CN116056587A

CN116056587A - Recombinant antimicrobial peptides as feed supplements for improving growth performance and immune response

Info

Publication number: CN116056587A
Application number: CN202180016669.8A
Authority: CN
Inventors: 陈志毅
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-02-24
Filing date: 2021-02-24
Publication date: 2023-05-02
Also published as: WO2021173670A2; WO2021173670A3; TW202146654A

Abstract

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

Description

Recombinant antimicrobial peptides as feed supplements for improving growth performance and immune response

Technical Field

The present invention relates to a novel recombinant antimicrobial peptide useful as a dietary feed supplement for improving growth performance and immune response.

Background

Antibiotics can inhibit or kill pathogens that can have a negative impact on broiler health, so that broiler chickens are grown to a maximum. Breeders often seek methods to increase the abundance, uniformity, and growth rate of animals, including livestock, poultry, and aquatic animals. Thus, it is common practice to control disease outbreaks by typical administration of antibiotics. Antibiotics have been widely used in animal husbandry for many years to reduce bacterial infections [1]. However, antibiotic treatment during certain stages of life severely destroys intestinal microorganisms, resulting in delayed development of the immune system and immune dysfunction. For example, improper antibiotic treatment may lead to broiler chickens becoming susceptible to infection by pathogens at a later developmental stage [2]. Furthermore, overuse of antibiotics promotes antibiotic resistance in pathogens, which may have a negative impact on animal and human health (https:// www.danmap.org/downloads/reports. Aspx) [3]. Since 2006, europe has banned the use of antibiotics to promote growth [4,5].

Thus, there is a need for novel antimicrobial agents that can be used as alternative feed additives in the production of poultry. Recently, antimicrobial peptides (AMPs) have emerged as equivalents of antibiotics due to their ability to disrupt the membrane integrity of bacteria and other pathogens [6]. AMP exhibits strong antimicrobial activity against diverse and diverse microorganisms, but hemolytic activity against host cells is still generally low [7]. Piscidin comprises one of the most widely studied AMP families. Piscidin isolated from several fish species has been shown to exhibit a wide range of biological functions including antibacterial, antifungal, antiparasitic, antinociceptive and antitumor functions [8-12]. Five piscidin-type AMPs (designated TP1-TP 5) from Ni Luo Wuguo fish (Oreochromis niloticus) have been cloned and characterized by some groups of study groups identical to the inventors of the present application [9]. Studies have shown that pathogens are less resistant to picidin than antibiotics. Toxicity of Piscidin-type AMPs may be due to the relative non-specificity of electrostatic interactions between Piscidin and membrane lipid components of pathogens such as klebsiella pneumoniae and acinetobacter baumannii [13]. There have been few reports describing the use of recombinant protein expression systems to produce piscidin [14, 15].

However, it is not known whether piscidin is effective in improving the growth performance and immune response of chickens. In the poultry farming industry, there is still a need to develop a diet to replace antibiotics as feed additives.

Disclosure of Invention

Accordingly, the present invention provides a novel dietary supplement as an alternative to antibiotics for improving growth performance and immune response in animal husbandry, wherein recombinant garrupa piscidin (recombinant Epinephelus lanceolatus piscidin, rEP) is used to prevent pathogen infection, as well as to avoid pathogen resistance.

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

(1)EP1:CIMKHLRNLWNGAKAIYNGAKAGWTEFK(SEQ ID NO:1)，

(2) EP2: CFFRHIKSFWRGAKAIFRGARQGWRE (SEQ ID No. 2)

(3)EP3:GFIFHIIKGLFHAGRMIHGLVNRRRHRHGMEE(SEQ ID NO:3)。

In a specific embodiment, the dietary supplement comprises an EP or a mixture thereof. In the present invention, garrupa piscidin (EP) can be prepared in the form of a feed supplement, which has been shown to have an effect on the growth performance and immune response of chickens (g.domisticus).

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

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will become apparent from the following detailed description of several specific embodiments, and from the appended claims.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

FIG. 1 provides the sequences of garrupa piscidin (E.lancenolatus piscidin) g6496.t1 (EP-1), g6497.t1 (EP-2) and g6498.t1 (EP-3); wherein the nucleotide (nt) sequence and predicted amino acid (aa) sequence are shown. Nucleotides are numbered starting from the first nucleotide. Asterisks indicate stop codon. The EP cDNA gene (g 6498. T1) was modified based on the preferential codon usage of the expression system of M.methanotrophic yeast (P.pastoris).

FIG. 2 shows an alignment of the sequences of Piscidin from Evodia rutaecarpa and Piscidin from Epinephelus lanceolatus. Multiple sequence alignment of the peptide of the Piscidin peptide of Evodia rutaecarpa with isolated Piscidin of Epinephelus lanceolatus (g 6496.t1, g6497.t1 and g 6498.t1); in which gaps are inserted to obtain maximum homology. All the coding sequences are input into a dendrogram for comparison. The same amino acids are represented by the same color, for example, methionine (M) is represented by yellow. Shows the results of piscidin lineage analysis from Evodia rutabaga (TP 1 to TP 5) and Epinephelus lanceolatus.

FIG. 3 shows the expression of the garrupa piscidin-6 XHis (rEP) protein in methylotrophic yeasts. FIG. 3 (a) shows a plastid map of the pPICZ alpha A-EP-his vector. FIG. 3 (b) provides methanol at various concentrations for induction and analysis of recombinant protein expression by Western blotting. FIG. 3 (c) shows that cells were collected and analyzed for total protein from supernatant and pellet by SDS-PAGE and Western blotting. Lane 1, low range rainbow marker; lane 2, synthesized EP; lane 3, protein expressed by ppiczαa vector; lanes 4-9, cells containing the EP expression vector after 0 hours of induction (no methanol induction), 1, 2, 3, 4 and 5 days.

FIG. 4 provides the effect of the culture medium composition on rEP performance in M.methanotrophic yeasts. The effect of nutrient content was assessed by comparing cultures of methanotrophic yeast X33 transformants grown in BMGY (flask cultures, circles) and BSM (fermenter cultures, squares) based on their (a) wet cell weight and (b) cell count.

Figure 5 shows the antimicrobial activity of rEP produced in methanotrophic yeasts by the flask and fermenter method. FIG. 5 (a) shows rEP concentrations in yeast culture supernatants before and after induction with methanol in flask culture for 24 to 120 hours. After 5 days of induction, the yeast culture supernatant produced inhibition zones largely aligned in width for klebsiella acidogens (k.xyoca), escherichia coli (e.coli), pseudomonas aeruginosa (p.aeromonas) and staphylococcus aureus (s.aureus). The label "-" indicates gram negative; "+" indicates gram positive. Fig. 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. By OD ₆₀₀ The supernatant showed a high yield to E.coli, pseudomonas aeruginosa and golden yellowInhibitory Activity of staphylococci. The flask supernatant exhibited greater antimicrobial activity than the fermenter supernatant.

Figure 6 shows the effect of rEP on gram positive and gram negative bacteria by co-incubation analysis. Measuring OD ₆₀₀ . Lower OD compared to control group ₆₀₀ The expression shows that growth was inhibited. Vector control group represents empty ppiczαa vector. EP refers to the protein expressed by the pPICZ alpha A-EP-his vector.

FIG. 7 shows the effect of rEP administration on chickens by ELISA to determine the concentration 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).

Figure 8 shows the effect of oral administration rEP on posture, body weight, intestinal morphology and intestinal flora. FIG. 8 (a) shows that chickens receiving 1.5% rEP were larger than spirulina-A and basal diet control group after 28 days of feeding. FIG. 8 (b) provides representative images of hematoxylin-and eosin-stained intestinal villi and crypts. FIG. 8 (c) shows the measurement of intestinal villus length and crypt depth. FIG. 8 (d) shows the effect of feeding 1.5% rEP on body weight. After 28 days of the trial, the body weight of group rEP increased significantly. FIG. 8 (e) shows the effect of oral administration of spirulina-A, basal diet and 1.5% rEP on intestinal microbiota of chickens. Shows the relative proportions of the bacterial families. In the duodenum, the total viable count of intestinal bacteria and staphylococci decreases, while the abundance of Lactobacillaceae (Lactobacillaceae) and Enterococcaceae (Enterococcaceae) increases.

Detailed Description

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.

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

The terms "comprises" or "comprising" are used generally in the sense of including/comprising, which means that one or more features, ingredients or components are allowed to exist. The words "comprise" or "comprising" encompass the words "consisting of" or "consisting of".

As used herein, the words "about", "about" or "about" may generally refer to 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 words "about", "about" or "about" can be inferred if not explicitly stated.

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

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

In the present invention, a novel recombinant garrupa piscidin (Epinephelus lanceolatus piscidin, EP) is provided which has been shown to have an effect on the growth performance of chickens (Gallus gallus domesticus) (chickens).

In the present invention, the gene encoding EP is isolated, sequenced, codon optimized and cloned into the expression system. Test performance recombinant EP for use as a dietary supplement for chickens; overall health, growth performance and immunity. According to the inhibition zone diameter (mm) analysis, rEP exhibited that the supernatant of yeast showed in vitro antimicrobial activity against gram positive bacteria and gram negative bacteria. Furthermore, the antimicrobial peptide function of rEP is independent of temperature. The broth yielded a spray-dried powder formulation comprising 262.9 μg of EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed rEP molecular weight of 4279Da as expected for the 34 amino acid peptide; the DNA sequence of the expression vector was also confirmed. Next, we evaluated rEP as a feed additive for chickens. The treatment group included control, basal diet and different doses (0.75%, 1.5%, 3.0%, 6.0% and 12%) of rEP. rEP supplementation significantly increased the weight gain, feed efficiency, IL-10 and FN-gamma production in chickens compared to the control group. Our results indicate that crude rEP can provide a substitute for traditional antibiotic feed additives for chickens for enhancing animal growth and health.

Accordingly, the present invention provides a dietary supplement extracted from cultures exhibiting AMP. In one embodiment of the invention, the dietary supplement may be prepared in the form of an agricultural feed supplement.

In the present invention, three cDNAs encoding putative antimicrobial piscidin peptides are isolated and characterized from Epinephelus lanceolatus. Transcripts were designated g6496.t1 (encoded as EP-1), g6497.t1 (encoded as EP-2) and g6498.t1 (encoded as EP-3) and encoded putative AMPs of 76, 76 and 69 amino acid residues, respectively. Based on the sequence alignment between the Piscidin from Evodia rutaecarpa and EP1 (g 6496. T1), EP2 (g 6497. T1) and EP-3 (g 6498. T1), it was found that EP-3 (g 6498. T1) showed a high degree of similarity to the highly active TP3 and TP4 peptides from Evodia rutaecarpa.

In the examples of the present invention, it was shown that the peptide EP-3 (g 6498. T1) has a better activity than EP-1 (g 6496. T1) or EP-2 (g 6497. T1), which also has antimicrobial or growth inhibitory activity in gram-negative and gram-positive bacteria.

In the present invention, EP 21 is expressed in the pPICZ alpha A expression vector of methylotrophic yeast using a gene having an optimized yeast codon. Since EP is a fish gene, it is expected that optimizing the gene for Pichia (Pichia) codon usage will increase yield. Notably, other researchers reported that it was difficult to produce high concentrations of AMP [15] due to degradation of the host proteolytic enzyme. In the present invention, no proteolytic inhibition was found to prevent substantial accumulation of rEP in pichia. His-tagged rEP exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria in methanotrophic yeasts.

In the present invention, it was found that EP from garrupa expressed in methanotrophic yeast after incubation under extreme temperature conditions (100 ℃) was relatively stable in vitro, still retaining some antimicrobial activity against staphylococcus aureus (BCRC 10780). The stability of rEP may be related to its high arginine content. From this point, it has been previously reported that increasing the arginine composition of peptides increases antimicrobial activity and enhances the ability of peptides to intercalate into membranes [22]. Increased arginine content may also enhance translocation and membrane permeability functions [23].

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

1. Materials and methods

All steps involving the animal were performed as required and approved by the animal care and related authorities.

1.2. Molecular cloning, construction and transformation of expression plastids and selection of positive transformants

Primer sequences for amplifying EP were designed according to previous transcriptome analysis and experimental methods [9, 16 ]. Briefly, piscidin was isolated from Epinephelus lanceolatus by RT-PCR. Extracting mRNA from the liver of the grouper and carrying out reverse transcription; the primers used to amplify the cDNA are listed in Table 1. Three recombinant clones (g 6496.T1, g6497.T1, g6498. T1) were selected for sequencing.

Table 1: sequence listing of primer

¹ g6496.t1 piscidin-1 from Epinephelus lanceolatus

² g6497.t1 piscidin-2 from Epinephelus lanceolatus

³ g6498.t1, piscidin-3 of Epinephelus lanceolatus

Multiple sequence alignments were performed with the peptide piscidin from Epinephelus lanceolatus and Ni Luo Wuguo fish (O.niloticus). GL Biochem Inc. (Shanghai, china) synthesized the following peptides, and the antimicrobial activity of each peptide was determined as Minimal Inhibitory Concentration (MIC) of the mini-broth dilution series (microbroth dilution series) [9, 13 ]:

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

(2) EP-2 g6497.t1 (Ac-CFFRHIKSFWRGAKAIFRGARQGWRE-NH 2)

(3)EP-3:g6498.t1(Ac-GFIFHIIKGLFHAGRMIHGLVNRRRHRHGMEE-NH2)。

A102 bp codon-optimized sequence was designed corresponding to the mature EP cDNA gene (g6498.t1). The sequences were based on preferential codon usage by M.methanotrophic yeasts according to the figure decryption codon usage analyzer (http:// gcua. Schoedl. De /), and were synthesized by Omics Bio (Taipei, taiwan, china). The DNA was cloned into EcoRI/XbaI digested pPICZαA from Omics Bio (Taibei, taiwan, china) to produce the recombinant vector pPICZαA-EP-his. The sequence of the EP cDNA gene (g6498.t1) in the vector was then confirmed by sequencing.

pPICZαA-EP-his was linearized with SacI and subsequently transformed into Methanotorula X-33 by electroporation (1.5 kV, 25 μF, 200Ω; ECM 399 electroporation system, BTX Harvard apparatus). As previously reported, identification of positive transformants and selection of rEP expressing clones with high expression were performed without modification [17]. Briefly, recombinant methylotrophic yeast strains containing the rEP gene were incubated in 3ml YPD medium (containing 300. Mu.g Zeocin). After 24 hours (30 ℃), 30mL of BMGY medium was inoculated with 1mL of methanotrophic yeast (P.pastoris) for 24 hours (30 ℃).

Cells were then collected by centrifugation at 6000 Xg for 15 minutes. The cell pellet was resuspended in 50mL BMMY medium. Supernatants and pellet were checked by SDS-PAGE and Western blotting using His-tag antibodies (Abcam, ab 213204). Recombinant proteins were identified by MALDI-TOF (matrix assisted laser Desorption free 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.

Expression of rEP in fermentation tanks

To optimize performance conditions in 5000ml fermenters, 200ml BMGY was inoculated with PTM4 medium at 28℃and 200rpm using a single rEP colony for 36 hours. The culture was then transferred to a 5000ml fermentor (windact, major Science, taiwan peach orchard) containing 3000ml commercial fermentation medium (BMGY with PTM 4) [19]. During fermentation, the temperature was maintained at 30 ℃. The pH was adjusted to 6.0 with 14% ammonia and 0.1N H2SO4, and dissolved oxygen was maintained at a saturation level above 20%. To produce rEP as a livestock feed for supplementing the feed of chickens, rEP [20] was expressed in the fermenter using basal salt medium and PTM 1. After complete consumption of glycerol (19 hours) 50% w/v glycerol was added for 360 minutes. Next, 100% methanol was started to be added and maintained for 24 to 96 hours. The rEP expressing yeast culture was then centrifuged at 6000rpm for 30 minutes and the supernatant was spray dried (YC-500,Pilotech Instrument&Equipment Co.) and then mixed into feed. Prior to feed production, rEP performance in each culture was verified by western blotting and levels of rEP were assessed by comparison to synthetic EP peptides.

Antimicrobial activity of rEP and feed preparation

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 (Riemerella anatipestifer) (RA 3, RA9, RA16, CFC27, CFC363, CFC 437). Bacterial cultures were derived from individual strains that had been amplified and stored at-70 ℃. The culture was inoculated in a liquid medium and cultured at 37℃overnight 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) was mixed with 100 μl of rdep solution and incubated overnight at 37 ℃. The supernatant of the vector-only transformed methanotrophic yeast was used as a control group. After 24 hours, by OD ₆₀₀ The cultures were assessed for growth. All assays were performed in triplicate. The composition analysis of the food fed to the chickens is shown in table 2.

Table 2: approximate analysis of basal feed and commercial feed compositions with additives

1.5. Maintenance and diet therapy for chickens

A total of 189 male or female chickens, 2 days old, were randomly assigned to seven dietary treatment groups including rEP (0.75%, 1.5%, 3.0%, 6.0% and 12%), spirulina-a (rui chemical pharmaceutical co.ltd, new north urban three isthmus region, taiwan) and the basal diet group. Each treatment group consisted of a cage with 27 chickens. The compositions of the feeds are listed in tables 3-5.

Table 3: early basal diet preparation ¹

Table 4: preparation of medium-term basic diet ¹

Table 5: preparation of later-stage basic diet ¹

¹ The fermentation supernatant was added to the food under consumption of cellulose, spray-dried powder to provide a concentration of 0, 0.75, 1.5, 3.0, 6.0 and 12g/100g food.

² Antibiotics: each gram contains: 30mg (can) of spiramycin adipate, 30mg (can) of streptomycin sulfate, 2,500I.U., 15 mg of vitamin B, 10mg of vitamin B, 62 mg of vitamin B, 12 mcg of vitamin B, 2mg of vitamin E, 3 500I.U., 41mg of vitamin K, 0.2mg of folic acid, 5mg of calcium pantothenate, 10mg of nicotinic acid and 20mg of lysine

During the trial, animals were allowed to access feed and water ad libitum, starting three days old. The total time of feeding was 35 days. Chickens were kept in cages (animal containers) under constant conditions (28.9 to 37.8 ℃,12/12 hours of light and shade cycle). rEP the feed and fecal sewage are not directly discharged to the outside.

1.6. Sample collection and enzyme-linked immunosorbent assay

Body weight and survival were monitored daily during the experiment. Weight gain, feed Efficiency (FE), protein Efficiency Ratio (PER) and percent survival were calculated. After 35 days of treatment, the chickens were euthanized, blood samples were collected and serum was obtained by centrifugation (3000×g,4 ℃,15 minutes). Serum was kept at-80 ℃ until analysis. Enzyme-linked immunosorbent assays (ELISA) were performed to determine the concentration of immune factors, including TNF- α, interleukin-1 β, interleukin-6, interleukin-10, lysozyme, immunoglobulin-G (IgG), and interferon- γ, following standard procedures of the manufacturer. Chicken serum samples were analyzed with an ELISA kit of abclon corporation (Woburn, ma, usa). After the reaction is completed, the micro-disk reader is arranged

i3, molecular Devices, lagehausstrasse, wals, austria) at 450 nm.

1.7. Statistical analysis

Data were analyzed with Prism 7 software (GraphPad, inc., la Jolla, california, usa). Values represent mean ± Standard Deviation (SD). One-way variance analysis (ANOVA) was performed using Tukey's multiple comparison test, P <0.05 was considered significant.

2. Results

2.1. Novel piscidins from garrupa exhibit antimicrobial activity

cDNA coding regions of different piscidin sequences (SEQ ID NOS: 10, 12 and 14; FIG. 1) were isolated and characterized from Epinephelus lanceolatus. The three identified cDNA sequences were designated g6496.T1, g6497.T1 and g6498.T1 and encode 76, 76 and 69 amino acids (SEQ ID NOS: 11, 13 and 15), respectively. The results of the alignment of garrupa piscidin (g 6496.t1, g6497.t1 and g 6498.t1) and of Ni Luo Wuguo piscidin (TP 1-TP 5) showed significantly higher sequence similarity (FIG. 2). The lineage tree shows that g6498.t1 corresponds to TP4 and TP3, which shows the best antimicrobial activity characterized so far (fig. 2).

Next, the antimicrobial activity of the synthetic g6496.t1, g6497.t1 and g6498.t1 peptides was determined. As shown in Table 6, all three peptides had activity against both gram positive and gram negative bacteria.

Table 6: in vitro activity of antimicrobial peptides against gram-positive and gram-negative bacteria.

¹ g6496.t1:piscidin-1 of Epinephelus lanceolatus (EP-1)

² g6497.t1: garrupa piscidin-2 (EP-2)

³ g6498.t1: garrupa piscidin-3 (EP-3)

(MIC unit is μg/ml)

The g6498.T1 and g6497.T1 peptides also have activity against Methicillin-resistant Staphylococcus aureus (MRSA) with MIC of 5.6 μg/ml and 60 μg/ml, respectively. These two peptides are also toxic to Vibrio enteritis (Vibrio parahaemolyticus) at similar MICs. Since g6498.T1 has stronger antimicrobial activity than g6496.T1 and g6497.T1, we therefore named the peptide as EP and further produced the peptide in the methanotrophic yeast protein expression system.

Methanotrophic yeast expression System of rEP peptides

As shown in FIG. 3 (a), the persistent expression vector pPICZ. Alpha.A-EP-his contains the methanol-inducible AOX promoter, the. Alpha. -factor signal peptide and the STE13 gene of dipeptidyl aminopeptidase A. rEP the amino acid sequence is encoded by a gene insert optimized for the methanotrophic yeast codon. The methanotrophic yeast (X-33) transformants were grown on Zeocin plates (25. Mu.g/ml) and screened by colony hybridization with anti-His antibodies; high performing strains were selected for subsequent experiments. After identification of the germ, the presence of the correct expression cassette was confirmed by PCR amplification and DNA sequencing. During the initial performance experiment, the optimal methanol concentration was determined by providing different concentrations of methanol over 24 hours. The supernatant and the pellet (after centrifugation) were collected and analyzed by western blot. The results showed that 1% methanol induced a strong manifestation of rEP (fig. 3 (b)). The time-dependent effect of 1% methanol induction is shown in FIG. 3 (c). Thus, 1% methanol induction was chosen for the subsequent fermenter experiments.

2.3. Influence of time and culture Medium on rEP fermentation tank

To evaluate the effect of induction time and medium on rEP performance, transformants were grown in fermentation tanks with Basal Salt Medium (BSM) or BMGY. Large-scale production of rEP from methylotrophic yeast (X-33) is probably the most efficient using high-density culture methods. Thus, induction of rEP was assessed in a 5000mL fermentation tank. When the wet weight of cells in the glycerol feed batch reached 200g/L (16 to 18 hours after inoculation), 1% methanol was added for 24 to 120 hours. After methanol induction, the yeast number was reduced (FIGS. 4 (a) and 4 (b)). After 48 hours of induction in BSM medium, the total rEP protein concentration reached a maximum of 0.8mg/L in the supernatant and a maximum of 5.63mg/L in the pellet. On the other hand, at 48 hours, the maximum rEP total protein concentration in the BMGY medium was 3.6mg/L in the supernatant and 7.74mg/L in the pellet. The antimicrobial activity of rEP was also monitored and gradually increased to its maximum concentration after 120 hours of methanol induction (fig. 5 (a), fig. 5 (b), fig. 6).

Antimicrobial Activity of EP

Antimicrobial activity was assessed by measuring the ability of rEP in flask cultures to prevent the growth of gram positive and gram negative bacteria by a paper spindle diffusion assay. Tables 7 and 8 show the effect of antimicrobial activity and temperature of rEP. Shake flasks were used to produce rEP in methanotrophic yeast. Supernatants were collected 120 hours after induction and applied to the paper ingots at maximum load, where the paper ingots were left to stand on bacterial culture plates for 16 hours at 37 ℃. The zone of inhibition diameter was measured. Representative radial diffusion analysis. rEP exhibit broad antimicrobial activity against the test strain. rEP and temperature effects. Shake flasks were used to produce rEP in methanotrophic yeast. Supernatants were collected 120 hours after induction and applied to the paper ingots at maximum load, where the paper ingots were left to stand on bacterial culture plates for 16 hours at 37 ℃. The zone of inhibition diameter was measured. Representative radial diffusion analysis. The most susceptible pathogen was Riemerella anatipestifer (R.ananticlamp) (CFC 27) and Riemerella anatipestifer (CFC 437) (Table 7). Next, the thermostability of flask fermentation rEP against staphylococcus aureus (BCRC 10780), escherichia coli (BCRC 10675) and pseudomonas aeruginosa (ATCC 19660) was studied. Empty vector without insert in methanotrophic yeast was used as control group. After incubation of the cultures at 40, 60, 80 or 100 ℃ for 5 minutes, the thermal stability was measured by means of a paper spindle diffusion assay (table 8).

Table 7: antimicrobial activity of rEP

Table 8: effect of temperature on rEP antimicrobial Activity

¹ NI, no inhibition

² The control group was a fermentation supernatant from a transformant containing empty bodies. Mu.l ampicillin (2 mg/ml) was applied to the paper pig.

We have found that the inhibition zone diameter (mm) value decreases with increasing pretreatment temperature, but in Staphylococcus aureus (BCRC 10780), the antimicrobial activity is not affected by different temperatures. This finding suggests that the tertiary (or secondary) structure of rEP is important for peptide stability and that it should be protected from high temperatures to maintain activity.

rEP supplementation improves growth performance and immune response

The growth performance of chickens was evaluated on the basis of weight gain and Feed Efficiency (FE). Animals fed with 1.5% and 3.0% rsep had significantly higher growth performance than spirulina-a and basal diet at the end of the 35 day experimental period (table 9). Next, the physiological effects of the chicken administration rEP per day were evaluated by ELISA to determine the concentration 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 (fig. 7 (a) to fig. 7 (G)). No significant changes in TNF- α, II-1β, II-6 or Lyz concentrations were observed between the groups (fig. 7a, c, d and g). The group supplemented with 1.5% EP showed significantly increased IFN- γ concentration compared to the antibiotic and control group (fig. 7 (b), p=0.0043 compared to the antibiotic group, and p=0.0073 compared to the control group). Furthermore, the rEP supplemented group had significantly higher IL-10 concentration than the control group (p=0.0341), but no difference from the antibiotic group (fig. 7 (e)). Chickens raised with antibiotics showed higher serum IgG concentrations (p=0.0394) compared to the control group (fig. 7 (f)).

Table 9: chickens were kept on diets containing rEP (0.75, 1.5, 3.0, 6.0 and 12%) fermented supernatant spray-dried powder for 4 weeks with weight gain, feed Efficiency (FE), protein Efficiency Ratio (PER) and survival rate ¹

¹ The values in the same column with different superscripts are significantly different (p<0.05). Data are expressed as mean ± SD from chicken group (n=27)

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

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

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

As described above, rEP was found in the present invention to improve the growth performance of chickens compared to spirulina-A supplemented feeds. These results indicate that EP can potentially replace antibiotics to improve growth performance (fig. 8 (a) and 8 (d)). Histological analysis showed that the duodenal villi height was significantly higher in the rEP animals than in the control group (fig. 8 (b) and 8 (c)). In the present invention, it was found that 1.5% rEP significantly increased the number of Lactobacillaceae (Lactobacillus) and Enterobacteriaceae (Enterobacteriaceae) in the duodenum, and decreased the number of Enterobacteriaceae and Vitaceae (Staphylococcus) compared to spirulina-A and basal diet (FIG. 8 (e)). No Salmonella (Salmonella) was detected in the blood after challenge (data not shown). After rEP administration per day, the rEP-supplemented group had significantly higher IL-10 than 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. Higher IFN- γ production in rEP supplemented animals may increase cytokines that induce antimicrobial pathways against extracellular and intracellular pathogens.

It was concluded that recombinant EP has a beneficial use as a supplement, replacing antibiotics in chicken feed. The results show that the addition of 1.5% or 3% rEP to chicken feed can improve the growth performance, intestinal morphology, microbiota and immunity of broiler chickens and other animals.

Reference to the literature

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Sequence listing

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His His

Claims

1. A recombinant garrupa piscidin (Epinephelus lanceolatus piscidin, EP) comprising an amino acid molecule consisting of the amino acid sequence of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

2. The EP of claim 1, which is an amino acid molecule consisting of the amino acid sequence consisting of SEQ ID NO. 1.

3. The EP of claim 1, which is an amino acid molecule consisting of an amino acid sequence consisting of SEQ ID NO. 2.

4. The EP of claim 1, which is an amino acid molecule consisting of an amino acid sequence consisting of SEQ ID NO. 3.

5. A dietary supplement for improving the growth performance and immune response of chickens comprising an EP as defined in any one of claims 1 to 4.

6. The dietary supplement of claim 5 in the form of a feed supplement.

7. A feed supplement for improving the growth performance and immune response of chickens comprising an EP as defined in any one of claims 1 to 4.

8. A method of improving the growth performance and immune response in an animal comprising administering to the animal an effective amount of an EP as defined in any one of claims 1 to 4.

9. The method of claim 8, wherein the animal is livestock, poultry or aquatic animal.

10. Use of a composition for improving the growth performance and immune response of an animal, wherein the composition comprises an effective amount of an EP as defined in any one of claims 1 to 4.

11. The use of claim 9, wherein the animal is livestock, poultry or aquatic animal.