Antifreeze polypeptide, cattle semen freezing diluent and application thereof

Abstract

The present invention provides an antifreeze polypeptide derived from bovine collagen, which can reduce the difference in solute concentration inside and outside the cell during the cooling process, thereby inhibiting the formation of ice crystals inside and outside the sperm cell, and further reducing ice crystal damage to cell membranes and organelles during deep cryopreservation and recovery, thereby improving the survival rate of sperm cells after deep cryopreservation and recovery. The frozen sperm has a higher in vitro fertilization cell development rate and fertility rate, and can reproduce rapidly. In addition, the semen freezing diluent provided by the present application does not contain any exogenous protein, which can effectively reduce exogenous protein interference and immunosuppression during in vitro fertilization, and further improve the fertilization rate and fertility rate of frozen semen.

Description

Technical Field

[0001] The invention belongs to the field of gene proteins, and in particular relates to an antifreeze polypeptide, a nucleotide, and the application of the polypeptide in a bovine semen freezing diluent. Background Art

[0002] With the rapid development of biotechnology, gamete and embryo cryopreservation is gradually becoming the most effective way to replace live animal seed preservation. Semen cryopreservation is to store semen at ultra-low temperatures (usually liquid nitrogen -196℃) after special treatment and adding protective agents to achieve long-term preservation. Its biggest advantage is that it can be stored for a long time and its use is not limited by time, region and the life span of male animals. At the same time, it can greatly improve the utilization efficiency of breeding bulls.

[0003] However, ice crystal damage, cryoprotectant toxicity and oxidative stress in the freezing process of semen are the main reasons for the low quality of semen after thawing and the low conception rate after insemination. Low temperature can inhibit all biochemical activities in cells, thereby reducing the biochemical reaction rate in cells and prolonging the cell activity time. Antifreeze protein is a class of polypeptides or glycopeptides produced by organisms to adapt to extreme cold environments. It can reduce the freezing point of the solution in a non-colligative form, but has little effect on the melting point. Antifreeze protein was first discovered in polar fish. So far, humans have isolated a variety of antifreeze proteins from organisms such as marine fish, terrestrial insects, plants, bacteria and fungi. Although the structures of different types of antifreeze proteins are different, they all have the characteristics of preventing ice crystal formation, regulating the growth of extracellular ice crystals, inhibiting ice recrystallization, and maintaining the non-colligative state of liquids. The special antifreeze mechanism of antifreeze protein has attracted widespread attention in the fields of food processing and preservation, freezing surgery, organ preservation and transplantation. However, the production of antifreeze proteins is still costly and difficult to industrialize, so finding a suitable new source of antifreeze proteins has become an urgent problem to be solved. Secondly, although bovine collagen has been found to have certain osmotic pressure regulation activity, its active fragments only exist in local specific polypeptide chain domains rather than the entire protein. Even purified bovine collagen often has low antifreeze activity, and it is still necessary to explore its active domain to further improve the antifreeze efficiency. Summary of the invention

[0004] The purpose of the present invention is to provide an antifreeze polypeptide, nucleotide and application derived from bovine collagen, so as to solve the problems of low cell permeability and toxicity to cells caused by the existing bovine collagen as a cryoprotectant.

[0005] First, the present application provides an antifreeze polypeptide derived from bovine collagen, whose amino acid sequence is: PYPGAANVPGPAATAGAGG (SEQ ID NO.1). The protein is obtained by screening using bovine collagen as raw material, using an enzymatic hydrolysis method, and establishing an antifreeze activity detection system according to the international antifreeze protein activity detection standard.

[0006] Furthermore, the present application provides a nucleotide encoding the above-mentioned antifreeze polypeptide.

[0007] Furthermore, the present application provides an application of the above-mentioned antifreeze protein in the preparation of a bovine semen freezing diluent, wherein the bovine semen freezing diluent comprises: 15-28 g / L of fructose, 30-500 μmol / L of antifreeze protein, 100-1000 mg / L of vitamin B12, 5-100 mg / L of adenosine triphosphate, 50,000-100,000 IU of penicillin, and 50,000-100,000 IU of streptomycin.

[0008] Furthermore, the present application provides an application of the above-mentioned bovine semen freezing diluent in the cryopreservation of bovine semen.

[0009] Beneficial Effects

[0010] The present invention provides an antifreeze polypeptide derived from bovine collagen. The antifreeze polypeptide derived from bovine collagen can be autonomously integrated into the lipid bilayer of the cell membrane, promote the translocation of water and cryoprotectant molecules across the cell membrane to maintain the equilibrium homeostasis of the cell fluid, and respond to the osmotic gradient to reduce the difference in solute concentration inside and outside the cell during the cooling process. Thereby, the formation of ice crystals inside and outside the sperm cells is inhibited, and the ice crystal damage to the cell membrane and organelles during deep cryopreservation and recovery is reduced, and the survival rate of sperm cells after deep cryopreservation and recovery is improved. The frozen sperm has a higher in vitro fertilization cell development rate and fertility rate, and can reproduce rapidly. Moreover, the semen freezing diluent provided by the present application does not contain any exogenous protein, which can effectively reduce the interference of exogenous proteins and immunosuppression in the process of in vitro fertilization, and further improve the fertilization rate and fertility rate of frozen semen. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the analysis of the antifreeze activity of the crude extract of bovine collagen polypeptide in the examples of this application;

[0012] Figure 2 This is an analysis of the antifreeze activity of the refined bovine collagen polypeptide component in the examples of this application. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0014] Example 1 Analysis of bovine collagen dissociated polypeptides and their antifreeze activity

[0015] Take the finished bovine collagen product (Sigma), prepare it into a 1mmol / L stock solution with ultrapure water, mix it, add ficin and alkaline protease (enzyme activity ratio of 2:1) at a ratio of 3% of dry weight, stir it evenly, and perform enzymolysis at 50°C for 3-5h. After the enzymolysis is completed, centrifuge it at 12000rpm, collect the supernatant, filter the supernatant with a 10kDa membrane, filter the filtrate with a 1000Da membrane, and collect the retentate; obtain a polypeptide liquid with a molecular weight in the range of 1000Da-10kDa, and spray-dry the polypeptide liquid to obtain a crude bovine collagen polypeptide extract.

[0016] Take 10g of crude bovine collagen polypeptide powder and dissolve it in 100mL pure water to make a 100mg / mL polypeptide solution. Use macroporous adsorption resin (LSP-21: LSC200 = 1:1) to separate bovine collagen polypeptide and elute with methanol or ethanol. Collect 20% methanol elution part F1, 40% methanol elution part F2, and 80% methanol elution part F3 respectively. After drying the collected different parts, perform component analysis and activity detection.

[0017] Determination of thermal hysteresis activity (THA) of enzymatic hydrolysis products by differential scanning calorimetry (DSC)

[0018] Weigh a certain weight of the enzymatic hydrolysate, seal it in an aluminum dish, and place it in the DSC instrument. When the instrument is filled with liquid nitrogen and stabilized, first cool it down to -20°C at a rate of 10°C / min and keep it for 5 minutes; then heat it up at a rate of 5°C / min until the sample is partially melted, that is, reaches its retention temperature (Th), keep it for 15 minutes, and then cool it down from Th to -20°C at a rate of 1°C / min. Repeat the above heating and cooling procedures and keep it at different Th for 15 minutes. Record the initial crystallization temperature (T0) of the sample at different Th, and calculate THA according to formula (1). Full-length bovine serum albumin (BSA) without antifreeze activity was used as a control.

[0019] THA=Th-T0 (1)

[0020] from Figure 1 The results show that the curves of BSA before and after the temperature change are smoothly connected, and there is no freezing hysteresis phenomenon, indicating that there is no thermal hysteresis effect in the BSA solution. The heat flow-temperature curves of F1 and F2 are similar to BSA, and there is no freezing hysteresis phenomenon, so there is no thermal hysteresis effect. The crystallization peak of F3 is significantly delayed, and the thermal hysteresis activity is 2.48℃, indicating that the F3 polypeptide at the 80% methanol elution site has antifreeze activity.

[0021] 3) Refining of bovine collagen peptides

[0022] The F3 polypeptide was prepared into a 100 mg / mL solution and purified by preparative liquid chromatography (Dubhe C18 preparative column (250×20 mm, 10 μm), flow rate 8 mL / min, injection volume 1 mL, 100 mg / mL, column temperature at room temperature, detection wavelength 220 nm). The mobile phase was a mixed solution of water and methanol containing 0.1% trifluoroacetic acid, and the elution conditions were: 0 min, 5% methanol; 5 min, 5% methanol; 180 min, 80% methanol. The components Fn-1 / 2 / 3 / 4 were collected separately, and after drying, activity detection and component analysis were performed. The F3-1 component was eluted with 5% methanol and 0.1% trifluoroacetic acid in water, the F3-2 component was eluted with 5%-15% methanol and 0.1% trifluoroacetic acid in water, the F3-3 was eluted with 15%-30% methanol and 0.1% trifluoroacetic acid in water, and the F3-4 was eluted with 30%-45% methanol and 0.1% trifluoroacetic acid in water.

[0023] like Figure 2 As shown, the heat flow-temperature curves of the refined bovine collagen polypeptide components F3-2 and F3-3 are smoothly connected, and there is no freezing hysteresis phenomenon, so there is no thermal hysteresis activity. The crystallization peaks of the refined components F3-1 and F3-4 are significantly delayed, and the thermal hysteresis activities are 2.84℃ and 3.15℃ respectively; therefore, F3-4 was selected for purification and then submitted to BGI for sequencing, and its amino acid sequence was obtained as: PYPGAANVPGPAATAGAGG (SEQ ID NO.1).

[0024] Example 2 Effect of bovine collagen antifreeze polypeptide on bovine frozen semen

[0025] Ultrapure water was used to prepare a storage solution containing bovine collagen antifreeze peptides at a concentration of 10 mmol / L for testing. Semen diluents were prepared according to the final concentrations of fructose 25 g / L, antifreeze peptides 0, 50, 100, 200, 500 μmol / L, vitamin B12 200 mg / L, adenosine triphosphate 50 mg / L, penicillin 50,000 IU, and streptomycin 50,000 IU, for a total of 5 backups.

[0026] The collection, vitality test, density measurement, packaging, freezing and storage of Simontal bull semen were strictly carried out in accordance with the requirements of the Technical Regulations for the Production of Frozen Semen of Cattle (NY / T1234-2018). The semen of 10 Simontal bulls was taken, the volume was measured by weighing, and each semen was divided into 5 equal parts with a pipette, and diluted with the above-mentioned different polypeptide concentration diluents, respectively. After packaging and freezing, the vitality and deformity rate of the frozen semen were tested, and 1,000 frozen semen were produced for each concentration.

[0027] The frozen semen was thawed in 40℃ warm water for 15s, the two ends were cut off and dripped into the coverslip, the mixed semen was sucked with SQA-Vb sperm sampling tube, and the sperm sampling tube was inserted into the SQA-Vb measurement chamber for detection. Each treatment group was tested 3 times and the results were saved. Various sperm movement-related parameters are as follows: active sperm rate (%), forward motility sperm rate (%), forward motility sperm average velocity (μm·s -1 ). The sperm tail hypoosmotic swelling test (HOST) was used to detect the integrity of the sperm plasma membrane. The peanut agglutinin fluorescent labeling (FITC-PNA) staining method was used to detect the integrity of the sperm acrosome. Rhodamine (Rh123) fluorescent dye co-staining was used to distinguish whether the sperm mitochondria were active.

[0028] The results are shown in Table 1, from which it can be seen that compared with the control group, bovine collagen antifreeze polypeptide significantly improved various parameters of Qinchuan cattle sperm (P<0.05). -1 When compared with the control group, the viability and forward motility of Qinchuan bovine sperm after freezing and thawing were significantly increased (P<0.05), and both reached the highest level. The average rate of forward motility of sperm tended to increase. Later, with the increase of the concentration of added peptides, the viability, vitality and average rate of forward motility of bovine sperm after freezing and thawing did not increase significantly, and the average rate of forward motility of sperm decreased. Moreover, the bovine collagen antifreeze peptide added in the diluent had a significant protective effect on the plasma membrane integrity of Qinchuan bovine sperm after freezing and thawing with the increase of concentration. The plasma membrane integrity of sperm was significantly higher than that of the control group (concentration 0 group). Moreover, the sperm acrosome integrity rate and sperm mitochondrial activity after freezing and thawing were positively correlated with the concentration of added bovine collagen antifreeze peptides.

[0029] Table 1 Effect of added peptide concentration on frozen semen

[0030]

[0031] The freezing process of semen can cause oxidative stress in sperm. All components in sperm cells, including lipids, proteins, nucleic acids and sugars, are potential targets of oxidative stress. The proportion of changes caused by oxidative stress depends on the nature and concentration of reactive oxygen species (ROS), the time of exposure to ROS, but also on extracellular factors such as temperature, oxygen and environmental composition (ions, proteins, ROS scavengers). In frozen and thawed semen, the plasma membrane and the outer acrosomal membrane are most sensitive to low temperature. Oxidative stress usually leads to loss of sperm motility, plasma membrane swelling and acrosomal membrane blistering, and changes in the permeability of sperm plasma membrane. During the freezing and thawing process, when ROS are produced in large quantities, structural damage to the plasma membrane increases sensitivity to lipid peroxidation. Seminal plasma contains a large amount of non-enzymatic antioxidants such as ascorbic acid, α-tocopherol, pyruvate, glutathione, taurine, etc. These are the most important protective elements used by sperm cells to resist ROS attacks, but these elements are limited and insufficient to protect sperm from damage during the freezing process. In the in vitro experiment of this study, before the semen was cryopreserved, a certain concentration of bovine collagen antifreeze peptide was added to the bovine semen freezing diluent, which had a more favorable protection on the vitality, mitochondrial activity, physiological structure (plasma membrane, acrosome) and sperm antioxidant capacity of Qinchuan bull sperm after freezing and thawing. -1 The effect is best when it is used, which can promote the permeability of cell membranes and promote transmembrane transport, reduce the difference in solute concentration inside and outside cells during cooling, and be used to reduce cell freezing damage. It not only reduces the cell apoptosis rate, but also has good prospects for practical application.

[0032] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An antifreeze polypeptide derived from bovine collagen, characterized in that: The amino acid sequence of the antifreeze polypeptide is: PYPGAANVPGPAATAGAGG (SEQ ID NO.1).

2. A nucleic acid molecule encoding the antifreeze polypeptide derived from bovine collagen as claimed in claim 1.

3. A bovine semen freezing diluent containing the antifreeze polypeptide according to claim 1.

4. The bovine semen freezing diluent as claimed in claim 3, comprising: Fructose 15-28 g / L, the antifreeze polypeptide described in claim 1 30-500 μmol / L, vitamin B12 100-1000 mg / L, adenosine triphosphate 5-100 mg / L, penicillin 50,000-100,000 IU, and streptomycin 50,000-100,000 IU.

5. Use of the bovine semen freezing diluent according to claim 3 or 4 in the cryopreservation of bovine semen.

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