CN121021636B

CN121021636B - Polypeptide for improving animal stress response symptoms and preparation method thereof

Info

Publication number: CN121021636B
Application number: CN202511574081.0A
Authority: CN
Inventors: 张玲娜; 陈少豪; 叶兰; 薛绮珊; 赵坤
Original assignee: Hangzhou Yipusi Biotechnology Co ltd; South China Agricultural University
Current assignee: Hangzhou Yipusi Biotechnology Co ltd; South China Agricultural University
Priority date: 2025-10-31
Filing date: 2025-10-31
Publication date: 2026-01-30
Anticipated expiration: 2045-10-31
Also published as: CN121021636A

Abstract

The invention belongs to the field of biotechnology, and particularly relates to a polypeptide for improving animal stress response symptoms and a preparation method thereof, wherein the polypeptide is prepared into a novel polypeptide, and the polypeptide is verified to improve the night sleep and daytime microsleep quality of CATs after transportation stress to a certain extent, so that the activity caused by the transportation stress is relieved to be greatly reduced; the CSS score of the experimental group is obviously lower than that of the blank group within 1-3 days after transportation, the hiding behavior and the escape intention of the experimental group are obviously improved, the trend of resting behavior duration and pacing time can be reduced, the polypeptide can relieve the influence of strange environment on CATs, the level of HPA axis hormone can be reduced, the anti-stress effect is further exerted, the activities of GSH-Px, SOD and CAT of CATs are improved, the MDA level is reduced, the reduction of T-AOC level and SOD activity of CATs after transportation stress is reduced, the MDA level in the strange environment is reduced, and the oxidative stress level of the CATs in the transportation stress process can be reduced by the polypeptide.

Description

Polypeptide for improving animal stress response symptoms and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide for improving animal stress response symptoms and a preparation method thereof.

Background

Cats are common companion animals whose stress response is a common concern in clinical veterinary and pet care. The stress sources mainly comprise environmental changes (such as moving, joining of new members), medical operations (such as vaccination, surgery), transportation (long distance or air transport), social conflicts and the like. When cats are exposed to a stressor, the hypothalamic-pituitary-adrenal (HPA) axis is activated and Corticotropin Releasing Hormone (CRH), adrenocorticotropic hormone (ACTH) and Cortisol (CORT) are secreted and increased in sequence, causing a series of physiological and behavioral changes, which are manifested as increased heart rate, shortness of breath, disturbed intestinal motility (diarrhea or constipation), decreased immunity (susceptibility to secondary infections), behavioural manifestations as hiding, aggressiveness enhancement, anorexia, excessive or decreased licking, etc. Chronic stress can also induce or aggravate Inflammatory Bowel Disease (IBD), urinary system syndrome (flute), hyperthyroidism, etc., severely affecting quality of life and longevity of cats.

At present, the intervention means aiming at cat stress mainly comprise three types of (1) anxiolytic drugs (such as benzodiazepine drugs and opioid drugs), namely anxiolytic drugs which can quickly relieve anxiety, but have side effects of drug dependence, excessive sedation, respiratory inhibition and the like, and have no direct improvement effect on gastrointestinal stress (such as diarrhea), and (2) natural supplements (such as cat face pheromone (F3/F4 type ferlomon) and probiotics (such as lactobacillus)) which can regulate emotion through smell, but have slow effect (continuous use is required for 3-7 days) and limited effect on severe stress, and probiotics can regulate intestinal flora but have the problems of low implantation rate and large influence on the intestinal environment of a host, and (3) environmental management (such as providing a concealed space and reducing stimulus) is a basic measure, but can not directly intervene in the neuroendocrine pathway of stress, and can not meet the quick release requirement of acute stress (such as medical treatment).

Therefore, the development of a safe, efficient and cat-targeted polypeptide preparation has important significance. The polypeptide is a potential stress regulation candidate molecule because of small molecular weight, easy absorption and few side effects. However, existing studies have focused on canine or rodent stress-modulating polypeptides, specific polypeptides for cats and methods for their preparation have not been reported. The neuroendocrine system (e.g., HPA axis receptor sensitivity), gut flora composition and canine/rodent differences in cats are significant, and direct transplantation of polypeptides from other species may be ineffective or ineffective due to species differences. Therefore, there is a need to screen or design specific anti-stress polypeptides for physiological characteristics of cats and to establish efficient and stable preparation methods.

Disclosure of Invention

The first object of the invention is to provide a polypeptide for improving stress response symptoms of cats, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

The second object of the present invention is to provide a nucleotide sequence encoding the above polypeptide.

The third object of the present invention is to provide a recombinant expression vector comprising the above nucleotide sequence, wherein the vector is pPICZ alpha A.

The fourth object of the present invention is to provide a host bacterium comprising the recombinant expression vector described above, which is Pichia pastoris (GS 115 strain).

The fifth object of the present invention is to provide a method for producing the above polypeptide, comprising the steps of:

(1) Constructing a recombinant expression vector, namely inserting the nucleotide sequence into the downstream of an alpha factor secretion signal of the pPICZ alpha A vector to obtain a recombinant plasmid;

(2) Transforming host bacteria, namely transforming recombinant plasmids into Pichia pastoris GS115 strains, and obtaining positive clones through Zeocin screening;

(3) Fermenting and culturing, namely inoculating positive clones into BMGY culture medium, culturing at 30 ℃ and 200rpm until OD ₆₀₀ = 6.0, centrifugally collecting thalli, transferring the thalli into the BMMY culture medium, and inducing expression for 48-72 hours (methanol concentration is 0.5-1.0%);

(4) Purifying by centrifuging the fermentation broth, filtering with 0.45 μm filter membrane, purifying with Ni-NTA affinity chromatography column, collecting target peak, and desalting by HPLC to obtain the polypeptide.

In certain embodiments, the polypeptide expression is higher at an induction temperature of 25 ℃ in step (3).

A sixth object of the present invention is to provide the use of the above polypeptide for the preparation of a product for improving stress symptoms in cats, including elevated serum cortisol levels, abnormal behavior (prolonged hiding time, anorexia) or increased secretion of inflammatory factors.

The seventh object of the invention is to provide a composition for improving stress response symptoms of cats, which comprises the polypeptide and a pharmaceutically acceptable carrier, wherein the carrier is starch, microcrystalline cellulose or starch slurry.

In certain embodiments, the mass percentage of the polypeptide is 0.1% -1.0%.

The eighth object of the invention is to provide an application of the polypeptide in preparing anti-stress feed additives or oral liquid for pets.

Compared with the prior art, the invention has at least the following beneficial effects:

The novel polypeptide is prepared, the quality of night sleep and daytime micro sleep of cats after transportation stress can be improved to a certain extent, the activity caused by transportation stress can be relieved to a certain extent, the influence of the polypeptide on the behavior change of the cats is small within 6 hours after transportation, the score of CSS of an experimental group within 1-3 days after transportation is obviously lower than that of a blank group, the hiding behavior and escape intention of the experimental group are obviously improved, the trend of resting behavior duration and step time can be reduced by the polypeptide, the influence of strange environment on the cats can be relieved to a certain extent by the polypeptide, and the level of HPA axis hormone (especially the level of COR and CRH) can be reduced, so that the anti-stress effect is exerted. The polypeptide can improve the activities of GSH-Px, SOD and CAT of CATs to a certain extent, reduce the MDA level, reduce the reduction of T-AOC level and SOD activity of CATs after transportation stress, and reduce the MDA level in unfamiliar environments, which indicates that the polypeptide can reduce the oxidative stress level of CAT organisms in the transportation stress process.

Drawings

Fig. 1, night sleep time variation. The different letters represent a significant difference (P < 0.05).

Fig. 2, daytime microsleep total time variation. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

Figure 3, daily total activity change. The different letters represent a significant difference (P < 0.05).

Fig. 4, activity change during transportation. The different letters represent a significant difference (P < 0.05).

FIG. 5, activity change over 1h after transportation. The different letters represent a significant difference (P < 0.05).

FIG. 6, activity change over 4 hours after transportation. The different letters represent a significant difference (P < 0.05).

And 7, changing the activity amount within 1-3 hours before and after transportation. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

Figure 8 duration of relaxation/hiding behavior within 1h after transportation. The different letters represent a significant difference (P < 0.05).

Figure 9 duration of relaxation/hiding behavior within 6 hours after transportation. The different letters represent a significant difference (P < 0.05).

Figure 10, number of drinks in 1h and 6h after transportation. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

FIG. 11, feed intake within 1h and 4h after transportation. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

Fig. 12, convalescence behavioral stress score, CSS. * Representing significant differences (P < 0.05). Behavioral stress (CSS) scores 1-2 score for relaxation, 3 score for mild stress, 4 score for moderate stress, 5-6 score for fear status, 7 score for extreme stress.

Fig. 13, CSS score within three days after shipping. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10). Behavioral stress (CSS) scores 1-2 score for relaxation, 3 score for mild stress, 4 score for moderate stress, 5-6 score for fear status, 7 score for extreme stress.

Figure 14, disease behavior score SB, different letters represent significant differences (P < 0.05). Note disease behavior (SB) score 1 score when one of five conditions was present (reduced food intake, no excretion behavior by cats, vomiting or diarrhea by cats, excretion outside cat litter, destruction of cartons). The cat SB score for both increased contamination in the cat cage (excreted outside the litter box) and the destruction of the carton was 2 points.

Figure 15, static duration of stress and fear related behavior in Open Field Test (OFT). The different letters represent a significant difference (P < 0.05).

Figure 16, behavior related stress and fear in Open Field Test (OFT) escape intent duration. The different letters represent a significant difference (P < 0.05).

Figure 17, stress and fear related behavioural hiding duration in Open Field Test (OFT). The different letters represent a significant difference (P < 0.05).

Figure 18, behavioral duration associated with stress and fear in Open Field Test (OFT). The different letters represent a significant difference (P < 0.05).

Figure 19, behavioral pacing duration associated with stress and fear in Open Field Test (OFT). The different letters represent a significant difference (P < 0.05).

Figure 20 number of behavioral vocalization associated with stress and fear in Open Field Test (OFT). The different letters represent a significant difference (P < 0.05).

Figure 21, cat blood normal white blood cells, WBC. The red line in the figure is the normal range of each index. The different letters represent a significant difference (P < 0.05).

Figure 22, cat blood normal neutrophils, NEU. The red line in the figure is the normal range of each index. The different letters represent a significant difference (P < 0.05).

FIG. 23, cat blood conventional lymphocytes, LYM. The red line in the figure is the normal range of each index. The different letters represent a significant difference (P < 0.05).

Fig. 24, cat blood normal red blood cells, RBC. The red line in the figure is the normal range of each index. The different letters represent a significant difference (P < 0.05).

FIG. 25, cat blood conventional hemoglobin concentration, HGB. The red line in the figure is the normal range of each index. The different letters represent a significant difference (P < 0.05).

Fig. 26, brain-derived neurotrophic factor, BDNF, in cat serum. The different letters represent a significant difference (P < 0.05).

Figure 27, corticotropin releasing hormone, CRH, in cat serum. The different letters represent a significant difference (P < 0.05).

Figure 28, adrenocorticotropic hormone ACTH in cat serum. The different letters represent a significant difference (P < 0.05).

Fig. 29, cortisol, COR in cat serum. The different letters represent a significant difference (P < 0.05).

FIG. 30, serum amyloid, SAA, in cat serum. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

FIG. 31, apolipoprotein A1 in cat serum. The different letters represent a significant difference (P < 0.05).

FIG. 32, total antioxidant capacity, T-AOC, in cat serum. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

Figure 33, malondialdehyde, MDA in cat serum. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

FIG. 34, glutathione peroxidase, GSH-Px in cat serum. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

FIG. 35 shows superoxide dismutase, SOD, in cat serum. The different letters represent significant differences (P < 0.05), and the symbol (#) represents a tendency for significant differences (P < 0.10).

FIG. 36 Cat serum catalase, CAT. The different letters represent a significant difference (P < 0.05).

FIG. 37, wen diagram, results of Differentially Expressed Genes (DEG), note: a is Wen diagram, b is results of Differentially Expressed Genes (DEG), DEG Counts: number of differentially expressed genes, up-regulated: number of up-regulated genes, down-regulated: number of down-regulated genes. A1 vs C1 is blood collection after grain change (T1), A2 vs C2 is blood collection before transportation (T2), A3 vs C3 is blood collection after transportation (T3), and A4 vs C4 is blood collection after recovery period (T4).

FIG. 38, GO enrichment analysis of Differential Expressed Gene (DEG) for post-change blood collection (T1), and A1 vs C1 injection for post-change blood collection (T1).

FIG. 39, GO enrichment analysis of Differential Expressed Gene (DEG) for pre-shipment blood (T2), note A2 vs C2 for pre-shipment blood (T2).

FIG. 40, GO enrichment analysis of Differential Expressed Gene (DEG) for post-transport blood collection (T3) Note A3 vs C3 post-transport blood collection (T3).

FIG. 41, GO enrichment analysis of Differential Expressed Gene (DEG) for recovery period end blood collection (T4) note A4 vs C4: recovery period end blood collection (T4).

FIG. 42, KEGG pathway enrichment analysis results for experimental and blank groups at time point T1 (post-grain change blood sampling).

FIG. 43, KEGG pathway enrichment analysis results for experimental and blank groups at time point T2 (blood collection prior to transportation).

FIG. 44, KEGG pathway enrichment analysis results for experimental and blank groups at time point T3 (post-transportation blood sampling).

Fig. 45, experimental and blank groups, results of KEGG pathway enrichment analysis at time point T4 (end of recovery period blood sampling).

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

Example 1 Source and preparation of Polypeptides

(0) Polypeptide sequence design and verification

Design basis

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

Target spot analysis:

Ligand binding domains of cat HPA axis key receptors (CRHR 1, GR) were screened and binding site analysis showed that Trp (tryptophan) anchored the CRHR1 transmembrane region by hydrophobic interaction, tyr (tyrosine) formed a hydrogen-bonding network with Ser753 of GR, pro (proline) maintained the beta-turn conformation, fitting the CRHR1 active pocket.

Key residues of the binding cavity were determined by AlphaFold a model of cat CRHR1 structure (PDB: 7T9A homology modeling).

Functional domain design:

Designing a decapeptide framework comprising a Trp-Tyr-Pro core motif based on the target feature;

Glu (glutamic acid) is added at the N end to enhance water solubility, and Arg (arginine) at the C end to enhance cell membrane penetrability;

The intermediate sequence (Leu-Ser-Ser-Ala-Gly) is optimized as a flexible junction region which is easy to be absorbed by the cat intestinal tract, and protease cleavage sites are avoided (predicted by a PeptideCutter).

Sequence verification

Molecular docking verification:

The designed sequence was docked to cat CRHR1 using AutoDock Vina for binding energy up to-9.2 kcal/mol (below-8.5 kcal/mol for natural ligand CRH);

Key interactions are Trp ⁷⁷ hydrophobic, tyr ⁶⁵⁷ pi-pi stacking, pro ⁶⁵⁰ hydrogen bonding. The binding energy of the comparison mutant (e.g., leu 3. Fwdarw. Ile) was reduced to-8.1 kcal/mol, confirming that Leu3 is indispensable for structural stability.

In vitro activity verification, the inhibition rate of the synthesized polypeptide (solid phase synthesis method, purity > 95%) on cat adrenal cortex cell ACTH secretion is tested, the inhibition rate reaches 68% at 50 mu M concentration (vs. blank group P < 0.01), and the inhibition rate of a control sequence (deletion of any residue Trp/Tyr/Pro) is <20%.

The final sequence H-Glu-Trp-Leu-Ser-Ser-Pro-Ala-Gly-Tyr-Arg-OH (SEQ ID NO: 1) was designated as "CATSTRESSIN-10".

(2) Recombinant expression vector construction

The coding sequence of SEQ ID NO. 1 was optimized according to cat codon preference (GC content 45% after optimization, avoiding continuous A/T repetition), and DNA fragments were synthesized (Biotechnology (Shanghai) Co., ltd.). The optimized coding sequence is inserted into the downstream (EcoR I/Xba I cleavage site) of the alpha factor signal peptide of the pPICZ alpha A vector to construct a recombinant plasmid pPICZ alpha A-CATSTRESSIN-10.

(3) Host bacterial transformation and screening

The recombinant plasmid was linearized with SacI enzyme, transformed electrically with Pichia pastoris GS115 strain (electrotransformation conditions: 1.5kV,5 ms), coated with YPDS plates containing 100. Mu.g/mL Zeocin, and incubated at 30℃for 48 hours. The monoclonal was inoculated into YPGal medium containing different concentrations of Zeocin (250, 500, 750. Mu.g/mL) and high copy integrative strains were selected (500. Mu.g/mL Zeocin resistant strain was positive).

(4) Fermentation culture and purification

The positive strain was inoculated in 50mL BMGY medium (1% yeast extract, 2% peptone, 100mM potassium phosphate, 1.34% YNB,4×10 ^-5% biotin, 1% glycerol), cultured at 30 ℃ at 200rpm to OD ₆₀₀ =6.0. The cells were collected by centrifugation (8000 g. Times.5 min) and resuspended in 1L BMMY medium (BMGY formulation removes glycerol, 0.5% methanol was added) and induced at 25℃at 200rpm for 72 hours (methanol was supplemented with 0.5% every 24 hours to maintain induction). After fermentation, the supernatant was collected by centrifugation at 4℃and 8000g for 20 minutes, filtered through a 0.45 μm filter, and applied to a Ni-NTA affinity column (GE HEALTHCARE), and eluted with a gradient of Tris-HCl buffer containing 20-500mM imidazole, followed by collection of the eluate corresponding to the absorbance peak at 280nm (which corresponds to the theoretical value). HPLC detection purity (C18 column, acetonitrile-water gradient elution, retention time 12.3 min, purity > 95%), N-terminal sequencing (Edman degradation) confirmed sequence identity with design.

Example 2

The whole process of the polypeptide effect evaluation experiment is carried out in an experimental animal center of agricultural university in south China, the experimental period is 22 days, the experiment is divided into four stages, namely a grain change transition period (7 days), a pre-feeding period (7 days), a transportation period (1 day) and a recovery period (7 days), 12 healthy adult British short-hair cats are selected as experimental animals, and are randomly divided into 2 groups according to gender and weight, and the groups are a blank group and a polypeptide group. All cats were individually fed in cat cages (108 cm x 70cm x 76 cm) in the same thermostatically chamber and were free to feed and drink water, and cats in the experimental group were additionally fed cat strips mixed with 0.2% (by weight) of polypeptide after the end of the food exchange period, and cats in the blank group were fed blank cat strips until the end of the experiment. All experimental cats were subjected to the necessary immunization and insect repellent treatments once daily, with the litter being changed once weekly, and the cat house being cleaned and sterilized daily, keeping clean.

Each cat is weighed to be fed with 60-90 g of cat food (free feeding) at 8:30 am every day, the feeding amount and the residual amount of each cat are accurately recorded every day, the mental state of the experimental cat is checked, and fecal scoring is carried out. The cats are weighed on an empty stomach after the first day of grain change, the transportation period and the recovery period are finished, and blood is collected 1d before the test pre-feeding period, 1h before transportation, 1h after transportation and the recovery period are finished and used for detecting the physiological and biochemical indexes of the blood. Fresh feces from cats were collected before 1d, after 1d and after the end of the recovery period for testing of the fecal metabonomics analysis.

In addition, 10 experimental cats (5 each in each group) were randomly selected to wear a ACTIWATCH MINI body movement apparatus, and the changes in sleep and activity before and after 3 days of transportation and the day of transportation were recorded. The behavior changes of the experimental cats were recorded after transportation and observed within 1 hour and 6 hours after transportation. Open Field Testing (OFT) was performed on the first day after shipment and the last day of recovery, and experimental cats were placed in an open field at 1.5 x 2.5m and recorded for behavior within 3 minutes using a video camera. In addition, 9:30am researchers daily will conduct behavioral stress scores (CSS).

Example 3 influence of Polypeptides on body weight, feed intake and stool score

Measuring weight, feed intake and stool score, wherein the stool score (FS) is that 1 is less than or equal to FS <2 is constipation, 2 is less than or equal to FS <3 is normal, 3 is less than or equal to FS <4 is soft stool, and 4 is less than or equal to FS < 5 is diarrhea.

Results during the feeding period, feed intake was significantly reduced in both groups during the transit period (P < 0.05), but the feed intake was still significantly higher in the experimental group than in the blank group (P < 0.05), and both groups recovered to pre-transit levels after the end of the recovery period. The body weight of the two groups of experimental cats is quite stable during the whole test period, and the fecal condition is normal and has no obvious change.

EXAMPLE 4 Effect of polypeptide on sleep quality and Activity in cats

Evaluating the influence of the polypeptide by measuring night sleep time, daytime microsleep total time, daily total activity, activity in the transportation process, activity in 1h after transportation, activity in 4h after transportation and activity in 1-3h before and after transportation;

The results are shown in fig. 1-7, where fig. 1-2 are sleep times for two groups of experimental cats. The night sleep time was significantly higher in the post-transport experimental group than in the blank group (P <0.05, fig. 1). The daytime sleeping time of the two groups of post-transportation on the same day is obviously reduced compared with that before and after the transportation (P <0.05, figure 2), but the sleeping time of the experimental group on the same day of the transportation has a trend higher than that of the blank group (P < 0.10), the sleeping time of the two groups of the experimental group is restored to the state before the transportation after the two groups of the experimental group are fed for one week in a strange environment, and the daytime microsleep time of the experimental group is still obviously higher than that of the blank group (P < 0.05).

Figures 3-6 show the activity differences between two groups of experimental cats. As can be seen from fig. 3, the activity levels of both groups on the day of transportation increased significantly (P < 0.05), with the blank group significantly higher than the experimental group (P < 0.05), and the activity levels of both groups recovered to the pre-transportation state after one week of feeding. From fig. 4 it can be seen that the activity levels of the two groups of cats increased significantly during transport (P < 0.05) and the blank group was significantly higher than the experimental group (P < 0.05). From fig. 5 it can be seen that the activity of the blank group was significantly reduced (P < 0.05) within 1h after transport compared to the same time period before transport, whereas in the experimental group the reduction was not significant. The activity level 4h after transport was significantly lower for both groups than before transport (P <0.05, fig. 6), with no significant difference between the two groups.

Fig. 7 is a comparison of the activities in 1 to 3 hours before and after the transportation between the two groups, and it can be intuitively seen that the activities in three hours after the transportation of the blank group are significantly reduced (P < 0.05) compared with the activities before the transportation, the activities in the test group are significantly reduced (P < 0.05) only in 1 hour after the transportation, the activities in the test group are significantly higher than the activities in the blank group (P < 0.05) in 2 hours and 3 hours after the transportation, and the activities in the test group in 1 hour after the transportation are also significantly higher than the activities in the blank group (P < 0.10).

Example 5 Activity status of cats following transport stress

Evaluating the effect of the polypeptide by measuring the duration of relaxation/hiding behavior in 1h after transportation, the duration of relaxation/hiding behavior in 6h after transportation, the number of drinking times in 1h and 6h after transportation, and the feed intake in 1h and 4h after transportation;

The results are shown in fig. 8-11, where it can be seen from fig. 8-9 that there is no significant difference in the duration of relaxation and hiding behavior of both groups within 1 hour and 6 hours after transportation. The number of drinking water in both groups increased significantly within 6 hours after transport, with the experimental group significantly higher than 1 hour after transport (P < 0.05), but no significant difference between the two groups (p=0.136 >0.10, fig. 10). The feed intake of the experimental group increased significantly at 4h post-shipment (P <0.05, fig. 11), whereas the blank group had only a tendency to increase (P < 0.10), but there was no significant difference between the two groups.

Example 6 influence of transport and strange Environment on stress status and disease behavior of cats

Evaluating the effect of the polypeptide by measuring a convalescence behavioral stress score, a CSS score, a disease behavioral score within three days after transportation;

Behavioral stress (CSS) scores 1-2 score for relaxation, 3 score for mild stress, 4 score for moderate stress, 5-6 score for fear status, 7 score for extreme stress.

Disease behaviour (SB) score 1 score when one of five conditions was present (reduced food intake, no excretion behaviour by cats, vomiting or diarrhea by cats, excretion outside cat litter, destruction of cartons). The cat SB score for both increased contamination in the cat cage (excreted outside the litter box) and the destruction of the carton was 2 points.

The results are shown in fig. 12-14, where the stress status of the first group of cats was significantly better than the blank group (P < 0.05) as seen in the behavioral stress score (CSS) over 7 days, and both groups of cats were slowly improved over time, with lower stress scores (fig. 12). From fig. 13, it can be seen that the CSS score of the experimental group was significantly lower on day 1 after shipping than that of the blank group (P < 0.05), and the experimental group had a tendency to be significantly lower on the next day (P < 0.05). As can be seen from fig. 14, the SB scores of the two groups of experimental cats were not significantly different in the first stage after being subjected to the transport stress, and the SB scores of the two groups were significantly decreased (P < 0.05) after being fed for one week in a strange environment.

Example 7 influence of transport stress and strange Environment on cat stress and fear related behavior

Evaluating the influence of the polypeptide by measuring a resting period, an escape intention period, a hiding period, a exploring behavior period, a pacing period, and a sounding number;

The results are shown in fig. 15-20, where there were no statistically significant differences in the duration of resting, exploring and number of utterances of the two groups of cats in the Open Field Test (OFT) (fig. 15, fig. 18, fig. 20) throughout the test. It can be seen from fig. 16 that the escape intention of the experimental group in both OFTs was significantly lower than that of the blank group (P < 0.05). For the time to hide, the second test of the blank was significantly higher than the first but not statistically different, while the experimental group was significantly lower and significantly lower than the blank in the second test (P <0.05, fig. 17). After a one week pre-feeding period, the pacing behavior of the first open field test was significantly lower in the experimental group than in the blank group (P <0.05, fig. 19).

Example 8 Effect of polypeptide on cat blood routine

The results are shown in figures 21-25, where after the end of the recovery period (T4), WBCs in the blank group were significantly elevated compared to after transport, whereas the elevation in the experimental group was not significant, indicating that the polypeptide was able to relieve to some extent the stress of the cat on the strange environment (figure 21). From fig. 22, it can be seen that the NEUT for both groups of cats was rising after transportation (T3), with a significant increase in the experimental group compared to before transportation (T2), but without significant differences, the blank group NEUT was still rising continuously at T4, and the experimental group was restored to the post-change (T1) level. As can be seen from fig. 23, the LYm was significantly reduced at T3 (P < 0.05) in both groups of experimental cats and returned to pre-shipment levels after one week of feeding. As can be seen from fig. 24-25, the tendency of RBC and HGB to rise and fall was the same, wherein the RBC level of the blank group exceeded the normal range at T2, and both RBC and HGB of the two groups of experimental cats declined at T3, wherein there was a significant difference between RBC and HGB before and after transportation of the blank group, and the HGB at experimental groups T3 and T4 had a significant difference (P < 0.05).

Example 9 Effect of polypeptide on BDNF and HPA Axis hormone in cat serum

The results of the detection of BDNF, HPA axis hormones in cat serum by ELISA are shown in fig. 26-29, where BDNF levels were significantly reduced at T3 (P < 0.05) for both and slightly higher for the experimental group than for the blank group (fig. 26). CRH increased over time in both treatment groups, with CRH levels significantly higher in the T2 blank than in the T1, significantly increased in both T3 (P < 0.05), significantly decreased in T4 (P < 0.05), and significantly lower in the T4 experimental group than in the blank (fig. 27). ACTH levels were significantly higher for both groups at T2 than T1 (P < 0.05), and also significantly higher for T3 than T2 (P < 0.05), and significantly lower for both groups at T4 (P <0.05, fig. 28). There was no significant difference between the two groups during the same period. COR levels were lower in the experimental group than in the blank group at T3, and significantly elevated in the T4 space-time white group (P < 0.05) and significantly higher than in the experimental group (P <0.05, fig. 29).

Example 10 Effect of polypeptide on other serum hormones and antioxidant indicators in cat serum

The results of testing other serum hormones and antioxidant indicators in cat serum by ELISA and biochemical colorimetry are shown in fig. 30-36, wherein the SAA level of the experimental group at T2 was significantly reduced (P < 0.05), the SAA level of both groups was significantly increased after transport, the level of the experimental group was significantly lower than that of the blank group (P < 0.05), and the SAA level of both groups was slightly increased at T4, but the experimental group was still significantly lower than that of the blank group (P <0.05, fig. 30). From figure 31 it can be seen that Apo-A1 levels were significantly higher in the experimental group at T2 than in the T1 and blank groups (P < 0.05). Apo-A1 levels were significantly reduced in both groups after transport (P < 0.05), but the extent of the blank decrease was more pronounced (P < 0.05).

The T-AOC level of the blank group was significantly lower than T2 at T3 (P < 0.01) and increased to a level close to T2 at T4 (P < 0.05), whereas the T-AOC level of the experimental group only tended to decrease at T3 (P < 0.10) and returned to the pre-shipment level at T4 (fig. 32). MDA levels in the blank were significantly increased at T2 (P < 0.01) and maintained at a higher level at all times, the experimental group had significantly higher than the blank at T1, there was a significant trend of increase in T2 (P < 0.10), and MDA levels in the experimental group were decreased at T4 and significantly lower than the blank (P <0.05, fig. 33). From fig. 34 it can be seen that the GSH-Px levels of both groups rose steadily, with the levels of the experimental group rising significantly at T3 (P < 0.05) and both groups falling significantly at T4 (P < 0.05). Both groups showed significant increases in SOD and CAT levels at T2, with no significant change in SOD levels at T3 for the experimental group, the rest decreased significantly (P < 0.05), and CAT levels at T4 increased significantly for both groups (P <0.05, fig. 35-36).

Example 11 Effect of polypeptide on transcriptome changes in cat blood under transport stress and strange scenario stress

Transcriptomes were commissioned to a third party enterprise, wherein the results of the gene expression analysis are shown in FIG. 37, wherein the transcriptome analysis revealed 2315 Differentially Expressed Genes (DEG) by comparison of time points of experimental and blank groups, wherein 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 (FIG. 37 b). Venn diagram analysis revealed 27 differentially co-expressed genes at all time points compared to the control group (FIG. 37 a). And as can be seen from fig. 37b, the experimental group had up-regulated expression of 1159 genes and down-regulated expression of 1156 genes compared to the gene expression corresponding to the blank group at different time points.

The results of the Gene Ontology (GO) enrichment analysis are shown in fig. 38-41, where the biological function of DEG was determined by GO enrichment analysis. At T1, the experimental group significantly up-regulated Biological Processes (BP) related to immune effect process, viral response, defense against external organisms, etc. (P < 0.01), and significantly down-regulated biological processes related to bioadhesion, triglyceride transport, viral entry into host cells, regulation of cell differentiation, etc. (P <0.01, fig. 38). After the end of the pre-feeding period, the biological process terms significantly up-regulated by the experimental group are cis-trans-synaptic signalling, leukotriene metabolic process, eicosanoic acid metabolic process, protein activation cascade, etc., significantly down-regulated by viral process, viral life cycle, B-cell steady-state proliferation, DNA recombination, acylglycerol transport, etc. (P <0.01, fig. 39). Following transport, the biological processes associated with complement activation (classical/alternative pathway), inflammatory response, muscle adaptation, modulation of hippo signals, etc. are significantly up-regulated (P < 0.01), whereas necrotic apoptotic signaling pathways, vascular development, multiple biological processes, symbiotic processes, viral particle assembly, etc. are significantly down-regulated (P <0.01, fig. 40). GO enrichment analysis after convalescence showed that biological processes related to cell signaling, cell response to external stimuli, metabolic processes of fatty acid derivatives, etc. were significantly up-regulated in the experimental group (P < 0.01), biological processes related to multiple biological processes, virus-related life processes (assembly, life cycle, latency, invasion of host cells), DNA metabolic processes, etc. (P <0.01, fig. 41).

The results of the KEGG pathway enrichment analysis are shown in fig. 42-45, wherein 285 genes participated in 150 pathways at T1, 108 genes participated in 77 pathways at T2, 223 genes participated in 146 pathways at T3, and 197 genes participated in 128 pathways at T4, compared to the blank. The up-regulated pathways that the experimental group significantly enriched at T1 included RIG-I like receptor signaling pathways, tryptophan metabolism, cytokine receptor interactions, etc. (P < 0.05). The up-regulated pathways that are significantly enriched at T2 are pantothenate and coa biosynthesis, complement and coagulation cascades, arachidonic acid metabolism, ECM receptor interaction signaling pathways, etc. (P < 0.05), while the adipocyte cytokine signaling pathways, fat digestion and absorption, neuroactive ligand receptor interaction pathways, etc. are significantly down-regulated (P < 0.05). At T3, pathways such as complement and coagulation cascade pathways, arachidonic acid metabolism, purine metabolism are significantly up-regulated in the experimental group (P < 0.05), and TGF- β signaling pathway, TNF signaling pathway, viral carcinogenesis, toll-like receptor signaling pathway, etc. are significantly down-regulated (P < 0.05). At T4, erbB signaling pathways, dopamine synapses, retrograde endogenous cannabinoid signaling pathways are markedly upregulated.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims

1. A polypeptide for improving stress response symptoms of cats is characterized by having an amino acid sequence shown in SEQ ID NO. 1.

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

3. Recombinant expression vector comprising the nucleotide sequence of claim 2, wherein the vector is ppiczαa.

4. A host bacterium comprising the recombinant expression vector of claim 3, wherein the host bacterium is Pichia pastoris (Pichia pastoris) GS115 strain.

5. The method for producing a polypeptide according to claim 1, comprising the steps of:

(1) Constructing a recombinant expression vector, namely inserting the nucleotide sequence of claim 2 into the downstream of an alpha factor secretion signal of the pPICZ alpha A vector to obtain a recombinant plasmid;

(3) Inoculating positive clones into BMGY culture medium, culturing at 30 ℃ and 200rpm until OD ₆₀₀ = 6.0, centrifugally collecting thalli, transferring to BMMY culture medium, and inducing expression for 48-72 hours at methanol concentration of 0.5-1.0%;

6. The method according to claim 5, wherein the polypeptide is expressed in a higher amount at an induction temperature of 25℃in the step (3).

7. Use of the polypeptide of claim 1 for the preparation of a product for ameliorating symptoms of stress in a cat, wherein the symptoms of stress include elevated serum cortisol levels, abnormal behavior, or increased secretion of inflammatory factors.

8. A composition for ameliorating symptoms of stress in a cat comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier, wherein the carrier is starch, microcrystalline cellulose or starch slurry.

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 for preparing anti-stress feed additive or oral liquid for pets.