A fusion protein, nucleic acid molecule, biological material, application and product co-expressing porcine EGF, Ghrelin and IGF-1

Abstract

The present invention relates to a fusion protein, nucleic acid molecule, biological material, application and product co-expressing porcine EGF, Ghrelin and IGF-1, belonging to the field of biotechnology, including porcine EGF, Ghrelin and IGF-1. The fusion protein is as follows: (a1) consisting of the amino acid sequence shown in SEQ ID NO:1; (a2) obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence; (a3) having an identity of 80-99% or more with (a1) or (a2); (a4) substituting, deleting and / or adding one or several amino acid residues to (a1) or (a2). The present invention constructs a fusion gene of porcine EGF, Ghrelin and IGF-1, integrates it into an expression system to obtain a fusion protein, and explores the immunobiomodulatory effect of the fusion protein in vitro, the anti-infection activity in mice and the immunomodulation in pigs and other biological effects; it is found that the fusion protein can significantly enhance the immune barrier function of the animal digestive tract mucosa, improve its systemic immune level and antibacterial infection ability, and has good biological effects in promoting animal growth and development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a fusion protein, nucleic acid molecule, biological material, application and product co-expressing porcine EGF, Ghrelin and IGF-1. Background Art

[0002] Establishing an efficient modern prevention and treatment system for animal infectious diseases that combines prevention and treatment, and developing food or feed additives that promote the healthy growth of animals and enhance the immune capacity of animal bodies are of great significance for ensuring the sustainable development of the aquaculture industry in China. In view of this, the present invention provides a fusion protein, nucleic acid molecule, biological material, application and product co-expressing porcine EGF, Ghrelin and IGF-1. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a fusion protein, nucleic acid molecule, biological material, application and product co-expressing porcine EGF, Ghrelin and IGF-1. The aim is to provide a feed additive that promotes the healthy growth of animals and enhances the immune capacity of animal bodies.

[0004] To solve the above technical problem, the first aspect of the present invention is to provide a fusion protein co-expressing porcine EGF, Ghrelin and IGF-1, including porcine EGF, Ghrelin and IGF-1. The fusion protein is as follows (a1), (a2), (a3) or (a4):

[0005] (a1) Composed of the amino acid sequence shown in SEQ ID NO: 1;

[0006] (a2) Obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence in (a1);

[0007] (a3) A protein having 80-99% or more identity with (a1) or (a2) and having the function of enhancing the immune capacity of animals;

[0008] (a4) A protein obtained by substituting, deleting, and / or adding one or several amino acid residues to (a1) or (a2) and having the function of enhancing the immune capacity of animals.

[0009] In (a1), positions 1 - 53 of the amino acid sequence shown in SEQ ID NO:1 are porcine EGF, positions 54 - 59 are a histidine tag, positions 60 - 62 are a linker peptide (GSG), positions 63 - 81 are a P2A self - cleaving peptide, positions 82 - 96 are a secretion signal peptide (XPR2Pre), positions 97 - 124 are porcine Ghrelin, positions 125 - 131 are a linker peptide, positions 132 - 201 are porcine IGF - 1, and positions 202 - 207 are a histidine tag; preferably in (a3), a protein having more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99% identity with (a1) or (a2) and having the function of enhancing the immune ability of animals.

[0010] Based on the above - mentioned technical solution, the present invention can also be improved as follows.

[0011] The second aspect of the present invention is to provide a nucleic acid molecule, which encodes the fusion protein as described above.

[0012] Furthermore, the nucleic acid molecule is as follows (b1), (b2) or (b3):

[0013] (b1) Composed of the nucleotide sequence shown in SEQ ID NO:2;

[0014] (b2) Hybridizes with the nucleotide sequence defined by (b1) under stringent conditions and encodes the fusion protein;

[0015] (b3) Has 80 - 99% or more homology with the nucleic acid molecule defined by (b1) or (b2) and encodes the fusion protein.

[0016] Among them, 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, etc.; in (b1), nucleotides 1 - 159 of the nucleotide sequence shown in SEQ ID NO:2 encode porcine EGF, nucleotides 160 - 177 encode a histidine tag, nucleotides 178 - 186 encode a linker peptide, nucleotides 187 - 243 encode a P2A self - cleaving peptide, nucleotides 244 - 288 encode a secretion signal peptide XPR2Pre, nucleotides 289 - 372 encode porcine Ghrelin, nucleotides 373 - 393 encode a Linker, nucleotides 394 - 603 encode porcine IGF - 1, nucleotides 604 - 621 encode a histidine tag, and nucleotides at positions 622 - 624 are stop codons.

[0017] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid or amino acid sequence; identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. Homologous sequences refer to different sequences that have evolved through divergent evolution from a common ancestor.

[0018] The third aspect of the present invention is to provide a biological material, which is any one of the following (c1) to (c6):

[0019] (c1) An expression cassette containing the nucleic acid molecule according to claim 2 or 3;

[0020] (c2) A recombinant vector containing the nucleic acid molecule according to claim 2 or 3;

[0021] (c3) A transgenic cell line containing the nucleic acid molecule according to claim 2 or 3;

[0022] (c4) A recombinant microorganism containing the nucleic acid molecule according to claim 2 or 3;

[0023] (c5) A culture product of the transgenic cell line;

[0024] (c6) A fermentation product of the recombinant microorganism.

[0025] The culture product of the transgenic cell line or the fermentation product of the recombinant microorganism can be prepared by a method comprising the following steps: culturing the transgenic cell line or the recombinant microorganism to express the coding gene to obtain the culture product of the transgenic cell line or the fermentation product of the recombinant microorganism. For example, the culture product of the transgenic cell line is the supernatant expressed by it; the fermentation product of the recombinant microorganism can be the supernatant of its fermentation broth.

[0026] The fourth aspect of the present invention is to provide an application, which is to use the fusion protein as described above, the nucleic acid molecule as described above, or the biological material as described above in the preparation of a product for improving the immune ability of animals.

[0027] Furthermore, the improvement of the immune ability of animals is at least one of the following (d1) to (d6):

[0028] (d1) Promote effector target cell immunity and / or humoral immunity;

[0029] (d2) Promote the establishment of the immune barrier of effector target animals;

[0030] (d3) Promote animal development and growth;

[0031] (d4) Promote the increase of animal immune cells;

[0032] (d5) Promote animal cell-mediated immunity and humoral immunity;

[0033] (d6) Resist pathogenic microorganism infections.

[0034] Furthermore, the target animals are pigs and mice, the target cells are small intestinal mucosal epithelial cells; the immune cells are lymphocytes (such as T lymphocytes); the pathogenic microorganisms are Salmonella typhimurium and / or Staphylococcus aureus. The antibodies produced after the above immunization are IgG and sIgA.

[0035] Furthermore, the animal is a pig or a mouse.

[0036] The fifth aspect of the present invention is to provide a product, including the fusion protein as described above, the nucleic acid molecule as described above, or the biological material as described above. The product can have the fusion protein, nucleic acid molecule, or biological material as the active ingredient, or can also have a composition in which they are combined with other substances that can enhance the immune ability of animals as the active ingredient; additionally, carriers and / or excipients acceptable on the product can also be added.

[0037] Furthermore, the product is a vaccine or a biological agent material. The vaccine can specifically be a vaccine against the above-mentioned pathogenic microorganisms. The biological agent is a biological drug that enhances the immune ability of animals and treats diseases caused by the above-mentioned pathogenic microorganisms.

[0038] The sixth aspect of the present invention is to provide a method for enhancing the immune ability of animals, including the following steps: administering the product to the animal to enhance the immune ability of the animal; the product includes the fusion protein as described above, the nucleic acid molecule as described above, or the biological material as described above.

[0039] Experimental results have shown that the co-expressed fusion protein of porcine EGF, Ghrelin and IGF-1 and the fermentation product of recombinant yeast of the present invention have the following effects: (1) Through lymphocyte proliferation experiment - CCK8, it is shown that the fusion protein EGF / Ghrelin-IGF synthesized in the present invention has immunobiological activity; (2) Through the study of the biological activity of recombinant Yarrowia lipolytica Po1h-EGF / Ghrelin-IGF in mice, it is proved that: (a) The recombinant yeast has reliable biosafety; (b) Adjust the proportion of each cell subset in T lymphocytes and B lymphocytes in the peripheral blood of mice; (c) The fermentation product of recombinant yeast Po1h-EGF / Ghrelin-IGF adjusts the changes of immune-related genes in the small intestine tissue of mice (Jak-1, STAT1, IL-1β, IL-8, BD1, S100A8, Reg3, TGF-β and TNF-α genes), can stimulate mice to produce more non-specific antibody IgG, and significantly improve the immunity of mice to Staphylococcus aureus or Salmonella typhimurium; (d) Under the condition of challenge with Staphylococcus aureus or Salmonella typhimurium, the height of intestinal villi in the Po1h-EGF / Ghrelin-IGF group was significantly higher than that in the PBS group (P<0.01), and the crypt depth in the Po1h-EGF / Ghrelin-IGF group was significantly higher than that in the PBS group (P<0.001), improving the survival rate of mice after challenge; (3) The study of biological activity in pigs shows that: (a) It can effectively promote the growth and development of pigs and the increase of body weight, significantly improve the digestion and utilization rate of feed by piglets, and reduce the feed-to-meat ratio; (b) The numbers of white blood cells, neutrophils, lymphocytes and platelets in the peripheral blood of pigs are significantly or extremely significantly higher than those in the blank control group, indicating that the recombinant yeast Po1h-EGF / Ghrelin-IGF has high biosafety; (c) It can enhance the cellular immunity and humoral immunity of pigs, and promote the generation of immune memory-related factors, thereby improving the immunity of pigs.

[0040] Cytokines are important molecules that synergistically and efficiently regulate the immune system and play an indispensable key regulatory role in the differentiation, development, maturation, and activation of active cells in various animal tissues. In addition, the synergistic effect of co-expression of multiple cytokines has a greater regulatory role in immunobiology and application value in anti-infection defense. Therefore, in the prevention and treatment of infectious diseases in livestock and poultry and ensuring the normal growth and development of animals, multi-cytokine co-expression preparations are a highly potential new biological molecular preparation that can replace antibiotics to promote animal growth and development and enhance the immune level of animals. In this invention, a fusion gene of porcine EGF, Ghrelin, and IGF-1 was constructed and integrated into the eukaryotic expression system of Yarrowia lipolytica to obtain a fusion protein, and then the immunobiological regulatory effect of the fusion protein in vitro and the anti-infection activity of the recombinant yeast in mice and its biological effects on improving the immune level and promoting growth and development in pigs were further explored. It was found that the fusion expression of porcine EGF, Ghrelin, and IGF-1 can significantly enhance the immune barrier function of the animal digestive tract mucosa, improve its systemic immune level and antibacterial infection ability, and has a good biological effect on promoting animal growth and development. Brief Description of the Drawings

[0041] Figure 1 It is a control chart of the expression level of the recombinant protein of the recombinant yeast of the present invention; wherein, a is the standard curve of EGF, b is the standard curve of Ghrelin-IGF, c is the expression level of EGF, and d is the expression level of Ghrelin-IGF;

[0042] Figure 2 It is a CCK-8 cell proliferation experiment chart of the present invention;

[0043] Figure 3 It is a control chart of the dynamic change of the body weight of mice in the present invention;

[0044] Figure 4 It is a chart showing the change in the proportion of each cell subset of T lymphocytes in the peripheral blood of mice in the present invention at different time points; wherein, a is cytotoxic T cells, b is helper T cells, c is naive T cells, d is central memory T cells, e is effector memory T cells, and f is regulatory T cells;

[0045] Figure 5 It is a chart showing the change in the proportion of each cell subset of B lymphocytes in the peripheral blood of mice in the present invention at different time points; wherein, a is plasma cells, b is naive B cells, c is non-switched memory B cells, and d is switched memory B cells;

[0046] Figure 6 It is a control chart of the change in total IgG in the plasma of mice in the present invention;

[0047] Figure 7Shows the changes in immune-related genes in the small intestine tissues of mice after challenging with the toxins of the present invention; among them, a represents challenge with Staphylococcus aureus, and b represents challenge with Salmonella typhimurium.

[0048] Figure 8 Is the control chart of the sIgA level in the feces of mice of the present invention.

[0049] Figure 9 Is the control chart of the morphological changes in the small intestine tissues of mice of the present invention after the challenge is over.

[0050] Figure 10 Is the statistical chart of the morphological changes in the small intestine tissues of the present invention; among them, a is under the condition of challenge with Staphylococcus aureus, and b is under the condition of challenge with Salmonella typhimurium.

[0051] Figure 11 Is the survival rate chart of mice of the present invention after challenge; among them, a is under the condition of challenge with Staphylococcus aureus, and b is under the condition of challenge with Salmonella typhimurium.

[0052] Figure 12 Shows the changes in the growth performance of piglets during the nursery period under different treatments of the present invention; among them, a is the change in the average total weight gain of piglets, b is the change in the average daily weight gain of piglets, c is the average daily feed intake of piglets, and d is the feed conversion ratio of piglets.

[0053] Figure 13 Is the dynamic change chart of the number of white blood cells, neutrophils, lymphocytes, red blood cells, hemoglobin concentration and platelet count in the peripheral blood of piglets of the present invention; among them, a is white blood cells, b is neutrophils, c is lymphocytes, d is red blood cells, e is hemoglobin, and f is platelets.

[0054] Figure 14 Is the dynamic change chart of the expression level of Th1-type cytokine IL-2 in porcine PBMC of the present invention.

[0055] Figure 15 Is the dynamic change chart of the expression levels of Th2-type cytokines IL-4 and IL-6 in porcine PBMC of the present invention; among them, a is the dynamic change of the expression level of Th2-type cytokine IL-4, and b is the dynamic change of the expression level of Th2-type cytokine IL-6.

[0056] Figure 16 Is the dynamic change chart of the expression levels of immune memory-related cytokines IL-15 and IL-23 in porcine PBMC of the present invention; among them, a is the dynamic change of the expression level of immune memory-related cytokine IL-15 in porcine PBMC, and b is the dynamic change of the expression level of immune memory-related cytokine IL-23 in porcine PBMC.

[0057] Figure 17This is a dynamic change graph of the contents of EGF and IGF-1 in porcine peripheral blood plasma in the present invention; wherein, a is the dynamic change of the content of EGF in porcine peripheral blood plasma, and b is the dynamic change graph of the content of IGF-1 in porcine peripheral blood plasma. Detailed implementation manners

[0058] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. The recombinant plasmids have all been verified by sequencing. Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, and the results are averaged.

[0059] The Kunming mice in the following examples: Experimental Animal Center of Sichuan University, production license number: SCXK(Sichuan)2018-026. The Chuanxiang black pigs in the following examples: A new pig breed cultivated and provided by the Pig Research Institute of Sichuan Academy of Animal Science. 100× penicillin-streptomycin mixture: HyClone company. The vector pINA1297 (vector pINA1297) and the Yarrowia lipolytica Po1h (Po1h strain) were both kindly donated by Professor Madzak of the French Academy of Agricultural Sciences and are all recorded in the following literature: Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol. 2004 Apr 8;109(1-2):63-81. doi:10.1016 / j.jbiotec.2003.10.027. PMID:15063615.

[0060] Example 1: Construction of a co-expression system of porcine EGF, Ghrelin and IGF-1 fusion genes in Yarrowia lipolytica and in vitro activity research

[0061] I. Design of the fusion protein EGF / Ghrelin-IGF-1 and its encoding gene

[0062] The fusion protein co-expressing porcine EGF, Ghrelin and IGF-1 (denoted as fusion protein EGF / Ghrelin-IGF-1) is shown as SEQ ID NO:1 in the sequence listing. In the amino acid sequence shown in SEQ ID NO:1, positions 1-53 are porcine EGF, positions 54-59 are histidine tags, positions 60-62 are linker peptides (GSG), positions 63-81 are P2A self-cleaving peptides, positions 82-96 are secretion signal peptides (XPR2Pre), positions 97-124 are porcine Ghrelin, positions 125-131 are linker peptides, positions 132-201 are porcine IGF-1, and positions 202-207 are histidine tags.

[0063] The DNA molecule shown as SEQ ID NO:2 in the sequence listing encodes the fusion protein EGF / Ghrelin-IGF-1, and this DNA molecule is named the EGF / Ghrelin-IGF-1 fusion gene. In the nucleotide sequence shown in SEQ ID NO:2, nucleotides 1-159 encode porcine EGF, nucleotides 160-177 encode histidine tags, amino acids 178-186 encode linker peptides, nucleotides 187-243 encode P2A self-cleaving peptides, nucleotides 244-288 encode the secretion signal peptide XPR2Pre, nucleotides 289-372 encode porcine Ghrelin, nucleotides 373-393 encode Linker, nucleotides 394-603 encode porcine IGF-1, nucleotides 604-621 encode histidine tags, and nucleotides 622-624 are stop codons.

[0064] Among them, the EGF / Ghrelin-IGF-1 fusion gene fragment was obtained by total gene synthesis from Nanjing Genscript Biotechnology Co., Ltd. and was constructed into the self-cloning shuttle vector pINA1297. The recombinant shuttle vector was named pINA1297-EGF / Ghrelin-IGF-1.

[0065] II. Transformation of the recombinant shuttle vector into Escherichia coli and large-scale plasmid extraction

[0066] 1. Transformation of the recombinant shuttle vector into Escherichia coli

[0067] (1) The constructed recombinant shuttle vector pINA1297-EGF / Ghrelin-IGF-1 was transformed into Escherichia coli competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) Top10. The bacterial solution was spread on a Luria-Bertani (LB) medium plate containing kanamycin, placed face up for 30 min, and then incubated overnight in an inverted position in a 37°C constant temperature incubator.

[0068] (2) Pick a single colony on the LB plate and inoculate it into 1 mL of LB liquid medium containing 50 mg / ml kanamycin. Incubate it on a shaker for 2 h, then transfer the bacterial solution to 5 mL of fresh LB liquid medium and culture it overnight for 12 - 16 h. Preserve the bacterial solution for the next experiment.

[0069] 2. Screening and identification of positive clones

[0070] Identify the above positive transformants by colony PCR and sequencing.

[0071] 3. Extraction of recombinant self-cloning vector pINA1297 - EGF / Ghrelin - IGF-1

[0072] Extract the recombinant self-cloning vector pINA1297 - EGF / Ghrelin - IGF-1 from the bacterial solution in step 1(2) according to the operation manual of the plasmid miniprep kit from OMEGA. Detect the extraction product by 1.5% agarose gel electrophoresis.

[0073] III. Construction of recombinant Yarrowia lipolytica Po1h - pINA1297 - EGF / Ghrelin - IGF-1

[0074] 1. Linearization and transformation of recombinant plasmid pINA1297 - EGF / Ghrelin - IGF-1

[0075] (1) Linearize the pINA1297 - EGF / Ghrelin - IGF-1 recombinant plasmid with the restriction enzyme Not I. Transform the recovered linearized fragment into the competent cells of Yarrowia lipolytica Po1h to obtain recombinant yeast, named Po1h - EGF / Ghrelin - IGF-1 (also known as Po1h - EGF / Ghrelin - IGF).

[0076] (2) Take the pINA1297 empty plasmid and also linearize it with the restriction enzyme NotⅠ. Transform the obtained linearized fragment into the competent cells of Yarrowia lipolytica Po1h to obtain recombinant yeast, named Po1h - pINA1297.

[0077] IV. Expression of EGF / Ghrelin - IGF-1 fusion gene in Yarrowia lipolytica and in vitro activity study

[0078] 1. Protein level detection

[0079] Inoculate Po1h-EGF / Ghrelin-IGF and recombinant yeast Po1h-pINA1297 into 250 ml shake flasks containing 100 ml of YPD liquid medium (10 g / L yeast extract, 20 g / L tryptone, and 20 g / L glucose) respectively, and culture them in an air bath shaker at 28 °C and 200 rpm for 48 h. Take the fermentation broth and perform ultrasonic lysis treatment (power 200 W, ultrasound for 3 s, interval 10 s, repeat 30 times). Centrifuge at 4 °C and 10,000 rpm to collect the supernatant for the determination of the recombinant protein expression level. Refer to the instructions of the porcine EGF ELISA Kit (Wuhan Huamei Biological Engineering Co., Ltd., CSB-E06788p) and IGF-1 ELISA Kit (Wuhan Huamei Biological Engineering Co., Ltd., CSB-E06829p) to detect the expression of recombinant proteins in the supernatant of the lysed recombinant yeast.

[0080] The results are shown in Figure 1 , where a is the standard curve of EGF, b is the standard curve of Ghrelin-IGF, c is the expression level of EGF, and d is the expression level of Ghrelin-IGF. The expression of the target protein was detected in the supernatant of the lysed recombinant yeast Po1h-EGF / Ghrelin-IGF, and the contents of porcine EGF and Ghrelin-IGF were 2.851 ng / mL and 206.147 ng / mL respectively. Only trace amounts of the corresponding target protein expression were detected in the supernatant of the lysed recombinant yeast Po1h-pINA1297.

[0081] 2. Lymphocyte proliferation assay - CCK8

[0082] (1) Preparation of lymphoblasts

[0083] Under sterile conditions, collect 5 mL of peripheral blood from the anterior vena cava of pigs, anticoagulate with EDTA-2K (the molecular formula of EDTA-2K is C 10 H 14 K2N2O8·2H2O), and separate porcine lymphocytes according to the instructions of the porcine peripheral blood lymphocyte separation liquid KIT (Tianjin Haoyang Huake Biotechnology Co., Ltd., China). Dilute the separated cells with complete 1640 culture medium (containing 10% fetal bovine serum, 100 μg / mL ampicillin, and 100 μg / mL streptomycin) to a final cell concentration of 2×10 6 cells / mL, dispense them into cell culture dishes with a diameter of 10 cm, 10 mL per dish, and finally add Con A (L7647, Merck KGaA, Germany) with a final concentration of 10 μg / mL for stimulation, and culture them in a 37 °C, 5% CO2 cell culture incubator for 24 h.

[0084] (2) Biological activity detection

[0085] After culturing for 24 h, collect the porcine lymphoblasts in the culture dish into a clean centrifuge tube, and centrifuge at 1500 rpm for 15 min to collect the cells; wash the cells twice with 1640 complete medium (containing double antibodies and serum), and centrifuge at 1500 rpm for 15 min.

[0086] Adjust the cells to about 6×10 6 cells / mL with 1640 medium containing 20 mg / mL α-MM. Add 100 μL of target cells (porcine lymphoblasts) and the same volume of the broken supernatant of Po1h-EGF / Ghrelin-IGF and the broken supernatant of Po1h-pINA1297 to the experimental wells of a 96-well plate respectively; set 3 replicate wells for each sample, and set control wells, and culture in a 37 °C, 5% CO2 cell culture incubator for 48 h; after 48 h, take out the 96-well plate, add 10 μL of CCK8 to each well, gently blow and mix evenly, and continue to culture for 2 h; take out the 96-well plate, and use a Bio-Reader680 to detect the OD 450 .

[0087] The results are shown in Figure 2 . The ability of the broken supernatant of recombinant yeast Po1h-EGF / Ghrelin-IGF to promote the proliferation of porcine lymphoblasts was detected using a CCK-8 kit. It was found that the broken supernatant of Po1h-EGF / Ghrelin-IGF significantly increased the proliferation of lymphoblasts compared with the empty vector group PBS and the blank control group Po1h-pINA1297 (P<0.05), indicating that the recombinant protein has biological activity.

[0088] Example 2: Study on the biological activity of recombinant Yarrowia lipolytica Po1h-EGF / Ghrelin-IGF in mice I. Preparation of recombinant yeast fermentation broth

[0089] 1. After inoculating and reviving Po1h-pINA1297 by streaking on a YPD plate, pick a single colony and inoculate it into a 100 mL flask containing 30 mL of YPD medium, and culture it in an air bath shaker at 28 °C and 220 rpm for 48 h until the OD 600 is about 10 to obtain the Po1h-pINA1297 fermentation broth.

[0090] 2. According to the above method, replace Po1h-pINA1297 with Po1h-EGF / Ghrelin-IGF, and keep other steps unchanged to obtain the recombinant yeast fermentation broth of Po1h-EGF / Ghrelin-IGF.

[0091] II. Mouse experiment protocol

[0092] 1. Grouping and experimental treatment of mice

[0093] (1) Thirty 4- to 5-week-old SPF-grade healthy female Kunming mice, weighing approximately 18 grams, were randomly divided into three groups: a blank control group, a vector control group, and an experimental group, with 10 mice in each group.

[0094] (2) Blank control group:

[0095] The mice were gavaged with phosphate buffered saline (PBS) at a dose of 100 μL / mouse / time, once every 3 days, for a total of 10 times.

[0096] (3) Vector control group:

[0097] The mice were gavaged with the fermentation broth of Po1h-pINA1297 recombinant yeast at a dose of 4×10 8 CFU / mouse / time (100 μL), once every 3 days, for a total of 10 times.

[0098] (4) Experimental group:

[0099] The mice were gavaged with the fermentation broth of Po1h-EGF / Ghrelin-IGF recombinant yeast at a dose of 4×10 8 CFU / mouse / time, once every 3 days, for a total of 10 times.

[0100] (5) Challenging

[0101] Challenging began 28 days after the gavage treatment. Procedure: The concentrated Salmonella typhimurium (ATCC 14028) was resuspended in fresh liquid LB medium to obtain a bacterial suspension of 1.0×10 10 CFU / mL, which was the challenging liquid. Each mouse was gavaged once every other day, for a total of 3 times, with 300 μL of the bacterial suspension per mouse each time (5 mice in each group). The mice were fasted but allowed to drink water 2 h before each gavage. The day of gavage was defined as day 0 after challenging. The morbidity of the mice was observed every 24 h, and the survival rate of the mice was counted. The internal organs of the dead mice were observed by dissection. The Staphylococcus aureus (ATCC 25923) challenging experiment was carried out in the same way.

[0102] 2. Sample and data collection

[0103] (1) Body weight index

[0104] The mice were weighed once a week for four consecutive weeks, and the dynamic changes in the body weights of the mice in each group were recorded.

[0105] The results are shown in Figure 3 . There were no significant differences in the body weights of the mice in each group from Day 7 to Day 28 (P>0.05), indicating that the recombinant yeast had reliable biosafety.

[0106] (2) Blood immune indexes

[0107] Before the first gavage, on the 7th, 14th, 21st, and 28th days after gavage, and on the third day after challenge, peripheral blood was collected by cutting the tail vein of the mice.

[0108] (3) Fecal / intestinal immune indexes

[0109] On the 28th day after gavage and on the third day after challenge, fresh feces of the mice were collected; after the mice were sacrificed at the end of the challenge, small intestine tissues were collected for morphological analysis and transcriptional level analysis.

[0110] 3. Flow cytometry analysis of the changes in immune cells in the peripheral blood of mice

[0111] Anticoagulated blood collected on the 7th, 14th, and 28th days after gavage was analyzed by flow cytometry. After the peripheral blood of the mice at each time point was labeled with fluorescent antibodies, flow cytometry was used to analyze the changes in the number of lymphocytes in the PBS group, Po1h-pINA1297 group, and Po1h-EGF / Ghrelin-IGF group. The immune typing of T lymphocytes could obtain Th, Tc, Naive T Cell, Tcm, Tem, Teff, Treg, etc., and the immune typing of B lymphocytes could obtain Plasma Cell, Naive B Cell, Switched Memory BCell, Non-switched Memory B Cell, etc. The markers for lymphocyte typing are shown in Table 1.

[0112] Table 1 Markers for lymphocyte typing

[0113] Lymphocyte subsets Immune cell markers T Cells CD3+ Cytotoxic T Cells(Tc) CD3+ / CD8+ Helper T Cells(Th) CD3+ / CD4+ Th Central Memory(TCM) CD3+ / CD4+ / CD44+ / CD62L+ Th Effector Memory(TEM) CD3+ / CD4+ / CD44+ / CD62L2- Naive T Cells CD3+ / CD4+ / CD44- / CD62L+ Regulatory T Cells(Treg) CD3+ / CD4+ / CD25+ / FoxP3+ B Cells CD19+ B Unswitched Memory(BUSM) CD19+ / CD27+ / IgD+ B Switched Memory(BSM) CD19+ / CD27+ / IgD- Naive B Cells CD19+ / CD27- / IgD+ Plasma Cells CD27+ / CD38+

[0114] Figure 4 The changes in the proportions of each cell subset of T lymphocytes in the peripheral blood of mice at different time points are as follows: (a) cytotoxic T cells, (b) helper T cells, (c) naive T cells, (d) central memory T cells, (e) effector memory T cells, (f) regulatory T cells. Figure 4 (a) There was no significant difference in the number of cytotoxic T cells among the PBS group, Po1h-pINA1297 group, and Po1h-EGF / Ghrelin-IGF group (P>0.05); Figure 4 (b) The number of helper T cells in the Po1h-EGF / Ghrelin-IGF group was significantly more than that in the PBS group and Po1h-pINA1297 group at 7-14 days (P<0.05); there was no significant difference at 28 days. Figure 4(c) The initial T cell numbers in all three groups increased, and the initial T cell numbers in the Po1h-EGF / Ghrelin-IGF group were significantly more than those in the other two groups at D14 and D28; Figure 4 (d) There was no significant difference in the central memory T cell numbers among the three groups (P>0.05); Figure 4 (e) In the PBS group and the Po1h-pINA1297 group, the effector memory T cell numbers showed a trend of first decreasing and then increasing, while the TEM cell numbers in the Po1h-EGF / Ghrelin-IGF group continuously decreased; however, the proportion of TEM cells at D28 was significantly lower than that at D14. Figure 4 (f) The Treg numbers in the PBS group and the Po1h-pINA1297 group were significantly higher than those in the Po1h-EGF / Ghrelin-IGF group at 7-14 days (P<0.05); at 28 days, the regulatory T cell numbers in the Po1h-EGF / Ghrelin-IGF group exceeded those in the other two groups (P>0.05).

[0115] Figure 5 The proportion changes of each cell subset of B lymphocytes in the peripheral blood of mice at different time points: (a) plasma cells, (b) naive B cells, (c) non-switched memory B cells, (d) switched memory B cells. Figure 5 (a) The plasma cell numbers in all three groups first increased and then decreased, and at the D14 time point, the plasma cell numbers in the Po1h-EGF / Ghrelin-IGF group were the highest, significantly more than those in the control group (P<0.05); Figure 5 (b) The three groups of naive B cells maintained an increasing trend in proportion, with no significant difference (P>0.05); Figure 5 (c) In the PBS group and the Po1h-EGF / Ghrelin-IGF group, the non-switched memory B cell numbers first increased and then decreased, and the difference was significant at 14 days (P<0.05); while the non-switched memory B cell numbers in the Po1h-pINA1297 group gradually increased; the difference was significant from the other groups at 28 days (P<0.05); Figure 5 (d) The changing trends of the switched memory B cell numbers in the three groups were basically the same, all gradually decreasing, with no significant difference among them (P>0.05).

[0116] 4. Changes in total IgG in the plasma of mice

[0117] On the 7th, 14th, and 28th days after intragastric administration, and on the 3rd day after challenge, 200 μL of EDTA-anticoagulated peripheral blood was collected from the tail vein of mice. (Under the condition of 4000 rpm, centrifuge for 20 min to obtain the supernatant, which is the plasma. According to the instruction manual of the mouse immunoglobulin G (IgG) kit (ELISA) (RX202736M, Ruixin Biotech), the change of total IgG in the mouse plasma was measured.)

[0118] The results are shown in Figure 6 . Among the first 3 time points before challenge, the content of IgG in the peripheral blood plasma of mice in the Po1h-EGF / Ghrelin-IGF group was extremely significantly higher than that of mice in the blank control group (PBS) (P < 0.01); on the 28th day and after challenge with Staphylococcus aureus, the content of IgG in the plasma of mice in the Po1h-EGF / Ghrelin-IGF group was extremely significantly and significantly higher than that in the Po1h-pINA1297 group (P < 0.01, P < 0.05); the results indicate that the fermentation product of recombinant yeast Po1h-EGF / Ghrelin-IGF can stimulate mice to produce more non-specific antibody IgG and effectively enhance the ability of mice to resist Staphylococcus aureus.

[0119] 5. Changes in immune-related genes in mouse small intestine tissue

[0120] (1) Sample processing of mouse small intestine tissue

[0121] Collect 25 mg of mouse small intestine tissue after the end of challenge, grind it with liquid nitrogen, extract total RNA and reverse transcribe it into cDNA, and use fluorescence quantitative PCR to detect the expression of Jak-1, STAT1, IL-1β, IL-8, BD1, S100A8, Reg3, TGF-β, and TNF-α genes in the intestinal tissue. The primers for detecting immune-related genes in mouse small intestine tissue are shown in Table 2.

[0122] Table 2 Primers for detecting immune-related genes in mouse small intestine tissue

[0123] Figure 7For the changes in immune-related genes in the small intestine tissues of mice after detoxification, in (a), under the condition of Staphylococcus aureus challenge, the expression levels of various immune-related genes (including Jak-1, STAT1, IL-1β, IL-8, BD1, S100A8, Reg3, TGF-β, and TNF-α genes) in the small intestine of mice in the Po1h-EGF / Ghrelin-IGF group were extremely significantly higher than those in the empty vector group (Po1h-pINA1297) and the blank control group (PBS) (P<0.01), indicating that the fermentation product of recombinant yeast Po1h-pINA1297-EGF / Ghrelin-IGF-1 could significantly improve the immunity of mice against Staphylococcus aureus; in (b), under the condition of Salmonella typhimurium challenge, among the immune-related genes in the small intestine of mice in the Po1h-EGF / Ghrelin-IGF group, only the expression level of the S100A8 gene was significantly higher than that in the empty vector group (Po1h-pINA1297) and the blank control group (PBS) (P<0.01), while the expression level of the TGF-β gene was extremely significantly lower than that in the empty vector group (Po1h-pINA1297) and the blank control group (PBS) (P<0.001).

[0124] 6. sIgA level in mouse feces

[0125] Collect the fresh feces of mice on the 28th day after gavage and on the 3rd day after challenge, suspend them with 4 mL / g of 0.01M PBS and 0.05M EDTA buffer, shake on ice for 15 min, centrifuge at 10000g at 4℃ for 5 min, and store the supernatant at -80℃ for later detection. Detect the content of fecal sIgA according to the instruction manual of the mouse secretory immunoglobulin A (sIgA) quantitative detection kit (ELISA) (RX-G202950M, Ruixin Bio).

[0126] Figure 8 For the sIgA level in mouse feces, under D28 and different challenge conditions, the content of sIgA in the feces of mice in the Po1h-EGF / Ghrelin-IGF group was extremely significantly higher than that in the PBS group and the empty vector group Po1h-pINA1297 (P<0.001), and the content of fecal sIgA in both the Po1h-pINA1297 group and the PBS group was relatively low, indicating that the fusion protein EGF / Ghrelin-IGF-1 had a high effect of enhancing the expression level of sIgA.

[0127] 7. Morphological changes in small intestine tissues of mice after challenge

[0128] After the challenge, take the small intestine tissues of mice in each group, stain them with hematoxylin and eosin (H&E), observe and measure the villus height, crypt depth, and intestinal wall thickness to evaluate the structure and function of the small intestine.

[0129] Figure 9 It shows the morphological changes of small intestinal tissues after mouse challenge with pathogens. Figure 10 It is a statistical chart of the morphological changes of small intestinal tissues. (a) Under the condition of challenge with Staphylococcus aureus, the height of small intestinal villi in the Po1h-EGF / Ghrelin-IGF group was significantly higher than that in the PBS group (P<0.01); (b) Under the condition of challenge with Salmonella typhimurium, the height of small intestinal villi in the Po1h-EGF / Ghrelin-IGF group was significantly higher than that in the PBS group and the blank control group Po1h-pINA1297 (P<0.001), and the depth of crypts in the Po1h-EGF / Ghrelin-IGF group was significantly higher than that in the PBS group (P<0.001).

[0130] 8. Survival rate of mice after pathogen challenge

[0131] On the 28th day after intragastric administration to mice, 5 mice in each group were challenged with Staphylococcus aureus, and another 5 mice were challenged with Salmonella typhimurium. After observation for two weeks, they were sacrificed, the daily change in the number of mice after challenge was recorded, the survival days of mice were counted, the survival rate was calculated, and the survival curve of mice after challenge was plotted.

[0132] Figure 11 It shows the survival rate of mice after pathogen challenge. (a) Under the condition of challenge with Staphylococcus aureus; (b) Under the condition of challenge with Salmonella typhimurium. Under both challenge conditions, there were significant differences in the survival rates of mice between the Po1h-EGF / Ghrelin-IGF group and the PBS group and the empty vector group Po1h-pINA1297 (P<0.05), indicating that intragastric administration of the Po1h-EGF / Ghrelin-IGF fermentation broth to mice for immunization for 28 days significantly increased the survival days after challenge and significantly improved and increased the survival rate of mice after challenge. After two weeks of challenge under both conditions, only 40% of the mice in the PBS group survived, 60% in the Po1h-pINA1297 group, while the survival rate in the Po1h-EGF / Ghrelin-IGF group was 100%.

[0133] Example 3: Biological activity study of porcine EGF, Ghrelin and IGF-1 fusion protein in pigs

[0134] I. Preparation of fermentation product of recombinant yeast Po1h-pINA1297-EGF / Ghrelin-IGF-1

[0135] 1. Inoculate the recombinant yeast Po1h-pINA1297-EGF / Ghrelin-IGF-1 into 2.5 mL of liquid YPD medium and culture it overnight at 28°C with shaking in an air bath at 200 rpm;

[0136] 2. Take the bacterial liquid obtained in step 1 and inoculate it into a 2 L flask containing 1 L of liquid YPD medium, and culture it at 28°C with shaking in an air bath at 220 rpm until OD600 is 20 (about 24 h);

[0137] 3. Take the bacterial liquid obtained in step 2 and inoculate it into a 15 L fermenter containing 10 L of BSM fermentation medium (for 1 L of BSM fermentation medium: 85% phosphoric acid (26.7 ml), calcium sulfate 0.93 g, potassium sulfate 18.2 g, magnesium sulfate heptahydrate 14.9 g, potassium hydroxide 4.13 g, glycerol 40.0 g, make up to 1 L with distilled water; for 1 L of PTM1: cupric sulfate anhydrous 6.0 g, sodium iodide 0.08 g, manganese sulfate monohydrate 3.0 g, sodium molybdate dihydrate 0.2 g, boric acid 0.02 g, cobalt chloride 0.5 g, zinc chloride 20.0 g, ferrous sulfate heptahydrate 65.0 g, biotin 0.2 g, concentrated sulfuric acid 5.0 ml, sterilize by microporous filtration with 0.22 μm at room temperature; add 40 mL of PTM1 to 1 L of BSM medium), and culture with stirring at 28 °C and 400 rpm until OD 600 is 80 (about 48 h), and the whole fermentation system obtained is named Po1h-pINA1297-EGF / Ghrelin-IGF-1 fermentation product.

[0138] II. Grouping and treatment of experimental animals

[0139] 1. 38 healthy Chuanxiang black pigs, with a birth weight of about 2.4 kg, are randomly divided into 2 groups (20 in the experimental group and 19 in the control group);

[0140] 2. Experimental group (Po1h-pINA1297-EGF / Ghrelin-IGF-1 group): Starting from 10 days old, the Po1h-pINA1297-EGF / Ghrelin-IGF-1 yeast liquid is added to the creep feed at 20 ml per piglet every two days until the end of the 28-day lactation period; after 28 days old, the yeast liquid is added to the nursery feed at 30 ml per head (the commercial feed is a piglet compound feed (620) produced by Cargill Feed (Chongqing) Co., Ltd., and the raw material composition includes expanded corn, corn, soybean meal, white sugar, whey powder, stone powder, calcium dihydrogen phosphate, sodium chloride, vitamins and vitamin-like substances, mineral elements and their complexes (chelates), zinc chloride, L-lysine, DL-methionine, threonine, and mold inhibitor (calcium propionate), etc.), and fed once every two days until the end of the 56-day nursery period; the control group is fed with an equal amount of PBS added to the conventional creep feed and nursery feed, without adding other raw materials;

[0141] 3. On the 14th, 28th, 42nd, and 56th days after birth, collect jugular vein blood from each pig for the following experimental procedures: 2.5 mL of anticoagulated blood is used for routine blood tests and the detection of the expression of immune-related genes in PBMCs, and the remaining anticoagulated blood is separated to detect the plasma for changes in the content of related cytokines; weigh the experimental pigs in each group at birth and at the end of the nursery period.

[0142] III. Detection of Each Index

[0143] 1. Growth Performance Index of Piglets

[0144] Figure 12 The changes in the growth performance of piglets during the nursery period under different treatments are as follows. Among them, a is the change in the average total weight gain of piglets, b is the change in the average daily weight gain of piglets, c is the average daily feed intake of piglets, and d is the feed conversion ratio of piglets. The average total weight gain and average daily weight gain of the Po1h-pINA1297-EGF / Ghrelin-IGF-1 experimental group were extremely significantly higher than those of the control group (PBS) (P < 0.01); while the average daily feed intake and feed conversion ratio of the Po1h-pINA1297-EGF / Ghrelin-IGF-1 experimental group were extremely significantly lower than those of the control group (PBS) (P < 0.01). The results show that the fermentation product of recombinant yeast Po1h-pINA1297-EGF / Ghrelin-IGF-1 can effectively promote the weight gain of piglets, significantly improve the digestion and utilization rate of feed by piglets, and reduce the feed-to-meat ratio.

[0145] 2. Routine Blood Test

[0146] Figure 13 (a, b, c, f) show the dynamic changes in the numbers of white blood cells, neutrophils, lymphocytes, and platelets in the peripheral blood of piglets. During the entire feeding period with mixing, there were no significant differences in the numbers of white blood cells, neutrophils, lymphocytes, and platelets in the peripheral blood of pigs in the Po1h-EGF / Ghrelin-IGF group and those in the blank control group (PBS) (P > 0.05). The results indicate that recombinant yeast Po1h-EGF / Ghrelin-IGF has high biosafety; Figure 13 (d and e) show the dynamic changes in the numbers of red blood cells and hemoglobin concentrations in the peripheral blood of piglets. On the 14th day of feeding with mixing, the numbers of red blood cells and hemoglobin concentrations in the peripheral blood of pigs in the Po1h-EGF / Ghrelin-IGF group were significantly higher than those in the blank control group (PBS) (P < 0.05); while at the other three time points, there were no significant differences in the numbers of red blood cells and hemoglobin concentrations in the peripheral blood between the experimental group and the control group (P > 0.05).

[0147] 3. Detection of the Expression of Immune-Related Genes in PBMCs by Fluorescent Quantitative PCR

[0148] Figure 14 It is the dynamic changes of the expression level of Th1 cytokine IL-2 in porcine PBMC. Th1 cytokines mainly mediate immune responses related to cytotoxicity and local inflammation, assist antibody production, and participate in the occurrence of cellular immunity and delayed hypersensitivity inflammation. On the 56th day after mixing and feeding, the expression level of IL-2 in porcine PBMC of the Po1h-EGF / Ghrelin-IGF group was extremely significantly higher than that of the blank control group (PBS) (P<0.01). The results prove that the fermentation product of recombinant yeast Po1h-EGF / Ghrelin-IGF can enhance the cellular immunity of pigs.

[0149] Figure 15 It is the dynamic changes of the expression levels of Th2 cytokines IL-4 and IL-6 in porcine PBMC. Among them, a is the dynamic changes of the expression level of Th2 cytokine IL-4, and b is the dynamic changes of the expression level of Th2 cytokine IL-6; the main function of Th2 cytokines is to stimulate the proliferation of B cells and produce IgG, and participate in humoral immunity. On the 56th day after mixing and feeding, the expression levels of both IL-4 and IL-6 in porcine PBMC of the Po1h-EGF / Ghrelin-IGF group were significantly higher than those of the blank control group (PBS) (P<0.05). The results show that the fermentation product of recombinant yeast Po1h-EGF / Ghrelin-IGF can enhance the humoral immunity of pigs.

[0150] Figure 16 It is the dynamic changes of the expression levels of immune memory-related cytokines IL- fifteen and IL- twenty-three in porcine PBMC; among them, a is the dynamic changes of the expression level of immune memory-related cytokine IL- fifteen in porcine PBMC, and b is the dynamic changes of the expression level of immune memory-related cytokine IL- twenty-three in porcine PBMC. Immune memory-related factors can promote the differentiation and development of immune memory-related cells, promote the formation of immune memory in the body, and enhance the immune response of the body. On the 56th day after mixing and feeding, the expression levels of both IL- fifteen and IL- twenty-three in porcine PBMC of the Po1h-EGF / Ghrelin-IGF group were extremely significantly higher than those of the blank control group (PBS) (P<0.01). The results indicate that the fermentation product of recombinant yeast Po1h-EGF / Ghrelin-IGF can promote the production of immune memory-related factors, thereby improving the immunity of pigs.

[0151] 4. ELISA was used to detect the contents of EGF and IGF-1 in porcine peripheral blood plasma

[0152] Figure 17Dynamic changes in the contents of EGF and IGF-1 in porcine peripheral blood plasma; among them, a is the dynamic change in the content of EGF in porcine peripheral blood plasma, and b is the graph of the dynamic change in the content of IGF-1 in porcine peripheral blood plasma. On the 42nd and 56th days after mixing and feeding, the content of EGF in the porcine peripheral blood plasma of the Po1h-EGF / Ghrelin-IGF group was significantly higher than that of the blank control group (PBS) (P<0.05); on the 28th and 42nd days after mixing and feeding, the content of IGF-1 in the porcine peripheral blood plasma of the Po1h-EGF / Ghrelin-IGF group was extremely significantly higher than that of the blank control group (PBS) (P<0.01).

[0153] In summary, the present invention constructs a fusion gene of porcine EGF, Ghrelin and IGF-1, integrates it into the eukaryotic expression system of Yarrowia lipolytica to obtain a fusion protein, and then further explores the immunobiomodulatory effect of the fusion protein in vitro and the anti-infective activity of the recombinant yeast in mice and its biological effects of improving the immune level and promoting growth and development in pigs. It is found that the fusion-expressed porcine EGF, Ghrelin and IGF-1 can significantly enhance the immune barrier function of the animal digestive tract mucosa, improve its systemic immune level and antibacterial infection ability, and have good biological effects of promoting animal growth and development.

[0154] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A fusion protein co-expressing porcine EGF, Ghrelin and IGF-1, characterized in that, Comprising porcine EGF, Ghrelin and IGF-1, the fusion protein is as follows (a1) or (a2): (a1) Composed of the amino acid sequence shown in SEQ ID NO:1; (a2) Obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence described in (a1).

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein as claimed in claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleic acid molecule is as follows (bI): (b1) Composed of the nucleotide sequence shown in SEQ ID NO:

2.

4. A biological material, characterized in that, The biological material is any one of the following (c1) to (c6): (c1) An expression cassette containing the nucleic acid molecule as claimed in claim 2 or 3; (c2) A recombinant vector containing the nucleic acid molecule as claimed in claim 2 or 3; (c3) A transgenic cell line containing the nucleic acid molecule as claimed in claim 2 or 3; (c4) A recombinant microorganism containing the nucleic acid molecule as claimed in claim 2 or 3; (c5) The culture product of the transgenic cell line; (c6) The fermentation product of the recombinant microorganism.

5. An application, characterized in that, Using the fusion protein as claimed in claim 1, the nucleic acid molecule as claimed in claim 2 or 3, or the biological material as claimed in claim 4 in the preparation of a product for enhancing the immune ability of animals; the animals are pigs or mice.

6. The application according to claim 5, characterized in that, The enhancement of the immune ability of animals is at least one of the following (d1) to (d6): (d1) Promoting effector target cell immunity and / or humoral immunity; (d2) Promoting the establishment of the immune barrier of effector target animals; (d3) Promoting animal development and growth; (d4) Promoting the increase of animal immune cells; (d5) Promoting animal cell immunity and humoral immunity; (d6) Resisting pathogenic microorganism infection; The effector target animals are pigs and mice, the target cells are small intestinal mucosal epithelial cells; the immune cells are lymphocytes; the pathogenic microorganisms are Salmonella typhimurium and / or Staphylococcus aureus.

7. A product, characterized in that, Comprising the fusion protein as claimed in claim 1, the nucleic acid molecule as claimed in claim 2 or 3, or the biological material as claimed in claim 4; the product is a vaccine.

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