CN115960170A - Antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus and feed - Google Patents
Antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus and feed Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus and a feed. The amino acid sequence of the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus is shown in SEQ ID NO 1. The invention designs an antibacterial peptide LR18 to evaluate the inhibition effect of the antibacterial peptide on the porcine epidemic diarrhea virus, and the antibacterial peptide can effectively inhibit the replication of the porcine epidemic diarrhea virus from the outside of a body. The inhibition occurs during the replication phase of the porcine epidemic diarrhea virus infection. Furthermore, at any time during viral replication, more precisely mainly during the first 18h of the replication phase, the antimicrobial peptide LR18 has an inhibitory effect on the virus. In addition, the antibacterial peptide LR18 has a clear and single component, which lays a foundation and provides convenient conditions for applying the antibacterial peptide LR18 to the molecular or cytological research of porcine epidemic diarrhea viruses and the research of antiviral additives.
Description
Technical Field
The invention relates to the field of feed additives, in particular to an antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus and a feed.
Background
Since the 21 st century, serious epidemic diseases of livestock and poultry caused by viruses are in great momentum all over the world, so that a series of public health accidents are caused, great harm is caused to human health and social development, the healthy development of animal husbandry is seriously hindered, and serious economic loss is caused. The Porcine Epidemic Diarrhea (PED) can cause diarrhea and emaciation of a swinery, the death rate of infected piglets can reach 80-100%, huge loss is caused to the pig industry all over the world, effective measures are urgently needed for specific prevention and control, but the disease is mainly immune prevention at present, and no proper and efficient medicine or feed additive exists. With the development of molecular biology technology, antibacterial peptides with a wide spectrum of antibacterial, antiviral and other biological activities become one of the important additives for developing feed anti-bacterial products.
The antibacterial peptide is a micromolecular polypeptide which is derived from organisms and has biological activities such as antibiosis and the like, is an important component of natural immunity of the organisms, can resist invasion of foreign pathogens, regulates the immunity of the organisms and promotes wound healing. The antibacterial peptide generally consists of 20-60 amino acids, has the molecular weight of about 2-7 kDa, high isoelectric point and good thermal stability, not only has broad-spectrum antibacterial activity, but also has antiviral, antifungal and antitumor activities, does not influence the activities such as physiological metabolism of normal cells, does not cause damage to organisms, is not easy to generate drug resistance due to the unique antibacterial mechanism, and has wide application prospect in the aspects of disease prevention and treatment and the like.
Porcine Epidemic Diarrheal Virus (PEDV), which belongs to members of the genera niidodales, coronaviridae, and α -coronaviruses, is the etiological agent of PED, a highly contagious enteric infectious disease characterized by diarrhea, vomiting, dehydration, and high lethality to suckling piglets. Since 2011, PEDV has a variant strain, the fatality rate to newborn piglets reaches 80-100%, and the damage to the pig industry is huge. At present, no medicament for treating PED (porcine epidemic diarrhea) is available in the market, and under the background that PED causes great harm to the pig industry, a novel antibacterial peptide is designed to be used as an additive of a novel green feed, so that the novel antibacterial peptide is particularly important for preventing viral diseases.
Disclosure of Invention
The invention mainly aims to provide an antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus and a feed, and aims to design a novel antibacterial peptide LR18 to be applied to the feed for preventing viral diseases.
The technical scheme for solving the technical problems is to provide the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus, wherein the amino acid sequence of the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus is shown as SEQ ID NO. 1.
In order to solve the technical problem, the invention also provides a feed, which comprises: the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus has the ratio of 200 g/ton.
In one embodiment of the invention, the feed comprises the following components:
60-70 parts of corn;
8-12 parts of puffed soybean meal;
7-10 parts of corn distillers' grains;
3-5 parts of germ meal;
4-8 parts of rice bran meal;
1-2 parts of stone powder;
2-3 parts of calcium hydrophosphate;
0.5-1 part of sodium chloride;
0.5-1.5 parts of lysine;
0.5 to 1.5 portions of bentonite;
0.5 to 4.3 portions of premix;
the total amount is 100 parts;
wherein the premix comprises the anti-porcine epidemic diarrhea virus antibacterial peptide LR18.
The invention has the beneficial effects that:
1. the invention relates to an antibacterial peptide LR18 for resisting porcine epidemic diarrhea, which is used for evaluating the inhibition effect of the antibacterial peptide LR18 on porcine epidemic diarrhea viruses and can effectively inhibit the replication of the porcine epidemic diarrhea viruses in vitro. The inhibition occurs during the replicative phase of porcine epidemic diarrhea virus infection. Furthermore, at any time during viral replication, more precisely mainly during the first 18h of the replication phase, the antimicrobial peptide LR18 has an inhibitory effect on the virus. In addition, the antibacterial peptide LR18 has a clear and single component, which lays a foundation and provides convenient conditions for applying the antibacterial peptide LR18 to the molecular or cytological research of porcine epidemic diarrhea viruses and the research of antiviral additives.
2. According to the antibacterial peptide for resisting the porcine epidemic diarrhea virus, disclosed by the invention, the antibacterial peptide LR18 can inhibit the replication of the porcine epidemic diarrhea virus, and when the concentration of the antibacterial peptide LR18 is as high as 110 mu g/mL, the activity rate of normal cells can be still ensured to be more than 80%. When the concentration of the antibacterial peptide is 20 mug/mL (namely the ratio of the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus is 200 g/ton), the virus titer of the porcine epidemic diarrhea virus is 10 or less compared with that of a negative control group -4.46 Down to 10 -2.44 . Compared with the common vaccine immunity, the functional feed additive for resisting the porcine epidemic diarrhea is simple to prepare, convenient to use, low in cost and long in action period.
3. According to the antibacterial peptide for resisting the porcine epidemic diarrhea, disclosed by the invention, the antibacterial peptide LR18 can inhibit the replication of the porcine epidemic diarrhea virus, the antibacterial peptide LR18 can still play an in-vitro antiviral role at 37 ℃, the antibacterial peptide LR18 is incubated for 60min under the optimal condition, and the effect of resisting the porcine epidemic diarrhea virus is the best. When the antibacterial peptide LR18 is incubated with the porcine epidemic diarrhea virus, the inhibition rate of the virus is 56%; when the antibacterial peptide LR18 is added firstly and then the porcine epidemic diarrhea virus is added, the inhibition rate to the virus is 40 percent; when the porcine epidemic diarrhea virus is added firstly and then the antibacterial peptide LR18 is added, the inhibition rate of the antibacterial peptide LR18 on the virus is 35 percent, which proves that the antibacterial peptide LR18 has the inhibition effect on the porcine epidemic diarrhea virus. Thus laying a theoretical foundation for preventing and treating porcine epidemic diarrhea virus infection by using the antibacterial peptide LR18.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a normal Vero E6 cell growth curve;
FIG. 2 is a diagram of toxicity test of the antibacterial peptide LR18 against porcine epidemic diarrhea virus of the invention on Vero E6 cells;
FIG. 3 is a graph showing the toxicity of the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 against Vero E6 cells at different concentrations according to the present invention;
FIG. 4 is a graph showing the dose dependence of the activity of the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 of the present invention against porcine epidemic diarrhea virus;
FIG. 5 is a graph showing the in vitro aging observation result of the antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus;
FIG. 6 is an electron microscope result diagram of the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 of the invention against the direct-acting porcine epidemic diarrhea virus;
FIG. 7 is a graph showing the indirect immunofluorescence result of the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 directly acting on the porcine epidemic diarrhea virus;
FIG. 8 is a graph showing the effect of LR18, an antibacterial peptide against porcine epidemic diarrhea virus, on IL-17;
FIG. 9 is a graph showing the effect of LR18, an antibacterial peptide against porcine epidemic diarrhea virus, on IL-1 β;
FIG. 10 is a graph showing the effect of the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 of the present invention on TNF- α;
fig. 11 is a graph showing the effect of LR18, an antimicrobial peptide against porcine epidemic diarrhea virus, of the present invention, on the copy number of PEDV nucleic acid.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all directional indicators (such as up, down, left, right, front, and back \8230;) in the embodiments of the present invention are only used to explain the relative positional relationship between the components, the motion situation, etc. in a specific posture (as shown in the attached drawings), and if the specific posture is changed, the directional indicators are changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise explicitly stated or limited, the terms "connected", "fixed", and the like are to be understood broadly, for example, "fixed" may be fixedly connected, may be detachably connected, or may be integrated; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be interconnected within two elements or in a relationship where two elements interact with each other unless otherwise specifically limited. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of the technical solutions by those skilled in the art, and when the technical solutions are contradictory to each other or cannot be realized, the combination of the technical solutions should be considered to be absent and not to be within the protection scope of the present invention.
The invention provides an antibacterial peptide LR18 for resisting porcine epidemic diarrhea virus and feed, and aims to design a novel antibacterial peptide LR18 to be applied to feed for preventing viral diseases.
Experimental Material of the present invention
1. Antibacterial peptides, strains and cell sources
Vero E6 cells and porcine epidemic diarrhea virus CV777 strain, preserved by the clinical veterinary laboratory of Jilin agricultural university. The antibacterial peptide LR18 is chemically synthesized by adopting a stationary phase, the purity is 98%, and the amino acid sequence of the antibacterial peptide is as follows: LRRLLRLPRRPLRLLRRL-NH 2 。
2. Biochemical reagent
DMEM high-glucose liquid medium, 20 XPBS buffer, bovine Serum Albumin (BSA), MTT and dimethyl sulfoxide were purchased from Biotechnology engineering (Shanghai) GmbH; fetal bovine serum was purchased from Gibco reagent; triple-resistant (penicillin-streptomycin-amphotericin B) solution and pancreatin were purchased from beijing holo-type gold biotechnology limited; hydroxymethyl cellulose and crystal violet are available from Shanghai Michelin Biochemical technology, inc.
3. Preparation of the principal solution
(1) Preparation of cell culture solution
Cell growth solution: DMEM high glucose medium, 10% fetal bovine serum, 1% triantibody (penicillin-streptomycin-amphotericin B);
cell maintenance solution: DMEM high-glucose medium, 2% fetal bovine serum, 1% tris (penicillin-streptomycin-amphotericin B).
(2) Preparation of antibacterial peptide diluent
0.2% BSA solution with 0.01% acetic acid: 0.1g of BSA was weighed, dissolved in 50mL of distilled water, filtered through a 0.22. Mu.M aqueous filter, dispensed in 1mL portions, and frozen in a freezer at-80 ℃ for use.
(3) MTT method solution preparation
Preparation of 5mg/mL MTT solution: 0.1g of MTT was weighed, dissolved in 20mL of PBS, filtered through a 0.22. Mu.M aqueous filter, dispensed every 5mL, and frozen in a freezer at-20 ℃ in the dark for use.
(4) Preparation of plaque experimental solution
2% hydroxymethyl cellulose: weighing 30g of hydroxymethyl cellulose, adding into 1000mL of double distilled water, stirring thoroughly, autoclaving at 121 deg.C for 30min, and storing at room temperature.
(5) Preparation of artificial gastric juice (SGF)
Referring to United states Pharmacopeia 2004 edition, concentrated hydrochloric acid 16.4mL is taken, distilled water of about 800mL and pepsin of 10g are added, after stirring, the distilled water is added to a constant volume of 1000mL, and then the artificial gastric juice is obtained.
(6) Preparation of artificial intestinal juice (SIF)
Referring to United states Pharmacopeia 2004 edition, taking 6.8g of monopotassium phosphate, adding 500mL of distilled water, adjusting the pH value to 6.8 by using 0.4% sodium hydroxide solution, taking 10g of trypsin, and adding a proper amount of distilled water for dissolving; and stirring the two solutions, mixing, adding water to a constant volume of 1000mL to obtain the artificial intestinal juice.
(7) Preparation of enteric microcapsules
Chitosan, sodium alginate, calcium chloride and the like are used as raw materials, and the pH value of the solution is adjusted to 5.0. The mass fractions are 3%, 1% and 2%, respectively. Stirring evenly, pouring into a mould, putting into a drying box at 50 ℃, taking out and drying in the air.
4. Main instrument
TABLE 1 Main instruments used in the test
| Name of instrument | Type number | Manufacturer of the product |
| Super clean bench | VS-840-1 | Shanghai Bocheng Shi medical equipment factory |
| Electronic balance | ME204E | Germany Mettler-toledo |
| Vertical pressure steam sterilizing pot | YXQ-LS-50S II | Shanghai Bocheng medical equipment factory |
| Ultra-low temperature refrigerator | DW-86L626 | Haier refrigerator |
| Enzyme-linked immunosorbent assay (ELISA) instrument | Multiskan GO 1510 | Shanghai Bajiu Kogyo Co Ltd |
Example 1: detection of LR18 cytotoxicity as antibacterial peptides
1. Determination of Vero E6 cell growth curve
The experiment is based on an MTT colorimetric method, the time is taken as a horizontal axis, the OD value is taken as a vertical axis, and a cell growth curve is drawn, wherein the specific experimental method comprises the following steps:
(1) Digesting monolayer Vero E6 cells with good form in T25 culture flask with pancreatin, adding growth liquid, blowing to obtain single cell suspension, diluting cell density to 1 × 10 5 one/mL.
(2) Inoculating to a 96-well plate, wherein each well is 0.2mL, and 3 multiple wells are provided, and 7 groups and a blank control group are arranged; standing at 37 deg.C, 5% CO 2 Culturing in an incubator (two liquid changes of cells maintain the original culture liquid amount).
(3) One group was taken out every 24h for 7 days, 20. Mu.L of MTT solution was added to each well, incubation was carried out at 37 ℃ for 4h, supernatant was aspirated from the wells, 150. Mu.L of DMSO was added to each well, and shaking was carried out for 10min to dissolve sufficiently.
(4) Setting 490nm wavelength measurement light absorption value on enzyme-linked immunoassay instrument, detecting, recording result, repeating experiment for 3 times, and taking average value as result.
(5) According to the results, a cell growth curve is drawn, the abscissa represents time, and the ordinate represents the measured cell OD value.
As can be seen from FIG. 1, the passage cells grow rapidly in 1-3 days, slowly in 3-5 days, and uniformly in 6-7 days.
2. Inoculating the single cell suspension into a 96-well plate, wherein each well is 0.1mL, and the cell concentration of each well is ensured to be consistent; standing at 37 deg.C, 5% CO 2 In the incubator, the cells are grown into a monolayer.
3. Setting a cell experimental group (with different concentrations of antibacterial peptide), a cell control group (without antibacterial peptide) and a cell-free withering group; and (3) diluting the antibacterial peptide with cell maintenance solution to different concentrations, abandoning the growth solution, adding the antibacterial peptide with different concentrations, wherein each well is 0.1mL, and each concentration is 3 multiple wells.
4. And after 48h, adding 20 mu L of MTT solution into each hole, continuously culturing for 4h at 37 ℃, discarding supernatant, adding 150 mu L of DMSO into each hole, oscillating for 10min, setting a 490nm wavelength on an enzyme-linked immunoassay tester to measure a light absorption value, and recording results.
Cell protection rate = cell drug group OD value/cell control group OD value-no cell wither group OD value.
As can be seen from FIG. 2, the highest concentration of the LR18 antimicrobial peptide was 110 μ g/mL under the condition that the cell protection rate reached 80%, and then the concentrations of the experimental antimicrobial peptides were all within the safe concentration range. As can be seen from FIG. 3, the cell morphology shrank from normal to some extent as the concentration of the antimicrobial peptide increased under light.
Example 2: dose dependence of antimicrobial peptide LR18 against PEDV
(1) Half infection of PEDV (TCID) 50 ) Measurement of (2)
Inoculating the cell suspension into 96-well plate, culturing at 37 deg.C in 5% cell culture boxTo a monolayer of cells. Setting a virus experimental group and a normal cell negative control group; the virus solution was diluted 10-fold continuously with the cell maintenance solution (10) -1 ～10 -9 ) The cell growth medium in the 96-well plate was aspirated off, and 0.1mL of virus solution at different concentrations was added to each well, 8 replicates per concentration. Continuously observing for 72h, recording CPE of each hole, and calculating TCID according to Reed-Muench formula 50 。
(2) Antiviral dose dependence of antimicrobial peptides
Inoculating the Vero E6 single cell suspension into a 96-well plate, placing the 96-well plate in a cell culture box, and culturing the plate until a monolayer cell is obtained; respectively diluting the antibacterial peptide LR18 to 20 mu g/mL, 15 mu g/mL, 10 mu g/mL and 5 mu g/mL by using PEDV virus solution, and incubating for 1h at 37 ℃; the mixed virus solution was serially diluted by multiple (10) -1 ～10 -9 ) Observing and recording the number of holes with CPE, and calculating TCID according to Reed-Muench formula 50 。
Half the infected dose of PEDV (TCID) 50 ) The results of the measurement are shown in Table 2, and TCID of PEDV was calculated by the Reed-Muench method 50 Number 10 -4.5 。
TABLE 2 half infection amount of PEDV (TCID) 50 ) Measurement result of (2)
| Degree of dilution | With CPE | Without CPE | Total CPE | Without total CPE | Percent (%) |
| 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 |
As can be seen from fig. 4, LR18 has an effect of inhibiting viral activity, and the viral titer is significantly decreased, and the inhibitory effect increases with increasing concentration within a certain range, which is dose-dependent. Under the condition that the concentration of the antibacterial peptide is 20 mu g/mL, the virus titer can be made to be 10 -4.46 Down to 10 -2.44 . Namely, the ratio of the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus in the feed is 200 g/ton.
Example 3: in vitro aging observation of antibacterial peptide LR18
(1) Adding the cell suspension to a 12-well plate, standing at 37 ℃,5% CO 2 The cells in the cell culture chamber grow to a monolayer. The antibacterial peptide is placed in an incubator at 37 ℃ for 0h, 24h and 48h.
(3) The antibacterial peptide was diluted to 20. Mu.g/mL with a virus solution, incubated at 37 ℃ for 1 hour, and a virus control was set.
(4) The mixture and virus control were diluted to infect cells per well at a virus concentration of 600PFU/mL.
(5) And (3) co-culturing the virus liquid and the cells for 1h, then sucking and removing the supernatant, washing the supernatant for 2 times by PBS (phosphate buffer solution), mixing 2% hydroxymethyl cellulose with an equivalent amount of DMEM (DMEM), and adding 1mL of mixed liquid into each hole after fully and uniformly mixing.
(6) After 72h, adding 1ml of 10% formaldehyde solution into each hole, fixing the cells for 30min, removing the liquid by suction, adding 1mL of crystal violet into each hole, dyeing for 10min, and slowly washing by running water.
As shown in FIG. 5, the antibacterial peptide LR18 still can exert the effect of resisting PEDV in vitro at 37 ℃, so the accuracy and the reliability of the test are not influenced by the culture condition at 37 ℃.
Example 4: in vitro optimal conditions of antibacterial peptide LR18
(1) Inoculating the cell suspension into a 96-well plate, standing at 37 deg.C, 5% 2 And (5) in the cell incubator, growing a monolayer of cells.
(2) Different incubation times and incubation temperatures were set. The incubation time is 60min, 30min, 15min and 5min, and the temperature and other variables are the same; the incubation temperatures were set at 37 deg.C, 25 deg.C and 4 deg.C, and the incubation time and other variables were the same.
(3) Diluting the mixture by multiple times (10) -1 ～10 -9 ) Inoculating cells, 0.1mL per well, continuously observing for 72h, recording the number of lesion wells, and calculating TCID 50 。
As can be seen from table 3, comparing the results of the combined action of the antimicrobial peptide LR18 and PEDV at different temperatures, the temperature has no significant effect on the in vitro anti-PEDV effect of the antimicrobial peptide LR18. As can be seen from table 4, the optimal duration of action of the antimicrobial peptide LR18 on PEDV was 60min.
TABLE 3 optimal temperature results for LR18 antibacterial peptide in vitro
TABLE 4 results of optimal time for LR18 antibacterial peptide to act in vitro
Example 5: antibacterial peptide LR18 in vitro antiviral assay
(1) The Vero E6 single cell suspension is inoculated in a 96-well plate, 0.1mL of the suspension is added into each well, and an in-vitro antiviral experiment is carried out after monolayer cells are formed by culture.
(2) Different times of adding the antimicrobial peptide were set, and the test was performed according to the following three methods: (1) LR18 has effects of adsorbing PEDV and blocking invasion cells; antibacterial peptide LR18 and Vero cells at 37 ℃ C. And 5% CO 2 After incubation in the incubator for 1h, the cells were infected with PEDV. (2) Direct inactivation of PEDV by LR 18; the antibacterial peptide LR18 infected cells after 1h of incubation with the virus. (3) The inhibitory effect of the antibacterial peptide LR18 on the proliferation of PEDV; the cells are infected with virus and then added with antibacterial peptide.
(3) The 3 methods are carried out, 3 wells are compounded in each group, an MTT colorimetric method is carried out, the cell survival rate and the inhibition rate of LR18 on PEDV are calculated according to a formula, and the test is repeated for 3 times.
Equation 1: cell viability (%) = OD value of sample-treated group/OD value of normal cell control group × 100%.
Equation 2: PEDV inhibition (%) = (sample treatment OD value-PEDV control OD value)/PEDV control OD value × 100%.
As shown in table 5, the in vitro inhibitory rate of the antimicrobial peptide LR18 against PEDV was 35% to 56%.
TABLE 5 results of the in vitro antiviral action of the antimicrobial peptide LR18
Example 6: effect of antibacterial peptide LR18 on PEDV through electron microscope negative staining observation
(1) Inoculating virus in a cell culture bottle, freezing and thawing for 3 times after obvious lesion is generated to fully crack cells, and centrifuging liquid at 4000r/min for 30min to remove cell debris.
(2) Adding the supernatant into a super-separation tube, and filling the super-separation tube with a PBS solution; and adding a PBS solution into the super-centrifugal tube, weighing, balancing, and centrifuging for 2.5 hours at 26000r/min by using a low-temperature super-centrifugal machine.
(3) Discarding supernatant, reversing the filter paper, sucking water in the tube, adding 100 μ L PBS to resuspend virus particles, and sucking into EP tube
(4) Setting an antibacterial peptide control group and a virus experimental group; mixing the resuspended virus particles with antibacterial peptide, incubating at 37 deg.C for 1h, dripping the sample on copper mesh, naturally drying, dripping coloring agent for negative staining, and observing virus particle change by electron microscope.
As seen in FIG. 6, in contrast to the normally cultured PEDV group, the envelope of PEDV added with the antimicrobial peptide group was exfoliated, demonstrating that the antimicrobial peptide can destroy the envelope of PEDV.
Example 7: indirect immunofluorescence experiment observation of effect of antibacterial peptide LR18 on PEDV
(1) Inoculating 12-pore plate with single cell suspension, and culturing in culture box until it grows to single layer
(2) The virus solution dilutes the antibacterial peptide to 5 mug/mL and 20 mug/mL, the mixed solution incubator incubates for 1h at 37 ℃, and the mixed solution dilutes to 800PFU/mL to inoculate cells.
(3) The supernatant was removed by suction overnight, washed 3 times, fixed with-20 ℃ glacial ethanol for 20min, and washed 3 times.
(4) 1.
(5) 1:100 dilution of FITC-IgG fluorescent secondary antibody, 300 mu L per well, incubation for 1h in the dark and repeating the previous step.
(6) The sample was observed under a fluorescent microscope.
As can be seen from FIG. 7, the antibacterial peptide LR18 with the concentration of 5. Mu.g/mL and 20. Mu.g/mL was observed under immunofluorescence electron microscopy after being incubated with PEDV, and the antibacterial peptide can act on the envelope of the virus.
Example 8: enteric microcapsule coated with antibacterial peptide LR18 for resisting artificial gastric juice and artificial intestinal juice
The enteric microcapsule coated with the antimicrobial peptide LR18 is treated in artificial gastric juice for 3 hours, then transferred into artificial intestinal juice for 7 hours, the treatment time is determined according to the digestion and absorption time of food in stomach and intestine, and the stability of the enteric microcapsule is judged by taking the expansion rate of the enteric microcapsule as an index. Method for measuring swelling ratio: extracting 10 small enteric-coated capsules at random, acting on artificial gastric juice for 3h, acting on artificial intestinal juice for 3h, placing into phosphate buffer solution to stop reaction, measuring the volume with a vernier caliper, calculating the swelling rate, and repeating for 3 times to calculate the average value. Calculation formula of expansion ratio: sw = DT/DO × 100%, sw: the expansion rate; DO: volume of enteric capsule before treatment; DT: volume of enteric-coated capsules after treatment.
As can be seen from tables 6 and 7, when the enteric microcapsule coated with the antimicrobial peptide LR18 is placed in the artificial gastric juice for 3 hours, the swelling rate of the enteric microcapsule is reduced very slowly, almost no swelling occurs in 0-0.5 hours, and the swelling rate is reduced by 16.2% only after 3 hours, which indicates that the enteric microcapsule material has higher resistance to gastric acid and pepsin. When the microcapsule is placed in artificial intestinal juice, the swelling rate is obviously reduced within 1 hour, and the microcapsule is completely dissolved within 3 hours, so that the enteric microcapsule can be well and slowly released in the intestinal environment, the antibacterial peptide LR18 can be absorbed after reaching intestinal field planting, and the therapeutic effect is exerted.
TABLE 6 swelling ratio of enteric-coated capsules in artificial gastric juice
| Time (h) | Swelling ratio (%) |
| 0 | 100 |
| 0.5 | 98.3 |
| 1 | 97.6 |
| 2 | 89.5 |
| 3 | 83.8 |
TABLE 7 swelling Rate of enteric capsules in Artificial intestinal juice
| Time (h) | Swelling ratio (%) |
| 0 | 100 |
| 0.5 | 75.5 |
| 1 | 34.3 |
| 2 | 9.8 |
| 3 | 0 |
Example 9: ELISA method for detecting content changes of IL-17, IL-1 beta and TNF-alpha in blood plasma
10 piglets with the weight of 20-25Kg are averagely divided into 2 groups, wherein one group is added with the functional additive of the antibacterial peptide LR18, and the other group is a normal feeding group. And (3) using 5mL of 1.0MOI PEDV to perform toxin attacking on each pig, collecting a blood sample of the pig from the anterior vena cava after 7 days of toxin attacking, adding the blood sample into a sterile EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, mixing for 10-20 min, and then placing the mixture into a centrifuge for centrifuging at 4000r/min for 10min. Changes in inflammatory factors IL-17, IL-1 beta, TNF-alpha in peripheral blood of 2 groups of pigs were compared using a commercial ELISA kit.
As is clear from fig. 8, 9 and 10, the pig peripheral blood inflammatory factors IL-17, IL-1 β and TNF- α in the feed containing LR18 coated antimicrobial peptide were reduced, and the difference was significant (P < 0.05) compared to the normal group, indicating that the pig intestinal inflammation could be reduced by adding LR18 coated antimicrobial peptide.
Example 10: qPCR method for detecting toxin expelling condition of offending pigs
By utilizing a pig PEDV challenge model established in the research laboratory, 3 rd after the pig is challenged, anus swabs of test pigs are collected, total virus RNA is extracted and cDNA is subjected to reverse transcription, the toxin expelling amount is detected by utilizing a commercial qPCR kit, and the effect of PEDV prevention of enteric microcapsules coated with antibacterial peptide LR18 is evaluated.
As can be seen from fig. 11, the pig manure toxin expelling amount of the feed added with LR 18-coated antimicrobial peptide is significantly reduced, and the difference is very significant (P < 0.01) compared with the normal group, which indicates that the viral load of PEDV in pig manure infected with the addition of LR 18-coated antimicrobial peptide functional feed additive can be reduced.
Example 11: field experiment
As can be seen from Table 8, the effective rate of the anti-PEDV functional feed additive developed by the invention for preventing PED is 99%, and the application effect is proved to be good, so that the anti-PEDV functional feed additive can be applied to clinical practice.
TABLE 8 results of field experiments
| Group of | Number of pig heads tested | Number of heads with diarrhea | Diarrhea Rate (%) | Effective rate (%) |
| LR18 group supplemented with antibacterial peptides | 500 | 5 | 1％ | 99％ |
| LR18 group without addition of antimicrobial peptide | 500 | 60 | 12％ | --- |
From the above examples 1-12, it is clear that 20. Mu.g/mL antimicrobial peptide LR18 has a good in vitro anti-PEDV effect. The enteric-coated microcapsule is prepared and is subjected to pig feeding tests, and the effective rate of reducing pig intestinal inflammation and preventing porcine epidemic diarrhea by adding the LR18 antibacterial peptide functional additive group is 99%. Through the research on the antibacterial peptide LR18 for inhibiting the replication of PEDV, theoretical basis and technical conditions are provided for the development of the functional feed additive for resisting the porcine epidemic diarrhea.
Example 12: feed
The feed comprises: the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus has the ratio of 200 g/ton.
In one embodiment of the invention, the feed comprises the following components:
606kg of corn;
105kg of puffed soybean meal;
80kg of corn distillers' grains;
40kg of germ meal;
57.5kg of rice bran meal;
15kg of stone powder;
25kg of calcium hydrophosphate;
8kg of sodium chloride;
lysine (70%) 10kg;
10.5kg of bentonite;
43kg of premix;
1000kg in total;
the premix comprises the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus, and the antibacterial peptide LR18 is 0.2kg.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (3)
1. The antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus is characterized in that the amino acid sequence of the antibacterial peptide LR18 for resisting the porcine epidemic diarrhea virus is shown as SEQ ID NO. 1.
2. A feed, characterized in that it comprises:
the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 of claim 1, wherein the ratio of the anti-porcine epidemic diarrhea virus antibacterial peptide LR18 is 200 g/ton.
3. The feed of claim 2, wherein the feed comprises the following components:
60-70 parts of corn;
8-12 parts of puffed soybean meal;
7-10 parts of corn distillers' grains;
3-5 parts of germ meal;
4-8 parts of rice bran meal;
1-2 parts of stone powder;
2-3 parts of calcium hydrophosphate;
0.5-1 part of sodium chloride;
0.5-1.5 parts of lysine;
0.5 to 1.5 portions of bentonite;
0.5 to 4.3 portions of premix;
the total amount is 100 parts;
wherein the premix comprises the anti-porcine epidemic diarrhea virus antibacterial peptide LR18.
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