Preparation method and application of cattle specific stress blocker

Abstract

The invention is applicable to the field of anti-stress therapy, and provides a preparation method and application of a bovine specific stress blocker, and the preparation method comprises the following steps: synthesizing a coding bovine immune checkpoint extracellular region sequence with double restriction enzyme cutting sites by taking an amino acid sequence of a bovine immune checkpoint as a template; the amino acid sequence of the bovine immune checkpoint is as shown in SEQ ID NO. 1; the method comprises the following steps: constructing a coding bovine immune checkpoint extracellular region sequence into a plasmid vector by virtue of an enzyme digestion connection method, so as to obtain a recombinant plasmid; transforming the recombinant plasmids into competent cells, then inducing target protein expression, and purifying to obtain recombinant protein; after animals are immunized by using the recombinant protein, a positive hybridoma cell strain for blocking bovine immune checkpoints is obtained through cell fusion, cloning and screening; after the positive hybridoma cell strain is injected into an animal pretreated by liquid paraffin, ascites is collected and purified, and the bovine specific stress blocking agent is obtained and has high specificity and affinity to bovine immune checkpoints.

Description

Technical Field

[0001] The invention belongs to the field of anti-stress treatment, and in particular relates to a preparation method and application of a cattle-specific stress blocker. Background Art

[0002] Stress refers to a type of non-specific reaction caused by various stressful stimuli (stressors) in the internal and external environment, also known as stress response. In veterinary clinics, when animals are in a harsh environment such as long-term fixed-term feeding, crowding, hunger, abnormal pen temperature, and continuous noise, they are prone to stress syndrome, which seriously damages the health of animals and hinders the healthy development of animal husbandry. When animals are stimulated by a series of internal and external stressors, the initial physiological stress response activates the sympathetic nervous system and the hypothalamus-pituitary-adrenal axis (HPA axis), causing the body to initiate defense adaptive mechanisms to restore the homeostasis of the internal environment. However, as the intensity of the stressor stimulation increases and the time is prolonged, the long-term and strong stimulation destroys the homeostasis of the body's internal environment, and the HPA axis is overactivated, causing the adrenal cortex to release a large amount of glucocorticoids (GC), mainly cortisol (corticosterone in rodents). Sustained high levels of GC can cause rapid activation and morphological changes in M1 microglia in the central nervous system, with branches becoming shorter and thicker, and cell bodies becoming larger, showing proinflammatory and neurotoxic effects. Polarized M1 microglia often cause hyperactivity of the HPA axis, thereby promoting the development of stress, while polarized M2 microglia can alleviate the stress state. Stress can cause abnormal activation of microglia, significantly enhance the expression of immune checkpoints, and cause microglia to transform into M1 type, leading to instability of the central nervous system and aggravating hyperactivity of the HPA axis. Previous studies have shown that immune checkpoints on microglia can promote their transformation into M1 microglia, and knocking out or silencing this immune checkpoint can inhibit and reverse abnormal activation of microglia.

[0003] Therefore, the present invention intends to prepare immune checkpoint blockers, which can specifically block immune checkpoints through in vivo experiments to inhibit abnormal activation of microglia M1, significantly reverse the morphology and function of microglia, restore the homeostasis of the central nervous system, and at the same time reduce stress damage, enhance the body's stress resistance, and improve the production performance of livestock and poultry, thereby promoting the sustainable, green and healthy development of my country's animal husbandry. Summary of the invention

[0004] The purpose of the embodiments of the present invention is to provide a method for preparing a bovine-specific stress blocker, aiming to solve the problems raised in the background technology.

[0005] The embodiment of the present invention is achieved by a method for preparing a cattle-specific stress blocker, which comprises the following steps:

[0006] Using the amino acid sequence of the bovine immune checkpoint as a template, synthesizing the extracellular region sequence encoding the bovine immune checkpoint with double restriction sites; the amino acid sequence of the bovine immune checkpoint is shown in the sequence table SEQ ID NO.1;

[0007] The extracellular region sequence encoding the bovine immune checkpoint is constructed into a plasmid vector by restriction digestion and ligation to obtain a recombinant plasmid;

[0008] The recombinant plasmid is transformed into competent cells, and then the expression of the target protein is induced, and the recombinant protein is obtained after purification;

[0009] After immunizing animals with recombinant proteins, positive hybridoma cell lines that block bovine immune checkpoints were obtained through cell fusion and cloning screening;

[0010] After the positive hybridoma cell lines are injected into animals pretreated with liquid paraffin, the ascites is collected and purified to obtain a bovine-specific stress blocker.

[0011] Preferably, the animal is a mouse.

[0012] Preferably, the primer set used for the synthesis includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.2 in the sequence listing and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.3 in the sequence listing.

[0013] Preferably, the double restriction enzyme cleavage site includes Hind III and EcoR I restriction enzyme cleavage sites.

[0014] Preferably, the plasmid vector is pET-28a.

[0015] Another object of the present invention is to provide a cattle-specific stress blocker prepared by the above preparation method.

[0016] Another object of the present invention is to provide a use of the above-mentioned cattle-specific stress blocker in the preparation of anti-stress therapeutic drugs.

[0017] Preferably, the cattle-specific stress blocker is administered by intramuscular injection.

[0018] The present invention provides a method for preparing a bovine specific stress blocker. The prepared bovine specific stress blocker has strong specificity and high affinity for bovine immune checkpoints, wherein the bovine immune checkpoint blocker is verified by in vivo experiments to be useful for stress treatment with significant treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the bovine immune checkpoint extracellular region gene fragment with signal peptide deleted for PCR amplification;

[0020] Figure 2This is the result of double enzyme digestion identification;

[0021] Figure 3 This is a diagram showing the SDS-PAGE identification results of the induced expressed bovine immune checkpoint recombinant protein;

[0022] Figure 4 This is a graph showing the results of mouse behavior and blood-related factor testing;

[0023] Figure 5 This is a graph showing the results of bovine blood-related factor testing. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] In one embodiment of the present invention, a method for preparing a cattle-specific stress blocker is provided, comprising the following steps:

[0026] The amino acid sequence of the bovine immune checkpoint (published in NCBI, sequence number: NM_001245949.1, specifically: MLWEAWFQVWLFLQLLWAAAVEAPEPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLPRTRQVTWQHVPESGSAAPTPRGPGPRRYTVLRLAPGGLRIGKLPLQPRVQLEEMGLQRGDFSLWLRPARRADAGEYHAAVRFGNRALACRLRLRVGQAAVTASPPGPLWTSSWVVLNCSFSRPDLPASVHWFRGPGRVPVQESPHHHLVGNFLFLPQVSSLDSGTWGCSLTYRDGFNVSITY NLAVLGLEPRAPLTVYAGAGSKVELPCRLPPGVGIQSSLTAMWTPPGEGPDLLVAGDRNNFTLRLEAVGQAQAGTYTCRVHLQGRQLSATVTLAVITVTPKPYGSSGSLRKPFCEVTPASGQERFVWSPLDKRSQRRSPGPW LLTPDARPLSQPWQCHLYQGERLLGTAVYLTELSHPGAQRFGRALGAGRTAHLPLLILGLLFLLLLVTGASSFHLWRRQWRPRRFSALEHGTHPSQASSKTGELEPELEPEPDPEVEPEPEPEPESQPQLQPEQP, such as the sequence table SEQ IDNO.1) was used as a template to synthesize the extracellular region sequence encoding the bovine immune checkpoint with Hind III and EcoR I restriction sites, which was constructed into the pET-28a vector by restriction ligation. The plasmid was extracted and transformed into Trans5α competent cells (full gold), and the recombinant protein with stable expression was obtained by MSX pressure screening. After purification, the bovine immune checkpoint recombinant protein was obtained. The recombinant protein was used to immunize mice, and the titer reached 1:256000 for cell fusion until a positive hybridoma cell line that secreted efficient and stable blocking of the immune checkpoint was screened. The positive hybridoma cell line was injected into BALB / c mice pretreated with liquid paraffin (Sigma), and the ascites was collected and purified to obtain a specific stress blocker with high concentration and good purity.

[0027] The primer set used in the above synthesis includes an upstream primer (F: 5'-CGGAATTCATGCCAGAGCCCGGGGCAGAG-3') with a nucleotide sequence as shown in SEQ ID NO.2 of the sequence listing and a downstream primer (R: 5'-CAAGCTTGTGAGCTGTTCTCCCCGCC-3') with a nucleotide sequence as shown in SEQ ID NO.3 of the sequence listing.

[0028] In another embodiment of the present invention, a method for establishing a mouse stress model is also provided, which is as follows:

[0029] Homology comparison analysis of bovine immune checkpoint proteins and mouse immune checkpoint proteins was performed using NCBI online software, and the results showed that the homology of the two proteins was 84.47%. To verify the effect of specific stress blockers in anti-stress, the test selected 8-week-old male and female BALB / c mice, weighing about 18-22 g, and randomly divided into 3 groups according to baseline test results: 1-normal control group (n=6), 2-stress group (n=6) and 3-stress treatment group (n=6). The normal control group was given a corresponding volume of normal saline. The eyes of the experimental mice in each group were enucleated and blood was collected. The mice in the room temperature stress group were randomly exposed to two stressors every day, including restraint (4 h), water / food deprivation (24 h), cold stress (4℃, 1 h), tail clamping (1 min), cold water swimming (18℃, 5 min), turning on the light during the day (12 h), turning off the light at night (3 h), strobe (12 h), day and night reversal (24 h), cage tilting (24 h), crowding (24 h), and wet cage (24 h). The modeling process lasted for 21 days; the control group was kept in a conventional manner. After modeling, 30 ng / mouse of bovine specific stress blocker was given by intraperitoneal injection for 3 consecutive days, and the mice were left to stand for 0.5 h, centrifuged at 3500 rpm for 10 min, and the serum was separated to detect the levels of serum hormones and cytokines IL-6 and TNF-α in each group of mice.

[0030] In another embodiment of the present invention, a method for establishing a cow stress model is provided, which is as follows: the experimental cows are divided into 1-normal control group, 2-stress group and 3-stress treatment group, each group has 15 cows, the 15 cows in the stress group are placed in a smaller straw bed corral, the cows are transferred to an unfamiliar corral twice a day, and subjected to noise (barking) stress treatment, each lasting 2 hours, and the stress time is 3 weeks. The 15 cows in the control group remain in the original cowshed, and each cow has an activity area of ​​10 m 2The above, sufficient feed and drinking water were given, free access to food. After modeling, 3 g / cow specific stress blocker was given by intramuscular injection for 3 consecutive days, and the normal control group was given a corresponding volume of normal saline. 10 mL of jugular vein blood was collected from each group of experimental cattle, left to stand at room temperature for 0.5 h, centrifuged at 3500 rpm for 10 min, and serum was separated to detect serum hormone levels of cattle in each group; cytokine IL-6, TNF-α and other levels.

[0031] The bovine-specific stress blocker disclosed in the embodiment of the present invention can specifically bind to an immune checkpoint with high affinity, restore the animal's stress state and central nervous immune system homeostasis, and thus alleviate the activation state of the hypothalamus-pituitary-adrenal (HPA) axis.

[0032] The embodiment of the present invention provides a method for preparing a bovine-specific stress blocker, wherein the prepared bovine-specific stress blocker has strong specificity and high affinity for bovine immune checkpoints, wherein the bovine immune checkpoint blocker is verified by in vivo experiments to be useful for stress treatment with significant therapeutic effects.

[0033] Example 1: This example provides a method for preparing a cattle-specific stress blocker, which specifically comprises the following steps:

[0034] S1. Bovine immune checkpoint gene cloning: Using the amino acid sequence of the bovine immune checkpoint as shown in SEQ ID NO.1 in the sequence list as a template, the lentiviral expression vector is pClone007 as an empty vector, and synthetic primers (as shown in SEQ ID NO.2-3 in the sequence list) are designed to amplify the bovine immune checkpoint gene fragment by PCR. The amplified product is subjected to 1% agarose gel electrophoresis and recovered by gel. Figure 1 As shown; the plasmid was extracted using a plasmid mini-extraction kit, and the obtained target plasmid was frozen and stored in a -20°C refrigerator to avoid repeated freezing and thawing.

[0035] S2, construction of recombinant plasmid: the product obtained in step S1 and the pET-28a empty vector were digested and connected overnight, the ligation product was transformed into competent cells to obtain recombinant bacteria, and then plated and cultured and single colonies were picked for expansion and identification; wherein the double enzyme digestion identification result is as follows Figure 2 shown.

[0036] S3. Preparation of recombinant protein: 1.0 mmol / L IPTG was added to the recombinant bacteria obtained in step S2 to induce the expression of the target protein. After purification, dialysis, renaturation and concentration, the purified bovine immune checkpoint recombinant protein with good purity and high concentration was obtained by detection with a BCA protein quantification kit. The SDS-PAGE identification results of the induced expressed bovine immune checkpoint recombinant protein are as follows: Figure 3 shown.

[0037] S4, mouse immunization: the bovine immune checkpoint recombinant protein purified in step S3 was mixed with Freund's adjuvant in equal volumes, and female BALB / c mice were immunized after emulsification. After immunization for 3 times, blood was collected from the tail vein, and the serum was separated and the serum antibody titer was detected by indirect ELISA method;

[0038] S5. Cell fusion: After the mice treated in step S4 were killed by cervical dislocation, spleen cells were separated in a sterile operating table, and spleen cells were fused with SP2 / 0 cells using PEG1450. The cells were then suspended in preheated culture medium containing 1×HAT 1640 and plated in a 96-well cell culture plate, which was cultured in an incubator at 37°C and 5% CO2. The cell supernatant was aspirated after 7-9 days, and the indirect ELISA method was used for detection to screen the positive cell wells.

[0039] S6. Positive cell screening: The hybridoma cells in the positive cell wells of step S4 are diluted by limiting dilution method and then plated, and 3-5 rounds of subcloning are performed continuously until a 100% positive hybridoma cell line is screened, and the hybridoma cells are expanded and frozen in time to finally obtain a hybridoma cell line that stably secretes immune checkpoint-specific blockers;

[0040] S7. Preparation of bovine-specific stress blocker: After the positive hybridoma cell line was injected into BALB / c mice pretreated with liquid paraffin (Sigma), the ascites was collected and purified to obtain the bovine-specific stress blocker, and the affinity was detected by indirect ELISA method.

[0041] Example 2: This example provides a method for determining the affinity of a specific stress blocker, which specifically includes:

[0042] The ELISA plate was coated with 400 ng bovine immune checkpoint recombinant protein per well, and the bovine specific stress blocker was obtained for ELISA detection. 200 µL of blocking solution containing 5% skimmed milk powder in PBS'T was added to each well, and the plate was placed in a 37°C incubator for 1 hour. 100 µL / well of bovine immune checkpoint recombinant protein was incubated at 37°C for 1 hour. The primary antibody was discarded, and the ELISA plate was washed with PBS'T for 3 times. The secondary antibody was added with HRP-labeled goat anti-mouse IgG secondary antibody and incubated at 37°C for 1 hour. The secondary antibody was discarded, and the ELISA plate was washed with PBS'T for 3 times. 100 µL TMB color development solution was added and the plate was color developed at room temperature in the dark for 15 min. The color development reaction was terminated with H2SO4 solution, and the OD was read with an ELISA reader. 450nm The test results are shown in Table 1, where con is the control group.

[0043] Table 1

[0044]

[0045] Example 3: This example provides a method for constructing a stress mouse (CUMS) model, as follows:

[0046] The animals used in the experiment were 8-week-old male and female BALB / c mice, weighing about 18-22 g. After one week of adaptive feeding, the baseline body weight and sugar water preference of the mice were measured. According to the results of the baseline test, they were randomly divided into 3 groups: 1-normal control group (Con, n=6), 2-stress group (CUMS, n=6) and 3-stress treatment group (CUMS+Blockader, n=6). The mice in the stress group were randomly exposed to two stressors every day, including restraint (4 h), water / food deprivation (24 h), cold stress (4℃, 1 h), tail clamp (1 min), cold water swimming (18℃, 5 min), turning on the lights during the day (12 h), turning off the lights at night (3 h), strobe light (12 h), day and night reversal (24 h), cage tilt (24 h), crowding (24 h), and wet cage (24 h). The modeling process lasted for 21 days; the control group was fed conventionally. After modeling, 30 ng / bovine specific stress blocker was intraperitoneally injected for 3 consecutive days, and the normal control group was given a corresponding volume of normal saline.

[0047] Example 4: This example provides a method for detecting mouse behavior, as follows:

[0048] The sugar water preference test is a classic experiment for detecting anhedonia symptoms in depression. It is mainly divided into two stages: sugar water adaptation stage and sugar water test stage. First, two drinking bottles containing 1% sucrose solution and clean water were placed on both sides of the BALB / c mouse cage for 48 hours. To prevent the animals from forming a position preference, the position of the bottle was changed after 24 hours. After the adaptation, the mice were deprived of water and food for 6 hours, and the sugar water intake of BALB / c mice within 1 hour was measured (sugar water preference value = 1 hour sugar water consumption / (1 hour total consumption of clean water + sugar water) × 100%).

[0049] In the tail suspension test, tape was attached to the tip of the mouse's tail 2 cm away, and the tape was fixed to the tail suspension instrument, so that the mouse was suspended upside down with its head about 20 cm from the ground. The behavior of the mouse was recorded with a camera for 5 minutes. The immobility time of the mouse from the 2nd minute to the 5th minute was recorded by an experimenter who was unaware of the experiment. The time when the mouse stopped struggling and its body was suspended vertically and motionless was defined as the immobility time.

[0050] The forced swimming test is mainly used to evaluate the desperation behavior of rats. At the beginning of the experiment, BALB / c mice were placed in a transparent acrylic barrel (water depth 30 cm, diameter 20 cm, water temperature 23±1) for 5 minutes. Clean water was replaced after each test. The immobility time of BALB / c mice within 5 minutes was analyzed. The results of the above test are shown in Figure 2. Figure 4 shown.

[0051] Example 5: This example provides a method for detecting related factors in bovine blood, as follows:

[0052] The experimental cows were divided into 1-normal control group (Con), 2-stress group (CUMS) and 3-stress treatment group (CUMS+Blockader), with 15 cows in each group. The 15 cows in the stress group were placed in a smaller straw bed corral. The cows were transferred to an unfamiliar corral twice a day and subjected to noise (barking) stress treatment for 2 hours each time. The stress time was 3 weeks. The 15 cows in the control group stayed in the original cowshed, and each cow had an activity area of ​​10 m 2 After the model was established, 3 g / cow of specific stress blocker was given by intramuscular injection for 3 consecutive days, and the normal control group was given a corresponding volume of saline. After 3 days of treatment, 10 mL of jugular venous blood was collected from each group of experimental cattle, left to stand at room temperature for 0.5 h, centrifuged at 3500 rpm for 10 min, and serum was separated. The ELISA method was used to detect the serum hormone levels, cytokine IL-6, TNF-α and other levels of cattle in each group in strict accordance with the instructions of the detection kit. The results are shown in Figure 5 shown.

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a cattle-specific stress blocker, characterized in that: The following steps are involved: Using the amino acid sequence of the bovine immune checkpoint as a template, synthesizing the extracellular region sequence encoding the bovine immune checkpoint with double restriction sites; the amino acid sequence of the bovine immune checkpoint is shown in the sequence table SEQ ID NO.1; The sequence encoding the extracellular region of the bovine immune checkpoint is constructed into a plasmid vector by restriction digestion and ligation to obtain a recombinant plasmid; The recombinant plasmid is transformed into competent cells, and then the expression of the target protein is induced, and the recombinant protein is obtained after purification; After immunizing animals with recombinant proteins, positive hybridoma cell lines that block bovine immune checkpoints were obtained through cell fusion and cloning screening; After the positive hybridoma cell lines were injected into animals pretreated with liquid paraffin, the ascites was collected and purified to obtain a bovine-specific stress blocker.

2. The method for preparing the cattle-specific stress blocker according to claim 1, characterized in that: The animal is a mouse.

3. The method for preparing the cattle-specific stress blocker according to claim 1, characterized in that: The primer set used for the synthesis includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.2 in the sequence listing and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.3 in the sequence listing.

4. The method for preparing the cattle-specific stress blocker according to claim 1, characterized in that: The double restriction enzyme cutting sites include Hind III and EcoR I restriction enzyme cutting sites.

5. The method for preparing the cattle-specific stress blocker according to claim 1, characterized in that: The plasmid vector is pET-28a.

6. A cattle-specific stress blocker prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the cattle-specific stress blocker according to claim 6 in the preparation of anti-stress therapeutic drugs.

8. The use according to claim 7, characterized in that: The cattle-specific stress blocker is administered by intramuscular injection.

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