Disordered protein cocktail extracted from Antarctic nematodes and application of disordered protein cocktail in low-temperature protection of cells

Abstract

The invention belongs to the field of medicine application, and particularly relates to disordered protein cocktail extracted from Antarctic nematodes and application of the disordered protein cocktail in cell low-temperature protection. The disordered protein disclosed by the invention can effectively reduce low-temperature damage and improve the survival rate of cells under a low-temperature condition by preventing ice crystal formation and stabilizing a cell membrane structure. Compared with a traditional low-temperature protective agent, the low-temperature protective agent can play a protective role at low concentration, and the toxicity risk to cells is remarkably reduced. Meanwhile, the natural anti-oxidation characteristic is achieved, oxidative stress damage induced by low temperature can be relieved, and therefore cells are further protected. The method is wide in application range, can be applied to low-temperature preservation of various types of cells, tissues and organs, and is suitable for multiple fields of medicine, agriculture, scientific research and the like.

Description

Technical Field

[0001] The invention belongs to the field of medical applications, and in particular relates to a disordered protein cocktail extracted from Antarctic nematodes and an application thereof in cell cryoprotection. Background Art

[0002] With the advancement of biotechnology, the importance of cell cryopreservation technology in the fields of medicine, agriculture and biological research is increasing. Cryopreservation can effectively prolong the preservation time of cells, tissues and organs. It is a key means of modern biotechnology and is widely used in the fields of medicine, agriculture and biological research. For example, in medicine, the long-term preservation of cells, tissues and organs is crucial for transplantation surgery, the establishment of biological sample banks and drug development.

[0003] However, low temperature environments can lead to ice crystal formation and osmotic pressure changes in cells, which can cause cell damage or even death, which is the main challenge facing cryopreservation technology. Although traditional cryoprotectants, such as glycerol and dimethyl sulfoxide (DMSO), can alleviate cryoprotective damage to a certain extent, they are often toxic to cells at high concentrations, limiting their clinical and industrial applications. Therefore, the development of new, low-toxic cell-protective biological agents has become an important direction of current research. Summary of the invention

[0004] The present invention aims to provide a disordered protein cocktail extracted from Antarctic nematodes and application thereof in cell cryoprotection, and improves the effect of cell cryoprotection by using the disordered protein cocktail isolated from Antarctic nematodes.

[0005] In order to achieve the above technical purpose, the technical solution provided by the present invention is as follows:

[0006] A disordered protein cocktail extracted from Antarctic nematodes, comprising one or more of the following compounds:

[0007] Idp1:GNLATLISSLLHDFQSQL;

[0008] Idp2:FCSKLPFFRNLHNLAQKGPFDVLGPGQFCGQSCVIDTVYTESPK;

[0009] Idp3:KIVFILCIVTRKT.

[0010] The preparation method of the above compound is: synthesized by using Fmoc solid phase synthesis method, reacting the pre-made symmetrical anhydride of N-protected amino acid and catalyst DMAP with hydroxy resin, forming the first peptide bond and then repeating the deprotection and condensation steps, after obtaining the target product, blowing dry the detergent to obtain a crude peptide; the crude peptide is purified and then freeze-dried.

[0011] Antarctic nematodes are tiny organisms that live in extremely cold environments and have a unique mechanism for adapting to low temperatures. Since the Antarctic region is covered with ice and snow all year round and the temperature is extremely low, organisms need to have special adaptability to survive in this environment. Through evolution, Antarctic nematodes have developed the ability to hibernate in low temperatures, allowing them to survive in extreme conditions. Low temperature hibernation is a physiological state in which the metabolic activity of Antarctic nematodes almost completely stops, thereby reducing energy consumption and increasing survival rate.

[0012] The ability of Antarctic nematodes to survive in extremely cold environments depends mainly on their ability to hibernate at low temperatures and the intrinsically disordered proteins (IDPs) in their bodies. These proteins are highly flexible due to their lack of a fixed three-dimensional structure, and can prevent ice crystal formation, stabilize cell membranes, and provide antioxidant protection under low temperature conditions. These properties make intrinsically disordered proteins ideal cryoprotectants.

[0013] These proteins can prevent the formation of ice crystals in cells at low temperatures while maintaining the integrity and function of cell membranes. In addition, the disordered proteins in Antarctic nematodes have strong antioxidant capacity, which can alleviate low-temperature-induced oxidative stress. These proteins can maintain a flexible structure at low temperatures, thereby adapting to changes in the internal and external environment of the cell and providing multiple protective effects.

[0014] The present invention has the advantages that:

[0015] 1. The present invention effectively reduces low-temperature damage and improves the survival rate of cells under low-temperature conditions by preventing ice crystal formation and stabilizing cell membrane structure.

[0016] 2. Compared with traditional cryoprotectants, the novel disordered protein cocktail disclosed in the present invention can exert a protective effect at low concentrations, significantly reducing the risk of toxicity to cells.

[0017] 3. The disordered protein disclosed in the present invention has natural antioxidant properties, which can alleviate the oxidative stress damage induced by low temperature, thereby further protecting cells.

[0018] 4. The present invention uses naturally extracted protein as a protective agent, which has good biocompatibility and environmental friendliness and is safer.

[0019] 5. The present invention has a wide range of applications and can be applied to the cryopreservation of various types of cells, tissues and organs, and is suitable for many fields such as medicine, agriculture and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The following is a statistical chart of the survival of BJ cells and HaCaT cells in a low temperature environment (4°C) after 6h, 24h and 48h under the co-intervention of the peptide mixture. The groups are: simple culture medium (DMEM) group, solvent control (DMEM+ddH2O) group and peptide mixture (Idps) group. The peptide mixture was dissolved in ddH2O, and the concentration of each peptide in the solution was 10μM. The content of ddH2O in the solution was 6.5%, and the content of ddH2O in the solvent control group was 6.5%.

[0021] Figure 2 Laser confocal scanning microscopy was used to detect the peptide mixture entering BJ cells and HaCaT cells after 48 hours in a 4°C environment, where the cell nucleus was labeled with blue fluorescence, the cytoskeleton was labeled with red fluorescence (E-cadherin), and the peptide was labeled with a green fluorescent label (FITC). DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with 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.

[0023] Example 1

[0024] This embodiment discloses a disordered protein cocktail extracted from Antarctic nematodes, including one or more of the following compounds:

[0025] Idp1:GNLATLISSLLHDFQSQL;

[0026] Idp2:FCSKLPFFRNLHNLAQKGPFDVLGPGQFCGQSCVIDTVYTESPK;

[0027] Idp3:KIVFILCIVTRKT.

[0028] The preparation method of this embodiment is introduced as follows:

[0029] In this embodiment, the raw materials and related reagents for peptide synthesis include:

[0030] 1. Protected amino acid raw materials: 20 kinds of Fmoc protected amino acids, Fmoc-(Acp)-OH, 5-FITC; 2. Condensation reagents: HBTU, DIEA; 3. Solvents: DMF, DCM, methanol, acetonitrile; 4. Resin: 2-ChlorotritylChloride resin with a degree of substitution of 1.1 mmol / g; 5. Deprotection reagent: piperidine; 6. Detection reagents: phenol reagent, pyridine reagent, ninhydrin reagent; 7. Cutting reagents: TFA, TIS, EDT, anhydrous ether; 8. Nitrogen; 9. Precision electronic balance.

[0031] Instruments and equipment: 1. Twelve-channel semi-automatic peptide synthesizer; 2. High performance liquid chromatograph; 3. Freeze dryer; 4. Centrifuge.

[0032] Synthesis process:

[0033] First, the resin is swollen in DMF, and then Fmoc-protected amino acids, DIEA, and DMF are added in sequence for reaction, followed by a deprotection step in which the Fmoc group is removed using a piperidine solution. During the synthesis process, the washing, condensation, and detection steps need to be repeated many times until the polypeptide chain is completely synthesized.

[0034] Finally, 5-FITC was added for labeling and confirmed by ninhydrin detection. After the synthesis was completed, the resin was washed with a series of solvents, followed by cleavage and blow-drying washing steps to purify and collect the synthesized peptides.

[0035] Identification: Take a small amount of the finished peptide for molecular weight identification by mass spectrometry (MS) and purity identification by high performance liquid chromatography (HPLC).

[0036] Dissolution: Weigh the required amount of peptides and dissolve them in ddH2O to prepare a mixed solution.

[0037] The physicochemical properties of the three peptides are shown in Table 1.

[0038] Table 1 Physicochemical properties of the three peptides

[0039] Peptides Molecular weight Solvents Molar concentration at maximum solubility (μM) Idp1 2459.59 <![CDATA[ddH2O]]> 580.82 Idp2 5393.03 <![CDATA[ddH2O]]> 370.85 Idp3 2036.37 <![CDATA[ddH2O]]> 491.07

[0040] The protective effect of the peptide mixture (cocktail) on HaCaT and BJ cell lines in a low temperature environment (4°C) was verified by pharmacological experiments as follows.

[0041] The specific steps are as follows:

[0042] 1) HaCaT and BJ cell lines (purchased from Zhejiang Meisen Cell Technology Co., Ltd., the cell information used in the experiment is shown in Table 2) were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U / ml) in a 5% CO2, 37°C incubator.

[0043] Table 2 Cell information used in the experiment

[0044] type HaC BJ Species Human Human Organization Source skin skin disease normal normal age 62 years old Newborns gender male male Cell Type Keratinocytes Fibroblasts Growth characteristics Adherent growth Adherent growth

[0045] 2) Inoculate 100 μL of cell suspension into each well of a 96-well plate (approximately 5,000 cells / well) and culture in a 37°C incubator.

[0046] 3) When the cell confluence reached 70-80%, fresh 10% FBS+DMEM medium+peptide solution (the concentration of the three peptides was 10 μM) was added to the experimental group, and fresh 10% FBS+DMEM medium+ddH2O in the same proportion as the experimental group was added to the control group.

[0047] 4) After standing in a 37°C incubator for 1 hour, take out the 96-well plate, seal the 96-well plate with a sealing film, and place it in a 4°C refrigerator for cooling.

[0048] 5) After incubation for 6, 24, and 48 h, the culture medium was removed and 10 μL of CCK-8 solution was added to each well of the plate.

[0049] 6) After incubating the culture plate in an incubator for 1-4 hours, use a microplate reader to measure the absorbance at 450 nm.

[0050] Cell survival rate (%) = [(As-Ab) / (Ac-Ab)] × 100

[0051] As = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8 and test compounds)

[0052] Ab = absorbance of blank well (absorbance of wells containing culture medium and CCK-8)

[0053] Ac = absorbance of control well (absorbance of wells containing cells, culture medium and CCK-8)

[0054] Results Figure 1

[0055] After 6 h, the number of cells in the DMEM+ddH2O group was significantly less than that in the DMEM group and the Idps group (p<0.001);

[0056] After 24 hours, the number of cells in the Idps group of the BJ cell line was greater than that in the DMEM group and the DMEM-ddH2O group, and the trend of the HaCaT cell line was opposite to that of the BJ cell line.

[0057] After 48 hours, the number of cells in the Idps group of the BJ cell line was greater than that in the DMEM group and the DMEM-ddH2O group, while the trend in the HaCaT cell line was the opposite.

[0058] This shows that the present invention has a good cryoprotective effect on cells in a short time (6h). In a long time (24h and 48h), especially in the BJ cell line, the Idps group of the present invention can effectively perform cryoprotection, showing better effects than the DMEM and DMEM+ddH2O groups. On the contrary, in the HaCaT cell line, the cell proliferation effect of the Idps group is not as good as that of the DMEM group and the DMEM+ddH2O group, which shows that the present invention has differences in the effects on different cell lines.

[0059] Peptide mixture cell entry detection

[0060] The specific steps are as follows:

[0061] 1) Place a slide in a 12-well cell culture plate, plate cells at a density of 50%, and add fresh 10% FBS+DMEM medium+peptide solution (the concentration of the three peptides is 10 μM).

[0062] 2) After the cells have attached to the wall for 12-24 hours, add the polypeptide mixed solution, place the cells in a 37°C incubator for 1 hour, and then place them in a 4°C refrigerator for cooling.

[0063] 3) After 48 h, the culture medium was discarded and the cells were washed three times with PBS (30 s / time, the same below).

[0064] 4) Add 500 μL of 4% paraformaldehyde, incubate at room temperature for 10 min, and then wash three times with PBS.

[0065] 5) Add 500 μL of 0.2% TnitonX-100 (prepared in PBS) to each well, incubate at room temperature for 10 min, and then wash three times with PBS.

[0066] 6) Add 500 μL of 5% BSA (prepared in PBS) to each well and let stand at room temperature for 30 min.

[0067] 7) Incubate with primary antibody: Prepare antibody diluent (5% BSA, PBS), dilute E-cadherin antibody at 1:2000, add 50 μL of diluted antibody to each slide, and incubate at room temperature for 1 hour.

[0068] 8) Wash three times with PBS (5 min / time), add red fluorescent secondary antibody dilution solution (1:500 dilution), incubate at room temperature in the dark for 1 hour, and then wash three times with PBS (5 min / time).

[0069] 9) DAPI staining: dilute DAPI with PBS (1:1000), incubate at room temperature for 1 min, and wash with PBS three times (5 min / time).

[0070] 10) Add 20 μl of 90% glycerol (propylene glycol) to the slide for sealing, place the cell slide upside down on the 90% glycerol drop, seal the slide with nail polish, and observe using a confocal microscope after drying.

[0071] Results Figure 2 :

[0072] Under 37℃ and 4℃ environments, Idp1, Idp2 and Idp3 interventions could observe the peptides entering the cells after 48h.

[0073] From the above results, it can be seen that in a low-temperature environment of 4°C, the polypeptide mixture can enter the cells, effectively reduce low-temperature damage, and improve the survival rate of cells under low-temperature conditions.

[0074] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A disordered protein cocktail extracted from Antarctic nematodes, characterized in that: Includes one or more of the following compounds: Idp1:GNLATLISSLLHDFQSQL; Idp2:FCSKLPFFRNLHNLAQKGPFDVLGPGQFCGQSCVIDTVYTESPK; Idp3:KIVFILCIVTRKT.

2. A method for preparing a disordered protein cocktail extracted from Antarctic nematodes, characterized in that: The Fmoc solid phase synthesis method is used to react the pre-made symmetrical anhydride of the N-protected amino acid and the catalyst DMAP with the hydroxy resin to form the first peptide bond, and then the deprotection and condensation steps are repeated to obtain the target product and then blow dry the detergent to obtain the crude peptide; the crude peptide is purified and freeze-dried.

3. A pharmaceutical composition, characterized in that The invention comprises the three compounds or pharmaceutically acceptable salts thereof as claimed in claim 1.

4. A pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition may be in the form of tablets, capsules, pills, granules, oral liquids or suspensions.

5. A pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

6. Use of any one of the compounds or compositions of claims 1 to 5 for improving the survival rate of cells under low temperature conditions.

7. Use of any one of the compounds or compositions of claims 1 to 5 in the preparation of cryoprotectant preparations.

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