Anti-swine epidemic diarrhea virus antibacterial peptide LR18 and feed

Abstract

The application discloses an antibacterial peptide LR18 against porcine epidemic diarrhea virus and feed. The amino acid sequence of the antibacterial peptide LR18 against the porcine epidemic diarrhea virus is shown as SEQ ID NO:1. The application designs an antibacterial peptide LR18, evaluates the inhibiting effect of the antibacterial peptide on the porcine epidemic diarrhea virus, and finds that the antibacterial peptide can effectively inhibit the replication of the porcine epidemic diarrhea virus in vitro. The inhibiting effect occurs in the replication stage of the porcine epidemic diarrhea virus infection. Furthermore, the antibacterial peptide LR18 has the inhibiting effect on the virus at any time of the virus replication, more accurately, mainly in the first 18 hours of the replication stage. In addition, the antibacterial peptide LR18 component is clear and single, which lays a foundation and provides convenient conditions for applying the antibacterial peptide LR18 to the molecular or cytological research of the porcine epidemic diarrhea virus and the research of the antiviral additive.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of feed additives, in particular to an antibacterial peptide LR18 against porcine epidemic diarrhea virus and a feed. BACKGROUND

[0002] Since the 21st century, major viral diseases of livestock and poultry have been occurring globally, which not only caused a series of public health emergencies, but also caused great harm to human health and social development, and seriously hindered the healthy development of animal husbandry, causing serious economic losses. Porcine epidemic diarrhea (PED) can cause diarrhea and wasting in pig herds, and the mortality rate of infected piglets can reach 80% to 100%, causing huge losses to the global pig industry, and effective measures are urgently needed for specific prevention and control, but at present the disease is mainly prevented by immunization, and there is no suitable and efficient drug or feed additive. With the development of molecular biology technology, antibacterial peptides with broad-spectrum antibacterial, antiviral and other biological activities have gradually become one of the important additives for the development of feed alternative products.

[0003] Antibacterial peptides refer to small molecular polypeptides with antibacterial and other biological activities derived from organisms, which are important components of natural immunity of organisms and can resist invasion of foreign pathogens, regulate immune system and promote wound healing. Antibacterial peptides are generally composed of 20 to 60 amino acids, with a molecular weight of about 2 to 7 kDa, a high isoelectric point and good thermal stability. They not only have broad-spectrum antibacterial activity, but also have antiviral, antifungal and antitumor activity, and do not affect the physiological metabolism of normal cells and other activities, and will not cause damage to the body. Due to its unique antibacterial mechanism, it is not easy to develop drug resistance, and has broad application prospects in disease prevention and treatment.

[0004] Porcine epidemic diarrhea virus (PEDV) belongs to the family of Nidovirales, the family of Coronaviridae, the subfamily of Coronavirinae and the genus of alpha-coronavirus, and is the pathogen of PED. The disease is a highly contagious enteric disease characterized by diarrhea, vomiting, dehydration and high mortality rate in suckling piglets. Since 2011, the PEDV has emerged as a variant strain, with a mortality rate of 80% to 100% in newborn piglets, causing great harm to the pig industry. At present, there is no particularly effective drug for treating PED in the market, and in the background of PED causing great harm to the pig industry, it is particularly important to design a new type of antibacterial peptide as a new green feed additive for preventing viral diseases. SUMMARY

[0005] The main purpose of the embodiment of the present application is to provide an antibacterial peptide LR18 and feed against porcine epidemic diarrhea virus, aiming to design a new antibacterial peptide LR18 to be applied to feed to prevent viral diseases.

[0006] The technical solution of the present application to solve the above technical problems is to provide an antibacterial peptide LR18 against porcine epidemic diarrhea virus, wherein the amino acid sequence of the antibacterial peptide LR18 against porcine epidemic diarrhea virus is shown as SEQ ID NO: 1.

[0007] To solve the above technical problems, the present application further provides a feed, which comprises the antibacterial peptide LR18 against porcine epidemic diarrhea virus described above, and the proportion of the antibacterial peptide LR18 against porcine epidemic diarrhea virus is 200g / ton.

[0008] In an embodiment of the present application, the feed comprises the following components:

[0009] 60-70 parts of corn;

[0010] 8-12 parts of puffed soybean meal;

[0011] 7-10 parts of corn alcohol dregs;

[0012] 3-5 parts of germ meal;

[0013] 4-8 parts of rice bran meal;

[0014] 1-2 parts of stone powder;

[0015] 2-3 parts of calcium hydrogen phosphate;

[0016] 0.5-1 part of sodium chloride;

[0017] 0.5-1.5 parts of lysine;

[0018] 0.5-1.5 parts of bentonite;

[0019] 0.5-4.3 parts of premix;

[0020] 100 parts in total;

[0021] The premix comprises the antibacterial peptide LR18 against porcine epidemic diarrhea virus.

[0022] The present application has the following beneficial effects:

[0023] 1. The anti-swine epidemic diarrhea virus antibacterial peptide according to the present application, a kind of antibacterial peptide LR18 is designed to evaluate its inhibitory effect on swine epidemic diarrhea virus, and the antibacterial peptide can effectively inhibit the replication of swine epidemic diarrhea virus in vitro. The inhibitory effect occurs in the replication stage of swine epidemic diarrhea virus infection. Further, the antibacterial peptide LR18 has inhibitory effect on the virus at any time of viral replication, more specifically, mainly in the first 18 hours of the replication stage. In addition, the antibacterial peptide LR18 component is clear and single, which lays a foundation and provides convenient conditions for the application of antibacterial peptide LR18 to molecular or cellular studies of swine epidemic diarrhea virus and the study of antiviral additives.

[0024] 2. The anti-swine epidemic diarrhea virus antibacterial peptide according to the present application, the antibacterial peptide LR18 can inhibit the replication of swine epidemic diarrhea virus, and when the concentration of antibacterial peptide LR18 is as high as 110 μg / mL, it can still ensure that the normal cell activity rate is more than 80%. When the concentration of antibacterial peptide is 20 μg / mL (i.e. the proportion of antibacterial peptide LR18 for anti-swine epidemic diarrhea virus is 200 g / ton), the virus titer of swine epidemic diarrhea virus is reduced from 10 -4.46 to 10 -2.44 . Compared with the commonly used vaccine immunization, the preparation of functional feed additive for anti-swine epidemic diarrhea is simple, convenient to use, low in cost and long in action period.

[0025] 3. The anti-swine epidemic diarrhea virus antibacterial peptide according to the present application, the antibacterial peptide LR18 can inhibit the replication of swine epidemic diarrhea virus, and the antibacterial peptide LR18 can still play an antiviral role in vitro under the condition of 37 DEG C. The antibacterial peptide is incubated for 60 min under the optimum condition, and the effect of anti-swine epidemic diarrhea virus is the best. When the antibacterial peptide LR18 is incubated with swine epidemic diarrhea virus, the inhibition rate of virus is 56%; when the antibacterial peptide LR18 is added first and then the swine epidemic diarrhea virus is added, the inhibition rate of virus is 40%; when the swine epidemic diarrhea virus is added first and then the antibacterial peptide LR18 is added, the inhibition rate of virus is 35%, which proves that the antibacterial peptide LR18 has inhibitory effect on swine epidemic diarrhea virus. This lays a theoretical foundation for the use of antibacterial peptide LR18 to prevent and treat swine epidemic diarrhea virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings from the structures shown in the drawings without creating any creative labor.

[0027] Figure 1The growth curve of normal Vero E6 cells;

[0028] Figure 2 The toxicity detection diagram of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application to Vero E6 cells;

[0029] Figure 3 The toxicity result diagram of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application at different concentrations to Vero E6 cells;

[0030] Figure 4 The dose-dependent result diagram of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application to the activity of anti-swine epidemic diarrhea virus;

[0031] Figure 5 The in-vitro time-effect observation result diagram of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application;

[0032] Figure 6 The electron microscope result diagram of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application to directly acting swine epidemic diarrhea virus;

[0033] Figure 7 The indirect immunofluorescence result diagram of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application to directly acting swine epidemic diarrhea virus;

[0034] Figure 8 The diagram of the influence of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application on IL-17;

[0035] Figure 9 The diagram of the influence of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application on IL-1β;

[0036] Figure 10 The diagram of the influence of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application on TNF-α;

[0037] Figure 11 The diagram of the influence of the anti-swine epidemic diarrhea virus antibacterial peptide LR18 of the present application on the nucleic acid copy number of PEDV. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0039] It should be noted that all the directionality indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present application are only used to explain the relative position relationship, motion condition, etc. between components in a certain specific posture (as shown in the drawings), and if the specific posture changes, the directionality indications will also change accordingly.

[0040] In addition, the descriptions such as "first", "second" and the like in the present application are only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second" can be explicitly or implicitly included at least one of the features. In the description of the present application, the meaning of "several", "a plurality of" is at least two, for example, two, three, etc., unless otherwise explicitly specified.

[0041] In the present application, unless otherwise explicitly specified and limited, the terms "connection", "fixation" and the like should be understood in a broad sense, for example, "fixation" can be fixed connection, or detachable connection, or integral; can be mechanical connection, or electrical connection; can be direct connection, or indirect connection through intermediate medium, can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise explicitly limited. For ordinary skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0042] In addition, the technical solutions of various embodiments of the present application can be combined with each other, but it must be based on the fact that ordinary skilled in the art can realize it, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is not within the protection scope required by the present application.

[0043] The present application provides an antibacterial peptide LR18 against porcine epidemic diarrhea virus and a feed, aiming to design a new antibacterial peptide LR18 to be applied to the feed to prevent viral diseases.

[0044] Experimental materials of the present application

[0045] 1. Source of antibacterial peptide, strain and cell

[0046] African green monkey kidney cells (Vero E6 cells) and porcine epidemic diarrhea virus CV777 strain were preserved by the Clinical Veterinary Medicine Laboratory of Jilin Agricultural University. The antibacterial peptide LR18 was synthesized by fixed phase chemistry, with a purity of 98%, and the amino acid sequence of the antibacterial peptide was: LRRLLRLPRRPLRLLRRL-NH2.

[0047] 2. Biochemical reagents

[0048] DMEM high-sugar liquid medium, 20x PBS buffer, bovine serum albumin (BSA), MTT and dimethyl sulfoxide were purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.; fetal bovine serum was purchased from Gibco Reagent Company; the three-antibiotic (penicillin-streptomycin-amphotericin B) solution and trypsin were purchased from Beijing Quanshi Gold Biotechnology Co., Ltd.; hydroxymethyl cellulose and crystal violet were purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.

[0049] 3. Preparation of main solutions

[0050] (1) Preparation of cell culture solution

[0051] Cell growth solution: DMEM high-sugar medium, 10% fetal bovine serum, 1% three-antibiotic (penicillin-streptomycin-amphotericin B);

[0052] Cell maintenance solution: DMEM high-sugar medium, 2% fetal bovine serum, 1% three-antibiotic (penicillin-streptomycin-amphotericin B).

[0053] (2) Preparation of antibacterial peptide diluent

[0054] 0.2% BSA solution containing 0.01% acetic acid: 0.1 g of BSA was weighed into 50 mL of distilled water, filtered with a 0.22 μM water filter, and each 1 mL was aliquoted and stored in a -80°C freezer for use.

[0055] (3) Preparation of MTT solution

[0056] 5 mg / mL MTT solution preparation: 0.1 g of MTT was weighed into 20 mL of PBS, filtered with a 0.22 μM water filter, and each 5 mL was aliquoted and stored in a -20°C freezer for use in the dark.

[0057] (4) Preparation of plaque assay solution

[0058] 2% hydroxymethyl cellulose: 30 g of hydroxymethyl cellulose was weighed into 1000 mL of double-distilled water, stirred thoroughly, autoclaved at 121°C for 30 min, and stored at room temperature.

[0059] (5) Preparation of artificial gastric juice (SGF)

[0060] According to the United States Pharmacopoeia 2004 edition, 16.4 mL of concentrated hydrochloric acid was taken, about 800 mL of distilled water was added, 10 g of pepsin was added, stirred, and then distilled water was added to make up to 1000 mL to obtain artificial gastric juice.

[0061] (6) Preparation of artificial intestinal juice (SIF)

[0062] According to the United States Pharmacopoeia 2004, take 6.8 g of potassium dihydrogen phosphate, add 500 mL of distilled water, adjust the pH value to 6.8 with 0.4% sodium hydroxide solution, take 10 g of trypsin and dissolve in appropriate amount of distilled water; after stirring, mix the two solutions, and then add water to 1000 mL, to obtain artificial intestinal fluid.

[0063] (7) Preparation of enteric microcapsules

[0064] Chitosan, sodium alginate and calcium chloride were used as raw materials, and the pH value of the solution was adjusted to 5.0. The mass fractions were 3%, 1% and 2% respectively. After stirring uniformly, pour into the mold, put into the 50°C drying oven, take out and dry again.

[0065] 4. Main instruments

[0066] Table 1 Main instruments used in the test

[0067] Instrument name Model Manufacturer Clean bench VS-840-1 Shanghai Boshen Medical Equipment Factory Electronic balance ME204E Mettler-toledo, Germany Vertical pressure steam sterilization pot YXQ-LS-50S II Shanghai Boshen Medical Equipment Factory Ultralow temperature refrigerator DW-86L626 Haier refrigerator Microplate reader Multiskan GO 1510 Shanghai Baju Industrial Company

[0068] Example 1: Detection of cytotoxicity of antibacterial peptide LR18

[0069] 1. Determination of Vero E6 cell growth curve

[0070] Based on MTT colorimetric method, the cell growth curve was drawn with time as the horizontal axis and OD value as the vertical axis. The specific experimental method is as follows:

[0071] (1) Single layer Vero E6 cells with good morphology in T25 culture bottle were trypsinized, added with growth solution and blown, to prepare single cell suspension, and the cell density was diluted to 1 x 10 5 / mL.

[0072] (2) Inoculate in 96-well plate, 0.2 mL per well, 3 replicates, set 7 groups and blank control group; place in 37°C, 5% CO2 incubator for culture (cell two liquid maintenance original culture liquid amount).

[0073] (3) Take out one group every 24 h, continuously for 7 d, add 20 μL of MTT solution to each well, incubate at 37°C for 4 h, remove the supernatant in the well, add 150 μL of DMSO to each well, shake for 10 min, and dissolve thoroughly.

[0074] (4) Set 490 nm wavelength on enzyme-linked immunosorbent assay instrument to detect absorbance value, record the results, repeat the experiment 3 times, and take the average value as the result.

[0075] (5) Draw the cell growth curve according to the results, with time as the horizontal coordinate and the measured cell OD value as the vertical coordinate.

[0076] From Figure 1It can be seen that passaged cells grow rapidly from 1 to 3 days, grow slowly from 3 to 5 days, and grow at the same rate from 6 to 7 days.

[0077] 2. Seed the single-cell suspension into a 96-well plate, 0.1 mL per well, to ensure a consistent cell concentration in each well; place in a 37°C, 5% CO2 incubator until the cells grow into a monolayer.

[0078] 3. Set up cell experimental groups (different concentrations of antimicrobial peptide), cell control groups (no antimicrobial peptide), and cell apoptosis-free groups; dilute the antimicrobial peptide with cell maintenance medium to different concentrations, discard the growth medium and add different concentrations of antimicrobial peptide, 0.1 mL per well, with 3 replicates for each concentration.

[0079] 4. After 48 hours, add 20 μL of MTT solution to each well and continue incubation at 37°C for 4 hours. After 4 hours, discard the supernatant, add 150 μL of DMSO to each well, shake for 10 minutes, and measure the absorbance at 490 nm on the ELISA reader. Record the results.

[0080] Cell protection rate = OD value of cell drug group / OD value of cell control group - OD value of cell apoptosis-free group.

[0081] Depend on Figure 2 It is evident that, with a cell protection rate of 80%, the highest concentration of the LR18 antimicrobial peptide was 110 μg / mL, and the concentrations of antimicrobial peptides used in subsequent experiments were all within the safe range. Figure 3 As can be seen under a light microscope, as the concentration of antimicrobial peptides increases, the cell morphology changes from normal to a certain degree of shrinkage.

[0082] Example 2: Dose-dependent effect of antimicrobial peptide LR18 against PEDV

[0083] (1) PEDV median infectious dose (TCID) 50 Determination of )

[0084] Cell suspensions were seeded into 96-well plates and cultured at 37°C in a 5% cell culture incubator until a monolayer of cells was formed. A virus experimental group and a normal cell negative control group were set up; the virus solution was serially diluted 10-fold with cell maintenance medium (10... -1 ~10 -9 The cell growth medium in the 96-well plate was aspirated, and 0.1 mL of virus solution of different concentrations was added to each well, with 8 replicates per concentration. Observations were performed continuously for 72 hours, and the cell prophylaxis (CPE) of each well was recorded. TCID was calculated using the Reed-Muench formula. 50 .

[0085] (2) Antimicrobial peptide antiviral dose-dependent

[0086] Vero E6 single cell suspension was inoculated into 96-well plates and placed in a cell incubator for culture to a single layer of cells; the antibacterial peptide LR18 was diluted to 20 μg / mL, 15 μg / mL, 10 μg / mL and 5 μg / mL respectively with PEDV virus liquid, and incubated at 37°C for 1 h; the mixed virus liquid was serially diluted by 10 -1 ~10 -9 times, the number of CPE wells was observed and recorded, and the TCID 50 was calculated according to the Reed-Muench formula.

[0087] The determination results of the median infectious dose (TCID 50 ) of PEDV are shown in Table 2, and the TCID 50 value of PEDV is 10 -4.5 according to the Reed-Muench method.

[0088] Table 2 Determination results of the median infectious dose (TCID 50 ) of PEDV

[0089] Dilution CPE No CPE Total CPE No total CPE Percentage (%) 10 -1 ]] 8 0 32 0 100 10 -2 ]] 8 0 24 0 100 10 -3 ]] 8 0 16 0 100 10 -4 ]] 6 2 8 2 80 10 -5 ]] 2 6 2 12 14.5 10 -6 ]] 0 8 0 20 0 10 -7 ]] 0 8 0 28 0 10 -8 ]] 0 8 0 36 0 10 -9 ]] 0 8 0 40 0

[0090] As can be seen from Figure 4 , the LR18 has the effect of inhibiting the activity of the virus, and the virus titer decreases obviously, and the inhibition effect increases with the increase of the concentration within a certain range, showing a dose-dependent manner. Under the condition that the concentration of the antibacterial peptide is 20 μg / mL, the virus titer can be reduced from 10 -4.46 to 10 -2.44 . That is, the proportion of the antibacterial peptide LR18 in the feed for resisting porcine epidemic diarrhea virus is 200 g / ton.

[0091] Example 3: In vitro time observation of antibacterial peptide LR18

[0092] (1) The cell suspension was added to a 12-well plate and placed in a 37°C, 5% CO2 cell incubator for culture to a single layer of cells. The antibacterial peptide was placed in a 37°C incubator for 0 h, 24 h and 48 h.

[0093] (3) The above antibacterial peptide was diluted to 20 μg / mL with virus liquid and incubated at 37°C for 1 h, and a virus control was set.

[0094] (4) The diluted mixed liquid and the virus control were infected with each well of cells, and the virus concentration was 600 PFU / mL.

[0095] (5) The virus liquid was co-cultured with the cells for 1 h, and then the supernatant was aspirated and washed with PBS for 2 times, 2% hydroxymethyl cellulose was mixed with an equal amount of DMEM, and then 1 mL of the mixed liquid was added to each well after thorough mixing.

[0096] (6) After 72 hours, add 1 ml of 10% formaldehyde solution to each well, fix the cells for 30 minutes, aspirate the liquid, add 1 ml of crystal violet to each well for staining for 10 minutes, and rinse slowly with running water.

[0097] like Figure 5 It is evident that the antimicrobial peptide LR18 can still exert its in vitro anti-PEDV effect at 37℃. Therefore, the accuracy and reliability of the experiment will not be affected by the culture conditions at 37℃.

[0098] Example 4: Optimal in vitro conditions for antimicrobial peptide LR18

[0099] (1) Inoculate the cell suspension into a 96-well plate and place it in a 37°C, 5% CO2 cell culture incubator until it grows into a monolayer of cells.

[0100] (2) Set different incubation times and incubation temperatures. The incubation times were 60 min, 30 min, 15 min, and 5 min, with the same temperature and other variables; the incubation temperatures were 37℃, 25℃, and 4℃, with the same incubation time and other variables.

[0101] (3) Dilute the above mixture by 10 times (10 times) -1 ~10 -9 Cells were seeded at 0.1 mL per well, and observed continuously for 72 hours. The number of wells with lesions was recorded, and TCID was calculated. 50 .

[0102] Table 3 shows that, comparing the combined effects of antimicrobial peptide LR18 and PEDV at different temperatures, temperature had no significant effect on the in vitro anti-PEDV activity of antimicrobial peptide LR18. Table 4 shows that the optimal action time of antimicrobial peptide LR18 against PEDV is 60 min.

[0103] Table 3 Results of the optimal temperature for the in vitro action of antimicrobial peptide LR18

[0104]

[0105] Table 4 Results of the optimal in vitro action time of antimicrobial peptide LR18

[0106]

[0107] Example 5: In vitro antiviral assay of antimicrobial peptide LR18

[0108] (1) Vero E6 single-cell suspension was seeded into 96-well plates, with 0.1 mL added to each well. After forming a monolayer of cells, in vitro antiviral experiments were performed.

[0109] (2) Set the different time of adding antibacterial peptide, the test is carried out according to the following three methods: ① The blocking effect of LR18 on PEDV adsorption and invasion of cells; antibacterial peptide LR18 is incubated with Vero cells at 37℃, 5% CO2 incubator for 1h, and then the cells are infected with PEDV. ② The direct inactivation effect of LR18 on PEDV; the cells are infected after antibacterial peptide LR18 is co-incubated with the virus for 1h. ③ The inhibition effect of antibacterial peptide LR18 on the proliferation of PEDV; the cells are infected with the virus, and then the antibacterial peptide is added.

[0110] (3) MTT colorimetric method is carried out for each group of triplicates, the cell survival rate and the inhibition rate of LR18 on PEDV are calculated according to the formula, and the test is repeated three times.

[0111] Formula 1: Cell survival rate (%) = OD value of sample treatment group / OD value of normal cell control group x 100%.

[0112] Formula 2: PEDV inhibition rate (%) = (OD value of sample treatment group-OD value of PEDV control group) / OD value of PEDV control group x 100%.

[0113] As shown in Table 5, the in vitro inhibition rate of antibacterial peptide LR18 on PEDV is 35%-56%.

[0114] Table 5 Results of in vitro antiviral effect of antibacterial peptide LR18

[0115]

[0116]

[0117] Example 6: Observation of the effect of antibacterial peptide LR18 on PEDV by negative staining electron microscopy

[0118] (1) Virus is inoculated in a cell culture bottle, and after obvious cytopathic effect is generated, the cells are fully lysed by freezing and thawing 3 times, and the cell debris is removed by centrifugation at 4000r / min for 30min.

[0119] (2) The supernatant is added to an ultracentrifuge tube, and PBS solution is added to fill it; another ultracentrifuge tube is added with PBS solution, weighed and leveled, and then centrifuged at 26000r / min for 2.5h by low-temperature ultracentrifuge.

[0120] (3) Discard the supernatant, invert the tube on filter paper to absorb the water in the tube, add 100μL PBS to resuspend the virus particles, and then suck them into an EP tube

[0121] (4) Set the antibacterial peptide control group and the virus experimental group; resuspend the virus particles and mix with the antibacterial peptide, incubate at 37℃ for 1h, then drop the sample on a copper mesh, dry naturally, add a staining agent for negative staining, and observe the changes of virus particles by electron microscopy.

[0122] By Figure 6 It can be seen that compared with the normal culture PEDV group, the PEDV envelope of the antibacterial peptide group falls off, which proves that the antibacterial peptide can destroy the envelope of PEDV.

[0123] Example 7: Effect of antibacterial peptide LR18 on PEDV observed by indirect immunofluorescence experiment

[0124] (1) Single cell suspension was inoculated into a 12-well plate and placed in an incubator until it grew to a monolayer

[0125] (2) The virus solution was diluted with antibacterial peptide to 5 μg / ml and 20 μg / ml, and the mixture was incubated in an incubator at 37°C for 1 h. The mixture was diluted to 800 PFU / mL and inoculated into the cells.

[0126] (3) The supernatant was aspirated and washed 3 times with -20°C ice alcohol for 20 min, and then washed 3 times.

[0127] (4) PEDV-S primary antibody was diluted 1:2000, 500 μL was added to each well, incubated for 1 h, and washed 5 times, each time for 5 min on a shaking table.

[0128] (5) FITC-IgG fluorescent secondary antibody was diluted 1:100, 300 μL was added to each well, and incubated in the dark for 1 h. The above step was repeated.

[0129] (6) The sample was observed under a fluorescence microscope.

[0130] By Figure 7 It can be seen that when the antibacterial peptide LR18 with a concentration of 5 μg / mL and 20 μg / mL is co-incubated with PEDV, it is found that the antibacterial peptide can act on the envelope of the virus under the observation of immunofluorescence electron microscopy.

[0131] Example 8: Effect of enteric microcapsules coated with antibacterial peptide LR18 on artificial gastric juice and artificial intestinal juice

[0132] After the enteric microcapsules coated with antibacterial peptide LR18 were treated in artificial gastric juice for 3 h and then transferred to artificial intestinal juice for 7 h, the above treatment time was determined according to the digestion and absorption time of food in the stomach and intestine. The stability of the enteric microcapsules was determined by the swelling rate. The swelling rate was determined by measuring the volume of 10 randomly selected small enteric microcapsules with a vernier caliper, and the average value was calculated by repeating the measurement 3 times. The swelling rate was calculated by the formula: Sw = DT / DO x 100%, Sw: swelling rate; DO: volume of enteric microcapsules before treatment; DT: volume of enteric microcapsules after treatment.

[0133] From Table 6 and Table 7, it can be seen that the swelling rate of the enteric microcapsule coated with antibacterial peptide LR18 is reduced very slowly in artificial gastric juice for 3 h, almost no swelling in 0-0.5 h, and the swelling rate is only reduced by 16.2% at 3 h, indicating that the enteric microcapsule material has high resistance to gastric acid and pepsin. When it is placed in artificial intestinal juice, the swelling rate is significantly reduced within 1 h, and it is completely dissolved at 3 h, indicating that the enteric microcapsule can be well released in the intestinal environment, so that the antibacterial peptide LR18 reaches the intestinal colonization and is absorbed to exert the therapeutic effect.

[0134] Table 6 Swelling rate of enteric microcapsule in artificial gastric juice

[0135] Time (h) Swelling rate (%) 0 100 0.5 98.3 1 97.6 2 89.5 3 83.8

[0136] Table 7 Swelling rate of enteric microcapsule in artificial intestinal juice

[0137] Time (h) Swelling rate (%) 0 100 0.5 75.5 1 34.3 2 9.8 3 0

[0138] Example 9: ELISA method for detecting changes in IL-17, IL-1β, TNF-α content in plasma

[0139] Ten piglets with a body weight of 20-25 Kg were divided into two groups, one group was added with antibacterial peptide LR18 functional additive, and the other group was a normal feeding group. Each pig was infected with 5 mL of 1.0 MOI PEDV, and 7 days after infection, the blood samples of the pigs were collected from the anterior vena cava into a sterile EDTA anticoagulant tube, mixed for 10-20 min, and then centrifuged in a centrifuge at 4000 r / min for 10 min. Commercial ELISA kit was used to compare the changes of inflammatory factors IL-17, IL-1β, TNF-α in the peripheral blood of the two groups of pigs.

[0140] From Figure 8 , Figure 9 and Figure 10 , it can be seen that the addition of antibacterial peptide LR18 in feed can reduce the inflammatory factors IL-17, IL-1β, TNF-α in the peripheral blood of pigs, and the difference is significant compared with the normal group (P<0.05), indicating that the addition of antibacterial peptide LR18 can reduce the inflammation in the intestinal tract of pigs.

[0141] Example 10: Detection of virus shedding in infected pigs by qPCR method

[0142] Using the PEDV infection model of pigs established in the laboratory, the anal swabs of the test pigs were collected on the 3rd day after the pigs were infected, the total viral RNA was extracted and reverse transcribed into cDNA, and the virus shedding amount was detected by using a commercial qPCR kit to evaluate the effect of enteric microcapsule coated with antibacterial peptide LR18 in preventing PEDV.

[0143] FromFigure 11 It can be seen that the amount of pig feces detoxification containing coated antibacterial peptide LR18 added in feed is significantly reduced, and the difference is extremely significant compared with the normal group (P<0.01), which shows that the addition of functional feed additive containing coated antibacterial peptide LR18 can reduce the viral load of PEDV in infected pig feces.

[0144] Example 11: Field experiment

[0145] As shown in Table 8, the effective rate of the anti-PEDV functional feed additive developed by the present application for preventing PED is 99%, which proves that the application effect is good and can be applied to clinical practice.

[0146] Table 8: Field experiment results

[0147] Group Number of experimental pigs Number of diarrhea pigs Diarrhea rate (%) Efficiency (%) Add antibacterial peptide LR18 group 500 5 1％ 99％ No antibacterial peptide LR18 group 500 60 12％ ---

[0148] As shown in the above examples 1-12, 20 μg / mL antibacterial peptide LR18 has good in vitro anti-PEDV effect. By preparing enteric microcapsules and conducting pig feeding experiments, it is proved that the addition of antibacterial peptide LR18 functional additive group can reduce pig intestinal inflammation, and the effective rate of preventing porcine epidemic diarrhea is 99%. Through the study of antibacterial peptide LR18 inhibiting PEDV replication, a theoretical basis and technical conditions are provided for the development of anti-porcine epidemic diarrhea functional feed additive.

[0149] Example 12: A feed

[0150] The feed comprises the above antibacterial peptide LR18 against porcine epidemic diarrhea virus, and the proportion of the antibacterial peptide LR18 against porcine epidemic diarrhea virus is 200 g / ton.

[0151] In an embodiment of the present application, the feed comprises the following components:

[0152] Corn 606 kg;

[0153] Puffed soybean meal 105 kg;

[0154] Corn alcohol dregs 80 kg;

[0155] Embryo meal 40 kg;

[0156] Rice bran meal 57.5 kg;

[0157] Stone powder 15 kg;

[0158] Calcium hydrogen phosphate 25 kg;

[0159] Sodium chloride 8 kg;

[0160] Lysine (70%) 10 kg;

[0161] Bentonite 10.5kg;

[0162] Premix 43kg;

[0163] Total 1000kg;

[0164] The premix comprises the anti-swine epidemic diarrhea virus antibacterial peptide LR18, and the antibacterial peptide LR18 is 0.2kg.

[0165] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

[0166]

Claims

1. Application of the antibacterial peptide LR18 in the preparation of a feed against porcine epidemic diarrhea virus, characterized in that, The antibacterial peptide LR18 against porcine epidemic diarrhea virus, the proportion of the antibacterial peptide LR18 against porcine epidemic diarrhea virus is 200g / ton; the amino acid sequence of the antibacterial peptide LR18 against porcine epidemic diarrhea virus is shown as SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The feed against porcine epidemic diarrhea virus comprises the following components: 60-70 parts of corn; 8-12 parts of puffed soybean meal; 7-10 parts of corn alcohol dregs; 3-5 parts of germ meal; 4-8 parts of rice bran meal; 1-2 parts of stone powder; 2-3 parts of calcium hydrogen phosphate; 0.5-1 part of sodium chloride; 0.5-1.5 parts of lysine; 0.5-1.5 parts of bentonite; 0.5-4.3 parts of premix; 100 parts in total; The premix comprises the antibacterial peptide LR18 against porcine epidemic diarrhea virus.

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