CN108558998A - Porcine IL-4/6 co-express the preparation and application of recombination yeast bacteria preparation with pig antibacterial peptide is merged - Google Patents

Abstract

The invention discloses the preparation and application of a recombinant yeast preparation co-expressed with porcine interleukin 4/6 and fusion porcine antimicrobial peptide. The antimicrobial peptide fusion cytokine FPAPIL46 provided by the present invention is as follows A1), A2) or A3): A1) the amino acid sequence is the protein of sequence 1 in the sequence listing; A2) the amino acid sequence shown in sequence 1 in the sequence listing is passed through a or several amino acid residue substitutions and/or deletions and/or additions and proteins with the same function; A3) A fusion protein obtained by linking tags at the N-terminal or/and C-terminal of A1) or A2). Experiments have proved that FPAPIL46 of the present invention can promote the proliferation of lymphocytes, red blood cells and white blood cells, inhibit the growth of pathogenic microorganisms, promote the secretion of non-specific antibodies (IgG, IgG1, IgG2a) and disease-specific antibodies, and improve the immunity and survival of animals. Rate.

Description

Co-expression of porcine interleukin 4/6 and fusion porcine antimicrobial peptides in recombinant yeast preparations Preparation and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of a recombinant yeast preparation co-expressed with porcine interleukin 4/6 and fusion porcine antimicrobial peptide.

Background technique

At present, China's livestock and poultry breeding industry is under the pressure of controlling the spread of more than 30 kinds of infectious pathogens, especially severe diseases such as avian influenza and respiratory reproductive syndrome, as the degree of intensification increases, and the scale and density of livestock and poultry breeding are increasing day by day. Diarrhea caused by drug-resistant bacteria in animal breeding, Salmonella, Escherichia coli, streptococcus, PRRSV (PRRSV), circovirus (PCV2), swine fever (CSFV) and other bacterial and viral diseases, For a long time, it has been a bottleneck problem in animal husbandry, which seriously hinders the development of animal husbandry; the abuse of drug additives such as antibiotics in feed is very serious, and the pollution problems of livestock and poultry diseases and antibiotic feed additives have become restrictions on the development of China's livestock and poultry industry. and the bottleneck of economic efficiency improvement. The abuse of feed antibiotic additives not only increases the cost of feeding, but also leads to the obvious enhancement of the drug resistance and pathogenicity of pathogenic microorganisms, which seriously damages the micro-ecological balance of the animal digestive tract. The health level of the body is weakened due to the toxic and side effects of drugs, and the immune resistance against diseases is significantly reduced. In such a vicious circle, various severe infectious diseases of livestock and poultry frequently occur, which is difficult to control and treat. On the other hand, drug residues and the increasingly serious food safety problems of livestock and poultry products not only directly threaten the health of human beings themselves, but also hinder the development of the breeding industry.

Adding antibiotics to animal feed, long-term or inappropriate use will not only induce drug resistance in bacteria, but also cause drug residues in animal products, causing serious food safety hazards, damaging human health and inducing the failure of antibiotics. Developed regions such as the European Union and the United States began to fully restrict antibiotics as feed additives in 2006. my country also strictly restricts the addition of antibiotics to feed. The modern aquaculture industry urgently needs to develop new bio-feeds and their additives that replace traditional antibiotics without residue, without induction of resistance, and are safe and pollution-free.

As a cytokine regulating animal humoral immunity, IL4 can activate resting B cells to enter the proliferation stage, induce differentiation and produce high levels of IgG1 and IgE; it has strong macrophage activation activity, enhances the expression of MHC-II molecules, antigen Presentation and phagocytosis; can stimulate the proliferation of bone marrow hematopoietic stem cells, enhance the body's hematopoietic function; also have a wider activation effect on T cells, can promote the growth of CD4 and CD3 T cells, and promote the continued growth of activated T cells; and stimulate thymocytes The hyperplasia is mature.

IL6 is one of the cytokines with the most extensive functions, which is secreted by T cells and macrophages, stimulates immune responses, and promotes the proliferation and differentiation of various cells. In inflammation, for example, IL-6 plays a role in fighting infection. At the site of local infection, endothelial cells, fibroblasts, and mononuclear macrophages secrete IL6 to fight the virus and promote the proliferation and growth of locally activated T cells, thereby inducing the differentiation and maturation of T cells into cytotoxic T cells; when IL6 is released, enter After blood circulation, it can activate resting hematopoietic stem cells, promote the division and proliferation of blood cells, induce or promote other immune cells to produce cytokines and play a role; induce a rise in body temperature, and awaken a series of defensive physiological responses of the body to fight infection.

The molecular weight of antimicrobial peptides is small, and there are certain difficulties in separation, purification and inspection. At present, the main source of antimicrobial peptides in scientific research is chemical synthesis, but the high cost and small amount of chemical synthesis are not conducive to large-scale application. Therefore, by using genetic engineering methods, multiple antimicrobial peptide genes are recombined and connected to obtain highly efficient recombinant antimicrobial peptide molecules with broad-spectrum anti-microbial (G+, G-, viruses, fungi, parasites) and immunomodulatory activities.

At present, there are few reports on recombinant cloning and co-expression of porcine interleukin fusion antimicrobial peptide gene.

Contents of the invention

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

In order to solve the above technical problems, the present invention provides a protein comprising porcine antimicrobial peptide and interleukin 4/6.

The above-mentioned protein is any protein in the following a)-e):

a) a protein whose amino acid sequence includes the amino acid sequence shown in Sequence 1 in the sequence listing;

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

c) A protein whose amino acid sequence defined in a) or b) has undergone substitution and/or deletion and/or addition of one or several amino acid residues and has the function of improving animal immunity;

d) A protein that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the amino acid sequence defined in a) or b) and has the function of improving animal immunity;

e) a fusion protein obtained after the N-terminal and/or C-terminal of the protein defined in any one of a)-d) is linked with a tag.

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

Above-mentioned nucleic acid molecule is the nucleic acid molecule shown in any one of following 1)-4):

1) its coding sequence includes sequence 2 in the sequence listing;

2) Its coding sequence is sequence 2 in the sequence listing;

3) A DNA molecule that hybridizes to the DNA molecule defined in 1) or 2) under stringent conditions and encodes the above-mentioned protein;

4) A DNA molecule that has 80% or more or 90% homology with the DNA molecule defined in 1) or 2) and encodes the above-mentioned protein.

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

Among them, sequence 2 encodes FPAPIL46 shown in sequence 1.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding FPAPIL46 of the present invention. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of FPAPIL46 isolated in the present invention, as long as they encode FPAPIL46 and have the function of FPAPIL46, are all derived from the nucleotide sequence of the present invention And is equivalent to the sequence of the present invention.

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

In the above-mentioned application, the stringent conditions are in a solution of 2×SSC and 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5×SSC and 0.1% SDS, in Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

The identity of 75% or more may be 80%, 85%, 90% or more.

Any biological material in the following 1)-3) is also within the protection scope of the present invention:

1) an expression cassette containing the above-mentioned nucleic acid molecule;

2) A recombinant vector containing the above-mentioned nucleic acid molecule;

3) Recombinant bacteria or transgenic cell lines containing the above-mentioned nucleic acid molecules;

4) The fermentation product of the recombinant bacteria.

In the above-mentioned application, the expression cassette (FPAPIL46 gene expression cassette) described in B2) that contains the nucleic acid molecule encoding FPAPIL46 refers to the DNA capable of expressing FPAPIL46 in the host cell, and the DNA can not only include the promoter that starts the transcription of the FPAPIL46 gene, A terminator that terminates transcription of the FPAPIL46 gene may also be included. Further, the expression cassette may also include an enhancer sequence.

An existing vector can be used to construct a recombinant vector containing the FPAPIL46 gene expression cassette.

In the above application, the vector can be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid can be a pGAPZαA vector.

B3) The recombinant vector may contain the DNA sequence shown in sequence 2 for encoding FPAPIL46. Further, the recombinant vector can specifically be pG-46P. The pG-46P is a recombinant vector obtained by replacing the DNA fragment between the EcoR I and Xba I recognition sequences of the pGAPZαA vector with the FPAPIL46 gene shown in Sequence 2.

In the above applications, the microorganisms can be yeast, bacteria, algae or fungi. Wherein, the yeast can be Pichia pastoris SMD1168.

In the above application, the recombinant microorganism can be a recombinant microorganism obtained by introducing an expression cassette containing a nucleic acid molecule encoding FPAPIL46 into yeast. Specifically, the recombinant microorganism can be a recombinant microorganism obtained by introducing a recombinant vector containing the FPAPIL46 expression cassette into yeast. When introducing the recombinant vector into the yeast, the recombinant vector can be directly introduced into the yeast, or the recombinant vector can be linearized before being introduced into the yeast. The yeast may be Pichia pastoris SMD1168.

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

In the above application, the fermentation product of the recombinant microorganism can be prepared according to a method comprising the following steps: cultivating the recombinant microorganism, expressing the gene encoding FPAPIL46, and obtaining the fermentation product of the recombinant microorganism.

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

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

C1. Improving animal immunity;

C2. Preparation of products for improving animal immunity.

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

M1, inhibit the growth of pathogenic microorganisms;

M2, promote the increase of immune cells;

M3, promote vaccine-induced immune response;

M4. Promote cellular immunity and/or humoral immunity;

M5, improve animal development and growth and weight gain;

And/or, the pathogenic microorganism is specifically Escherichia coli, Staphylococcus aureus, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus or swine fever virus, such as Escherichia coli standard bacteria (G ⁻ ), Escherichia coli drug-resistant bacteria (G ^- ), Staphylococcus aureus standard bacteria (G ⁺ ), Staphylococcus aureus resistant bacteria (G ⁺ ).

And/or, the immune cells are specifically lymphocytes (such as CD4+ or CD8+), red blood cells or white blood cells;

And/or, the antibody is specifically IgG, IgG1 and/or IgG2a. The antibody can specifically be an antibody of the pathogenic microorganism.

Any product of following X1 or X2 is also the scope of protection of the present invention:

X1. Biological preparations containing the following X3a, X3b or X3c:

X3a, the above-mentioned protein;

X3b, the above-mentioned nucleic acid molecules;

X3c. The biological materials mentioned above;

X2. A kit of reagents for improving animal immunity, consisting of the above-mentioned X1 and antibiotics.

In the above products, the biological preparation can be X3a, X3b or X3c as the active ingredient, and a composition combining X3a, X3b or X3c with other substances that can improve animal immunity can also be used as the active ingredient.

Or, a method for improving the immunity of an animal, comprising administering the above-mentioned protein or the above-mentioned nucleic acid molecule or the above-mentioned biological material or the biological preparation or the kit of reagents to the animal to improve the immunity of the animal.

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

H1, Mammals;

H2, pig;

H3, mice.

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

In order to solve the above technical problems, the present invention also provides a method for improving the immunity of animals, which may include administering FPAPIL46, the biological material or the biological preparation to the animals to improve the immunity of the animals.

In the present invention, the improvement of animal immunity can be any of the following M1-M5:

M1, inhibit the growth of pathogenic microorganisms;

M2, promote the increase of immune cells;

M3, promote vaccine-induced immune response;

M4. Promote cellular immunity and/or humoral immunity;

M5, improve animal development and growth and weight gain.

The pathogenic microorganism can be Escherichia coli, Staphylococcus aureus, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus (PRRSV) or swine fever virus (HCV or CSFV), such as Escherichia coli standard bacteria (G ⁻ ), Escherichia coli drug-resistant bacteria (G ^- ), Staphylococcus aureus standard bacteria (G ⁺ ), Staphylococcus aureus drug-resistant bacteria (G ⁺ ).

The immune cells can be lymphocytes (such as CD4+ or CD8+), red blood cells or white blood cells.

The antibodies may be IgG, IgGl and/or IgG2a. The antibody can specifically be an antibody of the pathogenic microorganism.

The vaccine can specifically be a vaccine against the pathogenic microorganism.

In the present invention, the improvement of animal immunity can be specifically improving the immunity of the animal to the pathogenic microorganism, such as improving the immunity of Mycoplasma hyopneumoniae, which causes porcine asthma (MP).

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

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

Experiments have proved that the co-expression FPAPIL46 molecule of the present invention and the fermentation product of recombinant yeast containing the FPAPIL46 gene have the following effects: promote the increase of lymphocytes and leukocytes in animals, and the contents of lymphocytes and leukocytes in animals administered with FPAPIL46 can be increased by more than 10%, obviously Inhibit the growth of pathogenic microorganisms; the amount of pathogenic microorganisms administered with FPAPIL46 can be reduced by more than 20%; promote the secretion of immunoglobulins (IgG, IgG1, IgG2a) and disease-specific antibodies, non-specific antibodies and diseases in animals administered with FPAPIL46 The content of specific antibodies can be increased by 40%-60%, and the expression of immune-related genes can be promoted, thereby improving the immune and anti-infection ability of animals. The survival rate of animals treated with FPAPIL46 is significantly increased by at least 60%, and the growth and weight gain rate is more than 10%.

Description of drawings

Figure 1 is the RT-PCR electrophoresis results of the target genes FPAP and IL-4/6 in SMDpG-46P.

Figure 2 is the detection result of the protein expression level of the target gene in SMDpG-46P.

Figure 3 is the effect of the fermentation supernatant of yeast strain SMDpG-46P on the proliferation of porcine lymphoblastoid cells.

Figure 4 shows the inhibitory effect of SG46P on Escherichia coli standard bacteria.

Figure 5 shows the inhibitory effect of SG46P on Escherichia coli drug-resistant bacteria.

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

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

Figure 8 shows the changes of peripheral blood leukocytes in mice under different treatments.

Figure 9 shows the change of CD4 ⁺ T lymphocytes per 10,000 cells in peripheral blood of mice.

Figure 10 shows the changes of CD8 ⁺ T lymphocytes per 10,000 cells in peripheral blood of mice.

Figure 11 shows the changes of IgG content in peripheral blood of mice in different groups.

Figure 12 shows the changes of IgG1 content in peripheral blood of mice in different groups.

Figure 13 shows the changes of IgG2a content in peripheral blood of mice in different groups.

Figure 14 shows the levels of MP-specific antibodies in the peripheral blood of mice in different groups.

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

Figure 16 shows the gene expression levels of Th2 type cytokine IL4 in the peripheral blood of different groups of mice.

Figure 17 shows the expression levels of TLR genes in peripheral blood of mice in different groups.

Figure 18 shows the gene expression levels of IL-7 and IL-23 in the peripheral blood of mice in different groups.

Fig. 19 shows the survival rate of mice in different groups after being challenged with Escherichia coli and Staphylococcus aureus.

Figure 20 is the net weight gain of piglets in each group.

Figure 21 shows the dynamic changes of white blood cells during piglet growth.

Figure 22 shows the dynamic changes of CD4+T and CD8+T lymphocytes in peripheral blood of piglets during growth.

Figure 23 shows the dynamic changes of CSF-specific antibodies in peripheral blood of piglets during growth.

Figure 24 shows the dynamic changes of PRRSV-specific antibodies in peripheral blood of piglets during growth.

Figure 25 shows the expression levels of TLR-4 and TLR-7 genes in peripheral blood of piglets during growth.

Figure 26 shows the expression levels of CD45 and CD62L genes in peripheral blood of piglets during growth.

Figure 27 shows the expression levels of cytokine genes in peripheral blood of piglets during growth.

Detailed ways

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The pGAPZαA vector in the following examples is a product of Invitrogen Company, and the product catalog number is V20020.

Pichia pastoris SMD1168 in the following examples is a product of Invitrogen Company, and the product catalog number is C17500.

The Landrace pigs in the following examples are products from the Jianyang Base of Sichuan Breeding Pig Performance Testing Center.

The Tibetan pigs in the following examples are products from the Jianyang Base of Sichuan Breeding Pig Performance Testing Center.

Escherichia coli standard bacteria (G ⁻ ) in the following examples are ATCC (America Type Culture Collection) products, and the product catalog number is 25922.

The drug-resistant Escherichia coli (G ^- ) in the following examples was provided by the Sichuan Key Laboratory of Animal Disease Prevention and Control and Food Safety, Sichuan University. The public can obtain the biological material from the applicant, and the biological material is only a duplicate It is used for related experiments of the invention and cannot be used for other purposes.

The Staphylococcus aureus standard bacteria (G ⁺ ) in the following examples is a product of ATCC (America Type Culture Collection), the catalog number of which is 29213.

The drug-resistant Staphylococcus aureus (G ⁺ ) in the following examples was provided by Sichuan University Key Laboratory of Animal Disease Prevention and Control and Food Safety, and the public can obtain the biological material from the applicant. The biological material is only It is used for repeating related experiments of the present invention and cannot be used for other purposes.

The female ICR mice in the following examples are products of the Experimental Animal Research Institute of Sichuan Provincial People's Hospital.

Embodiment 1, porcine antimicrobial peptide interleukin 4/6 fusion protein (FPAPIL46) and its coding gene

1. Acquisition of the fusion protein FPAPIL46 and its coding gene

The porcine antimicrobial peptide and interleukin 4/6 are fused to obtain a fusion protein named FPAPIL46.

The amino acid sequence of the fusion protein is shown as sequence 1 in the sequence listing, the fusion gene encoding the fusion protein FPAPIL46 is named FPAPIL46, and the nucleotide sequence of the fusion gene is sequence 2.

Among them, the 387-475th position of the sequence 1 is a porcine antimicrobial peptide, the 277-386th position is a connecting peptide, and the 1-276th position is an interleukin 4/6;

The 1159-1428th position of sequence 2 is the porcine antimicrobial peptide encoding nucleic acid, the 829-1158th position is the connecting peptide encoding nucleic acid, and the 1-828th position is the interleukin 4/6 encoding nucleic acid.

The above-mentioned proteins can be obtained by artificially synthesizing encoding nucleic acids and prokaryotic expression.

2. Preparation of recombinant vector expressing fusion protein FPAPIL46

The recombinant vector pGAPZαA-46B (pG-46P for short) is to replace the DNA fragment between the EcoR I and Xba I recognition sequences of the pGAPZαA vector with the FPAPIL46 gene shown in sequence 2, and keep other sequences of the vector unchanged to obtain a recombinant vector. The vector expresses the porcine antimicrobial peptide interleukin 4/6 fusion protein (FPAPIL46) shown in Sequence 1.

3. Preparation of recombinant bacteria expressing fusion protein FPAPIL46

1. Preparation of recombinant bacteria

pG-46P was digested with AvrII to obtain linearized pG-46P; the linearized pG-46P was introduced into Pichia pastoris SMD1168 to obtain recombinant yeast, which was named SMDpG-46P.

Using the same method, pGAPZαA was digested with AvrII to obtain linearized pGAPZαA; the linearized pGAPZαA was introduced into Pichia pastoris SMD1168 to obtain the control recombinant strain SMDpG.

2. Detection of recombinant bacteria

1) Expression detection at the RNA level

The recombinant bacterium SMDpG-46P was inoculated in YPD medium containing 100 μg/ml Zeocin at 28° C., cultured overnight at 200 rpm, and centrifuged to obtain SMDpG-46P bacterium. Extract the total RNA of SMDpG-46P cells with 2 pairs of primers (IL-4/6F: 5'-ATGAGAAGTGTGAAAACCAGC-3' and R: 5'-CGCATGTTAGAAGACTTCCCCTG-3'; FPAP: F: 5'-GAAGCTGAATTCAGGAGACGTCCCCG-3'; R: 5'-AAACGGGCCCTCTAGACTAATGGT-3' detects the expression level of the gene.

The results are shown in Figure 1, shown in A and B (A and B are the RT-PCR electrophoresis figures of fusion porcine antimicrobial peptide and porcine IL-4/6 respectively), A: Lane 1-2: fusion porcine antimicrobial peptide FPAP RT-PCR amplification band (FPAP primer amplification); M: DNA Marker; B: Lane 1-2: RT-PCR amplification band of pig IL-4/6 (IL-4/6 primer amplification); It indicated that the porcine IL-4/6 and antimicrobial peptide fusion gene FPAP in SMDpG-46P was transcribed and expressed at the RNA level.

Using the same method to detect the control recombinant strain SMDpG, no fusion gene FPAP and IL-4/6 transcription expression was found.

2) Detection of protein expression level

The recombinant strain SMDpG-46P (SG46P) was inoculated in YPD medium containing 100μg/ml Zeocin at 28°C and 200rpm for fermentation, and the fermentation broth was taken at different fermentation times for centrifugation, and the supernatant was taken and tested by HIS-Tag ELISA The kit (all antibody detection reagents are inside) detects the fusion protein in the supernatant. The supernatant obtained from the fermentation of the control recombinant Pichia pastoris SMD1168 was used as a negative control (the control recombinant SMDpG was used as a control, and the results were shown in Figure 2, and its OD ₄₅₀ <0.02 was much lower than that of the SG46P fermentation supernatant).

The results are shown in Figure 2. Compared with Pichia pastoris SMD1168, the recombinant strain SMDpG-46P (SG46P) produced the fusion protein FPAPIL46, and the expression level was the highest after 72 hours of fermentation.

The above results indicated that the recombinant strain SMDpG-46P expressed the fusion protein FPAPIL46.

Embodiment 2, the effect of porcine antimicrobial peptide interleukin 4/6 fusion protein (FPAPIL46) on lymphocyte proliferation

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

(1) the recombinant bacterium SMDpG-46P (hereinafter referred to as SG46P) that embodiment 1 obtains is inoculated in 3mL substratum 1 (substratum 1 is the liquid medium that adds bleomycin (Zeocin) to obtain in YPD substratum, and The concentration of bleomycin was 100mg/mL), and the activated strain was cultivated overnight at 28°C and 200rpm.

(2) Inoculate 300 μL of the bacterial solution obtained in step (1) into a 100 mL Erlenmeyer flask containing 30 mL of YPD medium, and ferment at 28°C for 48 h on a shaking table at 200 rpm (OD ₆₀₀ is 25).

(3) Take 5 mL of the bacterial liquid obtained in step (2), centrifuge at 12 000×g for 2 min, and name the obtained supernatant as SG46P fermentation supernatant.

2. Protease treatment of the fermentation supernatant of recombinant bacteria SMDpG-46P

First use pH test paper to measure the pH of the SG46P fermentation supernatant obtained in the above 1, and then use 2mol/L NaOH and 1mo/L HCl to adjust the SG46P fermentation supernatant to the optimum pH of trypsin and pepsin respectively. To pH 2.0, add trypsin solution (Beijing Soleibao Technology Co., Ltd., Trypsin-EDTA Solution (Trypsin-EDTA Solution) containing 0.25% trypsin and 0.02% EDTA, catalog number: 9002-07-7) and stomach Protease solution (Beijing Suolaibao Technology Co., Ltd., 0.1% aqueous solution pH 4.0, enzyme activity: 3000-3500NFU/g, catalog number: 9001-75-6,) simulates the effect of digestive tract enzymes to degrade proteins, so that the final concentration of enzymes is equal to 0.5 mg/mL, 37 ° C water bath enzyme reaction for 1 h, to obtain trypsin-treated SG46P fermentation supernatant and pepsin-treated SG46P fermentation supernatant, and store them in a -20 ° C refrigerator for later use.

According to the above method, replace SMDpG-46P with SMDpG (hereinafter referred to as SG), and other steps are all unchanged, and respectively obtain SG fermentation supernatant, trypsin-treated SG fermentation supernatant and pepsin-treated SG fermentation supernatant , Store in -20°C refrigerator for later use.

3. Effect of fusion protein FPAPIL46 on lymphocyte proliferation

1), Preparation of Landrace pig lymphocytes

Under sterile conditions, use a blood collection tube (containing EDTA-2K anticoagulant) to collect 5mL of peripheral blood from the anterior vena cava of Landrace pigs, and follow the operation steps of the porcine lymphocyte separation solution (preheat the separation solution at 37°C and fully shake and mix before use) Separation of Landrace pig lymphocytes was performed.

2) In vitro biological activity detection of SMDpG-46P fermentation products

(1) After culturing the porcine lymphocytes isolated in the above 1) for 24 hours, transfer the Landrace porcine lymphoblasts in the culture dish to a clean 15 mL sterile centrifuge tube, and centrifuge at 1500 rpm for 15 minutes at room temperature to collect cell bodies.

(2) The cells were washed with RPMI1640 complete medium (containing penicillin and streptomycin, 10% fetal calf serum), repeated twice, and the cell pellet was collected by centrifugation at 1500 rpm for 15 minutes at room temperature.

(3) Reselect the cells with RPMI1640 complete medium containing 20 mg/mL α-MM and adjust the number of cells to about 6×10 ⁶ cells/mL to obtain a cell suspension.

(4) Add 75 μL of the cell suspension in step (3), 45 μL of the sample solution, and 30 μL of RPMI1640 complete medium containing 20 mg/mL α-MM (methylmannoside) to each well of the 96-well cell plate according to the layout.

The sample liquids are the SG46P fermentation supernatant obtained in steps 1 and 2, the SG46P fermentation supernatant treated with trypsin, the SG46P fermentation supernatant treated with pepsin, the SG fermentation supernatant, and the SG fermentation supernatant treated with trypsin. Supernatant and pepsin-treated SG fermentation supernatant; one sample solution per well, and three replicate wells for each sample solution.

The RPMI1640 complete medium containing only 20 mg/mL α-MM, PBS and the cell suspension in step (3) were respectively used as blank controls.

Place them in a 5% CO ₂ , 37°C cell culture incubator for 48 hours.

(5) Take out the 96-well cell plate, add 15 μL of CCK8 (Guangzhou Yiyuan Biotechnology Co., Ltd.) to each well and mix gently, then put it into a 5% CO ₂ , 37°C cell incubator to continue culturing for 2 hours, take out the 96-well cell plate, The OD ₄₅₀ of each well was detected with a microplate reader (Bio-Reader3350).

The results are shown in Figure 3 (in the cell suspension of step (3) as a blank control), it can be seen that no matter whether trypsin or pepsin is processed, compared with the recombinant bacterial SG fermentation supernatant of the control group, the SG46P fermentation The lymphocytes obtained by stimulating porcine lymphoblasts with the supernatant increased significantly (P<0.05), indicating that the fusion protein FPAPIL46 can significantly stimulate the proliferation of porcine lymphocytes (P<0.05).

In Fig. 3, untreated means the fermentation supernatant without trypsin treatment and pepsin treatment.

Example 3, Antibacterial Activity Detection of Porcine Antibacterial Peptide Interleukin 4/6 Fusion Protein (FPAPIL46)

Determination of porcine antimicrobial peptide interleukin 4/6 fusion protein FPAPIL46 against Escherichia coli standard bacteria (G ^- ) (hereinafter referred to as SG ^- ), Escherichia coli resistant bacteria (G ^- ) (hereinafter referred to as RG ^- ), Bacteriostatic status of coccus standard bacteria (G ⁺ ) (hereinafter referred to as SG ⁺ ), Staphylococcus aureus drug-resistant bacteria (G ⁺ ) (hereinafter referred to as RG ⁺ ), the specific methods are as follows:

First inoculate and activate the four bacterial strains and cultivate them in the exponential growth phase (OD ₆₀₀ is about 0.5), then dilute them with LB culture medium to OD ₆₀₀ about 0.005, and inoculate the diluted bacterial solution on a 96-well cell culture plate, 100 μL/well, one type of bacteria per 96-well cell culture plate.

For each 96-well cell culture plate containing bacteria, it was processed as follows: 100 μL of sample solution was added to the experimental well and mixed gently, and each sample was set in 3 replicate wells;

Wherein the sample liquid is the SG46P fermentation supernatant obtained in steps 1 and 2 of Example 2 (i.e. the untreated SG46P fermentation supernatant), the SG46P fermentation supernatant treated with trypsin, the SG46P fermentation supernatant treated with pepsin , and the SG fermentation supernatant obtained in steps 1 and 2 of Example 2, the SG fermentation supernatant treated with trypsin and the SG fermentation supernatant treated with pepsin were used as corresponding empty controls.

For each type of bacteria, four antibiotic gradients were set as positive controls (antibiotics and LB culture solution); for each type of bacteria, only LB culture solution was used as a negative control, and LB culture solution without bacteria was set as a blank control.

After incubating the 96-well cell culture plate in a 37°C incubator for 2 h and 16 h, the OD ₆₀₀ value of each well was detected with a microplate reader (Bio-Reader 3350).

The four antibiotic gradients of ^SG- and SG ⁺ are shown in Table 1, and the four antibiotic gradients of RG- ^and RG ⁺ are shown in Table 2.

Table 1. Standard bacterial strain antibiotic gradient

Note: In Table 1, "—" means no kanamycin.

Table 2. Antibiotic gradient of drug-resistant strains

The results showed that for each of these four bacteria, there was no significant difference between the OD ₆₀₀ of various sample solutions of SG and the OD ₆₀₀ of the negative control at 2h and 16h, indicating that SG had no effect on these four bacteria. Inhibitory effect; the OD ₆₀₀ of various sample solutions of SG46P was significantly lower than that of the blank control at the corresponding time at 2h and _16h (P<0.05).

The results at 16 hours are shown in Figure 4-Figure 7 (both the blank control is the SG fermentation supernatant), and the SG46P fermentation supernatant has a significant inhibitory effect on these four bacteria (P<0.05), indicating that the fusion protein FPAPIL46 can inhibit Escherichia coli standard bacteria (G ^- ) (hereinafter referred to as SG ^- ), Escherichia coli drug-resistant bacteria (G ^- ) (hereinafter referred to as RG ^- ), Staphylococcus aureus standard bacteria (G ⁺ ) (hereinafter referred to as SG ⁺ ), Staphylococcus aureus drug-resistant bacteria (G ⁺ ).

Example 4. Study on the Biological Activity of Porcine Antibacterial Peptide Interleukin 4/6 Fusion Protein (FPAPIL46) in Mice

1. Preparation of fermentation products

Fermentation: Activate the recombinant bacteria SMDpG-46P (hereinafter referred to as SG46P) obtained in Example 1 and inoculate it in a 100mL Erlenmeyer flask containing 30mL of LYPD medium, cultivate it at 30°C and 220rpm for 48h, so that the _OD600 is about 25, and obtain SG46P fermentation broth.

According to the above method, the control recombinant bacterium SMDpG was fermented to obtain SG fermentation broth.

2. Grouping of experimental ICR mice

Take 50 18-20g 3-week-old healthy female ICR mice, randomly divide them into 5 groups, 10 mice in each group, and the group numbers are 1-5, in which group 1 and group 4 are SG negative control groups, and group 3 is SG negative control group. Vaccine negative control group, group 2 and group 5 are experimental groups.

3. Mice feeding and vaccination

According to the grouping situation, the fresh fermented liquid was delivered to the stomach of the mice with a gavage needle, 0.6 mL/only, and the first gavage was recorded as the 0th day of gavage, and gavage was performed once every two days for 4 consecutive weeks ( That is, on the 0th day, the 3rd day, the 6th day, the 9th day, the 12th day, the 15th day, the 18th day, the 21st day, the 24th day and the 27th day, the corresponding fermentation broth was administered orally , and each gavage volume is 0.6mL/only). In order to ensure that the recombinant protein is not metabolized, fermentation of fresh recombinant Pichia pastoris must be carried out before each animal gavage.

When vaccinating, only intramuscular injection (muscular injection) on the 7th day of gavage, 0.2mL/bird.

The specific operation is as follows (Table 3), wherein the porcine asthma (MP) vaccines are all products produced by Huapai Bioengineering Group Co., Ltd. (catalogue number: 19200003).

Table 3, mouse gavage, vaccination dose

In Table 3, intragastric administration is the inoculation method of fermentation broth, and 0.6mL/only is the inoculation dose of fermentation broth; intramuscular injection is the inoculation method of vaccine, and 0.2mL/only is the inoculation dose of vaccine.

Before gavage, on the 7th day of gavage, on the 14th day of gavage, on the 21st day of gavage, and on the 28th day of gavage, the tail vein blood of each mouse was collected, and the following experiments were carried out: 30 μL whole blood and 30 μL normal saline After mixing, do blood routine on a blood cell counter; 50 μL whole blood for flow cytometry; 100 μL whole blood for RNA extraction and real-time quantification of immune-related genes; 100 μL whole blood plus 1 mL TRIZOL vigorously mixed and stored at -80 °C for later use; 200 μL whole blood at low speed Plasma was collected by centrifugation for detection of antibodies.

4. Antivirus experiment

Highly drug-resistant lethal Escherichia coli (provided by the Sichuan Provincial Key Laboratory of Animal Disease Control and Food Safety, Sichuan University, catalog number: SCSU-ECOLI-HRL-1012) was inoculated in a culture medium containing 0.1 mg/ml ampicillin and 0.1 mg/ml ampicillin. Activated in kanamycin LB liquid medium, inoculate the activated fresh bacterial liquid in LB liquid medium at 37°C, culture at 1500rpm until the logarithmic phase and centrifuge to collect the bacteria, resuspend it with fresh LB liquid medium to 5.0×10 ⁵ CFU/ml to obtain Escherichia coli fermentation broth. The pre-experiment explored that the semi-lethal dose of E. coli fermentation broth to 7-week-old healthy female ICR mice was 0.1ml. On the 28th day of intragastric administration, 5 mice were randomly selected in each group for intraperitoneal injection of Escherichia coli fermentation broth. The injection volume of each mouse was a semi-lethal dose, and the day of intraperitoneal injection was the 0th day after challenge. The incidence of the mice was observed every 24 hours, and the survival rate of the mice was counted, and the internal organs of the dead mice were dissected to observe the changes.

The highly resistant lethal Staphylococcus aureus (provided by Sichuan Key Laboratory of Animal Disease Prevention and Control and Food Safety, Sichuan University, catalog number: SCSU-STREPC—HRL-2026) was inoculated with 0.1mg/ml ampicillin and 0.1mg /ml Kanamycin activated in LB liquid medium, inoculate the activated fresh bacterial liquid in LB liquid medium at 37°C, cultivate at 1500rpm until the logarithmic phase and centrifuge to collect the bacteria, and use fresh LB liquid medium to inoculate it Resuspend to 5.0×10 ⁵ CFU/ml to obtain Staphylococcus aureus fermentation broth. The pre-experiment explored that the semi-lethal dose of Staphylococcus aureus fermentation broth to 3-week-old healthy female ICR mice was 0.2ml. On the 28th day of intragastric administration, the remaining 5 mice in each group that were not injected with Escherichia coli fermentation broth were intraperitoneally injected with Staphylococcus aureus fermentation broth. The injection amount of each mouse was a semi-lethal dose. Day 0 after poisoning. The incidence of the mice was observed every 24 hours, and the survival rate of the mice was counted, and the internal organs of the dead mice were dissected to observe the changes.

5. Analysis of experimental results

The results of blood routine detection of peripheral blood leukocytes in each group of mice are shown in Figure 8. SG represents the fermentation broth of blank control bacteria, and the content of peripheral blood leukocytes in the experimental group mice is significantly higher than that of the SG negative control group and the vaccine negative control group (P <0.05); SMDpG-46P fermentation product can effectively stimulate the proliferation of immune cells, indicating that the fusion protein FPAPIL46 can effectively stimulate the proliferation of immune cells.

Process the samples according to the flow cytometry operation steps, and detect the number of Th (CD4+ lymphocytes) and Tc (CD8+ lymphocytes) cells on the computer at low temperature in the dark. Changes of CD4+ and CD8+ lymphocytes per 10,000 cells; as can be seen from the figure, the contents of CD4+ and CD8+ in the peripheral blood of the experimental mice after immunization were significantly higher than those of the SG negative control group and the vaccine negative control group (P<0.05) , both peaked at 21 or 28 days after immunization. It shows that the SMDpG-46P fermentation product has the function of stimulating the immune response of experimental mice, indicating that the fusion protein FPAPIL46 can effectively stimulate the proliferation of immune cells.

The plasma collected by low-speed centrifugation was tested for non-specific antibodies IgG, IgG1, and IgG2a according to the operation steps of the ELISA kit, and the antibody titer of the specific antibody MP antibody was detected. The kits used were mouse immunoglobulin G1 (IgG1) ELISA Kit (Cat. No. 69-210245), Mouse Immunoglobulin G (IgG) ELISA Kit (Cat. No. 59-20037), Mouse Immunoglobulin G (IgG2a) ELISA Kit (Cat. No. 69-210250), Mycoplasma Suis Antibody ELISA kit (Cat. No. 69-40349), all of the above kits are products of Wuhan Mercak Biotechnology Co., Ltd.

The results are shown in Figures 11, 12, and 13, showing that IgG, IgG1, and IgG2a levels in the peripheral blood serum of the experimental mice after immunization were significantly increased (P<0.05) compared with the SG negative control group and the vaccine negative control group. It peaked 14 or 28 days after immunization. It shows that SMDpG-46P fermentation product can stimulate immune mice to produce more IgG, IgG1, IgG2a antibodies. Figure 14 shows the antibody titer of MP (mycoplasma) in the peripheral blood serum of experimental mice after immunization, as time increases, the antibody titer of each group declines gradually, but the experimental group is significantly higher than the SG negative control group Compared with the vaccine negative control group (P<0.05). It shows that the SMDpG-46P fermentation product can significantly improve the immune response effect induced by the vaccine, indicating that the fusion protein FPAPIL46 can effectively stimulate the proliferation of immune cells.

Detect the expression of immune-related genes in mice at the RNA level, use RNA extracted from whole blood as a template, and use the primers shown in Table 4 to amplify to detect the expression of immune-related genes in mice. The internal reference gene is actin β- actin.

Table 4 is the primer

TNF-α is an important Th1-type cytokine, mainly involved in Th1 cell differentiation and cellular immunity, its dynamic changes are shown in Figure 15, the experimental group was significantly higher than the SG negative control group and the vaccine negative control group (P<0.05), And reached the peak on the 21st day after immunization. Th2 cells secrete IL4 cytokines and participate in the body's humoral immunity. The dynamic changes are shown in Figure 16. The experimental group was significantly higher than the SG negative control group and the vaccine negative control group (P<0.05), and reached the peak at 14 to 21 days . The results showed that the SMDpG-46P fermentation product could promote both cellular immunity and humoral immunity, that is, the fusion protein FPAPIL46 could simultaneously promote cellular immunity and humoral immunity.

Figure 17 shows the dynamic changes of TLR gene expression levels. The results showed that after immunization, the expression levels of TLR1 gene and TLR4 gene increased significantly (P<0.05), and the TLR gene expression level of mice in the experimental group was higher than that of the SG negative control group and the vaccine. The negative control group increased significantly (P<0.05), reaching the peak at 21 days after immunization.

Figure 18 is the dynamic change of the expression level of immune memory-related genes, generally showing that after immunization, the expression levels of IL-23 gene and IL-7 gene of the mice in the experimental group were significantly increased compared with the SG negative control group and the vaccine negative control group ( P<0.05).

On the 5th day after the challenge, for the mice injected intraperitoneally with E. coli fermentation broth, the survival rate of the mice in the experimental group was significantly higher than that of the SG negative control group and the vaccine negative control group (Figure 19). The survival rate of the mice in the experimental group was significantly higher than that of the SG negative control group and the vaccine negative control group, indicating that the SMDp46P in the experimental group can effectively protect the mice against Escherichia coli and Staphylococcus aureus. Significant enhancement, that is, the fusion protein FPAPIL46 can improve the survival rate of mice challenged by pathogenic bacteria. After dissecting the dead mice, it was found that the digestive tract of the mice killed by Escherichia coli had obvious lesions in the abdominal cavity, and the spleen and liver were blackened. , the liver is normal.

Example 5. Research on biological activity of fusion protein FPAPIL46 in piglets

1. Preparation of fermentation products

Fermentation: Activate the recombinant strain SMDpG-46P (hereinafter referred to as SG46P) obtained in Example 1 and inoculate it in a 100mL Erlenmeyer flask containing 30mL of LYPD medium, cultivate it at 30°C and 220rpm for 48h, so that the _OD600 is about 40, and obtain SG46P fermentation broth.

According to the above method, the control recombinant bacterium SMDpG was fermented to obtain SG fermentation broth.

2. Experimental animals were fed fermentation broth

Eighteen 45-day-old healthy Tibetan pigs weighing about 8 kg were selected from the Jianyang Base of Sichuan Breeding Pig Performance Testing Center, and randomly divided into the experimental group (nine pigs) and the control group (nine pigs). Each pig in the experimental group was fed with the SG46P fermented liquid in step 1, and each pig in the control group was fed with the SG fermented liquid in step 1. The feeding amount was 12.5ml/kg body weight, fed for 28 days, and fed every other day. Feed once, and record the day before the first feeding as feeding 0 day. On day 0, day 7, day 14, day 28, and day 42 of feeding, 3-4 mL of vena cava blood was collected from all piglets in vacuum tubes containing EDTA-K2, and then used for immune cell changes in blood samples. , 28 days, 56 days to weigh all piglets.

All experimental pigs received the same attenuated classical swine fever vaccine (China Animal Husbandry Chengdu Pharmaceutical Equipment Factory, catalog number; 220051001) and PRRS inactivated vaccine (China Animal Husbandry Chengdu Pharmaceutical Equipment Factory, catalog number; 22003) at the age of 6 weeks. Intramuscular vaccination.

3. Body weight change of experimental piglets

The experimental piglets of each group were weighed at 0 days, 14 days, 28 days and 42 days of feeding, and the results (Fig. 1.35, 1.16 and 1.13 times of , the difference reached a significant level (P<0.05); indicating that SG46P effectively promotes the growth of piglets.

4. The dynamic changes of the number of peripheral blood leukocytes in experimental piglets

The blood samples taken on the 7th, 14th, 28th and 42nd day of feeding were mixed together in groups, and the number of white blood cells in the blood samples was measured with a conventional blood cell analyzer. The results ( FIG. 21 ) showed that the number of white blood cells in the SG46P experimental group was significantly higher than that in the SG control group after feeding (P<0.05). It shows that the fusion protein FPAPIL46 can effectively increase the number of peripheral blood immune cells of the subject, which is beneficial to enhance immunity.

5. Detection of peripheral blood CD8+ T lymphocyte subsets of experimental piglets

The blood samples taken at 7 days, 14 days, 28 days and 42 days of feeding were mixed together in groups, and used for flow cytometry to detect the number of CD4+T and CD8+T lymphocyte subsets in peripheral blood, using CD4 and CD8 antibody was carried out with 1 μl of Mouse Anti-Porcine CD4-PE (Southern Biotech, Cat. No. 4515-09) and Mouse Anti-Porcine CD8a-SPRD (Southern Biotech, Cat. No. 4520-13), respectively, and the specific steps were as follows:

(1) Take 100 μl of fresh anticoagulated piglet venous blood (the number of white blood cells is about 10 ⁵ -10 ⁷ ), add 60 μl of normal saline;

(2) Pipette 2 μl Mouse Anti-Porcine CD8a-SPRD and 1 μl Mouse Anti-Porcine CD4-PE into a 1.5ml EP tube, mix well, and incubate for 20 minutes;

(3) Add 0.2ml 10x erythrocyte lysate into the special test tube for flow cytometry, then add 1.8ml PBS, add the incubated blood into the lysate, lyse for 5min, and wait until the blood cells are completely lysed;

(4) Centrifuge at 1500rpm for 5min, discard the supernatant. Add 2ml of PBS, mix by pipetting, and suspend the cells;

(5) Centrifuge at 1500rpm for 5min, discard the supernatant, leave about 150μl PBS and mix gently by pipetting, and wash 1-2 times in total. The amount of washing liquid should usually be at least 5 times the volume of the cell pellet;

(6) Mix the cells with 150 μl PBS by blowing and blowing for detection.

The results are shown in Figure 22. After feeding for 7 days, 14 days, 28 days and 42 days, the numbers of CD4+T and CD8+T lymphocytes in the SG46P experimental group were significantly higher than those in the SG control group (P<0.05). It shows that the fusion protein FPAPIL46 can effectively improve the level of cellular immune response in piglets.

6. ELISA detection of CSF and PRRSV specific antibodies:

The plasma collected by low-speed centrifugation was used to detect specific antibodies according to the operation steps of ELISA kits (CSF antibody (CSF Ab) ELISA kit, porcine blue ear virus ELISA kit).

Fig. 23 and Fig. 24 are respectively the dynamic change figure of the specific antibody of classical swine fever (CSF) and the specific antibody of blue ear disease (PRRS) in the peripheral blood of piglets during the growing period, it can be clearly seen in the figure that feeding 0 days, 14 days, 28 days On day 1 and day 42, the amounts of the two specific antibodies in the SG46P experimental group were significantly higher than those in the SG control group (P<0.05). It shows that the fusion protein FPAPIL46 can significantly enhance the immune response induced by the vaccine, thereby increasing the protection rate of the vaccine.

7. Expression changes of immune-related genes

The expression of immune-related genes in piglets at the RNA level was detected. The immune-related genes and primers are shown in Table 5, and the internal reference gene was PPIA.

Table 5. Quantitative primers

Figure 25 shows the dynamic changes of TLR gene expression levels, the results showed that after immunization, the expression levels of TRL-4 gene and TRL-7 gene increased significantly (P<0.05), and the TLR gene expression level of experimental pigs was higher than that of SG negative control group Significantly increased (P<0.05), the most obvious difference was 7, 14 or 42 days after immunization.

Figure 26 shows the dynamic changes in the expression levels of immune memory-related genes. The results show that after immunization, the expression levels of CD45 gene and CD62L gene in experimental pigs increased significantly (P<0.05), and reached a peak 14 days after immunization.

The changes of immune cytokine genes are shown in Figure 27. The results show that after immunization, the expression levels of IL-2, IFN-γ, IL-10 and IL-23 genes in the experimental pigs were significantly increased compared with the SG negative control group (P<0.05) .

The results indicated that the fusion protein FPAPIL46 could increase the expression levels of genes related to innate immunity and acquired immunity (humoral and cellular immunity) after immunization with experimental swine fever (CSF) and blue ear disease (PRRS) vaccines.

sequence listing

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Claims (10)

1. a kind of protein, including pig antibacterial peptide and interleukin-4/6.

2. protein according to claim 1, it is characterised in that：The protein be following a)-e) in any albumen Matter：

A) amino acid sequence includes the protein of amino acid sequence shown in sequence 1 in sequence table；

B) amino acid sequence amino acid residue shown in sequence in sequence table 1 forms；

C) by amino acid sequence defined by a) or b) by the substitution of one or several amino acid residues and/or missing and/or Addition and the protein with raising animal immune ability function；

D) amino acid sequence defined by and a) or b) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology and the protein with raising animal immune ability function；

E) a)-d) in it is any defined by protein N-terminal and/or C-terminal connection label after obtained fusion protein.

3. encoding the nucleic acid molecules of albumen described in claims 1 or 2.

4. nucleic acid molecules according to claim 3, it is characterised in that：The nucleic acid molecules are following 1) -4) in it is any Shown in nucleic acid molecules：

1) its coded sequence includes sequence 2 in sequence table；

2) its coded sequence is sequence 2 in sequence table；

1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and encode the DNA molecular of albumen described in claim 1；

1) or 2) 4) with the DNA molecular that limits with described in 80% or more or 90% or more homology and coding claim 1 The DNA molecular of albumen.

5. following 1) -3) any one of biomaterial：

1) expression cassette containing the nucleic acid molecules of claim 3 or 4；

2) recombinant vector containing the nucleic acid molecules of claim 3 or 4；

3) recombinant bacterium or transgenic cell line containing the nucleic acid molecules of claim 3 or 4；

4) tunning of the recombinant bacterium.

6. the biological material described in protein or claim 3 or 4 nucleic acid molecules or claim 5 described in claims 1 or 2 Expect the application in following C1 or C2：

C1, animal immune ability is being improved；

C2, it prepares and improves animal immune ability product.

7. applying according to claim 6, it is characterised in that：The raising animal immune ability be following M1-M5 in extremely Few one kind：

M1, the growth for inhibiting pathogenic microorganisms；

M2, the increase for promoting immunocyte；

M3, promote vaccine-induced immune response；

M4, promote cellular immunity and/or humoral immunity；

M5, animal development and growth-weight gain are improved；

And/or the pathogenic microorganisms is specially Escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, pig breeding and exhales Inhale disorders syndrome virus or swine fever virus；

And/or the immunocyte is specially lymphocyte, red blood cell or leucocyte；

And/or the antibody specific is IgG, IgG1 and/or IgG2a.

8. any products of following X1 or X2：

X1, biological agent contain following X3a, X3b or X3c：

Protein described in X3a, claims 1 or 2；

X3b, claim 3 or 4 nucleic acid molecules；

Biomaterial described in X3c, claim 5；

X2, the reagent set for improving animal immune ability, by above-mentioned X1 and antibiotic group at.

9. a kind of method improving animal immune ability, including protein or right described in claims 1 or 2 are applied to animal and wanted Biomaterial described in 3 or 4 nucleic acid molecules or claim 5 or the biological agent or the reagent set are asked, is improved The immunocompetence of the animal.

10. applied described according to claim 6 or 7 or claim 9 described in method, it is characterised in that：The animal is H1- Any one of H3：

H1, mammal；

H2, pig；

H3, mouse.