A recombinant antimicrobial peptide as a feed supplement to improve growth performance and immune response

Abstract

The present invention relates to a recombinant antimicrobial peptide that can be expressed in transformed yeast. The present invention also provides a dietary supplement for improving growth performance and immune response in animals, comprising the recombinant peptide.

Description

Technical Field

[0001] The present invention relates to a novel recombinant antimicrobial peptide that can be used as a dietary feed supplement to improve growth performance and immune response. Background Art

[0002] Antibiotics inhibit or kill pathogens that can negatively impact broiler health in order to maximize broiler growth. Producers often seek ways to increase the abundance, uniformity, and growth rate of their animals, including livestock, poultry, and aquatic animals. Therefore, it is common practice to control disease outbreaks through the typical administration of antibiotics. For many years, antibiotics have been widely used in animal husbandry to reduce bacterial infections [1]. However, antibiotic treatment at certain stages of life can severely disrupt intestinal microorganisms, leading to delayed immune system development and immune dysfunction. For example, inappropriate antibiotic treatment can cause broilers to become susceptible to pathogens at later stages of development [2]. In addition, excessive use of antibiotics can promote antibiotic resistance in pathogens, which can have negative consequences for animal and human health (https: / / www.danmap.org / downloads / reports.aspx) [3]. Since 2006, the use of antibiotics for growth promotion has been banned in Europe [4, 5].

[0003] Therefore, there is a need for novel antimicrobial agents that can replace antibiotics in poultry production as alternative feed additives. Recently, antimicrobial peptides (AMPs) have emerged as antibiotic equivalents due to their ability to disrupt the membrane integrity of bacteria and other pathogens [6]. AMPs exhibit strong antimicrobial activity against different and diverse microorganisms, but their hemolytic activity against host cells is generally still low [7]. Piscidin comprises one of the most extensively studied AMP families. Piscidins isolated from several fish species exhibit a wide range of biological functions, including antibacterial, antifungal, antiparasitic, antinociceptive and antitumor functions [8–12]. Five piscidin-like AMPs (designated TP1–TP5) from Nile tilapia (Oreochromis niloticus) have been selected and characterized by the same research groups as the inventors of the present application [9]. Studies have shown that pathogens are less resistant to picidin than to antibiotics. The toxicity of piscidin-type AMPs may be due to the relatively non-specific electrostatic interactions between piscidin and membrane lipid components of pathogens such as Klebsiella pneumoniae and Acinetobacter baumannii

[13] . Few reports describe the production of piscidin using recombinant protein expression systems [14, 15].

[0004] However, it is unknown whether piscidin can effectively improve the growth performance and immune response of chickens. In the poultry industry, there is still a need to develop a diet to replace antibiotics as feed additives. Summary of the Invention

[0005] Therefore, the present invention provides a novel dietary supplement for improving growth performance and immune response in livestock farming as an alternative to antibiotics, wherein recombinant Epinephelus lanceolatus piscidin (rEP) is used to prevent pathogen infection and avoid drug resistance in pathogens.

[0006] According to the present invention, Epinephelus lanceolatus piscidin (EP) comprises an amino acid sequence selected from the group consisting of:

[0007] (1)EP1:CIMKHLRNLWNGAKAIYNGAKAGWTEFK(SEQ ID NO:1),

[0008] (2)EP2:CFFRHIKSFWRGAKAIFRGARQGWRE (SEQ ID No: 2), and

[0009] (3) EP3: GFIFHIIKGLFHAGRMIHGLVNRRRHRHGMEE (SEQ ID NO: 3).

[0010] In one embodiment, the dietary supplement comprises one EP or a mixture thereof. In the present invention, Epinephelus piscidin (EP) can be prepared in the form of a feed supplement, which has been shown to have an effect on growth performance and immune response in G. domesticus (chicken).

[0011] In another aspect, the present invention provides a method for improving growth performance and immune response in an animal, comprising administering to the animal an effective amount of the rEP of the present invention.

[0012] The following description sets forth details of one or more specific embodiments of the present invention. Other features and advantages of the present invention will become apparent from the following detailed description of several specific embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing summary of the invention and the following embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise configuration and means shown.

[0014] Figure 1 Provided are the sequences of Epinephelus lanceolatus piscidin g6496.t1 (EP-1), g6497.t1 (EP-2), and g6498.t1 (EP-3); the nucleotide (nt) and predicted amino acid (aa) sequences are shown. Nucleotides are numbered starting with the first nucleotide. Asterisks (*) indicate stop codons. The EP cDNA gene (g6498.t1) was modified based on the preferred codon usage of the P. pastoris expression system.

[0015] Figure 2 Shown is a sequence alignment of tilapia piscidin and saddle grouper piscidin. Tilapia piscidin peptides are aligned with multiple sequences of isolated saddle grouper piscidin (g6496.t1, g6497.t1, and g6498.t1); gaps were inserted to maximize homology. All coding sequences were entered into the dendrogram for alignment. Identical amino acids are represented by the same color, for example, methionine (M) is represented by yellow. Results of a lineage analysis of piscidin from tilapia (TP1 to TP5) and saddle grouper are shown.

[0016] Figure 3 shows the expression of the Epinephelus piscidin-6×His (rEP) protein in methanol-loving yeast. Figure 3(a) shows the plasmid map of the pPICZαA-EP-his vector. Figure 3(b) shows the application of different concentrations of methanol for induction, and analysis of recombinant protein expression by Western blot. Figure 3(c) shows the collection of cells, and analysis of total protein from the supernatant and precipitate by SDS-PAGE and Western blot. Lane 1, low-range rainbow marker; lane 2, synthetic EP; lane 3, protein expressed from the pPICZαA vector; lanes 4–9, cells containing the EP expression vector at 0 hour (no methanol induction), 1, 2, 3, 4, and 5 days after induction.

[0017] Figure 4 provides the effect of media composition on rEP expression in Saccharomyces methylotrophicus. The effects of nutrient levels were assessed by comparing cultures of Saccharomyces methylotrophicus X33 transformants grown in BMGY (flask cultures, circles) and BSM (fermenter cultures, squares) based on (a) wet cell weight and (b) cell counts.

[0018] Figure 5 shows the antimicrobial activity of rEP produced in methanol-loving yeast by the flask and fermenter methods. Figure 5(a) shows the concentration of rEP in the yeast culture supernatant before and after induction with methanol for 24 hours to 120 hours in flask culture. After 5 days of induction, the yeast culture supernatant produced inhibition zones of K. oxytoca, E. coli, Pseudomonas aeruginosa and Staphylococcus aureus (S. aureus) arranged in width. The mark "-" indicates Gram-negative; "+" indicates Gram-positive. Figure 5(b) shows the antimicrobial activity of rEP produced by the fermenter method. Yeast was induced with methanol in the fermenter for 24 hours to 120 hours. The OD 600 The supernatant showed inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The flask supernatant showed greater antimicrobial activity than the fermentation tank supernatant.

[0019] Figure 6 The effect of rEP on Gram-positive and Gram-negative bacteria was shown by co-incubation assay. OD was measured. 600 Compared with the control group, the lower OD 600 Indicates growth inhibition. The vector control group represents the empty pPICZαA vector. EP refers to the protein expressed by the pPICZαA-EP-his vector.

[0020] Figure 7 shows the effects of rEP administration on chickens evaluated by measuring the concentrations of immune factors including (a) tumor necrosis factor (TNF)-α, (b) interferon (IFN)-γ, (c) interleukin (IL)-1β, (d) IL-6, (e) IL-10, (f) immunoglobulin G (IgG), and (g) lysozyme (Lyz) by ELISA.

[0021] Figure 8 shows the effects of oral rEP on body shape, body weight, intestinal morphology and intestinal flora. Figure 8(a) shows that chickens receiving 1.5% rEP were larger than the Spirulina-A and basal diet control groups after 28 days of feeding. Figure 8(b) provides representative images of intestinal villi and crypts stained with hematoxylin and eosin. Figure 8(c) shows the measurement of intestinal villus length and crypt depth. Figure 8(d) shows the effect of feeding 1.5% rEP on body weight. After 28 days of the experiment, the body weight of the rEP group increased significantly. Figure 8(e) shows the effects of oral administration of Spirulina-A, basal diet and 1.5% rEP on the intestinal microbiota of domestic chickens. The relative proportions of bacterial families are shown. In the duodenum, the total viable counts of enterobacteria and Staphylococci decreased, while the abundance of Lactobacilli and Enterococci increased. DETAILED DESCRIPTION

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0024] The words "comprise" or "comprising" are generally used in the sense of include / including, which means that one or more features, ingredients or components are allowed to be present. The words "comprise" or "comprising" encompass the words "consists" or "consisting of".

[0025] As used herein, the terms "around," "about," or "approximately" may generally mean within 20%, specifically within 10%, and more specifically within 5% of a given value or range. The numerical quantities given herein are approximate, meaning that the terms "around," "about," or "approximately" can be inferred if not expressly stated.

[0026] As used herein, the term "animal" or "animals" refers to livestock, poultry or aquatic animals.

[0027] The present invention provides a novel dietary supplement that can be used as feed and mixed with antibiotics. When administered to animals (including livestock, poultry, and aquatic animals), the supplement improves survival rates and prevents disease outbreaks, thereby avoiding the overuse of antibiotics, which can promote the development of drug-resistant bacteria.

[0028] In the present invention, a novel recombinant Epinephelus lanceolatus piscidin (EP) is provided, which is shown to have an effect on the growth performance of Gallus gallus domesticus (chicken).

[0029] In the present invention, the gene encoding EP was isolated, sequenced, codon-optimized, and cloned into an expression system. Recombinant EP was tested as a dietary supplement for chickens, assessing overall health, growth performance, and immunity. Supernatants from rEP-expressing yeast demonstrated in vitro antimicrobial activity against both Gram-positive and Gram-negative bacteria, as measured by inhibition zone diameter (mm). Furthermore, the antimicrobial peptide function of rEP was independent of temperature. The fermentation broth yielded a spray-dried powder formulation containing 262.9 μg EP / g powder, and LC-MS / MS (tandem MS) analysis confirmed the molecular weight of rEP to be 4279 Da, as expected for a 34-amino acid peptide. The DNA sequence of the expression vector was also confirmed. Next, rEP was evaluated as a feed additive for chickens. Treatment groups included a control group, a basal diet, and rEP at varying doses (0.75%, 1.5%, 3.0%, 6.0%, and 12%). Compared with the control group, rEP supplementation significantly increased weight gain, feed efficiency, and IL-10 and FN-γ production in chickens. Our results suggest that crude rEP may offer an alternative to traditional antibiotic feed additives for chickens to enhance animal growth and health.

[0030] Thus, the present invention provides a dietary supplement extracted from a culture expressing AMP. In one embodiment of the present invention, the dietary supplement may be prepared in the form of an agricultural feed supplement.

[0031] In the present invention, three cDNAs encoding putative antimicrobial piscidin peptides were isolated and characterized from the tilapia grouper. The transcripts, designated g6496.t1 (encoding EP-1), g6497.t1 (encoding EP-2), and g6498.t1 (encoding EP-3), encode putative AMPs of 76, 76, and 69 amino acid residues, respectively. Sequence alignment of tilapia piscidin with EP-1 (g6496.t1), EP-2 (g6497.t1), and EP-3 (g6498.t1) revealed that EP-3 (g6498.t1) exhibited high similarity to the highly active TP3 and TP4 peptides from tilapia.

[0032] In the examples of the present invention, it was shown that peptide EP-3 (g6498.t1) has better activity than EP-1 (g6496.t1) or EP-2 (g6497.t1), and it also has antimicrobial or growth inhibitory activity against Gram-negative and Gram-positive bacteria.

[0033] In the present invention, EP was expressed in the pPICZαA expression vector in methanolic yeast using a gene with optimized yeast codon usage

[21] . Since EP is a pichia gene, it was expected that the gene optimized for Pichia codon usage would increase production. Of note, other researchers have reported that high concentrations of AMP are difficult to produce due to degradation by host proteolytic enzymes

[15] . In the present invention, proteolysis was not found to prevent the accumulation of rEP in Pichia. His-tagged rEP expressed in methanolic yeast showed antimicrobial activity against both Gram-positive and Gram-negative bacteria.

[0034] In the present invention, we found that after incubation at extreme temperature conditions (100°C), the EP from Epinephelus saddlebackii expressed in methanolic yeast was relatively stable in vitro and still retained some antimicrobial activity against Staphylococcus aureus (BCRC 10780). The stability of rEP may be related to its high arginine content. In line with this view, it has been previously reported that increasing the arginine composition of peptides increases antimicrobial activity and enhances the ability of peptides to embed into membranes

[22] . Increased arginine content may also enhance translocation and membrane permeability

[23] .

[0035] The present invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Based on this disclosure, those skilled in the art will appreciate that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0036] Example

[0037] 1. Materials and Methods

[0038] All procedures involving animals were performed as required and approved by the animal care and relevant departments.

[0039] 1.2. Molecular cloning, construction and transformation of expression plasmids, and selection of positive transformants

[0040] Primer sequences for EP amplification were designed based on previous transcriptome analyses and experimental methods [9, 16]. Briefly, piscidin was isolated from Epinephelus saddleback by RT-PCR. mRNA was extracted from Epinephelus saddleback liver and reverse transcribed; primers used for cDNA amplification are listed in Table 1. Three recombinant clones (g6496.t1, g6497.t1, and g6498.t1) were selected for sequencing.

[0041] Table 1: Primer sequence list

[0042]

[0043] 1 g6496.t1: Epinephelus piscidin-1

[0044] 2 g6497.t1: Epinephelus piscidin-2

[0045] 3 g6498.t1: Epinephelus piscidin-3

[0046] Multiple sequence alignments of selected peptides from piscidin of grouper and Nile tilapia (O. niloticus) were performed. The following peptides were synthesized by GL Biochem Co., Ltd. (Shanghai, China), and the antimicrobial activity of each peptide was determined as the minimum inhibitory concentration (MIC) in a microbroth dilution series [9, 13]:

[0047] (1)EP-1:g6496.t1(Ac-CIMKHLRNLWNGAKAIYNGAKAGWTEFK-NH2),

[0048] (2)EP-2:g6497.t1(Ac-CFFRHIKSFWRGAKAIFRGARQGWRE-NH2), and

[0049] (3)EP-3:g6498.t1(Ac-GFIFHIIKGLFHAGRMIHGLVNRRRHRHGMEE-NH2).

[0050] A 102-bp codon-optimized sequence corresponding to the mature EP cDNA gene (g6498.t1) was designed. This sequence was based on the preferred codon usage of Saccharomyces methylotrophicus using a graphical codon usage analyzer (http: / / gcua.schoedl.de / ) and synthesized by Omics Bio (Taipei, Taiwan). The DNA was cloned into EcoRI / XbaI-digested pPICZαA, obtained from Omics Bio (Taipei, Taiwan), to generate the recombinant vector pPICZαA-EP-his. The EP cDNA gene (g6498.t1) sequence in the vector was then confirmed by sequencing.

[0051] pPICZαA-EP-his was linearized with SacI and then transformed into methanolic yeast X-33 by electroporation (1.5 kV, 25 μF, 200 Ω; ECM 399 electroporation system, BTX Harvard Apparatus). Identification of positive transformants and selection of high-expressing rEP-expressing strains were performed as previously reported without modification

[17] . Briefly, recombinant methanolic yeast strains containing the rEP gene were incubated in 3 ml of YPD medium (containing 300 μg of Zeocin). After 24 hours (30°C), 30 mL of BMGY medium was inoculated with 1 ml of methanolic yeast (P. pastoris) for 24 hours (30°C).

[0052] The cells were then collected by centrifugation at 6000 × g for 15 minutes. The cell pellet was resuspended in 50 mL of BMMY medium. The supernatant and precipitate were examined by SDS-PAGE and Western blot analysis using a His-tag antibody (Abcam, ab213204). The recombinant protein was identified by MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry. The antimicrobial activity of rEP was analyzed using the inhibition zone assay at different treatment temperatures

[18] . All assays were performed in triplicate.

[0053] 1.3. rEP performance in fermentation tanks

[0054] To optimize expression conditions in a 5000 ml fermenter, a single rEP colony was used to inoculate 200 ml of BMGY with PTM4 medium at 28°C and 200 rpm for 36 h. The culture was then transferred to a 5000 ml fermenter (Winpact, Major Science, Taoyuan, Taiwan) containing 3000 ml of commercial fermentation medium (BMGY with PTM4)

[19] . During fermentation, the temperature was maintained at 30°C. The pH was adjusted to 6.0 with 14% ammonia and 0.1 N H2SO4, and the dissolved oxygen was maintained above 20% saturation. To produce rEP for livestock feed supplementation of chicken feed, rEP was expressed in a fermenter using basal salt medium and PTM1

[20] . After complete consumption of glycerol (19 h), 50% w / v glycerol was added for 360 min. Subsequently, 100% methanol was added and maintained for 24 to 96 h. The rEP-expressing yeast cultures were then centrifuged at 6,000 rpm for 30 minutes, and the supernatant was spray-dried (YC-500, Pilotech Instrument & Equipment Co., Ltd.) before being incorporated into the feed. Prior to feed production, rEP expression in each culture was verified by Western blot analysis, and rEP content was assessed by comparison with synthetic EP peptides.

[0055] 1.4. Antimicrobial activity of rEP and feed preparation

[0056] The antimicrobial activity of rEP was tested on cultures of Staphylococcus aureus (BCRC 10780), Escherichia coli (BCRC 10675), Pseudomonas aeruginosa (ATCC 19660) and Riemerella anatipestifer (RA3, RA9, RA16, CFC27, CFC363, CFC437). Bacterial cultures were derived from single strains that had been amplified and stored at -70°C. The cultures were inoculated in liquid medium and incubated overnight at 37°C on a shaker at 180 rpm. Subsequently, the bacteria were diluted in fresh medium (1:1000) and incubated under the same conditions. Four types of bacteria (10 4 CFU / ml) were mixed with 100 μl of rEP solution and incubated at 37°C overnight. The supernatant of methanol-loving yeast transformed with the vector alone was used as a control group. After 24 hours, the supernatant was measured by OD 600 The culture growth was assessed. All assays were performed in triplicate. The composition of the diet fed to the chickens is shown in Table 2.

[0057] Table 2: Proximate analysis of basic feed and commercial feed compositions with additives

[0058]

[0059] 1.5. Chicken care and dietary therapy

[0060] A total of 189 2-day-old male or female chickens were randomly assigned to seven dietary treatments, including rEP (0.75%, 1.5%, 3.0%, 6.0%, and 12%), Spirulina-A (Solitary Chemical Pharmaceutical Co., Ltd., Sanxia District, New Taipei City, Taiwan), and a basal diet. Each treatment group consisted of 27 chickens per cage. The diet compositions are listed in Tables 3-5.

[0061] Table 3: Preparation of early basal diet 1

[0062]

[0063] Table 4: Preparation of medium-term basal diet 1

[0064]

[0065] Table 5: Preparation of late basal diet 1

[0066]

[0067] 1 The fermentation supernatant was added to the diet at the expense of cellulose to provide concentrations of 0, 0.75, 1.5, 3.0, 6.0, and 12 g / 100 g diet.

[0068] 2 Antibiotics: Each gram contains: Spiramycin adipate 30mg (can), Streptomycin sulfate 30mg (can), Vitamin A 2,500IU, Vitamin B1 5mg, Vitamin B2 10mg, Vitamin B6 2mg, Vitamin B12 5mcg, Vitamin E 2mg, Vitamin D3 500IU, Vitamin K 41mg, Folic acid 0.2mg, Calcium pantothenate 5mg, Niacin 10mg, Lysine 20mg

[0069] During the experimental period, starting at three days of age, the animals were allowed ad libitum access to feed and water. The total duration of rearing was 35 days. The chickens were housed in cages (animal enclosures) in a constant temperature environment (28.9 to 37.8°C, 12 / 12 hour light / dark cycle). The feed and fecal wastewater were not directly discharged to the outside.

[0070] 1.6. Sample collection and enzyme-linked immunosorbent assay

[0071] Body weight and survival rate were monitored daily during the experiment. Weight gain, feed efficiency (FE), protein efficiency ratio (PER) and survival percentage were calculated. After 35 days of treatment, the chickens were euthanized, blood samples were collected and serum was obtained by centrifugation (3000×g, 4°C, 15 minutes). The serum was kept at -80°C until analysis. Enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's standard procedures to determine the concentrations of immune factors, including TNF-α, interleukin-1β, interleukin-6, interleukin-10, lysozyme, immunoglobulin-G (IgG), and interferon-γ. Chicken serum samples were analyzed using an ELISA kit from ABclonal, Inc. (Woburn, MA, USA). After the reaction was completed, the serum was analyzed on a microplate reader ( The optical density (OD) was measured at 450 nm on a ELISA kit (i3, Molecular Devices, Lagerhausstrasse, Wals, Austria).

[0072] Statistical analysis

[0073] Data were analyzed using Prism 7 software (GraphPad Inc., La Jolla, CA, USA). Values are mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed using Tukey's multiple comparison test, and P < 0.05 was considered significant.

[0074] 2. Results

[0075] 2.1. Novel piscidin from Epinephelus saddleback exhibits antimicrobial activity

[0076] Different piscidin sequences (SEQ ID NOs: 10, 12, and 14; Figure 1 ) cDNA coding region. The three identified cDNA sequences were named g6496.t1, g6497.t1, and g6498.t1, encoding 76, 76, and 69 amino acids, respectively (SEQ ID NO: 11, 13, and 15). Comparison of piscidin from Epinephelus saddleback (g6496.t1, g6497.t1, and g6498.t1) and piscidin from Nile tilapia (TP1–TP5) revealed significant high sequence similarity ( Figure 2 The phylogenetic tree showed that g6498.t1 corresponds to TP4 and TP3, which exhibit the best antimicrobial activity characterized to date ( Figure 2 ).

[0077] Next, the antimicrobial activities of the synthesized peptides g6496.t1, g6497.t1, and g6498.t1 were determined. As shown in Table 6, all three peptides exhibited activity against both Gram-positive and Gram-negative bacteria.

[0078] Table 6: In vitro activity of antimicrobial peptides against Gram-positive and Gram-negative bacteria.

[0079]

[0080] 1 g6496.t1: Epinephelus piscidin-1 (EP-1)

[0081] 2 g6497.t1: Epinephelus piscidin-2 (EP-2)

[0082] 3 g6498.t1: Epinephelus piscidin-3 (EP-3)

[0083] (MIC unit is μg / ml)

[0084] Peptides g6498.t1 and g6497.t1 also exhibited activity against methicillin-resistant Staphylococcus aureus (MRSA) at MICs of 5.6 μg / ml and 60 μg / ml, respectively. These two peptides were also toxic to Vibrio parahaemolyticus at similar MICs. Because g6498.t1 exhibited stronger antimicrobial activity than g6496.t1 and g6497.t1, we named this peptide EP and further produced it in a methanolic yeast protein expression system.

[0085] 2.2. rEP peptide expression system in methanol-loving yeast

[0086] As shown in Figure 3(a), the constitutive expression vector pPICZαA-EP-his contains the methanol-inducible AOX promoter, the α-factor signaling peptide, and the STE13 gene for dipeptidyl aminopeptidase A. The amino acid sequence of rEP is encoded by a gene insert optimized for the Saccharomyces methylotrophicus (X-33) codon. Transformants of Saccharomyces methylotrophicus (X-33) were grown on Zeocin plates (25 μg / ml) and screened by colony hybridization with an anti-His antibody; high-expressing colonies were selected for subsequent experiments. After colonies were identified, the presence of the correct expression cassette was confirmed by PCR amplification and DNA sequencing. During the initial expression experiment, the optimal methanol concentration was determined by providing different concentrations of methanol for 24 hours. The supernatant and precipitate (after centrifugation) were collected and analyzed by Western blotting. The results showed that 1% methanol induced robust expression of rEP (Figure 3(b)). The time-dependent effect of 1% methanol induction is shown in Figure 3(c). Therefore, 1% methanol induction was selected for subsequent fermentation tank experiments.

[0087] 2.3. Effects of time and culture medium on rEP fermentation

[0088] To evaluate the effect of induction time and culture medium on rEP expression, transformants were grown in fermenters with basal salt medium (BSM) or BMGY. Large-scale production of rEP from methanolophilic yeast (X-33) may be most efficient using high-density culture methods. Therefore, induction of rEP was evaluated in 5000 mL fermenters. When the wet weight of cells in the glycerol-fed batch reached 200 g / L (16 to 18 hours after inoculation), 1% methanol was added for 24 to 120 hours. After methanol induction, the yeast count decreased ( Figure 4(a) and 4(b)After 48 hours of induction in BSM medium, the total rEP protein concentration reached a maximum of 0.8 mg / L in the supernatant and 5.63 mg / L in the precipitate. On the other hand, at 48 hours, the maximum rEP total protein concentration in BMGY medium was 3.6 mg / L in the supernatant and 7.74 mg / L in the precipitate. The antimicrobial activity of rEP was also monitored and gradually increased to its highest concentration after 120 hours of methanol induction (Figures 5(a), 5(b), Figure 6 ).

[0089] Antimicrobial activity of EP

[0090] The antimicrobial activity of rEP was evaluated by measuring its ability to inhibit the growth of Gram-positive and Gram-negative bacteria in flask cultures using a paper diffusion assay. Tables 7 and 8 show the antimicrobial activity of rEP and the effect of temperature. rEP was produced in methanolic yeast using shake flasks. The supernatant was collected after 120 hours of induction and applied to paper tablets at maximum loading, wherein the paper tablets were placed on bacterial culture plates at 37°C for 16 hours. The inhibition zone diameter was measured. Representative radial diffusion assay. rEP showed broad antimicrobial activity against the test strains. Antimicrobial activity of rEP and the effect of temperature. rEP was produced in methanolic yeast using shake flasks. The supernatant was collected after 120 hours of induction and applied to paper tablets at maximum loading, wherein the paper tablets were placed on bacterial culture plates at 37°C for 16 hours. The inhibition zone diameter was measured. Representative radial diffusion assay. The most susceptible pathogens were R. anatipestifer (CFC27) and R. anatipestifer (CFC437) (Table 7). Next, we investigated the thermostability of rEP in flask fermentations against Staphylococcus aureus (BCRC 10780), Escherichia coli (BCRC 10675), and Pseudomonas aeruginosa (ATCC 19660). An empty vector containing no insert was used as a control in methanolic yeast. After incubation at 40, 60, 80, or 100°C for 5 minutes, thermostability was measured by paper diffusion assay (Table 8).

[0091] Table 7: Antimicrobial activity of rEP

[0092]

[0093] Table 8: Effect of temperature on rEP antimicrobial activity

[0094]

[0095] 1 NI, no inhibition

[0096] 2The control group was the fermentation supernatant from the transformant containing empty plasmids. 10 μl of ampicillin (2 mg / ml) was applied to the paper disk.

[0097] We found that the inhibition zone diameter (mm) decreased with increasing pretreatment temperature, but antimicrobial activity against Staphylococcus aureus (BCRC10780) was unaffected by varying temperatures. This finding suggests that the tertiary (or secondary) structure of rEP is important for peptide stability and should be protected from high temperatures to maintain activity.

[0098] 2.5. rEP supplementation improves growth performance and immune response

[0099] The growth performance of chickens was evaluated based on weight gain and feed efficiency (FE). At the end of the 35-day experimental period, animals fed with 1.5% and 3.0% rEP had significantly higher growth performance than the Spirulina-A and basal diet groups (Table 9). Next, the concentrations of immune factors in serum, including immunoglobulin G (IgG), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10, lysozyme (Lyz), and interferon (IFN)-γ, were measured by ELISA to evaluate the physiological effects of daily rEP administration in chickens (Figures 7(a) to 7(g)). No significant changes in TNF-α, II-1β, II-6, or Lyz concentrations were observed between groups (Figures 7a, c, d, and g). The group supplemented with 1.5% EP showed significantly increased IFN-γ concentrations compared to the antibiotic and control groups (Figure 7(b), P = 0.0043 compared to the antibiotic group and P = 0.0073 compared to the control group). In addition, the IL-10 concentration in the rEP-supplemented group was significantly higher than that in the control group (P = 0.0341), but not different from that in the antibiotic group (Figure 7(e)). Chickens fed with antibiotics showed higher serum IgG concentrations compared to the control group (P = 0.0394) (Figure 7(f)).

[0100] Table 9: Weight gain, feed efficiency (FE), protein efficiency ratio (PER) and survival rate of chickens fed a diet containing rEP (0.75, 1.5, 3.0, 6.0 and 12%) fermentation supernatant spray-dried powder for 4 weeks 1

[0101]

[0102] 1 Values with different superscripts in the same column are significantly different (p<0.05). Data are expressed as mean ± SD from chicken groups (n=27)

[0103] 2Weight gain (%) = [final weight (g) - initial weight (g)] / initial weight (g) x 100

[0104] 3 Feed efficiency = [final body weight (g) - initial body weight (g)] / feed intake (g)

[0105] 4 Protein efficiency ratio = [final body weight (g) - initial body weight (g)] / protein intake (g)

[0106] As described above, in the present invention, it was found that rEP improved the growth performance of chickens compared to feed supplemented with Spirulina-A. These results suggest that EP can potentially replace antibiotics to improve growth performance ( Figure 8(a) and 8(d) Histological analysis showed that the height of duodenal villi in rEP animals was significantly higher than that in the control group ( Figure 8(b) and 8(c) ). In the present invention, it was found that 1.5% rEP significantly increased the number of Lactobacillus and Enterobacteriaceae in the duodenum, while reducing the number of Enterobacteriaceae and Staphylococcaceae compared to the Spirulina-A and basal diet groups (Figure 8(e)). It was found that Salmonella was not detected in the blood after challenge (data not shown). After daily rEP administration, IL-10 was significantly higher in the rEP-supplemented group compared to the control group. IL-10 is a pro-inflammatory cytokine that is produced by different cells (such as Th2 cells, macrophages and monocytes) and acts as an immunomodulator during infection with bacteria, fungi and viruses. The higher IFN-γ production in rEP-supplemented animals may increase cytokines that induce antimicrobial pathways to resist invasion by extracellular and intracellular pathogens.

[0107] The results showed that the addition of 1.5% or 3% rEP to chicken feed improved growth performance, intestinal morphology, microbiota, and immunity in broilers and other animals.

[0108] The present invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Based on this disclosure, those skilled in the art will appreciate that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0109] References

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[0131] 23. Schmidt NW, Tai KP, Kamdar K, Mishra A, Lai GH, Zhao K, et al. Argininein alpha-defensins: differential effects on bactericidal activity correspondto geometry of membrane curvature generation and peptide-lipid phase behavior. J Biol Chem. 2012; 287(26): 21866-72. Epub 2012 / 05 / 09.doi:10.1074 / jbc.M112.358721.PubMed PMID:22566697;PubMed Central PMCID:PMCPMC3381149. Sequence Listing <110> Liu Fudong <120> A recombinant antimicrobial peptide as a feed supplement to improve growth performance and immune response <130> ACA0139WO <150> US 62 / 980,730 <151> 2020-02-24 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 28 <212> PRT <213> Artificial sequence <220> <223> EP1 <400> 1 Cys Ile Met Lys His Leu Arg Asn Leu Trp Asn Gly Ala Lys Ala Ile 1 5 10 15 Tyr Asn Gly Ala Lys Ala Gly Trp Thr Glu Phe Lys 20 25 <210> 2 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> EP2 <400> 2 Cys Phe Phe Arg His Ile Lys Ser Phe Trp Arg Gly Ala Lys Ala Ile 1 5 10 15 Phe Arg Gly Ala Arg Gln Gly Trp Arg Glu 20 25 <210> 3 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> EP3 <400> 3 Gly Phe Ile Phe His Ile Ile Lys Gly Leu Phe His Ala Gly Arg Met 1 5 10 15 Ile His Gly Leu Val Asn Arg Arg Arg His Arg His Gly Met Glu Glu 20 25 30 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> g6496.t1-f <400> 4 atggcaagtg agcctgacga ac 22 <210> 5 <211> 25 <212> DNA <213> Artificial sequence <220> <223> g6496.t1-r <400> 5 ttagcctttg gtttttcctc ctgct 25 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <220> <223> g6497.t1-f <400> 6 atgggtgtgg cggtgcag 18 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> g6497.t1-r <400> 7 tcatttctgc cagtatggcg g 21 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> g6498.t1-f <400> 8 atgaggtgca tcatcctctt tc 22 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> g6498.t1-r <400> 9 tcaggcaaaa gctttctctc gttc 24 <210> 10 <211> 231 <212> DNA <213> Artificial sequence <220> <223> g6496.t1 <400> 10 atgaggtgca ccatgatctt tctcgtgttg tcgctggtcg tcctcatggc tgaacctggg 60 gagtgtatta tgaaacacct tcgaaattta tggaatgggg ccaaggccat atacaatggt 120 gccaaggcgg gatggacaga gtttaaaaac agcctccccg ctgaagagac ggctccaggg 180 cctggatcga accagcagag gcctccagca ggaggaaaaa ccaaaggcta a 231 <210> 11 <211> 76 <212> PRT <213> Epinephelus lanceolatus <400> 11 Met Arg Cys Thr Met Ile Phe Leu Val Leu Ser Leu Val Val Leu Met 1 5 10 15 Ala Glu Pro Gly Glu Cys Ile Met Lys His Leu Arg Asn Leu Trp Asn 20 25 30 Gly Ala Lys Ala Ile Tyr Asn Gly Ala Lys Ala Gly Trp Thr Glu Phe 35 40 45 Lys Asn Ser Leu Pro Ala Glu Glu Thr Ala Pro Gly Pro Gly Ser Asn 50 55 60 Gln Gln Arg Pro Pro Ala Gly Gly Lys Thr Lys Gly 65 70 75 <210> 12 <211> 231 <212> DNA <213> Artificial sequence <220> <223> g6497.t1 <400> 12 atgaagtttg ttatgatctt tctggtgctg tcgctggtcg tcctcatggc cgaacccggg 60 gagtgttttt ttagacacat aaaatctttc tggagagggg ccaaggctat attcaggggt 120 gccaggcagg gatggagaga acacagagcc ttgtctaagc agcgcaagat ggatcaaggg 180 ggtggaggaa acgtagtgga caatggaact ccgccatact ggcagaaatg a 231<6000439><210> 13 <211> 76 <212> PRT <213> Epinephelus lanceolatus <400> 13 Met Lys Phe Val Met Ile Phe Leu Val Leu Ser Leu Val Val Leu Met 1 5 10 15 Ala Glu Pro Gly Glu Cys Phe Phe Arg His Ile Lys Ser Phe Trp Arg 20 25 30 Gly Ala Lys Ala Ile Phe Arg Gly Ala Arg Gln Gly Trp Arg Glu His 35 40 45 Arg Ala Leu Ser Lys Gln Arg Lys Met Asp Gln Gly Gly Gly Gly Asn 50 55 60 Val Val Asp Asn Gly Thr Pro Pro Tyr Trp Gln Lys 65 70 75 <210> 14 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> g6498.t1 <400> 14 atgaggtgca tcatcctctt tcttgtgttg tcgctggtgg tcctcatggc tgaacccggg 60 gagggtttta tcttccacat catcaaagga ctctttcacg ctggcaggat gatccatgga 120 cttgtcaaca ggagacgaca tcgacatggc atggaagagc tgcaagacct ggaccaacgt 180 gcctttgaac gagagaaagc ttttgcctga 210 <210> 15 <211> 69 <212> PRT <213> Epinephelus lanceolatus <400> 15 Met Arg Cys Ile Ile Leu Phe Leu Val Leu Ser Leu Val Val Leu Met 1 5 10 15 Ala Glu Pro Gly Glu Gly Phe Ile Phe His Ile Ile Lys Gly Leu Phe 20 25 30 His Ala Gly Arg Met Ile His Gly Leu Val Asn Arg Arg Arg His Arg 35 40 45 His Gly Met Glu Glu Leu Gln Asp Leu Asp Gln Arg Ala Phe Glu Arg 50 55 60 Glu Lys Ala Phe Ala 65 <210> 16 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> piscidin-6xHis <400> 16 Glu Phe Phe Ile Phe His Ile Ile Lys Gly Leu Phe His Ala Gly Arg 1 5 10 15 Met Ile His Gly Leu Val Asn Arg Arg Arg His Arg His His His His 20 25 30 His His

Claims

1. A recombinant Epinephelus lanceolatus piscidin (rEP) having the ability to improve animal growth performance, intestinal morphology, microbiota, and immunity, comprising the amino acid sequence of SEQ ID NO:

15.

2. A dietary supplement for improving growth performance and immune response in chickens, comprising recombinant Epinephelus saddleback peptide (rEP) as defined in claim 1.

3. A dietary supplement according to claim 2, in the form of a feed supplement.

4. A feed supplement for improving growth performance and immune response in animals, comprising the recombinant Epinephelus saddleback peptide (rEP) as defined in claim 1.

5. Use of the recombinant Epinephelus saddleback peptide (rEP) as defined in claim 1 for preparing a feed supplement for improving growth performance and immune response in animals.

6. The feed supplement according to claim 4, wherein the animal is livestock, poultry or aquatic animal.

7. The method according to claim 5, wherein the animal is livestock, poultry or aquatic animal.

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