Parasite protein peptide with immunological enhancement effect and application thereof

Abstract

The invention relates to identification and application of haemonchus contortus protein peptide with an immunomodulatory effect. The invention identifies a parasite protein peptide capable of being used as an immunopotentiator, and the amino acid sequence of the parasite protein peptide is shown as SEQ ID NO.4. The polypeptide can up-regulate the expression of IL-12, IFN-gamma, IL-17, IL-6, IL-1 and TNF-alpha of mice and goats and inhibit the expression of IL-4 and TGF-beta, has a stimulation effect on Th1 and Th17 type immunoreactions of animals, and has an inhibition effect on Th2 and Treg type immunoreactions. The parasite protein peptide is combined with a foot-and-mouth disease vaccine for use, shows a relatively good immune enhancement effect and has a potential application prospect. The parasitic protein peptide has the advantages of easiness in preparation, high yield, good stability, low production cost and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary immunology and molecular biology, and relates to a parasite protein peptide with immune enhancement effect and application thereof. Background Art

[0002] Parasites are a common type of pathogens that have acquired the ability to adapt to and rely on their hosts during the long process of evolution. The establishment of parasitic life in the host is the result of the interaction between the parasite and the host immune system. The host attempts to prevent parasitic infection through immune response, while the parasite evades the attack of the host immune system through various mechanisms. During parasitic infection, reducing immunosuppressive effects or increasing immunostimulatory effects can disrupt the balance between "parasite-host", thereby improving the host's ability to resist infection.

[0003] Haemonchus contortus is a nematode of the genus Haemonchus in the family Trichodinae. It parasitizes in the abomasum of ruminants such as cattle and sheep, sucks blood, and causes anemia in animals. Studies have found that during infection with Haemonchus contortus, a variety of excretory secretory proteins (ESPs) can bind to host mononuclear cells (PBMCs). Further research on the ES-15 protein found that the ESP can promote goat PBMCs to produce cytokines such as IL-17, thereby inducing the host to produce anti-insect immunity; but at the same time, the protein can also inhibit the expression of host cytokines such as IFN-γ, showing an immunosuppressive effect. Similar results were also found in excretory secretory proteins such as ARF1 and ES-24 of Haemonchus contortus. These results indicate that Haemonchus contortus regulates the host immune system through its parasite proteins, so that the host immune system is not strongly activated, which is conducive to the parasitism of the parasite.

[0004] In order to screen peptides in ES-15 protein that can stimulate host immune response without immunosuppression, the inventors identified the ES15-1 peptide through software analysis and animal experiments, and found that the peptide can upregulate the expression of host IL-12, IFN-γ, IL-17, IL-1, IL-6, TNF-α and other cytokines, indicating that the peptide has a good immunomodulatory effect. In order to further apply the peptide, it was used in combination with foot-and-mouth disease vaccine, and the potential of the parasite protein peptide as an animal vaccine adjuvant was analyzed. Summary of the invention

[0005] The purpose of the present invention is to provide a parasite protein peptide having an immunoenhancing effect.

[0006] Another object of the present invention is to provide the application of the parasite protein peptide.

[0007] Another object of the present invention is to provide nanoparticles prepared from the parasite protein peptide and applications thereof.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] The amino acid sequence of the Haemonchus contortus polypeptide ES15-1 is shown in SEQ ID NO.1.

[0010] The ES15-1 peptide fragment of the Haemonchus contortus is prepared by the following method: using the recombinant HcES-15 plasmid stored in the laboratory as a template, performing PCR amplification with primers shown in SEQ ID NO.8 and SEQ ID NO.9, obtaining a gene fragment and inserting it into the pET-32a vector to obtain a recombinant expression plasmid pET32a-ES15-1 containing the ES15-1 gene; transforming the plasmid into Escherichia coli to induce expression and separating and purifying to obtain the ES15-1 peptide fragment of the Haemonchus contortus.

[0011] The gene encoding the Haemonchus contortus polypeptide ES15-1 preferably has a nucleotide sequence as shown in SEQ ID NO.4.

[0012] Any one or more of the following applications of the Haemonchus contortus polypeptide ES15-1:

[0013] (A1) preparing animal immune enhancers;

[0014] (A2) Preparation of nanoparticles with immune-enhancing effects.

[0015] As a preferred embodiment of the present invention, the animal is selected from mice, goats or pigs.

[0016] Any one or more of the following applications of the gene encoding the Haemonchus contortus polypeptide ES15-1:

[0017] (B1) Preparation of recombinant Haemonchus contortus polypeptide ES15-1;

[0018] (B2) Preparation of animal immunopotentiators;

[0019] (B3) Preparation of nanoparticles with immune-enhancing effects.

[0020] As a preferred embodiment of the present invention, the animal is selected from mice, goats or pigs.

[0021] The invention discloses a nanoparticle with immune enhancement effect, which is PLGA-coated Haemonchus contortus polypeptide ES15-1.

[0022] The method for preparing the nanoparticles comprises the following steps:

[0023] PLGA is dissolved in dichloromethane to prepare a 3%-5% PLGA solution, and PVA and polypeptide ES15-1 are added in an amount of 1-2 mL of 5% PVA solution and 3-7 mg of polypeptide ES15-1 per 1 mL of the PLGA solution; a primary emulsion is formed after stirring and ultrasonic treatment; a 5% PVA solution equal to the first added PVA solution is added to the primary emulsion, and ultrasonic treatment is used to form a double emulsion; after the organic solvent in the double emulsion is completely evaporated, ultracentrifugation is performed, and the precipitate is dissolved in pure water and freeze-dried to obtain nanoparticles loaded with recombinant polypeptide ES15-1.

[0024] Preferably, the method comprises the following steps: dissolving PLGA in dichloromethane to prepare a 5% solution, adding 2 mL of 5% PVA and 5 mg of recombinant ES15-1 to every 1 mL of 5% PLGA solution; forming a primary emulsion by stirring and ultrasonic treatment; adding 2 mL of 5% PVA to the primary emulsion and ultrasonic treatment to form a double emulsion; performing ultracentrifugation after the organic solvent in the double emulsion is completely evaporated, dissolving the precipitate with 2 mL of pure water, and freeze-drying, thereby obtaining nanoparticles loaded with the recombinant polypeptide ES15-1.

[0025] The ultrasonic treatment conditions are as follows: ultrasonic power 40W, ultrasonic 5s, interval 5s.

[0026] Any of the following applications of the nanoparticles:

[0027] (C1) In the preparation of animal immune enhancers;

[0028] (C2) Preparation of animal vaccines.

[0029] As a preferred embodiment of the present invention, the animal is selected from mice, goats or pigs.

[0030] The present invention is based on the following discoveries:

[0031] It was found that the excretory secretory protein ES-15 of Haemonchus contortus has multiple immunomodulatory effects on goats, and the ES15-1 fragment was screened out to have the effect of stimulating the host immune system.

[0032] The peptide segment coated with PLGA has a good sustained-release effect in animals and can stimulate the immune system for a longer period of time.

[0033] Beneficial effects:

[0034] 1. Through segmented expression, peptides with immune-enhancing effects are screened out, which have good immune-regulating effects.

[0035] 2. Compared with natural proteins, recombinant proteins have higher expression levels and are easier to obtain.

[0036] 3. Recombinant ES15-1 has good safety.

[0037] 4. The recombinant protein ES15-1 can enhance the immune protection effect of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Protein characteristics analysis of ES15-1, 2, and 3 peptides

[0039] 1: ES15-1; 2: ES15-2; 3: ES15-3

[0040] Figure 2 Double enzyme digestion identification of recombinant expression plasmids pET32a-ES15-1, 2, and 3

[0041] M: DNA Mark DL2000; 1: pET32a-ES15-1; 2: pET32a-ES15-2; 3: pET32a-ES15-3

[0042] Figure 3 Purification of recombinant ES15-1, 2, and 3

[0043] M: protein standard molecular weight; 1: purified recombinant ES15-1; 2: purified recombinant ES15-2; 3: purified recombinant ES15-3

[0044] Figure 4 The immunomodulatory effects of ES15-1, 2, and 3 peptides on different Th subtypes

[0045] A: Effects of pET-32a tag protein, PBS and water (BLANK) on the transcription levels of IL-17 and other cytokines

[0046] B: Effects of ES15-1, 2, and 3 peptides on the transcriptional levels of IL-17 and other cytokines

[0047] Figure 5 The scanning electron microscopy image of recombinant ES15-1 coated with PLGA

[0048] A: PLGA-pET-32a tagged protein control; B: PLGA-ES15-1

[0049] Figure 6 Expression levels of various cytokines in mice after PLGA-ES15-1 injection

[0050] A: IL-12; B: IFN-γ; C: IL-4; D: TGF-β;

[0051] E: IL-17; F: IL-6; G: IL-1; H: TNF-α

[0052] Figure 7 Expression levels of various cytokines after PLGA-ES15-1 injection in goats

[0053] A: IL-12; B: IFN-γ; C: IL-4; D: TGF-β;

[0054] E: IL-17; F: IL-6; G: IL-1; H: TNF-α

[0055] Figure 8 The immunopotentiation effect of PLGA-ES15-1 on foot-and-mouth disease vaccine DETAILED DESCRIPTION

[0056] Base Materials:

[0057] 1. Recombinant HcES-15 plasmid (pET32a-ES15): Based on the gene encoding the ES-15 protein of Haemonchus contortus (GenBank accession number AY821552.1), the open reading frame of ES-15 was obtained by RT-PCR and cloned into the restriction sites between BamH Ⅰ and EcoR I of the pET32a(+) vector. The primer sequences used for RT-PCR amplification are shown in SEQ ID NO.6 and SEQ ID NO.7, and the gene sequence encoding the ES-15 protein is shown in SEQ ID NO.5.

[0058] 2. Experimental animals: 60 BALB / c mice, 4-6 weeks old, purchased from Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing, and kept in the barrier animal room of Nanjing Agricultural University. 12 goats, 5 months old, purchased from Yuanyuan Family Farm, Lai'an County, and kept in the animal room of Nanjing Agricultural University.

[0059] 3. PCR primers: The sequences of the upstream and downstream primers (F, R) for amplifying ES15-1 are listed in SEQ ID NO.8 and SEQ ID NO.9, respectively; the sequences of the upstream and downstream primers (F, R) for amplifying ES15-2 are listed in SEQ ID NO.10 and SEQ ID NO.11, respectively; the sequences of the upstream and downstream primers (F, R) for amplifying ES15-3 are listed in SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0060] 4. Enzymes and reagents: The endonucleases BamH Ⅰ, Hind Ⅲ and DNA Marker were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; PrimeSTAR® Max DNA Polymerase was purchased from Takara Bio; the plasmid extraction kit and agarose gel recovery kit were purchased from EZNA Biosciences, USA. .™ The company's products, BCA protein quantitative analysis kit is a product of Thermo Company in the United States; HisTrap ™ FF protein affinity chromatography column was purchased from GE, USA; poly(lactic-co-glycolic acid, PLGA) was purchased from Merck Sigma-Aldrich Biotechnology Co., Ltd.; mouse interleukin 17 (IL-17) ELISA kit, mouse interleukin 12 (IL-12) ELISA kit, mouse tumor necrosis factor α (TNF-α) ELISA kit, mouse interleukin 6 (IL-6) ELISA kit, mouse interleukin 4 (IL-4) ELISA kit, mouse gamma interferon (IFN-γ) ELISA kit, mouse interleukin 1β (IL-1β) ELISA kit, mouse transforming growth factor β1 (TGF-β1 ) ELISA kit, goat interleukin 17 (IL-17) ELISA kit, goat interleukin 12 (IL-12) ELISA kit, goat tumor necrosis factor α (TNF-α) ELISA kit, goat interleukin 6 (IL-6) ELISA kit, goat interleukin 4 (IL-4) ELISA kit, goat gamma interferon (IFN-γ) ELISA kit, goat interleukin 1β (IL-1β) ELISA kit, goat transforming growth factor β1 (TGF-β1) ELISA kit were purchased from Nanjing Jinyibai Biological Co., Ltd.

[0061] 5. Main instruments and equipment: PCR amplifier (TaKaRa), desktop refrigerated centrifuge (Eppendorf), electric pressure steam sterilizer (Shanghai Shen'an Medical Equipment Factory), ultrasonic disruptor (Ningbo Xinzhi Scientific Research Instrument Institute); gel imaging system, protein electrophoresis system and microplate reader (Bio-Red).

[0062] Example 1 Preparation of parasite protein peptides

[0063] 1.1 Primer synthesis

[0064] The protein characteristics of the ES-15 protein of Haemonchus contortus were analyzed using DNAstar software ( Figure 1), divided into three peptide segments, namely ES15-1, ES15-2 and ES15-3. According to the coding gene of ES-15 protein in GenBank (AY821552.1), the upstream primers and downstream primers for amplifying ES15-1, 2 and 3 were designed using PrimerPrimer 5.0 software. The primer sequences for amplifying ES15-1 are shown in SEQ ID NO.8 and SEQ ID NO.9, the primer sequences for amplifying ES15-2 are shown in SEQ ID NO.10 and SEQ ID NO.11, and the primer sequences for amplifying ES15-3 are shown in SEQ ID NO.12 and SEQ ID NO.13. Among them, the gene sequence of ES15-1 is shown in SEQ ID NO.4, and the gene encodes the peptide segment ES15-1, whose amino acid sequence is shown in SEQ ID NO.1, and consists of 47 amino acids. The amino acid sequence of ES15-2 is shown in SEQ ID NO.2, and consists of 46 amino acids. The amino acid sequence of ES15-3 is shown in SEQ ID NO.3, and consists of 44 amino acids.

[0065] 1.2 Gene cloning and expression of ES15-1, 2, and 3 ( Figure 2 )

[0066] The following reaction system was used to amplify ES15-1, 2, and 3 genes by PCR. The reaction system included 1.0 μL of HcES-15 plasmid template, 25 μL of PrimeSTAR Max Premix (2X), 2 μL of upstream primer F (10 pM), 2 μL of downstream primer R (10 pM), and 20 μL of sterilized ultrapure water. The mixture was mixed thoroughly. The reaction was pre-denatured at 94°C for 1 min, denatured at 98°C for 10 s, annealed at 55°C for 5 s, and extended at 72°C for 5 s for 35 cycles, and extended at 72°C for 1 min.

[0067] Take 50 μL of the PCR product obtained above, run it on a 2.5% agarose gel, cut the agarose gel at the target band under ultraviolet light, and use the gel recovery kit of Dalian Bao Biotechnology to recover and purify the target fragment. Refer to the instructions for the method. Double-digest the target gene and pET-32a plasmid with BamH Ⅰ and Hind III, respectively, recover the target gene and pET-32a large fragment again, and connect at 16℃ for 2h. Transform the ligation product into competent Escherichia coli BL21, pick the positive clones, extract the plasmid, and identify it with double-digestion of BamH Ⅰ and Hind III.

[0068] 1.3 Purification of expression products ( Figure 3 )

[0069] The recombinant bacteria were cultured to OD 600When the pH is equal to 0.6, use 1 mM IPTG to induce for 5 hours, centrifuge at 8000g for 5 minutes to collect the cells, use about 40ml of supernatant Binding Buffer to resuspend the cells, and use 600W ultrasonic intensity, work for 2s, interval for 3s, and break for 25 minutes. The suspension after breaking is centrifuged at 8000g, 4℃ for 20 minutes, and the supernatant is taken. After filtering with 0.45μm and 0.22μm filter membranes respectively, the sample preparation is completed.

[0070] The protein sample was slowly passed through a His Tag affinity chromatography column (5 mL) stored in 20% ethanol at 4°C at a flow rate of 0.5 mL / min, and the column was washed with 5 column volumes of Binding Buffer. The column was washed with 5-10 column volumes of Binding Buffer at a flow rate of 2 mL / min. The target protein was then eluted with Elution Buffer at a flow rate of 0.5 mL / min to obtain peptides ES15-1, ES15-2, and ES15-3, respectively.

[0071] Example 2. Screening of peptides with immunoenhancing effects

[0072] 2.1 Isolation of goat PBMCs

[0073] (1) Collect 50 mL of goat blood using a vacuum anticoagulated (trisodium citrate) blood collection tube.

[0074] (2) Mix the anticoagulant blood with PBS in equal proportions under sterile conditions.

[0075] (3) Pipette 5 mL of the mixed solution in (2) and slowly add it along the wall of the tube into the centrifuge tube containing 5 mL of lymphocyte separation solution.

[0076] (4) Centrifuge at room temperature (2400 rpm) for 25 min and collect the ring lymphocyte layer (the first layer is the plasma layer, the second layer is the milky white ring lymphocyte layer, the third layer is the transparent separation fluid layer, and the fourth layer is the red blood cell layer).

[0077] (5) Wash the cells collected in (4) twice with approximately 5 volumes of PBS (pH 7.4), centrifuging at room temperature for 15 min each time (1700 rpm).

[0078] (6) Trypan blue staining was used to determine if the cell viability was greater than 95%, and cell counting was performed.

[0079] (7) Resuspend the cells in RPMI 1640 medium and adjust the cell concentration to 5 × 10 6 / mL, and add 1% penicillin-streptomycin double antibody and 10% fetal bovine serum.

[0080] 2.2 Co-incubation of peptides with goat PBMC

[0081] 1 mL PBMC (5 × 10 6 100 cells were plated in 24-well cell culture plates, and peptides (ES5-1, 2, 3) were added at a final concentration of 40 μg / mL, and blank controls, PBS and empty carrier protein controls were set up, and incubated at 37 °C for 24 h. The cells were collected, added to a centrifuge tube, washed 3 times with PBS, and centrifuged (2000 rpm) for 5 min each time. The cell pellet was used for subsequent RNA extraction.

[0082] 2.3 Quantitative PCR detection of different types of cytokines

[0083] Total RNA of PBMC was extracted, and cDNA was amplified using a reverse transcription kit with gDNA removal function. Real-time quantitative PCR was used to detect cytokines such as IL-17, IFN-γ, IL-4, IL-5, IL-13, IL-9, IL-21, IL-22, TGF-β, and IL-10. The quantitative PCR reaction system was: cDNA 1.0μL, SYBR Green ProTaq HS premix 5μL, upstream primer (10pM) 0.2μL, downstream primer (10pM) 0.2μL, supplemented with ddH2O to 10μL, and mixed thoroughly. The reaction procedure was: pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 10s, annealing at 60℃ for 30s, and 35 cycles. 2 -ΔΔCt The changes of cytokine transcription levels were analyzed by

[0084] result( Figure 4 ) found that the ES15-1 peptide can significantly increase the levels of cytokines such as IL-17, IFN-γ, IL-4, and IL-13, indicating that the peptide ES15-1 has an immunoenhancing effect on the immune response of goats' Th17, Th1, Th2 and other subtypes. The peptide ES15-2 has an inhibitory effect on Th17 and Th1, and ES15-3 has a promoting effect on Treg immune response. ES15-2 and ES15-3 generally show immunosuppression.

[0085] Example 3. Preparation of PLGA-ES15-1 Nanoparticles

[0086] 3.1 Solution preparation

[0087] 5% PVA: Dissolve 1 g PVA in 20 mL deionized water, heat at 95°C and stir to dissolve.

[0088] 3.2 Preparation of Nanoparticles

[0089] ⑴ Weigh 50 mg of PLGA and place it in a 10 mL EP tube. Add 1 mL of dichloromethane (operate in a fume hood) to dissolve PLGA to prepare a 5% PLGA solution.

[0090] ⑵ Add 2 mL of 5% PVA dropwise to the dissolved PLGA while vortexing. Vortex and mix for 1 min. Ultrasonicate for 5 min in an ice bath (ultrasonic power 40 W, ultrasonication 5 s, interval 5 s).

[0091] ⑶ Add 5 mg of recombinant polypeptide ES15-1 dropwise while vortexing, and vortex mix for 1 min; ultrasonically disrupt for 3 min (ultrasonic power 40 W, ultrasonic 5 s, interval 5 s) in an ice bath to form a milky primary emulsion.

[0092] (4) Add 2 mL of 5% PVA to the primary emulsion while vortexing, and perform ultrasonic disruption in an ice bath for 5 min (ultrasonic power 40 W, ultrasonication 5 s, interval 5 s) to form a double emulsion.

[0093] ⑸ Place the ultrasonic emulsion in a fume hood and evaporate it while stirring until the organic solvent is completely evaporated.

[0094] (6) Centrifuge at 4℃ refrigerated ultracentrifugation at 30,000 r / min for 30 min; after centrifugation, collect the supernatant and precipitate separately.

[0095] ⑺ Measure the supernatant volume, detect the protein content in the supernatant using the BCA protein quantification kit, and calculate the embedding rate of the recombinant protein encapsulated by PLGA.

[0096] ⑻ Resuspend the precipitate obtained after ultracentrifugation with 2 mL of deionized water, place it in a 5 mL vial, place it at -80°C for 2 h, then transfer it to a vacuum freeze dryer and freeze-dry it for 24 h.

[0097] ⑼ Take out the freeze-dried sample and store the PLGA nano subunit vaccine at 4°C for later use. At the same time, use the same method to prepare PLGA nanoparticles of pET-32a tag protein.

[0098] 3.3 Electron microscopy observation of nanoparticles Figure 5 )

[0099] A small amount of PLGA-ES15-1 and PLGA-pET-32a tag protein freeze-dried powder was taken out and observed using a scanning electron microscope.

[0100] 3.4 Determination of protein embedding efficiency and protein loading in nanoparticles

[0101] Measure the volume of the supernatant collected after ultracentrifugation, detect the protein content in the supernatant using the BCA protein detection kit, and calculate the embedding rate according to the following formula:

[0102]

[0103] The total mass of the nanoparticles obtained after freeze-drying was weighed using an electronic balance, and the amount of protein loaded was calculated according to the following formula:

[0104]

[0105] Example 4. Immunomodulatory effect of recombinant ES15-1 on mice

[0106] 4.1 Subcutaneous injection of mice

[0107] Sixty mice aged 4-6 weeks were divided into two groups, with 30 mice in each group. PLGA-ES15-1 and PLGA-pET-32a tag protein freeze-dried powder were dissolved in sterile PBS, and 100 μg of protein was injected subcutaneously into each mouse.

[0108] 4.2 Collection of serum samples

[0109] After protein injection, 5 mice were randomly selected from each group every week to remove their eyeballs and collect their whole blood. The blood was centrifuged at 3000g and 4°C for 10 min, and the supernatant was collected and stored at -80°C after aliquoting.

[0110] 4.3 Effects of PLGA-ES15-1 on different types of cytokines in mice ( Figure 6 )

[0111] According to the instructions of the ELISA kit (Nanjing Jinyibai Biotechnology Co., Ltd.), the levels of cytokines such as IL-12, IFN-γ, IL-4, TGF-β, IL-17, IL-6, IL-1 and TNF-α in mouse serum were detected 1w-6w after protein injection. The results are shown in Tables 1-8.

[0112] Table 1 Results of ELISA detection of IL-12 (pg / mL) in mouse serum at different times

[0113] Table 2 Results of ELISA detection of IFN-γ (pg / mL) in mouse serum at different times

[0114]

[0115] Table 3 Results of ELISA detection of IL-4 (pg / mL) in mouse serum at different times

[0116]

[0117] Table 4 Results of ELISA detection of TGF-β (pg / mL) in mouse serum at different times

[0118]

[0119] Table 5 Results of ELISA detection of IL-17 (pg / mL) in mouse serum at different times

[0120]

[0121] Table 6 Results of ELISA detection of IL-6 (pg / mL) in mouse serum at different times

[0122]

[0123] Table 7 Results of ELISA detection of IL-1 (pg / mL) in mouse serum at different times

[0124]

[0125] Table 8 Results of ELISA detection of TNF-α (pg / mL) in mouse serum at different times

[0126]

[0127] As can be seen from Table 1-8, the peptide can upregulate the expression of IL-12, IFN-γ, IL-17, IL-1, IL-6, TNF-α, and downregulate the expression of cytokines such as TGF-β and IL-4 in mice, indicating that the peptide has a good immunomodulatory effect.

[0128] Example 5 Immunomodulatory effect of recombinant ES15-1 on goats ( Figure 7 )

[0129] Twelve goats aged 5 months, 6 in each group, were used to dissolve PLGA-ES15-1 and PLGA-pET-32a tagged protein freeze-dried powder in sterile PBS, and each goat was subcutaneously injected with 500 μg of protein. The second immunization was performed 2 weeks after the first immunization.

[0130] The goats were bled through the jugular vein to collect non-anticoagulated whole blood, centrifuged at 3000g, 4℃, for 10min, and the supernatant was collected and stored at -80℃ after aliquoting. Blood was collected before immunization as a 0w sample, and blood was collected 1w, 2w, and 3w after immunization to separate serum.

[0131] According to the instructions of the ELISA kit (Nanjing Jinyibai Biotechnology Co., Ltd.), the contents of cytokines such as IL-12, IFN-γ, IL-4, TGF-β, IL-17, IL-6, IL-1 and TNF-α in goat serum were detected. The results are shown in Tables 9-16.

[0132] Table 9 Results of ELISA detection of IL-12 (pg / mL) in goat serum at different times

[0133]

[0134] Table 10 Results of ELISA detection of IFN-γ (pg / mL) in goat serum at different times

[0135]

[0136] Table 11 Results of ELISA detection of IL-4 (pg / mL) in goat serum at different times

[0137]

[0138] Table 12 Results of ELISA detection of TGF-β (pg / mL) in goat serum at different times

[0139]

[0140] Table 13 Results of ELISA detection of IL-17 (pg / mL) in goat serum at different times

[0141]

[0142] Table 14 Results of ELISA detection of IL-6 (pg / mL) in goat serum at different times

[0143]

[0144] Table 15 Results of ELISA detection of IL-1 (pg / mL) in goat serum at different times

[0145]

[0146] Table 16 Results of ELISA detection of TNF-α (pg / mL) in goat serum at different times

[0147]

[0148] As can be seen from Tables 9-16, it was found that the ES15-1 peptide segment can upregulate the expression of cytokines such as IFN-γ, IL-12, IL-17, IL-1, IL-6, and TNF-α in goats, indicating that the peptide segment has a good immunomodulatory effect.

[0149] Example 6 Immunoenhancing Effect of Recombinant ES15-1 on Foot-and-Mouth Disease Vaccine

[0150] Eight 80-day-old pigs were randomly divided into two groups, 4 in each group, and divided into a control group and an experimental group. The control group was injected with a commercial foot-and-mouth disease vaccine (containing foot-and-mouth disease vaccine antigen and a commercial immunoenhancer). In the experimental group, PLGA-ES15-1 was dissolved in PBS, and 300 μg was injected subcutaneously into each pig, and foot-and-mouth disease vaccine antigen (commercial foot-and-mouth disease vaccine, without commercial immunoenhancer) was injected at the same time. Pig serum was collected before (0 days) and after (14 days) injection, and the titer of anti-foot-and-mouth disease virus A antigen antibodies in the serum was detected using an ELISA kit. The results showed that the antibody titer of the parasite protein peptide ES15-1 group was 9.0, which was equivalent to the immune effect of the commercial immunoenhancer group ( Figure 7 ).

Claims

1. Haemonchus contortus polypeptide ES15-1, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the Haemonchus contortus polypeptide ES15-1 according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

4.

4. Any one or more of the following uses of the Haemonchus contortus polypeptide ES15-1 according to claim 1: (A1) preparing animal immune enhancers; (A2) Preparation of nanoparticles with immune-enhancing effects.

5. The use according to claim 4, characterized in that: The animal is selected from mice, goats or pigs.

6. Any of the following uses of the gene according to claim 2: (B1) Preparation of recombinant Haemonchus contortus polypeptide ES15-1; (B2) Preparation of animal immunopotentiators; (B3) Preparation of nanoparticles with immune-enhancing effects.

7. The use according to claim 6, characterized in that: The animal is selected from mice, goats or pigs.

8. A nanoparticle having an immunoenhancing effect, characterized in that: The ES15-1 polypeptide of Haemonchus contortus according to claim 1 is coated with PLGA.

9. Any one or more of the following uses of the nanoparticles according to claim 8: (C1) In the preparation of animal immune enhancers; (C2) Preparation of animal vaccines.

10. The use according to claim 9, characterized in that: The animal is selected from mice, goats or pigs.

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