A parasitic protein peptide with immunopotentiating effect and its application

Abstract

The present invention relates to the identification and application of a Haemonchus contortus protein peptide with immunomodulatory effects. The present invention has identified a parasite protein peptide that can be used as an immune enhancer, and its amino acid sequence is shown in SEQ ID NO.4. This polypeptide can up-regulate the expression of IL-12, IFN-γ, IL-17, IL-6, IL-1 and TNF-α in mice and goats, inhibit the expression of IL-4 and TGF-β, stimulate the Th1 and Th17 immune responses in animals, and inhibit the Th2 and Treg immune responses. When this parasite protein peptide is used in combination with a foot-and-mouth disease vaccine, it shows good immune enhancement effects and has potential application prospects. This parasite protein peptide has the advantages of being easy to prepare, high in yield, good in stability, and low in production cost.

Description

Technical Field

[0001] The present invention belongs to the technical fields of veterinary immunology and molecular biology, and relates to a parasite protein peptide with immune-enhancing effects and its applications. Background Art

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

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

[0004] To screen for peptide segments in the ES-15 protein that can stimulate the host immune response without immunosuppressive effects, the inventors identified the ES15-1 peptide segment through software analysis and animal experiments, and found that this peptide segment can up-regulate the expression of cytokines such as IL-12, IFN-γ, IL-17, IL-1, IL-6, and TNF-α in the host, indicating that this peptide segment has good immune regulatory effects. To further apply this peptide segment, it was used in combination with the foot-and-mouth disease vaccine, and the potential of this parasite protein peptide segment 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 with immune-enhancing effects.

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

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

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] The Haemonchus contortus polypeptide ES15-1 has an amino acid sequence as shown in SEQ ID NO.1.

[0010] The said Haemonchus contortus ES15-1 peptide segment is prepared by the following method: Using the recombinant HcES-15 plasmid stored in the laboratory as a template, PCR amplification is carried out with the primers shown in SEQ ID NO.8 and SEQ ID NO.9, and the obtained gene fragment is inserted into the pET-32a vector to obtain the recombinant expression plasmid pET32a-ES15-1 containing the ES15-1 gene; this plasmid is transformed into Escherichia coli for induced expression and separation and purification to obtain the said Haemonchus contortus polypeptide ES15-1.

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

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

[0013] (A1) Preparing an animal immune enhancer;

[0014] (A2) Preparing nanoparticles with immune enhancing effects.

[0015] As a preference of the present invention, the said animals are selected from mice, goats or pigs.

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

[0017] (B1) In the preparation of recombinant Haemonchus contortus polypeptide ES15-1;

[0018] (B2) Preparing an animal immune enhancer;

[0019] (B3) Preparing nanoparticles with immune enhancing effects.

[0020] As a preference of the present invention, the said animals are selected from mice, goats or pigs.

[0021] A kind of nanoparticle with immune enhancing effects, which is the Haemonchus contortus polypeptide ES15-1 wrapped by PLGA.

[0022] The preparation method of the said nanoparticles comprises the following steps:

[0023] Dissolve PLGA in dichloromethane to prepare a 3%-5% PLGA solution, and add PVA and polypeptide ES15-1 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; form a primary emulsion by stirring and ultrasonic treatment; add 5% PVA solution equal to the amount of PVA solution added for the first time to the primary emulsion, and form a double emulsion by ultrasonic treatment; after the organic solvent in the double emulsion is completely volatilized, perform ultracentrifugation, dissolve the precipitate in pure water, and freeze-dry to obtain nanoparticles loaded with recombinant polypeptide ES15-1.

[0024] Preferably, it includes the following steps: dissolve PLGA in dichloromethane to prepare a 5% solution, and add 2 mL of 5% PVA and 5 mg of recombinant ES15-1 per 1 mL of 5% PLGA solution; form a primary emulsion by stirring and ultrasonic treatment; add 2 mL of 5% PVA to the primary emulsion, and form a double emulsion by ultrasonic treatment; after the organic solvent in the double emulsion is completely volatilized, perform ultracentrifugation, dissolve the precipitate in 2 mL of pure water, and freeze-dry to obtain nanoparticles loaded with recombinant polypeptide ES15-1.

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

[0026] Any one 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 preference of the present invention, the animals are selected from mice, goats or pigs.

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

[0031] It is found that the excretory-secretory protein ES-15 of Haemonchus contortus has multiple immunomodulatory effects on goats, and it is screened that the ES15-1 fragment among them has 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 more persistently.

[0033] Beneficial effects:

[0034] 1. Peptide segments with immune-enhancing effects are screened out by segmental expression, and have good immunomodulatory effects.

[0035] 2. Compared with natural proteins, recombinant proteins have high expression levels and are easily obtained.

[0036] 3. The recombinant ES15-1 has good safety.

[0037] 4. The recombinant protein ES15-1 can improve the immune protection effect of vaccines. Description of the Drawings

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

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

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

[0041] M: DNA Marker 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 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 transcriptional levels of cytokines such as IL-17

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

[0047] Figure 5 Scanning electron micrograph of PLGA-coated recombinant ES15-1

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

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

[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 injecting goats with PLGA-ES15-1

[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 Immunopotentiating effect of PLGA-ES15-1 on foot-and-mouth disease vaccine Detailed implementation manners

[0056] Basic materials:

[0057] 1. Recombinant HcES-15 plasmid (pET32a-ES15): Obtained by using RT-PCR method to obtain the open reading frame of ES-15 according to the coding gene of Haemonchus contortus ES-15 protein (GenBank accession number: AY821552.1) and cloning it between the BamH I and EcoR I restriction enzyme cleavage sites 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 coding gene sequence of 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 in Jiangning District, Nanjing, and raised in the barrier animal house of Nanjing Agricultural University. 12 goats, 5 months old, purchased from Laian County Source Family Farm and raised in the animal house of Nanjing Agricultural University.

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

[0060] 4. Tool Enzymes and Reagents: Restriction endonucleases BamH I, Hind III, and DNA Marker were purchased from Nanjing Novozymes Biotech Co., Ltd.; PrimeSTAR® Max DNA Polymerase was purchased from Takara Bio Inc.; Plasmid Extraction Kit and Agarose Gel Recovery Kit were products of E.Z.N.A .™ Company; BCA Protein Quantification Assay Kit was a product of Thermo Company in the United States; HisTrap ™ FF Protein Affinity Chromatography Column was purchased from GE Company in the United States; Poly(lactic-co-glycolic acid) (PLGA) was purchased from Merck Sigma-Aldrich Biotech Company; 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 interferon γ (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 interferon γ (IFN-γ) ELISA Kit, Goat interleukin 1β (IL-1β) ELISA Kit, Goat transforming growth factor β1 (TGF-β1) ELISA Kit were purchased from Nanjing Jin Yibai Biotech Co., Ltd.

[0061] 5. Main Instrumentation and Equipment: PCR Amplifier (TaKaRa Company), Desktop Refrigerated Centrifuge (Eppendorf), Electric Pressure Steam Sterilizer (Shanghai Shen'an Medical Instrument Factory), Ultrasonic Disruptor (Ningbo Xinzhi Scientific Research Instrument Research Institute); Gel Imaging System, Protein Electrophoresis System, and Microplate Reader (Bio-Red).

[0062] Example 1 Preparation of Parasite Protein Peptides

[0063] 1.1 Synthesis of Primers

[0064] The protein characteristics of Haemonchus contortus ES-15 protein were analyzed using DNAstar software ( Figure 1),(divided into three peptide segments: ES15-1, ES15-2, and ES15-3. According to the coding gene of ES-15 protein in GenBank (AY821552.1), upstream 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 as shown in SEQ IDNO.4, which encodes the peptide segment ES15-1, and its amino acid sequence is as shown in SEQ ID NO.1, consisting of 47 amino acids. The amino acid sequence of ES15-2 is as shown in SEQ ID NO.2, consisting of 46 amino acids. The amino acid sequence of ES15-3 is as shown in SEQID NO.3, consisting 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 for PCR amplification of ES15-1, 2, and 3 genes. 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, and was thoroughly mixed. Pre-denaturation was carried out at 94 °C for 1 min on a PCR instrument; denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 5 s, for 35 cycles; extension at 72 °C for 1 min.)

[0067] Take 50 μL of the above-obtained PCR product, electrophorese it on a 2.5% agarose gel, cut the agarose gel at the position of the target band under ultraviolet light, and recover and purify the target fragment using the gel recovery kit from TaKaRa Biotechnology (Dalian) Co., Ltd. according to the instructions. The target gene and pET-32a plasmid were double-digested with BamH Ⅰ and Hind III respectively, and the target gene and the large fragment of pET-32a were recovered again and ligated at 16 °C for 2 h. The ligation product was transformed into competent Escherichia coli BL21, positive clone bacteria were picked, plasmids were extracted, and double-digestion with BamH Ⅰ and Hind III was used for identification.)

[0068] 1.3 Purification of the expression product ( Figure 3 )

[0069] The recombinant bacteria were cultured until OD 600When it is equal to 0.6, after inducing with 1 mM IPTG for 5 h, the bacterial cells were collected by centrifugation at 8000 g for 5 min, and the bacterial cells were resuspended with about 40 ml of supernatant Binding Buffer. The ultrasonic intensity was 600 W, working for 2 s and interval for 3 s, and disrupted for 25 min. The disrupted suspension was centrifuged at 8000 g and 4 °C for 20 min, and the supernatant was taken. The samples were prepared after filtering with 0.45 μm and 0.22 μm filter membranes respectively.

[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 chromatography column was washed with 5 - 10 column volumes of Binding Buffer at a flow rate of 2 mL / min. Then the target protein was eluted with Elution Buffer at a flow rate of 0.5 mL / min, and polypeptides ES15 - 1, ES15 - 2, and ES15 - 3 were obtained respectively.

[0071] Example 2. Screening of peptides with immune - enhancing effects

[0072] 2.1 Isolation of goat PBMC cells

[0073] (1) 50 mL of goat blood was collected using a vacuum anticoagulant (sodium citrate) blood collection tube.

[0074] (2) The anticoagulated blood was mixed with PBS in equal proportion under sterile conditions.

[0075] (3) 5 mL of the mixed solution in (2) was slowly added along the tube wall to a centrifuge tube containing 5 mL of lymphocyte separation solution.

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

[0077] (5) The cells collected in (4) were washed 2 times with about 5 - fold volume of PBS (pH 7.4), and centrifuged at room temperature for 15 min (1700 rpm) each time.

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

[0079] (7) The cells were resuspended with RPMI 1640 medium and the cell concentration was adjusted to 5×10 6 cells / mL, and 1% penicillin - streptomycin double antibody and 10% fetal bovine serum were added.

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

[0081] Spread 1 mL of PBMC (5×10 6 cells) on a 24-well cell culture plate, add polypeptides (ES5-1, 2, 3) with a final concentration of 40 μg / mL respectively, and set up blank control, PBS and empty vector protein control, and incubate at 37 °C for 24 h. Collect the cells, add them to a centrifuge tube, wash 3 times with PBS, centrifuge at 2000 rpm for 5 min each time, and use the cell pellet for subsequent RNA extraction.

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

[0083] Extract the total RNA of PBMC, use a reverse transcription kit with gDNA removal function to amplify and obtain cDNA. Detect cytokines such as IL-17, IFN-γ, IL-4, IL-5, IL-13, IL-9, IL-21, IL-22, TGF-β, IL-10 by real-time quantitative PCR. The quantitative PCR reaction system is as follows: 1.0 μL of cDNA, 5 μL of SYBR Green ProTaq HS premix, 0.2 μL of upstream primer (10 pM), 0.2 μL of downstream primer (10 pM), supplement ddH2O to 10 μL, and mix well. The reaction procedure is: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, for 35 cycles. Use the 2 -ΔΔCt method to analyze the changes in the transcriptional levels of cytokines.

[0084] Results ( Figure 4 ) found that the ES15-1 peptide segment could significantly increase the levels of cytokines such as IL-17, IFN-γ, IL-4, IL-13, indicating that the polypeptide ES15-1 has an immune-enhancing effect on the immune responses of goat Th17, Th1, Th2 and other subtypes. The polypeptide ES15-2 showed an inhibitory effect on Th17 and Th1, ES15-3 showed a promoting effect on the immune response of Treg, and ES15-2 and ES15-3 generally showed immunosuppression.

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

[0086] 3.1 Solution preparation

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

[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 and prepare a 5% PLGA solution.

[0090] ⑵ Slowly add 2 mL of 5% PVA dropwise to the dissolved PLGA while vortexing. Vortex for 1 min; under ice bath conditions, sonicate for 5 min (ultrasonic power 40 W, sonication for 5 s, interval for 5 s).

[0091] ⑶ Slowly add 5 mg of recombinant polypeptide ES15-1 dropwise while vortexing. Vortex for 1 min; under ice bath conditions, sonicate for 3 min (ultrasonic power 40 W, sonication for 5 s, interval for 5 s) to form a milky white primary emulsion.

[0092] ⑷ Add 2 mL of 5% PVA to the primary emulsion while vortexing. Sonicate under ice bath for 5 min (ultrasonic power 40 W, sonication for 5 s, interval for 5 s) to form a double emulsion.

[0093] ⑸ Place the sonicated double emulsion in a fume hood and stir while volatilizing until the organic solvent is completely volatilized.

[0094] ⑹ Centrifuge at 30,000 r / min for 30 min at 4℃; after centrifugation, collect the supernatant and precipitate respectively.

[0095] ⑺ Measure the volume of the supernatant and detect the protein content in the supernatant using a BCA protein quantification kit to calculate the encapsulation efficiency of PLGA-encapsulated recombinant protein.

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

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

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

[0099] Take out a small amount of freeze-dried powder of PLGA-ES15-1 and PLGA-pET-32a tag protein and observe it with a scanning electron microscope.

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

[0101] Measure the volume of the supernatant collected after ultracentrifugation, detect the protein content in the supernatant using a BCA protein assay kit, and calculate the encapsulation efficiency according to the following formula:

[0102]

[0103] Weigh the total mass of the nanoparticles obtained after lyophilization using an electronic balance, and calculate the protein loading amount 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 4- to 6-week-old mice were divided into 2 groups, with 30 mice in each group. Dissolve the lyophilized powders of PLGA-ES15-1 and PLGA-pET-32a tag protein with sterile PBS, and subcutaneously inject 100 μg of the protein into each mouse.

[0108] 4.2 Collection of serum samples

[0109] After protein injection, randomly select 5 mice from each group every week to collect whole blood from the eyeballs of the mice. Centrifuge at 3000 g at 4 °C for 10 min, collect the supernatant, and store it 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.), detect the contents of cytokines such as IL-12, IFN-γ, IL-4, TGF-β, IL-17, IL-6, IL-1, and TNF-α in the sera of mice from 1 w to 6 w 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 sera at different times

[0113]

[0114] Table 2 Results of ELISA detection of IFN-γ (pg / mL) in mouse sera at different times

[0115]

[0116] Table 3 Results of ELISA detection of IL-4 (pg / mL) in mouse sera at different times

[0117]

[0118] Table 4 Results of ELISA detection of TGF-β (pg / mL) in mouse serum at different times

[0119]

[0120] Table 5 Results of ELISA detection of IL-17 (pg / mL) in mouse serum at different times

[0121]

[0122] Table 6 Results of ELISA detection of IL-6 (pg / mL) in mouse serum at different times

[0123]

[0124] Table 7 Results of ELISA detection of IL-1 (pg / mL) in mouse serum at different times

[0125]

[0126] Table 8 Results of ELISA detection of TNF-α (pg / mL) in mouse serum at different times

[0127]

[0128] As can be seen from Tables 1-8, this peptide segment can up-regulate the expression of IL-12, IFN-γ, IL-17, IL-1, IL-6, TNF-α and down-regulate the expression of cytokines such as TGF-β and IL-4 in mice, indicating that this peptide segment has good immunomodulatory effects.

[0129] Example 5 Immunomodulatory effect of recombinant ES15-1 on goats( Figure 7 )

[0130] Twelve 5-month-old goats, 6 in each group, were used. The freeze-dried powders of PLGA-ES15-1 and PLGA-pET-32a tag protein were dissolved in sterile PBS, and each goat was subcutaneously injected with a dose of 500 μg of protein. The second immunization was carried out 2 weeks after the first immunization.

[0131] Goats were bled through the jugular vein to collect non-anticoagulated whole blood, centrifuged at 3000 g at 4 °C for 10 min, the supernatant was collected, aliquoted and stored at -80 °C. Blood samples were collected before immunization as samples at 0 w, and blood was collected and serum was separated at 1 w, 2 w, and 3 w after immunization.

[0132] 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, and the results are shown in Table 9-16.

[0133] Table 9 Results of ELISA detection of IL-12 (pg / mL) in goat serum at different times

[0134]

[0135] Table 10 Results of ELISA detection of IFN-γ (pg / mL) in goat serum at different times

[0136]

[0137] Table 11 Results of ELISA detection of IL-4 (pg / mL) in goat serum at different times

[0138]

[0139] Table 12 Results of ELISA detection of TGF-β (pg / mL) in goat serum at different times

[0140]

[0141] Table 13 Results of ELISA detection of IL-17 (pg / mL) in goat serum at different times

[0142]

[0143] Table 14 Results of ELISA detection of IL-6 (pg / mL) in goat serum at different times

[0144]

[0145] Table 15 Results of ELISA detection of IL-1 (pg / mL) in goat serum at different times

[0146]

[0147] Table 16 Results of ELISA detection of TNF-α (pg / mL) in goat serum at different times

[0148]

[0149] As can be seen from Table 9-16, it was found that the ES15-1 peptide segment could up-regulate the expression of cytokines such as goat IFN-γ, IL-12, IL-17, IL-1, IL-6, and TNF-α, indicating that this peptide segment has good immunomodulatory effects.

[0150] Example 6 Immunopotentiating Effect of Recombinant ES15-1 on Foot-and-Mouth Disease Vaccine

[0151] Eight 80-day-old pigs were randomly divided into 2 groups, with 4 pigs in each group, namely the control group and the 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 immunopotentiator). The experimental group dissolved PLGA-ES15-1 with PBS, and each pig was subcutaneously injected with 300 μg, and at the same time, foot-and-mouth disease vaccine antigen (commercial foot-and-mouth disease vaccine, without a commercial immunopotentiator) was injected. Porcine sera were collected before injection (day 0) and after injection (day 14), and the titers of antibodies against foot-and-mouth disease virus A antigen in the sera were detected using an ELISA kit. It was found that the antibody titer of the parasite protein peptide ES15-1 group was 9.0, which was equivalent to the immunization effect of the commercial immunopotentiator 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. Use of the Haemonchus contortus polypeptide ES15-1 according to claim 1 in the preparation of an immune adjuvant.

5. Use of the gene according to claim 2 in preparing an immune adjuvant.

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

7. Use of the nanoparticles according to claim 6 in preparing an immune adjuvant for swine foot-and-mouth disease vaccine.

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