Application of recombinant humanized collagen

Abstract

The invention discloses application of recombinant humanized collagen, and relates to the field of application of recombinant humanized collagen. The invention provides an application of an amino acid sequence of recombinant humanized collagen in preparation of an implantable medical device material. Compared with the prior art, the recombinant humanized collagen provided by the invention has the advantages of higher biological activity and processability, no virus hidden danger and immunogenicity risk, higher hydrophilicity and suitability for the field of medical engineering. According to the method disclosed by the invention, the recombinant humanized collagen with high purity and high efficiency can be prepared, and the safety and effectiveness of the recombinant humanized collagen in biomedical application are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of humanized collagen, and more specifically to an application of recombinant humanized collagen. Background Art

[0002] Human collagen is the main structural protein in the extracellular matrix and plays a variety of important biological functions. Recombinant collagen is a new type of biomaterial that uses cutting-edge structural biology, genetic engineering and other technologies, with the gene encoding of the functional region of a specific type of human collagen as a template for screening and preparation to obtain the same or similar amino acid sequence as human collagen. Among them, recombinant humanized collagen that is consistent with the full-length or partial amino acid sequence of human collagen does not contain sequence fragments of non-human collagen and has a low risk of immunogenicity. At present, recombinant humanized collagen is gradually being promoted and applied in the biomedical field.

[0003] Collagen from xenogeneic and allogeneic sources has the following significant problems: the presence of pathogen residues, strong acid and alkali residues, low biological activity, low water solubility, viral contamination and immune rejection. It is not suitable for use as a biomaterial and is difficult to apply in the medical field.

[0004] Therefore, how to provide a recombinant humanized collagen that is free of viral hazards, non-immunogenic, and highly hydrophilic is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides an application of recombinant humanized collagen.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The recombinant gene, the sequence is shown in SEQ ID NO.3.

[0008] Preferably, the recombinant gene is recombined from type III collagen gene.

[0009] As an inventive concept identical to the above technical solution, the present invention also requests protection for the use of the recombinant gene in the preparation of re-humanized collagen.

[0010] As an inventive concept identical to the above technical solution, the present invention also requests protection for a recombinant humanized collagen protein encoded by the sequence shown in SEQ ID NO.3, the amino acid sequence of the protein being shown in SEQ ID NO.2.

[0011] As the same inventive concept as the above technical solution, the present invention also requests protection for the use of the recombinant humanized collagen in the preparation of implantable medical device materials.

[0012] From the above technical solutions, we can know that compared with the existing technology, the recombinant humanized collagen we produce is the collagen produced by transferring the human collagen gene into the engineered bacteria / engineered cells for expression after specific enzyme cleavage and splicing. Compared with animal-derived collagen, it has the following outstanding advantages:

[0013] 1) Strong biological activity and processability. The recombinant humanized collagen prepared by the present invention can form a triple-repeating helical structure, which not only enables it to have biological activity under natural conditions, but also has processability under the condition that the molecular weight and physicochemical properties remain unchanged. Animal-derived collagen has a triple-cross helical structure, and the molecules are easily broken during the processing process, causing changes in the physicochemical properties.

[0014] 2) No virus risk and no immunogenicity make recombinant human collagen more widely used in the field of medical engineering

[0015] 3) Through genetic design, recombinant collagen can be made more hydrophilic / water-soluble than natural molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 The attached figure is a structural diagram of the recombinant plasmid.

[0018] Figure 2 The attached figure is a diagram of the plasmid linearization results. M: DNA Marker; 1: before linearization; 2: after linearization.

[0019] Figure 3 The attached figure is a diagram of PCR identification results.

[0020] Figure 4 The attached figure is a diagram of protein identification results.

[0021] Figure 5 The attached figure shows the results of the implantation experiment; the left is before implantation, and the right is after implantation. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The materials and reagents used in the examples are:

[0024] Agarose, plasmid extraction kit, endonuclease, primers, E. coli DH5α, LB medium, kanamycin, nucleic acid electrophoresis marker

[0025] Example 1 Construction of recombinant genes

[0026] This sequence uses the 486-879 amino acids in the type III collagen sequence (https: / / www.uniprot.org / uniprotkb / P02461 / entry) as monomers, and a 6His histidine tag is fused to the C-terminus. Its sequence is as shown in SEQ ID NO.1:

[0027] GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGE RGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARG

[0028] The unstable GER in the GXY triplet was mutated to GAR, and its sequence is shown in SEQ ID NO.2:

[0029] GAPGFRGPAGPNGIPGEKGPAGARGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDT GPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGARGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGARGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDK GEGGAPGLPGIAGPRGSPGARGETGPPGPAGFPGAPGQNGEPGGKGARGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGARGSPGGPGAAGFPGARG

[0030] The amino acid sequence was optimized for Pichia pastoris codons to obtain its corresponding gene sequence, which is shown in SEQ ID NO.3:

[0031]

[0032] Example 2 Construction of recombinant yeast strains

[0033] This example uses the recombinant gene obtained in Example 1 to construct a recombinant yeast strain. The specific steps are as follows:

[0034] 1. Preparation of yeast engineering strains: Purchase ATCC engineering strains and ensure that their test reports are complete.

[0035] 2. Construction of recombinant plasmid:

[0036] Insert the gene fragment shown in SEQ ID NO.1 into an appropriate expression vector (such as pPICZ(alpha)A), perform double restriction digestion and ligation with EcoRI and XbaI ( Figure 1 ).

[0037] 3. Plasmid linearization:

[0038] Select the appropriate linearization site SacI and perform single enzyme digestion according to the following system:

[0039] Enzyme digestion system (40 μl):

[0040] 4μl FD Buffer (enzyme digestion buffer)

[0041] 3 μl FastDigest TM SacI endonuclease

[0042] 33 μl plasmid

[0043] Incubate in a 37°C water bath for 1 hour, and run a gel sample to verify the linearization effect ( Figure 2 ).

[0044] 4. Plasmid transformation into yeast:

[0045] Add 10 μl of the linearized plasmid to 100 μl of the X33 competent cells prepared in advance, mix well, transfer to a pre-cooled electroporation cup (2 mm), and perform electroporation (the electroporator is set to 1.8 kV, 4.5 ms to 5 ms).

[0046] Immediately after the electroporation, 300 μl of 1 M sorbitol was added, followed by 200 μl of YPD medium. After 3 hours of recovery at 30°C, the culture was spread on a YPD plate containing Zeocin antibiotics and cultured at 30°C for 2 to 3 days until single colonies appeared.

[0047] 5. PCR identification of positive clones:

[0048] Dissolve the single clones on the plate with 10 μl ddH2O, take 0.5 μl each as a template, and use the corresponding universal primers for PCR identification. Primer sequence:

[0049] 5ˋAOX1: GACTGGTTCCAATTGACAAGC, such as SEQ ID NO.4;

[0050] 3ˋAOX1: GCAATGGCATTCTGACATCC, such as SEQ ID NO.5;

[0051] PCR system (10 μl):

[0052] 5μl enzyme mix

[0053] 0.2 μl Primer F

[0054] 0.2 μl Primer R

[0055] 0.5 μl template

[0056] 4.1 μl ddH2O

[0057] PCR procedure:

[0058] 95℃ 10min

[0059] 95℃ 30s

[0060] 55℃ 30s(30cycles)

[0061] 72℃ 1min / kb

[0062] 72℃ 5min

[0063] PCR identification results are shown in Figure 3

[0064] Example 3 Preparation of recombinant humanized collagen

[0065] 1. Seed culture

[0066] 1) Prepare YPD medium: 1% yeast extract, 2% tryptone, 1% glycerol, add deionized water to a total volume of 500 mL, and sterilize by high pressure.

[0067] 2) Take the yeast strain expressing recombinant humanized collagen out of the -80°C refrigerator, slowly dissolve it at room temperature, and inoculate 10 μL of the bacterial solution into a 100 mL shake flask containing 15 mL of YPD medium. Incubate at 28°C and 225 rpm for 18 hours, and measure the OD 600 The value should be between 2-6.

[0068] 2. Bacterial expansion

[0069] Take 20 μL of the recovered bacterial solution and inoculate it into a 5L shake flask containing 500 mL of YPD medium. Incubate at 28°C and 225 rpm until the OD 600 The value reaches 5 or more.

[0070] 3. Fermentation culture

[0071] 1) Prepare BSM (1 / 4) fermentation medium: 6.67 mL phosphoric acid × 4, 0.23 g calcium sulfate × 4, 4.55 g potassium sulfate × 4, 3.72 g magnesium sulfate × 4, 1.03 g potassium hydroxide × 4, 50 g glycerol × 4, 10 g tryptone × 4, add deionized water to a total of 4 L, transfer to a fermenter and sterilize under high pressure for 30 minutes.

[0072] 2) Calibrate the pH electrode of the fermenter (calibrate to pH 6.86 and 4.00 with pH calibration solution before sterilizing the fermenter) and the dissolved oxygen electrode (calibrate after the culture medium in the fermenter is sterilized and cooled).

[0073] 3) After the culture medium is sterilized and cooled, set the fermentation parameters: temperature 30°C, rotation speed 800 rpm, ventilation volume 12 L / min, culture medium pH 5.5. Aseptically add Biotin (2 mL / L) and PTM1 trace elements (4.35 mL / L) sterilized by 0.22 μm filter membrane.

[0074] 4) Aseptic operation: Connect the inoculation port of the fermenter to the seed liquid feeding port, add 500 mL of YPD bacterial solution into the fermenter through a peristaltic pump, and the total volume is 4.5 L for fermentation culture. The fermenter parameters are set to a stirring speed of 800 rpm, a temperature of 30°C, and a DO value of more than 20%. Pure oxygen is introduced when necessary. Samples are taken every 6 hours to determine the wet weight of cells.

[0075] 5) Add 12 mL / L PTM1 trace elements to 50% glycerol after high-pressure sterilization and add it to the fermentation tank through a peristaltic pump.

[0076] 6) When the wet weight of the cells reached 209 g / L, stop adding glycerol and the DO value quickly returns to 100%, indicating that the glycerol has been exhausted. Continue to maintain the "glycerol starvation" state for 30 minutes, and then enter the methanol-induced expression stage.

[0077] 7) Add 12 mL / L PTM1 trace elements to methanol and add it to the fermenter via a peristaltic pump. After the start of methanol induction, take samples every 6 hours, centrifuge at 12,000 rpm for 10 minutes to determine the wet weight of the cells and retain the supernatant.

[0078] 8) After the fermentation was completed, the fermentation broth was collected and centrifuged at 8000 rpm for 15 minutes. The supernatant was collected and stored at -20°C.

[0079] 4. Centrifugation

[0080] The bacterial cells are removed by a high-speed centrifuge to obtain a supernatant. The bacterial cell waste residue after centrifugation is inactivated and dried by passing high-temperature steam.

[0081] 5. Ultrafiltration concentration

[0082] The supernatant obtained by centrifugation is diluted 8-15 times, and then concentrated 10-15 times by ultrafiltration to prepare a concentrate.

[0083] 6. Composite chromatography

[0084] Phenyl Sepharose 6FF (HS) gradient elution purification:

[0085] Wash the column with deionized water to remove the 20% ethanol storage solution in the column.

[0086] Equilibrate the column with equilibration solution (20 mM sodium phosphate, 1 M ammonium sulfate, pH 7.

[0087] Load 80 mL of sample at a flow rate of 3 mL / min. Wash the column with the equilibrium solution. Start collecting the flow-through peak when the UV peak rises and stop collecting when the UV peak ends.

[0088] · 35%, 60% gradient elution, flow rate 3mL / min, collect when the UV peak starts and stop collecting when the UV peak ends.

[0089] Wash the column with NaOH and regenerate it with the equilibrated solution.

[0090] ·Preserve the column in 20% ethanol.

[0091] Rigose MMC HiRes Gradient Purification:

[0092] The chromatography column was washed with deionized water to remove the 20% ethanol storage solution in the column.

[0093] The chromatography column was equilibrated with equilibration solution (20 mM sodium acetate, pH 4.5).

[0094] Load 96 mL of sample at a flow rate of 3 mL / min, flush the column with the equilibrium solution, start collecting the flow-through peak when the UV peak rises, and stop collecting when the UV peak ends.

[0095] 40%, 100% gradient elution, flow rate 3mL / min, collection at the beginning of the UV peak and stop collection at the end.

[0096] The column was cleaned with NaOH and then regenerated with the equilibrated solution.

[0097] The chromatography column was preserved in 20% ethanol.

[0098] 7. Ultrafiltration concentration

[0099] The liquid purified by chromatography is diluted and concentrated by ultrafiltration again to prepare a sterile collagen solution.

[0100] 8. Lyophilization of stock solution

[0101] · Add 2% mannitol to the purified sample and dispense into 2 ml bottles for freeze drying. The freeze drying process is divided into four stages:

[0102] Pre-freezing stage: freeze dryer is set to 20℃, sample is placed in it, balanced at -5℃ for 30 minutes, cooled to -45℃ and maintained for 150 minutes.

[0103] Primary drying stage: the pressure was reduced to 0.1 mbar, the pressure was maintained, and the shelf temperature was increased to -10°C and maintained for 900 minutes.

[0104] Secondary drying stage: Maintain pressure, raise temperature to 0°C and maintain for 180 minutes, raise temperature to 5°C and maintain for 120 minutes.

[0105] Final drying stage: the temperature is raised to 28°C, the pressure is reduced to 0.05 mbar, and maintained for 240 minutes, and the freeze drying is completed.

[0106] Example 4 Identification of recombinant protein

[0107] This example describes in detail the identification process of the recombinant protein, including using SDS-PAGE electrophoresis to confirm the protein molecular weight and purity, and using gel imaging equipment to record and analyze the electrophoresis results.

[0108] 1. Confirmation of protein molecular weight and purity

[0109] SDS-PAGE electrophoresis:

[0110] 1. Sample preparation:

[0111] Take an appropriate amount of recombinant protein sample and add an equal volume of SDS sample buffer (4X).

[0112] Boil in a water bath at 100°C for 5 minutes to ensure complete denaturation of the protein.

[0113] 2. Gel preparation:

[0114] Prepare a 12% SDS polyacrylamide gel.

[0115] The gel consists of two parts: separation gel and stacking gel.

[0116] 3. Sample loading and electrophoresis:

[0117] Load the boiled protein samples and standard molecular weight markers into the gel wells.

[0118] Set the voltage to 80V, and adjust it to 120V after the sample enters the separation gel for electrophoresis separation.

[0119] 4. Dyeing and bleaching:

[0120] After electrophoresis, stain the gel in Coomassie Brilliant Blue for 30 minutes.

[0121] Then destain with a destaining solution (40% methanol, 10% acetic acid) until the background is clear.

[0122] 2. Recording and Analysis of Electrophoresis Results

[0123] Gel imaging equipment:

[0124] 1. Gel imaging:

[0125] Place the destained gel in a gel imager and set appropriate exposure time and parameters for imaging.

[0126] 2. Image analysis:

[0127] The molecular weight and band intensity of the electrophoresis bands were quantitatively analyzed using image analysis software. The molecular weight of the recombinant protein was confirmed by calibration using standard protein molecular weight markers. The purity of the protein was evaluated by comparing the clarity of the sample bands and the background.

[0128] Identification results

[0129] 1. Molecular weight confirmation:

[0130] The electrophoresis pattern showed that the main band of the recombinant protein was around 37 kDa, which was consistent with the expected molecular weight, indicating that the molecular weight of the recombinant protein was correct. Figure 4 )

[0131] 2. Purity assessment:

[0132] The main bands in the electrophoresis pattern were clear and there were no obvious miscellaneous bands, indicating that the recombinant protein had a high purity.

[0133] Through SDS-PAGE electrophoresis and gel imaging equipment identification, it can be confirmed that the prepared recombinant protein has the expected molecular weight and high purity, verifying the quality and expression effect of the recombinant protein. These results provide a reliable basis for subsequent applications.

[0134] Example 5 Application Test

[0135] Implant biocompatibility testing

[0136] This example aims to evaluate the biocompatibility of recombinant collagen as a material for implantable medical devices. Healthy adult mice (weight 20-25 g) were selected for the experiment and divided into an experimental group and a control group, with 10 mice in each group.

[0137] Experimental design

[0138] 1. Animal Selection

[0139] Healthy adult mice, weighing 20-25 g, 10 mice per group.

[0140] 2. Sample Preparation

[0141] Experimental group: recombinant collagen solution.

[0142] Control group: an equal volume of normal saline.

[0143] 3. Pre-experimental treatment

[0144] One day before the experiment, the hair on both sides of the mouse spine was shaved with a shaver and then thoroughly removed with a depilatory cream to reduce skin irritation.

[0145] 4. Injection and disinfection

[0146] Before injection, the back skin of the mouse was wiped with iodine tincture and alcohol for disinfection.

[0147] Five injection points were placed on each side of the mouse spine, with 0.2 mL of solution injected into each point. The left side was injected with recombinant collagen solution, and the right side was injected with phosphate buffer as a control.

[0148] 5. Injection regimen

[0149] Experimental group: subcutaneous injection of recombinant collagen solution, the dose is 10mL / kg, once a week, for a total of 3 times.

[0150] Control group: injected with an equal volume of saline at the same frequency and time interval.

[0151] Recording and Observation

[0152] Observe the local and systemic reactions of the mice and record the redness, swelling, nodules and other allergic reactions at the injection site. Figure 5 )

[0153] result

[0154] There was no obvious inflammation or rejection reaction in the mice in the experimental group and the control group during the entire observation period, and there was no significant difference in the injection site.

[0155] Both groups of mice showed no abnormal behavior and their body weight and food intake were normal throughout the observation period.

[0156] Histological examination of the injection site showed no obvious pathological changes.

[0157] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0158] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of recombinant humanized collagen in the preparation of implantable medical device materials, It is characterized in that The sequence of the recombinant humanized collagen is shown in SEQ ID NO.2; the encoding gene is shown in SEQ ID NO.3.

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