Recombinant human collagen III with high thermal stability and high transdermal property

Abstract

The invention provides a recombinant human collagen III with high thermal stability and high transdermal property, and the recombinant collagen III comprises a collagen III part and a transdermal peptide part, has high transdermal absorption capacity, has the advantage of high thermal stability, and is suitable for preparing cosmetics, medicines and medical instruments.

Description

Field of the Invention

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

[0002] The largest part of the human body is the skin, which occupies the largest area in the living body. With age and external stimulation, 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 with strong elasticity, and the monomers assembled into collagen fibers are collagen. Among them, type I collagen accounts for the highest content of total collagen, followed by type III collagen. Usually, type III collagen has a significant increase in new tissues. Type III collagen is the main component of reticular fibers and exists together with type I collagen to provide support, water retention, and elasticity for skin and other tissues. Type III collagen is more flexible and is more beneficial for maintaining the elasticity of skin and tissues. Type III collagen is a homotrimer formed by three α chains forming a superhelical structure.

[0003] The skin is the first line of defense for the human body, and the outermost layer of the epidermis is the stratum corneum (composed of stratum corneum cells). Macromolecules such as nucleic acids, peptides, proteins and hydrophilic substances cannot enter. Substances that need to enter through the skin require auxiliary means, such as chemical methods (dissolving sebum, expanding hair follicles) and physical methods (electroporation, ultrasound, heating). These technologies still have many limitations, such as irritating the skin will cause strong stinging, some methods cause damage to skin cells, and are toxic to cells.

[0004] Studies have shown that peptides containing 5-30 amino acids can introduce hydrophilic peptides, 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 injections, and can control the dosage and time of administration by applying it to the skin, greatly improving people's acceptance of drug administration. Collagen is a large molecule that is not easy to pass through the skin. By adding transdermal peptides to the front end of collagen, the permeability of type III collagen in human skin can be increased, thereby making up for the lack of type III collagen in the human body. Summary of the invention

[0005] In order to solve the technical problem that the current collagen application is not easy to penetrate the skin, the inventors conducted a lot of research and obtained a highly permeable type III recombinant collagen composed of a transdermal peptide and a human type III collagen amino acid sequence fragment. The highly permeable type III recombinant collagen has a high transdermal absorption capacity and has the advantages of high thermal stability, thereby 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, wherein the recombinant type III collagen comprises a type III collagen portion and a transdermal peptide portion.

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

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

[0009] Furthermore, 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 with anti-wrinkle and / or repair functions.

[0011] On the other hand, the present application provides cosmetics with anti-wrinkle and / or repair functions, wherein 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 a drug or medical device for skin repair, wherein the drug or medical device comprises the above-mentioned recombinant type III collagen.

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

[0015] Furthermore, the preparation method also includes dialysis and chromatography steps.

[0016] Those skilled in the art can select other auxiliary materials and functional ingredients for cosmetics, medicines or medical treatments according to common sense in the field and product requirements. The dosage forms of cosmetics and medicines can be liquid preparations such as skin lotions, toners, gels, facial masks, creams, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The SDS-PAGE protein electrophoresis and molecular sieve map of the purified recombinant collagen;

[0018] Figure 2 Representative results for the recombinant collagen transdermal assay analysis;

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

[0020] Figure 4 Representative results of recombinant collagen repairing HaCaT cell damage;

[0021] Figure 5 These are the results of the thermal stability test of recombinant collagen. DETAILED DESCRIPTION

[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. Unless otherwise specified, the materials and reagents used in the steps can be purchased from merchants, among which the transdermal diffusion instrument (TP-6) was purchased from Tianjin Jingtuo Instrument Technology Co., Ltd.

[0023] Example 1 Preparation of recombinant type III collagen

[0024] 1. Construction of yeast expression strain:

[0025] The sequence of human type III collagen was queried through the NCBI database, and the types of transdermal peptides were searched in the literature. Transdermal peptides were selected according to the properties of human type III collagen. EcoRI (GAATTC) and NotI (GCGGCCGC) restriction sites were added to both ends of the target gene and sent to General Biotechnology Co., Ltd. to synthesize the target gene. After obtaining the synthesized target gene, it was transformed into Top 10 competent cells, and 8 monoclones were picked respectively, and their plasmids were extracted and verified by double restriction digestion with EcoR I and Not I. After verifying the correct plasmid, the following transformation steps were performed.

[0026] Recombinant collagen amino acid sequence (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, use Sal I enzyme to linearize it, transfer it into Pichia pastoris GS115 competent cells by chemical transformation, and apply it to basic glucose medium (MD) for growth. After growth, use YPD liquid medium containing geneticin (G418) resistance to screen multi-copy transformants, and take samples at 24h and 48h for SDS-PAGE analysis of protein expression. The strain with bands at the target protein size position and able to be cleaved by collagenase is the selected type III recombinant collagen GS115 expression strain.

[0040] 3. Shake flask induced expression and tank fermentation of target protein:

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

[0042] (2) The clones were picked and placed into 10 mL YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose) at 30°C, 250 rpm, for 24 h until the OD600 reached 2-6 to prepare seed solution.

[0043] (3) The seed solution was inoculated at a 1% inoculum into 200 mL of BMGY (1% yeast extract, 2% peptone, 1% glycerol, 10 mM phosphate buffer, 1% YNB, 0.02% biotin) liquid medium, cultured at 30°C, 220 rpm until OD600 was 6.0-9.0, and then methanol (final concentration 1%) was added to induce expression of the target protein.

[0044] (4) After 24 hours, methanol was added to a final concentration of 1%.

[0045] (5) After 48 h of induction, 200 μL of the culture medium was centrifuged at 12,000 rpm for 5 min. The supernatant was prepared as an SDS-PAGE sample and placed at 55°C for 10 min before SDS-PAGE electrophoresis.

[0046] (6) After shake flask fermentation, seed liquid is prepared for tank fermentation. The highly permeable type III recombinant collagen produced by this invention is produced using 2L and 15L fermentation tanks.

[0047] (7) Bacteria culture stage: Add 50% w / v glycerol for feeding. When the DO value rises, add glycerol, 50mL or 100mL. During the culture stage, control the OD 600 of Pichia cells at around 50, and then add methanol to enter the methanol induction stage.

[0048] (8) Methanol induction stage: After the yeast cells adapt to methanol, methanol containing the trace element PTM1 is added to induce them after the DO value rises, and the induction lasts for about 72 hours.

[0049] (9) Tank removal: disassemble the electrodes, collect the fermentation liquid, clean the fermentation tank, and collect the supernatant of the fermentation liquid by centrifugation.

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

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

[0052] (2) A fixed volume of crude protein was measured, 0.5% activated carbon was added, and adsorption was carried out at 200 rpm and 4°C for 6 h. The mixture was centrifuged at 10,000 rpm and 4°C for 1 h using a refrigerated centrifuge, and the supernatant was collected.

[0053] (3) Dialysis: Place the supernatant from the previous centrifugation step into a 3 kDa dialysis bag and place it in a pre-cooled 10 mM sodium acetate solution at 200 rpm for dialysis to remove salt. Generally, leave it overnight and replace it with new dialysis solution the next morning for a second dialysis.

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

[0055] (5) Equilibration of chromatography columns (Q+SP): Connect the Q column to the SP column, replace the ethanol in the column with ddH2O, rinse with 5 column volumes of ddH2O, and then use 10 mM sodium acetate solution (filtered with a 0.22 μm filter membrane and ultrasonically degassed) to equilibrate the Q+SP series column for 10 column volumes.

[0056] (6) Loading: The filtered dialysate was pumped through the Q+SP tandem column with a flow rate of 2 mL / min and a temperature of 16°C. After the first loading was completed, the flow-through was loaded onto the Q+SP tandem column again with a flow rate of 2 mL / min.

[0057] (7) Linear gradient elution: Use 10 mM sodium acetate and 1 M sodium chloride solution, set up a program, 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, the collected collagen is tested for purity and concentration by SDS-PAGE.

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

[0059] (9) Freeze drying: Freeze the packaged collagen at -20°C, freeze it in a -80°C ultra-low temperature refrigerator for 6 hours before freeze drying, and then place it in a freeze dryer for freeze drying (usually 24 hours).

[0060] Figure 1 The medium molecular sieve (S200increase) exclusion experiment showed that the collagen formed a triple helix structure.

[0061] Example 2 Transdermal Detection

[0062] (1) Preparation of mouse skin: ICR mice weighing 20-22 g were killed, and the abdominal hair was removed. The skin of the hairless area was then peeled off, the fat and tendons were removed, and the skin was repeatedly rinsed with distilled water. The mouse skin was washed and rinsed with physiological saline, and then treated with 10% glycerol solution to prevent the formation of ice crystals during storage, and stored in a -20°C low-temperature refrigerator for use (used within 7 days).

[0063] (2) Assembly of the detection device: The mouse skin is taken out and thawed at room temperature, and then cleaned with PBS solution. The mouse skin is directly fixed between the sample pool and the receiving pool (the patch side faces the supply chamber, and the skin side faces the receiving chamber). The sample pool contains PBS dissolved collagen, and PBS is used as a blank control. The receiving pool is filled with PBS solution, the capacity is 15mL, and constant stirring and constant temperature are maintained. The water bath temperature is 32±0.5℃, and the speed is 100r / min. (Add PBS solution to the receiving chamber and preheat it to 32℃ to exhaust all bubbles. To reduce interference, without administering the drug, first contact the inner surface of the mouse skin with the receiving solution, replace all the receiving solution, and then inject 2mL of collagen solution into the supply chamber close to the mouse skin).

[0064] (3) Detection: Take 0.2 mL of sample (using a pipette) at 1, 2, 4, 6, and 8 hours respectively, and add an equal amount of PBS solution. Determine the protein concentration in the taken solution, and calculate the cumulative permeation per unit area according to the following formula.

[0065]

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

[0067] The results are as follows Figure 2 As shown, compared with recombinant type III collagen without transdermal peptide, the permeation of transdermal peptide-hCOL3A increased by 94% at 2 hours, and increased by 56%, 46%, and 43.7% at 4, 6, and 8 hours, respectively.

[0068] Example 3 Proliferation Experiment

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

[0070] A certain concentration of highly permeable recombinant type III collagen was prepared and filtered using a sterile 0.22 μm filter membrane. The mother liquor was diluted to different concentrations of collagen using culture medium, and the fully grown HaCaT cells were digested with trypsin. The cultured cells were counted and diluted to 1×10 5, plated on a 96-well plate, and 100 μL was added to each well. The absorbance at 450 nm was measured using a multifunctional microplate reader. Each group of experiments was measured three times in parallel. 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. 0Cell survival rate = (At-A 0 ) / (Ac-A 0 )×100%.

[0071] Figure 3 This indicates that collagen has the ability to promote cell proliferation

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

[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 on the bottom of the well plate in a biosafety cabinet. After the cell growth is stable, trypsinize it into a HaCaT single cell suspension. Plate 5×10 5 into a 6-well plate, each well contains 2mL, and the cells are cultured in a 37°C, 5% CO2 incubator after inoculation. After growing for 24 hours and fully growing, use a 200μL pipette tip to scratch, wash with PBS, wash out the cell clumps remaining in the 6-well plate, and take pictures with an inverted microscope. Prepare recombinant type III collagen mother solution (3.0mg / ml), filter it at 0.22μm, and dilute it with low serum culture medium to different concentrations of collagen. Add 2mL of collagen serum culture medium to each well. Place it in a 37°C, 5% CO2 incubator and culture it for 24 hours, then take pictures with an inverted microscope. After the photography was completed, the cells were placed in a 37°C, 5% CO2 incubator and photographed using an inverted microscope after 48 hours. The photographs were processed using Image J software to calculate the scratch area.

[0074] Image J was used to measure the area of ​​the scratch and calculated 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 24h, the concentration of highly permeable type III recombinant collagen was 0.5mg / mL, which had the greatest effect on the migration of HaCaT cells. At 48h, the concentrations of 0.25 and 0.5mg / mL had a more obvious effect.

[0077] Example 5 Thermal Stability Experiment

[0078] The purified recombinant transdermal peptide-hCOL3A stock solution was made into a 10 mg / mL solution, placed in a water bath at (57±0.5°C), (67±0.5°C), and (77±0.5°C) for 4 hours, and then observed with the naked eye to see if there was gelation or floccules, and samples were taken for SDS-PAGE.

[0079] The results are as follows Figure 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. 2 . The recombinant type III collagen according to claim 1 , wherein the amino acid sequence of the transdermal peptide portion is SEQ ID NO.

2. 3 . The recombinant type III collagen according to claim 1 , wherein the amino acid sequence of the type III collagen portion is SEQ ID NO.

3. The recombinant type III collagen according to claim 1 , wherein the amino acid sequence of the recombinant type III collagen is SEQ ID NO.

1.

5. Use of the recombinant type III collagen according to any one of claims 1 to 4 in the preparation of cosmetics with anti-wrinkle and / or repair functions.

6. A cosmetic having anti-wrinkle and / or repairing function, characterized in that: The cosmetic comprises the recombinant type III collagen according to any one of claims 1 to 4.

7. Use of the recombinant type III collagen according to any one of claims 1 to 4 in the preparation of drugs or medical devices for skin repair.

8. A drug or medical device for skin repair, characterized in that: The medicine or medical device comprises the recombinant type III collagen according to any one of claims 1 to 4.

9. The method for preparing recombinant type III collagen according to any one of claims 1 to 4, characterized in that: The preparation method uses Pichia pastoris as a host to express the recombinant type III collagen.

10. The preparation method according to claim 9, further comprising dialysis and chromatography steps.

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