, and 4 respectively. °C, -20 °C, samples were taken for SDS-PAGE detection on days 3, 7, and 15. The results are shown in Figure 6. There is no obvious difference between the recombinant collagen placed at -20 °C and 4 °C and the original one. M4 The stability under liquid conditions of 60°C, 40°C, and 25°C is significantly higher than the original sequence and other variants.
  • Recombinant protein M2, M4, M5, M6 freeze-dried sponge and UP water are mixed into a 1mg/ml solution, and the pH is adjusted to acidic (pH4-5), neutral (pH7-7.5), alkaline (pH9-10), 0.22
  • acidic pH4-5
  • neutral pH7-7.5
  • alkaline pH9-10
  • 0.22 After filtration with a ⁇ m filter membrane, place it in a sterile centrifuge tube and place it at 4°C.
  • the original sequence recombinant collagen under neutral and alkaline conditions has no main band (results not shown).
  • Figure 7 shows that the original sequence result on day 10 is consistent with that on day 5, and has been completely degraded. Other variants have main bands. The proportion still exceeds 60%, and the stability of each variant under neutral conditions is significantly higher than that of the original
  • Absorbance represents the cell adhesion activity of collagen samples: the higher the absorbance, the more cells the protein adheres to, the higher the adhesion activity, and the more collagen can help cells adhere to the wall or adhere to the extracellular matrix in a short period of time. , which is more conducive to building a suitable extracellular environment.
  • the adhesion activities of M2, M4, M5, and M6 were close to those of the original sequence collagen, with no significant difference, and were all significantly higher than those of the control group.
  • CCIC Huatongwei International Inspection (Suzhou) Co., Ltd. was entrusted to use in vitro cultured mammalian L-929 cells to test the potential cytotoxic effect of recombinant collagen M4 in accordance with the method requirements of GB/T16886.5-2017.
  • the test samples and control samples were placed in MEM medium containing 10% fetal calf serum, and extracted in a 37°C incubator for 24 hours. After the extraction, the cell culture medium in the 96-well plate (10 4 cells/well) cultured for 24 hours was removed, replaced with the corresponding extraction solution, and incubated in a cell culture incubator (37°C, 5% CO2, >90% humidity ) for 24 hours.
  • the cell morphology and cell lysis were observed under the microscope, and the MTT method was used to determine the cytotoxicity value of the test product.
  • the results showed that the cells in the blank control group and the negative control group (high-density polyethylene) had intact and normal morphology during the entire test process, and showed no cytotoxic reaction.
  • the positive control group ZDEC
  • the cell morphology of the 100% concentration extract of the test sample was basically intact after incubating the cells for 24 hours, and the cell viability value was 83.6%.
  • Recombinant collagen M4 has no potential cytotoxic effects in the MTT cytotoxicity test conditions.
  • CCIC Huatongwei International Inspection (Suzhou) Co., Ltd. was entrusted to observe the potential intradermal reaction test of recombinant collagen M4 using New Zealand rabbits in accordance with the method requirements of GB/T16886.10-2017.
  • the samples were extracted using 0.9% sodium chloride injection and sesame oil. 18 hours before the test, a sufficient area of hair on both sides of the animal's back spine was completely removed to prepare for injection of the extract solution.
  • CCIC Huatongwei International Inspection (Suzhou) Co., Ltd. was entrusted to observe the potential skin sensitization effect of recombinant collagen M4 using the guinea pig maximum dose test method in accordance with the method requirements of GB/T16886.10-2017.
  • the samples were extracted using 0.9% sodium chloride injection and sesame oil.
  • the prepared extract solution was mixed with Freund's complete adjuvant to form a stable emulsifier, and the emulsifier was injected intradermally into the inner part of the scapula of each animal for intradermal induction and local induction. 14 days after local induction, a provocation test was performed on the untested sites during the induction phase.
  • Recombinant collagen 10 (g), sodium chloride: 9 (g), disodium hydrogen phosphate: 1 (g), water for injection: 1000ml (packed into 20 bottles).
  • Preparation process Measure the prescribed volume of water for injection into the dispensing tank, weigh the prescribed amount of sodium chloride and disodium hydrogen phosphate and add it to the water. Stir for 15 minutes to completely dissolve. Add the prescribed amount of collagen and stir for 15 minutes. It dissolves completely. The solution was filtered through a 0.45 ⁇ m filter, and the filtrate was filtered through a 0.22 ⁇ m filter. Dispense the filtrate (50ml/bottle) and seal it.
  • Efficacy test A mouse model of interstitial cystitis was constructed to evaluate the therapeutic effect of recombinant collagen repair agent on cystitis. Studies have shown that recombinant collagen repair agent can significantly improve bladder bleeding, and the bladder mucosa after repair agent treatment The epithelium can remain intact without obvious edema and shedding damage. In addition, the collagen fibers under the bladder mucosa The arrangement is tight and the collagen content is significantly improved.

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Abstract

本发明公开了高稳定性重组胶原蛋白、构建方法及其应用，在原始胶原蛋白序列的基础上进行包括选自避免不稳定GXY三联体、去除潜在的MMP酶切位点、消除化学不稳定因素、增加序列中RGD含量中一种或多种的突变。该方法可以扩展到毕赤酵母以外的表达系统。由该方法获得的重组胶原蛋白与原始序列有类似的理化性质和生物学功能，作为生物材料使用时由于更高的稳定性，批间一致性高，便于质量控制，有利于产品稳定性。

Description

高稳定性重组胶原蛋白、构建方法及其应用 技术领域
本发明涉及胶原蛋白技术领域，尤其涉及高稳定性重组胶原蛋白、构建方法及其应用。
背景技术
胶原蛋白是动物机体中含量最丰富的一类蛋白质，是重要的细胞外基质成分，在细胞迁移、细胞代谢、细胞信号通路应答、血小板凝聚、细胞、组织、器官的正常生理功能的维护、调节及损伤修复等方面有重要作用。胶原蛋白有良好的生物相容性、生物活性和可降解性，现已广泛应用于化工、医药、食品、化妆品等众多领域。
动物组织中提取胶原蛋白的技术已较为成熟、且该类胶原蛋白已有悠久的应用历史。但传统胶原生产过程中使用的酸碱等原料对环境不友好，提取的胶原肽性质不均、批间差异大且有病毒感染的安全隐患，鉴于此类原因基因工程技术生产的重组胶原蛋白日益受到重视。重组胶原的研究及应用已有超过30年的历史，现有的文献及专利主要集中在不同宿主中人源胶原蛋白单链的表达、截短的胶原蛋白基因单链的表达，另有少量关注胶原蛋白与相关表达后修饰酶的共表达获得三螺旋结构的胶原蛋白。
现有技术涉及的重组胶原蛋白的表达多使用人源胶原蛋白序列(CN201010527766.7，CN201510535383.7，CN201911093124.8)，表达产物多为无规卷曲，较少有规则的二级结构，更少有三、四级结构等高级结构，另外由于水溶性蛋白易于加工、使用，故氨基酸序列的选择多倾向于高水溶性。这就导致此类线状重组胶原蛋白在宿主内易被蛋白酶降解，在纯化、加工过程中，高水溶性蛋白由于疏水区域含量很少，无法折叠成稳定的高级结构，不稳定的侧链、残基、肽键易于发生脱酰胺、氧化、水解，最终可能导致产生与过程相关的杂质和降解产物，增加免疫原性。
获得高稳定性的重组胶原蛋白是本领域的迫切需求。
发明内容
为克服上述问题，本发明旨在通过对现有氨基酸序列的突变或对氨基酸序列的选择获得稳定性提高的重组胶原蛋白序列，简化纯化工艺，提高蛋白收率以及储存稳定性。
具体地，本发明第一方面提供了一种稳定性提高的胶原蛋白变体构建方法，在原始胶原蛋白序列的基础上进行包括选自避免不稳定GXY三联体、去除潜在的MMP酶切位点、消除化学不稳定因素、增加序列中RGD含量中一种或多种的突变。
在某些实施方式中，所述原始胶原蛋白包含SEQ ID NO：1所示的氨基酸序列或包含与SEQ ID NO：1具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
在某些实施方式中，所述GXY三联体中G为甘氨酸，X和Y可为任意氨基酸，胶原蛋白由GXY基序构成。
在某些实施方式中，所述避免不稳定GXY三联体包括在初始胶原蛋白序列中进行 选自GPL→GPS、GIA→GPA、GDR→GER、GAA→GPA的突变。
在某些实施方式中，所述去除潜在的MMP酶切位点包括去除潜在的MMP2和/或MMP9酶切位点；优选地，进行GLA→GPA和/或GIK→GEK的突变。
在某些实施方式中，所述化学不稳定因素包括易于脱酰胺、易于水解、或易于氧化的位点；优选地，进行选自如下突变：1)NG→QG；2)F→P；3)MPGPR→PPGPR；4)在保留原有RGD序列情况下，将其他R突变为K；5)在保留原有RGD序列情况下，将其他D突变成E。
本发明第二方面提供了根据本发明第一方面所述方法获得的稳定性提高的胶原蛋白变体。
在某些实施方式中，所述胶原蛋白变体的序列包含选自与SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
在某些实施方式中，所述胶原蛋白变体的序列包含选自SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6所示的氨基酸序列。
本发明第三方面提供了高稳定性重组胶原蛋白，所述重组胶原蛋白包含选自SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6所示的氨基酸序列或包含选自与SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
本发明第四方面提供了分离的多核苷酸，所述多核苷酸编码本发明第二方面所述的稳定性提高的胶原蛋白变体或本发明第三方面所述的高稳定性重组胶原蛋白。
在某些实施方式中，所述多核苷酸包含选自与SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的核酸序列。
在某些实施方式中，所述多核苷酸包含选自与SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示的核酸序列。
本发明第五方面提供了载体，所述载体包含本发明第四方面所述的分离的多核苷酸。
在某些实施方式中，所述载体为真核载体。
在某些实施方式中，所述载体为原核载体。
本发明第五方面提供了宿主细胞，所述细胞包含本发明第四方面所述的分离的多核苷酸或本发明第五方面所述的载体。
在某些实施方式中，所述宿主细胞为真核细胞。优选地，所述宿主细胞为毕赤酵母。更优选地，所述毕赤酵母保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.24687、CGMCC No.24688、CGMCC No.24689、CGMCC No.24690。
在某些实施方式中，所述宿主细胞为原核细胞。优选地，所述原核细胞为基因工程菌。
本发明第六方面提供了组合物，所述组合物包括本发明第二方面所述的稳定性提高的胶原蛋白变体或本发明第三方面所述的高稳定性重组胶原蛋白。
本发明第七方面提供了制品，所述制品包括本发明第二方面所述的稳定性提高的胶原蛋白变体、本发明第三方面所述的高稳定性重组胶原蛋白、本发明第六方面提供的组 合物。
本发明第八方面提供了本发明第二方面所述的稳定性提高的胶原蛋白变体、本发明第三方面所述的高稳定性重组胶原蛋白、本发明第六方面提供的组合物或本发明第七方面所述的制品在制备医疗器材、生物材料、组织工程产品或化妆品中的应用。
与现有技术相比，本发明的有益效果在于：
(1)本发明提供了一种高稳定性胶原蛋白变体的构建方法，包括避免不稳定GXY三联体、去除潜在的MMP酶切位点、消除化学不稳定因素、增加序列中RGD含量。该方法可以扩展到毕赤酵母以外的表达系统。
(2)本发明涉及的重组蛋白稳定性提高，在发酵阶段主条带占85％以上，纯化过程中不易发生降解，大大提高了全长蛋白的收率；
(3)本发明涉及的重组蛋白经纯化后获得的样品在稳定性实验中的表现优于原始蛋白，水溶液在4℃存放1个月未见明显降解条带，冻干海绵可在室温长期保存，减小了储存难度，延长了存储时间；
(4)本发明涉及的重组蛋白与原始序列有类似的理化性质和生物学功能，作为生物材料使用时由于更高的稳定性，批间一致性高，便于质量控制，有利于产品稳定性。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更显著：
图1显示重组胶原蛋白质谱检测结果。
图2显示重组胶原蛋白摇瓶表达上清(诱导48h)的SDS-PAGE。
图3显示各重组胶原5L罐发酵上清的SDS-PAGE图。
图4显示重组胶原蛋白海绵的SDS-PAGE。
图5显示重组胶原蛋白海绵的M4反相色谱结果。
图6显示重组胶原液体在不同温度下的稳定性。
图7显示重组胶原液体在不同pH下的稳定性。
图8显示细胞粘附活性检测。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例，都属于本发明保护的范围。
除非另作定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
本发明提供了一种高稳定性胶原蛋白变体的制备方法、由该方法获得的胶原蛋白变体的性能鉴定及其应用。包括如下方法步骤：
(1)设计胶原蛋白变体序列：
以CN201310033299.6(该专利氨基酸序列选自3型胶原)中的胶原序列为基础设计 了一系列变体，其中：M2更改不稳定的GXY三联体(Bachinger，H.P.and J.M.Davis，Sequence specific thermalstability of the collagen triple helix.Int J Bio1Macromol，1991.13(3)：p.152-6.)，去除潜在的MMP酶切位点，易于脱酰胺的位点NG突变为QG，光氧化敏感性F突变为P，与原始序列同源性为95％；M4以M2为基础，保留原有RGD序列，将其他R突变成K，与原始序列同源性为91.7％；M5以M2为基础，保留原有RGD，其他D突变成E，点突变使总序列含8个RGD基序，MPGPR→PPGPR，与原始序列同源性为92％；选取原序列中不含上述不稳定GXY三联体、潜在MMP酶切位点、化学不稳定因素的稳定序列拼接形成单体，进行5次重复形成M6。
(2)构建重组表达载体：
合成含M2、M4、M5、M6的DNA序列，将外源DNA连接入表达载体pPIC9K中，分别构建表达M2、M4、M5、M6的重组表达载体。
(3)构建重组工程菌株、诱导表达和菌株筛选；
以PmeI线性化重组表达载体，电转入毕赤酵母感受态细胞，涂布至MD平板初筛后，再经过含有不同浓度G418的YPD平板筛选，挑取菌落接入BMGY培养基中，再以BMMY培养基诱导表达；挑取多株菌种，选择表达量高的一株工程菌株进行后续实验。
(4)蛋白质表达鉴定；
以SDS-PAGE电泳对表达的蛋白质进行初步鉴定，表明：M2、M4、M5、M6能够高效分泌表达于胞外，目的条带占90％以上，降解片段较少，大小符合胶原蛋白的表观迁移特征。液相分析结果与SDS-PAGE一致，目的条带含量达90％以上。
(5)高密度发酵、培养和蛋白质纯化；
采用发酵罐进行高密度发酵实验，SDS-PAGE验证主条带均占85％以上；经一步阳离子交换纯化后获得的目的条带占比达95％。
(6)体外细胞实验验证重组胶原蛋白细胞粘附活性；
使用体外培养的NIH/3T3细胞进行M2、M4、M5、M6与原序列的细胞粘附，实验表明：M2、M4、M5、M6与原序列的粘附活性无明显差别，与商品化的人胶原蛋白基本一致。
(7)重组胶原稳定性验证；
以原序列蛋白为对照，重组蛋白M2、M4、M5、M6冻干海绵进行不同温度(-20℃、4℃、25℃、40℃、60℃)、不同pH(4、7、9)条件下的稳定性检测，以SDS-PAGE和液相手段监测目的条带含量变化，M2、M4、M5、M6的稳定性显著高于原序列。
(8)重组胶原蛋白安全性实验；
委托中检华通威国际检验(苏州)有限公司按照GB/T16886.5-2017的方法要求，使用体外培养哺乳动物L-929细胞，测试重组胶原蛋白M4的潜在细胞毒性作用。按照GB/T16886.10-2017的方法要求，使用新西兰兔观察重组胶原蛋白M4的潜在皮内反应试验。按照GB/T16886.10-2017的方法要求，使用豚鼠最大剂量试验法观察重组胶原蛋白M4的潜在皮肤致敏作用。结果显示无细胞毒性、皮内反应和皮肤致敏作用。
(9)重组胶原蛋白膀胱修复剂的制备与检测
以下结合具体实施例对本发明的高稳定性重组胶原蛋白、其生产方法、性能验证进行阐述。
实施例1：胶原蛋白变体氨基酸序列的设计
原序列为CN201310033299.6中序列，该序列以3型胶原(https：//www.uniprot.org/uniprot/P02461)序列中第908-1136AA为单体进行串联，C端融合6His标签，其序列如SEQ ID NO：1：
M2将稳定性得分为0.0的三联体更改为得分为2.0或1.0的三联体，具体更改序列：GPLGIA(38-43，270-275)→GPSGPA，GDR(125-127，155-157，357-359，387-389)→GER，GAA(194-196，426-428)→GPA。删除D462、连接序列EFT(230-232)、6His后序列TGLARF(469-474)。突变GLA(潜在的MMP2、MMP9识别位点，49-51，94-96，277-279，322-324)→GPA，GIK(潜在的MMP2识别位点，196-198，424-426)→GEK。去除化学不稳定因素，NG(易于脱酰胺的位点，78-79，129-130，306-307，357-358)→QG，F(光诱导氧化敏感，203，431)→P。其序列如SEQ ID NO：2：
M4以M2为基础，保留原有RGD序列，将其他R突变成K(毕赤酵母多种内源蛋白酶识别碱性氨基酸，在其C端裂解，酶切位点最为常见的碱性氨基酸为精氨酸)。其序列如SEQ ID NO：3：
M5以M2为基础，保留原有RGD序列，其他D突变成E(谷氨酰胺处易发生水解)，点突变使总序列含8个RGD基序，MPGPR→PPGPR(M位于Kex2识别位点-5位上，-4位为阻碍酶切的P，M易发生氧化，故突变)，其序列如SEQ ID NO：4：

选取原序列中不含MMP酶切位点，不含不稳定三联体的序列(2-37aa、62-85aa、134-154aa、176-190aa)拼接形成单体，其序列如SEQ ID NO：5：GNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGET
M6为SEQ ID NO.5重复5次，序列如SEQ ID NO：6：
编码M2、M4、M5、M6的多核苷酸序列分别如SEQ ID NO：7-10所示。
实施例2：DNA序列的合成与重组表达载体的构建
委托南京金斯瑞生物科技股份有限公司完成：合成表达M2、M4、M5、M6的DNA片段，将合成后的基因片段克隆至pPIC9K空载体(购自赛默飞世尔科技公司)中，使目的片段准确插入到含有分泌信号α-因子的分泌型载体读码框内，获得表达M2、M4、M5、M6的重组质粒。
实施例3：重组工程菌株的构建、菌种筛选
将上述重组表达质粒10μg用PmeI(购自大连TaKaRa公司，具体操作按试剂盒说明书进行)37℃酶切消化过夜，使其线性化，再使用PCR产物纯化试剂盒(购自生工生物工程(上海)股份有限公司)回收线性化质粒，控制体积在10μL左右。
将线性化质粒电转化入宿主菌种毕赤酵母SMD1168(购自赛默飞世尔科技公司)感受态细胞，将电转后的菌液涂布于MD平板上，每100μL～200μL涂布一块平板，室温静置10min，于30℃倒置培养2-5天，直至单菌落(阳性转化子)出现。
向MD平板表面加入2mL无菌双蒸水，然后用无菌三角涂布器轻轻刮下平板表面的His+转化子，并转移到50mL离心管中。以无菌双蒸水稀释菌悬液，105个细胞涂布于含有0.5mg/mLG418的YPD平板上，倒置，30℃培养3～4d后至单菌落出现。从YPD平板上挑取菌落至无菌96孔板中(200μLYPD/孔)，混匀，于30℃培养48h；混匀孔中菌液，各取10μL接入至一块新的无菌96孔板，于30℃培养24h后再重复一次此操作；24h后，从第三块96孔板中取出1μL分别点在含有1.0mg/mL和4mg/mLG418的YPD平板上，于30℃继续培养96h～120h。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酵母体内并通过同源重组整合到酵母的染色体上。经过这一步筛选可得到的高拷贝、可高效表达的重组酵母工程菌种。
构建的4种工程菌样本均送至中国微生物菌种保藏管理委员会普通微生物中心，菌种保藏编号分别是：No.24687、No.24688、No.24689、No.24690。地址：北京市朝阳区北辰 西路1号院3号；保藏日期：2022年4月18日。分类命名：巴斯德毕赤酵母Komagataella phaffii。
实施例4：诱导表达与重组胶原蛋白的鉴定
分别取表达M2、M4、M5、M6的毕赤酵母工程菌种，同时取专利CN201310033299.6中的工程菌株置于装有10mLBMGY培养基的100mL三角瓶中，于28-30℃、220rpm培养至OD600为2～6(16-18h)。室温下1500～3000g离心5min，收集菌体，用BMMY培养基重悬菌体，使OD600为2左右，放置于28-30℃、220rpm的摇床上继续生长3天，每24h向培养基中添加100％甲醇至终浓度为1.0％。加甲醇诱导16h以上，就可收取菌液样品，取样量为1mL，置于1.5mL EP管中，4℃下以12000g离心5min，收集表达上清，待检测样品于-80℃保存备用。
收取的表达上清，加入5×上样缓冲液(250mMTris-HCl、pH6.8，10％SDS，0.5％溴酚蓝，50％甘油，5％β-巯基乙醇)，置于100℃金属浴加热10min，进行SDS-PAGE检测。结果如图2所示，各胶原变体均能高效分泌于培养基上清，目的条带均占80％以上。
将M4、M5、M6在SDS-PAGE上的预期条带切割下来，用胰蛋白酶将其酶解，Nano-HPLC-MS/MS质谱检测重组胶原的胰蛋白酶解后肽段(交由苏州普泰生物技术有限公司完成)，并将检测到肽段与理论序列进行比对。如图1所示：M4、M5、M6被酶解后检测到的肽段均属于对应胶原蛋白变体的理论序列，说明各胶原蛋白变体成功表达。
实施例5：高密度发酵与纯化试验
(1)对基因工程菌进行高密度发酵试验，重组M2、M4、M5、M6胶原蛋白规模化表达生产，获取含有重组胶原蛋白的发酵液。
种子培养基YPG(酵母粉10g/L、蛋白胨20g/L、甘油10g/L)；发酵培养基(NH4H2PO4190.4g/L、KH2PO410.06g/L、CaSO4·2H2O1.18g/L、K2SO418.2g/L、MgSO4·7H2O14.9g/L、甘油40g/L)；补料培养基(50％W/V甘油，每升加12mLPTM1微量元素)；诱导培养基(100％甲醇，每升加入12mLPTM1微量元素)；PTM1：用0.22μm的滤膜过滤除菌，4℃保存。发酵培养基高温灭菌后待温度降至室温加入PTM1，用氨水调节PH至5.0。
工程菌株分批培养条件和诱导表达条件为：采用分批补料培养方法，培养温度30℃。工程菌接入含200ml种子培养基YPG的1L摇瓶，220rpm、30℃，培养18-20h，至OD600＝2～10。使用5L发酵罐(保兴生物)，装液量2L发酵培养基，2％甘油分开灭菌，接种前调节转速300rpm，通气量4L/min，温度30℃，用浓氨水配制好的碱液调pH，设置pH为4.5。然后先接入0.9mLPTM1，再将制备好的200ml种子液接入罐内(火焰圈接种)，然后点击溶氧电极校百，校百后开始发酵。待生长溶氧第一次掉至30％，采用溶氧串级转速功能，保持30％；等待甘油耗完，溶氧反弹、溶氧大于70％(OD600值约20)，取消溶氧串级转速，调高搅拌650rpm，甘油采用30％联动补料，补料80ml。停止补甘油，溶氧反弹至70％以上后，设置pH4、温度29℃，以甲醇、甘油混合碳源(甲醇∶50％甘油＝7∶3)进行诱导培养。手动补加5ml，待溶氧反弹至70％以上后，设定补料速度为8ml/h，一小时后提高到为10ml/h，一小时后再次提高设定到20ml/h。待溶氧值低于30％，停止补料，等待溶氧反弹，溶氧回升至30％后联动补料。诱导40～60h，UV测量蛋白浓度增长幅度不明显或下降即可放罐。UV蛋白定量公式：C(mg/mL)＝0.144*(A215-A225)，A215＜1.5。
收集发酵上清进行SDS-PAGE电泳检测，如图3所示，在高密度发酵条件下，各变体胶原蛋白在诱导24h以内均几乎只有目的条带，M4、M5在48h有轻微降解，光密度分析主条带 占比均超过85％。
(2)纯化
缓冲液A：20mMKH2PO4，pH4.0；
缓冲液B：20mMKH2PO4、1MNaCl，pH4.0。
收集发酵液，2000g、30min、4℃离心分离菌体和发酵上清。以缓冲液A平衡阳离子交换介质(层析填料为苏州纳微产UniGel-80sp，装载于利穗科技产XK50/30层析柱，使用GEAKTAPure蛋白质分离层析纯化系统)至A215吸光值和电导率值都保持不变后，设置100us/cm的流速上样，上样体积0.5L/次，检测紫外A215吸光值，当其上升时，开始接样。待上样结束后，关闭接样，再以缓冲液A平衡阳离子层析介质，当A215吸光值下降时，打开接样，直至紫外和电导降至最低且不再变化，停止接样。收集洗脱液，分别检测确定好组份后，进行透析(透析液为超纯水)，随后浓缩、冷冻干燥，收集冻干胶原蛋白海绵。取纯化后冻干海绵溶于超纯水，进行SDS-PAGE电泳，如图4所示。使用Image Lab软件(Bio-Rad Gel Doc XR+成像仪)测算：M2、M4、M5、M6纯度分别达到97.1％、96.7％、90.6％、97.6％。M4经反向色谱法测定纯度达99.78％，结果如图5所示。
实施例6：重组胶原蛋白稳定性试验
重组蛋白M2、M4、M5、M6冻干海绵加UP水配置成1mg/ml的溶液，0.22μm滤膜过滤后分装于无菌离心管，分别置于60℃、40℃、25℃、4℃、-20℃，第3天、7天、15天取样进行SDS-PAGE检测，结果如图6所示，-20℃、4℃条件下放置的各重组胶原蛋白与初始无明显区别，M4在60℃、40℃、25℃液体条件下的稳定性显著高于原序列及其他变体。
重组蛋白M2、M4、M5、M6冻干海绵加UP水配置成1mg/ml的溶液，调节pH为酸性(pH4-5)、中性(pH7-7.5)、碱性(pH9-10)，0.22μm滤膜过滤后分装于无菌离心管，置于4℃，第1天、5天、10天、30天取样，进行SDS-PAGE检测，仅显示第10天取样检测结果，第5天时中性和碱性条件下的原序列重组胶原蛋白已无主条带(结果未显示)，图7显示第10天的原序列结果和第5天一致，已全部降解，其他变体主条带占比仍超过60％，各变体在中性条件下的稳定性显著高于原序列蛋白。
实施例7：重组胶原蛋白细胞黏附活性实验
正常培养NIH/3T3细胞(购自中国科学院细胞库，货号GNM6，培养、传代方法参照细胞说明书执行)。取重组M2、M4、M5、M6胶原蛋白冻干海绵、对照人胶原蛋白(Sigma，货号C7774)及牛血清白蛋白(BSA，购自生工生物(上海)股份有限公司)溶解(超纯水或1MHCl溶液)，以UV蛋白定量经验公式：C(mg/mL)＝0.144*(A215-A225)测定蛋白浓度，再以PBS(pH7.4)稀释至0.5mg/mL。向96孔细胞培养板中加入100μL各种蛋白溶液和空白PBS溶液对照，室温静置60min；再向每孔中加入105个培养状态良好的3T3细胞，37℃、5％CO2孵育60min。以PBS清洗4次孔中细胞。使用LDH检测试剂盒(Roche，04744926001)检测570nm的吸光度值(具体操作参照说明书执行)。
吸光度表征胶原蛋白样品的细胞粘附活性：吸光度越高，说明蛋白粘附的细胞越多，黏附活性越高，胶原蛋白越能在短时间内帮助细胞贴壁或粘附于细胞外基质之上，更利于构建合适的细胞外环境。如图8所示，M2、M4、M5、M6与原序列胶原粘附活性接近，无显著差异，均显著高于对照组。
实施例8：重组胶原蛋白安全性实验
委托中检华通威国际检验(苏州)有限公司按照GB/T16886.5-2017的方法要求，使用体外培养哺乳动物L-929细胞，测试重组胶原蛋白M4的潜在细胞毒性作用。将试验样品与对照样品分别放在含有10％胎牛血清的MEM培养基中，于37℃培养箱浸提24小时。在浸提结束后将培养24小时的96孔板(104个/孔)内细胞培养基去除，换成相应浸提液，并在细胞培养箱(37℃，5％CO2，＞90％湿度)中培养24小时。培养结束后镜下观察细胞形态和细胞裂解情况，并采用MTT法测定供试品的细胞毒性值。结果显示，空白对照组和阴性对照组(高密度聚乙烯)中细胞在整个试验过程中形态完好正常，没有显示细胞毒性反应。阳性对照组(ZDEC)中显示严重的细胞毒性反应。测试样品100％浓度浸提液在孵育细胞24小时后细胞形态基本完好，细胞活力值为83.6％。重组胶原蛋白M4在MTT法细胞毒性试验条件中没有潜在的细胞毒性影响。
委托中检华通威国际检验(苏州)有限公司按照GB/T16886.10-2017的方法要求，使用新西兰兔观察重组胶原蛋白M4的潜在皮内反应试验。使用0.9％氯化钠注射液和芝麻油浸提样品。在试验前18h，彻底除去动物背部脊柱两侧足够面积的被毛，以备注射浸提液。在试验当天，在每只兔脊柱一侧皮内注射10个位点，五个点注射0.2mL极性浸提液，五个点注射0.2mL非极性浸提液；在脊柱另一侧分别注射极性和非极性对照溶液，操作步骤同试验样品浸提液。根据“皮内反应评分系统”，观察并记录(24±2)h，(48±2)h和(72±2)h各注射点部位状况。结果显示，试验过程中动物未出现异常症状或死亡。试验组一侧皮肤反应未超过阴性对照组一侧皮肤反应，供试品与对照组动物皮肤完好，未出现红斑和水肿等反应。重组胶原蛋白M4在新西兰兔体内没有潜在皮内反应。
委托中检华通威国际检验(苏州)有限公司按照GB/T16886.10-2017的方法要求，使用豚鼠最大剂量试验法观察重组胶原蛋白M4的潜在皮肤致敏作用。使用0.9％氯化钠注射液和芝麻油浸提样品。将配制好的浸提液与弗氏完全佐剂混合成稳定性乳化剂，在每只动物去毛的肩胛骨内侧部位皮内注射乳化剂进行皮内诱导和局部诱导。局部诱导后14天，在诱导阶段未试验部位进行激发试验。激发后24小时和48小时，分别观察供试品组和对照组动物激发部位皮肤反应情况，按Magnusson和Kligman分级标准对每一激发部位的皮肤红斑和水肿反应进行评分。结果显示，阴性对照组(0.9％氯化钠注射液与芝麻油)中动物在试验过程中，皮肤完好，未出现皮肤红斑和水肿反应。阳性对照组(DNCB)动物出现明显皮肤红斑和水肿反应。重组胶原蛋白M4浸提液组动物皮肤完好，未出现皮肤红斑和水肿反应。重组胶原蛋白M4在致敏试验中没有皮肤致敏作用。
实施例9：重组胶原蛋白膀胱修复剂的制备与检测
重组胶原蛋白膀胱修复剂配方：
规格：50ml：0.5g，浓度1％。配方：重组胶原蛋白：10(g)，氯化钠：9(g)，磷酸氢二钠：1(g)，注射用水：1000ml(分装20瓶)。
制备过程：量取处方量体积注射用水于配液罐中，称取处方量氯化钠、磷酸氢二钠加至水中，搅拌15分钟使完全溶解后，加入处方量胶原蛋白，搅拌15分钟使其完全溶解。溶液过0.45μm滤膜，滤液过0.22μm滤膜。将滤液分装(50ml/瓶)、熔封。
功效检测：通过构建小鼠间质性膀胱炎模型，来评估重组胶原蛋白修复剂对膀胱炎的治疗效果，研究表明组胶原蛋白修复剂可以明显改善膀胱出血情况，而且经过修复剂治疗后膀胱粘膜上皮能够保持完整，无明显的水肿和脱落损伤，此外膀胱粘膜下胶原纤维 排列紧密，胶原蛋含量有明显提升。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点，对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这中叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。

Claims (13)

  1. 稳定性提高的胶原蛋白变体构建方法，其特征在于，在原始胶原蛋白序列的基础上进行包括选自避免不稳定GXY三联体、去除潜在的MMP酶切位点、消除化学不稳定因素、增加序列中RGD含量中一种或多种的突变。
  2. 根据权利要求1所述的方法，其特征在于，所述原始胶原蛋白包含SEQ ID NO：1所示的氨基酸序列或包含与SEQ ID NO：1具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  3. 根据权利要求1所述的方法，其特征在于，所述GXY三联体中G为甘氨酸，X和Y可为任意氨基酸；优选地，所述避免不稳定GXY三联体包括在初始胶原蛋白序列中进行选自GPL→GPS、GIA→GPA、GDR→GER、GAA→GPA的突变。
  4. 根据权利要求1所述的方法，其特征在于，所述去除潜在的MMP酶切位点包括去除潜在的MMP2和/或MMP9酶切位点；优选地，进行GLA→GPA和/或GIK→GEK的突变。
  5. 根据权利要求1所述的方法，其特征在于，所述化学不稳定因素包括易于脱酰胺、易于水解、或易于氧化的位点；优选地，进行选自如下突变：1)NG→QG；2)F→P；3)MPGPR→PPGPR；4)在保留原有RGD序列情况下，将其他R突变为K；5)在保留原有RGD序列情况下，将其他D突变成E。
  6. 根据权利要求1-5任一项所述的方法获得的稳定性提高的胶原蛋白变体；优选地，所述胶原蛋白变体的序列包含选自与SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列；更优选地，所述胶原蛋白变体的序列包含选自SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6所示的氨基酸序列。
  7. 高稳定性重组胶原蛋白，其特征在于，所述重组胶原蛋白包含选自SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6所示的氨基酸序列，或包含选自与SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：6具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  8. 分离的多核苷酸，所述多核苷酸编码权利要求6所述的稳定性提高的胶原蛋白变体或权利要求7所述的高稳定性重组胶原蛋白；优选地，所述多核苷酸包含选自与SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10具有80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的核酸序列；更优选地，所述多核苷酸包含选自SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示的核酸序列。
  9. 载体，所述载体包含权利要求8所述的分离的多核苷酸；优选地，所述载体为真核载体或原核载体。
  10. 宿主细胞，所述细胞包含权利要求8所述的分离的多核苷酸或权利要求9所述的载体；优选地，所述宿主细胞为真核细胞或原核细胞；更优选地，所述宿主细胞为毕赤酵母；更优选地，所述毕赤酵母保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.24687、CGMCC No.24688、CGMCC No.24689、CGMCC No.24690。
  11. 组合物，所述组合物包括权利要求6所述的稳定性提高的胶原蛋白变体或权利要求7所述的高稳定性重组胶原蛋白。
  12. 制品，所述制品包括权利要求6所述的稳定性提高的胶原蛋白变体或权利要求7所述的高稳定性重组胶原蛋白或权利要求11所述的组合物。
  13. 根据权利要求6所述的稳定性提高的胶原蛋白变体、权利要求7所述的高稳定性重组胶原蛋白、权利要求11所述的组合物或权利要求12所述的制品在制备医疗器材、生物材料、组织工程产品、化妆品中的应用。
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