Polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium and preparation method thereof

Abstract

The invention relates to the technical field of bovine embryo culture media, in particular to a polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium and a preparation method thereof.The polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium comprises the following components: 200-300 [mu] g / mL of SS-31, 0.1-0.3 mg / mL of superoxide dismutase, 0.5-1.0 mg / mL of heat shock protein mimetic peptide, 20-50 mg / mL of amino acid complex and a basic culture medium, and an efficient mitochondrial protection system is established. The targeting effect of SS-31 and the cooperation of an antioxidant network enable the mitochondrial membrane potential to be maintained at the optimal level, and the ATP yield is increased by nearly 100% and is far superior to that in the prior art. And multi-level defense of oxidative stress is realized. Experimental data show that the ROS level in the culture system is reduced by 45% compared with that of a control group, and lipid peroxidation and protein oxidative damage are remarkably relieved. The protection effect derives from the synergistic effect of multiple antioxidant components.

Description

Technical Field

[0001] The present invention relates to the technical field of bovine embryo culture medium, and in particular to a polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium and a preparation method thereof. Background Art

[0002] Bovine embryo in vitro culture technology has important applications in animal breeding and biomedical research. With the deepening of the understanding of the microenvironment of embryo development, people have gradually realized the key role of mitochondrial function and oxidative stress defense in embryo culture. Existing technologies mainly improve the culture environment by adding a single antioxidant or growth factor. Although this method improves the survival rate of embryos to a certain extent, it still has many limitations.

[0003] First, the traditional culture system ignores the specific needs of mitochondrial function regulation. Relying solely on ordinary antioxidants cannot effectively target the mitochondrial inner membrane, leading to mitochondrial dysfunction and energy metabolism disorders. Secondly, existing antioxidant strategies often remain at a single level and lack a systematic synergistic protection mechanism. This one-sided treatment method cannot cope with the complex oxidative stress damage during in vitro culture. Thirdly, the composition ratio of conventional culture media lacks theoretical guidance, fails to fully consider the molecular interactions between the components, and it is difficult to construct a stable culture microenvironment.

[0004] It is particularly noteworthy that the existing technology has obvious deficiencies in protecting protein conformation. During in vitro culture, proteins in embryonic cells are very prone to misfolding and aggregation, and the traditional heat shock protein addition scheme is not very effective and has high costs. In addition, the supply mode of amino acids is also relatively extensive, failing to achieve precise regulation of the cell metabolic network. Summary of the invention

[0005] In response to the above problems, the present invention provides an innovative bovine embryo culture medium that achieves all-round protection of embryonic development through multiple synergistic mechanisms. Its core innovation lies in the construction of a trinity protection system of "mitochondrial targeting-antioxidant network-metabolic regulation".

[0006] The object of the present invention is to provide a polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium, comprising the following components:

[0007] 200-300 μg / mL SS-31, 0.1-0.3 mg / mL superoxide dismutase, 0.5-1.0 mg / mL heat shock protein mimetic peptide, 20-50 mg / mL amino acid complex and basal culture medium;

[0008] Wherein, the basal culture medium comprises:

[0009] 6.0-7.0g / L sodium chloride, 0.3-0.4g / L potassium chloride, 0.15-0.25g / L sodium dihydrogen phosphate, 0.15-0.25g / L magnesium sulfate, 0.15-0.25g / L calcium chloride, 2.0-2.5g / L sodium bicarbonate, 3.5-4.5g / L D-glucose, 0.08-0.12g / L sodium pyruvate, 0.5-0.8g / L sodium lactate, 0.001-0.003g / L folic acid , 0.0001-0.0003g / L biotin, 0.0001-0.0003g / L vitamin B12, 0.02-0.04g / L inositol, 0.002-0.004g / L thiamine, 0.0001-0.0003g / L riboflavin, 0.0001-0.0003g / L copper sulfate, 0.0001-0.0003g / L zinc sulfate and 0.00001-0.00003g / L sodium selenate.

[0010] Preferably, the SS-31 is a sequence as shown in SEQ ID NO.1: D-Arg-2',6'-dimethylTyr-Lys-Phe-NH2.

[0011] Preferably, the heat shock protein mimetic peptide comprises:

[0012] 35-40% of the sequence TKDNNLLGRFELSG (SEQ ID NO. 2),

[0013] 30-35% of the sequence YCVQQLKEFDGKSLVS (SEQ ID NO. 3),

[0014] 25-30% of the sequence is AKVVLDSKHWDVL (SEQ ID NO. 4).

[0015] Preferably, the amino acid complex comprises by weight:

[0016] 15-20% L-leucine, 8-12% L-isoleucine, 8-12% L-valine, 10-15% L-lysine, 8-12% L-phenylalanine, 5-8% L-methionine, 8-12% L-threonine, 2-4% L-tryptophan, 8-12% L-arginine, 4-6% L-histidine, 6-8% L-alanine, 5-7% L-aspartic acid, 12-15% L-glutamine, 4-6% L-glycine, 4-6% L-proline, 4-6% L-serine, 4-6% L-tyrosine, 2-3% L-carnitine, 2-3% taurine and 1-2% beta-alanine.

[0017] Preferably, the superoxide dismutase is human recombinant Cu / Zn-SOD, with a specific activity of ≥3000 U / mg protein and a purity of ≥95%.

[0018] The method for preparing the polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium is characterized by comprising the following steps:

[0019] (1) preparing a basal culture medium;

[0020] (2) preparing heat shock protein mimetic peptides;

[0021] (3) Final preparation of the culture medium.

[0022] Preferably, in step (2), the heat shock protein mimetic peptide is prepared by solid phase synthesis, comprising:

[0023] Wang resin was used as a carrier, and the Fmoc strategy was adopted for amino acid coupling. 20% piperidine / DMF was used for Fmoc deprotection, and TFA / TIS / H2O (95:2.5:2.5) was used for cleavage. Three peptide sequences were obtained by HPLC purification. The steps were carried out at 20-25° C. under nitrogen protection.

[0024] Preferably, in step (1), the preparation of the basal culture medium comprises:

[0025] Inorganic salt components, energy substances, vitamin components and trace elements are added in sequence at 20-25° C., and the pH value is maintained at 7.2-7.4 during the preparation process.

[0026] Preferably, in step (3), the final preparation comprises:

[0027] SS-31, superoxide dismutase, heat shock protein mimetic peptide and amino acid complex were added to the basal medium in sequence and mixed at 25±2°C.

[0028] Preferably, the final prepared culture medium needs to be treated as follows:

[0029] The pH was adjusted to 7.2-7.4, the osmotic pressure was adjusted to 280-300 mOsm / kg, and filtered through 0.45 μm and 0.22 μm membranes in sequence. Finally, 2.5 mL was dispensed into each culture dish.

[0030] From the perspective of molecular mechanism, the SS-31 peptide of the present invention specifically interacts with the inner membrane of mitochondria through its unique secondary structure. The 2',6'-dimethyltyrosine residues in its molecule can form π-π stacking with cardiolipin, while the positively charged arginine residues stabilize this binding through electrostatic interaction. This precise molecular recognition ensures the targeting of the protective effect.

[0031] In terms of antioxidant network construction, the present invention creatively combines superoxide dismutase with a modified selenoprotein complex. Through the synergistic effect, the two realize a complete scavenging chain from superoxide anions to hydrogen peroxide and then to common free radicals. In particular, the redox cycle of selenium atoms in the modified selenoprotein complex significantly improves the antioxidant efficiency.

[0032] The most groundbreaking is the heat shock protein mimetic peptide system designed by the present invention. The three different sequences of mimetic peptides simulate the functional domain structure of natural heat shock proteins through precise spatial arrangement. Among them, the TKDNNLLGRFELSG sequence is responsible for substrate recognition, the YCVQQLKEFDGKSLVS sequence mediates ATPase activity, and the AKVVLDSKHWDVL sequence participates in oligomer formation. This unique molecular design achieves efficient maintenance of cellular protein homeostasis.

[0033] Through the above innovative design, the present invention achieves the following significant technical effects:

[0034] First, an efficient mitochondrial protection system has been established. The targeted action of SS-31 and the synergy of the antioxidant network maintain the mitochondrial membrane potential at an optimal level, and the ATP production is increased by nearly 100%, which is far superior to existing technologies.

[0035] Second, it achieves multi-level defense against oxidative stress. Experimental data show that the ROS level in the culture system of the present invention is 45% lower than that of the control group, and lipid peroxidation and protein oxidative damage are significantly reduced. This protective effect is due to the synergistic effect of multiple antioxidant components.

[0036] Third, the embryonic development potential was significantly improved. In the culture system of the present invention, the blastocyst formation rate was increased by 25 percentage points, and the embryo quality was comprehensively improved. It is particularly worth mentioning that metabolomics analysis found that the present invention also unexpectedly induced a beneficial metabolic reprogramming effect.

[0037] Fourth, epigenetic homeostasis is maintained. This is an unexpected discovery that the culture system of the present invention can affect the DNA methylation pattern, making it closer to the in vivo development state. This epigenetic stability is of great significance to the long-term development of the embryo.

[0038] Fifth, a unique stress adaptive regulation mechanism was discovered. Moderate stress response enhances the embryo's ability to adapt to the environment. This "resilience culture" effect is a feature that traditional culture systems do not have.

[0039] In general, the present invention not only solves the key problems in the prior art through the exquisite coordination of multiple molecular mechanisms, but also achieves a number of unexpected technical effects, opening up a new path for the development of embryo in vitro culture technology. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] Example 1

[0042] This example provides a polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium, the components of which are as follows:

[0043] SS-31 with the sequence of D-Arg-2',6'-dimethylTyr-Lys-Phe-NH2 (SEQ ID NO.1) was selected at a concentration of 200 μg / mL, which can ensure that it can effectively penetrate the cell membrane and reach the inner membrane of the mitochondria to achieve targeted protection of the mitochondria. Superoxide dismutase was selected from human recombinant Cu / Zn-SOD, with a specific activity of 3000U / mg protein, a purity of 95% (SEC-HPLC method), and a concentration of 0.1mg / mL, which can effectively remove superoxide anion free radicals generated during embryo culture.

[0044] The heat shock protein mimetic peptide was selected at 0.5 mg / mL and contained the following three sequences and their proportions: sequence TKDNNLLGRFELSG (SEQ ID NO.2) accounted for 40%, sequence YCVQQLKEFDGKSLVS (SEQ ID NO.3) accounted for 30% and sequence AKVVLDSKHWDVL (SEQ ID NO.4) accounted for 30%. This combination can simulate the function of heat shock protein to the greatest extent and maintain the correct conformation of embryonic protein.

[0045] The total concentration of amino acid complex is 20mg / mL, including 15% L-leucine, 8% L-isoleucine, 8% L-valine, 10% L-lysine, 8% L-phenylalanine, 5% L-methionine, 8% L-threonine, 2% L-tryptophan, 8% L-arginine, 4% L-histidine, 6% L-alanine, 5% L-aspartic acid, 12% L-glutamine, 4% L-glycine, 4% L-proline, 4% L-serine, 4% L-tyrosine, 2% L-carnitine, 2% taurine and 1% beta-alanine. This amino acid ratio can provide comprehensive nutritional support for embryonic development.

[0046] The preparation method comprises the following steps:

[0047] (1) Preparation of basal culture medium

[0048] At 20°C, the inorganic salt components are first dissolved in ultrapure water in sequence, followed by the addition of energy substances, then vitamin components, and finally trace elements. During the stirring process, the pH is adjusted to 7.2 with a 1 mol / L sodium hydroxide solution. Preferably, this step is performed in a laminar flow bench to ensure a sterile environment. The temperature is maintained at 20±0.5°C throughout the process, and the change in pH is monitored in real time by an online pH monitor.

[0049] (2) Preparation of heat shock protein mimetic peptides

[0050] Under nitrogen protection, Wang resin was used as a carrier and the Fmoc strategy was used for solid phase synthesis. First, Fmoc deprotection was performed with 20% piperidine / DMF solution (volume ratio), and the temperature was controlled at 20°C; secondly, Fmoc-amino acid (4 equivalents), HBTU (3.9 equivalents) and DIEA (8 equivalents) were dissolved in DMF and activated for 2 minutes, and then reacted with the resin for 30 minutes; then, the above deprotection and coupling steps were repeated until the target sequence was synthesized; finally, TFA / TIS / H2O (volume ratio 95:2.5:2.5) was used for cutting at room temperature for 4 hours. The peptides of the three sequences were purified by reversed-phase high-performance liquid chromatography, and the purity was greater than 98%.

[0051] (3) Final preparation of culture medium

[0052] Final preparation was performed at 25°C. First, 200 μg / mL of SS-31 was dissolved in sterile saline; second, 0.1 mg / mL of superoxide dismutase was added; then, 0.5 mg / mL of heat shock protein mimetic peptide mixture was added; third, 20 mg / mL of amino acid complex was added; finally, it was mixed with basal medium at a ratio of 1:1. The final pH was adjusted to 7.2 and the osmotic pressure was adjusted to 280 mOsm / kg. Filtered through 0.45 μm and 0.22 μm membranes in sequence, and 2.5 mL was dispensed into each culture dish.

[0053] Example 2

[0054] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0055] This embodiment uses the intermediate concentration value of each component, and the detailed components are as follows:

[0056] SS-31 with the sequence of D-Arg-2',6'-dimethylTyr-Lys-Phe-NH2 (SEQ ID NO.1) was selected at a concentration of 250 μg / mL. Human recombinant Cu / Zn-SOD was selected as superoxide dismutase with a specific activity of 3500 U / mg protein, a purity of 97% (SEC-HPLC method), and a concentration of 0.2 mg / mL.

[0057] The heat shock protein mimetic peptide was selected at 0.75 mg / mL and contained the following three sequences and their proportions: sequence TKDNNLLGRFELSG (SEQ ID NO. 2) accounted for 37.5%, sequence YCVQQLKEFDGKSLVS (SEQ ID NO. 3) accounted for 32.5% and sequence AKVVLDSKHWDVL (SEQ ID NO. 4) accounted for 30%.

[0058] The total concentration of the amino acid complex is 35 mg / mL, including L-leucine 17.5%, L-isoleucine 10%, L-valine 10%, L-lysine 12.5%, L-phenylalanine 10%, L-methionine 6.5%, L-threonine 10%, L-tryptophan 3%, L-arginine 10%, L-histidine 5%, L-alanine 7%, L-aspartic acid 6%, L-glutamine 13.5%, L-glycine 5%, L-proline 5%, L-serine 5%, L-tyrosine 5%, L-carnitine 2.5%, taurine 2.5% and beta-alanine 1.5%.

[0059] In the basic culture medium, inorganic salt components are selected: sodium chloride 6.5g / L, potassium chloride 0.35g / L, sodium dihydrogen phosphate 0.2g / L, magnesium sulfate 0.2g / L, calcium chloride 0.2g / L and sodium bicarbonate 2.25g / L. Energy substances are selected: D-glucose 4.0g / L, sodium pyruvate 0.1g / L and sodium lactate 0.65g / L. Vitamin components are selected: folic acid 0.002g / L, biotin 0.0002g / L, vitamin B12 0.0002g / L, inositol 0.03g / L, thiamine 0.003g / L and riboflavin 0.0002g / L. Trace elements are selected: copper sulfate 0.0002g / L, zinc sulfate 0.0002g / L and sodium selenate 0.00002g / L.

[0060] The preparation method comprises the following steps:

[0061] (1) Preparation of basal culture medium

[0062] At 22°C, the inorganic salt components are first dissolved in ultrapure water in sequence, followed by the addition of energy substances, then vitamin components, and finally trace elements. A 1 mol / L sodium hydroxide solution is used to adjust the pH to 7.3. Preferably, the entire process is carried out in a laminar flow bench, the temperature is maintained at 22±0.5°C, and the change in pH is monitored in real time by an online pH monitor.

[0063] (2) Preparation of heat shock protein mimetic peptides

[0064] Under nitrogen protection, Wang resin was used as a carrier and the Fmoc strategy was used for solid phase synthesis. First, Fmoc deprotection was performed with 20% piperidine / DMF solution (volume ratio), and the temperature was controlled at 22°C; secondly, Fmoc-amino acid (4.5 equivalents), HBTU (4.4 equivalents) and DIEA (9 equivalents) were dissolved in DMF and activated for 2.5 minutes, and then reacted with the resin for 35 minutes; then, the above deprotection and coupling steps were repeated until the target sequence was synthesized; finally, TFA / TIS / H2O (volume ratio 95:2.5:2.5) was used for cutting at room temperature for 5 hours. The peptides of the three sequences were purified by reversed-phase high-performance liquid chromatography, and the purity was greater than 98%.

[0065] (3) Final preparation of culture medium

[0066] Final preparation was performed at 25°C. First, 250 μg / mL of SS-31 was dissolved in sterile saline; second, 0.2 mg / mL of superoxide dismutase was added; then, 0.75 mg / mL of heat shock protein mimetic peptide mixture was added; third, 35 mg / mL of amino acid complex was added; finally, it was mixed with basal medium at a ratio of 2:1. The final pH was adjusted to 7.3 and the osmotic pressure was adjusted to 290 mOsm / kg. Filtered through 0.45 μm and 0.22 μm membranes in sequence, and 2.5 mL was dispensed into each culture dish.

[0067] Example 3

[0068] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0069] This embodiment uses the high end value range of each component, and the detailed components are as follows:

[0070] SS-31 with the sequence of D-Arg-2',6'-dimethylTyr-Lys-Phe-NH2 (SEQ ID NO.1) was selected at a concentration of 300 μg / mL. Human recombinant Cu / Zn-SOD was selected as superoxide dismutase with a specific activity of 4000 U / mg protein, a purity of 99% (SEC-HPLC method), and a concentration of 0.3 mg / mL.

[0071] The heat shock protein mimetic peptide was selected at 1.0 mg / mL and contained the following three sequences and their proportions: sequence TKDNNLLGRFELSG (SEQ ID NO. 2) accounted for 40%, sequence YCVQQLKEFDGKSLVS (SEQ ID NO. 3) accounted for 35% and sequence AKVVLDSKHWDVL (SEQ ID NO. 4) accounted for 25%.

[0072] The total concentration of the amino acid complex is 50 mg / mL, including L-leucine 20%, L-isoleucine 12%, L-valine 12%, L-lysine 15%, L-phenylalanine 12%, L-methionine 8%, L-threonine 12%, L-tryptophan 4%, L-arginine 12%, L-histidine 6%, L-alanine 8%, L-aspartic acid 7%, L-glutamine 15%, L-glycine 6%, L-proline 6%, L-serine 6%, L-tyrosine 6%, L-carnitine 3%, taurine 3% and beta-alanine 2%.

[0073] In the basic culture medium, the inorganic salt components are: sodium chloride 7.0g / L, potassium chloride 0.4g / L, sodium dihydrogen phosphate 0.25g / L, magnesium sulfate 0.25g / L, calcium chloride 0.25g / L and sodium bicarbonate 2.5g / L. Energy substances are: D-glucose 4.5g / L, sodium pyruvate 0.12g / L and sodium lactate 0.8g / L. Vitamin components are: folic acid 0.003g / L, biotin 0.0003g / L, vitamin B12 0.0003g / L, inositol 0.04g / L, thiamine 0.004g / L and riboflavin 0.0003g / L. Trace elements are: copper sulfate 0.0003g / L, zinc sulfate 0.0003g / L and sodium selenate 0.00003g / L.

[0074] The preparation method comprises the following steps:

[0075] (1) Preparation of basal culture medium

[0076] At 25°C, the inorganic salt components are first dissolved in ultrapure water in sequence, followed by the addition of energy substances, then vitamin components, and finally trace elements. A 1 mol / L sodium hydroxide solution is used to adjust the pH to 7.4. Preferably, the entire process is carried out in a laminar flow bench, the temperature is maintained at 25±0.5°C, and the change in pH is monitored in real time by an online pH monitor.

[0077] (2) Preparation of heat shock protein mimetic peptides

[0078] Under nitrogen protection, Wang resin was used as a carrier and the Fmoc strategy was used for solid phase synthesis. First, Fmoc deprotection was performed with 20% piperidine / DMF solution (volume ratio), and the temperature was controlled at 25°C; secondly, Fmoc-amino acid (5 equivalents), HBTU (4.9 equivalents) and DIEA (10 equivalents) were dissolved in DMF and activated for 3 minutes, and then reacted with the resin for 40 minutes; then, the above deprotection and coupling steps were repeated until the target sequence was synthesized; finally, TFA / TIS / H2O (volume ratio 95:2.5:2.5) was used for cutting at room temperature for 6 hours. The peptides of the three sequences were purified by reversed-phase high-performance liquid chromatography, and the purity was greater than 99%.

[0079] (3) Final preparation of culture medium

[0080] Final preparation was performed at 25°C. First, 300 μg / mL of SS-31 was dissolved in sterile saline; second, 0.3 mg / mL of superoxide dismutase was added; then, 1.0 mg / mL of heat shock protein mimetic peptide mixture was added; third, 50 mg / mL of amino acid complex was added; finally, it was mixed with basal medium at a ratio of 5:1. The final pH was adjusted to 7.4 and the osmotic pressure was adjusted to 300 mOsm / kg. Filtered through 0.45 μm and 0.22 μm membranes in sequence, and 2.5 mL was dispensed into each culture dish.

[0081] Example 4

[0082] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0083] SS-31 with the sequence of D-Arg-2',6'-dimethylTyr-Lys-Phe-NH2 (SEQ ID NO.1) was selected at a concentration of 275 μg / mL. Human recombinant Cu / Zn-SOD was selected as superoxide dismutase, with a specific activity of 3700 U / mg protein, a purity of 98% (SEC-HPLC method), and a concentration of 0.25 mg / mL.

[0084] The heat shock protein mimetic peptide was selected at 0.85 mg / mL and contained the following three sequences and their proportions: sequence TKDNNLLGRFELSG (SEQ ID NO. 2) accounted for 38%, sequence YCVQQLKEFDGKSLVS (SEQ ID NO. 3) accounted for 33% and sequence AKVVLDSKHWDVL (SEQ ID NO. 4) accounted for 29%.

[0085] The total concentration of the amino acid complex is 42.5 mg / mL, including L-leucine 18%, L-isoleucine 11%, L-valine 11%, L-lysine 13%, L-phenylalanine 11%, L-methionine 7%, L-threonine 11%, L-tryptophan 3.5%, L-arginine 11%, L-histidine 5.5%, L-alanine 7.5%, L-aspartic acid 6.5%, L-glutamine 14%, L-glycine 5.5%, L-proline 5.5%, L-serine 5.5%, L-tyrosine 5.5%, L-carnitine 2.7%, taurine 2.7% and beta-alanine 1.8%.

[0086] In the basic culture medium, inorganic salt components are selected: sodium chloride 6.7g / L, potassium chloride 0.37g / L, sodium dihydrogen phosphate 0.22g / L, magnesium sulfate 0.22g / L, calcium chloride 0.22g / L and sodium bicarbonate 2.35g / L. Energy substances are selected: D-glucose 4.2g / L, sodium pyruvate 0.11g / L and sodium lactate 0.7g / L. Vitamin components are selected: folic acid 0.0025g / L, biotin 0.00025g / L, vitamin B12 0.00025g / L, inositol 0.035g / L, thiamine 0.0035g / L and riboflavin 0.00025g / L. Trace elements are selected: copper sulfate 0.00025g / L, zinc sulfate 0.00025g / L and sodium selenate 0.000025g / L.

[0087] The preparation method comprises the following steps:

[0088] (1) Preparation of basal culture medium

[0089] At 23°C, the inorganic salt components are first dissolved in ultrapure water in sequence, followed by the addition of energy substances, then vitamin components, and finally trace elements. A 1 mol / L sodium hydroxide solution is used to adjust the pH to 7.35. Preferably, the entire process is carried out in a laminar flow bench, the temperature is maintained at 23±0.5°C, and the change in pH is monitored in real time by an online pH monitor.

[0090] (2) Preparation of heat shock protein mimetic peptides

[0091] Under nitrogen protection, Wang resin was used as a carrier and the Fmoc strategy was used for solid phase synthesis. First, Fmoc deprotection was performed with 20% piperidine / DMF solution (volume ratio), and the temperature was controlled at 23°C; secondly, Fmoc-amino acid (4.7 equivalents), HBTU (4.6 equivalents) and DIEA (9.5 equivalents) were dissolved in DMF and activated for 2.7 minutes, and then reacted with the resin for 37 minutes; then, the above deprotection and coupling steps were repeated until the target sequence was synthesized; finally, TFA / TIS / H2O (volume ratio 95:2.5:2.5) was used for cutting at room temperature for 5.5 hours. The peptides of the three sequences were purified by reversed-phase high-performance liquid chromatography, and the purity was greater than 98.5%.

[0092] (3) Final preparation of culture medium

[0093] Final preparation was performed at 25°C. First, 275 μg / mL of SS-31 was dissolved in sterile saline; second, 0.25 mg / mL of superoxide dismutase was added; then, 0.85 mg / mL of heat shock protein mimetic peptide mixture was added; third, 42.5 mg / mL of amino acid complex was added; finally, it was mixed with basal medium at a ratio of 3:1. The final pH was adjusted to 7.35 and the osmotic pressure was adjusted to 295 mOsm / kg. Filtered by 0.45 μm and 0.22 μm membranes in sequence, and 2.5 mL was dispensed into each culture dish.

[0094] Comparative Example 1 (corresponding to Example 1, verifying the synergistic effect of SS-31)

[0095] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0096] Except for not containing SS-31, the other components and contents of this comparative example are the same as those of Example 1. Through this comparative design, the key role of SS-31 in the present invention can be verified. Studies have shown that in the culture system lacking SS-31, the mitochondrial targeted protection effect is significantly reduced, resulting in an increase of about 45% in oxidative stress damage to the embryo during in vitro culture. This result confirms that SS-31 forms a significant synergistic effect with other antioxidant components through its specific mitochondrial targeting effect. At the same time, flow cytometry analysis found that the mitochondrial membrane potential stability of the group without SS-31 decreased by 30%, and ATP production decreased by 25%, indicating that the presence of SS-31 is essential for maintaining the energy metabolism of embryonic cells.

[0097] The preparation method is basically the same as that of Example 1, except that SS-31 is not added in the final preparation step. Preferably, during the final preparation of the culture medium, the effect of the lack of SS-31 on cellular energy metabolism can be intuitively observed by real-time monitoring of mitochondrial membrane potential and ATP content.

[0098] Comparative Example 2 (corresponding to Example 2, verifying the importance of the ratio of heat shock protein mimetic peptides)

[0099] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0100] In this comparative example, the ratio of the three sequences of the heat shock protein mimetic peptide was changed, and the sequence TKDNNLLGRFELSG (SEQ ID NO.2) accounted for 45%, the sequence YCVQQLKEFDGKSLVS (SEQ ID NO.3) accounted for 40% and the sequence AKVVLDSKHWDVL (SEQ ID NO.4) accounted for 15%, and the total concentration was still 0.75 mg / mL. Circular dichroism analysis showed that this ratio change caused a significant change in the spatial conformation of the mimetic peptide, an increase in the β-folded structure content, and a decrease in the α-helix content, which affected its ability to bind to the target protein. Proteomic analysis showed that this ratio change led to a significant decrease in HSP70-like activity, an increase in protein misfolding rate, and an increase in abnormal protein aggregation in cells.

[0101] The preparation method is basically the same as that in Example 2, but in the synthesis step of the heat shock protein mimetic peptide, the feed ratio of each sequence needs to be adjusted. It is worth noting that this change in ratio affects the solubility of the peptide chain during the synthesis process. Preferably, the reaction temperature needs to be increased to 24°C during the synthesis process to ensure complete dissolution of sequence 1. Real-time circular dichroism monitoring shows that the conformational stability of the heat shock protein mimetic peptide under this ratio is significantly reduced, and the conformation retention time at 37°C is significantly shortened.

[0102] Comparative Example 3 (corresponding to Example 2, verifying the synergistic effect of amino acid complex)

[0103] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0104] This comparative example simplifies the amino acid complex into a traditional mixture of 20 natural amino acids in equal amounts, with a total concentration of 35 mg / mL. Metabolomics analysis showed that in the simplified formula, the energy metabolism of the embryos was significantly abnormal: the content of intermediates in the tricarboxylic acid cycle decreased, the activity of the electron transport chain decreased, and the accumulation of lactic acid increased. Isotope tracing experiments further confirmed that the lack of synergistic effects of L-carnitine and β-alanine led to reduced efficiency of fatty acid β-oxidation and insufficient utilization of energy substrates.

[0105] The preparation method of this comparative example is basically the same as that of Example 2, but special attention should be paid to the dissolution order in the amino acid mixing step: first dissolve the hydrophilic amino acid in ultrapure water, and then gradually add the hydrophobic amino acid, maintaining the temperature at 24±1°C and the pH value between 7.2-7.4 during the process. Compared with Example 2, the amino acid solution mixed in equal amounts exhibits poor solubility and stability, and the stirring time needs to be extended to 2 hours. At the same time, the osmotic pressure of the simplified formula fluctuates greatly and needs to be adjusted more frequently.

[0106] Comparative Example 4 (corresponding to Example 3, verifying the importance of superoxide dismutase activity)

[0107] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0108] This comparative example uses superoxide dismutase (specific activity is 2000U / mg protein) with reduced activity, and other components are consistent with Example 3. Electron spin resonance detection shows that the scavenging efficiency of superoxide anion free radicals by SOD with reduced activity significantly decreases. The reduction of this activity destroys the balance of the antioxidant defense network, causes the level of mitochondrial membrane lipid peroxidation to increase, protein carbonylation modification to increase, and DNA oxidative damage to increase. Real-time fluorescence imaging observation shows that SOD with reduced activity cannot maintain the steady-state level of reactive oxygen species in embryonic cells.

[0109] The preparation method is basically the same as that of Example 3, but SOD pretreated at 37°C for 48 hours is used, resulting in reduced activity. During the final preparation process, the actual activity of SOD needs to be confirmed by real-time activity detection to ensure that it is maintained at a level of 2000±100U / mg protein.

[0110] Comparative Example 5 (corresponding to Example 4, verifying the importance of the distribution ratio of the basic culture medium components)

[0111] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0112] In this comparative example, the ratio of energy substances in the basal culture medium was adjusted, the concentration of D-glucose was increased to 5.0 g / L, and sodium pyruvate was reduced to 0.05 g / L, and the other components were consistent with Example 4. Cell metabolic flux analysis showed that this ratio change led to overactivation of the glycolysis pathway, increased lactic acid accumulation, and decreased mitochondrial oxidative phosphorylation efficiency. 13C-NMR tracing experiments confirmed that excessive glucose supply inhibited fatty acid oxidation and amino acid metabolism, disrupting the balanced utilization of multiple energy substrates.

[0113] The preparation method is basically the same as that of Example 4, but the amount of energy substance added needs to be adjusted when preparing the basal culture medium. Preferably, a 48-hour stability observation is required after the final preparation to ensure that no precipitation of the culture medium components occurs under high sugar conditions.

[0114] Comparative Example 6 (corresponding to Example 1, verifying the overall synergistic effect of the components)

[0115] Preparation of peptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium

[0116] This comparative example adopts a step-by-step addition method, and the functional components in Example 1 are added to the basal medium separately, and the components are added every 12 hours. This addition method breaks the synergistic timeliness between the components. Although the final component content is the same as that in Example 1, the omics analysis shows that the metabolite spectrum related to mitochondrial function has changed significantly, the synergy of the antioxidant network has decreased, and the cell stress response has increased. Through real-time metabolomics monitoring, it was found that the step-by-step addition caused obvious fluctuations in various metabolic indicators and failed to form a stable metabolic network.

[0117] The basic framework of the preparation method is the same as that of Example 1, but a step-by-step addition strategy is adopted: first, the basic culture medium components are added, SS-31 is added 12 hours later, superoxide dismutase is added 12 hours later, and so on. After each addition of a new component, the pH value and osmotic pressure need to be readjusted, and the effect of each component is evaluated by real-time cell metabolism monitoring.

[0118] Through the above six comparative examples, the synergistic mechanism of the components in the present invention is systematically verified, which fully proves the innovation and superiority of the overall formula. In particular, the ratio optimization and synergistic time effectiveness of each functional component are of great significance for maintaining the steady-state environment of embryo in vitro culture.

[0119] In order to comprehensively evaluate the performance of the culture medium of the present invention, the present invention designed a systematic test experiment, which mainly includes the following aspects:

[0120] 1. Evaluation of mitochondrial function

[0121] The JC-1 probe was used to detect the mitochondrial membrane potential, and the ATP fluorescence detection kit was used to determine the ATP content. The experiment was carried out at 37°C and 5% CO2. First, embryos cultured for 48 hours were collected, washed with PBS, and 2μM JC-1 dye was added and incubated for 30 minutes. The red-green fluorescence ratio was observed by confocal microscopy to reflect the functional status of mitochondria. Secondly, the ATP detection kit was used to determine the embryonic ATP content by chemiluminescence.

[0122] 2. Assessment of oxidative stress level

[0123] The DCFH-DA probe was used to detect the level of reactive oxygen species, and the oxidative stress state was comprehensively evaluated by indicators such as lipid peroxide content (MDA) and protein carbonylation level. During the experiment, the embryos were placed in a culture medium containing 10μM DCFH-DA for 20 minutes, and the fluorescence intensity was detected by flow cytometry. At the same time, the culture medium was collected to determine the MDA content and protein carbonylation level.

[0124] 3. Developmental Potential Evaluation

[0125] The embryonic development potential was evaluated by blastocyst formation rate, cell count, apoptosis index, etc. The number of blastocysts formed was counted on the 7th day of embryo culture, and the cell viability was evaluated by Hoechst / PI double staining.

[0126] The experimental results are as follows:

[0127] Table 1 Results of mitochondrial function evaluation

[0128]

[0129] Table 2 Evaluation of oxidative stress level

[0130]

[0131] Table 3 Evaluation of embryonic development potential

[0132] Sample set Blastocyst formation rate (%) Total cell number Cell apoptosis index (%) Example 1 45±3 125±10 4.5±0.5 Example 2 47±3 128±10 4.2±0.5 Example 3 50±3 132±10 4.0±0.5 Example 4 48±3 130±10 4.1±0.5 Comparative Example 1 25±3 85±10 8.5±0.8 Comparative Example 2 30±3 95±10 7.5±0.8 Comparative Example 3 32±3 98±10 7.2±0.8 Comparative Example 4 28±3 90±10 8.0±0.8 Comparative Example 5 29±3 92±10 7.8±0.8 Comparative Example 6 33±3 100±10 7.0±0.8

[0133] The results analysis and unexpected technical effects are as follows:

[0134] 1. Mitochondrial function maintenance effect: The present invention achieves comprehensive protection of mitochondrial function through the synergistic effect of SS-31 and other components. In particular, in Example 3, the mitochondrial membrane potential is maintained at an optimal level (red / green fluorescence ratio 3.0), which is nearly 100% higher than that of Comparative Example 1. This significant improvement effect is due to the "dual targeting" protection mechanism formed between SS-31 and the improved selenoprotein complex, which not only maintains the structural stability of the mitochondrial inner membrane, but also significantly increases ATP production.

[0135] 2. Synergistic effect of antioxidant network: The present invention constructs a multi-level antioxidant defense network. Experimental data show that the ROS level of the example group is maintained at the baseline level, while the comparative group is significantly increased. It is particularly noteworthy that the MDA content (0.40nmol / mg) in Example 3 is reduced by about 53% compared with that in Comparative Example 1. This significant antioxidant effect is attributed to the synergistic effect between superoxide dismutase and heat shock protein mimetic peptide.

[0136] 3. Comprehensive improvement of developmental potential: The most surprising thing is the significant promotion of embryo development potential by the present invention. The blastocyst formation rate of Example 3 reached 50%, which was 25 percentage points higher than that of Comparative Example 1. More importantly, through the analysis of cell apoptosis index, it was found that the cell quality of the Example group was significantly better than that of the Comparative Example group, which shows that the present invention not only improves the development rate of embryos, but also significantly improves the quality of embryos.

[0137] The present invention also has the following unexpected technical effects:

[0138] 1. Metabolic reprogramming effect: Metabolomics analysis revealed that the culture system of the present invention can induce beneficial metabolic reprogramming in embryos. In the example group, the glycolysis / oxidative phosphorylation ratio was maintained at an optimal level, and this balanced energy metabolism pattern is difficult to achieve in traditional culture systems.

[0139] 2. Epigenetic regulation: It was unexpectedly found that the culture system of the present invention can affect the epigenetic modification pattern of embryos. The DNA methylation level of the example group is closer to that of the in vivo developing embryo than that of the control group. The maintenance of this epigenetic stability is of great significance to the long-term development of the embryo.

[0140] 3. Adaptive regulation of stress response: Experimental data show that the culture system of the present invention can induce a moderate "stress response" in the embryo. This mild stress response actually enhances the embryo's adaptability and developmental potential, which is an unexpected discovery.

[0141] Based on the above test results, Example 3 was determined to be the best implementation scheme, which not only performed best in various indicators, but also had the best repeatability and stability. These test results fully demonstrated the innovation and superiority of the present invention, especially the significant effects in maintaining mitochondrial function, building antioxidant networks and improving developmental potential.

[0142] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium, characterized in that: The invention comprises the following components: 200-300 μg / mL SS-31, 0.1-0.3 mg / mL superoxide dismutase, 0.5-1.0 mg / mL heat shock protein mimetic peptide, 20-50 mg / mL amino acid complex and basal culture medium; Wherein, the basal culture medium comprises: 6.0-7.0g / L sodium chloride, 0.3-0.4g / L potassium chloride, 0.15-0.25g / L sodium dihydrogen phosphate, 0.15-0.25g / L magnesium sulfate, 0.15-0.25g / L calcium chloride, 2.0-2.5g / L sodium bicarbonate, 3.5-4.5g / L D-glucose, 0.08-0.12g / L sodium pyruvate, 0.5-0.8g / L sodium lactate, 0.001-0.003g / L folic acid , 0.0001-0.0003g / L biotin, 0.0001-0.0003g / L vitamin B12, 0.02-0.04g / L inositol, 0.002-0.004g / L thiamine, 0.0001-0.0003g / L riboflavin, 0.0001-0.0003g / L copper sulfate, 0.0001-0.0003g / L zinc sulfate and 0.00001-0.00003g / L sodium selenate.

2. The polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium according to claim 1, characterized in that: The SS-31 is a sequence as shown in SEQ ID NO. 1: D-Arg-2',6'-dimethylTyr-Lys-Phe-NH 2.

3. The polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium according to claim 1, characterized in that: The heat shock protein mimetic peptide comprises: 35-40% of the sequence TKDNNLLGRFELSG (SEQ ID NO. 2), 30-35% of the sequence YCVQQLKEFDGKSLVS (SEQ ID NO. 3), 25-30% of the sequence is AKVVLDSKHWDVL (SEQ ID NO. 4).

4. The polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium according to claim 1, characterized in that: The amino acid complex comprises by weight: 15-20% L-leucine, 8-12% L-isoleucine, 8-12% L-valine, 10-15% L-lysine, 8-12% L-phenylalanine, 5-8% L-methionine, 8-12% L-threonine, 2-4% L-tryptophan, 8-12% L-arginine, 4-6% L-histidine, 6-8% L-alanine, 5-7% L-aspartic acid, 12-15% L-glutamine, 4-6% L-glycine, 4-6% L-proline, 4-6% L-serine, 4-6% L-tyrosine, 2-3% L-carnitine, 2-3% taurine and 1-2% beta-alanine.

5. The polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium according to claim 1, characterized in that: The superoxide dismutase is human recombinant Cu / Zn-SOD, with a specific activity of ≥3000 U / mg protein and a purity of ≥95%.

6. The method for preparing the polypeptide-mitochondrial targeting-antioxidant synergistic bovine embryo culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) preparing a basal culture medium; (2) preparing heat shock protein mimetic peptides; (3) Final preparation of the culture medium.

7. The preparation method according to claim 6, characterized in that: In the step (2), the heat shock protein mimetic peptide is prepared by solid phase synthesis, comprising: Wang resin was used as a carrier, and the Fmoc strategy was adopted for amino acid coupling. 20% piperidine / DMF was used for Fmoc deprotection, and TFA / TIS / H2O (95:2.5:2.5) was used for cleavage. Three peptide sequences were obtained by HPLC purification. The steps were carried out at 20-25° C. under nitrogen protection.

8. The preparation method according to claim 6, characterized in that: In the step (1), the preparation of the basal culture medium comprises: Inorganic salt components, energy substances, vitamin components and trace elements are added in sequence at 20-25° C., and the pH value is maintained at 7.2-7.4 during the preparation process.

9. The preparation method according to claim 6, characterized in that: In the step (3), the final preparation includes: adding SS-31, superoxide dismutase, heat shock protein mimetic peptide and amino acid complex to the basal culture medium in sequence, and mixing at 25±2°C.

10. The preparation method according to claim 9, characterized in that: The final prepared culture medium needs to be processed as follows: The pH was adjusted to 7.2-7.4, the osmotic pressure was adjusted to 280-300 mOsm / kg, and filtered through 0.45 μm and 0.22 μm membranes in sequence. Finally, 2.5 mL was dispensed into each culture dish.

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