Candidate antigen combinations of echinococcus granulosus and uses thereof

Abstract

The application relates to the field of biotechnology, in particular to a candidate antigen combination of Echinococcus granulosus and application thereof. In the present application, bioinformatics and immunology methods are used to screen and identify protective antigens in different stages, and a recombinant subunit vaccine candidate antigen with simple immunization procedure, good protection effect and high stability is developed, which has commercial vaccine development value. Meanwhile, compared with three immunizations in the previous research, the present application adopts a twice subcutaneous injection procedure to immunize dogs, so that the cost and difficulty are reduced, and the present application is more in line with the actual application requirements and is convenient to implement.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to candidate antigen combinations of Echinococcus granulosus and their applications. Background Technology

[0002] Echinococcus granulosus is a tapeworm that parasitizes the intestines of dogs. Its larvae can parasitize the parenchymal organs of hosts such as humans, cattle, and sheep, causing cysticercosis, an important zoonotic parasitic disease worldwide. This disease poses a serious threat to human and animal health, especially in remote areas lacking sanitation and infrastructure, causing severe economic losses and public health problems.

[0003] Dogs are the definitive host of *Echinococcus granulosus* and the main source of echinococcosis infection. The primary routes of infection for humans and animals are oral contact with dog feces carrying echinococcosis eggs, or consumption of food contaminated with these eggs, and contact with contaminated items. Currently, the prevention and control of *Echinococcus granulosus* in dogs mainly relies on the regular use of deworming medication. However, this method is difficult to implement long-term and faces problems such as drug resistance, drug residues, and environmental pollution. On the other hand, the control of intermediate hosts primarily relies on vaccination. Due to the complexity of echinococcosis epidemics and the diversity of intermediate host species and numbers, simply immunizing sheep to block transmission is insufficient. Dogs, as the main definitive host of echinococcosis, and far fewer in number than major intermediate hosts such as cattle and sheep, may offer a more cost-effective option for infection control and are a key measure to reduce disease transmission.

[0004] In recent years, research on Echinococcus granulosus antigen genes has largely focused on screening diagnostic antigens, with only a few genes used in vaccine research. Existing studies have shown that some candidate vaccines can affect egg maturation and reduce the parasite load in dogs. Among them, antigens such as the EgM family, tropomyosin, paramyosin, and 3-hydroxyacyl-CoA dehydrogenase have shown some protective effects. However, due to the low parasite reduction rate reported for candidate antigen proteins (only about 70%), they are not yet sufficient to meet the requirements for developing commercial vaccines. Summary of the Invention

[0005] In view of this, the present invention provides candidate antigen combinations for Echinococcus granulosus and their applications. The present invention uses bioinformatics and immunology methods to screen and identify protective antigens at different stages, developing recombinant subunit vaccine candidate antigens with simple immunization procedures, good protective efficacy, and high stability, possessing commercial vaccine development value. Furthermore, compared to the three immunizations used in previous studies, this study uses a two-subcutaneous injection procedure to immunize dogs, reducing the cost and difficulty of use, better meeting practical application needs, and facilitating implementation.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a candidate antigen combination for Echinococcus granulosus, the candidate antigen combination comprising EgEno protein, EgSev protein and EgCyc protein;

[0008] The EgEno protein, EgSev protein, and EgCyc protein sequentially possess the following characteristics:

[0009] (I) Amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15; or

[0010] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0011] (III) Sequences that are more than 90% homologous to the amino acid sequences shown in (I) or (II).

[0012] In some specific embodiments of the present invention, the UniProtKB accession numbers of the Severin protein, Enolase protein, and Cyclophilin protein are U6IX85, D0VLV3, and P14088, respectively.

[0013] The present invention also provides a nucleic acid molecule encoding the candidate antigen combination, the nucleic acid molecule comprising the EgENO gene, the EgSev gene and the EgCyc gene.

[0014] In some specific embodiments of the present invention, the GenBank accession numbers of the EgENO gene, EgSev gene and EgCyc gene are GU080332.1, XM_024497312.1 and AF430707.1, respectively.

[0015] The present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecule.

[0016] In some specific embodiments of the present invention, the recombinant vector includes pET series vectors, pGEX series vectors or pMAL series vectors, preferably pET32a(+).

[0017] The present invention also provides a host, including the aforementioned recombinant vector.

[0018] In some specific embodiments of the present invention, the host includes Escherichia coli BL21(DE3).

[0019] The present invention also provides primer pairs for amplifying the said nucleic acid molecules, comprising:

[0020] The primer pair used to amplify the EgENO gene has an upstream primer sequence as shown in SEQ ID NO:1 and a downstream primer sequence as shown in SEQ ID NO:2.

[0021] The primer pair used to amplify the EgSev gene has the upstream primer sequence as shown in SEQ ID NO:3 and the downstream primer sequence as shown in SEQ ID NO:4.

[0022] The primer pair used to amplify the EgCyc gene has an upstream primer sequence as shown in SEQ ID NO:5 and a downstream primer sequence as shown in SEQ ID NO:6.

[0023] This invention also provides the application of any of the following in the preparation of Echinococcus granulosus vaccine:

[0024] (I) the aforementioned candidate antigen combinations; and / or

[0025] (II) the aforementioned nucleic acid molecules; and / or

[0026] (III) the recombinant vector; and / or

[0027] (IV) The host mentioned above; and / or

[0028] (V) The primer pair described above.

[0029] This invention also provides the application of any of the following in the preparation of a kit for detecting Echinococcus granulosus:

[0030] (I) the aforementioned candidate antigen combinations; and / or

[0031] (II) the aforementioned nucleic acid molecules; and / or

[0032] (III) the recombinant vector; and / or

[0033] (IV) The host mentioned above; and / or

[0034] (V) The primer pair described above.

[0035] The present invention also provides a method for obtaining a vaccine against Echinococcus granulosus, comprising the following steps: using the primer pair described above, amplifying the nucleic acid molecule, expressing and purifying it to obtain rEgENO, rEgSev and rEgCyc, and mixing them to obtain the vaccine against Echinococcus granulosus.

[0036] In some specific embodiments of the present invention, the purification method includes Ni 2+ Affinity chromatography.

[0037] In some specific embodiments of the present invention, the mass ratio of rEgENO, rEgSev, and rEgCyc in the Echinococcus granulosus vaccine is 1:1:1.

[0038] The immunization dose of the Echinococcus granulosus vaccine ranges from 150 μg to 450 μg per dose.

[0039] The present invention also provides a vaccine against Echinococcus granulosus, comprising a vaccine prepared by the method described above.

[0040] This invention provides the following beneficial effects:

[0041] This study employed bioinformatics and immunology methods to screen and identify protective antigens at different stages, developing recombinant subunit vaccine candidate antigens with simple immunization procedures, good protective efficacy, and high stability, demonstrating commercial vaccine development value. Furthermore, compared to the three-immunization procedure used in previous studies, this study employed a two-subcutaneous injection procedure to immunize dogs, reducing costs and complexity, better meeting practical application needs, and facilitating implementation. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0043] Figure 1 The diagram shows adult worm exosome proteins; where A: distribution of unique peptide number; B: distribution of peptide length; C: distribution of protein percentage; and D: distribution of protein molecular weight.

[0044] Figure 2 Functional annotation analysis of GO protein in adult worm exosomes;

[0045] Figure 3 The diagram shows Venn, representing exosome proteins from adult worms; where A represents adult secretory proteins identified in this study; and B represents adult secretory proteins identified in previous studies.

[0046] Figure 4 The comparative transcriptome analysis included: A: analysis of the difference in gene transcription levels between the protocercariae and adults of Echinococcus granulosus; B: analysis of the major differentially expressed genes in key functional categories using multiple volcano plots; and C: GO clustering analysis of genes upregulated in adults relative to protocercariae.

[0047] Figure 5 The results of multi-omics joint analysis and antigenicity prediction are shown; among them, A: adult secretory protein set; B: protocercarial secretory protein set; C: adult-to-protocercarial upregulated gene set; D: Echinococcus granulosus protein set identified by positive serum of canine echinococcosis infection;

[0048] Figure 6 The specific bands of the candidate genes are shown; where lane M: DNA molecular quality standard (MarkerD2000); 1: EgENO; 2: EgSev; 3: EgCyc; 4: EgFABP1; 5: EgCaM; 6: EgSrp1; 7: negative control;

[0049] Figure 7 The specific bands of the recombinant protein are shown; where lane M: protein molecular weight standard marker; 1: rEgENO; 2: rEgSev; 3: rEgCyc; 4: rEgFABP1; 5: rEgCaM; 6: rEgSrp1; 7: pET-32a(+) empty vector bacteria;

[0050] Figure 8 The changes in serum-specific antibody IgG in experimental dogs were observed after initial immunization (week 0), booster immunization (week 2), and challenge immunization (week 4) with rEgENO, rEgSev, rEgCyc, rEgFABP1, rEgCaM, and rEgSrp1.

[0051] Figure 9 The changes in serum cytokines IL-2, IL-4, IL-5, IL-10 and IFN-γ in experimental dogs were observed after initial immunization (a), booster immunization (b), challenge immunization (c), and challenge immunization (d) with PBS + QuilA (A), rEgFABP1&rEgCaM&rEgSrp1 (B), and rEgENO&rEgSev&rEgCyc (C).

[0052] Figure 10 The changes in serum-specific antibody IgG in experimental dogs were observed after initial immunization (week 0), booster immunization (week 2), and challenge immunization (week 4).

[0053] Figure 11 The changes in serum cytokines IL-2, IL-4, IL-5, IL-10 and IFN-γ in experimental dogs after initial immunization (a) with PBS + QuilA (A) and rEgENO & rEgSev & rEgCyc (B), one week after booster immunization (b), one week after challenge (c) and four weeks after challenge (d) were shown.

[0054] Figure 12 The changes in serum-specific antibody IgG in experimental dogs were observed after initial immunization (week 0), booster immunization (week 2), and challenge immunization (week 4).

[0055] Figure 13The changes in serum cytokines IL-2, IL-4, IL-5, IL-10 and IFN-γ in experimental dogs after initial immunization (a) with PBS+QuilA (A) and rEgENO&rEgSev&rEgCyc (B), one week after booster immunization (b), one week after challenge (c), and four weeks after challenge (d) were shown. Detailed Implementation

[0056] This invention discloses candidate antigen combinations for *Echinococcus granulosus* and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0057] The main reagents and instruments used in this invention are as follows:

[0058] 1. Main reagents

[0059] Goat anti-canine IgG (HRP-labeled) was purchased from Beyotime Biotechnology Co., Ltd.; IPTG and Quil A (saponin) were purchased from Sigma-Aldrich; 96-well microplates were purchased from Corning; horseradish peroxidase-labeled TMB substrate chromogenic solution was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Mini Profinity Ni-charged IMAC pre-packed columns were purchased from Bio-Rad. Cytokine kits IL-2, IL-4, IL-5, IL-10, and IFN-γ ELISA kits were purchased from Solarbio.

[0060] 2. Main Instruments

[0061] NanoDrop One ultra-micro spectrophotometer (Thermo Fisher Scientific, USA); refrigerated high-speed centrifuge (Thermo Fisher Scientific, USA); micropipette (Thermo Fisher Scientific, USA); -80℃ ultra-low temperature freezer (Thermo Fisher Scientific, USA); PCR amplification instrument (Bio-Rad, USA); mini vertical electrophoresis system (Bio-Rad, USA); fully automated medium-high pressure chromatography system (Bio-Rad, USA); ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.); constant temperature incubator (Shanghai Qixin Scientific Instruments Co., Ltd.); digital pathology scanner (OLYMPUS); nucleic acid electrophoresis instrument (Liuyi Instruments), etc.

[0062] Unless otherwise specified, the candidate antigen combinations for Echinococcus granulosus provided in this invention and the raw materials and reagents used in their application are all commercially available.

[0063] The present invention will be further illustrated below with reference to the embodiments:

[0064] Example

[0065] 1. Materials and Animals

[0066] Forty-two Beagles, aged 6-12 months (half male and half female), were provided by the Dujiangyan Experimental Beagle Breeding Center of the Sichuan Musk Deer Research Institute. Prior to the experiment, parasitological testing showed no tapeworms or other intestinal parasites. During the experiment, each dog was randomly assigned to a group and strictly fed according to laboratory animal husbandry and management standards.

[0067] Liver infections caused by *Echinococcus granulosus* were collected from sheep. Cysts were aseptically isolated and identified as *Echinococcus granulosus* G1 type. Protocercariae survival was assessed using trypan blue staining. Protocercariae were cultured in vitro and subjected to canine challenge experiments, provided the cyst genotype was G1 and the protocercariae survival rate was greater than 95%.

[0068] 2. Bioinformatics Analysis

[0069] 2.1 Proteomics analysis of adult worm exosomes

[0070] Each dog was orally administered 150,000 protocercariae and euthanized 28 days later. The small intestine was completely isolated and longitudinally cut, then incubated in PBS solution at 37°C for 1 hour. After the worms detached, the supernatant was discarded, and the liquid containing the segmented worms was repeatedly replaced with 5% triple-antibiotic DMEM high-glucose medium until clear. The collected segmented worms were assessed for contamination, and the culture supernatant was collected. Under aseptic conditions, small vesicles were separated by ultracentrifugation. The vesicles were resuspended in PBS solution containing albumin and trehalose, and proteins were purified using two-dimensional chromatography. Peptides were analyzed by mass spectrometry. Mascot software was used to generate a peak list from the raw files, and the echinococcosis proteome was screened in the Swiss-Pro database. The screening criteria were peptide confidence ≥95% and the presence of at least one unique peptide.

[0071] Protein function annotation and analysis: GO enrichment and pathway annotation were performed on each functional subgroup using the Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO terms were screened using an adjusted P-value ≤ 0.05.

[0072] 2.2 Transcriptome-Proteomics Analysis

[0073] (1) Sequencing data acquisition: The read2 data in the FASTQ files obtained by high-throughput sequencing were analyzed using the fastqc software, and the transcriptome sequencing data were statistically analyzed using the BSCMatrix software.

[0074] (2) Functional gene annotation and differential analysis: GO enrichment and pathway annotation were performed on each functional subgroup using the Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO terms were screened with an adjusted P value ≤ 0.05, and the GO enrichment results of worm genes at different developmental stages were integrated.

[0075] 2.3 Prediction of protein antigenicity

[0076] For prediction of protein antigenicity, please refer to the following website and operating instructions for ANTIGENpro.

[0077] (http: / / scratch.proteomics.ics.uci.edu / ).

[0078] 3. Prokaryotic expression and protein purification of candidate genes

[0079] The recombinant proteins rEgENO, rEgSev, rEgCyc, rEgFABP1, rEgCaM, and rEgSrp1 from *Echinococcus granulosus* were expressed and purified using the following method. Specific primers were designed using Primer Premier 5.0 software, referencing the *Echinococcus granulosus* gene sequences published in GenBank (Table 1). The amplified target gene products were seamlessly cloned and ligated into the pET32a(+) plasmid, which was then transformed into *Escherichia coli* BL21. The expression host bacteria containing the recombinant plasmid were then induced at 37°C with 1 mM IPTG for 6 h. Finally, the recombinant proteins were expressed using Ni... 2+ The protein was purified using affinity chromatography. The concentration of the purified protein was determined using a BCA protein assay kit.

[0080] Table 1. PCR primer names and sequences

[0081]

[0082]

[0083] >EgEno amino acid sequence (SEQ ID NO:13)

[0084] MSILKIHARQIFDSRGNPTVEVDLTTSKGLFRAAVPSGASTGVHEAVELRDADKNAYMGKGVLNAVKNVNEVIAPALIKEKFVVTDQQRIDEFMIKLDGSPNKGKLGANAILGVSLAVCKAGAAEKGVPLYRHVADLAGNKDVVLPVPSFNVLNGGSHAGNKLAMQEFMILPTGAKSFSEAMKMGTEVYHHLKSVIKGKYGLDACNVGDEGGFAPNIQDNMEGLELLKTAIDKAGYTGKVKIGMDVAASEFYQDGNYNLDFKNPKAAASSIVSGSKLSDIYSEMISKYPIVSIEDPFDQDDWAAWTEFNAKAGIQIVGDDLTVTNPERVQQAIDRKACNALLLKVNQIGSVTESIKACKMSRAAGWGVMVSHRSGETEDSTIADIVVGLRTGQIKTGAPCRSERLAKYNQLLRIEEELGPKAVYAGEHFRNPL

[0085] >Amino acid sequence of EgSev (SEQ ID NO: 14)

[0086] MAGLVKAKDYDWKDSNMELFGSSKDRQVKKESAMTEKCWEPVGRATSPFLMVW RVNQFTLEPVPSDEIGNFYNGDPYVICKATRSPGGDKLLYNVHFWIGKHSTADEYGTAAYKTVELDTFLDDAAVQHREVEGYESQLFKSYFDKLVILKGGYASGFRHVKPDEYRPRLLRFCKEGKTTYMRQVAFSKQSVHSGDVFILDLGSRAYQFNGSKCSAFEKSSAAAFLQDLESKRNGRCNTSVLDEADTPQDHEFWTALPDVPVKELEPPKEVIKSLYKLSDSSGKLELTIVSEGSASKHDIKPDDVYIILTKEGLFVYIGKDCSVLEKRNALSNAHKFLQTCPNPFLPITVVTDEQAESFLKGIWDE

[0087] >Amino acid sequence of EgCyc (SEQ ID NO: 15)

[0088] MGVKCFFDISIGGKPAGRIVFALFDDVPKTVENFRALCTGEKGFGYKGSKFHRIIPGFMCQGGDFTAGNGTGGKSIYGSKFEDENFNHKHSKPMMLSMANAGKNTNGSQFFITTAVTSWLDGKHVVFGEVESGEDVVKDMEAVGSSSGKTSQEVLITDCGQL

[0089] >Amino acid sequence of EgFABP1 (SEQ ID NO: 16)

[0090] MEAFLVTWKMEKSEGFDKIMERLGVDFVTRKMGNLVKPNLIVTDLGGGKYKMRSESTFKTTECSFKLGEKFKEVTPDSREVTSLITVENGVMKHEQDDKTKVTYIERVVEGNELKATVKVDEVVCVRNYSKVA

[0091] >Amino acid sequence of CaM (SEQ ID NO: 17)

[0092] MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTESELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGLISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVTMMQSK

[0093] >Amino acid sequence of EgSrp1 (SEQ ID NO: 18)

[0094] MSPLSVYSALSLALAGSESETREELVSVLGLAPGKDIDTIVKSLGEDLQAVADGDAKKTLVEANGVFIQAGSRIRETYTSAVSKHLKADMKQVTALFPADNVVFQLDFGGDCEGSRVSINRWIAEKTREKIKDLLAQGSITPMTHVVLANAVYFKGVWKCKFEKSKTDRNGVFHSLESGDVRV SMMTQKASYPMADFVDLEVRALKVPFETHEMVIVLPEKNDGLPNLLKQLSANAKHLEEMLTSDQYFDTEVVLKLPRFSLGGHNMKLKEPLHRMGLKSAFDAERADFSGITSDRSLAVSDVYHQAVIDVDEEGAEAAAATAMPMMVHCMPAPPVDFIVDHPFIFFIVTKTGIPVFMGHVVHPESK

[0095] Example 1: Multi-omics combined analysis and antigenicity prediction

[0096] 1. Bioinformatics screening of candidate antigens

[0097] Figure 1 The results showed that, using Q-Exactive HF X liquid chromatography-mass spectrometry, 1916 adult exosome proteins and 13207 peptides were identified, of which 343 had a unique peptide number of more than 10.

[0098] We performed GO functional annotation analysis on all identified proteins. Figure 2 For each GO entry in the three ontologies (cellular component, biological process, and molecular function), the IDs and number of corresponding proteins were listed, and statistical graphs were generated. GO entries without corresponding proteins were omitted. Most of the exosome proteins identified in this study are involved in binding transport, the immune system, and the metabolic process.

[0099] This study collected proteins identified from the secretory mesotomy of early-stage adults and constructed a Venn diagram. Figure 3 The study found that 1,916 adult exosome proteins identified included 17 previously identified proteins.

[0100] 2. Comparative transcriptomics analysis

[0101] Existing transcriptome data of protocercariae and adults were compared using differential volcano plots. Figure 4 The study found 960 genes whose transcription levels were significantly increased from protocercariae to adults, of which 328 were genes expressed only in adults.

[0102] Then, using multiple sets of volcano maps and GO cluster analysis, we compared 960 genes with significantly increased transcription levels from protocercariae to adults and found that the main candidate antigen genes with increased levels focused on 7 key categories, including extracellular proteins and Immune regulation.

[0103] 3. Results of multi-omics combined analysis and antigenicity prediction

[0104] Combining transcriptomics (genes upregulated in adults), secretomics (adults and protocercariae), and immunomicomics (somatic antigens recognized by positive serum of canine echinococcosis infection), with adult secreted proteins as the core reference, the intersection was taken, resulting in 90 candidate antigens. Figure 5 ).

[0105] From the above 90 candidate antigens, combined with antigenicity prediction results, literature research on protein function, and prokaryotic expression probability analysis, 6 candidate antigens were finally selected as candidate antigens for prokaryotic expression genetic engineering subunit vaccines.

[0106] Table 2 Candidate antigens for prokaryotic expression genetically engineered subunit vaccines

[0107]

[0108] Example 2: Recombinant Protein Expression and Purification

[0109] This study successfully amplified specific bands of six candidate genes, namely EgENO, EgSev, EgCyc, EgFABP1, EgCaM, and EgSrp1, from the mixed cDNA of Echinococcus granulosus PSC and segmented worms. Figure 6 Sequencing revealed that the amplified fragments matched the corresponding gene sequences in the GenBank data.

[0110] Recombinant proteins rEgENO, rEgSev, rEgCyc, rEgFABP1, rEgCaM, and rEgSrp1 were all successfully expressed in BL21(DE3) in soluble protein form. Figure 7 ).

[0111] Example 3: Immunoprotection test

[0112] 1. Immunization grouping

[0113] Eighteen beagle dogs were randomly divided into three groups of six each, with half males and half females, for an immunization test.

[0114] 2. Immunization program

[0115] Each dog in each experimental group received a subcutaneous injection of 1 mL of the immune mixture in the neck.

[0116] Group A (Control Group 1): 1 mg Quil A + 1 mL PBS;

[0117] Group B (immune group 1, rEgFABP1&rEgCaM&rEgSrp1): 100μgrEgFABP1+100μgrEgCaM+100μgrEgSrp1+1mg Quil A+1mLPBS;

[0118] Group C (immunogroup 2, rEgENO&rEgSev&rEgCyc): 100μgrEgENO+100μgrEgSev+100μgrEgCyc+1mg Quil A+1mL PBS;

[0119] The patient was immunized twice, with a second immunization given two weeks after the first.

[0120] 3. Artificial infection

[0121] Four weeks after the second immunization, artificial infection was carried out, with each beagle in both the experimental and control groups receiving 150,000 protocercariae orally.

[0122] 4. Blood collection

[0123] Blood was collected from the cephalic vein of the forearm of each beagle before and after immunization and weekly after infection. The serum was separated by centrifugation at 3500 rpm for 10 min and stored in a -20°C freezer.

[0124] 5. Autopsy count

[0125] Four weeks after artificial infection, all beagle dogs in all groups underwent necropsy to record the number of worms in the small intestine of each dog and to measure the length and width of the worms.

[0126] 6. Serum antibody indirect ELISA detection

[0127] Specific IgG antibodies in the serum of immunized beagle dogs were detected using an indirect ELISA method. The method was as follows: ELISA plates were coated with recombinant proteins rEgENO, rEgSev, rEgCyc, rEgFABP1, rEgCaM, and rEgSrp1, incubated overnight at 4°C, washed three times with PBST, incubated with 5% skim milk at 37°C for 2 hours, washed three times with PBST and dried, and then incubated with serum from each group (immunized group and blank control group) at 37°C for 1 hour. Subsequently, HRP-labeled goat anti-canine IgG antibody was added and incubated at 37°C for 1 hour. After washing with PBST, soluble TMB substrate was added for color development, and the color development was terminated with 2M H2SO4. The absorbance was measured at 450 nm using an ELISA reader.

[0128] 7. Serum cytokine detection

[0129] The detection of canine IL-2, IL-4, IL-5, IL-10, and IFN-γ cytokines was performed according to the instructions of the ELISA kit (Solepro).

[0130] 8. Data Analysis

[0131]

[0132] The Mann–Whitney U test was used for testing. SPSS 20.0 was also used for statistical analysis of the number of cephalopods in each group.

[0133] 9. Results of insect protection

[0134] Four weeks after oral infection with protocercariae, all beagle dogs in the experimental and control groups underwent necropsy. The infection status of each group is shown in Table 3. The control group had an average number of worms bearing 100,451, with an average worm length of 1.199 mm and an average worm width of 0.199 mm. The rEgENO&rEgSev&rEgCyc immunization group had an average number of worms bearing 19,511, with an average worm length of 0.901 mm and an average worm width of 0.155 mm, resulting in a worm reduction rate of 81% (P<0.05). The worm length and width decreased by 25% and 22%, respectively (P<0.05). The rEgFABP1&rEgCaM&rEgSrp1 immunization group had an average number of worms bearing 52,313, with an average worm length of 1.013 mm and an average worm width of 0.218 mm, resulting in a worm reduction rate of 48% (P<0.05). The worm length decreased by 16% (P<0.05), while there was no significant difference in worm width.

[0135] Table 3. Post-mortem examination results of beagle dogs after parasite challenge immunization.

[0136]

[0137]

[0138] 10. Serum-specific IgG detection

[0139] Compared with the control group, the specific IgG levels of rEgENO, rEgSev, and rEgCyc in the serum of immunized dogs reached a higher level one week after the second immunization; antibody levels remained at a higher level for six weeks after the second immunization. In the experimental group, the specific IgG levels of rEgFABP1, rEgCaM, and rEgSrp1 in the serum of dogs reached a higher level one week after the second immunization; antibody levels showed a significant decrease in the sixth week after the second immunization, and the differences in antibody levels between individuals were substantial. Figure 8 ).

[0140] 11. Serum cytokine detection

[0141] The results are as follows Figure 9 As shown, in the control group, IL-2 significantly increased and IL-4 significantly decreased one week after the second immunization (PBS + QuilA), exhibiting a Th1-biased immune response. IL-10 decreased one week after challenge and returned to normal levels by the fourth week; its suppression may be one of the reasons for intestinal hemorrhagic injury. IL-4 increased to normal levels after challenge, indicating that worm colonization induces IL-4-type immune protection. INF-γ was significantly suppressed one week after challenge, which may have hindered the early immune response; by the fourth week, the host immune system returned to a balanced state.

[0142] One week after the second immunization with rEgFABP1, rEgCaM, and rEgSrp1, IL-2, IL-4, and INF-γ significantly increased. One week after challenge, similar to the control group, INF-γ was significantly suppressed, and IL-2 and IL-4 also decreased, indicating that the host's immunity returned to a balanced state. Four weeks after challenge, IL-4 and IL-10 significantly increased, indicating the formation of Th2-biased immunity.

[0143] One week after the second immunization with rEgENO, rEgSev, and rEgCyc, there were no significant changes in cytokines. The participation of these antigens helps maintain immune homeostasis in the host. Compared to the other two experimental groups, IL-2 and IL-4 showed no significant increase. One week after challenge, IL-2 decreased, while IL-4 and IL-10 increased significantly, shifting towards a Th2-biased pattern. Notably, INF-γ significantly increased after challenge, and unlike the other two groups where it was suppressed, the early immune response in this group was not inhibited. Furthermore, unlike the other two groups, IL-10 levels increased after challenge, while the control group showed a decrease. IL-5 showed a significant decrease at the sixth week after challenge.

[0144] Example 4: Immunoprotection test

[0145] 1. Immunization grouping

[0146] Twelve beagle dogs were randomly divided into two groups of six each, with half males and half females, for an immunization test.

[0147] 2. Immunization program

[0148] Each dog in each experimental group received a subcutaneous injection of 1 mL of the immune mixture in the neck.

[0149] Group A (Control Group 2): 1 mg Quil A + 1 mL PBS;

[0150] Group B (immunogroup 3, rEgENO&rEgSev&rEgCyc): 50μg rEgENO+50μg rEgSev+50μg rEgCyc+1mg Quil A+1mL PBS;

[0151] The patient was immunized twice, with a second immunization given two weeks after the first.

[0152] 3. The remaining processing is carried out in the same way as shown in 3 to 8 of Example 3.

[0153] 4. Results of insect protection

[0154] Four weeks after oral infection with protocercariae, all beagle dogs in both the experimental and control groups underwent necropsy. Infection status for each group is shown in Table 4. The control group had an average worm count of 98,350, an average worm length of 1.185 mm, and an average worm width of 0.192 mm. The control group immunized with rEgENO (50 μg), rEgSev (50 μg), and rEgCyc (50 μg) had an average worm count of 19,521, an average worm length of 0.903 mm, and an average worm width of 0.150 mm, resulting in a worm reduction rate of 80.15% (P<0.05). Worm length and width decreased by 23.8% and 21.9%, respectively (P<0.05).

[0155] Table 4. Post-mortem examination results of beagle dogs after parasite challenge immunization.

[0156]

[0157]

[0158] 5. Serum-specific IgG detection

[0159] Compared with the control group, the specific IgG levels of rEgENO, rEgSev, and rEgCyc in the serum of immunized dogs reached a higher level one week after the second immunization; antibody levels remained at a higher level for six weeks after the second immunization. Figure 10 )

[0160] 6. Serum cytokine detection

[0161] In the control group, one week after the second immunization (PBS + QuilA), IL-2 significantly increased, while IL-4 significantly decreased, exhibiting a Th1-biased immune response. IL-10 decreased one week after challenge but returned to normal levels by week 4 post-challenge; its suppression may be one of the reasons for intestinal hemorrhagic injury. IL-4 increased to normal levels after challenge, indicating that worm colonization induces IL-4-type immune protection. INF-γ was significantly suppressed one week after challenge, which may have hindered the early immune response; host immunity returned to equilibrium by week 4 post-challenge.

[0162] One week after the second immunization with rEgENO, rEgSev, and rEgCyc, there were no significant changes in cytokines. The participation of these antigens helps maintain immune homeostasis in the host. Compared to the control group, IL-2 and IL-4 showed no significant increase. One week after challenge, IL-2 decreased, while IL-4 and IL-10 significantly increased, shifting towards a Th2-biased pattern. Notably, INF-γ significantly increased after challenge, and unlike the suppressed response in the control group, the early immune response in this group was not inhibited. Simultaneously, unlike the control group, IL-10 levels increased after challenge, while the control group showed a decrease. IL-5 showed a significant decrease in the fourth week after challenge. Figure 11 )

[0163] Example 5: Immunoprotective test

[0164] 1. Immunization grouping

[0165] Twelve beagle dogs were randomly divided into two groups of six each, with half males and half females, for an immunization test.

[0166] 2. Immunization program

[0167] Each dog in each experimental group received a subcutaneous injection of 1 mL of the immune mixture in the neck.

[0168] Group A (control group 3): 1 mg Quil A + 1 mL PBS;

[0169] Group B (immunogroup 4, rEgENO&rEgSev&rEgCyc): 150μg rEgENO+150μg rEgSev+150μg rEgCyc+1mg Quil A+1mL PBS;

[0170] The patient was immunized twice, with a second immunization given two weeks after the first.

[0171] 3. The remaining processing is carried out in the same way as shown in 3 to 8 of Example 3.

[0172] 4. Results of insect protection

[0173] Four weeks after oral infection with protocercariae, all beagle dogs in the immunized and control groups underwent necropsy. The infection status of each group is shown in Table 5. The average number of worms in the control group was 100,852, with an average worm length of 1.199 mm and an average worm width of 0.199 mm. The average number of worms in the immunized group (rEgENO (150 μg), rEgSev (150 μg), and rEgCyc (150 μg)) was 19,368, with an average worm length of 0.901 mm and an average worm width of 0.155 mm. The worm reduction rate was 80.8% (P<0.05), and the worm length and width decreased by 24.85% and 22.1%, respectively (P<0.05).

[0174] Table 5. Post-mortem examination results of beagle dogs after parasite challenge immunization.

[0175]

[0176]

[0177] 5. Serum-specific IgG detection

[0178] Compared with the control group, the specific IgG levels of rEgENO, rEgSev, and rEgCyc in the serum of immunized dogs reached a higher level one week after the second immunization; antibody levels remained at a higher level for six weeks after the second immunization. Figure 12 )

[0179] 6. Serum cytokine detection

[0180] In the control group, one week after the second immunization (PBS + QuilA), IL-2 significantly increased, while IL-4 significantly decreased, exhibiting a Th1-biased immune response. IL-10 decreased one week after challenge but returned to normal levels by week 4 post-challenge; its suppression may be one of the reasons for intestinal hemorrhagic injury. IL-4 increased to normal levels after challenge, indicating that worm colonization induces IL-4-type immune protection. INF-γ was significantly suppressed one week after challenge, which may have hindered the early immune response; host immunity returned to equilibrium by week 4 post-challenge.

[0181] One week after the second immunization with rEgENO, rEgSev, and rEgCyc, there were no significant changes in cytokines. The participation of these antigens helps maintain immune homeostasis in the host. Compared to the control group, IL-2 and IL-4 showed no significant increase. One week after challenge, IL-2 decreased, while IL-4 and IL-10 significantly increased, shifting towards a Th2-biased pattern. Notably, INF-γ significantly increased after challenge, and unlike the suppressed response in the control group, the early immune response in this group was not inhibited. Simultaneously, unlike the control group, IL-10 levels increased after challenge, while the control group showed a decrease. IL-5 showed a significant decrease in the fourth week after challenge. Figure 13 )

[0182] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A candidate antigen combination for Echinococcus granulosus, characterized in that, The candidate antigen combination includes EgEno protein, EgSev protein and EgCyc protein; The EgEno protein, EgSev protein, and EgCyc protein have the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

2. A nucleic acid molecule encoding a candidate antigen combination as described in claim 1, wherein the nucleic acid molecule comprises the EgENO gene, the EgSev gene, and the EgCyc gene.

3. A recombinant vector, characterized in that, Includes the nucleic acid molecules as described in claim 2.

4. The host, characterized in that, Includes the recombinant vector as described in claim 3.

5. The following are applications in the preparation of Echinococcus granulosus vaccine: (I) The candidate antigen combination as described in claim 1; and / or (II) The nucleic acid molecule as described in claim 2; and / or (III) The recombinant vector as described in claim 3; and / or (IV) The host as described in claim 4.

6. Any of the following applications in the preparation of a kit for detecting Echinococcus granulosus: (I) The candidate antigen combination as described in claim 1; and / or (II) The nucleic acid molecule as described in claim 2; and / or (III) The recombinant vector as described in claim 3; and / or (IV) The host as described in claim 4.

7. A method for preparing a vaccine against Echinococcus granulosus, characterized in that, The steps include: using primer pairs to amplify the nucleic acid molecule as described in claim 2, expressing and purifying it to obtain rEgENO, rEgSev and rEgCyc, and mixing them to obtain the Echinococcus granulosus vaccine; The primer pair is: The primer pair used for amplifying the EgENO gene has the upstream primer sequence as shown in SEQ ID NO:1 and the downstream primer sequence as shown in SEQ ID NO:2; and The primer pair used for amplifying the EgSev gene has the upstream primer sequence as shown in SEQ ID NO:3 and the downstream primer sequence as shown in SEQ ID NO:4; and The primer pair used to amplify the EgCyc gene has an upstream primer sequence as shown in SEQ ID NO:5 and a downstream primer sequence as shown in SEQ ID NO:

6.

8. The preparation method according to claim 7, characterized in that, The mass ratio of rEgENO, rEgSev, and rEgCyc in the Echinococcus granulosus vaccine is 1:1:

1.

9. A vaccine against Echinococcus granulosus, characterized in that, It is prepared by the preparation method as described in claim 7 or 8.

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