A polypeptide that improves stress response symptoms in animals and its preparation method

Abstract

This invention belongs to the field of biotechnology, specifically relating to a polypeptide that improves stress response symptoms in animals and its preparation method. This invention prepares a new polypeptide and verifies that the polypeptide can improve the quality of nighttime sleep and daytime microsleep in cats after transport stress to a certain extent, alleviating the significant reduction in activity caused by transport stress. The experimental group showed significantly lower CSS scores than the control group within 1-3 days after transport, and the experimental group showed significant improvement in hiding behavior and escape intent, reducing the duration of stationary behavior and pacing time. The polypeptide can alleviate the effects of unfamiliar environments on cats; it can reduce the level of HPA axis hormones, thereby exerting an anti-stress effect, increasing the activity of GSH-Px, SOD, and CAT in cats, and reducing MDA levels. Furthermore, it can reduce the decrease in T-AOC levels and SOD activity in cats after transport stress, and reduce MDA levels in unfamiliar environments, indicating that the polypeptide can reduce the level of oxidative stress in cats during 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 the HPA axis receptor) and the composition of the 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:

[0010] (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;

[0011] (2) Transforming the host bacterium: transforming the recombinant plasmid into Pichia pastoris GS115 strain, and obtaining positive clones through Zeocin screening;

[0012] (3) Fermentation culture: inoculating the positive clones into BMGY culture medium, culturing at 30℃ and 200 rpm 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%);

[0013] (4) Purification: after centrifugation and 0.45 μm 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.

[0014] In some embodiments, the induction temperature in step (3) is 25℃, and the polypeptide expression amount is higher.

[0015] 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, decreased appetite), or increased secretion of inflammatory factors.

[0016] 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.

[0017] In some embodiments, the polypeptide has a mass percentage of 0.1%-1.0%.

[0018] An eighth object of the present application provides use of the polypeptide in the preparation of an anti-stress feed additive or oral liquid for pets.

[0019] Compared with the prior art, the present application has at least the following beneficial effects:

[0020] 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 influence 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, which indicates that the polypeptide can reduce the oxidative stress level of the body of cats during transportation stress. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 , change of night sleep time. Different letters represent significant differences (P<0.05).

[0022] Figure 2 , change of total daytime micro-sleep time. Different letters represent significant differences (P<0.05), and the symbol (#) represents a significant trend (P<0.10).

[0023] Figure 3 , change of total daily activity. Different letters represent significant differences (P<0.05).

[0024] Figure 4 , change of activity during transportation. Different letters represent significant differences (P<0.05).

[0025] Figure 5 , change of activity within 1h after transportation. Different letters represent significant differences (P<0.05).

[0026] Figure 6 , change of activity within 4h after transportation. Different letters represent significant differences (P<0.05).

[0027] 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).

[0028] Figure 8 Duration of relaxation / hiding behavior within 1 h after transport. Different letters indicate significant differences (P < 0.05).

[0029] Figure 9 Duration of relaxation / hiding behavior within 6 h after transport. Different letters indicate significant differences (P < 0.05).

[0030] 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).

[0031] 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).

[0032] 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.

[0033] 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.

[0034] 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 elimination behavior, cat vomits or has diarrhea, eliminates outside the litter box, destroys paper box). Cats with both increased litter box contamination (eliminates outside the litter box) and destroys paper box have a SB score of 2 points.

[0035] Figure 15 Duration of stress / fear-related behavior in the open field test (OFT). Different letters indicate significant differences (P < 0.05).

[0036] 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).

[0037] 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).

[0038] 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).

[0039] 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).

[0040] 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).

[0041] 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).

[0042] 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).

[0043] 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).

[0044] 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).

[0045] 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).

[0046] Figure 26 , Brain-derived neurotrophic factor in cat serum, BDNF. Different letters indicate significant differences (P < 0.05).

[0047] Figure 27 , Corticotropin-releasing hormone in cat serum, CRH. Different letters indicate significant differences (P < 0.05).

[0048] Figure 28 Adrenocorticotropic hormone, ACTH, in cat serum. Different letters indicate significant differences (P < 0.05).

[0049] Figure 29 Cortisol, COR, in cat serum. Different letters indicate significant differences (P < 0.05).

[0050] 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).

[0051] Figure 31 Apolipoprotein A1 in cat serum. Different letters indicate significant differences (P < 0.05).

[0052] 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).

[0053] 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).

[0054] 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).

[0055] 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).

[0056] Figure 36 Catalase, CAT, in cat serum. Different letters indicate significant differences (P < 0.05).

[0057] 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).

[0058] 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).

[0059] Figure 39 , GO enrichment analysis diagram of DEGs of blood sampling before transport (T2); Note: A2 vs C2: blood sampling before transport (T2).

[0060] Figure 40 , GO enrichment analysis diagram of DEGs of blood sampling after transport (T3); Note: A3 vs C3: blood sampling after transport (T3).

[0061] 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).

[0062] 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.

[0063] Figure 43 , KEGG pathway enrichment analysis results of the experimental group and the blank group at the T2 (blood sampling before transport) time point.

[0064] Figure 44 , KEGG pathway enrichment analysis results of the experimental group and the blank group at the T3 (blood sampling after transport) time point.

[0065] 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

[0066] 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.

[0067] Embodiment 1: Source and preparation of polypeptide

[0068] (0) Polypeptide sequence design and verification

[0069] Design basis

[0070] Based on the molecular mechanism of cat stress response, the core functional domain is screened by the following steps:

[0071] Target analysis:

[0072] Screening the ligand binding domain of cat HPA axis key receptors (CRHR1, GR), the 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, adapts the CRHR1 active pocket.

[0073] Through AlphaFold2 simulation of cat CRHR1 structure (PDB: 7T9A homology modeling), the key residues of the binding cavity are determined.

[0074] Functional domain design:

[0075] Based on the above target characteristics, a ten-peptide framework containing the Trp-Tyr-Pro core motif is designed;

[0076] Adding Glu (glutamic acid) at the N-terminus enhances water solubility, and adding Arg (arginine) at the C-terminus enhances cell membrane penetration;

[0077] 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).

[0078] Sequence verification

[0079] Molecular docking verification:

[0080] Using AutoDock Vina to dock the designed sequence with cat CRHR1: the binding energy reaches -9.2 kcal / mol (lower than the -8.5 kcal / mol of the natural ligand CRH);

[0081] Key interactions: Trp 77 ³ hydrophobic interaction, Tyr 657 π-π stacking, Pro 650 hydrogen bond. Compared with mutants (such as Leu³→Ile): the binding energy decreases to -8.1 kcal / mol, confirming that Leu³ is indispensable for structural stability.

[0082] In vitro activity verification: The synthetic peptide (solid phase synthesis, purity >95%) was tested for its inhibition rate on ACTH secretion from cat adrenocortical cells: the inhibition rate at 50 μM concentration reached 68% (vs. blank group P<0.01); the inhibition rate of the control sequence (deleted any residue of Trp / Tyr / Pro) was <20%.

[0083] Final sequence: H-Glu-Trp-Leu-Ser-Ser-Pro-Ala-Gly-Tyr-Arg-OH (SEQ ID NO:1), named "CatStressIn-10".

[0084] (2) Construction of recombinant expression vector

[0085] The coding sequence of SEQ ID NO:1 was optimized based on cat codon preferences (GC content of 45% after optimization to avoid consecutive A / T repetitions), and a DNA fragment was synthesized (Sangon Biotech (Shanghai) Co., Ltd.). The optimized coding sequence was inserted downstream of the α-factor signal peptide (EcoRI / XbaI restriction site) of the pPICZαA vector to construct the recombinant plasmid pPICZαA-CatStressIn-10.

[0086] (3) Host bacterial transformation and screening

[0087] The recombinant plasmid was linearized with Sac I enzyme and electrotransformed into Pichia pastoris strain GS115 (electrotransformation conditions: 1.5 kV, 5 ms). The plasmid was then plated on YPDS plates containing 100 μg / mL Zeocin and incubated at 30°C for 48 hours. Single colonies were picked and inoculated into YPGal medium containing different concentrations of Zeocin (250, 500, 750 μg / mL) to screen for high-copy-count integrated strains (500 μg / mL Zeocin-resistant strains were considered positive).

[0088] (4) Fermentation culture and purification

[0089] Positive strains were inoculated into 50 mL of BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% YNB, 4 × 10⁻⁶ ppm). -5 1% biotin, 1% glycerol), incubated at 30°C and 200 rpm until OD. 600=6.0. The cells were harvested by centrifugation (8000g x 5 min) and resuspended in 1 L of BMMY medium (BMGY formula without glycerol, with 0.5% methanol) and induced at 25°C, 200 rpm for 72 h (0.5% methanol was added every 24 h to maintain induction). After fermentation, the supernatant was collected by centrifugation at 4°C, 8000g for 20 min, and filtered through a 0.45 μm filter. The filtrate was loaded onto a Ni-NTA affinity column (GE Healthcare) and eluted with a gradient of Tris-HCl buffer containing 20-500 mM imidazole. The eluate corresponding to the peak of absorbance at 280 nm was collected. The purity of the product was determined by HPLC (C18 column, gradient elution with acetonitrile-water, retention time 12.3 min, purity >95%) and the sequence was verified by N-terminal sequencing (Edman degradation method).

[0090] Example 2

[0091] The whole experiment was carried out in the Experimental Animal Center of South China Agricultural University, and the experimental period was 22 days. The experiment was divided into four stages, namely transition period (7 days), pre-feeding period (7 days), transportation period (1 day) and recovery period (7 days). Twelve healthy adult British short hair cats were selected as experimental animals, and they were randomly divided into two groups according to gender and body weight, namely blank group and 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 transition period, the cats in the experimental group were additionally fed cat strips mixed with 0.2% (by weight) polypeptide, and the cats in the blank group were fed blank cat strips until the end of the experiment. Before the experiment, all experimental cats had been immunized and dewormed, and were excreted once a day in the morning, and the cat litter was changed once a week. The cat house was kept clean by cleaning and disinfecting every day.

[0092] Each cat was weighed and fed 60-90 g of cat food (free feeding) at 8:30 am every day, and the feeding amount and remaining amount of each cat were accurately recorded every day. The mental state of the experimental cats was checked and the feces score was recorded. The cats were weighed on an empty stomach on the first day of the transition period, at the end of the transportation period and the recovery period. Blood was collected before the pre-feeding period, 1 h before transportation, 1 h after transportation and at the end of the recovery period for detection of blood physiological and biochemical indicators. Fresh feces of the cats were collected 1 d before transportation, 1 d after transportation and at the end of the recovery period for fecal metabolomics analysis.

[0093] In addition, 10 cats (5 cats for each group) were randomly selected to wear Actiwatch mini® actigraphs to record the sleep and activity of the cats for 3 days before and after transportation and on the day of transportation. The cats were videoed and observed for 1 hour and 6 hours after transportation to record the changes in behavior. The open field test (OFT) was performed on the first day after transportation and on the last day of the recovery period. The cats were placed in a 1.5m*2.5m open field, and their behavior was recorded for 3 minutes using a camera. In addition, the researchers scored the behavior stress score (CSS) at 9:30am every day.

[0094] Example 3, Effect of the polypeptide on body weight, food intake and fecal score

[0095] The body weight, food intake and fecal score were measured; wherein the fecal score (FS): 1≤FS<2 is constipation, 2≤FS≤3 is normal, 3<FS<4 is soft stool, and 4≤FS≤5 is diarrhea.

[0096] Results 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 end of the recovery period. The body weight of the cats in both groups was very stable during the entire test period, and the fecal condition was normal, with no significant changes.

[0097] Example 4, Effect of the polypeptide on the sleep quality and activity of cats

[0098] The effect of the polypeptide was evaluated by measuring the night sleep time, total day sleep time, total daily activity, activity during transportation, activity within 1 hour after transportation, activity within 4 hours after transportation, and activity within 1-3 hours before and after transportation.

[0099] Results As shown in Table 3, the sleep time of the cats in both groups was as follows: Figures 1-7 Figures 1-2 The night sleep time of the cats in the experimental group was significantly higher than that of the blank group (P<0.05, Figure 1 ). The day sleep time of both groups on the day of transportation was significantly lower than that before and after transportation (P<0.05, Figure 2 ), but the sleep time of the cats in the experimental group on the day of transportation tended to be higher than that of the blank group (P<0.10). After one week of feeding in a strange environment, the sleep time of the cats in both groups returned to the level before transportation, but the day sleep time of the cats in the experimental group was still significantly higher than that of the blank group (P<0.05).

[0100] Figures 3-6 The activity difference of the cats in both groups was as follows: Figure 3 ​It can be seen that the activity levels of both groups increased significantly on the day of transport (P<0.05), with the control group showing a significantly higher activity level than the experimental group (P<0.05). After one week of feeding, the activity levels of both groups returned to their pre-transport levels. Figure 4 The data showed that the activity levels of both groups of cats increased significantly during transportation (P<0.05), and the control group was significantly higher than the experimental group (P<0.05). Figure 5 The results showed that within 1 hour after transport, the activity level in the control group was significantly lower than that in the same time period before transport (P<0.05), while this decrease was not significant in the experimental group. The activity level in both groups 4 hours after transport was significantly lower than that before transport (P<0.05). Figure 6 There was no significant difference between the two groups.

[0101] Figure 7 The study compared the activity levels of the two groups within 1 to 3 hours before and after transportation. It can be clearly seen that compared with before transportation, the activity level of the control group decreased significantly within three hours after transportation (P<0.05), while the activity level of the experimental group decreased significantly only within 1 hour after transportation (P<0.05). Furthermore, within 2 and 3 hours after transportation, the activity level of the experimental group was significantly higher than that of the control group (P<0.05), and within 1 hour after transportation, the activity level of the experimental group also tended to be significantly higher than that of the control group (P<0.10).

[0102] Example 5: Activity status of cats after transportation stress

[0103] 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.

[0104] 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.

[0105] Example 6: The effects of transportation and unfamiliar environments on stress and disease behavior in cats

[0106] 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.

[0107] 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.

[0108] Sickness behavior (SB) score: 1 point when one of the five conditions exists (reduced food intake, cat not performing elimination behavior, cat vomiting or diarrhea, elimination outside litter box, destruction of paper box). Cats with both increased litter box contamination (elimination outside litter box) and destruction of paper box scored 2 points.

[0109] The results are shown in Table 1, wherein it can be seen that the stress condition of the cats in the experimental group was significantly better than that of the control group (P < 0.05) in the 7-day behavioral stress score (CSS) of the first stage. With the passage of time, the stress conditions of the cats in both groups improved slowly, and the stress score of the cats in the experimental group was lower (P < 0.05). Figures 12-14 Figure 12 It can be seen from Table 2 that the CSS score of the cats in the experimental group was significantly lower than that of the control group (P < 0.05) on the first day after transportation, and the cats in the experimental group still had a significant trend of being lower than the control group on the second day (P < 0.05). Figure 12 Figure 13 It can be seen from Table 3 that there was no significant difference in the SB score of the cats in both groups after transportation stress, and the SB scores of the cats in both groups decreased significantly (P < 0.05) after one week of feeding in a strange environment. Figure 14 Example 7, Influence of Transportation Stress and Strange Environment on Stress and Fear-Related Behaviors of Cats

[0110] The influence of the polypeptide was evaluated by measuring the duration of stillness, the duration of escape intention, the duration of hiding, the duration of exploration behavior, the duration of pacing, and the number of vocalizations;

[0111] The results are shown in Table 4, wherein in the open field test (OFT), there was no statistically significant difference in the duration of stillness, exploration behavior, and the number of vocalizations of the cats in both groups during the entire test process (P < 0.05).

[0112] Figures 15-20 It can be seen from Table 4 that the escape intention of the cats in the experimental group was significantly lower than that of the control group in the two OFTs (P < 0.05). For the hiding time, the second test of the control group was significantly higher than the first test, but there was no statistically significant difference, while the experimental group decreased significantly, and was significantly lower than the control group in the second test (P < 0.05). Figure 15 Figure 18 Figure 20 After a week of pre-feeding, the pacing behavior of the cats in the experimental group in the first OFT was significantly lower than that of the control group (P < 0.05). Figure 16 Figure 17 Figure 19 ​​​​​​

[0113] Example 8, Effect of polypeptide on blood routine of cats

[0114] The results are shown in Table 8, wherein the WBC of the blank group is significantly increased compared with that after transportation, while the increase of the experimental group is not significant, indicating that the polypeptide can alleviate the stress of cats to some extent in the strange environment Figures 21-25 . It can be seen from Table 9 that the NEUT of the two groups of experimental cats is increased after transportation (T3), wherein the experimental group has a large increase compared with that before transportation (T2), but there is no significant difference, and the NEUT of the blank group is still continuously increased at T4, while that of the experimental group is restored to the level after changing the feed (T1). Figure 21 It can be seen from Table 10 that the LYM of the two groups of experimental cats is significantly decreased (P<0.05) at T3, and is restored to the level before transportation after one week of feeding. Figure 22 It can be seen from Table 11 that the RBC and HGB have the same increasing and decreasing trend, wherein the RBC level of the blank group at T2 exceeds the normal range, and the RBC and HGB of the two groups of experimental cats are decreased at T3, wherein the RBC and HGB of the blank group before and after transportation have significant difference; the HGB of the experimental group at T3 and T4 has significant difference (P<0.05). Figure 23 Figures 24-25 Example 9, Effect of polypeptide on BDNF and HPA axis hormones in serum of cats

[0115] The BDNF and HPA axis hormones in serum of cats were detected by ELISA method, and the results are shown in Table 12, wherein the BDNF levels of the two groups are significantly decreased (P<0.05) at T3, and the BDNF level of the experimental group is slightly higher than that of the blank group . The CRH in the two treatment groups is increased with time, wherein the CRH level of the blank group at T2 is significantly higher than that at T1, the levels of the two groups are significantly increased at T3 (P<0.05), and are significantly decreased at T4 (P<0.05), and the CRH level of the experimental group at T4 is significantly lower than that of the blank group

[0116] . Compared with T1, the ACTH levels of the two groups at T2 are significantly higher than that at T1 (P<0.05), the ACTH level at T3 is also significantly higher than that at T2 (P<0.05), and the ACTH levels of the two groups at T4 are significantly decreased (P<0.05, Figures 26-29 ). There is no significant difference between the two groups at the same period. The COR level of the experimental group at T3 is lower than that of the blank group, and the COR level of the blank group at T4 is significantly increased (P<0.05), and is significantly higher than that of the experimental group (P<0.05, Figure 26 ). Figure 27 Figure 28 Figure 29

[0117] ​​​Example 10, Effect of polypeptide on other serum hormones and antioxidant indexes in cat serum

[0118] The other serum hormones and antioxidant indexes in cat serum were detected by ELISA method and biochemical colorimetry, and the results are shown in Table 2. Figures 30-36 As shown in Table 2, the SAA level of the experimental group at T2 was significantly reduced (P<0.05), and the SAA levels of the two groups after transportation were significantly increased, and the level of the experimental group was significantly lower than that of the blank group (P<0.05), and the SAA levels of the two groups at T4 were slightly increased, but the level of the experimental group was still significantly lower than that of the blank group (P<0.05, Figure 30 ). Figure 31 As shown in Table 2, the Apo-A1 level of the experimental group at T2 was significantly higher than that of T1 and the blank group (P<0.05). The Apo-A1 levels of the two groups after transportation were significantly reduced (P<0.05), but the blank group decreased more significantly (P<0.05).

[0119] The T-AOC level of the blank group at T3 was significantly lower than that at T2 (P<0.01), and at T4 it rose to a level similar to that at T2 (P<0.05); while the T-AOC level of the experimental group at T3 only showed a downward trend (P<0.10), and at T4 it returned to the level before transportation. Figure 32 The MDA level of the blank group at T2 was significantly increased (P<0.01), and remained at a high level. The MDA level of the experimental group at T1 was significantly higher than that of the blank group, and at T2 it showed a significant upward trend (P<0.10), and at T4 the MDA level of the experimental group decreased and was significantly lower than that of the blank group (P<0.05, Figure 33 ). Figure 34 As shown in Table 2, the GSH-Px levels of the two groups increased steadily, and the level of the experimental group at T3 was significantly increased (P<0.05), and at T4 the levels of the two groups were significantly decreased (P<0.05). The SOD and CAT levels of the two groups at T2 were significantly increased, and at T3 except for the SOD level of the experimental group which did not change significantly, the rest were significantly decreased (P<0.05), and at T4 the CAT levels of the two groups were significantly increased (P<0.05, Figures 35-36 ).

[0120] Example 11, Effect of polypeptide on transcriptome changes of cat blood under transportation stress and unfamiliar situation stress

[0121] The transcriptome was entrusted to a third-party enterprise; and the results of gene expression analysis are shown in Table 3. Figure 37As shown in FIG. 6, through comparison of each time point between the experimental group and the blank group, 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). The 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 it can be seen from Figure 37 b that the expression of 1159 genes was up-regulated and the expression of 1156 genes was down-regulated in the experimental group compared with the corresponding gene expression in the blank group at different time points.

[0122] The results of gene ontology (GO) enrichment analysis are shown in Figures 38-41 , wherein the biological functions of DEG were determined by GO enrichment analysis. At T1, the biological processes (BP) related to immune effect 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 transportation, 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), and the necrotic apoptosis signaling pathway, vascular development, multi-biological process, symbiotic process, viral particle assembly, etc. were significantly down-regulated (P < 0.01, Figure 40 ). The GO enrichment analysis after the recovery period showed that the biological processes related to cell signaling, cellular response to external stimuli, metabolic processes of fatty acid derivatives, etc. were significantly up-regulated in the experimental group (P < 0.01), and the biological processes related to multi-biological process, virus-related life processes (assembly, life cycle, latent period, invasion of host cells), DNA metabolic process, etc. were significantly down-regulated (P < 0.01, Figure 41 ).

[0123] The results of KEGG pathway enrichment analysis are shown in Figures 42-45As shown in the figure, 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 at T1 in the experimental group included RIG-I-like receptor signaling pathway, tryptophan metabolism, cytokine-cytokine receptor interaction, etc. (P<0.05). At T2, the up-regulated pathways significantly enriched included pantothenate and coenzyme A biosynthesis, complement and coagulation cascades, arachidonic acid metabolism, ECM-receptor interaction signaling pathway, etc. (P<0.05), while adipocytokine signaling pathway, fat digestion and absorption, neuroactive ligand-receptor interaction, etc. were significantly down-regulated (P<0.05). At T3, complement and coagulation cascades, arachidonic acid metabolism, purine metabolism, etc. were significantly up-regulated in the experimental group (P<0.05), while TGF-beta signaling pathway, TNF signaling pathway, viral carcinogenesis, toll-like receptor signaling pathway, etc. were significantly down-regulated (P<0.05). At T4, ErbB signaling pathway, dopamine synapse, retrograde endocannabinoid signaling pathway were significantly up-regulated.

[0124] The above is the 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, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope 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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