Compound preparation for improving animal immunity or feed additive containing compound preparation as well as preparation method and application of compound preparation

Abstract

The invention discloses a compound preparation for improving animal immunity or a feed additive containing the compound preparation as well as a preparation method and application of the compound preparation. The compound preparation is prepared from 10 to 30 parts of lucid ganoderma extract, 15 to 30 parts of herba andrographitis extract, 10 to 20 parts of rhizoma polygoni cuspidati extract, 1 to 6 parts of allicin and 0 to 5 parts of biological composition, the biological composition contains an Amuc1100 protein and an antibacterial peptide. The compound preparation can be mixed according to a certain proportion to be prepared into an oral medicine or a feed additive. The compound preparation has a synergistic effect, can stimulate proliferation and activation of immune cells, promote differentiation, maturation and function exertion of the immune cells, inhibit inflammatory response and pathogen infection and improve the immunity of animals, and is suitable for disease prevention and treatment in the fields of breeding industry, animal husbandry and the like.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition, and provides a compound preparation or feed additive containing the same to enhance animal immunity, as well as its preparation method and application. Background Technology

[0002] An animal's health and growth are closely related to the state of its immune system. However, during the breeding process, due to environmental changes, disease stress, and nutritional deficiencies, an animal's immunity is often affected to some extent. Improving the immunity of livestock and poultry can create significant economic value in several aspects: 1) Enhancing disease resistance: Improving animal immunity strengthens their disease resistance and reduces the chance of illness. When animals face pathogens, those with strong immunity can quickly and effectively resist them, preventing disease. 2) Promoting growth: Animal health is closely related to their immunity. Improving immunity can promote animal growth and development, improve production performance and feed conversion rate. 3) Preventing the spread of epidemics: In farms, improving animal immunity can effectively prevent the spread of epidemics. Once an epidemic occurs, animals with strong immunity can better resist pathogens, reducing the spread of the epidemic. 4) Improving economic efficiency: Improving animal immunity can reduce drug use and medical expenses, improving the economic efficiency of farms. At the same time, animals with strong immunity can better adapt to environmental changes and cope with various challenges, improving the survival ability of farms. 5) Meeting food safety requirements: Improving animal immunity can reduce problems such as drug residues and bacterial contamination, improve the quality and safety level of animal products, and meet consumers' health needs.

[0003] Therefore, it is of great significance to develop a formula, compound preparation or feed that can improve animal immunity.

[0004] Studies have found that animal immunity can be comprehensively enhanced through multiple mechanisms, including increasing the activity of immune cells and molecules, regulating the endocrine system, and boosting metabolism, thereby increasing the body's resistance to various pathogens and stress stimuli. Traditionally, traditional Chinese medicine (TCM) has been widely used. The active ingredients in TCM can stimulate the proliferation and activation of immune cells, enhancing the body's immune response and improving immunity. Furthermore, TCM can regulate the endocrine system, promote hormone secretion, and enhance metabolism and immunity.

[0005] Herbal extracts are generally more potent than the herbs themselves because they extract the essence of the herbs, obtaining higher purity active ingredients or substances, thus enhancing efficacy. Herbal extracts can also boost the body's immunity through the following mechanisms. 1) Regulating the immune system: Components in traditional Chinese medicine extracts, such as flavonoids, polysaccharides, and amino acids, can regulate the immune system and improve animal immunity. These components can enhance animal strength and endurance, reduce fatigue, and improve production performance. 2) Promoting digestion and absorption: Traditional Chinese medicine extracts can also promote digestion and absorption in animals, improve gut microbiota, and reduce digestive system diseases such as diarrhea. This helps improve the absorption and utilization of nutrients, further enhancing the body's immunity. 3) Antioxidant effects: Some traditional Chinese medicine extracts have antioxidant effects, which can scavenge free radicals and protect tissues from oxidative damage. This helps reduce oxidative stress in animals under stress and maintain their health. 4) Antiviral potential: Some traditional Chinese medicine extracts have antiviral potential, which can reduce histamine production and inhibit the generation of pro-inflammatory factors, thereby effectively regulating innate immunity and inducing cellular and humoral immune responses. This helps animals defend against viral diseases.

[0006] Traditional Chinese medicine extracts contain numerous active ingredients that strongly promote the body's immunity. However, some current formulations for enhancing animal immunity have several problems, including: 1) Nutritional imbalance: Insufficient or unbalanced nutrients in animal feed can lead to decreased immunity. For example, a lack of certain essential vitamins, minerals, or proteins can negatively impact the animal's immune system. 2) Inappropriate formulation: Some formulations may contain inappropriate drug combinations, leading to drug interactions and affecting efficacy. 3) Unstable quality: Some formulations may have inconsistent quality, resulting in varying drug quality and affecting treatment effectiveness. 4) Side effects: Some drugs may cause side effects, such as damage to organs like the liver and kidneys. 5) Cost: Some immunity-enhancing drugs are expensive, potentially increasing breeding costs and impacting farmers' economic benefits. 6) Drug resistance: Drugs added to animal feed, such as antibiotics and antibacterial agents, may cause animals to develop drug resistance, thereby reducing the therapeutic effect. Furthermore, long-term use of antibiotics can lead to dysbiosis in animals, thereby affecting their immunity. 7) Immunosuppression: Certain drugs or additives may suppress the animal's immune system, leading to decreased immunity. For example, some chemical drugs may interfere with the normal function of immune cells, thus reducing the animal's immunity.

[0007] In recent years, with the rise of biomedicine, researchers have discovered that some biological agents also have certain effects on animal immunity. For example, the Amuc_1100 protein from Akkermansia muciniphila (Akk bacteria) is a protein of great value in improving animal immunity. Amuc_1100 is the outer membrane protein of Akk bacteria. Akk bacteria can use intestinal mucus on the surface of the intestinal epithelium as their sole source of carbon, nitrogen, and energy for growth. Akk bacteria play an important role in regulating intestinal barrier function, homeostasis, and metabolism, and are associated with obesity, diabetes, and cardiovascular metabolic diseases. Therefore, Akk bacteria can be used as a probiotic to improve the body's condition; however, Akk bacteria are strict anaerobic bacteria and are difficult to culture. The Amuc_1100 protein, derived from *Hyalophora chinensis*, has been shown to have many similar functions to *Hyalophora chinensis*. Amuc_1100 is a bioactive peptide that can inhibit inflammatory and oxidative stress responses, protecting the body from damage. Furthermore, in mice, oral administration of inactivated *Hyalophora chinensis* or Amuc_1100 can improve colitis by regulating CD8+ cytotoxic T cells (CTLs) and prevent related colorectal cancer. However, there are few studies on directly administering Amuc_1100 orally to enhance animal immunity. Another example is cecropin (CP), a 37-amino acid cationic polypeptide isolated from the fat body of the hawk moth *Hyalophora cecropia*, which plays an important role in the host's defense system.

[0008] However, since both cephalosporin antimicrobial peptides and Amuc_1100 protein belong to the emerging field of biotechnology, while traditional Chinese medicine belongs to the field of traditional medicine, there are many technical barriers in both fields. For example, biological agents have advantages such as high activity, strong specificity, and low toxicity, but their oral activity is low due to the influence of the gastrointestinal environment; and existing technologies for biological products applied to animals generally require complex downstream purification processes. Therefore, new combinations of these two fields, namely traditional Chinese and Western medicine, urgently need to be researched and explored. Summary of the Invention

[0009] The purpose of this invention is to overcome the problems of unbalanced nutritional formulations, improper compatibility, unstable quality, side effects, high costs, drug resistance, and immunosuppression in existing technologies for improving animal immunity using traditional Chinese medicine extracts. Simultaneously, this invention overcomes the problem that most existing biological products have low oral activity and require expression and complex downstream purification to obtain pure products, resulting in high production and application costs. Biological agents have advantages such as high activity, strong specificity, and low toxicity, but their oral activity is low due to the influence of the gastrointestinal environment; and existing biological products used in animals generally require complex downstream purification. The inventors of this application, through numerous experiments, selected suitable combinations of biological proteins and refined the preparation method of these combinations, avoiding complex downstream purification steps and reducing costs; simultaneously, they provide orally administered, highly active biomolecules that can improve animal immunity. This invention provides a compound preparation for improving animal immunity, its preparation method, and its application. This compound preparation is nutritionally balanced and has immune-enhancing and antibacterial properties, and is low in cost. The compound preparation described in this invention has a synergistic effect, which can stimulate the proliferation and activation of immune cells, promote the differentiation, maturation and function of immune cells, inhibit inflammatory responses and pathogen infection, improve animal immunity, reduce animal morbidity, and promote animal growth. Moreover, the compound preparation described in this invention is nutritionally balanced and inexpensive, and is suitable for disease prevention and treatment in the fields of animal husbandry and animal husbandry.

[0010] The first aspect of the present invention provides a compound preparation or feed additive containing the same to enhance animal immunity, wherein the compound preparation or feed additive contains, by weight, 10-30 parts of Ganoderma lucidum extract, 15-30 parts of Andrographis paniculata extract, 10-20 parts of Reynoutria japonica extract, 1-6 parts of Allium sativum extract and 0-5 parts of a biological composition; wherein the biological composition contains Amuc_1100 protein and antimicrobial peptides.

[0011] According to the present invention, the compound preparation is preferably an oral preparation, which is convenient for making into a feed additive.

[0012] Preferably, the compound preparation or feed additive containing it comprises, by weight, 10-30 parts of Ganoderma lucidum extract, 15-30 parts of Andrographis paniculata extract, 10-20 parts of Reynoutria japonica extract, 1-6 parts of Allium sativum extract, and 0-5 parts of a biological composition; wherein the biological composition comprises Amuc_1100 protein and antimicrobial peptides.

[0013] In some embodiments, the compound preparation or feed additive containing it comprises, by weight: 10-15 parts of Ganoderma lucidum extract, 15-20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1-4 parts of allicin and 1-3 parts of biological composition.

[0014] In other embodiments, the compound preparation or feed additive containing it comprises, by weight:

[0015] 15 parts of Ganoderma lucidum extract, 15-22.5 parts of Andrographis paniculata extract, 10-15 parts of Polygonum cuspidatum extract, 1.5-3 parts of a biological composition and 1.5-6 parts of allicin.

[0016] In some preferred embodiments, the compound preparation or feed additive containing it comprises, by weight, 10-15 parts of Ganoderma lucidum extract, 15-20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1-4 parts of allicin and 1-3 parts of biological composition.

[0017] In some other preferred embodiments, the compound preparation or feed additive containing it comprises, by weight, 15 parts of Ganoderma lucidum extract, 15-22.5 parts of Andrographis paniculata extract, 10-15 parts of Polygonum cuspidatum extract, 1.5-3 parts of biological composition and 1.5-6 parts of allicin.

[0018] In some preferred embodiments of the present invention, the compound preparation or feed additive containing it comprises, by weight parts:

[0019] 10 parts of Ganoderma lucidum extract, 15 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1 part of a biological composition, and 1 part of allicin; or,

[0020] Contains: 15 parts Ganoderma lucidum extract, 20 parts Andrographis paniculata extract, 10 parts Polygonum cuspidatum extract, 3 parts of a biological composition, and 4 parts allicin; or,

[0021] Contains: 30 parts Ganoderma lucidum extract, 30 parts Andrographis paniculata extract, 20 parts Polygonum cuspidatum extract, 5 parts of a biological composition, and 6 parts allicin; or,

[0022] It contains: 15 parts Ganoderma lucidum extract, 20 parts Andrographis paniculata extract, 10 parts Polygonum cuspidatum extract and 6 parts Allicin.

[0023] In some other preferred embodiments, the compound preparation or feed additive containing it comprises, by weight, 10 parts of Ganoderma lucidum extract, 15 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1 part of a biological composition, and 1 part of allicin; or,

[0024] Composed of 15 parts Ganoderma lucidum extract, 20 parts Andrographis paniculata extract, 10 parts Polygonum cuspidatum extract, 3 parts of a biological composition, and 4 parts allicin; or,

[0025] It consists of 30 parts of Ganoderma lucidum extract, 30 parts of Andrographis paniculata extract, 20 parts of Polygonum cuspidatum extract, 5 parts of a biological composition, and 6 parts of allicin; or,

[0026] It consists of 15 parts of Ganoderma lucidum extract, 20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract and 6 parts of allicin.

[0027] According to the present invention, the extracts of the aforementioned traditional Chinese medicines, including Ganoderma lucidum, Andrographis paniculata, Polygonum cuspidatum, and allicin, are extracts obtained by conventional methods in the art, such as water extracts and / or alcohol extracts; commercially available extract products may also be used. The extracts of Ganoderma lucidum, Andrographis paniculata, and Polygonum cuspidatum in this invention are all prepared using a mixture of ethanol and water extracts, which are convenient and environmentally friendly to prepare.

[0028] In this invention, the weight unit of the number of weight parts can be conventionally determined by those skilled in the art as needed, for example, the weight of each part is mg, g or kg.

[0029] In some embodiments, the yeast is a transformant expressing the biological composition, and commonly used Pichia pastoris such as commercially available Pichia pastoris X33, GS115 or KM71H are selected.

[0030] A second aspect of the present invention provides a fusion protein comprising an antimicrobial peptide and an Amuc_1100 protein, wherein the antimicrobial peptide and the Amuc_1100 protein are functional proteins that perform the main biological activities.

[0031] In a preferred embodiment of the present invention, the bioactive functional protein of the fusion protein is composed of an antimicrobial peptide and an Amuc_1100 protein; of course, other proteins or antimicrobial peptides that enhance animal immunity may also be added.

[0032] In some preferred embodiments, the fusion protein further comprises a signal peptide and / or a cleavage tag. The cleavage tag of this invention enables yeast cells to automatically cleave the fusion protein during secretion to separate cephalosporin and Amuc_1100 protein.

[0033] In a preferred embodiment of the present invention, the fusion protein comprises an antimicrobial peptide, an Amuc_1100 protein, a signal peptide, and a cleavage tag.

[0034] In some implementations, the cleavage tag is an enzyme cleavage site.

[0035] In one specific embodiment of the present invention, the enzyme cleavage site is selected from the kex2 enzyme cleavage site present in the transformant itself for precursor protein processing.

[0036] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:5.

[0037] A third aspect of the present invention provides an isolated nucleic acid that encodes a fusion protein as described in the second aspect of the present invention.

[0038] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:1.

[0039] A fourth aspect of the present invention provides an expression vector comprising the isolated nucleic acid as described in the third aspect of the present invention.

[0040] In some implementations, the backbone of the expression vector is a plasmid backbone.

[0041] In some preferred embodiments of the present invention, the plasmid backbone is selected from pGAPZαA, pPICZαA or pPIC9K, which are suitable for Pichia pastoris transformants.

[0042] A fifth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in a third aspect of the present invention, or an expression vector as described in a fourth aspect of the present invention.

[0043] In some embodiments, the host cell used to construct the transformant is Pichia pastoris, such as Pichia pastoris X33, GS115 or KM71H.

[0044] A sixth aspect of the present invention provides a protein combination comprising an antimicrobial peptide and an Amuc_1100 protein.

[0045] In this invention, the antimicrobial peptide is an antimicrobial peptide known in the art, such as defensins, antibiotics, and antimicrobial peptide fragments. The antimicrobial peptide may be an antimicrobial peptide derived from microorganisms or a synthetically produced antimicrobial peptide.

[0046] In some embodiments, the antimicrobial peptide is preferably cephalosporin; cephalosporin is chosen because it has a strong killing effect on major pathogenic bacteria in livestock and poultry (such as Salmonella, Escherichia coli, Staphylococcus aureus, etc.), and can be co-expressed with Amuc_1100, so that the biological composition of the present invention can be obtained through a single fermentation, simplifying the preparation process of the biological composition.

[0047] In some embodiments, the biological composition is a yeast fermentation product expressing Amuc_1100 protein and antimicrobial peptides; the yeast fermentation product can be a freshly fermented wet product or a product that has undergone freeze-drying, and the freeze-drying process does not affect the efficacy of the yeast fermentation product; the freeze-dried yeast fermentation product is easy to transport and store.

[0048] In some embodiments, the cephalosporin has an amino acid sequence as shown in SEQ ID NO:8.

[0049] In some embodiments, the Amuc_1100 protein has an amino acid sequence as shown in SEQ ID NO:9.

[0050] In one embodiment, the nucleotide sequence encoding the cephalosporin is shown in SEQ ID NO:6.

[0051] In one embodiment, the nucleotide sequence encoding the Amuc_1100 protein is shown in SEQ ID NO:7.

[0052] A seventh aspect of the present invention provides a method for preparing a protein combination as described in the sixth aspect of the present invention or a fusion protein as described in the second aspect of the present invention, comprising culturing a transformant as described in the fifth aspect of the present invention to obtain a fermentation product.

[0053] In some embodiments, the obtained fusion protein is isolated upon secretion from the transformant to form a protein combination as described in the sixth aspect of the invention, as the biological composition of the invention.

[0054] The eighth aspect of the present invention provides the use of a compound preparation or feed additive containing the compound as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, or a protein combination as described in the sixth aspect of the present invention in the preparation of a preparation for enhancing animal immunity.

[0055] In some implementations, the animals are selected from poultry, livestock, and economically important aquatic animals.

[0056] In some implementations, the poultry is selected from chickens, ducks, geese, etc.

[0057] In some implementations, the livestock are selected from animals such as pigs, cattle, and sheep.

[0058] In some implementation schemes, the economically important aquatic animals are selected from fish, shellfish, and other animals.

[0059] In some implementations, the animal is a chicken (Gallus).

[0060] A ninth aspect of the present invention provides a composition of a compound preparation and an animal diet, wherein the amount of the compound preparation is 0.1-2% of the animal diet.

[0061] In some embodiments, the compound preparation is used at a dosage of 0.5% of the animal's daily diet.

[0062] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0063] The reagents and raw materials used in this invention are all commercially available.

[0064] The positive and progressive effects of this invention are as follows:

[0065] The compound preparation described in this invention has a synergistic effect, which can stimulate the proliferation and activation of immune cells, promote the differentiation, maturation and function of immune cells, inhibit inflammatory responses and pathogen infection, improve animal immunity, reduce animal morbidity, and promote animal growth. Moreover, the compound preparation described in this invention is nutritionally balanced and inexpensive, and is suitable for disease prevention and treatment in the fields of animal husbandry and animal husbandry. Attached Figure Description

[0066] Figure 1 The image shows the plasmid map of the pGAPZαA-Cecropin-Amuc_1100 co-expression vector.

[0067] Figure 2 The effect of different concentrations of yeast fermentation freeze-dried powder on the proliferation of mouse spleen lymphocytes; **p<0.01 indicates that each treatment group had a highly significant difference compared with the control group (Control).

[0068] Figure 3 Results of antibacterial experiment of yeast fermentation broth (CA1100); ***p<0.001 indicates comparison between CA1100 group and PBS; ## p<0.01 indicates a comparison between the CA1100 (25mg / mL) group and the 1mg / mL and 5mg / mL groups.

[0069] Figure 4 The effect of the compound on LPS-induced inflammation of RAW264.7 macrophages; *p<0.05, **p<0.01, ***p<0.001 indicate the comparison between each compound group and LPS. # p<0.05, ## p<0.01 indicates a comparison between each compound treatment group and compound D group.

[0070] Figure 5 The effect of cephalosporin and Amuc_1100 alone and in combination on serum IgG in CTX-induced immunosuppressed mice. ***p<0.001 indicates comparison between the CTX control group and the blank group; # p<0.05, ## p<0.01 indicates that each treatment group is compared with the CTX control group;& p<0.05 indicates a comparison between the CA1100 group and the cephalosporin group and the Amuc_1100 group.

[0071] Figure 6 The effects of each compound on organ indices in CTX-induced immunosuppressed mice are shown; A represents the thymus index; B represents the spleen index. **p<0.01 indicates a comparison between the CTX control group and the blank control group; # p<0.05, ## p<0.01 indicates a comparison between each compound treatment group and the CTX control group; & p<0.05 indicates a comparison between compound group D and the control compound.

[0072] Figure 7 The effect of each compound on serum IgG levels in CTX-induced immunosuppressed mice; **p<0.01 indicates a comparison between the CTX control group and the blank group; # p<0.05, ## p<0.01 indicates a comparison between each compound treatment group and the CTX control group; & p<0.05 indicates a comparison between compound group D and the control compound.

[0073] Figure 8 The effects of feeding the compound preparation on the weight of chicks and the IgG content in chick serum were investigated. A represents the effect of feeding the compound preparation on the weight of chicks, and B represents the effect of feeding the compound preparation on the IgG content in chick serum. Detailed Implementation

[0074] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0075] Example 1: Construction and expression of cephalosporin and Amuc_1100 expression vectors

[0076] The gene sequences (SEQ ID NO:1) of cecropin and Amuc_1100 were obtained by whole-genome synthesis.

[0077] An XhoI restriction site and a signal peptide cleavage sequence were added to the 5' end of the sequence shown in SEQ ID NO:1 using primer CA-F. The nucleotide sequence of primer CA-F is shown in SEQ ID NO:2. The 3' end of the nucleotide sequence of primer CA-F encodes the C-terminal LEKREAEA sequence of the Pichia pastoris signal peptide α-factor (SEQ ID NO:3), which is used for the cleavage of the signal peptide during secretion. A KpnI restriction site was added to the 3' end of the sequence shown in SEQ ID NO:1 using primer CA-R. The nucleotide sequence of primer CA-R is shown in SEQ ID NO:4.

[0078] A kex2 restriction site was inserted between the nucleotide sequences of cecropin and Amuc_1100, allowing them to be automatically cleaved during secretion to separate cecropin and Amuc_1100. The amino acid sequence of the cecropin and Amuc_1100 fusion protein is shown in SEQ ID NO:5.

[0079] The synthesized gene sequence was double-digested with XhoI and KpnI, and then ligated with the Pichia pastoris expression vector pGAPZαA, which had also been double-digested, using T4 DNA ligase to obtain the Pichia pastoris expression vector pGAPZαA-Cecropin-Amuc_1100. Figure 1 ).

[0080] The expression vector was transformed into Pichia pastoris X33 host cells via electroporation. The cells were then plated on agar plates containing bleomycin (Zeocin), with the zeocin concentration gradually increased from 100 μg / mL to 800 μg / mL. Three clones were selected from the 800 μg / mL zeocin plate and transferred to YPD medium for further culture. After 5 days of culture, the fermentation supernatant was analyzed by SDS-PAGE to determine the expression level of the Amuc_1100 protein. The strain with the highest expression level (CA1100-1) was selected for further studies.

[0081] SEQ ID NO:1 (corresponding nucleotide sequence): where nucleotides 1 to 108 encode cephalosporin; nucleotides 130 to 990 encode Amuc_1100 protein.

[0082] AAATGGAAATTATTTAAAAAAATTGAAAAAGTCGGACAGAATATCAGAGATGGTCACAAAGCCGGACCTGCTGTTGCAGTTGTGGGACAGGCTACGCAGATAGCAAAGGGTAAAAGAGAGGCTGAAGCCATTGTCAATAGTAAACGTTCTGAATTGGATAAAAAGATATCAATCGCTGCCAAGGAGATTAAGTCTGCAAATGCCGCCGAGATAACACCAAGTAGATCTTCTAACGAGGAGCTAGAGAAAGAACTTAACAGATACGCTAAGGCTGTTGGATCCTTGGAGACAGCCTATAAACCTTTCTTGGCATCATCTGCCTTGGTTCCCACTACACCAACCGCCTTCCAAAACGAACTGAAGACATTCAGAGACTCTTTAATATCCTCTTGTAAGAAGAAGAACATTTTGATTACCGATACCTCTTCCTGGCTGGGATTTCAGGTATACTCCACTCAAGCTCCCTCAGTCCAAGCTGCTTCTACCCTAGGTTTCGAGTTGAAGGCTATTAATTCTTTGGTAAACAAACTTGCAGAATGTGGCTTGAGTAAATTCATTAAG GTTTACAGACCTCAACTGCCTATAGAAACCCCTGCTAACAACCCTGAAGAGTCTGATGAAGCAGACCAGGCTCCTTGGACTCCTATGCCTCTGGAAATCGCTTTTCAGGGAGATAGAGAATCCGTTCTGAAGGCCATGAATGCTATTACAGGTATGCAAGATTACTTGTTTACAGTTAATTCCATCCGTATTAGAAACGAAAGAATGATGCCTCCTCCTATTGCTAACCCCGCAGCAGCTAAACCTGCTGCAGCTCAACCAGCTACTGGTGCTGCTTCCTTAACTCCAGCTGATGAGGCTGCAGCTCCAGCTGCCCCCGCTATCCAGCAAGTTATAAAGCCCTATATGGGAAAGGAACAAGTTTTTGTTCAAGTCTCTCTGAACCTTGTACACTTCAACCAACCTAAAGCACAAGAACCATCAGAAGAC

[0083] SEQ ID NO:6 (Castrodin nucleotide sequence)

[0084] AAATGGAAATTATTTAAAAAAATTGAAAAAGTCGGACAGAATATCAGAGATGG TCACAAAGCCGGACCTGCTGTTGCAGTTGTGGGACAGGCTACGCAGATAGCAAAGSEQ ID NO:7 (Amuc_1100 nucleotide sequence)

[0085] ATTGTCAATAGTAAACGTTCTGAATTGGATAAAAAGATATCAATCGCTGCCAAGGAGATTAAGTCTGCAAATGCCGCCGAGATAACACCAAGTAGATCTTCTAACGAGGAGCTAGAGAAAGAACTTAACAGATACGCTAAGGCTGTTGGATCCTTGGAGACAGCCTATAAACCTTTCTTGGCATCATCTGCCTTGGTTCCCACTACACCAACCGCCTTCCAAAACGAACTGAAGACATTCAGAGACTCTTTAATATCCTCTTGTAAGAAGAAGAACATTTTGATTACCGATACCTCTTCCTGGCTGGGATTTCAGGTATACTCCACTCAAGCTCCCTCAGTCCAAGCTGCTTCTACCCTAGGTTTCGAGTTGAAGGCTATTAATTCTTTGGTAAACAAACTTGCAGAATGTGGCTTGAGTAAATTCATTAAGGTTTACAGACCTCAACTGCCTATAGAAACCCCTGCTAACAACCCTGAAGAGTCTGATGAAGCAGACCAGGCTCCTTGGACTCCTATGCCTCTGGAAATCGCTTTTCAGGGAGATAGAGAATCCGTTCTGAAGGCCATGAATGCTATTACAGGTATGCAAGATTACTTGTTTACAGTTAATTCCATCCGTATTAGAAACGAAAGAATGATGCCTCCTCCTATTGCTAACCCCGCAGCAGCTAAACCTGCTGCAGCTCAACCAGCTACTGGTGCTGCTTCCTTAACTCCAGCTGATGAGGCTGCAGCTCCAGCTGCCCCCGCTATCCAGCAAGTTATAAAGCCCTATATGGGAAAGGAACAAGTTTTTGTTCAAGTCTCTCTGAACCTTGTACACTTCAACCAACCTAAAGCACAAGAACCATCAGAAGAC

[0086] SEQ ID NO: (The underlined nucleotide is the cleavage site of XhoI):

[0087] CTCGAG ​

[0088] SEQ ID NO:3:

[0089] LEKREAEA

[0090] SEQ ID NO:4 (underlined is the KpnI restriction site):

[0091] GGTACC TTAGTCTTCTGATGGTTCTTGTGCTTTAGG

[0092] SEQ ID NO:5 (underlined are the cleavage sites of kex2): Positions 1 to 36 are the amino acid sequences corresponding to cephalosporin; positions 44 to 330 are the amino acid sequences corresponding to Amuc_1100.

[0093] KWKLFKKIEKVGQNIRDGHKAGPAVAVVGQATQIAK GKR EAEAIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSK FIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED

[0094] SEQ ID NO:8 (Cybene amino acid sequence)

[0095] KWKLFKKIEKVGQNIRDGHKAGPAVAVVGQATQIAK

[0096] SEQ ID NO:9 (Amuc_1100 amino acid sequence)

[0097] IVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFI KVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED

[0098] Example 2: Freeze-drying

[0099] Freeze-drying stabilizes the active ingredients, allows for long-term storage at room temperature, and facilitates transportation and application, offering significant advantages. The CA1100-1 strain obtained in Example 1 was inoculated into YPD medium and cultured for 5 days. The bacterial cells and medium were then freeze-dried simultaneously. The freeze-drying conditions were as follows: freezing at -40°C for 3-5 hours; vacuuming and raising the temperature to -25°C, maintaining this temperature for 18-24 hours (first drying); raising the plate temperature to 25-30°C and maintaining this temperature for 5-8 hours (second drying). A dried powder containing cephalosporin antimicrobial peptides, Amuc_1100 protein, Pichia pastoris cells, and beneficial components of the fermentation medium was obtained. This dried powder can be added to feed in the required amount and can be used as a feed additive.

[0100] Example 3: Yeast fermentation broth stimulates lymphocyte proliferation

[0101] Male SPF-grade BALB / c mice (Experimental Animal Center, Academy of Military Medical Sciences), 6 to 8 weeks old, weighing 18-22g. Housing environment: room temperature 22±2℃, 12h light / dark cycle, free access to water and food. After euthanasia, the mice were disinfected and soaked in 75% alcohol for 10 min; the spleen was separated in a laminar flow hood, washed three times with PBS, and then ground and passed through a 100-mesh sieve; all cell suspensions were collected, centrifuged at 600g for 5 min, and the supernatant was discarded; the cells were resuspended in 5 mL of pre-chilled erythrocyte lysis buffer and centrifuged at 600g for 5 min; this process was repeated until all erythrocytes were removed; after centrifugation, the cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and cell counting was performed.

[0102] Yeast (CA1100-1) containing the expression vector pGAPZαA-Cecropin-Amuc_1100 was fermented to produce lyophilized powder. After fermentation, the fermentation broth (CA1100) was freeze-dried to obtain lyophilized powder. The powder was reconstituted with sterile water, centrifuged to remove insoluble matter, and the soluble supernatant was adjusted to concentrations of 1, 5, and 25 mg / mL. The prepared spleen lymphocytes were then cultured in RPMI-1640 medium to adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 6 Splenocytes were added to 96-well plates at a concentration of 100 μL per well. In the control group, 100 μL of RPMI-1640 medium was added; in the experimental groups, 100 μL of yeast fermentation product at concentrations of 1, 5, and 25 mg / mL were added; in the positive control wells, RPMI-1640 complete medium containing concanavalin A (ConA, final concentration 2.5 μg / mL) was added. The blank control group was added to 200 μL of RPMI-1640 medium. The plates were incubated at 37°C for 48 h in a 5% CO2 incubator. After incubation, the plates were centrifuged at 4°C for 10 min at 200 g, the supernatant was discarded, and 180 μL of serum-free medium and 20 μL of MTT solution were added to each well. Incubation continued for 4 h. After centrifugation at 4°C for 15 min at 200 g, the supernatant was discarded, and 150 μL of DMSO solution was added to each well. The plates were shaken for 10 min to dissolve the cells, and the absorbance at 490 nm was measured using a microplate reader.

[0103] Calculate the spleen lymphocyte growth rate using the following formula:

[0104] Lymphocyte proliferation rate (%) = [(As-Ab) / (Ac-Ab)] × 100% (As experimental wells; Ac control wells; Ab blank wells)

[0105] The results are as follows Figure 2 As shown, compared with the control group, all treatment groups significantly stimulated the proliferation of mouse splenic lymphocytes. ConA served as a positive control, and the treatment groups containing different yeast fermentation broth (CA1100) lyophilized powders showed similar effects to ConA, significantly stimulating the proliferation of mouse splenic lymphocytes. While the stimulatory effect increased with increasing CA1100 concentration among the treatment groups, the differences were not statistically significant. This experiment demonstrates that fermentation broth containing Amuc_1100 protein and cephalosporin antimicrobial peptides can stimulate the proliferation of mouse splenic lymphocytes and enhance mouse immunity.

[0106] Example 4: Fermentation broth inhibits bacterial growth

[0107] The antibacterial effect of cecropin produced by yeast fermentation was detected using the plate spread method. Yeast (CA1100-1) containing the expression vector pGAPZαA-Cecropin-Amuc_1100 was fermented. After fermentation, the fermentation broth (CA1100) was freeze-dried to obtain lyophilized powder. The powder was reconstituted with sterile water, centrifuged to remove insoluble matter, and the soluble supernatant was adjusted to concentrations of 1, 5, and 25 mg / mL. *Escherichia coli* (ATCC 25922) was cultured overnight in LB medium. The next day, fresh LB medium was used, and the OD600 was diluted to approximately 0.01. Different concentrations of CA1100 culture medium were then added, with the same volume of PBS added as a negative control. The culture was incubated at 37°C in a shaker for approximately 8 hours. The bacterial suspensions were then serially diluted 10⁻⁶ times. 4 10 5 10 6 The bacterial count was doubled, with 50 μL of each sample spread onto plates and incubated overnight at 37°C. The antibacterial effect was observed the next day, and the inhibition rate was calculated. Inhibition rate = [(number of colonies in control group - number of colonies in experimental group) / number of colonies in control group] × 100%; the inhibition rate of the negative control group was 0.

[0108] The results are as follows Figure 3 As shown, the yeast fermentation broth (CA1100) can inhibit bacterial growth in a dose-dependent manner, with the most significant antibacterial effect at 25 mg / mL; this indicates that yeast fermentation expresses cecropin, and that cecropin retains extremely high biological activity after freeze-drying.

[0109] Example 5: Inhibition of the release of pro-inflammatory cytokines

[0110] 1) Extraction method

[0111] The Ganoderma lucidum extract was prepared by the following method: 1 kg of Ganoderma lucidum raw material was crushed, and 10 L of 75% ethanol was added for reflux extraction three times, each extraction lasting 60 min at a temperature of 65℃; another 1 kg of Ganoderma lucidum raw material was added to 10 L of purified water and boiled for 2 h, then pressed and filtered; after extraction, all extracts were combined, filtered, and freeze-dried to obtain the Ganoderma lucidum extract.

[0112] Andrographis paniculata extract was prepared by the following method: 1 kg of Andrographis paniculata raw material was crushed, 10 L of 75% ethanol was added, and the mixture was refluxed and extracted 3 times, 90 min each time, at an extraction temperature of 45℃; another 1 kg of Andrographis paniculata raw material was added, 10 L of purified water was added, and the mixture was boiled and extracted for 1 h, then pressed and filtered; after extraction, all extracts were combined, filtered, and freeze-dried to obtain Andrographis paniculata extract.

[0113] Polygonum cuspidatum extract was prepared by the following method: 1 kg of Polygonum cuspidatum raw material was crushed, 15 L of 75% ethanol was added, and the mixture was refluxed and extracted 3 times, 60 min each time, at an extraction temperature of 55℃; another 1 kg of Polygonum cuspidatum raw material was added, 15 L of purified water was added, and the mixture was boiled and extracted for 2 h, then pressed and filtered; after extraction, all extracts were combined, filtered, and freeze-dried to obtain Polygonum cuspidatum extract.

[0114] Allicin is prepared by the following method: 1 kg of garlic is crushed and extracted by steam distillation for 2-4 hours to obtain garlic extract, which is then further concentrated under reduced pressure to obtain allicin.

[0115] 2) Prepare the compound preparation according to the following formula:

[0116] Compound A: Weigh 10 parts of Ganoderma lucidum extract, 15 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1 part of freeze-dried yeast fermentation broth containing Amuc_1100 and antimicrobial peptides, and 1 part of allicin, and mix them thoroughly to prepare a compound preparation.

[0117] Compound B: Weigh 15 parts of Ganoderma lucidum extract, 20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 3 parts of freeze-dried yeast fermentation broth containing Amuc_1100 and antimicrobial peptides, and 4 parts of allicin, and mix them thoroughly to prepare a compound preparation.

[0118] Compound C: Weigh 30 parts of Ganoderma lucidum extract, 30 parts of Andrographis paniculata extract, 20 parts of Polygonum cuspidatum extract, 5 parts of freeze-dried yeast fermentation broth containing Amuc_1100 and antimicrobial peptides, and 6 parts of allicin, and mix them thoroughly to prepare a compound preparation.

[0119] Compound D: Weigh 15 parts of Ganoderma lucidum extract, 20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, and 6 parts of allicin, and mix them thoroughly to prepare a compound preparation.

[0120] The extracts described above were extracted separately for research purposes. To save on processing time, they can also be extracted after mixing. Alternatively, commercially available extract products can be purchased.

[0121] Comparative compound: Patent CN104623541A discloses a composition of traditional Chinese medicine extracts for improving immunity. We prepared a comparative compound according to this patent: 65 parts of turmeric extract, 42 parts of Ganoderma lucidum extract, 15 parts of Dendrobium extract, 36 parts of Polygonum cuspidatum extract, 11 parts of Magnolia officinalis extract, 2 parts of Aloe vera extract, and 19 parts of Eucommia ulmoides extract; and mixed them thoroughly to make a compound preparation.

[0122] The "freeze-dried product of yeast fermentation broth containing Amuc_1100 and antimicrobial peptides" described in this embodiment was prepared in Example 2.

[0123] 3) Add 500mL of purified water to the powder (in this example, 1 part is calculated as 1g, and the volume of purified water can be scaled up proportionally), and stir thoroughly with a magnetic stirrer; use a 0.22μm filter membrane to filter and sterilize, and remove insoluble impurities, or use centrifugation to clarify.

[0124] 4) Inhibit the release of pro-inflammatory cytokines from RAW264.7 cells

[0125] RAW264.7 macrophages were cultured in DMEM medium until approximately 80% confluence. Then, the RAW264.7 macrophages were digested with trypsin to prepare a single-cell suspension. The cells were seeded into 96-well plates at a density of 5 × 10⁶ cells / well. 4 Cells were cultured overnight at 37°C and 5% CO2. Cells were divided into seven groups: blank group, lipopolysaccharide (LPS) group, compound A group, compound B group, compound C group, compound D group, and control compound group. Each group had four replicates, with 20 μL of PBS added to each group. The compound groups and LPS groups were treated with LPS at a final concentration of 200 ng / mL, while the blank control group received 20 μL of PBS. Cells were cultured for another 24 h at 37°C and 5% CO2. The cell culture supernatant was collected, and the inflammatory factor TNF-α secreted by the cells was detected using a tumor necrosis factor (TNF-α) ELISA kit.

[0126] Lipopolysaccharide (LPS) can induce macrophages to produce inflammatory factors, such as TNF-α. In this experiment, the results are as follows... Figure 4 As shown, the expression level of TNF-α increased significantly after the addition of LPS (compared to the blank group); however, the expression level of TNF-α was significantly reduced after the addition of certain proportions of the various compound preparations, and this reduction was dose-dependent; the reduction was greatest in compound C group. Compound D group, which did not contain CA1100 yeast fermentation broth, also significantly inhibited the expression level of TNF-α, but the inhibitory effect of compound D was significantly lower than that of compound C and compound B. This indicates that the components of these compound preparations can synergistically inhibit the secretion of inflammatory factors and have significant anti-inflammatory activity. While the comparative compound preparations also showed some inhibitory effect, their inhibitory effect was lower than that of compound A, B, C, and D, indicating that the compound preparations in this patent are more advantageous.

[0127] Example 6: Effects of cephalosporin and Amuc_1100 on IgG in CTX-induced immunosuppressed mice

[0128] 1) To characterize the effects of cephalosporin or Amuc_1100 on animal immunity, we used a 10kDa filter membrane to separate the fermentation products from Example 2. Cephalosporin, with its smaller molecular weight, could pass through the filter membrane; while Amuc_1100, with its larger molecular weight, could not pass through the filter membrane; thus, separation was achieved. Using the freeze-drying method described in Example 2, freeze-dried powders containing cephalosporin and Amuc_1100 were prepared, respectively. A 100mg / mL solution was prepared from the powder.

[0129] 2) Establishment and treatment of CTX-induced immunodeficiency mouse model

[0130] Cyclophosphamide (CTX) has a strong immunosuppressive effect, significantly impacting the body's normal immune system function. Therefore, CTX can be used to suppress the immunity of experimental animals and establish a low-immunity model. The developmental status of immune organs directly affects immune function and disease resistance. The spleen and thymus are important hematopoietic and immune organs in non-specific immunity, where lymphocytes differentiate, mature, and generate immune responses. Therefore, the thymus index and spleen index can be used to reflect the body's immune function.

[0131] 3) Preparation of CTX-induced immunosuppressed mice: Male KM mice (Experimental Animal Center, Academy of Military Medical Sciences) were divided into four groups: a blank control group, a CTX control group, a cephalosporin group, an Amuc_1100 group, and a CA1100 group (yeast CA1100-1 fermentation culture). Except for the blank control group, the other groups were intraperitoneally injected with CTX (80 mg / kg / d) continuously from day 1 to day 3; on days 4 and 6, a maintenance dose of CTX (40 mg / kg / d) was injected intraperitoneally; and on days 8 and 10, CTX (20 mg / kg / d) was injected intraperitoneally. This established an immunosuppressed mouse model. The blank control group was given the same volume of physiological saline or PBS. Starting from day 5 after successful model establishment, each experimental group was administered 0.5 mL / day by gavage for 20 consecutive days; the blank control group and the CTX control group were given the same volume of physiological saline.

[0132] Blood was collected from the eyes of mice in each treatment group and centrifuged at 4°C and 4000 rpm for 15 min; serum was collected. The level of immunoglobulin IgG in mouse serum was determined using an IgG ELISA kit.

[0133] The results are as follows Figure 5As shown, after CTX treatment, the serum IgG level decreased significantly compared with the normal control group; while feeding with cephalosporin or Amuc_1100 alone significantly increased the serum IgG level; and feeding with CA1100 had a stronger stimulating effect on IgG increase than cephalosporin or Amuc_1100 alone. This study shows that cephalosporin or Amuc_1100 alone can improve the immunity of animals, and cephalosporin and Amuc_1100 have a synergistic effect, and the combined use can further improve the immunity of animals.

[0134] Example 7: Organ index of compound preparation on mouse immune organs (thymus and spleen)

[0135] 1) Add 300 mL of purified water to the compound preparations of groups A, B, C, and D in Example 5 and the comparative compound powder, and stir thoroughly with a magnetic stirrer; filter using a 0.22 μm filter membrane to remove insoluble impurities or clarify by centrifugation.

[0136] 2) Preparation of CTX-immunocompromised mice: Male KM mice were divided into a blank group, a CTX control group, a compound A group, a compound B group, a compound C group, a compound D group, and a control compound group; the CTX-immunocompromised mouse model in this embodiment was treated according to the administration regimen described in Example 6.

[0137] Twenty-four hours after the last administration, the mice were accurately weighed and then sacrificed in each group. The spleen and thymus of the mice were then accurately weighed and the organ indices were calculated.

[0138] Organ index = organ mass (mg) / body weight (g);

[0139] Result: From Figure 6 It can be seen that after CTX treatment, the thymus and spleen indices of mice decreased significantly; while after 20 days of treatment with various compound preparations, both the thymus and spleen indices increased significantly. This demonstrates that each compound can improve the immunity of mice. Among them, the increase in thymus and spleen indices in compound group D was lower than that in compound groups A, B, and C, indicating that the freeze-dried yeast fermentation broth of Amuc_1100 and antimicrobial peptides has a significant effect on improving the immunity of mice; and the components in the compound have a synergistic effect. Although the comparative compound can also improve the organ indices of CTX-treated mice, it is significantly lower than that in compound group D; as can be seen from the figure, its effect on improving immunity is not as good as that of compound groups A, B, C, and D.

[0140] Example 8: Effect of the compound on serum immunoglobulin levels in immunocompromised mice

[0141] The antibodies produced by the body in response to viral, bacterial, or toxin stimulation are mainly IgG (immunoglobulin G). IgG is the main immunoglobulin produced by the humoral immune response and plays an important role in the body's resistance to external infections and enhancing immunity. Therefore, the level of IgG in the blood can directly reflect the strength of the body's immunity.

[0142] Before sacrifice, blood was collected from the eyes of mice in each treatment group in Example 5, and the blood was centrifuged at 4°C and 4000 rpm for 15 min; serum was collected. The content of immunoglobulin IgG in mouse serum was determined using an IgG ELISA kit.

[0143] The results are as follows Figure 7 As shown, after CTX treatment, the serum IgG level decreased significantly compared to the normal control group, proving that CTX effectively established an immunocompromised mouse model. After continuous treatment with each compound group, a significant increase was observed compared to CTX; among them, compound C group showed the greatest increase in IgG. While there was a gradual increasing trend among the different treatment groups of compounds A, B, C, and D, no significant difference was reached; this indicates that the compound preparation of the present invention can significantly enhance the immunity of mice. The comparative compound also increased the IgG level in CTX-treated mice, but significantly lower than that of compound D group; as can be seen from the figure, its effect on improving immunity is not as good as the other compound treatment groups in this embodiment.

[0144] Example 9: Effects of compound preparations on chick growth

[0145] Sixty 1-3 day old Gallus chicks, weighing approximately 30-35g, were selected. The chicks were randomly divided into two groups: a control group fed a normal basal diet (CP Group 521 chick feed: moisture ≤14.0%, crude protein ≥20.0%, calcium 0.60-1.2%, total phosphorus ≥0.5%, sodium chloride 0.2-0.8%, crude fiber ≤6.0%, crude ash ≤6.0%, methionine + cystine ≥0.74%); and an experimental group fed a basal diet supplemented with 5g / kg of compound preparation C, thoroughly mixed, for 30 consecutive days. After 30 days, the chicks were weighed, and 10 chicks were randomly selected from each group for serum IgG level testing. Results are as follows: Figure 8 As shown, after 30 days of continuous feeding, the weight of chicks in the compound C group was significantly higher than that in the control group, and the serum IgG level in chicks in the compound C group was also significantly higher than that in the control group. This indicates that the chicks fed a basal diet supplemented with the compound preparation showed better growth and development than the control group chicks, and that the feed supplemented with the compound preparation of this invention can significantly enhance the immunity of the chicks.

Claims

1. A compound preparation for enhancing animal immunity or a feed additive containing the same, characterized in that, The compound preparation or feed additive containing it comprises, by weight, 10-30 parts of Ganoderma lucidum extract, 15-30 parts of Andrographis paniculata extract, 10-20 parts of Polygonum cuspidatum extract, 1-6 parts of allicin and 0-5 parts of biological composition; the biological composition contains Amuc_1100 protein and antimicrobial peptides.

2. The compound preparation or feed additive containing it as described in claim 1, characterized in that, The antimicrobial peptide is cephalosporin; And / or, the biological composition is a yeast fermentation product expressing Amuc_1100 protein and antimicrobial peptides; And / or, the compound preparation or feed additive containing it comprises, by weight: 10-15 parts of Ganoderma lucidum extract, 15-20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1-4 parts of allicin and 1-3 parts of biological composition. Alternatively, the compound preparation or feed additive containing it may contain, by weight, the following: 15 parts of Ganoderma lucidum extract, 15-22.5 parts of Andrographis paniculata extract, 10-15 parts of Polygonum cuspidatum extract, 1.5-3 parts of a biological composition and 1.5-6 parts of allicin; Preferably, the compound preparation or feed additive containing it comprises, by weight parts: 10 parts of Ganoderma lucidum extract, 15 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 1 part of a biological composition and 1 part of allicin; or, 15 parts of Ganoderma lucidum extract, 20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract, 3 parts of a biological composition, and 4 parts of allicin; or, 30 parts of Ganoderma lucidum extract, 30 parts of Andrographis paniculata extract, 20 parts of Polygonum cuspidatum extract, 5 parts of a biological composition, and 6 parts of allicin; or, 15 parts of Ganoderma lucidum extract, 20 parts of Andrographis paniculata extract, 10 parts of Polygonum cuspidatum extract and 6 parts of allicin.

3. The compound preparation or feed additive containing it as described in claim 2, characterized in that, The yeast fermentation product is a freeze-dried fermentation product of Pichia pastoris, such as Pichia pastoris X33, GS115 or KM71H. Preferably, the amino acid sequence of the cephalosporin is as shown in SEQ ID NO:8; and / or, the amino acid sequence of the Amuc_1100 protein is as shown in SEQ ID NO:

9.

4. A fusion protein, characterized in that, The fusion protein contains an antimicrobial peptide and the Amuc_1100 protein; Preferably, the antimicrobial peptide is cephalosporin; and / or, the fusion protein further comprises a signal peptide and / or a cleavage tag; More preferably, the amino acid sequence of the cephalosporin is as shown in SEQ ID NO:8; the amino acid sequence of the Amuc_1100 protein is as shown in SEQ ID NO:9; and / or, the cleavage tag is a kex2 restriction enzyme site; More preferably, the fusion protein has an amino acid sequence as shown in SEQ ID NO:

5.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the fusion protein as described in claim 4; Preferably, the nucleotide sequence encoding the cephalosporin is shown in SEQ ID NO:6; and / or, the nucleotide sequence encoding the Amuc_1100 protein is shown in SEQ ID NO:7; More preferably, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

1.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid isolated as described in claim 5; Preferably, the backbone of the expression vector is a plasmid backbone; More preferably, the plasmid backbone is pGAPZαA, pPICZαA, or pPIC9K.

7. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 5, or the expression vector as described in claim 6; Preferably, the host cell used to construct the transformant is Pichia pastoris, such as Pichia pastoris X33, GS115 or KM71H.

8. A protein composition, characterized in that, The protein combination includes antimicrobial peptides and Amuc_1100 protein; Preferably, the antimicrobial peptide is cephalosporin; and / or, the Amuc_1100 protein has the amino acid sequence shown in SEQ ID NO:9; More preferably, the cephalosporin has an amino acid sequence as shown in SEQ ID NO:

8.

9. A method for preparing the fusion protein of claim 4 or the protein combination of claim 8, comprising culturing the transformant of claim 7 to obtain a fermentation product; Preferably, the obtained fusion protein is isolated upon secretion from the transformant to form the protein combination as described in claim 8.

10. The use of the compound preparation or feed additive containing the compound preparation as described in any one of claims 1-3, the fusion protein as described in claim 4, the isolated nucleic acid as described in claim 5, the expression vector as described in claim 6, the transformant as described in claim 7, or the protein combination as described in claim 8 in the preparation of a preparation to enhance animal immunity; Preferably, the animals are selected from poultry, livestock, and economically important aquatic animals; the poultry are selected from chickens, ducks, geese, etc.; the livestock are selected from pigs, cattle, sheep, etc.; and the economically important aquatic animals are selected from fish, shellfish, etc. More preferably, the animal is a chicken (Gallus).

Images