CN101182360B - A fusion protein with antibacterial function and its application - Google Patents

Abstract

The invention discloses a fusion protein with antibacterial function and an application thereof. The gene sequence compositions of the fusion protein are hepcidinl precursor antibacterial peptide gene which is obtained by cloning and synthetic magainin antibacterial peptide which are combined as a novel fusion protein cDNA and integrated into a yeast gene group by the gene engineering means; the fusion protein cDNA is induced to express at the yeast, and furthermore, the yeast ferments and cultivates to obtain a plurality of fusion protein. The fusion protein of the invention is detected having obvious antibacterial activity, is applied to prevent and remedy the bacterial disease of aquatic organisms and cultured animals, or the purified fusion protein is directly used as a drug to replace antibiotic to be applied to the treatment of the disease caused by bacteria and even virus.

Description

A fusion protein with antibacterial function and its application

technical field

The present invention relates to a fusion protein and its application, in particular to an animal antimicrobial peptide gene carrier established by genetic engineering technology and an antimicrobial peptide fusion protein obtained through a yeast expression system, and its application in the preparation of antibacterial drugs or disease prevention and control functions. Application in aquatic organisms or farmed livestock and poultry feed products.

Background technique

Antimicrobial peptides are the immune substances produced by the organism itself to resist the infection of external microorganisms, viruses and other pathogens, and are used for non-specific defense of the body. It widely exists in animals and plants, and has the characteristics of high bactericidal activity, rapid action, and no drug resistance. It is an effective means of multicellular biological immune defense and plays an important role in the survival and evolution of organisms. Antimicrobial peptides can not only directly kill pathogenic microorganisms, but also activate the immune system of the organism and increase the ability to resist the invasion of external pathogens. So far, a variety of antimicrobial peptides from lower animals to higher mammals and humans have been isolated and purified. For example: cecropin and cecatotoxin in silkworms, defensins in sharks, shellfish, and tunicates, pleurocidin in lampreys and flounder, magainin and dermasepin in amphibians, defensins in birds, and mammals α-defensin, β-defensin, thionin in plants, plant defensins, etc. More than 800 antimicrobial peptides have been discovered.

Marine biological antimicrobial peptides are an important part of the body's natural immune system. Since the immune system of most marine animals is an innate immune system, compared with mammals, the antimicrobial peptides that are members of the innate immune system have less effect on marine animals. appear more important. The research on antimicrobial peptides of marine organisms mainly focuses on some crustaceans, such as Limulus, crab, etc. In recent years, there have also been research reports on antimicrobial peptides or antimicrobial peptides from shrimp and shellfish, indicating that antimicrobial peptides from marine organisms and their role in aquatic products The application prospect of aquaculture is receiving more and more attention. However, the research on fish antimicrobial peptides started late, and the types of isolated antimicrobial peptides are limited.

defensin is an important family of antimicrobial peptides, and hepcidin is a member of the β-defensin protein family (Verga F, et al, Gene. 2005, 364: 37-44). Hepcidin was originally isolated from human plasma filtrate or urine, and it was confirmed to be a cysteine-rich antibacterial protein, and its antibacterial activity has been confirmed in many ways (Krause A, et al, FEBS Lett, 2000, 480:147-150; Park CH, et al, J Biol Chem, 2001, 276:7806-7810). While studying its antibacterial activity, it was found that Hepcidin also plays a role in iron metabolism (Nicolas G, et al, Proc Natl Acad, 2001, 98: 8780-8785). Subsequently, multiple homologous isoform genes of hepcidin were cloned. Shike et al obtained the hepcidin gene from hybrid grouper, which is the first hepcidin gene obtained from fish (Shike H, et al, Eur J Biochem, 2002, 269: 2232-2237). Magainin was originally isolated from the skin of toads. This antimicrobial peptide has an α-helical structure and is usually composed of L-type amphoteric amino acids. It has killing effects on Gram-negative bacteria and positive bacteria (Zasloff M, Proc Natl Acad Sci USA, 1987, 84:5449-5453; BarrellPJ, et al, Protein Expr Purif, 2004, 33:153-159).

Although antimicrobial peptides have remarkable antibacterial efficacy and broad application prospects, the content of natural antimicrobial peptides is very low and cannot be obtained in large quantities from organisms; chemically synthesized peptides are expensive, which limits their wide application. At present, due to serious environmental pollution, aquaculture diseases frequently occur, often causing relatively large economic losses. The widespread and extensive use of antibiotics such as chloramphenicol in the aquaculture industry has caused excessive accumulation of antibiotics in aquatic products, which has brought great harm to the health of consumers. At present, the European Union has banned the export of aquatic products with excessive antibiotics in my country to EU countries. Therefore, the use of genetic engineering technology to produce genetically engineered antibacterial peptides with high-efficiency bactericidal effects and apply them to the aquaculture industry, as well as to the breeding of various livestock and poultry animals, to replace the increasingly banned antibiotics has become a national aquaculture industry. And the inevitable choice of animal husbandry.

The use of yeast expression system to express eukaryotic genes is being more and more widely used in recombinant DNA technology. The Saccharomyces cerevisiae system was first used, and the Pichia pastoris expression system with excellent characteristics has attracted more and more attention from the scientific and industrial circles, and has gradually replaced the Saccharomyces cerevisiae system. The yeast expression system has unique biological characteristics, and has the advantages of strictly regulating the expression of exogenous eukaryotic cell proteins, processing and modifying expression products, high expression levels, and low nutritional requirements. Since antimicrobial peptides directly act on prokaryotic cell membranes, inhibit their growth and even kill bacteria, the expression system using eukaryotic yeast is almost the only choice for the production of genetically engineered recombinant antimicrobial peptides. In addition, the Pichia pastoris expression system can use methanol as the sole carbon source to synthesize proteins in large quantities, and has been used for the production of single-cell proteins on an industrial scale. The expression of genetically engineered antimicrobial peptides by yeast engineering bacteria can mass-produce antimicrobial peptides expressed by the animal's own genes that are urgently needed by aquatic organisms and animal feeding and can replace antibiotics, and provide the market with non-toxic, harmless, healthy and green products. And the further development and utilization of antimicrobial peptides will produce high economic value and huge social benefits.

Contents of the invention

The object of the present invention is to provide an animal antimicrobial peptide gene carrier established by genetic engineering technology and an antimicrobial peptide fusion protein obtained through a yeast expression system, and its use in the preparation of antibacterial drugs or aquatic organisms with disease prevention and control functions or livestock and poultry feed application in the product.

The technical idea of the present invention is to combine fish antimicrobial peptides and amphibian antimicrobial peptides into a novel fusion protein, use genetic engineering to construct an expression vector containing the cDNA sequence of the fusion protein, and integrate it into the yeast genome for expression. Its expression product can be applied to aquatic organisms or livestock and poultry animal breeding; it can also be purified or concentrated to obtain a high-purity fusion protein, which can be directly used as a substitute for antibiotics for the prevention and control of biological diseases, bacterial and even viral diseases Treatment.

The fusion protein with antibacterial function of the present invention is composed of modified fish antibacterial peptide hepcidin and amphibian antibacterial peptide magainin, and is characterized in that: the fusion protein is a short peptide containing the amino acid sequence shown in SEQ ID NO:1.

The gene of the above fusion protein consists of the cDNA sequence encoding the fish hepcidinl precursor mature peptide, the cDNA sequence encoding the amphibian antibacterial peptide magainin mature peptide, the cDNA sequence of the restriction endonuclease EcroR I site, and the cDNA sequence that conforms to the eukaryotic expression preference Gene sequence composition, it is characterized in that: the nucleotide sequence of described fusion protein gene is the nucleotide sequence shown in SEQ ID NO:2.

The application of the fusion protein of the present invention in the preparation of antibacterial drugs.

The application of the fusion protein of the present invention in the preparation of feed for aquatic organisms or breeding livestock and poultry with disease prevention and control functions.

Wherein: the fusion protein is directly added to aquatic organisms or livestock and poultry feed as an immune additive, or used directly as a disease control drug after purification or as a feed additive.

The yeast expression system of the fusion protein of the present invention is characterized in that the yeast intracellular expression vector pAO815 containing exogenous fusion protein cDNA or other types of yeast expression vectors, and using the vector to make the fusion protein gene in Pichia pastoris, Saccharomyces cerevisiae , Candida or other edible yeast for intracellular or extracellular expression. Its steps are:

Construction of the expression vector: the artificially synthesized antibacterial fusion protein cDNA sequence is designed with a restriction endonuclease site, the fusion protein cDNA sequence and Invitrogen's yeast cell expression plasmid pAO815 are digested and linked together, and then linked together by PCR The direction of insertion of the fusion protein gene was detected by reaction, and the clone containing the fusion protein gene inserted in the forward direction was screened out.

Construction of the yeast expression system: Use restriction endonucleases to digest the yeast expression plasmid to linearize it, and integrate the cDNA sequence of the fusion protein into the yeast genome by electroporation transformation. The transformed bacterial suspension was spread on the MD plate, cultured at 30°C, induced and screened until a single colony appeared. Pick a single colony and use it as a template to confirm positive clones by PCR reaction.

Induced expression and activity detection of fusion protein in cells or outside cells.

The detection method of expression product antibacterial activity is as follows:

Select the positive clone colony, cultivate it at 30°C, collect the bacteria, and resuspend the bacteria with a medium containing methanol, so that the exogenous gene can be induced to express. If using an intracellular expression vector, collect the bacteria, break up the cells and obtain the crude protein extract. If an extracellular expression vector is used, the cell culture fluid is collected, purified by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography, and separated by polyacrylamide gel electrophoresis to obtain an electrophoresis-pure fusion protein.

By coating the crude extract of the fusion protein on a petri dish and simultaneously inoculating pathogenic bacteria such as E. Fusion protein crude extract, detection of A600 absorbance value to detect its antibacterial activity.

The fusion protein of the present invention has obvious antibacterial activity after testing, and can be used for the prevention and treatment of bacterial diseases that are resistant to antibiotics in aquatic organisms and farmed animals, or the purified fusion protein can be directly used as medicine to treat bacteria and even viruses The treatment of the disease caused.

The beneficial effects of the present invention are:

(1) The application of genetic engineering technology can mass-produce antimicrobial peptides of new aquatic organisms and farmed animals.

(2) Solve the problems of long-standing disease hazards in the breeding industry and the accumulation of antibiotics in aquatic products and poultry products exceeding the standard.

(3) Use high technology to drive the technical progress of the aquaculture industry and the aquaculture bait industry.

(4) Provide healthy green aquatic products and poultry products to the society.

Description of drawings

Figure 1. Cloning of fish hepcidinl precursor gene.

Figure 2. Vector pAO815-antibac plasmid expressing fusion protein cDNA.

Figure 3. Detection of pAO815-antibac positive clones.

Figure 4. The antibacterial effect of the antibacterial fusion protein expressed by the Pichia pastoris expression system on Escherichia coli.

Figure 5. The antibacterial effect of the antibacterial fusion protein expressed by the Pichia pastoris expression system on Vibrio anguillarum.

Figure 6. The antibacterial effect of the antibacterial fusion protein expressed by the Pichia pastoris expression system on Vibrio alginolyticus.

Wherein: 1×, 2×, 3× in Fig. 4～6 are different concentrations of antibacterial fusion protein expression system protein bold solution, 0 is control (yeast expression system protein bold solution expressing β-Gal).

Detailed ways

Below with embodiment and in conjunction with accompanying drawing, content of the present invention will be further described:

1. Preparation of fish liver cDNA: 24 hours after giant flounder was injected with Vibrio anguillarum, the liver was taken out, quickly placed in liquid nitrogen, and the hard tissue pieces were frozen at -80°C for preservation. Refer to TRIZOL Reagent operation instructions, take about 100mg of frozen preserved tissue, add it to 1ml TRIZOL reagent, homogenize at room temperature for 5 minutes, add 200μl chloroform to shake vigorously for 15 seconds, and incubate at room temperature for 3 minutes. Centrifuge at 12,600 rpm for 15 min at 4°C. Add 250μl of high-concentration salt solution composed of 0.8M sodium acetate and 1.2M sodium chloride to obtain the upper layer of the interface, mix well, then add 250μl isopropanol and mix well, settle at room temperature for 10min, and then rotate at 12,600rpm at 4℃ Centrifuge for 10 min. After the supernatant was removed, the precipitate was rinsed with 1 ml of 70% ethanol, and centrifuged at 11,000 rpm at 4°C for 5 min. After dissolving the RNA pellet in 20 μl of DEPC-treated double distilled water (RNase-free) at 55°C for 10 min, take 2.0 μg of total RNA, add 1 μl of 50 pM random primers, and dilute to 17.1 μl with DEPC water. After destroying the secondary structure of RNA at 70°C for 5 minutes, place it on ice for quenching. Add 5 μl M-MLV 5× transcription buffer, 1.3 μl 10 mM dNTP, 24U rRNAse inhibitor and 200UM-MLV reverse transcriptase in sequence. After reacting at 42°C for 1 hour, maintain at 94°C for 5 minutes to terminate the reaction, and store at 4°C.

2. Cloning and sequence analysis of fish antimicrobial peptide hepcidinl precursor gene: Taking turbot liver cDNA as template, using primers WBHsp(5`-CAAACCCTCCTAAGATGAAG-3`), WBHap(5`-AATCCTCAGAACCTACAGCA-3`) for PCR reaction . The annealing temperature of the reaction was 52° C., and a PCR band of about 300 bp was obtained after 30 cycles (see FIG. 1 ). The obtained PCR product was recovered using a gel electrophoresis kit. It was confirmed by sequencing that the sequence was 293 bp in length, including the complete coding region, in which the open reading frame was 273 bp in length and encoded 90 amino acids. See GenBank sequence number AM113708 for details. . Nucleic acid sequences were compared for homology and similarity through NCBI's Blastn server. Most of the sequences with high homologous similarity are gene sequences of the Hepcidin family, among which the homologous similarity with flounder is 90% [Pseudosciaena crocea (DQ307050)], and the homologous similarity with white perch is 84% [Morone chrysops (AF394246)], with a homologous similarity of 83% to black sea bream [Acanthopagrusschlegelii (AY669380)]. The sequence was determined to be the hepcidinl precursor gene of turbot. The signal peptide region of the hepcidinl precursor protein sequence was predicted using the signalP server, and the 24 amino acids at the C-terminal of the hepcidinl precursor protein sequence were signal peptides. The secondary structure of the hepcidinl precursor protein sequence was predicted using the software Omiga2.0. The N-terminal 21 amino acids of the hepcidinl precursor protein sequence were β-sheet structures, and this part of the sequence contained eight cysteines. The most notable feature of the mature peptide of the Hepcidin protein family is the β-sheet structure formed by these eight cysteines, which is consistent with the characteristics of the Hepcidin protein family. Comparing the mature peptide of sea bass and zebrafish Hepcidin, it can be concluded that the mature peptide of turbot hepcidinl precursor has 21 amino acids.

3. Design and preparation of the fusion protein cDNA: the fusion protein cDNA sequence consists of the gene sequence encoding the fish hepcidinlprecursor mature peptide, the gene sequence encoding the amphibian antimicrobial peptide Magainin mature peptide, the sequence of the restriction endonuclease EcroR I site and Gene sequence composition that conforms to eukaryotic expression preference and Kozak rules. The site for genetic engineering modification is described as follows: (1) The preferred base at the 4th position is G; (2) There is no base T in the flanking sequence of about 15 bp at the 5' end of ATG; (3) At -3 , -6 and -9 positions, G is a preferred base; (4) Except for -3, -6 and -9 positions, in the entire flanking sequence region, C is a preferred base. Therefore, the protein sequence encoded by the fusion protein gene sequence is composed of two mature peptide sequences of hepcidinl precursor and Magainin, which are optimized and modified by genetic engineering methods. This gene sequence was artificially synthesized using the solid-phase phosphoramidite method. The sequence is 151bp long (see the nucleotide sequence shown in SEQ ID NO: 2), encoding 39 amino acids, and the protein expressed by the yeast expression system constructed subsequently is this A short peptide containing 39 amino acids (see the amino acid sequence shown in SEQ ID NO: 1).

4. Construction of yeast expression system pAO815-antibac vector: the artificially synthesized antibacterial fusion protein gene sequence is designed with a restriction endonuclease EcroR I enzyme cutting site, and the yeast intracellular expression plasmid vector pAO815 also has a single enzyme of EcroR I The cleavage site, the fusion protein gene and the pAO815 plasmid have the same cohesive ends after digestion. The fusion protein gene and the pAO815 plasmid were respectively digested with EcroR I, and the digested products were recovered using a gel electrophoresis recovery kit. Use the DNA Ligation Kit kit for ligation, take 11 μl of fusion protein gene, add 2 μl of pAO815 vector, 2.5 μl of ligation buffer, 4.5 μl of ligase, and ligate overnight at 16°C. Link them together and introduce them into E. coli DH5α. Using primers HMsp (5`-GGACAGCCACATCTCCCTTT-3`) and HMap (5`-GTGCGAATTCTCAGAACTTC-3`), the transformed Escherichia coli DH5α bacterial suspension was used as a template to perform PCR reaction at an annealing temperature of 53°C for 30 cycles. The ligation product detects the direction of insertion of the fusion protein gene. If the fusion protein gene is inserted forward into the plasmid pAO815, a PCR band of about 130 bp will appear, and if the fusion protein gene is inserted backward, no PCR band will appear. Clones containing the fusion protein gene inserted in the forward direction were screened. The resulting expression vector was named pAO815-antibac plasmid (Figure 2). This plasmid was introduced into Escherichia coli to form polyclones. Prepare the pAO815-antibac plasmid, and use the restriction endonuclease Sal I to digest the plasmid to linearize it. Mix approximately 20 μg of the linearized pAO815-antibac plasmid solution with 80 μl of GS115 competent cells, and transfer to a 0.2 ml ice-cooled electroporation cuvette. The electrotransformation cup was placed in an ice bath for 5 min and then transformed using an electrotransformer. The conditions are: a voltage of 1.5kV, electric shock for 10msec, to integrate the exogenous gene into the genome of yeast GS115. After transformation, add 1 ml of ice-cooled 1 mol sorbitol solution to the cells, mix well, and transfer to a 1.5 ml EP tube. The transformed bacterial suspension was then evenly spread on MD plates and cultured at 30°C until a single colony appeared. Pick a single colony, use it as a template, and use primers 5'AOX (5'-GACTGGTTCCAATTGACAAGC-3') and 3'AOX (5'-GCAAATGGCCATTCTGACATCC-3') to screen positive clones with pAO815-antibac by PCR reaction. A PCR band of about 600 bp appeared in the positive clone integrating the cDNA sequence of the fusion protein (Fig. 3). The colony containing pAO815-antibac was selected, placed in a shake flask filled with 25 ml of BMGY medium, and cultured at 30° C. until the OD ₆₀₀ of the bacterial solution = 2.0. Collect the cells and resuspend the cells with BMMY to make OD ₆₀₀ = about 1.0. The obtained bacterial solution was placed in a 1 L shake flask and cultured at 30° C., adding anhydrous methanol to the medium every 24 hours to a final concentration of 0.5% and culturing until OD ₆₀₀ =3.0. The bacteria were collected, and the crude protein extract was obtained after breaking the cells, which was rich in fusion proteins.

The protein crude extract can also be precipitated with gradient ammonium sulfate, and then the precipitate is collected and dissolved in phosphate buffer, and then treated by ion exchange chromatography and gel chromatography respectively to obtain electrophoretic pure fusion protein.

At the same time, the bold solution of yeast expression system protein that can express β-Gal cultured under the same conditions was used as a control.

5. Bacteriostasis test: Drop different concentrations of yeast protein crude extract or purified fusion protein on a plate plate coated with Escherichia coli, Vibrio anguillarum or Vibrio alginolyticus. Culture the petri dish overnight at 37°C, observe and record the antibacterial effect the next day.

Both the crude extract rich in fusion protein and the purified fusion protein have antibacterial activity (results are shown in Figures 4-6).

6. The application of fusion protein in the preparation of antibacterial drugs: the yeast containing the fusion protein secretory expression vector is conventionally cultivated and the culture medium is collected, and then the veterinary-grade fusion protein is obtained through conventional precipitation, centrifugation, desalting, chromatography and other steps ; or add histidine cDNA sequence and other tags at both ends of the fusion protein sequence, and use affinity chromatography to obtain a higher purity fusion protein.

The obtained fusion protein can be directly used as an antibacterial drug, and can be used in the prevention and treatment of aquatic organisms or other livestock diseases with conventional dosage.

7. Application of fusion protein in the preparation of feed for aquatic organisms or livestock and poultry with disease prevention and control functions: since yeast is rich in protein, vitamins and other nutrients, yeast containing intracellular expression vectors can be used as feed additives. According to the set ratio, the yeast containing the intracellular expression vector is added to the feed of aquatic organisms or livestock and poultry, so as to provide nutrients for the aquatic organisms or livestock and poultry while improving their disease defense ability.

If the above method is applied, use a 100L fermenter to expand and cultivate Pichia pastoris GS115 containing the nucleotide sequence shown in SEQ ID NO: 2 in the genome at 30°C, and induce the antibacterial fusion protein gene by adding 0.5% methanol to the fermentation broth After 3-5 days of culture and filtration, a large amount of GS115 yeast expressing the fusion protein with antibacterial activity can be obtained, the number of the yeast is not less than 5×10 ⁴ cfu/g, and the water content is ≤10%.

Select corn flour, bran, bran, bean cake, fish meal, and bone meal as the basic feed for aquatic organisms or livestock and poultry in a conventional manner, and use the yeast obtained above as a feed additive to directly add to the basic feed for aquatic organisms or livestock and poultry. Mixing in the feed, the added proportion of the feed additive is 20% of the base feed weight (the consumption of fish meal or other protein feed can be correspondingly reduced), so that the final nutritional components of the base feed mixed with the additive include crude protein ≥ 35%, crude protein Fat ≥ 3%, crude fiber ≥ 3%, crude ash ≥ 15%, calcium ≥ 2%, phosphorus ≥ 1%, lysine ≥ 1.8%, to produce feed for aquatic organisms or breeding livestock and poultry with disease prevention and control functions.

Alternatively, the whole fermented liquid obtained above is directly used for feeding livestock and poultry and aquaculture organisms at an addition ratio of 5% to 25% by weight of the basic feed.

Further, the yeast containing the fusion protein secretory expression vector is conventionally cultured and the culture medium is collected, and then conventional precipitation, centrifugation, desalting, chromatography and other steps are used to obtain the veterinary drug grade containing the protein shown in SEQ ID NO: 1. Fusion protein of amino acid sequence.

Select corn flour, bran, bran, bean cake, fish meal, and bone meal as the basic feed for aquatic organisms or livestock and poultry in a conventional manner, and use the fusion protein obtained above as a feed additive to directly add to the basic feed for aquatic organisms or livestock and poultry. Mixing in the feed, the added proportion of the feed additive is 10% of the weight of the basal feed, so that the final nutrient composition of the basal feed mixed with the additive is ≥ 35% of crude protein, ≥ 3% of crude fat, ≥ 3% of crude fiber, and ≥ 3% of crude fiber Ash content ≥ 15%, calcium ≥ 2%, phosphorus ≥ 1%, lysine ≥ 1.8%, to prepare feed for aquatic organisms or breeding livestock and poultry with disease prevention and control functions.

sequence listing

<110> First Institute of Oceanography, State Oceanic Administration

<120> A fusion protein with antibacterial function and its application

<141>2007-9-11

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<213> Artificial sequence

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Lys Phe Leu His Ser Ala Lys Lys Phe

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actctgccaa gaagttctga gaattcgcac c 151

Claims (5)

1. A fusion protein with antibacterial function, composed of modified fish antimicrobial peptide hepcidin and amphibian antimicrobial peptide magainin, characterized in that: the fusion protein is a short peptide of the amino acid sequence shown in SEQ ID NO:1 . 2. the gene of fusion protein described in claim 1, is made up of the cDNA sequence of coding fish hepcidin1 precursor mature peptide, the cDNA sequence of coding amphibian antimicrobial peptide magainin mature peptide, the cDNA sequence of restriction endonuclease EcroR I site, And it is a gene sequence conforming to eukaryotic expression preference, characterized in that: the nucleotide sequence of the fusion protein gene is the nucleotide sequence shown in SEQ ID NO:2. 3. The application of the fusion protein according to claim 1 in the preparation of antibacterial drugs. 4. The application of the fusion protein according to claim 1 in the preparation of feed for aquatic organisms or breeding livestock and poultry with disease prevention and control functions. 5. The application according to claim 4, characterized in that: the fusion protein is directly added to aquatic organisms or livestock and poultry feed as an immune additive.

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