CN108558998B - Preparation and application of recombinant yeast preparation co-expressed by pig interleukin 4/6 and fused pig antibacterial peptide - Google Patents

Abstract

The invention discloses preparation and application of a pig interleukin 4/6 and fused pig antibacterial peptide co-expression recombinant yeast preparation, wherein the antibacterial peptide fused cytokine FPAPI L46 provided by the invention is A1), A2) or A3), A1) amino acid sequence is protein of a sequence 1 in a sequence table, A2) amino acid sequence shown in the sequence 1 in the sequence table is substituted, deleted and/or added by one or more amino acid residues and is protein with the same function, A3) is fused protein obtained by connecting labels at N end or/and C end of A1) or A2), experiments prove that the FPAPI L46 of the invention can promote proliferation of lymphocytes, erythrocytes and leukocytes, inhibit growth of pathogenic microorganisms, promote secretion of nonspecific antibodies (IgG, IgG1, IgG2a) and disease specific antibodies, and improve the immunocompetence and survival rate of animals.

Description

Preparation and application of recombinant yeast preparation co-expressed by pig interleukin 4/6 and fused pig antibacterial peptide

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of a pig interleukin 4/6 and fused pig antibacterial peptide co-expression recombinant yeast preparation.

Background

At present, the livestock and poultry breeding industry in China is gradually enlarged along with the improvement of the intensification degree, the breeding scale and the breeding density of livestock and poultry are forced to control the pressure of infection transmission and diffusion of more than 30 infectious pathogens, particularly the successive cyclic outbreaks of virulent diseases such as avian influenza and respiratory reproduction syndrome, diarrhea caused by drug-resistant bacteria in animal breeding, various bacteria and viral diseases such as salmonella, escherichia coli, streptococcus, Porcine Reproductive and Respiratory Syndrome (PRRSV), circovirus (PCV2) and swine fever (CSFV), and the bottleneck limiting problem in animal breeding for a long time is a bottleneck limiting problem in animal breeding, and the development of animal breeding is seriously hindered; the abuse of pharmaceutical additives such as antibiotics in the feed is very serious, and livestock and poultry diseases and the pollution problem of the antibiotic feed additives become bottlenecks which restrict the development level and the economic benefit improvement of livestock and poultry breeding in China. The abuse of the feed antibiotic additive not only increases the feeding cost, but also obviously enhances the drug resistance and the pathogenicity of pathogenic microorganisms, seriously destroys the microecological balance of the digestive tract of animals, remains and enriches in the bodies of the animals after long-term use, weakens the health level of livestock organisms due to the toxic and side effects of the drugs, and obviously reduces the immunity and disease resistance. In such a vicious circle, various virulent infectious diseases of livestock and poultry frequently occur and are difficult to control and treat. On the other hand, the drug residue and the increasingly serious food safety problem of the livestock and poultry products not only directly threaten the health of human beings, but also hinder the development of the breeding industry.

The addition of antibiotics to animal feed, long-term or improper use of which not only induces drug resistance of pathogens, but also causes drug residues in animal products, which causes serious food safety hazards, damages to human health and incurs drug failure for the treatment of antibiotics. Developed regions such as the european union and the united states began to restrict antibiotics as feed additives comprehensively in 2006. The addition of antibiotics to the feed is also strictly limited in China. The modern breeding industry urgently needs to develop novel biological feed and additives thereof which replace the traditional antibiotics, have no residue, no resistance induction, safety and no pollution.

I L4 is used as cytokine for regulating animal humoral immunity, can activate static B cell to enter proliferation stage, induce differentiation and generate high-level IgG1 and IgE, has strong macrophage activating activity, enhances MHC-II molecule expression, antigen presentation and phagocytic capacity, can stimulate proliferation of bone marrow hematopoietic stem cells, enhances hematopoietic function of organism, has wide activating effect on T cells, can promote growth of CD4 and CD3T cells, promotes activated T cells to continue to grow, and stimulates thymic cell proliferation and maturation.

I L6 is one of the most widely functional cytokines, secreted by T cells and macrophages, stimulates immune response, promotes proliferation and differentiation of various cells, for example, in inflammation, I L-6 plays a role in resisting infection, in local infection, endothelial cells, fibroblasts and mononuclear macrophages secrete I L6 to resist virus, promote proliferation and growth of locally activated T cells, thereby inducing the T cells to differentiate and mature into cytotoxic T cells, when I L6 is released and enters blood circulation, can activate quiescent hematopoietic stem cells, promote division and proliferation of blood cells, induce or promote other immune cells to produce cytokines and play a role, induce rise of body temperature, and arouse a series of defensive physiological responses of the body to resist infection.

The molecular weight of the antibacterial peptide is small, and the separation, purification and detection have certain difficulty. At present, the main source of the antibacterial peptide in scientific research is chemical synthesis, but the chemical synthesis has high cost and small amount and is not beneficial to large-scale application. Therefore, a plurality of antibacterial peptide genes are recombined and connected by utilizing a genetic engineering means to obtain the high-efficiency recombinant antibacterial peptide molecules with broad-spectrum anti-various microorganisms (G +, G-, viruses, fungi and parasites) and immunoregulation activity.

At present, reports about recombinant cloning and co-expression of pig interleukin fusion antibacterial peptide genes are rarely seen.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the immunity of animals.

In order to solve the technical problems, the invention provides a protein which comprises pig antibacterial peptide and interleukin 4/6.

The protein is any one of the following proteins a) to e):

a) the amino acid sequence comprises the protein of the amino acid sequence shown in the sequence 1 in the sequence table;

b) the amino acid sequence consists of amino acid residues shown in a sequence 1 in a sequence table;

c) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined by a) or b) and has the function of improving the animal immunity;

d) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in a) or b) and having a function of enhancing the animal's immunity;

e) a fusion protein obtained by connecting a label to the N-terminal and/or the C-terminal of the protein defined in any one of a) to d).

Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.

The nucleic acid molecule is a nucleic acid molecule represented by any one of the following 1) to 4):

1) the coding sequence comprises a sequence 2 in a sequence table;

2) the coding sequence is sequence 2 in the sequence table;

3) DNA molecules which hybridize under stringent conditions with the DNA molecules defined in 1) or 2) and which code for the proteins mentioned above;

4) a DNA molecule which has more than 80% or more than 90% of homology with the DNA molecule defined in 1) or 2) and codes the protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 2 encodes the FPAPI L46 shown in sequence 1.

Those nucleotides that have been artificially modified to have 75% or greater identity to the nucleotide sequence of the isolated FPAPI L of the invention are derived from and identical to the nucleotide sequence of the invention as long as they encode FPAPI L and have the function of FPAPI L46.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above applications, the stringent conditions are hybridization and washing of the membrane at 68 ℃ for 2 times, 5min each, in a solution of 2 × SSC, 0.1% SDS, and hybridization and washing of the membrane at 68 ℃ for 2 times, 15min each, in a solution of 0.5 × SSC, 0.1% SDS, or at 65 ℃ in a solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

Any of the following biomaterials 1) -3) is also within the scope of the present invention:

1) an expression cassette comprising the nucleic acid molecule;

2) a recombinant vector comprising the nucleic acid molecule;

3) recombinant bacteria or transgenic cell lines containing the nucleic acid molecules;

4) and (3) a fermentation product of the recombinant bacterium.

In the above applications, the expression cassette containing a nucleic acid molecule encoding FPAPI L46 (FPAPI L46 gene expression cassette) described in B2) refers to a DNA capable of expressing FPAPI L46 in a host cell, and the DNA may include not only a promoter that initiates transcription of FPAPI L46 gene, but also a terminator that terminates transcription of FPAPI L46 gene.

The recombinant vector containing the FPAPI L46 gene expression cassette can be constructed using existing vectors.

In the above application, the vector may be a plasmid, a cosmid, a phage or a viral vector, and the plasmid may be pGAPZ α A vector.

B3) The recombinant vector can contain a DNA sequence shown in a sequence 2 and used for encoding FPAPI L46, and further can be pG-46P, wherein the pG-46P is obtained by replacing a DNA fragment between EcoR I and Xba I recognition sequences of a pGAPZ α A vector with an FPAPI L46 gene shown in the sequence 2.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the yeast is Pichia pastoris SMD 1168.

In the application, the recombinant microorganism can be obtained by introducing an expression cassette containing a nucleic acid molecule for coding FPAPI L46 into yeast, specifically, a recombinant vector containing an FPAPI L46 expression cassette can be introduced into the yeast, the recombinant vector can be directly introduced into the yeast, or the recombinant vector can be linearized and then introduced into the yeast, and the yeast can be Pichia pastoris SMD 1168.

In one embodiment of the present invention, the recombinant microorganism is a recombinant microorganism obtained by introducing the pG-46P into Pichia pastoris SMD 1168. The preparation method of the recombinant microorganism specifically comprises the following steps: linearizing the pG-P to obtain linearized pG-P; and introducing the linearized pG-P into pichia pastoris SMD1168 to obtain a recombinant microorganism, wherein the name of the recombinant microorganism is SMDpG-46P.

In the above application, the fermentation product of the recombinant microorganism may be prepared by a method comprising culturing the recombinant microorganism to express the gene encoding FPAPI L46 to obtain the fermentation product of the recombinant microorganism.

In the above applications, the transgenic cell line does not comprise propagation material.

The application of the above protein or the above nucleic acid molecule or the above biological material in the following C1 or C2 is also within the scope of the present invention:

c1, improving animal immunity;

c2, preparing the product for improving the animal immunity.

In the application, the animal immunity improving effect is at least one of the following M1-M5:

m1, inhibiting the growth of pathogenic microorganisms;

m2, promoting increase of immune cells;

m3, promoting vaccine-induced immune responses;

m4, promoting cellular and/or humoral immunity;

m5, increasing animal development and growth weight gain;

and/or the pathogenic microorganism is in particular Escherichia coli, Staphylococcus aureus, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus or classical swine fever virus, such as Escherichia coli standard (G)^-) Escherichia coli resistant bacterium (G)^-) Staphylococcus aureus (G)⁺) Staphylococcus aureus resistant bacterium (G)⁺)。

And/or, the immune cell is in particular a lymphocyte (such as CD4+ or CD8+), a red blood cell or a white blood cell;

and/or, the antibody is specifically an IgG, IgG1 and/or IgG2 a. The antibody may specifically be an antibody of the pathogenic microorganism.

The following products X1 or X2 are also within the scope of the invention:

x1, a biological agent comprising the following X3a, X3b or X3 c:

x3a, the above protein;

x3b, the above nucleic acid molecule;

x3c, the biomaterial described above;

x2, kit for improving animal immunity, which consists of the X1 and antibiotics.

In the product, the biological agent can be X3a, X3b or X3c as an active ingredient, and can also be a composition which combines X3a, X3b or X3c with other substances capable of improving the animal immunity as an active ingredient.

Or, a method for enhancing the immunocompetence of an animal, comprising administering to the animal the above protein or the above nucleic acid molecule or the above biological material or the above biological agent or the kit, thereby enhancing the immunocompetence of the animal.

In the above, the animal is any one of H1-H3:

h1, mammalian;

h2, pig;

h3, mouse.

In the above product, the antibiotic may be ampicillin and/or kanamycin.

In order to solve the above technical problems, the present invention also provides a method for enhancing the immunocompetence of an animal, which may comprise administering the FPAPI L46, the biological material or the biological agent to the animal, thereby enhancing the immunocompetence of the animal.

In the invention, the animal immunity improving capability can be any one of the following M1-M5:

m1, inhibiting the growth of pathogenic microorganisms;

m2, promoting increase of immune cells;

m3, promoting vaccine-induced immune responses;

m4, promoting cellular and/or humoral immunity;

m5, improving animal development and growth weight gain.

The pathogenic microorganism can be Escherichia coli, Staphylococcus aureus, Mycoplasma hyopneumoniae, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or classical swine fever virus (HCV or CSFV), such as Escherichia coli Standard (G)^-) Escherichia coli resistant bacterium (G)^-) Staphylococcus aureus (G)⁺) Staphylococcus aureus resistant bacterium (G)⁺)。

The immune cell may be a lymphocyte (e.g., CD4+ or CD8+), a red blood cell, or a white blood cell.

The antibody may be IgG, IgG1, and/or IgG2 a. The antibody may specifically be an antibody of the pathogenic microorganism.

The vaccine may in particular be a vaccine against the pathogenic microorganism.

In the present invention, the improvement of the animal's immunity may specifically be an improvement of the animal's immunity to the pathogenic microorganism, such as an improvement of the immunity to mycoplasma hyopneumoniae which causes swine enzootic pneumonia (MP).

In the present invention, the animal may be a mammal; the mammal may be specifically a pig or a mouse.

In the invention, the products for improving the animal immunity can be medicines for improving the animal immunity.

Experiments prove that the co-expression FPAPI L46 molecule and the fermentation product of the recombinant yeast containing the FPAPI L46 gene have the effects of promoting the increase of lymphocytes and leukocytes of animals, improving the content of the lymphocytes and the leukocytes in the animals with the FPAPI L46 gene by more than 10 percent, obviously inhibiting the growth of pathogenic microorganisms, reducing the amount of the pathogenic microorganisms with the FPAPI L46 gene by more than 20 percent, promoting the secretion of immunoglobulin (IgG, IgG1 and IgG2a) and disease-specific antibodies, improving the content of nonspecific antibodies and disease-specific antibodies in the animals with the FPAPI L46 gene by 40 to 60 percent, promoting the expression of immune-related genes, further improving the immune anti-infection capacity of the animals, obviously improving the virus attack survival rate of the animals with the FPAPI L46 gene by at least 60 percent and increasing the growth rate by more than 10 percent.

Drawings

FIG. 1 shows the results of RT-PCR electrophoresis of the target genes FPAP and I L-4/6 in SMDpG-46P.

FIG. 2 shows the results of the detection of the protein expression level of the target gene in SMDpG-46P.

FIG. 3 shows the effect of yeast strain SMDpG-46P fermentation supernatant on porcine lymphoblastoid cell proliferation.

FIG. 4 shows the inhibitory effect of SG46P on E.coli standard bacteria.

FIG. 5 shows the inhibitory effect of SG46P on E.coli-resistant bacteria.

FIG. 6 shows the inhibitory effect of SG46P on Staphylococcus aureus standard bacteria.

FIG. 7 shows the inhibitory effect of SG46P on drug-resistant bacteria of Staphylococcus aureus.

FIG. 8 shows the change of peripheral blood leukocytes of mice under different treatments.

FIG. 9 shows mouse peripheral blood CD4 per 10000 cells⁺Changes in T lymphocytes.

FIG. 10 shows mouse peripheral blood CD8 per 10000 cells⁺Changes in T lymphocytes.

FIG. 11 shows the variation of peripheral blood IgG content in mice of different groups.

FIG. 12 shows the variation of the content of IgG1 in peripheral blood of mice of different groups.

FIG. 13 shows the variation of IgG2a content in peripheral blood of mice of different groups.

FIG. 14 shows the content of MP-specific antibodies in peripheral blood of mice of different groups.

FIG. 15 shows the expression level of Th1 type cytokine TNF- α gene in peripheral blood of different groups of mice.

FIG. 16 shows the expression level of Th2 type cytokine I L4 gene in peripheral blood of mice of different groups.

FIG. 17 shows the expression level of T L R gene in peripheral blood of mice of different groups.

FIG. 18 shows the expression levels of I L-7 and I L-23 genes in peripheral blood of mice of different groups.

FIG. 19 shows the survival rate of different groups of mice after challenge experiments with E.coli and Staphylococcus aureus.

Fig. 20 shows net weight gain of piglets in each group.

FIG. 21 is the dynamic change of leukocytes during piglet growth.

Fig. 22 is a graph of the dynamic changes of CD4+ T and CD8+ T lymphocytes in peripheral blood during piglet growth.

FIG. 23 is a graph showing the dynamic change of CSF-specific antibodies in peripheral blood during piglet growth.

Fig. 24 is a graph showing the dynamic changes of PRRSV-specific antibodies in peripheral blood during piglet growth.

FIG. 25 shows the expression levels of T L R-4 and T L R-7 genes in peripheral blood during piglet growth.

FIG. 26 shows the expression levels of CD45 and CD 62L genes in peripheral blood during piglet growth.

FIG. 27 shows the expression levels of peripheral blood cytokine genes during piglet growth.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The pGAPZ α A vector in the examples described below is Invitrogen, catalog number V20020.

Pichia pastoris SMD1168 in the examples below is Invitrogen, catalog number C17500.

The Changbai pig in the following embodiment is a product of Jianyang base of the performance testing center of Sichuan province breeding pigs.

The Tibetan pigs in the following examples are products of Jianyang base of the performance testing center of Sichuan province breeding pigs.

Escherichia coli Standard bacteria (G) in the following examples^-) Is the ATCC (American Type CultureCollection) product, catalog number 25922.

Escherichia coli-resistant bacteria (G) in the following examples^-) The biological material is provided for animal epidemic prevention and control and food safety of Sichuan university in key laboratories of Sichuan province, and the public can obtain the biological material from the applicant, and the biological material is only used for repeating relevant experiments of the invention and cannot be used for other purposes.

Staphylococcus aureus Standard bacteria (G) in the following examples⁺) Is the ATCC (American Type CultureCollection) product, catalog number 29213.

Staphylococcus aureus resistant bacteria (G) in the following examples⁺) The biological material is provided for animal epidemic prevention and control and food safety of Sichuan university in key laboratories of Sichuan province, and the public can obtain the biological material from the applicant, and the biological material is only used for repeating relevant experiments of the invention and cannot be used for other purposes.

The female ICR mice in the following examples are products of the institute for laboratory animals in the national Hospital, Sichuan province.

Example 1 porcine antimicrobial peptide Interleukin 4/6 fusion protein (FPAPI L46) and Gene encoding same

First, fusion protein FPAPI L46 and obtaining of its coding gene

The pig antibacterial peptide and interleukin 4/6 are fused to obtain fusion protein, which is named as FPAPI L46.

The amino acid sequence of the fusion protein is shown as sequence 1 in the sequence table, the fusion gene for coding the fusion protein FPAPI L46 is named as FPAPI L46, and the nucleotide sequence of the fusion gene is sequence 2.

Wherein, the 387-475 site of the sequence 1 is the pig antibacterial peptide, the 277-386 site is the connecting peptide, and the 1-276 site is the interleukin 4/6;

the position 1159-1428 of the sequence 2 is the encoding nucleic acid of the pig antibacterial peptide, the position 829-1158 is the encoding nucleic acid of the connecting peptide, and the positions 1-828 are the encoding nucleic acid of the interleukin 4/6.

The protein can be obtained by artificially synthesizing coding nucleic acid and performing prokaryotic expression.

Secondly, preparation of recombinant vector for expressing fusion protein FPAPI L46

The recombinant vector pGAPZ α A-46B (pG-46P for short) is obtained by replacing a DNA fragment between EcoR I and Xba I recognition sequences of pGAPZ α A vector with FPAPI L46 gene shown in sequence 2 and keeping other sequences of the vector unchanged, and expresses the porcine antimicrobial peptide interleukin 4/6 fusion protein (FPAPI L46) shown in sequence 1.

Preparation of recombinant bacteria for expressing fusion protein FPAPI L46

1. Preparation of recombinant bacteria

Using AvrII to carry out enzyme digestion on pG-46P to obtain linearized pG-46P; the linearized pG-46P is introduced into Pichia pastoris SMD1168 to obtain a recombinant yeast, which is named as SMDpG-46P.

The same method is adopted, pGAPZ α A is cut by AvrII enzyme to obtain linearized pGAPZ α A, and the linearized pGAPZ α A is introduced into pichia pastoris SMD1168 to obtain the control recombinant strain SMDpG.

2. Detection of recombinant bacteria

1) Expression detection at RNA level

The recombinant strain SMDpG-46P was inoculated in YPD medium containing 100. mu.g/ml Zeocin, cultured overnight at 28 ℃ and 200rpm, and centrifuged to obtain SMDpG-46P cells, and the total RNA of the SMDpG-46P cells was extracted and the gene expression level was examined with 2 pairs of primers (I L-4/6F: 5'-ATGAGAAGTGTGAAAACCAGC-3' and R: 5'-CGCATGTTAGAAGACTTCCCCTG-3'; FPAP: F: 5'-GAAGCTGAATTCAGGAGACGTCCCCG-3'; R: 5'-AAACGGGCCCTCTAGACTAATGGT-3').

The results are shown in figure 1, A and B (A and B are RT-PCR electrophoretograms of fused pig antibacterial peptide and pig I L-4/6 respectively) show that A: L ane 1-2: RT-PCR amplification band of fused pig antibacterial peptide FPAP (FPAP primer amplification), M: DNA Marker, B: L ane 1-2: RT-PCR amplification band of pig I L-4/6 (I L-4/6 primer amplification), and show that pig I L-4/6 and antibacterial peptide fusion gene FPAP in SMDpG-46P are transcriptionally expressed at RNA level.

The control recombinant strain SMDpG is detected by the same method, and the transcriptional expression of fusion genes FPAP and I L-4/6 is not seen.

2) Protein expression level detection

Inoculating recombinant strain SMDpG-46P (SG46P) into YPD medium containing 100 μ g/ml Zeocin, fermenting at 28 deg.C and 200rpm, and respectively taking fermentation liquid at different timesCentrifuging, collecting supernatant, detecting fusion protein in the supernatant with HIS-Tag E L ISA kit (all antibody detection reagents are in the kit), fermenting with control recombinant Pichia pastoris SMD1168 to obtain supernatant as negative control (control recombinant bacterium SMDpG is shown in FIG. 2, and OD is shown in FIG. 2₄₅₀<0.02, well below SG46P fermentation supernatant).

As shown in FIG. 2, compared with Pichia pastoris SMD1168, the recombinant strain SMDpG-46P (SG46P) produces the target fusion protein FPAPI L46, and the expression level is the highest in 72 hours of fermentation.

The result shows that the recombinant strain SMDpG-46P expresses the target fusion protein FPAPI L46.

Example 2 Effect of the porcine antimicrobial peptide Interleukin 4/6 fusion protein (FPAPI L46) on lymphocyte proliferation

1. Preparation of recombinant strain SMDpG-46P fermentation supernatant

(1) The recombinant strain SMDpG-46P (hereinafter referred to as SG46P) obtained in example 1 was inoculated into 3m L medium 1 (medium 1 was a liquid medium obtained by adding bleomycin (Zeocin) to YPD medium and the concentration of bleomycin was 100mg/m L), and cultured at 28 ℃ and 200rpm for overnight activation of the strain.

(2) Inoculating 300 μ L bacterial solution obtained in step (1) into 100m L triangular flask containing 30m L YPD medium, performing shake fermentation at 28 deg.C and 200rpm for 48h (OD)₆₀₀Is 25).

(3) And (3) taking 5m of the bacterial liquid obtained in the step (2) L, centrifuging for 2min at 12000 × g, and naming the obtained supernatant as SG46P fermentation supernatant.

2. Protease treatment recombinant bacterium SMDpG-46P fermentation supernatant

Measuring the pH of the SG46P fermentation supernatant obtained in the step 1 by using a pH test paper, respectively adjusting the optimum pH value of the SG46P fermentation supernatant to 7.0 of Trypsin and the optimum pH value of 2.0 of pepsin by using 2 mol/L NaOH and 1 mol/L HCl, respectively adding a Trypsin solution (Trypsin-EDTA digestion solution (Trypsin-EDTA) containing 0.25% of Trypsin and 0.02% of EDTA, catalog number 9002-07-7) and a pepsin solution (Beijing Sorbabao technology Co., Ltd., 0.1% aqueous solution pH 4.0, enzyme activity: 3000-3500NFU/g, catalog number 9001-75-6) to simulate the effect of degrading proteins by using a digestive enzyme), so that the final enzyme concentration is 0.5mg/m L, performing water bath enzyme reaction for 1h at 37 ℃, obtaining the SG46P fermentation supernatant treated by the Trypsin and the SG46 treated by the digestive enzyme, and performing water bath enzyme reaction for 1h at the temperature of 37 ℃ in a refrigerator at 20-46P ℃.

According to the method, SMDpG-46P is replaced by SMDpG (hereinafter referred to as SG) and other steps are not changed, SG fermentation supernatant, trypsin-treated SG fermentation supernatant and pepsin-treated SG fermentation supernatant are obtained respectively and are stored in a refrigerator at the temperature of minus 20 ℃ for later use.

3. Effect of fusion protein FPAPI L46 on lymphocyte proliferation

1) Preparation of porcine lymphocytes

Under the aseptic condition, collecting 5m L peripheral blood of forechamber vein of long and white pig with blood collection tube (containing EDTA-2K anticoagulant), and separating the lymphocyte of long and white pig according to the operation steps of separating liquid of lymphocyte of pig (preheating the separating liquid at 37 ℃ before use, fully shaking and mixing uniformly).

2) SMDpG-46P fermentation product in vitro biological activity assay

(1) After culturing the porcine lymphocytes separated in the step 1) for 24h, the porcine lymphocytes are transferred to a clean 15m L sterile centrifuge tube, and centrifuged at 1500rpm for 15min at room temperature to collect cell bodies.

(2) The cells were washed with RPMI1640 complete medium (containing the penicillin-streptomycin double antibody, 10% fetal bovine serum) and repeated 2 times, and centrifuged at 1500rpm for 15min at room temperature to collect the cell pellet.

(3) Cells were selected and adjusted to about 6 × 10 cells by complete culture in RPMI1640 containing 20mg/m Lα -MM⁶At/m L, a cell suspension was obtained.

(4) 75 μ L of the cell suspension of step (3), 45 μ L of the sample liquid, and 30 μ L of RPMI1640 complete medium containing 20mg/m Lα -MM (methyl mannoside) were added to each well of a 96-well cell plate according to the layout.

Wherein the sample liquid is SG46P fermentation supernatant obtained in the steps 1 and 2, SG46P fermentation supernatant processed by trypsin, SG46P fermentation supernatant processed by pepsin, SG fermentation supernatant processed by trypsin and SG fermentation supernatant processed by pepsin respectively; one sample solution per well and three duplicate wells per sample solution.

RPMI1640 complete medium containing only 20mg/m Lα -MM, PBS and the cell suspension of step (3) were used as blanks, respectively.

Placing in 5% CO₂And culturing in a cell culture box at 37 ℃ for 48 hours.

(5) Taking out 96-well cell plate, adding 15 μ L CCK8 (Guangzhou Yiyuan Biotechnology Co., Ltd.) into each well, mixing, adding 5% CO₂Culturing in a 37 ℃ cell culture box for 2h, taking out a 96-well cell plate, and detecting OD of each well by using an enzyme-labeling instrument (Bio-Reader3350)₄₅₀。

The results are shown in FIG. 3 (as a blank control in the cell suspension of step (3)), and it can be seen that the lymphocytes obtained by stimulating porcine lymphoblasts with SG46P fermentation supernatant compared with the porcine lymphoblasts with control recombinant bacterium SG fermentation supernatant are significantly increased (P <0.05) regardless of whether the cells are treated with trypsin or pepsin, which indicates that the fusion protein FPAPI L46 can significantly stimulate porcine lymphocytes to proliferate (P < 0.05).

In FIG. 3, untreated means fermentation supernatants that were not trypsinized and not pepsin treated.

Example 3 detection of bacteriostatic Activity of the porcine antimicrobial peptide Interleukin 4/6 fusion protein (FPAPI L46)

Determination of porcine antimicrobial peptide interleukin 4/6 fusion protein FPAPI L46 on Escherichia coli standard bacteria (G)^-) (hereinafter abbreviated as S-G)^-) Escherichia coli resistant bacterium (G)^-) (hereinafter abbreviated as R-G)^-) Staphylococcus aureus (G)⁺) (hereinafter abbreviated as S-G)⁺) Staphylococcus aureus resistant bacterium (G)⁺) (hereinafter abbreviated as R-G)⁺) The specific method for inhibiting bacteria is as follows:

the 4 bacterial strains were first activated by inoculation and cultured in exponential growth phase (OD)₆₀₀About 0.5) and then diluted to OD with L B broth₆₀₀About 0.005, the diluted inoculum was inoculated into 96-well cell culture plates, 100. mu. L per well, one bacterium per 96-well cell culture plate.

For each 96-well cell culture plate containing bacteria, 100. mu. L sample solution was added to the experimental wells and gently mixed, 3 replicate wells per sample;

wherein the sample liquid was SG46P fermentation supernatant (i.e., untreated SG46P fermentation supernatant), trypsin-treated SG46P fermentation supernatant, pepsin-treated SG46P fermentation supernatant obtained in steps 1 and 2 of example 2, and SG fermentation supernatant, trypsin-treated SG fermentation supernatant and pepsin-treated SG fermentation supernatant obtained in steps 1 and 2 of example 2 were used as the respective empty controls.

Four antibiotic gradients were set for each strain as positive controls (antibiotic and L B medium), L B medium alone was set for each strain as negative control, and L B medium without any strain was set as blank control.

Incubating 96-well cell culture plates in an incubator at 37 ℃ for 2h and 16h, and detecting OD of each well by using an enzyme labeling instrument (Bio-Reader3350)₆₀₀The value is obtained.

S-G^-And S-G⁺The four antibiotic gradients are shown in Table 1, R-G^-And R-G⁺The four antibiotic gradients are shown in table 2.

TABLE 1 antibiotic gradient for Standard strains

Note: in Table 1, "-" indicates that kanamycin is not contained.

TABLE 2 antibiotic gradient of drug resistant strains

The results showed that the OD of each sample solution of SG was equal to that of each of the four types of bacteria₆₀₀OD with negative control₆₀₀The difference between 2h and 16h shows that SG has no inhibition effect on the four bacteria; OD of each sample solution of SG46P₆₀₀OD at 2h and 16h were significantly lower than that of the empty control at the corresponding times₆₀₀(P<0.05)。

The results at 16h are shown in FIGS. 4-7 (the empty control is SG fermentation supernatant), and SG46P fermentation supernatant has obvious inhibitory effect on the four bacteria (P)<0.05), indicating that the fusion protein FPAPI L46 can inhibit Escherichia coli standard bacteria (G)^-) (hereinafter abbreviated as S-G)^-) Escherichia coli resistant bacterium (G)^-) (hereinafter abbreviated as R-G)^-) Staphylococcus aureus (G)⁺) (hereinafter abbreviated as S-G)⁺) Staphylococcus aureus resistant bacterium (G)⁺)。

Example 4 study of the biological Activity of the porcine antimicrobial peptide Interleukin 4/6 fusion protein (FPAPI L46) in mice

1. Preparation of fermentation product

Fermentation the recombinant bacterium SMDpG-46P (hereinafter referred to as SG46P) obtained in example 1 was activated and inoculated into a 100m L flask containing 30m L YPD medium, and cultured at 30 ℃ and 220rpm for 48 hours to obtain OD₆₀₀About 25, SG46P fermentation broth was obtained.

And (3) fermenting the control recombinant bacterium SMDpG according to the method to obtain SG fermentation liquor.

2. Experimental ICR mice grouping

Taking 50 healthy female ICR mice of 18-20g and 3 weeks old, randomly grouping into 5 groups, wherein each group comprises 10 mice, the group number is 1-5, wherein group 1 and group 4 are SG negative control groups, group 3 is a vaccine negative control group, and group 2 and group 5 are experimental groups.

3. Mouse feeding and vaccination

According to grouping conditions, feeding fresh fermentation liquor into the stomach of a mouse by using a gavage needle, wherein each fermentation liquor is 0.6m L, marking the first gavage as the gavage day 0, and gavage once every two days for 4 weeks (namely, the corresponding fermentation liquor is respectively gavage on the gavage days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27, and the stomach feeding amount is 0.6m L/fermentation liquor every time).

When vaccination was performed, intramuscular injection (intramuscular injection) was performed only on day 7 of gavage, 0.2m L/mouse.

The specific operation is as follows (Table 3), wherein the swine enzootic pneumonia (MP) vaccines are all products produced by Wapai Biotech group, Inc. (catalog number: 19200003).

TABLE 3 mouse gavage, vaccination dose

In Table 3, the gavage was the inoculation method of the fermentation broth, 0.6m L/fermentation broth, the intramuscular injection was the inoculation method of the vaccine, and 0.2m L/fermentation broth was the inoculation dose of the vaccine.

The tail venous blood of each mouse is collected before, 7 days, 14 days, 21 days and 28 days respectively, and the experimental contents of the blood routine is carried out on a blood counting instrument after 30 mu L whole blood and 30 mu L physiological saline are mixed uniformly, the blood routine is carried out on 50 mu L whole blood for flow cytometry, real-time quantitative immunity related genes are obtained after RNA is extracted from 100 mu L whole blood, 100 mu L whole blood is mixed with 1m L TRIZO L violently and uniformly and is stored at 80 ℃ for standby, and blood plasma detection antibodies are collected from 200 mu L whole blood through low-speed centrifugation.

4. Toxicity attacking experiment

Inoculating high-drug-resistance lethal Escherichia coli (provided by key laboratories of Sichuan university for animal epidemic disease prevention and control and food safety, Sichuan province, catalog number: SCSU-ECO L I-HR L-1012) into liquid culture medium containing 0.1mg/ml ampicillin and 0.1mg/ml kanamycin L B for activation, inoculating activated fresh bacterial liquid into L B liquid culture medium at 37 ℃, culturing at 1500rpm until logarithmic phase, centrifuging to collect bacterial bodies, and re-suspending the bacterial bodies to 5.0 × 10 by using fresh L B liquid culture medium⁵CFU/ml, and obtaining the escherichia coli fermentation liquor. Preliminary experiments found that the half-lethal dose of E.coli fermentation broth to 7-week-old healthy female ICR mice was 0.1 ml. Randomly selecting 5 mice from each group to inject Escherichia coli fermentation broth in the intraperitoneal injection manner on the 28 th day of intragastric administration, wherein the injection amount of each mouse is half lethal dose, and the intraperitoneal injection day is the 0 th day after challengeAnd (5) day. The mice were observed every 24h for morbidity, and the survival rate of the mice was counted, and the organs inside the dead mice were dissected and observed for changes.

Inoculating high-drug-resistance lethal Staphylococcus aureus (provided by laboratory of animal epidemic prevention and control and food safety of Sichuan university, Sichuan province, and provided by key laboratory of Sichuan province, catalog number: SCSU-STREPC-HR L-2026) into liquid culture medium containing 0.1mg/ml ampicillin and 0.1mg/ml kanamycin L B for activation, inoculating activated fresh bacterial liquid into L B liquid culture medium at 37 ℃, culturing at 1500rpm until logarithmic phase, centrifuging to collect thallus, and re-suspending to 5.0 × 10 with fresh L B liquid culture medium⁵CFU/ml to obtain the staphylococcus aureus fermentation liquor. Preliminary experiments have found that the half-lethal dose of Staphylococcus aureus broth to 3-week-old healthy female ICR mice is 0.2 ml. And (3) injecting staphylococcus aureus fermentation liquor into the abdominal cavity of the 5 mice which are not injected with the escherichia coli fermentation liquor and remain in each group on the 28 th day of gastric lavage, wherein the injection amount of each mouse is half lethal dose, and the day of intraperitoneal injection is also the 0 th day after toxicity attack. The mice were observed every 24h for morbidity, and the survival rate of the mice was counted, and the organs inside the dead mice were dissected and observed for changes.

5. Analysis of Experimental results

The result of the routine detection of the change of peripheral blood leukocytes of each group of mice is shown in figure 8, SG represents blank control bacterium fermentation liquor, the content of peripheral blood leukocytes of experimental group mice is obviously higher than that of SG negative control group and vaccine negative control group (P is less than 0.05), and the SMDpG-46P fermentation product can effectively stimulate immune cell proliferation, which indicates that the fusion protein FPAPI L46 effectively stimulates immune cell proliferation.

The samples are processed by referring to the flow cytometry operation steps, the numbers of Th (CD4+ lymphocytes) and Tc (CD8+ lymphocytes) cells are detected on a computer at low temperature in a dark place, the flow cytometry results are shown in figures 9 and 10, the change of CD4+ and CD8+ lymphocytes in each 10000 cells of the peripheral blood of the randomly selected mice is shown, the graph shows that the content of CD4+ and CD8+ in the peripheral blood of the immunized mice is obviously higher than that of an SG negative control group and a vaccine negative control group (P <0.05), and the peak values are reached 21 or 28 days after immunization, which indicates that the SMDpG-46P fermentation product has the function of stimulating the immune response of the experimental mice, and the fusion protein pGAPI L46 effectively stimulates the proliferation of immune cells.

The plasma collected by low-speed centrifugation is used for detecting nonspecific antibodies IgG, IgG1 and IgG2a and the antibody titer of a specific antibody MP antibody according to the operation steps of an E L ISA kit, and the kits adopted are respectively a mouse immunoglobulin G1(IgG1) E L ISA kit (cargo number 69-210245), a mouse immunoglobulin G (IgG) E L ISA kit (cargo number 59-20037), a mouse immunoglobulin G (IgG2a) E L ISA kit (cargo number 69-210250) and a swine mycoplasma antibody E L ISA kit (cargo number 69-40349), and all the kits are products of Wuhan Shake biotechnology Limited.

The results are shown in fig. 11, 12 and 13, which show that the levels of IgG, IgG1 and IgG2a in peripheral blood serum of the immunized experimental mouse are significantly increased (P <0.05) compared with those of the SG negative control group and the vaccine negative control group, and both reach a peak value 14 or 28 days after immunization, indicating that the SMDpG-46P fermentation product can stimulate the immunized mouse to generate more IgG, IgG1 and IgG2a antibodies, fig. 14 shows that the antibody titer of MP (mycoplasma) in peripheral blood serum of the immunized experimental mouse is gradually reduced along with the increase of time, but the antibody titer of each group is significantly higher than that of the SG negative control group and the vaccine negative control group (P <0.05), indicating that the SMDpG-46P fermentation product can significantly improve the immune response effect induced by the vaccine, indicating that the fusion protein fp L46 effectively stimulates the immune cells to proliferate.

Detecting the expression of mouse immune related gene on RNA level, extracting RNA from whole blood as a template, amplifying by using primers shown in Table 4, and detecting the expression of mouse immune related gene, wherein the reference gene is actin β -actin.

Table 4 shows the primers

TNF- α is an important Th1 type cytokine, mainly involved in the differentiation and cellular immunity of Th1 cells, the dynamic change of the cytokine is shown in figure 15, the experimental group is significantly higher than SG negative control group and vaccine negative control group (P <0.05), and reaches a peak at 21 days after immunization, Th2 cells secrete I L4 cytokine and participate in the humoral immunity of the organism, the dynamic change of the cytokine is shown in figure 16, the experimental group is significantly higher than SG negative control group and vaccine negative control group (P <0.05), and reaches a peak at 14 to 21 days, the result shows that the SMDpG-46P fermentation product can simultaneously promote cellular immunity and humoral immunity, namely, the fusion protein FPAPI L46 can simultaneously promote cellular immunity and humoral immunity.

Fig. 17 shows the dynamic changes of the expression level of the T L R gene, and the results show that the expression levels of the T L R1 gene and the T L R4 gene are significantly increased (P <0.05) after immunization, and the expression level of the T L R gene of the mice in the experimental group is significantly increased (P <0.05) compared with the SG negative control group and the vaccine negative control group, and reaches a peak 21 days after immunization.

FIG. 18 is a graph showing the dynamic change of the expression level of the immunological memory-associated genes, which collectively shows that the I L-23 gene and I L-7 gene expression levels of the experimental group mice are significantly increased compared to the SG negative control group and the vaccine negative control group (P <0.05) after the immunization.

On the 5 th day after challenge, the survival rate of the mice injected with the escherichia coli fermentation liquid into the abdominal cavity is significantly higher than that of the SG negative control group and the vaccine negative control group (fig. 19), and the survival rate of the mice injected with the staphylococcus aureus fermentation liquid into the abdominal cavity is significantly higher than that of the SG negative control group and the vaccine negative control group, so that the SMDp46P of the experimental group can effectively protect the mice, the resistance of the mice to escherichia coli and staphylococcus aureus is significantly enhanced, namely the survival rate of the mice after being attacked by pathogenic bacteria can be improved by the fusion protein FPAPI L46.

Example 5 study of the biological Activity of the fusion protein FPAPI L46 in piglets

1. Preparation of fermentation product

Fermentation the recombinant bacterium SMDpG-46P (hereinafter referred to as SG46P) obtained in example 1 was activated and inoculated into a 30m L YPD-containing mediumCulture in 100m L Erlenmeyer flask of medium at 30 deg.C and 220rpm for 48h to OD₆₀₀About 40, SG46P fermentation broth was obtained.

And (3) fermenting the control recombinant bacterium SMDpG according to the method to obtain SG fermentation liquor.

2. Fermentation liquor for feeding experimental animals

Selecting 18 healthy Tibetan pigs with the weight of about 8kg and the age of 45 days, which are provided by a simple yang base of a Sichuan province breeding pig performance measuring center, randomly dividing the pigs into an experimental group (9 pigs) and a control group (9 pigs), feeding the SG46P fermentation liquor obtained in the step 1 to each pig in the experimental group, feeding the SG fermentation liquor obtained in the step 1 to each pig in the control group, wherein the feeding amount is 12.5ml/kg of the weight, feeding the pigs for 28 days, feeding the pigs once every 1 day, recording the day before the first feeding as 0 day of feeding, feeding all piglets with venous blood 3-4m L in a vacuum tube containing EDTA-K2 for 0 day, 7 days, 14 days, 28 days and 42 days before blood sampling, and then weighing all the piglets after feeding for immune cell change of blood sampling in 0 day, 28 days and 56 days.

All experimental pigs received the same classical intramuscular vaccination with the classical swine fever attenuated vaccine (Zhongmu Chengdu pharmaceutical machinery plant, catalog No.; 220051001) and the inactivated PRRS vaccine (Zhongmu Chengdu pharmaceutical machinery plant, catalog No.; 22003) at 6 weeks of age.

3. Experimental piglet weight change

The experimental piglets were weighed at 0, 14, 28 and 42 days of feeding, and the results (fig. 20) showed that the piglets in the experimental group gained weight 1.35, 1.16 and 1.13 times of the control group on 14, 28 and 42 days of feeding respectively, and the difference reached a significant level (P < 0.05); it is shown that SG46P effectively promoted piglet growth.

4. Dynamic change of peripheral blood leukocyte number of experimental piglet

The blood samples taken at 7 days, 14 days, 28 days and 42 days after feeding are mixed together according to groups, and the number of white blood cells in the blood samples is measured by a conventional blood cell analyzer, the result (figure 21) shows that the number of white blood cells in an SG46P experimental group is obviously higher than that in an SG control group (P <0.05) after feeding, and the fusion protein FPAPI L46 can effectively increase the number of peripheral blood immune cells of a using object and is beneficial to enhancing the immunity.

5. Detection of experimental piglet peripheral blood CD8+ T lymphocyte subset

Blood samples taken on days 7, 14, 28 and 42 of feeding were mixed together group by group for flow cytometry of CD4+ T and CD8+ T lymphocyte subpopulations in peripheral blood using CD4 and CD8 antibodies, respectively 1 μ l of Mouse Anti-Portone CD4-PE (Southern Biotech, Cat. 4515-09) and Mouse Anti-Portone CD8a-SPRD (Southern Biotech, Cat. 4520-13), following the specific procedure:

(1) fresh anticoagulated piglet venous blood 100. mu.l (leukocyte count of about 10)⁵-10⁷One), 60. mu.l of physiological saline was added;

(2) sucking 2 μ l of Mouse Anti-Portone CD8a-SPRD and 1 μ l of Mouse Anti-Portone CD4-PE into a 1.5ml EP tube, mixing uniformly, and incubating for 20 min;

(3) adding 0.2ml of 10x erythrocyte lysate into a special test tube for a flow cytometer, adding 1.8ml of PBS, adding the incubated blood into the lysate, and performing lysis for 5min until the blood cells are completely lysed;

(4) centrifuge at 1500rpm for 5min, and discard the supernatant. Adding 2ml of PBS, blowing, beating and mixing uniformly, and suspending cells;

(5) centrifuging at 1500rpm for 5min, discarding supernatant, slightly blowing and mixing with 150 μ l PBS, and washing for 1-2 times, wherein the amount of washing solution is at least 5 times of cell precipitation volume;

(6) the cells were blown down with 150. mu.l PBS and mixed well for detection.

The results are shown in fig. 22, and the SG46P experimental group showed significantly higher numbers of CD4+ T and CD8+ T lymphocytes than SG control group (P <0.05) at 7 days, 14 days, 28 days and 42 days of feeding, which indicates that the fusion protein FPAPI L46 can effectively increase the cellular immune response level of piglets.

6. E L ISA detection of CSF and PRRSV specific antibodies:

the plasma collected by low-speed centrifugation was subjected to detection of specific antibodies according to the procedures of the E L ISA kit (swine fever antibody (CSF Ab) E L ISA kit, porcine reproductive and respiratory syndrome virus E L ISA kit).

Fig. 23 and 24 are graphs showing the dynamic Changes of Swine Fever (CSF) -specific antibody and Porcine Reproductive and Respiratory Syndrome (PRRS) -specific antibody in peripheral blood during the growth period of piglets, respectively, and it is evident that the amounts of the two specific antibodies in the SG46P experimental group are significantly higher than those in the SG control group (P <0.05) at 0 days, 14 days, 28 days and 42 days of feeding, which indicates that the fusion protein FPAPI L46 can significantly enhance the immune response induced by the vaccine, thereby increasing the protection rate of the vaccine.

7. Variation of expression of immune-related genes

And (3) detecting the expression condition of the immune related genes of the piglets on the RNA level, wherein the immune related genes and primers are shown in the table 5, and the internal reference gene is PPIA.

TABLE 5 quantitative primers

Fig. 25 shows the dynamic change of the expression level of T L R gene, and the results show that after immunization, the expression levels of TR L-4 gene and TR L-7 gene are significantly increased (P <0.05), and the expression level of T L R gene of experimental swine is significantly increased (P <0.05) compared with SG negative control group, and the difference is most significant at 7, 14 or 42 days after immunization.

FIG. 26 shows the dynamic changes of the expression levels of the immunological memory-associated genes, and the results show that the expression levels of the CD45 gene and the CD 62L gene of experimental pigs are obviously increased (P <0.05) after immunization and reach a peak value 14 days after immunization.

The changes of the immunocytokine genes are shown in FIG. 27, and the results show that the expression levels of the I L-2, IFN-gamma, I L-10 and I L-23 genes of experimental pigs are remarkably increased compared with SG negative control group after immunization (P < 0.05).

The fusion protein FPAPI L46 can improve the expression level of innate immunity and acquired immunity (humoral immunity and cellular immunity) genes related to the immunity of experimental swine fever (CSF) and Porcine Reproductive and Respiratory Syndrome (PRRS) vaccines.

Sequence listing

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Claims (8)

1. A protein, the amino acid sequence of which consists of amino acid residues shown in a sequence 1 in a sequence table.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the coding sequence of the nucleic acid molecule is sequence 2 in the sequence table.

4. Any one of the following 1) -4):

1) an expression cassette comprising the nucleic acid molecule of claim 2 or 3;

2) a recombinant vector comprising the nucleic acid molecule of claim 2 or 3;

3) a recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3;

4) and (3) a fermentation product of the recombinant bacterium.

5. Use of a protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a biological material according to claim 4 for the preparation of a product for enhancing the immunocompetence of an animal.

6. Use according to claim 5, characterized in that: the animal immunity improving effect is at least one of the following M1-M5:

m1, inhibiting the growth of pathogenic microorganisms;

m2, promoting increase of immune cells;

m3, promoting vaccine-induced immune responses;

m4, promoting cellular and/or humoral immunity;

m5, increasing animal development and growth weight gain;

and/or the pathogenic microorganism is in particular escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus or classical swine fever virus;

and/or, the immune cell is in particular a lymphocyte, a red blood cell or a white blood cell;

and/or, the antibody is specifically an IgG, IgG1 and/or IgG2 a.

7. Use according to claim 5 or 6, characterized in that: the animal is any one of H1-H3:

h1, mammalian;

h2, pig;

h3, mouse.

8. Any one of the following X1 or X2:

x1, a biological agent comprising the following X3a, X3b or X3 c:

x3a, the protein of claim 1;

x3b, the nucleic acid molecule of claim 2 or 3;

x3c, the biomaterial of claim 4;

x2, kit for improving animal immunity, which consists of the X1 and antibiotics.

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