Polypeptide extracted from Antarctic nematode, polypeptide composition, extraction method and application in cell freezing resistance

Abstract

The invention belongs to the field of medicine application, and particularly relates to a polypeptide extracted from Antarctic nematode, a polypeptide composition, an extraction method and application in cell freezing resistance. Specific polypeptides (AF1, AF2) are extracted from frozen and naturally resuscitated Antarctic nematodes. The polypeptide or the polypeptide composition can be combined with a cell membrane, the stability of the cell membrane is enhanced, and physical damage caused by freezing is reduced. By stabilizing the membrane structure, the peptide fragments effectively prevent the leakage of cellular contents and maintain the integrity of cells. The polypeptide disclosed by the invention can effectively reduce the risk of ice crystal formation in cells, and the integrity of cell nucleuses is protected by regulating the environment in the cells; experiments prove that the polypeptide disclosed by the invention shows excellent biocompatibility and stability in various cell types, and has the advantages of multiple application scenes and wide application range.

Description

Technical Field

[0001] The invention belongs to the field of medical applications, and specifically relates to a polypeptide extracted from Antarctic nematodes, a polypeptide composition, an extraction method, and an application in cell antifreeze. Background Art

[0002] Cryopreservation technology plays a vital role in modern biomedicine, food industry and agriculture. This technology can prolong the storage time of biological materials and maintain their activity and function. It is of great significance in the fields of organ transplantation, cell therapy, biological sample bank construction, food preservation and crop seed preservation.

[0003] However, traditional cryopreservation methods face many challenges. During the freezing process, cells often suffer severe damage, including cell membrane rupture, intracellular ice crystal formation, organelle dysfunction, etc., which may lead to cell death or loss of function. Even the use of common cryoprotectants, such as dimethyl sulfoxide (DMSO), cannot completely avoid these problems, and some cryoprotectants themselves may be toxic to cells.

[0004] Therefore, a freezer that can effectively protect cells under freezing conditions and effectively reduce the impact of the drug itself on cells needs to be developed urgently. Summary of the invention

[0005] The present invention aims to provide a polypeptide extracted from Antarctic nematodes, a polypeptide composition, an extraction method and an application in cell antifreeze, which can provide significant protection for cell structure and function in the cell freezing state by extracting specific peptide segments (AF1 and AF2) from frozen and naturally revived Antarctic nematodes.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] A polypeptide extracted from Antarctic nematodes, comprising one or two of the following polypeptides:

[0008] AF1:ILVKLNFHQVSSKNMSLYHNRVMIICFGEHSPGFENRNKPNNKQCKPRNHFRKLMYVQWRTKSGHLVSQCST;

[0009] AF2:HSSIRRSFTQAQRSVVCTGRAGSNALFGKKVLTDGRSENFA*RTGVKRTYNRLGR A.

[0010] The extraction method of the invention comprises the following steps: swelling the resin in DMF, sequentially adding an Fmoc-protected amino acid, DIEA and DMF for reaction; performing a deprotection step, using a piperidine solution to remove the Fmoc group; then detecting whether the reaction is successful by using ninhydrin; during the synthesis process, repeatedly performing washing, condensation and detection steps until the polypeptide chain is completely synthesized; finally, adding 5-FITC for labeling, and confirming by using ninhydrin detection; after the synthesis is completed, using a solvent to wash the resin, and performing cutting and drying washing steps to purify and collect the synthesized polypeptide.

[0011] The present invention extracts specific polypeptides (AF1, AF2) from frozen and naturally revived Antarctic nematodes. These peptides provide significant protection for cell structure and function in the frozen state of cells. Through multiple mechanisms, these peptides can enhance the stability of cell membranes, reduce damage to cell membranes during freezing, reduce the risk of ice crystal formation in cells, and maintain the integrity of the cell nuclear structure. The protective effect of the present invention not only helps to maintain the integrity of cell membranes, but also provides support for the DNA of the cell nucleus and its related functions, ensuring that the cells maintain the stability of the genome during freezing and resuscitation.

[0012] The advantages of the present invention are:

[0013] 1. AF1 and AF2 in the present invention can bind to the cell membrane, enhance the stability of the cell membrane, and reduce the physical damage caused by freezing. By stabilizing the membrane structure, these peptides effectively prevent the leakage of cell contents and maintain cell integrity.

[0014] 2. The polypeptide disclosed in the present invention can effectively reduce the risk of ice crystal formation in cells, protect the integrity of the cell nucleus by regulating the intracellular environment; maintain the cell nucleus structure and DNA stability, and ensure the integrity of the genome during freezing and thawing

[0015] 3. Experiments have shown that the polypeptide of the present invention exhibits excellent biocompatibility and stability in a variety of cell types, and has the advantages of multiple application scenarios and a wide range of applications. In addition to the biomedical field, the present invention can also be applied to food preservation and low-temperature protection in agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The survival statistics of BJ cells and HaCaT cells after being frozen at -20℃ for 6h, 24h and 48h under the intervention of peptide mixture. The groups were: simple culture medium (DMEM) group, solvent control (DMEM+ddH2O) group and peptide mixture (AFs) group; the peptide mixture was dissolved in ddH2O, the concentration of each peptide in the solution was 10μM, and the content of ddH2O in the peptide solution was 6.5%. The content of ddH2O in the ddH2O control group was 6.5%.

[0017] Figure 2 Laser confocal scanning microscopy was used to detect the peptide mixture entering BJ cells and HaCaT cells after 48 hours in a -20°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

[0018] 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.

[0019] Example 1

[0020] A polypeptide extracted from Antarctic nematodes, comprising one or two of the following polypeptides:

[0021] AF1:ILVKLNFHQVSSKNMSLYHNRVMIICFGEHSPGFENRNKPNNKQCKPRNHFRKLMYVQWRTKSGHLVSQCST;

[0022] AF2:HSSIRRSFTQAQRSVVCTGRAGSNALFGKKVLTDGRSENFA*RTGVKRTYNRLGR A.

[0023] The preparation method of this embodiment is introduced as follows:

[0024] In this embodiment, the raw materials and related reagents for peptide synthesis include:

[0025] 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.

[0026] Instruments and equipment: 1. Twelve-channel semi-automatic peptide synthesizer; 2. High performance liquid chromatograph; 3. Freeze dryer; 4. Centrifuge.

[0027] Synthesis process:

[0028] 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, using a piperidine solution to remove the Fmoc group. Afterwards, the success of the reaction is detected by ninhydrin. During the synthesis process, the washing, condensation, and detection steps need to be repeated many times until the polypeptide chain is completely synthesized. Finally, 5-FITC is added for labeling and confirmed by ninhydrin detection. After the synthesis is completed, the resin is washed with a series of solvents, followed by a cutting and drying washing step to purify and collect the synthesized polypeptide.

[0029] 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).

[0030] Dissolution: Weigh the required amount of peptides and dissolve them in ddH2O to prepare a mixed solution.

[0031] The physicochemical properties of the polypeptide of the present invention are shown in Table 1.

[0032] Table 1 Physicochemical properties of peptides

[0033] Peptides Molecular weight Solvents Molar concentration at maximum solubility (μM) AF1 9047.35 <![CDATA[ddH2O]]> 221.06 AF2 6573.24 <![CDATA[ddH2O]]> 304.26

[0034] The protective effect of the peptide mixture (cocktail) on HaCaT and BJ cell lines in a frozen environment (-20°C) was verified by pharmacological experiments as follows.

[0035] The specific steps are as follows:

[0036] 1) HaCaT and BJ cell lines (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) were propagated in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U / ml) in a 5% CO2, 37°C incubator.

[0037] Table 2 Cell information used in the experiment

[0038] 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

[0039] 2) Inoculate 100 μL of cell suspension into each well of a 96-well plate (approximately 10,000 cells / well) and culture in a 37°C incubator.

[0040] 3) When the cell confluence reached 70-80%, fresh 10% FBS+DMEM medium+peptide solution (the concentration of both 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.

[0041] 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 -20°C refrigerator for cooling.

[0042] 5) After incubation for 6 h, 24 h, and 48 h, the culture medium was removed and 10 μL of CCK-8 solution was added to each well of the plate.

[0043] 6) After incubating the culture plate in an incubator for 1-4 hours, use a microplate reader to measure the absorbance at 450 nm.

[0044] Cell survival rate (%) = [(As-Ab) / (Ac-Ab)] × 100

[0045] As = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8 and test compounds)

[0046] Ab = absorbance of blank well (absorbance of wells containing culture medium and CCK-8)

[0047] Ac = absorbance of control well (absorbance of wells containing cells, culture medium and CCK-8)

[0048] Results Figure 1

[0049] After 6 hours, the cell mass in the AFs group of BJ cell line and HaCaT cell line was greater than that in the DMEM group and DMEM-ddH2O group (p<0.001); after 24 hours, the cell mass in the AFs group of BJ cell line and HaCaT cell line was greater than that in the DMEM group and DMEM-ddH2O group (p<0.05); after 48 hours, the cell mass in the AFs group of BJ cell line and HaCaT cell line was greater than that in the DMEM group and DMEM-ddH2O group (p<0.01).

[0050] This indicates that the present invention has significantly good protection for freezing (-20°C) of cells, whether in a short period of time (6 hours) or in a longer period of time (24 hours and 48 hours).

[0051] Peptide mixture cell entry detection

[0052] The specific steps are as follows:

[0053] 1) Place a slide in a 12-well cell culture plate one day in advance, spread cells at 50% density, and add fresh 10% FBS+DMEM culture medium+peptide solution (the concentration of both peptides is 10 μM).

[0054] 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.

[0055] 3) After 48 h, the culture medium was discarded and the cells were washed three times with PBS (30 s / time, the same below).

[0056] 4) Add 500 μL of 4% paraformaldehyde, incubate at room temperature for 10 min, and then wash three times with PBS.

[0057] 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.

[0058] 6) Add 500 μL of 5% BSA (prepared in PBS) to each well and let stand at room temperature for 30 min.

[0059] 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.

[0060] 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).

[0061] 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).

[0062] 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.

[0063] Results Figure 2 : Under -20℃ environment, after intervention with AF1 and AF2 (dissolved in DMEM, 10μM), the peptides could be observed entering the cells after 48h.

[0064] From the above results, it can be seen that after being frozen at -20°C, the polypeptide mixture can enter the cells, effectively reduce low-temperature damage, and improve the survival rate of cells under low-temperature conditions.

[0065] 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 polypeptide extracted from Antarctic nematodes, characterized in that: Includes one or two of the following polypeptides: AF1:ILVKLNFHQVSSKNMSLYHNRVMIICFGEHSPGFENRNKPNNKQCKPRNHFRKLMYVQWRTKSGHLVSQCST; AF2:HSSIRRSFTQAQRSVVCTGRAGSNALFGKKVLTDGRSENFA*RTGVKRTYNRLGRA.

2. A polypeptide composition extracted from Antarctic nematodes according to claim 1, characterized in that: It also includes two polypeptides, AF1 and AF2.

3. A method for extracting polypeptides extracted from Antarctic nematodes, characterized in that: The resin is swollen in DMF, and Fmoc-protected amino acids, DIEA and DMF are added in sequence for reaction; a deprotection step is performed, and the Fmoc group is removed using a piperidine solution; then the success of the reaction is detected by ninhydrin; during the synthesis process, the washing, condensation and detection steps are repeated many times until the polypeptide chain is completely synthesized; finally, 5-FITC is added for labeling, and the labeling is confirmed by ninhydrin detection; after the synthesis is completed, the resin is washed with a solvent, and the cutting and drying washing steps are performed to purify and collect the synthesized polypeptide.

4. A pharmaceutical composition, characterized in that The invention comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

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

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

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

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

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