Polypeptide for improving animal stress response symptoms and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to polypeptide for improving animal stress reaction symptoms and a preparation method of the polypeptide, new polypeptide is prepared, and it is verified that the polypeptide can improve night sleep and daytime micro sleep quality of cats after transportation stress to a certain extent and relieve great reduction of activity caused by transportation stress; the CSS score of the experimental group within 1-3 days after transportation is obviously lower than that of the blank group, the hiding behavior and the escape intention of the experimental group are obviously improved, and the trend of static behavior duration and pacing time can be reduced; the polypeptide can relieve the influence of an unfamiliar environment on cats; the polypeptide can reduce the level of HPA axis hormone so as to play an anti-stress role, improve the activity of GSH-Px, SOD and CAT of cats, reduce the MDA level, reduce the decrease of T-AOC level and SOD activity of cats after transport stress, and reduce the MDA level in an unfamiliar environment, which indicates that the polypeptide can reduce the oxidative stress level of cat bodies in the process of transport stress.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide for improving the symptoms of stress reaction in animals and a preparation method thereof. BACKGROUND

[0002] Stress reaction in cats, a common companion animal, is a common concern in clinical veterinarians and pet breeders. The main stressors include environmental changes (such as moving, new members joining), medical procedures (such as vaccination, surgery), transportation (long distance or air transportation), and social conflicts. When cats are exposed to stressors, the hypothalamic-pituitary-adrenal (HPA) axis is activated, and the secretion of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and cortisol (CORT) increases in turn, triggering a series of physiological and behavioral changes: physiologically, it shows rapid heart rate, rapid breathing, intestinal peristalsis disorder (diarrhea or constipation), and decreased immunity (easy to cause secondary infection); behaviorally, it shows hiding, increased aggressiveness, decreased appetite, excessive or reduced grooming, etc. Long-term chronic stress can also induce or aggravate inflammatory bowel disease (IBD), feline lower urinary tract syndrome (FLUTD), hyperthyroidism, and other diseases, which seriously affect the quality of life and lifespan of cats.

[0003] Currently, the main intervention methods for cat stress include three categories: (1) anti-anxiety drugs (such as benzodiazepines, opioid drugs): although they can quickly relieve anxiety, they have side effects such as drug dependence, excessive sedation, and respiratory depression, and have no direct improvement effect on gastrointestinal stress (such as diarrhea); (2) natural supplements (such as cat facial pheromones (F3 / F4 type felon), probiotics (such as lactobacillus)): felon regulates emotions through olfaction, but has a slow onset (requires continuous use for 3-7 days), and has limited effect on severe stress; probiotics can regulate intestinal flora, but have low colonization rate and are greatly affected by the host's intestinal environment; (3) environmental management (such as providing hiding spaces, reducing stimulation): although it is a basic measure, it cannot directly intervene in the neuroendocrine pathway of stress, and it is difficult to meet the rapid relief needs of acute stress (such as when going to the hospital).

[0004] Therefore, it is of great significance to develop a safe, efficient, and targeted polypeptide preparation for cat stress. Polypeptides, due to their small molecular weight, easy absorption, and few side effects, have become potential stress regulation candidate molecules. However, existing researches mainly focus on stress regulation polypeptides for dogs or rodents, and specific polypeptides for cats and their preparation methods have not been reported. The neuroendocrine system of cats (such as the sensitivity of HPA axis receptors) and the composition of intestinal flora are significantly different from those of dogs / rodents, and direct transplantation of polypeptides from other species may not be effective due to species differences. Therefore, it is urgent to screen or design specific anti-stress polypeptides according to the physiological characteristics of cats and establish an efficient and stable preparation method. SUMMARY

[0005] The first object of the present application is to provide a polypeptide for improving the stress reaction symptoms of cats, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0006] The second object of the present application is to provide a nucleotide sequence encoding the above polypeptide.

[0007] The third object of the present application is to provide a recombinant expression vector containing the above nucleotide sequence, the vector being pPICZαA.

[0008] The fourth object of the present application is to provide a host bacterium containing the above recombinant expression vector, the host bacterium being Pichia pastoris GS115 strain.

[0009] The fifth object of the present application is to provide a preparation method of the above polypeptide, comprising the following steps: (1) constructing a recombinant expression vector: inserting the above nucleotide sequence into the downstream of the α factor secretion signal of the pPICZαA vector to obtain a recombinant plasmid; (2) transforming a host bacterium: transforming the recombinant plasmid into Pichia pastoris GS115 strain, and obtaining a positive clone through Zeocin screening; (3) fermentation culture: inoculating the positive clone into BMGY culture medium, culturing at 30℃ and 200rpm until OD 600 =6.0, centrifuging to collect the bacterial cells and transferring to BMMY culture medium, and inducing expression for 48-72 hours (methanol concentration 0.5-1.0%); (4) purification: after centrifugation and 0.45μm filter membrane filtration of the fermentation broth, purifying by using a Ni-NTA affinity chromatography column, collecting the target peak, and desalting by HPLC to obtain the polypeptide.

[0010] In some embodiments, the induction temperature in step (3) is 25℃, and the polypeptide expression amount is higher.

[0011] The sixth object of the present application is to provide the use of the above polypeptide in the preparation of a product for improving the stress reaction symptoms of cats, the stress reaction symptoms including elevated serum cortisol level, abnormal behavior (prolonged hiding time, reduced appetite) or increased secretion of inflammatory factors.

[0012] The seventh object of the present application is to provide a composition for improving the stress reaction symptoms of cats, comprising the above polypeptide and a pharmaceutically acceptable carrier; the carrier being starch, microcrystalline cellulose or starch paste.

[0013] In some embodiments, the mass percentage of the polypeptide is 0.1%-1.0%.

[0014] The eighth object of the present application provides the use of the polypeptide in the preparation of an anti-stress feed additive or oral liquid for pets.

[0015] Compared with the prior art, the present application has at least the following beneficial effects: The present application prepares a new polypeptide, and verifies that the polypeptide can improve the quality of night sleep and daytime micro-sleep of cats after transportation stress to a certain extent, and can relieve the significant reduction in activity caused by transportation stress to a certain extent; the polypeptide has less effect on the behavior change of cats within 6h after transportation; the CSS score of the experimental group within 1-3 days after transportation is significantly lower than that of the blank group, and the hiding behavior and escape intention of the experimental group are significantly improved, and the polypeptide can reduce the trend of the duration of static behavior and pacing time; the polypeptide can relieve the influence of the strange environment on cats to a certain extent; the polypeptide can reduce the level of HPA axis hormones (especially the level of COR and CRH), thereby playing an anti-stress role. The polypeptide can increase the activity of GSH-Px, SOD and CAT of cats to a certain extent, reduce the level of MDA, and reduce the decrease in the level of T-AOC and the activity of SOD of cats after transportation stress, and reduce the level of MDA in a strange environment, indicating that the polypeptide can reduce the oxidative stress level of the body of cats during transportation stress. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 , night sleep time change. Different letters represent significant differences (P<0.05).

[0017] Figure 2 , daytime micro-sleep total time change. Different letters represent significant differences (P<0.05), and the symbol (#) represents a significant trend (P<0.10).

[0018] Figure 3 , daily total activity change. Different letters represent significant differences (P<0.05).

[0019] Figure 4 , activity change during transportation. Different letters represent significant differences (P<0.05).

[0020] Figure 5 , activity change within 1h after transportation. Different letters represent significant differences (P<0.05).

[0021] Figure 6 , activity change within 4h after transportation. Different letters represent significant differences (P<0.05).

[0022] Figure 7, Activity amount change within 1-3 h before and after transport. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0023] Figure 8 , Duration of relaxation / hiding behavior within 1 h after transport. Different letters indicate significant differences (P < 0.05).

[0024] Figure 9 , Duration of relaxation / hiding behavior within 6 h after transport. Different letters indicate significant differences (P < 0.05).

[0025] Figure 10 , Water intake within 1 h and 6 h after transport. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0026] Figure 11 , Food intake within 1 h and 4 h after transport. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0027] Figure 12 , Behavioral stress score, CSS, during the recovery period. * indicates a significant difference (P < 0.05). Behavioral stress (CSS) score: 1-2 points: relaxation, 3 points: mild stress, 4 points: moderate stress, 5-6 points: fear state, 7 points: extreme stress.

[0028] Figure 13 , CSS score within three days after transport. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10). Behavioral stress (CSS) score: 1-2 points: relaxation, 3 points: mild stress, 4 points: moderate stress, 5-6 points: fear state, 7 points: extreme stress.

[0029] Figure 14 , Disease behavior score SB; different letters indicate significant differences (P < 0.05). Note: Disease behavior (SB) score: 1 point when one of the five conditions exists (reduced food intake, cat does not perform excretion behavior, cat vomits or has diarrhea, excretion outside the litter box, destroys the paper box). Cats with both increased litter box contamination (excretion outside the litter box) and destruction of the paper box have a SB score of 2.

[0030] Figure 15 , Duration of relaxation / hiding behavior within 1 h after transport. Different letters indicate significant differences (P < 0.05).

[0031] Figure 16, The duration of escape intention related to stress and fear in the open field test (OFT). Different letters indicate significant differences (P < 0.05).

[0032] Figure 17 , The duration of hiding behavior related to stress and fear in the open field test (OFT). Different letters indicate significant differences (P < 0.05).

[0033] Figure 18 , The duration of exploration behavior related to stress and fear in the open field test (OFT). Different letters indicate significant differences (P < 0.05).

[0034] Figure 19 , The duration of pacing behavior related to stress and fear in the open field test (OFT). Different letters indicate significant differences (P < 0.05).

[0035] Figure 20 , The number of vocalizations related to stress and fear in the open field test (OFT). Different letters indicate significant differences (P < 0.05).

[0036] Figure 21 , White blood cells in cat routine blood, WBC. The red line in the figure is the normal range of each index. Different letters indicate significant differences (P < 0.05).

[0037] Figure 22 , Neutrophils in cat routine blood, NEU. The red line in the figure is the normal range of each index. Different letters indicate significant differences (P < 0.05).

[0038] Figure 23 , Lymphocytes in cat routine blood, LYM. The red line in the figure is the normal range of each index. Different letters indicate significant differences (P < 0.05).

[0039] Figure 24 , Red blood cells in cat routine blood, RBC. The red line in the figure is the normal range of each index. Different letters indicate significant differences (P < 0.05).

[0040] Figure 25 , Hemoglobin concentration in cat routine blood, HGB. The red line in the figure is the normal range of each index. Different letters indicate significant differences (P < 0.05).

[0041] Figure 26 , Brain-derived neurotrophic factor in cat serum, BDNF. Different letters indicate significant differences (P < 0.05).

[0042] Figure 27 , Corticotropin-releasing hormone in cat serum, CRH. Different letters indicate significant differences (P < 0.05).

[0043] Figure 28 Adrenocorticotropic hormone, ACTH, in cat serum. Different letters indicate significant differences (P < 0.05).

[0044] Figure 29 Cortisol, COR, in cat serum. Different letters indicate significant differences (P < 0.05).

[0045] Figure 30 Serum amyloid A, SAA, in cat serum. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0046] Figure 31 Apolipoprotein A1 in cat serum. Different letters indicate significant differences (P < 0.05).

[0047] Figure 32 Total antioxidant capacity, T-AOC, in cat serum. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0048] Figure 33 Malondialdehyde, MDA, in cat serum. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0049] Figure 34 Glutathione peroxidase, GSH-Px, in cat serum. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0050] Figure 35 Superoxide dismutase, SOD, in cat serum. Different letters indicate significant differences (P < 0.05), and the symbol (#) indicates a significant trend (P < 0.10).

[0051] Figure 36 Catalase, CAT, in cat serum. Different letters indicate significant differences (P < 0.05).

[0052] Figure 37, Venn diagram, DEG results; Note: a is Venn diagram; b is DEG results; DEG Counts: number of DEGs; up-regulated: number of up-regulated genes; down-regulated: number of down-regulated genes. A1 vs C1: blood sampling after diet change (T1); A2 vs C2: blood sampling before transport (T2); A3 vs C3: blood sampling after transport (T3); A4 vs C4: blood sampling at the end of recovery period (T4).

[0053] Figure 38 , GO enrichment analysis diagram of DEGs of blood sampling after diet change (T1); Note: A1 vs C1: blood sampling after diet change (T1).

[0054] Figure 39 , GO enrichment analysis diagram of DEGs of blood sampling before transport (T2); Note: A2 vs C2: blood sampling before transport (T2).

[0055] Figure 40 , GO enrichment analysis diagram of DEGs of blood sampling after transport (T3); Note: A3 vs C3: blood sampling after transport (T3).

[0056] Figure 41 , GO enrichment analysis diagram of DEGs of blood sampling at the end of recovery period (T4); Note: A4 vs C4: blood sampling at the end of recovery period (T4).

[0057] Figure 42 , KEGG pathway enrichment analysis results of the experimental group and the blank group at the T1 (blood sampling after diet change) time point.

[0058] Figure 43 , KEGG pathway enrichment analysis results of the experimental group and the blank group at the T2 (blood sampling before transport) time point.

[0059] Figure 44 , KEGG pathway enrichment analysis results of the experimental group and the blank group at the T3 (blood sampling after transport) time point.

[0060] Figure 45 , KEGG pathway enrichment analysis results of the experimental group and the blank group at the T4 (blood sampling at the end of recovery period) time point. DETAILED DESCRIPTION

[0061] In order to make the technical problems, technical solutions and advantages to be solved by the present application more clear, the following will be described in detail in combination with the drawings and specific embodiments.

[0062] Embodiment 1: Source and preparation of polypeptide (0) Polypeptide sequence design and verification Design rationale Based on the molecular mechanism of cat stress response, the core functional domain is screened by the following steps: Target analysis: Screening the ligand binding domain of cat HPA axis key receptors (CRHR1, GR). Binding site analysis shows that Trp (tryptophan) anchors the CRHR1 transmembrane region through hydrophobic interaction; Tyr (tyrosine) forms a hydrogen bond network with Ser753 of GR; Pro (proline) maintains the β-turn conformation, fitting the CRHR1 active pocket.

[0063] By AlphaFold2 simulation of cat CRHR1 structure (PDB: 7T9A homology modeling), the key residues of the binding cavity are determined.

[0064] Functional domain design: Based on the above target characteristics, a ten-peptide framework containing the Trp-Tyr-Pro core motif is designed; Glu (glutamic acid) is added at the N-terminus to enhance water solubility, and Arg (arginine) is added at the C-terminus to enhance cell membrane penetration; The middle sequence (Leu-Ser-Ser-Ala-Gly) is optimized as a flexible linker easily absorbed by the cat intestine, avoiding protease cleavage sites (predicted by PeptideCutter).

[0065] Sequence verification Molecular docking verification: Using AutoDock Vina to dock the designed sequence with cat CRHR1: binding energy reaches -9.2 kcal / mol (lower than the natural ligand CRH of -8.5 kcal / mol); Key interactions: Trp 77 ³ hydrophobic interaction, Tyr 657 π-π stacking, Pro 650 hydrogen bond. Compared with mutants (such as Leu³→Ile): binding energy decreases to -8.1 kcal / mol, confirming that Leu³ is indispensable for structural stability.

[0066] In vitro activity verification: Synthesize the polypeptide (solid-phase synthesis method, purity >95%), test its inhibition rate on cat adrenal cortical cell ACTH secretion: 50μM concentration, inhibition rate reaches 68% (vs. blank group P<0.01); control sequence (lack of any one of Trp / Tyr / Pro residues) inhibition rate <20%.

[0067] Final sequence: H-Glu-Trp-Leu-Ser-Ser-Pro-Ala-Gly-Tyr-Arg-OH (SEQ ID NO: 1), named "CatStressIn-10".

[0068] (2) Recombinant expression vector construction The coding sequence of SEQ ID NO: 1 was optimized according to the cat codon bias (optimized GC content 45%, avoiding continuous A / T repeats), and a DNA fragment was synthesized (Shanghai Genechem Co., Ltd.). The optimized coding sequence was inserted downstream of the alpha factor signal peptide of the pPICZαA vector (EcoR I / Xba I enzyme digestion site) to construct the recombinant plasmid pPICZαA-CatStressIn-10.

[0069] (3) Host strain transformation and screening The recombinant plasmid was linearized with Sac I enzyme and electroporated into Pichia pastoris GS115 strain (electroporation conditions: 1.5 kV, 5 ms), and plated on YPD S plates containing 100 μg / mL Zeocin and incubated at 30°C for 48 hours. Single colonies were inoculated in YPGal medium containing different concentrations of Zeocin (250, 500, 750 μg / mL) to screen for high copy integration strains (500 μg / mL Zeocin-resistant strains were positive).

[0070] (4) Fermentation and purification The positive strain was inoculated in 50 mL of BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% YNB, 4 x 10 -5 % biotin, 1% glycerol) and incubated at 30°C, 200 rpm until the OD 600 = 6.0. The bacterial cells were collected by centrifugation (8000 g x 5 min) and resuspended in 1 L of BMMY medium (BMGY formula without glycerol, with the addition of 0.5% methanol), and induced at 25°C, 200 rpm for 72 hours (0.5% methanol was added every 24 hours to maintain induction). After fermentation, the supernatant was collected by centrifugation at 4°C, 8000 g for 20 minutes, filtered with a 0.45 μm filter, and loaded onto a Ni-NTA affinity chromatography column (GE Healthcare). Elution was performed with a gradient of Tris-HCl buffer containing 20-500 mM imidazole, and the eluate corresponding to the peak of absorbance at 280 nm was collected (consistent with the theoretical value). The purity was detected by HPLC (C18 chromatographic column, acetonitrile-water gradient elution, retention time 12.3 minutes, purity > 95%), and the sequence was verified by N-terminal sequencing (Edman degradation method) to be consistent with the design.

[0071] Example 2 The polypeptide effect evaluation experiment was conducted in the experimental animal center of South China Agricultural University, and the test period was 22 days. The experiment was divided into four stages, namely, the transition period of changing food (7 days), the pre-feeding period (7 days), the transportation period (1 day) and the recovery period (7 days). In this experiment, 12 healthy adult British short hair cats were selected as experimental animals, and they were randomly divided into 2 groups according to gender and weight, namely, the blank group and the polypeptide group. All cats were individually housed in cat cages (108 cm*70 cm*76 cm) in the same constant temperature room, and could freely eat and drink water. After the end of the food change period, the cats in the experimental group were additionally fed with cat strips mixed with 0.2% (by weight) polypeptide, and the cats in the blank group were fed with blank cat strips until the end of the experiment. Before the experiment, all experimental cats had undergone necessary immunization and deworming treatment. The cats were excreted once a day in the morning, and the cat litter was changed once a week. The cat house was cleaned and disinfected every day to keep it clean.

[0072] At 8:30 am every day, each cat was weighed and fed with 60-90 g of cat food (free feeding). The feeding amount and the remaining amount of each cat were accurately recorded every day, and the mental state of the experimental cats was checked and the feces score was made. The cats were weighed on an empty stomach on the first day of changing food, at the end of the transportation period and the recovery period. Blood was collected before the pre-feeding period, 1 hour before transportation, 1 hour after transportation and at the end of the recovery period for the detection of blood physiological and biochemical indicators. Fresh feces of the cats were collected 1 day before transportation, 1 day after transportation and at the end of the recovery period for fecal metabolomics analysis.

[0073] In addition, 10 experimental cats (5 cats in each group) were randomly selected to wear Actiwatch mini®actigraphs to record the changes in sleep and activity of the experimental cats 3 days before and after transportation and on the day of transportation. The behavior changes of the experimental cats were recorded and observed 1 hour and 6 hours after transportation. The open field test (OFT) was conducted on the first day after transportation and on the last day of the recovery period. The experimental cats were placed in a 1.5 m*2.5 m open field, and their behavior was recorded for 3 minutes using a video camera. In addition, the researchers scored the behavior stress of the cats (CSS) at 9:30 am every day.

[0074] Example 3, Effect of Polypeptide on Body Weight, Food Intake and Fecal Score The body weight, food intake and fecal score were measured. The fecal score (FS) was 1≤FS<2 for constipation, 2≤FS≤3 for normal, 3<FS<4 for soft stool, and 4≤FS≤5 for diarrhea.

[0075] During the feeding period, the food intake of both groups decreased significantly during the transportation period (P<0.05), but the food intake of the experimental group was still significantly higher than that of the blank group (P<0.05), and the food intake of both groups returned to the level before transportation after the recovery period. The body weight of the experimental cats in both groups was very stable during the whole experiment, and the fecal condition was normal, with no significant changes.

[0076] Example 4, Effect of polypeptide on sleep quality and activity of cats The effect of polypeptide was evaluated by measuring the night sleep time, total daytime microsleep time, total daily activity, activity during transportation, activity within 1 h after transportation, activity within 4 h after transportation, and activity within 1-3 h before and after transportation. The results are shown in Table 2, wherein Figures 1-7 is the sleep time of cats in the two groups. The night sleep time of the experimental group was significantly higher than that of the blank group after transportation (P < 0.05, Figures 1-2 ). The daytime sleep time of both groups significantly decreased after transportation (P < 0.05, Figure 1 ), but the sleep time of the experimental group had a trend of being higher than that of the blank group on the day of transportation (P < 0.10). After feeding in a strange environment for one week, the sleep time of both groups returned to the state before transportation, but the daytime microsleep time of the experimental group was still significantly higher than that of the blank group (P < 0.05). Figure 2

[0077] Figures 3-6 is the activity difference of cats in the two groups. As can be seen from Table 3, Figure 3 the activity of both groups significantly increased on the day of transportation (P < 0.05), and the activity of the blank group was significantly higher than that of the experimental group (P < 0.05). After feeding for one week, the activity of both groups returned to the state before transportation. As can be seen from Table 4, Figure 4 the activity of cats in both groups significantly increased during transportation (P < 0.05), and the activity of the blank group was significantly higher than that of the experimental group (P < 0.05). Figure 5 As can be seen from Table 5, Figure 6 the activity of the blank group within 1 h after transportation significantly decreased compared with the same period before transportation (P < 0.05), while in the experimental group, the decrease was not significant. The activity of both groups within 4 h after transportation was significantly lower than that before transportation (P < 0.05, ), and there was no significant difference between the two groups.

[0078] Figure 7 is the comparison of activity within 1-3 h before and after transportation between the two groups. It can be directly seen that compared with before transportation, the activity of the blank group within three hours after transportation significantly decreased (P < 0.05), and the activity of the experimental group within 1 h after transportation significantly decreased (P < 0.05). Within 2 h and 3 h after transportation, the activity of the experimental group was significantly higher than that of the blank group (P < 0.05), and the activity of the experimental group within 1 h after transportation also had a trend of being significantly higher than that of the blank group (P < 0.10).

[0079] Example 5, Activity state of cats after transportation stress The effects of peptides were evaluated by measuring the duration of relaxation / hiding behavior within 1 hour after transport, the duration of relaxation / hiding behavior within 6 hours after transport, the number of times water was consumed within 1 hour and 6 hours after transport, and the amount of food consumed within 1 hour and 4 hours after transport. The results are as follows Figures 8-11 As shown, from Figures 8-9 The results showed no significant differences in the duration of relaxation and hiding behavior between the two groups within 1 hour and 6 hours after transport. The frequency of water intake increased significantly in both groups within 6 hours after transport, with the experimental group showing a significantly higher frequency than 1 hour after transport (P<0.05), but there was no significant difference between the two groups (P =0.136 >0.10). Figure 10 The experimental group showed a significant increase in feed intake 4 hours after transport (P<0.05). Figure 11 The control group showed an increasing trend (P<0.10), but there was no significant difference between the two groups.

[0080] Example 6: The effects of transportation and unfamiliar environments on stress and disease behavior in cats The effects of peptides were evaluated by measuring the recovery period behavioral stress score, CSS score, CSS score within three days after transport, and disease behavior score. Behavioral Stress (CSS) score: 1-2 points: Relaxed, 3 points: Mild stress, 4 points: Moderate stress, 5-6 points: Fearful state, 7 points: Extreme stress.

[0081] Disease Behavior (SB) Score: 1 point is awarded for any one of the five conditions present (reduced food intake, no defecation behavior, vomiting or diarrhea, defecation outside the litter box, or damage to the cardboard box). A cat with both increased cage contamination (defecation outside the litter box) and damage to the cardboard box has an SB score of 2 points.

[0082] The results are as follows Figures 12-14 As shown, the behavioral stress score (CSS) over the 7-day period indicates that the stress level of the experimental group cats was significantly better than that of the control group in the first phase (P<0.05). As time went on, the stress level of both groups of experimental cats gradually improved, with the experimental group having a lower stress score. Figure 12 ).from Figure 13 The results show that on the first day after transportation, the CSS score of the experimental group was significantly lower than that of the control group (P<0.05), and on the second day, the experimental group still showed a trend of being significantly lower than the control group (P<0.05). Figure 14 As can be seen, there was no significant difference in the SB scores of the two groups of experimental cats in the first stage after the transportation stress, and the SB scores of both groups decreased significantly after being raised in an unfamiliar environment for a week (P<0.05).

[0083] Example 7: The effects of transportation stress and unfamiliar environments on stress and fear-related behaviors in cats. The effects of peptides were evaluated by measuring duration of stillness, duration of escape intent, duration of hiding, duration of exploratory behavior, duration of pacing, and number of vocalizations. The results are as follows Figures 15-20 As shown, in the open field test (OFT), there were no statistically significant differences between the two groups of cats in the duration of stationary behavior, exploratory behavior, and number of vocalizations throughout the experiment. Figure 15 , Figure 18 , Figure 20 ).from Figure 16 The results show that the experimental group had a significantly lower escape intent in both OFT tests compared to the control group (P<0.05). Regarding hiding time, the control group showed a significant increase in the second test compared to the first, but the difference was not statistically significant, while the experimental group showed a significant decrease, and in the second test, it was significantly lower than the control group (P<0.05). Figure 17 After a one-week pre-feeding period, the pacing performance of the experimental group in the first open field test was significantly lower than that of the control group (P < 0.05). Figure 19 ).

[0084] Example 8: Effects of polypeptides on feline blood routine tests The results are as follows Figures 21-25 As shown, at the end of the recovery period (T4), the WBC count in the blank group was significantly higher than that after transport, while the increase in the experimental group was not significant, indicating that the peptide can alleviate the stress of cats to unfamiliar environments to some extent. Figure 21 ).from Figure 22 It can be seen that NEUT in both groups of experimental cats increased after transportation (T3). The experimental group showed a larger increase compared to before transportation (T2), but the difference was not statistically significant. At T4, NEUT in the control group continued to rise, while the NEUT in the experimental group returned to the level after the food change (T1). Figure 23 It can be seen that the LYM levels of both groups of experimental cats decreased significantly at T3 (P<0.05) and returned to pre-transport levels after one week of feeding. Figures 24-25 As can be seen, RBC and HGB showed the same upward and downward trends. At T2, the RBC level in the blank group exceeded the normal range. At T3, both RBC and HGB in the experimental cats decreased. There was a significant difference in RBC and HGB before and after transport in the blank group. There was a significant difference in HGB at T3 and T4 in the experimental group (P<0.05).

[0085] Example 9: Effects of peptides on BDNF and HPA axis hormones in cat serum The levels of BDNF and HPA axis hormones in feline serum were detected using an ELISA method. The results are as follows: Figures 26-29The BDNF levels of both groups were significantly decreased at T3 (P<0.05), and the BDNF level of the experimental group was slightly higher than that of the blank group Figure 26 CRH increased over time in both groups, and the CRH level of the blank group was significantly higher than that at T1 at T2, both levels were significantly increased at T3 (P<0.05), and both were significantly decreased at T4 (P<0.05), and the CRH level of the experimental group was significantly lower than that of the blank group at T4 (P<0.05) Figure 27 Compared with T1, the ACTH levels of both groups were significantly higher at T2 (P<0.05), and the ACTH level at T3 was also significantly higher than that at T2 (P<0.05), and the ACTH levels of both groups were significantly decreased at T4 (P<0.05, Figure 28 There was no significant difference between the two groups at the same period. The COR level of the experimental group was lower than that of the blank group at T3, and the COR level of the blank group was significantly increased at T4 (P<0.05), and was significantly higher than that of the experimental group (P<0.05, Figure 29

[0086] Example 10, Effect of Polypeptide on Other Serum Hormones and Antioxidant Indexes in Cat Serum The other serum hormones and antioxidant indexes in cat serum were detected by ELISA method and biochemical colorimetry, and the results are shown in Figures 30-36 The SAA level of the experimental group was significantly decreased at T2 (P<0.05), and the SAA levels of both groups were significantly increased after transportation, and the SAA level of the experimental group was significantly lower than that of the blank group (P<0.05), and the SAA levels of both groups were slightly increased at T4, but the SAA level of the experimental group was still significantly lower than that of the blank group (P<0.05, Figure 30 As can be seen from Figure 31 The Apo-A1 level of the experimental group was significantly higher than that at T1 and the blank group at T2 (P<0.05). The Apo-A1 levels of both groups were significantly decreased after transportation (P<0.05), but the decrease of the blank group was more significant (P<0.05).

[0087] The T-AOC level of the blank group was significantly lower than that at T2 at T3 (P<0.01), and increased to a level similar to that at T2 at T4 (P<0.05); while the T-AOC level of the experimental group only showed a decreasing trend at T3 (P<0.10), and returned to the level before transportation at T4 Figure 32 The MDA level of the blank group was significantly increased at T2 (P<0.01), and maintained at a high level, and the MDA level of the experimental group was significantly higher than that of the blank group at T1, and showed a significant increasing trend at T2 (P<0.10), and the MDA level of the experimental group was decreased at T4, and was significantly lower than that of the blank group (P<0.05, Figure 33 As can be seen from Figure 34 ​The GSH-Px levels of both groups were steadily increased, and the level of the experimental group was significantly increased at T3 (P<0.05), while the levels of both groups were significantly decreased at T4 (P<0.05). The SOD and CAT levels of both groups were significantly increased at T2, and except for the SOD level of the experimental group which did not change significantly, the rest were significantly decreased at T3 (P<0.05), and the CAT levels of both groups were significantly increased at T4 (P<0.05, Figures 35-36 ).

[0088] Example 11, Effect of polypeptides on the transcriptome changes of cat blood under transport stress and unfamiliar situation stress The transcriptome was outsourced to a third-party company; the gene expression analysis results are shown in Figure 37 , in which through the comparison of each time point of the experimental group and the blank group, the transcriptome analysis revealed 2315 differentially expressed genes (DEG), of which 534, 341, 672, and 768 genes were differentially expressed in A1 vs C1 (T1), A2 vs C2 (T2), A3 vs C3 (T3), and A4 vs C4 (T4), respectively Figure 37 b). Venn diagram analysis revealed that there were 27 differentially co-expressed genes at all time points compared with the control group Figure 37 a). And from Figure 37 b, it can be seen that compared with the gene expression of the blank group at different time points, the expression of 1159 genes in the experimental group was up-regulated, and the expression of 1156 genes was down-regulated.

[0089] The results of gene ontology (GO) enrichment analysis are shown in Figures 38-41 , in which through GO enrichment analysis, the biological functions of DEG were determined. At T1, the biological processes (BP) related to immune response process, viral response, defense against foreign organisms, etc. in the experimental group were significantly up-regulated (P<0.01), and the biological processes related to biological adhesion, triglyceride transport, virus entry into host cells, cell differentiation regulation, etc. were significantly down-regulated (P<0.01, Figure 38 ). After the end of the pre-feeding period, the biological process terms significantly up-regulated in the experimental group were forward trans-synaptic signaling, leukotriene metabolic process, eicosanoid metabolic process, protein activation cascade, etc., and the significantly down-regulated ones were viral process, viral life cycle, B cell homeostatic proliferation, DNA recombination, acylglycerol transport, etc. (P<0.01, Figure 39 ). After transport, the biological processes related to complement activation (classical / alternative pathway), inflammatory response, muscle adaptation, regulation of hippo signaling, etc. in the experimental group were significantly up-regulated (P<0.01), while the necrotic apoptosis signaling pathway, vascular development, multi-biological processes, symbiotic processes, viral particle assembly, etc. were significantly down-regulated (P<0.01, Figure 40). GO enrichment analysis after recovery showed that biological processes related to cell signaling, cell response to external stimuli, fatty acid derivative metabolic processes, etc. were significantly up-regulated in the experimental group (P<0.01), and biological processes related to multiple biological processes, virus-related life processes (assembly, life cycle, latency, invasion of host cells), DNA metabolic processes, etc. were significantly down-regulated (P<0.01, Figure 41 ).

[0090] KEGG pathway enrichment analysis results are shown in Figures 42-45 As shown in Table 2, compared with the blank group, 285 genes were involved in 150 pathways at T1, 108 genes were involved in 77 pathways at T2, 223 genes were involved in 146 pathways at T3, and 197 genes were involved in 128 pathways at T4. The up-regulated pathways significantly enriched in the experimental group at T1 included RIG-I-like receptor signaling pathway, tryptophan metabolism, cytokine receptor interaction, etc. (P<0.05). At T2, the up-regulated pathways significantly enriched included pantothenate and coenzyme A biosynthesis, complement and coagulation cascade pathway, arachidonic acid metabolism, ECM receptor interaction signal pathway, etc. (P<0.05), while the adipocytokine signaling pathway, fat digestion and absorption, and neuroactive ligand-receptor interaction pathways were significantly down-regulated (P<0.05). At T3, the complement and coagulation cascade pathway, arachidonic acid metabolism, and purine metabolism pathways were significantly up-regulated in the experimental group (P<0.05), and the TGF-β signaling pathway, TNF signaling pathway, virus carcinogenesis, and toll-like receptor signaling pathway were significantly down-regulated (P<0.05). At T4, the ErbB signaling pathway, dopamine synapse, and retrograde endocannabinoid signaling pathway were significantly up-regulated.

[0091] The above is a preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A polypeptide for ameliorating the symptoms of feline stress, characterized in that, The amino acid sequence is shown as SEQ ID NO:

1.

2. A nucleotide sequence encoding the polypeptide of claim 1.

3. A recombinant expression vector comprising the nucleotide sequence of claim 2, characterized in that, The vector is pPICZαA.

4. A host cell comprising the recombinant expression vector of claim 3, wherein, The host strain is Pichia pastoris GS115.

5. A method of producing the polypeptide of claim 1, comprising, The method comprises the following steps: (1) Constructing a recombinant expression vector: inserting the nucleotide sequence of claim 2 downstream of the α-factor secretion signal of the pPICZαA vector to obtain a recombinant plasmid; (2) Transforming the host strain: transforming the recombinant plasmid into Pichia pastoris GS115 strain, and obtaining positive clones through Zeocin screening; (3) Fermentation culture: positive clones were inoculated into BMGY medium, cultured at 30°C, 200 rpm until OD 600 = 6.0, centrifuged to collect the bacteria and transferred to BMMY medium, with methanol concentration of 0.5-1.0% to induce expression for 48-72 hours. (4) Purification: after centrifugation and 0.45 μm filter membrane filtration, the fermentation broth is purified by a Ni-NTA affinity chromatography column, and after collecting the target peak, desalination is performed by HPLC to obtain the polypeptide.

6. The preparation method according to claim 5, characterized in that, In step (3), when the induction temperature is 25℃, the polypeptide expression amount is higher.

7. Use of the polypeptide of claim 1 in the manufacture of a product for ameliorating the symptoms of feline stress, wherein the polypeptide is administered to the cat in an amount effective to ameliorate the symptoms of feline stress. The stress response symptoms include elevated serum cortisol levels, abnormal behavior, or increased secretion of inflammatory factors.

8. A composition for ameliorating the symptoms of feline stress, comprising, The polypeptide of claim 1, and a pharmaceutically acceptable carrier; the carrier is starch, microcrystalline cellulose, or starch paste.

9. The composition of claim 8, wherein, The mass percentage of the polypeptide is 0.1%-1.0%.

10. Use of the polypeptide of claim 1 in the preparation of an anti-stress feed additive or oral liquid for pets.

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