Design of novel antifreeze polypeptide and cell cryopreservation protection method

Abstract

The invention discloses a novel antifreeze polypeptide with a clear structure and excellent performance and a protection method of cryopreserved cells. The core structure of the polypeptide is formed by covalently connecting two functional modules, the N end is a unit with ice crystal binding performance, the C end is a super-hydrophilic unit, and the two functional modules are bridged through a flexible linker. Experimental verification shows that the synthetic polypeptide shows excellent anti-freezing activity, can significantly inhibit nucleation, growth and recrystallization processes of ice crystals, and has particularly outstanding ice recrystallization inhibition activity. The novel antifreeze polypeptide shows excellent biocompatibility, and is suitable for low-temperature cryopreservation of cells sensitive to freezing damage and other living microbial preparations. Results show that the polypeptide can greatly improve the freeze-drying survival rate of cells and the activity of the cells after recovery, is non-toxic to the cells, and shows the potential of replacing a traditional cryoprotectant. Therefore, the method has wide application prospects in food industry, biological medicine, agricultural breeding and low-temperature preservation technologies of biological sample libraries.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the design, construction method and application of a novel antifreeze polypeptide. Background Technology

[0002] Cryopreservation is a key technology for the long-term preservation of viable microbial materials such as cells. However, during freezing and thawing, the formation, growth, and recrystallization of ice crystals can cause mechanical damage to cell membranes, proteins, and other biomolecules, and trigger drastic changes in solution osmotic pressure, leading to cell dehydration and rupture, severely reducing cell viability and biological activity. Therefore, developing efficient and safe cryopreservation agents is crucial for maintaining cell viability and ensuring their application efficacy.

[0003] Currently, commonly used cryopreservation agents mainly include small-molecule permeable cryopreservatives (such as glycerol and dimethyl sulfoxide) and large-molecule non-permeable cryopreservatives (such as sucrose, polyvinylpyrrolidone, and serum albumin). Although small-molecule cryopreservatives can penetrate cells and lower the freezing point, they may have cytotoxicity and affect subsequent metabolic activity; large-molecule cryopreservatives mainly form a glassy matrix outside the cell to inhibit ice crystal growth, but their antifreeze activity is limited, and some synthetic polymers have biocompatibility issues.

[0004] In nature, certain organisms (such as polar fish, insects, and plants) can express antifreeze proteins or their active fragments (antifreeze peptides) with unique ice crystal binding and inhibition functions to adapt to frigid environments. These biomolecules can reversibly and specifically adsorb onto the surface of ice crystals, inhibiting ice crystal growth and recrystallization through the "Kelvin effect." This effectively modifies ice crystal morphology and reduces mechanical damage while noncollively lowering the freezing point. Compared to traditional chemical cryoprotectants, antifreeze proteins / peptides have advantages such as biodegradability, strong targeting, low dosage, and potential good cell compatibility, making them ideal candidates for next-generation bio-based cryoprotectants.

[0005] However, the direct application of natural antifreeze proteins faces several challenges: firstly, their sources are limited, and extraction and purification are costly; secondly, their large molecular weight may pose immunogenicity risks; and thirdly, their intact natural structure may not be entirely necessary for antifreeze activity, with some domains potentially introducing instability. Although existing studies have attempted to mimic the core active fragments of natural antifreeze proteins through genetic engineering expression or chemical synthesis, the designed peptides often suffer from problems such as limited antifreeze activity, insufficient stability, or a lack of sufficient hydrophilicity to function effectively at the ice-water interface. For applications such as probiotic, erythrocyte, and hematopoietic stem cell cryopreservation, peptides are needed that not only efficiently inhibit ice crystal formation in complex matrices but also interact well with cell surfaces to provide more comprehensive protection.

[0006] Therefore, there is an urgent need in this field to develop novel antifreeze peptides that are structurally simple, highly active, easy to synthesize, and optimized for cell cryopreservation requirements. By employing rational design strategies to construct novel antifreeze peptides with highly efficient antifreeze properties, it is hoped that the limitations of existing technologies can be overcome, providing a safer, more efficient, and specific solution for the cryopreservation of cells and other bioactive preparations. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a novel method for designing antifreeze peptides.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned novel antifreeze peptide.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: De novo design and application of a novel antifreeze peptide.

[0010] The specific steps for constructing the novel antifreeze peptide are as follows: S1: Screening and Optimization of Core Ice Crystal Integration Module Based on the consensus sequences and spatial conformations of known antifreeze proteins' ice crystal binding surfaces, bioinformatics tools were used to screen for the smallest active units with high binding affinity. Furthermore, molecular dynamics simulations were used to evaluate the binding stability with the primary ice crystal surface, and site-directed saturation mutagenesis was employed to optimize key amino acids, enhancing their hydrogen bond formation ability and structural rigidity, thereby obtaining a highly active core ice crystal binding module.

[0011] S2: Design and Construction of Structured Superhydrophilic Modules Unlike simple tandem hydrophilic amino acids, this invention designs hydrophilic modules with specific secondary structural tendencies (such as helices or turns). These modules are rich in electrically neutral amino acids with abundant side-chain hydroxyl or amide groups, such as serine (S), threonine (T), and glutamine (Q), aiming to form a highly hydrated "hydration shell." This module not only improves the overall solubility of the peptide but also reduces the non-specific aggregation of functional peptide molecules through steric hindrance, maintaining their monomeric activity at the working concentration.

[0012] S3: Functional assembly via rationally designed flexible linker peptides Computer-aided design was employed to optimize the sequence and length of the flexible linker connecting the two functional modules. This linker is typically composed of glycine (G, providing flexibility), serine (S, providing hydrophilicity), and proline (P, introducing a specific turn) repeated in a specific pattern. The design principle is to maintain the overall coherence of the polypeptide chain while ensuring the spatial orientation of the two functional modules is free and does not interfere with each other, and to avoid introducing protease-sensitive sites or immunogenic sequences.

[0013] Preferably, the IRI performance of the novel antifreeze peptide designed by S3 is tested using the following steps: A 10 μL droplet is dropped from a height of 1-1.5 m onto a crucible placed on liquid nitrogen, instantly forming a thin layer of ice; the crucible is then rapidly transferred to a pre-cooled liquid nitrogen platform, equilibrated for 3 min, heated at -20 ℃ / min to -8 ℃, and incubated for 30 min to allow recrystallization. The ice crystal morphology is recorded every 10 min, and the ice crystal images are observed and saved; the images are processed using the provided NIS-Elements BR software, and the mean grain area (MGA) of the ice crystals is obtained by calculating the number of ice crystals in the same area, and compared with the PBS positive control; the smaller the MGA, the stronger the IRI activity; the selected area is 1 / 3 of the field of view; all experiments are performed in parallel at least 3 times.

[0014] Preferably, the performance of the novel antifreeze peptide designed by S3 in inhibiting ice crystal growth rate is tested using the following steps: A directional ice crystal growth observation instrument equipped with a high-precision cooling stage and a high-speed camera system is used. The test solution containing the peptide and the control solution are injected into the sample cell, respectively. The system is cooled to a preset supercooling degree and stabilized, inducing the formation of individual ice crystals in the field of view. The ice crystals are controlled to grow directionally along a specific crystal axis, while the advancement process of the ice crystal growth front is continuously recorded using a high-speed camera. Using image analysis software, a linear fit is performed with time as the abscissa and the displacement of the growth front as the ordinate; the resulting slope is the ice crystal growth rate at that supercooling degree. The inhibitory effect of the antifreeze peptide is quantified by comparing the growth rates of the test group and the control group at different supercooling degrees. All experiments are independently repeated at least three times.

[0015] Preferably, the freezing point reduction performance of the novel antifreeze peptide designed by S3 is tested using the following steps: A differential scanning calorimeter equipped with a liquid nitrogen cooling system is used. The test solution containing the peptide and the control buffer are accurately weighed (10-15 mg each) and sealed in a standard aluminum sample crucible. An empty crucible is used as a reference. Under a dry nitrogen atmosphere, a preset temperature program is executed: first, the temperature is lowered from room temperature to -40 °C at a rate of -5 °C / min and held briefly to ensure complete freezing; then, the temperature is increased back to room temperature at a rate of +5 °C / min. By analyzing the heat flow curve during the cooling phase, the extrapolated temperature of the onset of the exothermic crystallization peak is determined as the freezing point of the sample. The freezing point reduction caused by the antifreeze peptide is calculated by comparing the freezing point difference between the test group and the control group. Each sample is tested in parallel at least three times to obtain the average value and standard deviation.

[0016] Preferably, the application of the above-mentioned novel antifreeze peptides includes the cryopreservation and preservation of probiotics, erythrocytes, hematopoietic stem cells, sperm, and egg cells. The specific steps are: after continuous passage culture of cells, the novel antifreeze peptide solution is added for cryopreservation at low temperature.

[0017] Beneficial effects: Compared with the prior art, the design solution provided by the present invention has the following significant advantages: 1. High efficiency: By focusing on and optimizing the smallest active unit, the peptide has higher antifreeze activity per unit mass.

[0018] 2. High stability: The structured hydrophilic module design effectively prevents the aggregation and precipitation of peptides during storage and application, thus extending the activity retention time.

[0019] 3. Customizability and scalability: The modular design allows for flexible adjustments to module combinations to suit different ice crystal types or application scenarios. Furthermore, the short peptide sequences are easier to chemically synthesize, resulting in high purity and low cost, which is beneficial for industrial production.

[0020] 4. Low immunogenicity risk: The use of rationally designed non-natural optimized sequences avoids the immunogenicity problems that may arise from the direct use of heterologous proteins.

[0021] 5. Wide range of applications: Novel antifreeze peptides can be used for cryopreservation of cells including probiotics, red blood cells, hematopoietic stem cells, sperm and egg cells. Attached Figure Description

[0022] Figure 1 Design ideas for novel antifreeze peptides; Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: The specific steps for obtaining the core ice crystal binding module are as follows: Using existing reports on the ice crystal binding surfaces of antifreeze proteins from fish, plants, insects, bacteria, and fungi as templates, and combining bioinformatics and deep learning techniques, core sequences containing key threonine clusters were screened. After identifying essential residues through alanine scanning, a series of mutants were synthesized using solid-phase synthesis technology. Through ice crystal morphology observation experiments, mutant sequences with the best inhibitory effect on ice crystal growth, including GE, GT, and TST, were selected as the core ice crystal binding module of this invention.

[0025] Example 2: The design and synthesis of structured, highly hydrophilic modules follow these steps: Based on 20 natural amino acids, a series of highly hydrophilic polypeptide sequences, including NNT, NNE, EKT, and ETK, were designed, with hydrophilic amino acids accounting for over 70%. Circular dichroism (CD) spectroscopy confirmed that the module indeed formed partial helical structures in buffer solution. Dynamic light scattering (DLS) testing showed that the module remained monodisperse at 4°C and high concentrations, without aggregation.

[0026] Example 3: The design of novel antifreeze peptides involves the following steps: The standard Fmoc solid-phase synthesis strategy was adopted, using Rink Amide AM resin as a carrier. Activation coupling was performed through the HOBt / HBTU / DIPEA system, and the desired amino acid sequences were sequentially linked. Finally, the target peptide was cleaved from the resin using a cleavage solution, and the side-chain protecting groups were removed. The resulting crude peptide was precipitated with cold diethyl ether and purified by reversed-phase high-performance liquid chromatography. The molecular weight of the product was identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirming the successful acquisition of the target antifreeze peptide.

[0027] Example 4: The specific steps for validating the IRI activity of the novel antifreeze peptide are as follows: This embodiment uses the "splat-cooling" assay to measure the ability of the novel antifreeze peptide to inhibit ice crystal recrystallization. The specific steps are as follows: S1: Drop 10 μL of liquid onto a crucible placed on liquid nitrogen from a height of 1-1.5 m, and a thin layer of ice will form instantly.

[0028] S2: Quickly transfer the crucible to a liquid nitrogen pre-cooled platform (-60 ℃), equilibrate for 3 min, raise the temperature to -8 ℃ at -20 ℃ / min and incubate for 30 min to recrystallize. Record the ice crystal morphology every 10 min, observe and save the ice crystal images.

[0029] S3: Images were processed using the provided NIS-Elements BR software. The mean grain area (MGA) of ice crystals was calculated by counting the number of ice crystals in the same region and compared with a PBS positive control. The smaller the MGA, the stronger the IRI activity. The selected region was 1 / 3 of the field of view. All experiments were performed in at least 3 replicates. 0.1%-1% of the novel antifreeze peptides showed 10%-20% IRI activity.

[0030] Example 5: The novel antifreeze peptide exhibits activity in inhibiting ice crystal growth rate, and the specific steps are as follows: S1: Procedure for determining the growth rate of single ice crystals: a) Sample loading and initial freezing: Inject approximately 5 μL of the sample solution to be tested into the capillary sample cell. Place the sample cell on a cold stage, program the temperature to -15 °C and hold it to induce supercooling of the solution, and generate a single ice crystal nucleus in the center of the field of view by touching or seeding.

[0031] b) Temperature equilibration and ice crystal shaping: Precisely adjust the stage temperature to a specific supercooling (ΔT). Maintain this temperature for a period of time to allow the ice crystal to grow into a single grain with a clear and stable hemispherical or hexagonal profile.

[0032] c) Directional Growth and Image Acquisition: By controlling the temperature gradient or using a mask, guide the ice crystals to grow unidirectionally along their a-axis or c-axis. Stabilize the system temperature at the set supercooling level (e.g., -0.1°C, -0.2°C, -0.3°C, -0.5°C). Start a high-speed camera and continuously record the advancement of the ice crystal growth front (usually the tip) at a rate of 5-10 frames per second for 1-3 minutes.

[0033] d) Multi-condition testing: Under the same supercooling, measure the same sample at least 3 times. Change the supercooling conditions and repeat steps b and c to obtain a series of growth data under supercooling.

[0034] S2: Data Processing and Analysis Image analysis software was used to track the position of the ice crystal growth tip frame by frame. A linear fit was performed with time on the x-axis and tip position on the y-axis; the slope of the resulting line represents the ice crystal growth rate (V, typically in μm / s) at that specific supercooling (ΔT). The average growth rate V of the negative control solution and different concentrations of the antifreeze peptide solution at different supercooling degrees was calculated. The relationship curve between ice crystal growth rate (V) and supercooling (ΔT) was plotted. The inhibitory effect of the antifreeze peptide was evaluated through comparative analysis. The ice crystal growth rate of 0.1-1% concentration of the novel antifreeze peptide was 6-8 μm / s at a supercooling of -0.3°C.

[0035] Example 6: The specific steps for verifying the freezing point lowering activity of the novel antifreeze peptide are as follows: S1: The freezing point was accurately determined using differential scanning calorimetry (DSC). A DSC equipped with an autosampler and liquid nitrogen cooling system was used. 10–15 mg of the sample solution was accurately weighed, placed in a standard aluminum DSC crucible, and sealed. An empty, sealed aluminum crucible was used as a reference. The test procedure was as follows: First, the system was equilibrated to 25 °C, then cooled to -40 °C at a rate of -5 °C / min, held for 2 minutes to allow for complete freezing, and then heated to 10 °C at a rate of +5 °C / min. All operations were performed under dry nitrogen purging at a flow rate of 50 mL / min. The freezing point temperature was determined by analyzing the heat flow curve during the cooling process, and the extrapolated temperature of the onset of the exothermic crystallization peak was taken as the freezing point (T0) of the sample solution. f Each concentration sample should be tested in parallel at least 3 times.

[0036] S2: Record and calculate the freezing point (T) of each concentration sample. f The mean and standard deviation of the peptide solution were calculated. Using the freezing point of PBS buffer (approximately -0.5°C, though this may vary slightly due to osmotic pressure) as a baseline, the freezing point depression (ΔT) of each peptide solution concentration relative to the buffer was calculated. f Experimental data show that the antifreeze peptides of this invention can significantly lower the freezing point of the solution in a dose-dependent manner. The novel antifreeze peptides at concentrations of 0.1-1% exhibit freezing point lowering activity of 2-5 °C.

[0037] Example 7: The application of novel antifreeze peptides in the cryopreservation of probiotics follows these steps: S1: After continuous subculturing of probiotics, novel antifreeze peptides and other cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol are added.

[0038] S2: After pre-freezing at -80℃ for 24 h and freeze-drying, the survival rate and morphological characteristics before and after freezing were measured, and the cells were observed to see if they were damaged. The results showed that the survival rate of probiotics containing novel antifreeze peptides after freezing was over 80%.

[0039] S3: The activity of enzymes such as lactate dehydrogenase and β-galactosidase in probiotics before and after freezing and thawing was detected. The results showed that the enzyme activity of the group containing antifreeze protein was more than 3 times that of the control group.

[0040] Example 8: The application of novel antifreeze peptides in red blood cell cryopreservation follows these steps: S1: Take an equal volume of concentrated red blood cells and each prepared cryopreservation solution, and slowly mix them at 4°C (adding the cryopreservation solution dropwise and gently shaking) to ensure that the red blood cells and cryopreservation solution are fully equilibrated for 30 minutes, finally obtaining a cryopreservation suspension with a hematocrit of approximately 35%. Aliquot the suspension into 2 mL sterile cryovials, 1.8 mL per tube. Cryopreservation is performed using the programmed cooling method: after equilibration at 4°C for 30 minutes, place the sample in a -80°C programmed cooling instrument, lowering the temperature to -40°C at a rate of -1°C / min, then to -80°C at a rate of -10°C / min, and finally transfer it to liquid nitrogen (-196°C) for long-term storage. After 4 weeks of cryopreservation, thawing is performed. The cryovials are quickly placed in a 37°C water bath and rapidly shaken until the ice crystals are completely melted. The thawed cell suspension is washed stepwise with a series of NaCl solutions of varying concentrations to gradually remove the cryopreservation agent, and finally resuspended in physiological saline.

[0041] S2: The quality of cryopreserved and recovered red blood cells is evaluated using the following key indicators: a) Red blood cell recovery rate: The red blood cell count in the suspension after resuscitation and washing and before freezing was measured using a fully automated blood cell analyzer, and the recovery rate percentage was calculated.

[0042] b) Hemolysis rate: Take the resuscitated and washed red blood cell suspension, centrifuge it, and measure the absorbance of the supernatant at 540 nm (representing the concentration of free hemoglobin). Compare the absorbance with that of a completely hemolyzed sample (treated with distilled water) to calculate the percentage of hemolysis.

[0043] c) Cell morphology observation: Prepare smears of resuscitated red blood cells, stain them with Wright-Gymsa, and observe the morphological changes of red blood cells (such as the proportion of spiny, spherical, and lobed cells) under an optical microscope to assess the integrity of the membrane structure.

[0044] d) Preliminary assessment of functional activity: The metabolic function of cells was assessed by measuring the ATP content (using the luciferase method) and 2,3-diphosphoglycerate (2,3-DPG) content in the erythrocyte suspension after resuscitation.

[0045] S3: Experimental results showed that red blood cells cryopreserved using a protective solution containing 0.1-1% of the novel antifreeze peptides of this invention achieved a recovery rate of 90.2-92.5% after thawing, with a hemolysis rate of only 1.5-1.8%. Morphological observation showed that the proportion of thrombocytopenic erythrocytes in the peptide group was high, with smooth and intact cell membranes, and the proportion of abnormal cells was significantly lower than that in the negative control group. ATP and 2,3-DPG content measurements showed that the peptide group could effectively maintain the levels of molecules related to energy metabolism and oxygen-carrying function of red blood cells.

[0046] Example 9: The application of novel antifreeze peptides in hematopoietic stem cell cryopreservation involves the following steps: S1: Mononuclear cells were collected from peripheral blood of healthy donors after mobilization, and high-purity CD34+ hematopoietic stem cells were obtained using immunomagnetic bead sorting (e.g., CD34+ positive sorting). The novel antifreeze peptide synthesized and purified in Example 3 was dissolved in a basal cryopreservation medium for hematopoietic stem cells as a positive control, and a solution containing only basal medium was used as a negative control.

[0047] S2: The sorted CD34+ cells were resuspended in basal medium and counted. Under pre-cooling conditions of 4 °C, equal volumes of cell suspension were slowly and dropwise mixed with pre-cooled cryopreservation solutions from each group, gently mixed, and the antifreeze peptide concentration was set. The mixed cell suspension was aliquoted into pre-cooled cryovials, 1 mL per tube. Programmed cooling was used: the cryovials were placed in a programmed cooling apparatus and cooled from 4 °C to -40 °C at a rate of -1 °C / min, then to -80 °C at a rate of -5 °C / min, and then quickly transferred to liquid nitrogen for long-term storage. After 2 weeks of cryopreservation, the cryovials were thawed by removing them from liquid nitrogen and immediately placing them in a 37 °C water bath with rapid shaking. After thawing, the cell suspension was diluted 10-fold with RPMI 1640 medium containing 10% FBS, then centrifuged at 300×g for 5 minutes, washed twice to remove the cryoprotectant, and finally resuspended in complete medium.

[0048] S3: The cryopreservation quality of hematopoietic stem cells was systematically evaluated using the following multi-level indicators: a) Cell viability and recovery rate: The immediate viability and apoptosis / necrosis ratio of cells after resuscitation were detected by trypan blue exclusion assay combined with flow cytometry (using 7-AAD or PI / Annexin V double staining). Cell recovery rate was calculated as: (number of viable cells after resuscitation / number of viable cells before cryopreservation) × 100%.

[0049] b) Community Forming Unit (CFU) Analysis: Resuscitated cells were seeded in a semi-solid methylcellulose medium formulated with a specific formulation (such as MethodCult™ H4435), with 500 cells per dish. After 14 days of incubation in a humidified incubator at 37 °C and 5% CO2, different types of colonies were counted and identified under an inverted microscope, including granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte burst colony-forming units (BFU-E), and mixed colony-forming units (CFU-GEMM). This assay was used to evaluate the proliferation and differentiation potential of cryopreserved hematopoietic progenitor cells.

[0050] c) Maintenance of stem / progenitor cell surface markers: Flow cytometry was used to detect the expression ratios of stem / progenitor cell markers such as CD34+, CD90+, and CD38- in the revived cell population to assess the impact of cryopreservation on stem cell phenotype.

[0051] d) Long-term in vitro culture starter cells (LTC-IC) assay: The revived cells were co-cultured with irradiated mesenchymal stem cell trophoblasts for 5-8 weeks, followed by colony formation experiments to assess the proportion of primitive stem cells with long-term proliferative capacity.

[0052] S4: Experimental results show that adding 0.1-1% of the novel antifreeze peptides significantly improves cryopreservation. The cell viability of the antifreeze peptide group after thawing was 92.5-93.7%, with a significantly reduced proportion of early apoptotic cells; the total colony formation efficiency (CFU / 500 seeded cells) was 145-158; and the CD34+ cell phenotype maintenance rate was higher. Preliminary results of in vivo transplantation experiments show that the human cell chimerism rate and multilineage differentiation capacity of the peptide group cells in mouse bone marrow are closer to those of fresh cells.

[0053] Example 10: The application of novel antifreeze peptides in sperm cryopreservation follows these steps: S1: Preparation of semen samples and cryopreservatives: Semen samples were collected from healthy donors, liquefied, and then high-motility, morphologically normal sperm were obtained using the upstream method or density gradient centrifugation. The sperm were resuspended in a balancing medium (such as Quinn's Sperm Washing Medium) and the concentration was adjusted. The novel antifreeze peptide synthesized and purified in Example 4 was dissolved in a commercially available or self-prepared basic sperm cryopreservation solution. Cryopreservation solutions containing different concentrations of the antifreeze peptide were prepared for the experimental groups. A basic cryopreservation solution without the antifreeze peptide served as a positive control (conventional cryopreservation group), and a solution containing only the balancing medium served as a negative control (expected to cause significant damage).

[0054] S2: Balancing, cryopreservation, and thaw procedures: Slowly mix the adjusted sperm suspension with an equal volume of pre-chilled cryopreservation solution from each experimental group and control group (stepwise addition is recommended). Equilibrate at 4°C for 2-4 hours to allow the cryoprotectant to fully penetrate. Dispense the equilibrated mixture into pre-chilled 0.5 mL or 1.25 mL sperm cryopreservation tubes. Use the liquid nitrogen vapor method for cryopreservation: suspend the cryopreservation tubes in the vapor layer at -80°C to -120°C above the opening of the liquid nitrogen tank for 15-20 minutes, then quickly immerse them in liquid nitrogen (-196°C) for long-term storage. Thaw after at least one week of cryopreservation. Remove the cryopreservation tubes from the liquid nitrogen and immediately place them in a 37°C water bath and shake rapidly to thaw (approximately 60-90 seconds) until completely thawed.

[0055] S3: Comprehensive evaluation of sperm quality after freeze-thaw: After resuscitation, the samples were incubated at 37°C for 10 minutes, and then several key functional indicators were tested: a) Vitality and Motion Parameter Analysis: Using a computer-aided sperm analysis system, total motility (PR+NP, according to WHO standards), the proportion of progressively motile sperm (PR) was assessed, and motility dynamic parameters such as curvilinear motility, linear motility, and head lateral amplitude were analyzed.

[0056] b) Plasma membrane integrity testing: Hypotonic swelling test or fluorescence staining method (such as SYBR-14 / PI double staining) is used. The HOST method involves placing sperm in a hypotonic solution and observing the proportion of sperm with swollen tails (indicating intact membrane function) under a microscope; the fluorescence method uses flow cytometry or fluorescence microscopy to distinguish between live cells (with intact membranes) and dead cells.

[0057] c) Acrosome integrity inspection: Staining with fluorescently labeled peanut lectin or fluorescein-labeled pea lectin, and then assessing the proportion of sperm with intact acrosome structure by fluorescence microscopy or flow cytometry, is crucial for sperm-egg recognition and fertilization.

[0058] d) Sperm morphology analysis: Prepare sperm smears, stain them with Diff-Quik or Giemsa, and evaluate the proportion of normal morphological sperm under a high-power microscope (evaluate at least 200 sperm) according to strict standards (such as the Tygerberg standard).

[0059] e) DNA fragmentation index detection: The degree of damage to sperm nuclear DNA was detected and the DNA fragmentation index was calculated using sperm chromatin structure analysis assay or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

[0061] S4: Experimental results showed that adding 0.1-1% of the novel antifreeze peptide to the basic cryopreservation solution significantly improved the cryopreservation effect. The total sperm motility after freeze-thaw in the antifreeze peptide group was 58.3-58.7%, with a significantly increased proportion of progressively motile sperm; the positive rate of the hypotonic swelling test was 61.7-65.2%; and the acrosome integrity rate was also significantly improved. More importantly, the DNA fragmentation index of the antifreeze peptide group was significantly lower than that of the conventional cryopreservation group, indicating that it can effectively reduce the damage to sperm genetic material during the cryopreservation process.

[0062] Example 11: The application of novel antifreeze peptides in oocyte cryopreservation follows these steps: S1: Preparation of laboratory animals, materials, and cryopreservation solutions: Mature oocytes (MII stage) were collected from superovulated female mice aged 6-8 weeks. The vitrification freezing base solutions included equilibration buffer and vitrification buffer. A novel, synthesized and purified antifreeze peptide was added to both ES and VS solutions. Three groups were set up: an experimental group (ES / VS solution containing the antifreeze peptide), a standard control group (ES / VS solution without the antifreeze peptide), and a fresh, unfrozen control group.

[0063] S2: Vitrification and thawing process: Oocytes were equilibrated at room temperature in ES solutions containing different concentrations of antifreeze peptides for 5-7 minutes, then transferred to the corresponding VS solutions and rapidly loaded into cryopreservation vectors (e.g., Cryotop). The vectors were then quickly immersed directly into liquid nitrogen. After cryopreservation for at least one week, the cells were thawed. The cryopreservation vectors were removed from the liquid nitrogen and rapidly immersed in preheated (37 °C) thawing medium (HTF / HEPES basal medium containing 1.0 M sucrose) for 1 minute. The oocytes were then sequentially transferred to decreasing concentrations of sucrose solutions (0.5 M, 0.25 M) for equilibration for 3 minutes each. Finally, the cells were washed twice in basal culture medium (e.g., HTF) and placed in an incubator (37 °C, 5% CO2) for at least 2 hours to recover.

[0064] S3: Comprehensive evaluation of oocyte quality and function after freeze-thaw: Two hours after resuscitation and incubation, a comprehensive assessment of the following indicators will be conducted: a) Morphological survival rate assessment: When observed under an inverted microscope, an oocyte with an intact morphology (smooth cell membrane, intact zona pellucida, and uniform cytoplasm without shrinkage or vacuoles) is considered to be alive.

[0065] b) Spindle and chromosome morphology detection: Immunofluorescence staining was used. After fixation and resuscitation, microtubules were labeled with anti-α-tubulin antibody and FITC-labeled secondary antibody, and chromosomes were stained with Hoechst 33342. The morphology of the spindle apparatus (whether it was a typical barrel shape) and the arrangement of chromosomes (whether they were neatly aligned at the equatorial plate) were observed using a laser confocal microscope.

[0066] c) Detection of cortical granule distribution: The distribution pattern of cortical granules was observed using fluorescently labeled pea lectin staining. In normal unfertilized MII oocytes, cortical granules should be evenly distributed beneath the plasma membrane. Cryotherapy damage may lead to premature release or abnormal distribution of these granules.

[0067] d) In vitro fertilization and embryonic developmental potential assessment: Morphologically normal, revived oocytes were co-cultured with capacitated mouse sperm. The fertilization rate (appearance of two pronuclei and a second polar body) was recorded. The fertilized eggs were transferred to embryo culture medium for in vitro culture, and their cleavage rate, blastocyst formation rate, blastocyst cell count, and inner cell mass / trophoblast cell ratio were observed and recorded.

[0068] S4: Experimental data showed that in the experimental group with 0.1-1% antifreeze peptides, the freeze-thaw morphology survival rate of oocytes was 90.2-91.4%. The rate of normal spindle morphology and normal chromosome alignment were also significantly improved, at 81.3-82.6% and 83.6-85.9%, respectively. More importantly, the blastocyst formation rate of oocytes in the experimental group after in vitro fertilization was not statistically different from that in the fresh control group, and was significantly higher than that in the conventionally frozen control group, indicating that their developmental potential was better preserved.

Claims

1. A polypeptide with antifreeze activity, characterized in that, The polypeptide comprises a first functional fragment and a second functional fragment, wherein the first functional fragment is an ice crystal binding module and the second functional fragment is a superhydrophilic module.

2. The polypeptide according to claim 1, characterized in that, The ice crystal binding module and the superhydrophilic module are connected by a flexible linker peptide.

3. A method for constructing the polypeptide as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Screen key ice crystal binding modules, including but not limited to GE, GT, TST, etc.; S2: Design of superhydrophilic modules, including but not limited to NNT, NNE, EKT, ETK, etc.; S3: The flexible linker combines the above two functional segments, including but not limited to GS4, G2S3, G3S2, etc.

4. The polypeptide according to claim 1 or 2, characterized in that, The polypeptide has antifreeze properties.

5. The antifreeze properties of the novel antifreeze polypeptide according to claim 4, characterized in that... Novel antifreeze peptides at concentrations of 0.1-1% exhibit 10-20% IRI performance.

6. The antifreeze properties of the novel antifreeze polypeptide according to claim 4, characterized in that... The novel antifreeze peptide at a concentration of 0.1-1% exhibits an ice crystal growth rate of 6-8 μm / s at a supercooling of -0.3 °C.

7. The antifreeze properties of the novel antifreeze polypeptide according to claim 4, characterized in that... Novel antifreeze peptides at concentrations of 0.1-1% exhibit freezing point reduction properties of 2-5 °C.

8. The application of the novel antifreeze polypeptide of claim 4 as a cell cryopreservation protectant.

9. The application according to claim 8, characterized in that: The specific steps are as follows: After continuous passage culture of cells, add novel antifreeze peptides (0.1-1% concentration) for cryopreservation at low temperature.

10. The application according to claim 8, characterized in that: The cells include probiotics, red blood cells, hematopoietic stem cells, sperm and egg cells, etc., with a survival rate of more than 90%.

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