Recombinant human type III collagen with high thermal stability and high transdermal permeability

Abstract

The present application provides a recombinant human type III collagen with high thermal stability and high transdermal permeability. The recombinant type III collagen comprises a type III collagen part and a transdermal peptide part, which has a high transdermal absorption capacity and has the advantage of high thermal stability, and is suitable for the preparation of cosmetics, drugs and medical devices.

Description

Field of the Invention

[0001] This application belongs to the field of biotechnology. Specifically, this application provides a recombinant human type III collagen with high thermal stability and high transdermal permeability. Background Art

[0002] The largest area of the human body is the skin, which occupies the largest area in the living body. With age and external stimuli, the epidermis and dermis of the skin will age. The main reason for fine lines or sagging in the epidermis is the reduction of collagen synthesis. The dermis is mainly composed of collagen fibers and elastic fibers to form a reticular layer structure, which has strong elasticity, and the monomers assembled into collagen fibers are collagen. Among them, type I collagen has the highest content in the total collagen content, and type III collagen content is the second. Usually, type III collagen significantly increases in new tissues. Type III collagen is the main component of reticular fibers, exists together with type I collagen, provides support, water retention, and maintains elasticity for tissues such as the skin. Type III collagen is more flexible and is more beneficial for maintaining the elasticity of the skin and tissues. Type III collagen forms a superhelical structure by three α-chains together and is a homotrimer.

[0003] As the first line of defense of the human body, the outermost layer of the epidermis of the skin is the stratum corneum (composed of stratum corneum cells). Macromolecules such as nucleic acids, polypeptides, proteins, and hydrophilic substances cannot enter. Substances that need to penetrate the skin require auxiliary means, such as chemical methods (dissolving sebum, enlarging hair follicles), physical methods (electroporation, ultrasound, heating). These technologies still have many limitations at present, such as strong stinging caused by skin irritation, some means causing damage to skin cells, and being toxic to cells.

[0004] Studies have shown that polypeptides containing 5 - 30 amino acids can introduce hydrophilic polypeptides, proteins, nucleic acids, and small molecule drugs into cells and are not prone to immune reactions. This method will greatly reduce the pain caused by traditional subcutaneous injection and can control the dosage and administration time by applying to the skin, greatly improving people's acceptance of drug administration. Collagen belongs to macromolecules and is not easy to pass through the skin. By adding a transdermal peptide at the front end of collagen, it is expected to improve the transdermal rate of type III collagen in human skin, thereby compensating for the lack of type III collagen in the human body. Summary of the Invention

[0005] In order to solve the technical problem that currently applied collagen is not easy to penetrate the skin. The inventor has conducted a large number of studies, and as a result, a highly transdermal type III recombinant collagen composed of a combination of a transdermal peptide and a human type III collagen amino acid sequence fragment is obtained. The highly transdermal type III recombinant collagen has a high transdermal absorption ability and has the advantage of high thermal stability, thus completing the present invention.

[0006] On the one hand, the present application provides a recombinant type III collagen with high thermal stability and high transdermal permeability, and the recombinant type III collagen comprises a type III collagen part and a transdermal peptide part.

[0007] Further, the amino acid sequence of the transdermal peptide part is SEQ ID NO.2.

[0008] Further, the amino acid sequence of the type III collagen part is SEQ ID NO.3.

[0009] Further, the amino acid sequence of the recombinant type III collagen is SEQ ID NO.1.

[0010] On the other hand, the present application provides the use of the above-mentioned recombinant type III collagen in the preparation of cosmetics having anti-wrinkle and / or repair functions.

[0011] On the other hand, the present application provides cosmetics having anti-wrinkle and / or repair functions, and the cosmetics contain the above-mentioned recombinant type III collagen.

[0012] On the other hand, the present application provides the use of the above-mentioned recombinant type III collagen in the preparation of drugs or medical devices for skin repair.

[0013] On the other hand, the present application provides drugs or medical devices for skin repair, and the drugs or medical devices contain the above-mentioned recombinant type III collagen.

[0014] On another aspect, the present application provides a preparation method of the above-mentioned recombinant type III collagen, and the preparation method uses Pichia pastoris as a host to express the above-mentioned recombinant type III collagen.

[0015] Further, the preparation method further includes dialysis and chromatography steps.

[0016] Those skilled in the art can select other excipients and functional components for cosmetics, drugs or other medical products according to the common knowledge in the art and product requirements. The dosage forms of cosmetics and drugs can be selected from liquid preparations such as skin care lotions, toners, gels, masks, creams, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the SDS-PAGE protein electrophoresis diagram and molecular sieve diagram of the purified recombinant collagen;

[0018] Figure 2 It is the representative result of the transdermal detection and analysis of the recombinant collagen;

[0019] Figure 3Representative results of recombinant collagen on the proliferation of HaCaT cells;

[0020] Figure 4 Representative results of recombinant collagen on the injury repair of HaCaT cells;

[0021] Figure 5 Results of the thermal stability test of recombinant collagen. Detailed implementation mode

[0022] The present invention will be described in detail through specific steps below. It should be particularly pointed out that the content presented in this step does not represent the scope of the present invention. The technical means used in the following steps are common means for those skilled in the art. Materials and reagents used in the steps can be purchased from commercial suppliers without special instructions. The transdermal diffusion instrument (TP-6) is purchased from Tianjin Jingtuo Instrument Technology Co., Ltd.

[0023] Example 1 Preparation of recombinant type III collagen

[0024] 1. Construction of yeast expression strains:

[0025] Query the sequence of human type III collagen through the NCBI database, and retrieve the literature to find the types of transdermal peptides. Select transdermal peptides according to the properties of human type III collagen. Add EcoRI (GAATTC) and NotI (GCGGCCGC) restriction enzyme sites at both ends of the target gene, and send it to General Biosystems Co., Ltd. for synthesis of the target gene. After obtaining the synthesized target gene, transform it into Top 10 competent cells, pick 8 monoclonal colonies respectively, extract their plasmids, and verify them by double digestion with EcoR I and Not I. The plasmids verified correctly are subjected to the following transformation steps.

[0026] Amino acid sequence of recombinant collagen (SEQ ID NO.1):

[0027] FEGPASRPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAG

[0028] ARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPG

[0029] INGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPG

[0030] VPGAKGEDGKDGSPGEPGANG

[0031] Transdermal peptide amino acid sequence (SEQ ID NO.2):

[0032] FEGPASR

[0033] Collagen amino acid sequence (SEQ ID NO.3):

[0034] PGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGA

[0035] RGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGG

[0036] KGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGE

[0037] DGKDGSPGEPGANG

[0038] 2. Screening of yeast expression strains:

[0039] After extracting the recombinant plasmid, it was linearized using Sal I enzyme and transformed into Pichia pastoris GS115 competent cells by chemical transformation method, and then spread on the basic glucose medium (MD) and waited for it to grow. After growth, multi-copy transformants were screened with YPD liquid medium containing geneticin (G418) resistance, and samples were taken at 24 h and 48 h respectively for SDS-PAGE analysis of protein expression. The III-type recombinant collagen GS115 expression strain was screened out if there was a band at the position of the target protein size and it could be digested by collagenase in the SDS-PAGE analysis.

[0040] 3. Shake flask induced expression and fermentation in bioreactor of the target protein:

[0041] (1) Streak the glycerol stock of Pichia pastoris strain on the YPD plate medium containing 0.5 mg / mL G418 resistance.

[0042] (2) Pick a single colony and inoculate it into icky YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), incubate at 30 °C, 250 rpm for 24 h until the OD600 reaches 2 - 6 to prepare the seed culture.

[0043] (3) Inoculate the seed liquid at an inoculation amount of 1% into 200 mL of BMGY (1% yeast extract, 2% peptone, 1% glycerol, 10 mM phosphate buffer, 1% YNB, 0.02% biotin) liquid medium, and culture at 30 °C and 220 rpm until the OD600 reaches 6.0 - 9.0, then add methanol (final concentration 1%) to induce the expression of the target protein.

[0044] (4) Add methanol after 24 h, and the final concentration is 1%.

[0045] (5) After inducing for 48 h, take 200 μL of the culture solution, centrifuge at 12,000 rpm for 5 min, take the supernatant to prepare SDS-PAGE samples, place them at 55 °C for 10 min, and then perform SDS-PAGE electrophoresis.

[0046] (6) After shake-flask fermentation, prepare the seed liquid for fermentation in a tank. The highly permeable type III recombinant collagen produced by this invention is carried out using 2 L and 15 L fermenters.

[0047] (7) Bacterial cell culture stage: Add 50% w / v glycerol for feeding. When the DO value rises, add glycerol, 50 mL or 100 mL. During the culture stage, control the OD600 of Pichia pastoris cells at about 50, and then add methanol to enter the methanol induction stage.

[0048] (8) Methanol induction stage: After the yeast cells adapt to methanol, when the DO value rises, add methanol containing trace element PTM1 to induce it for about 72 h.

[0049] (9) Take out of the tank: Disassemble the electrode, collect the fermentation broth, clean the fermenter, and centrifuge to collect the supernatant of the fermentation broth.

[0050] 4. Preparation of highly permeable recombinant type III collagen:

[0051] (1) Collect the fermentation broth in the tank, centrifuge at 5,000 rpm and 4 °C to collect the supernatant, which is the crude protein.

[0052] (2) Measure a fixed volume of the crude protein, weigh 0.5% of activated carbon, add activated carbon at 200 rpm and 4 °C for adsorption for 6 h, centrifuge at 10,000 rpm and 4 °C for 1 h using a refrigerated centrifuge, and collect the supernatant.

[0053] (3) Dialysis: Load the supernatant after centrifugation in the previous step into a 3 kDa dialysis bag, place it in a pre-cooled 10 mM sodium acetate solution, and perform dialysis for desalting at 200 rpm. Generally, it is overnight, and change to a new dialysis solution for secondary dialysis the next morning.

[0054] (4) Filtration: Filter the dialysate using 0.22 or 0.45 μm to remove large impurities to prevent clogging of the chromatography column.

[0055] (5) Equilibrate the chromatography column (Q+SP): Connect the Q column above the SP column, replace the ethanol in the column with ddH2O, rinse with 5 column volumes of ddH2O, and then equilibrate the Q+SP tandem column with 10 mM sodium acetate solution (filtered through a 0.22 μm filter membrane and degassed by sonication) for 10 column volumes.

[0056] (6) Loading: Load the filtered dialysate onto the Q+SP tandem column using a pump at a flow rate of 2 mL / min, maintaining the temperature at 16 °C. After the first loading is completed, load the flow-through again onto the Q+SP tandem column at a flow rate of 2 mL / min.

[0057] (7) Linear gradient elution: Use 10 mM sodium acetate and 1 M sodium chloride solutions, set the program to linearly increase the concentration of sodium chloride, elute the collagen enriched on the Q+SP tandem column, and collect by volume. After the program is completed, detect the purity and concentration of the collected collagen by SDS-PAGE.

[0058] (8) Dialysis and concentration: Collect the collagen according to the SDS-PAGE results, dialyze at 4 °C into 10 mM PBS solution, after dialysis, concentrate the collagen using a 10 kDa ultrafiltration concentrator tube, then aliquot into 1.5 mL EP tubes (0.5 mL / tube), seal with a sealing film, and punch holes.

[0059] (9) Freeze drying: Place the aliquoted collagen at -20 °C for freezing, place it in an -80 °C ultra-low temperature freezer 6 h before freeze drying to freeze solid, and then place it in a freeze dryer for freeze drying (usually 24 h).

[0060] Figure 1 The size exclusion experiment of the molecular sieve (S200increase) shows that the collagen forms a triple helix structure.

[0061] Example 2 Transdermal detection

[0062] (1) Preparation of mouse skin: ICR mice weighing 20 - 22 g are sacrificed, the abdominal hair is removed, then the skin at the hair-removed site is peeled off, fat and tendons are removed, and it is repeatedly rinsed with distilled water. After the mouse skin is washed, it is rinsed with physiological saline, then treated with 10% glycerol solution to prevent ice crystal formation during storage, and stored in a -20 °C low-temperature freezer for standby (to be used within 7 days).

[0063] (2) Assembly of the detection device: The mouse skin was taken out and thawed at room temperature, and then washed with PBS solution. The mouse skin was directly fixed between the sample cell and the receiving cell (the side with the drug facing the supply chamber and the skin side facing the receiving chamber). The sample cell contained collagen dissolved in PBS, with PBS used as the blank control. The receiving cell was filled with PBS solution with a volume of 15 mL, maintaining constant-speed stirring and constant temperature, with the water bath temperature at 32 ± 0.5 °C and the rotation speed at 100 r / min. (PBS solution was pre-warmed to 32 °C in the receiving chamber and air bubbles were exhausted. To reduce interference, without administration of the drug, first the inner surface of the mouse skin was contacted with the receiving fluid, the entire receiving fluid was replaced, and then 2 mL of collagen solution was injected into the supply chamber and closely adhered to the mouse skin).

[0064] (3) Detection: Samples of 0.2 mL were taken at 1, 2, 4, 6, and 8 h respectively (sampled with a pipette), and at the same time, an equal amount of PBS solution was added. The protein concentration in the taken-out solution was measured, and the cumulative permeation amount per unit area was calculated according to the following formula.

[0065]

[0066] In the formula: Cn - the collagen concentration measured at the nth sampling point; Ci - the collagen concentration measured at the ith sampling point; 1000 - unit conversion factor; V - the volume of the receiving cell, which is 15 mL; V0 - the sampling volume, which is 0.2 mL; A - the permeation area, which is 1.13 cm2;

[0067] The results are as Figure 2 shown, indicating that compared with the recombinant type III collagen without the transdermal peptide, the permeation amount of the transdermal peptide-hCOL3A increased by 94% at the 2nd h compared with the non-transdermal peptide-hCOL3A, and increased by 56%, 46%, and 43.7% at the 4th, 6th, and 8th h respectively.

[0068] Example 3 Proliferation experiment

[0069] Cultured cells, and the cytotoxicity of recombinant type III collagen solutions at different concentrations to human immortalized keratinocytes HaCaT was detected using Cell Counting Kit-8 (CCK8).

[0070] A high transdermal recombinant type III collagen with a certain concentration was prepared and filtered using a sterile 0.22 μm filter membrane. The mother liquor was diluted to different concentrations of collagen using the culture medium. The confluent HaCaT cells were digested with trypsin, and after counting the cultured cells, they were diluted to 1×105 cells and plated into a 96-well plate, with 100 μL added to each well. The absorbance at 450 nm was measured using a multi-functional microplate reader. Each group of experiments was measured in parallel three times. The absorbance value of the recombinant collagen group was At, the absorbance value of the control group was Ac, and the absorbance of the blank group was A 0. Cell viability = (At - A 0 ) / (Ac - A 0 ) × 100%.

[0071] Figure 3 It is shown that this collagen has the ability to promote cell proliferation

[0072] Example 4 Cell migration promotion experiment (cell scratch assay)

[0073] Human immortalized keratinocytes HaCaT were inoculated into DEME medium containing 10% FBS and 1% penicillin - streptomycin resistance for culture. Prepare a sterile 6 - well plate, and draw five parallel positioning lines at the bottom of the well plate in a biosafety cabinet. After the cells grew stably, they were digested into a single - cell suspension of HaCaT by trypsin. Plated at 5×10⁵ into the 6 - well plate, with 2 mL in each well. After inoculation, the cells were cultured in an incubator at 37°C and 5% CO₂. After growing for 24 h until completely confluent, a 200 - μL pipette tip was used to make a scratch, and PBS was used for washing to wash out the cell clumps remaining in the 6 - well plate, and then photographed using an inverted microscope. Prepare a stock solution of recombinant type III collagen (3.0 mg / ml), filter it through a 0.22 - μm filter, and dilute it with low - serum medium to different concentrations of collagen. Add 2 mL of collagen - serum culture medium to each well. Place it in an incubator at 37°C and 5% CO₂ for 24 h, and then photograph it using an inverted microscope. After photographing, continue to place it in an incubator at 37°C and 5% CO₂ for culture, and photograph it again after 48 h using an inverted microscope. Use Image J software to process the photographed images and calculate the scratch area.

[0074] Use Image J to measure the area of the scratch and calculate according to the following formula:

[0075] Wound healing percentage = (Initial scratch area - Scratch area at a certain time point) / Initial scratch area).

[0076] Figure 4 The results showed that: at 24 h, the concentration of highly transdermal type III recombinant collagen at 0.5 mg / mL had the greatest effect on the migration of HaCaT cells, and at 48 h, the concentrations at 0.25 and 0.5 mg / mL had relatively obvious effects.

[0077] Example 5 Thermal stability experiment

[0078] The stock solution of recombinant transdermal peptide - hCOL3A after separation and purification was made into a 10 - mg / mL solution, placed in a water bath at (57 ± 0.5°C), (67 ± 0.5°C), (77 ± 0.5°C) for 4 h, and then observed with the naked eye for gelation or flocculation, and samples were taken for SDS - PAGE.

[0079] The results are asFigure 5 As shown, the sample is stable at 57, 67, and 77 degrees.

Claims

1. A recombinant type III collagen with high thermal stability and high transdermal permeability, characterized in that: The recombinant type III collagen comprises a type III collagen portion and a transdermal peptide portion, and the amino acid sequence of the recombinant type III collagen is SEQ ID NO.

1.

2. Use of the recombinant type III collagen according to claim 1 in the preparation of cosmetics with anti-wrinkle and / or repair functions.

3. Cosmetics with anti-wrinkle and / or repairing functions, characterized in that: The cosmetic comprises the recombinant type III collagen according to claim 1.

4. The method for preparing recombinant type III collagen according to claim 1, wherein The preparation method uses Pichia pastoris as a host to express the recombinant type III collagen according to claim 1. The preparation method according to claim 4 , further comprising dialysis and chromatography steps.

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