Candidate antigen combination of echinococcus granulosus and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a candidate antigen combination of echinococcus granulosus and application of the candidate antigen combination. According to the research, protective antigens at different stages are screened and identified by adopting bioinformatics and immunology methods, and recombinant subunit vaccine candidate antigens which are simple in immune procedure, good in protection effect and high in stability are developed, so that the recombinant subunit vaccine candidate antigens have commercial vaccine development value. Meanwhile, compared with three times of immunization adopted in previous researches, the dogs are immunized by adopting a procedure of twice subcutaneous injection, so that the use cost and difficulty are reduced, the practical application requirements are better met, and the implementation is convenient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a candidate antigen combination of Echinococcus granulosus and its application. Background Art

[0002] Echinococcus granulosus is a tapeworm parasitizing in the intestine of dogs, and its larvae can parasitize in the parenchymal organs of hosts such as humans, cattle, and sheep, causing cystic echinococcosis, an important zoonotic parasitic disease worldwide. This disease poses a serious threat to the health of humans and animals, especially in remote areas lacking sanitation conditions and infrastructure, resulting in serious economic losses and public health problems.

[0003] Dogs are the definitive hosts of Echinococcus granulosus and the main sources of infection for echinococcosis. The main routes of infection of humans and animals with echinococcosis are oral contact with dog feces carrying eggs, or consumption of food contaminated with eggs and contact with contaminated items. At present, the prevention and control of Echinococcus granulosus in dogs mainly rely on regular use of anthelmintics. However, this method is difficult to implement in the long term, and there are problems such as drug resistance, drug residues, and environmental pollution. On the other hand, the prevention and control of intermediate hosts mainly rely on vaccination. Due to the complexity of the epidemic of echinococcosis and the diversity of the types and numbers of intermediate hosts, it is difficult to completely achieve the goal of blocking transmission only by immunizing sheep. As the main definitive host of echinococcosis, and the number of dogs is much lower than that of the main intermediate hosts such as cattle and sheep, the control of their infection may be more cost-effective and is one of the key measures to reduce the spread of the disease.

[0004] In recent years, the research on Echinococcus granulosus antigen genes has mainly focused on the screening of diagnostic antigens, and only a few genes have been used for vaccine research. Existing studies have shown that some candidate vaccines can affect the maturation of eggs and reduce the worm burden in dogs. Among them, antigens such as the EgM family, tropomyosin, paramyosin, and 3-hydroxyacyl-CoA dehydrogenase have shown certain protective effects. However, due to the low worm reduction rate of the reported candidate antigen proteins (only about 70%), it still cannot meet the requirements for the development of commercial vaccines. Summary of the Invention

[0005] In view of this, the present invention provides a candidate antigen combination of Echinococcus granulosus and its application. The present invention uses bioinformatics and immunological methods to screen and identify protective antigens at different stages, and develops recombinant subunit vaccine candidate antigens with a simple immunization program, good protective effect, and high stability, which have the value of commercial vaccine development. At the same time, compared with the three immunizations used in previous studies, this study uses a program of two subcutaneous injections to immunize dogs, reducing the cost and difficulty of use, better meeting the actual application requirements, and being convenient to implement.

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

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

[0008] The EgEno protein, EgSev protein and EgCyc protein successively have:

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

[0010] (II) sequences obtained by substituting, deleting, adding and / or replacing one or more amino acids on the basis of the amino acid sequences shown in (I); or

[0011] (III) sequences having a homology of more than 90% with 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, P14088 respectively.

[0013] The present invention also provides a nucleic acid molecule encoding the candidate antigen combination, and the nucleic acid molecule comprises EgENO gene, EgSev gene and 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, AF430707.1 respectively.

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

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

[0017] The present invention also provides a host comprising the recombinant vector.

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

[0019] The present invention also provides a primer pair for amplifying the nucleic acid molecule, comprising:

[0020] A primer pair for amplifying the EgENO gene, the upstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 1, and the downstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 2;

[0021] A primer pair for amplifying the EgSev gene, the upstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 3, and the downstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 4;

[0022] A primer pair for amplifying the EgCyc gene, the upstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 5, and the downstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 6.

[0023] The present invention also provides the use of any of the following in the preparation of an Echinococcus granulosus vaccine:

[0024] (I), the candidate antigen combination described above; and / or

[0025] (II), the nucleic acid molecule described above; and / or

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

[0027] (IV), the host described above; and / or

[0028] (V), the primer pair described above.

[0029] The present invention also provides the use of any of the following in the preparation of an Echinococcus granulosus detection kit:

[0030] (I), the candidate antigen combination described above; and / or

[0031] (II), the nucleic acid molecule described above; and / or

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

[0033] (IV), the host described above; and / or

[0034] (V), the primer pair described above.

[0035] The present invention also provides a method for preparing an Echinococcus granulosus vaccine, including the steps of: using the primer pair described above to amplify the nucleic acid molecule, and obtaining rEgENO, rEgSev and rEgCyc after expression and purification, and mixing them to obtain the Echinococcus granulosus vaccine.

[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 is 150 μg to 450 μg per time.

[0039] The present invention also provides an Echinococcus granulosus vaccine, which is prepared by the method described above.

[0040] The present invention has the following beneficial effects:

[0041] In this study, bioinformatics and immunological methods were used to screen and identify protective antigens at different stages, and recombinant subunit vaccine candidate antigens with simple immunization procedures, good protective effects and high stability were developed, which have the value of commercial vaccine development. At the same time, compared with the three immunizations used in previous studies, this study used a two - subcutaneous - injection procedure to immunize dogs, reducing the cost and difficulty of use, better meeting the actual application requirements and being convenient for implementation. Brief Description of the Drawings

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0043] Figure 1 Showing adult exosome proteins; where A: Distribution map of the number of unique peptides; B: Distribution map of peptide lengths; C: Distribution map of protein proportions; D: Distribution map of protein molecular weights;

[0044] Figure 2 Showing GO functional annotation analysis of adult exosome proteins;

[0045] Figure 3 Showing the Venn diagram of adult exosome proteins; where A: Proteins in the adult secretome identified in this study; B: Proteins in the adult secretome identified in previous studies;

[0046] Figure 4 Showing comparative transcriptome analysis; where A: Analysis of the difference in gene transcription levels between Echinococcus granulosus protoscoleces and adults; B: Analysis of key functional - category main transcriptional - difference genes by multi - group volcano plots; C: GO clustering analysis of genes up - regulated in adults relative to protoscoleces;

[0047] Figure 5 Showing the results of multi - omics joint analysis and antigenicity prediction; where A: Adult secretome protein set; B: Protoscolex secretome protein set; C: Gene set up - regulated in transcription level in adults compared with protoscoleces; D: Protein set of Echinococcus granulosus recognized by positive sera of dogs infected with echinococcosis;

[0048] Figure 6 Show the specific bands of candidate genes; among them, lane M: DNA molecular weight standard (Marker D2000); 1: EgENO; 2: EgSev; 3: EgCyc; 4: EgFABP1; 5: EgCaM; 6: EgSrp1; 7: negative control;

[0049] Figure 7 Show the specific bands of recombinant proteins; among them, lane M: protein molecular weight standard Marker; 1: rEgENO; 2: rEgSev; 3: rEgCyc; 4: rEgFABP1; 5: rEgCaM; 6: rEgSrp1; 7: pET-32a(+) empty bacteria;

[0050] Figure 8 Show the changes of specific antibody IgG in the sera of experimental dogs after the first immunization (0 week), booster immunization (2 weeks), and challenge (4 weeks) with rEgENO, rEgSev, rEgCyc, rEgFABP1, rEgCaM, and rEgSrp1;

[0051] Figure 9 Show the changes of cytokines IL-2, IL-4, IL-5, IL-10, and IFN-γ in the sera of experimental dogs after the first immunization (a), one week after booster immunization (b), one week after challenge (c), and four weeks after challenge (d) with PBS+QuilA (A), rEgFABP1&rEgCaM&rEgSrp1 (B), and rEgENO&rEgSev&rEgCyc (C);

[0052] Figure 10 Show the changes of specific antibody IgG in the sera of experimental dogs after the first immunization (0 week), booster immunization (2 weeks), and challenge (4 weeks) with rEgENO, rEgSev, and rEgCyc;

[0053] Figure 11 Show the changes of cytokines IL-2, IL-4, IL-5, IL-10, and IFN-γ in the sera of experimental dogs after the first immunization (a), one week after booster immunization (b), one week after challenge (c), and four weeks after challenge (d) with PBS+QuilA (A) and rEgENO&rEgSev&rEgCyc (B);

[0054] Figure 12 Show the changes of specific antibody IgG in the sera of experimental dogs after the first immunization (0 week), booster immunization (2 weeks), and challenge (4 weeks) with rEgENO, rEgSev, and rEgCyc;

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

[0056] The present invention discloses a candidate antigen combination of Echinococcus granulosus and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes, or appropriate changes and combinations, to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

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

[0058] 1. Main reagents

[0059] Goat anti-dog IgG (HRP-labeled) was purchased from Beyotime Biotechnology Co., Ltd.; IPTG, Quil A (saponin) were purchased from Sigma; 96-well enzyme-linked immunosorbent assay (ELISA) plates were purchased from Corning; Horseradish peroxidase-labeled TMB substrate chromogenic solution was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Mini Profinity Ni-charged IMAC pre-packed column was purchased from Bio-Rad. Cytokine kits for IL-2, IL-4, IL-5, IL-4, IL-10, IFN-γ ELISA kit were purchased from Solarbio.

[0060] 2. Main instruments

[0061] NanoDrop One ultra-micro spectrophotometer (Thermo Fisher Scientific, USA); refrigerated high-speed centrifuge (Thermo, USA); micropipette (Thermo, USA); -80°C ultra-low temperature freezer (Thermo, USA); PCR amplifier (Bio-Rad, USA); small vertical electrophoresis apparatus (Bio-Rad, USA); fully automatic 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 apparatus (Liuyi Instruments), etc.

[0062] Unless otherwise specified, the raw materials and reagents used in the candidate antigen combination of Echinococcus granulosus and its application provided by the present invention can be purchased from the market.

[0063] The present invention will be further described below in conjunction with embodiments:

[0064] Embodiment

[0065] 1. Materials and animals

[0066] Forty-two beagle dogs, 6 - 12 months old, with an equal number of males and females, were provided by the Dujiangyan Experimental Beagle Breeding Center of the Sichuan Musk Deer Research Institute. Before the experiment, they were tested parasitologically and found to be free of tapeworm and other intestinal parasite infections. During the experiment, each dog was randomly assigned to each group and raised strictly according to the experimental animal feeding and management standards.

[0067] Liver tissues infected with Echinococcus granulosus of sheep were collected. The cysts were isolated under aseptic conditions and molecularly identified as Echinococcus granulosus G1 type. The viability of protoscoleces was detected by trypan blue staining. In vitro culture of protoscoleces and canine infection experiments were carried out on the premise that the cyst genotype was G1 type and the viability of protoscoleces was greater than 95%.

[0068] 2. Bioinformatics analysis

[0069] 2.1 Proteomic analysis of adult exosomes

[0070] Each dog was orally infected with 150,000 protoscoleces. After 28 days of infection, the dog was euthanized. The small intestine was completely isolated and longitudinally cut open, then placed in PBS solution at 37°C and incubated for 1 hour. After the worms fell off, the supernatant was discarded, and the liquid containing segmented worms was repeatedly replaced with 5% triple-antibody DMEM high-glucose medium until it was clear. The collected segmented worms were identified for contamination and the culture supernatant was collected. Under aseptic conditions, small vesicles of the worms were isolated by ultracentrifugation. Finally, the small vesicles were resuspended in PBS solution containing albumin and trehalose, and two-dimensional chromatography was used for protein purification, and polypeptides were analyzed and detected by a mass spectrometer. The original file was used to generate a peak list by Mascot software to screen the hydatid proteome in the Swiss-Pro database, and the screening criteria were that the peptide confidence level was ≥95% and at least one unique peptide was included.

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

[0072] 2.2 Integrated transcriptome and proteome analysis

[0073] (1) Sequencing data acquisition: The read2 in the FASTQ file obtained by high-throughput sequencing was subjected to quality control analysis using the fastqc software, and the transcriptome sequencing data was 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 through the Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO terms were screened with the criterion of adjusted P value ≤ 0.05, and the GO enrichment results of genes in worms at different developmental stages were integrated.

[0075] 2.3 Prediction of protein antigenicity

[0076] For the prediction of protein antigenicity, ANTIGENpro was performed with reference to the following website and operation guidelines

[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 of Echinococcus granulosus were expressed and purified by the following method. Referring to the Echinococcus granulosus gene sequences published in GenBank (Table 1), specific primers were designed using Primer Premier 5.0 software. First, the amplified product of the target gene was ligated to the pET32a(+) plasmid by seamless cloning and transferred into Escherichia coli BL21. Then, the expression host bacteria containing the recombinant plasmid were induced at 37 °C for 6 h with 1 mM IPTG. Finally, the recombinant protein was purified by Ni 2+ affinity chromatography. The concentration of the purified protein was determined using a BCA protein assay kit.

[0080] Table 1 Names and sequences of PCR primers

[0081]

[0082]

[0083] > Amino acid sequence of EgEno (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] MSPLSVYSALSLALAGSESETREELVSVLGLAPGKDIDTIVKSLGEDLQAVADGDAKKTLVEANGVFIQAGSRIRETYTSAVSKHLKADMKQVTALFPADNVVFQLDFGGDCEGSRVSINRWIAEKTREKIKDLLAQGSITPMTHVVLANAVYFKGVWKCKFEKSKTDRNGVFHSLESGDVRVSMMTQKASYPMADFVDLEVRALKVPFETHEMVIVLPEKNDGLPNLLKQLSANAKHLEEMLTSDQYFDTEVVLKLPRFSLGGHNMKLKEPLHRMGLKSAFDAERADFSGITSDRSLAVSDVYHQAVIDVDEEGAEAAAATAMPMMVHCMPAPPVDFIVDHPFIFFIVTKTGIPVFMGHVVHPESK

[0095] Effect Example 1 Multi-omics Joint Analysis and Antigenicity Prediction

[0096] 1. Bioinformatics Screening of Candidate Antigens

[0097] Figure 1 The results showed that in this study, Q-Exactive HF X liquid chromatography-mass spectrometry was used to identify 1,916 adult exosome proteins and 13,207 peptides, among which 343 had a unique peptide number exceeding 10.

[0098] We performed GO functional annotation analysis on all the identified proteins ( Figure 2 ), and for the GO terms involved in the three ontologies (cellular component, biological process, molecular function), listed the IDs and the number of corresponding proteins, and at the same time made statistical charts, omitting the GO terms without corresponding proteins. Most of the exosome proteins identified in this study were involved in binding, transport, immune system, metabolic process, etc.

[0099] In this study, the proteins identified from the previous adult secretome were collected and made into a Venn diagram ( Figure 3 ), and it was found that the 1,916 adult exosome proteins identified already included 17 previously identified proteins.

[0100] 2. Comparative Transcriptomics Analysis

[0101] Comparing the existing transcriptome data of protoscoleces and adult worms through differential volcano plots ( Figure 4 ), it was found that there were 960 genes with significantly increased transcriptional levels from the protoscolex to the adult stage, among which 328 were genes expressed only in adults.

[0102] Then, using multiple sets of volcano plots and GO clustering analysis to compare the 960 genes with significantly increased transcriptional levels from the protoscolex to the adult stage, it was found that the main candidate antigen genes with increased levels were concentrated in 7 key categories, including Extracellular proteins, Immune regulation, etc.

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

[0104] Combining the transcriptome (genes with up - regulated expression in adults), secretome (adults and protoscoleces), and immunome (worm antigens recognized by positive sera of dogs infected with Echinococcus granulosus), taking the adult secreted proteins as the core reference and taking the intersection, a total of 90 candidate antigens were obtained ( Figure 5 ).

[0105] From the above 90 candidate antigens, combining the antigenicity prediction results, literature research on protein functions, and analysis of the possibility of prokaryotic expression, 6 candidate antigens were finally selected as candidate antigens for the prokaryotic - expressed genetic engineering subunit vaccine.

[0106] Table 2 Candidate antigens for the prokaryotic - expressed genetic engineering subunit vaccine

[0107]

[0108] Effect Example 2 Recombinant protein expression and purification

[0109] In this study, specific bands of 6 candidate genes, namely EgENO, EgSev, EgCyc, EgFABP1, EgCaM, and EgSrp1, were successfully amplified from the mixed cDNA of Echinococcus granulosus PSC and segmented worm bodies ( Figure 6 ). After sequencing, the amplified fragments were all consistent with the corresponding gene sequences in the GenBank data.

[0110] The recombinant proteins rEgENO, rEgSev, rEgCyc, rEgFABP1, rEgCaM, and rEgSrp1 were successfully expressed in BL21(DE3) in the form of soluble proteins ( Figure 7 ).

[0111] Effect Example 3 Immunoprotection test

[0112] 1. Immunization groups

[0113] Eighteen beagle dogs were randomly divided into 3 groups with 6 dogs in each group, half male and half female, and an immunization experiment was conducted.

[0114] 2. Immunization procedure

[0115] Each dog in each experimental group was subcutaneously injected with 1 mL of the immunization mixture in the neck:

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

[0117] Group B (Immunization group 1, rEgFABP1&rEgCaM&rEgSrp1): 100 μg rEgFABP1 + 100 μg rEgCaM + 100 μg rEgSrp1 + 1 mg Quil A + 1 mL PBS;

[0118] Group C (Immunization group 2, rEgENO&rEgSev&rEgCyc): 100 μg rEgENO + 100 μg rEgSev + 100 μg rEgCyc + 1 mg Quil A + 1 mL PBS;

[0119] The immunization was carried out twice in total. The second immunization was carried out two weeks after the first immunization.

[0120] 3. Artificial infection

[0121] Four weeks after the second immunization, artificial infection was carried out. Each beagle dog in the experimental group and the control group was orally administered 150,000 protoscoleces.

[0122] 4. Blood sampling

[0123] Before and after immunization and weekly after infection, each beagle dog was sampled from the cephalic vein of the forearm. The blood was centrifuged at 3500 rpm for 10 min, and the serum was separated and stored in a -20°C refrigerator.

[0124] 5. Postmortem examination and counting

[0125] Four weeks after artificial infection, all beagle dogs in all groups were subjected to postmortem examination. The number of worms in the small intestine of each dog was recorded, and the length and width of the worms were measured.

[0126] 6. Indirect ELISA detection of serum antibodies

[0127] The specific IgG antibody in the serum of immunized beagle dogs was detected by the indirect ELISA method. The method is as follows: ELISA enzyme-linked immunosorbent assay (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 h, washed and patted dry three times with PBST, then sera from each group (sera from the immunized group and the blank control group) were added and incubated at 37 °C for 1 h. Subsequently, HRP-labeled goat anti-dog IgG antibody was added and incubated at 37 °C for 1 h. After washing with PBST, soluble TMB substrate was added for color development, and the color development was terminated with 2 M H2SO4. The absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader.

[0128] 7. Serum cytokine detection

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

[0130] 8. Data analysis

[0131]

[0132] The Mann–Whitney U test was used for the test. At the same time, SPSS 20.0 was used for statistical analysis of the number of worms in each group.

[0133] 9. Results of worm challenge protection

[0134] Four weeks after oral infection with protoscoleces, all beagle dogs in the experimental group and the control group were dissected, and the infection conditions of each group are shown in Table 3. The average number of worms in the control group was 100,451, the average length of the worms was 1.199 mm, and the average width was 0.199 mm; the average number of worms in the rEgENO&rEgSev&rEgCyc immunized group was 19,511, the average length of the worms was 0.901 mm, and the average width was 0.155 mm, with a worm reduction rate of 81% (P < 0.05), and the length and width of the worms were reduced by 25% and 22% respectively (P < 0.05); the average number of worms in the rEgFABP1&rEgCaM&rEgSrp1 immunized group was 52,313, the average length of the worms was 1.013 mm, and the average width was 0.218 mm, with a worm reduction rate of 48% (P < 0.05), the length of the worms was reduced by 16% (P < 0.05), and there was no significant difference in width.

[0135] Table 3 Post-challenge immunization autopsy results of beagle dogs

[0136]

[0137]

[0138] 10. Serum specific IgG detection

[0139] Compared with the control group, the specific IgG of rEgENO, rEgSev, and rEgCyc in the sera of immunized dogs reached a relatively high level one week after the second immunization; the antibody level remained at a relatively high level within six weeks after the second immunization; the specific IgG of rEgFABP1, rEgCaM, and rEgSrp1 in the sera of experimental group dogs reached a relatively high level one week after the second immunization; the antibody level decreased significantly in the sixth week after the second immunization, and there were significant differences in antibody levels among individuals ( Figure 8 ).

[0140] 11. Serum cytokine detection

[0141] The results are as Figure 9 shown. In the control group, one week after the second immunization (PBS + QuilA), IL-2 increased significantly; IL-4 decreased significantly, showing a Th1-biased immunity. IL-10 decreased one week after worm challenge and returned to normal level in the fourth week. Its inhibition may be one of the reasons for the occurrence of intestinal hemorrhagic injury. IL-4 increased to the normal level after worm challenge, and worm colonization induced the body to produce IL-4-mediated immune protection. INF-γ was significantly inhibited one week after worm challenge, which could hinder the early immune response. In the fourth week, the host immunity returned to a balanced state.

[0142] One week after the second immunization with rEgFABP1, rEgCaM, and rEgSrp1, IL-2, IL-4, and INF-γ increased significantly. One week after worm challenge, similar to the control group, INF-γ was significantly inhibited, and IL-2 and IL-4 also decreased, and the host immunity returned to a balanced state. In the fourth week after worm challenge, IL-4 and IL-10 increased significantly, and Th2-biased immunity was formed.

[0143] One week after the second immunization with rEgENO, rEgSev, and rEgCyc, there were no significant changes in cytokines. The participation of these antigens kept the host in an immune balance state. Compared with the other two experimental groups, IL-2 and IL-4 showed insignificant increases. One week after worm challenge, IL-2 decreased, while IL-4 and IL-10 increased significantly, shifting to Th2 bias. It should be noted that INF-γ increased significantly after worm challenge. Different from the other two groups where it was inhibited, the early immune response in this group was not inhibited. At the same time, different from the other two groups, the level of IL-10 increased after worm challenge, while it decreased in the control group. IL-5 decreased significantly in the sixth week after worm challenge.

[0144] Effect Example 4 Immune protection test

[0145] 1. Immunization grouping

[0146] Twelve beagle dogs were randomly divided into two groups of six each, with an equal number of males and females, and an immunization experiment was conducted.

[0147] 2. Immunization schedule

[0148] Each dog in each experimental group was subcutaneously injected with 1 mL of the immunization mixture in the neck:

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

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

[0151] A total of two immunizations were carried out, and the second immunization was performed two weeks after the first immunization.

[0152] 3. The remaining treatments were carried out in the same manner as shown in 3 - 8 of Effect Example 3.

[0153] 4. Results of protection against challenge

[0154] Four weeks after oral infection with protoscoleces, all the beagle dogs in the experimental group and the control group were dissected, and the infection conditions of each group are shown in Table 4. The average number of worms in the control group was 98,350, the average length of the worms was 1.185 mm, and the average width was 0.192 mm; the average number of worms in the immunization group with rEgENO (50 μg)&rEgSev (50 μg)&rEgCyc (50 μg) was 19,521, the average length of the worms was 0.903 mm, and the average width was 0.150 mm. The worm reduction rate was 80.15% (P < 0.05), and the length and width of the worms were reduced by 23.8% and 21.9% respectively (P < 0.05).

[0155] Table 4 Results of dissection of beagle dogs after challenge immunization

[0156]

[0157]

[0158] 5. Detection of serum specific IgG

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

[0160] 6. Detection of serum cytokines

[0161] In the control group, one week after the second immunization (PBS + QuilA), IL-2 increased significantly; IL-4 decreased significantly, showing a Th1-biased immunity. IL-10 decreased one week after challenge, and returned to the normal level at the 4th week after challenge. Its inhibition may be one of the reasons for the occurrence of intestinal hemorrhagic injury. IL-4 increased to the normal level after challenge, and the colonization of worms induced the body to produce IL-4-mediated immune protection. INF-γ was significantly inhibited one week after challenge, which could hinder the early immune response, and the host immunity returned to the balanced state at the 4th week after challenge.

[0162] One week after the second immunization with rEgENO&rEgSev&rEgCyc, there were no significant changes in cytokines. The participation of these antigens enabled the host to maintain an immune balance state. Compared with the control group, IL-2 and IL-4 showed insignificant increases. One week after challenge, IL-2 decreased, while IL-4 and IL-10 increased significantly, shifting to a Th2-biased type. It is worth noting that INF-γ increased significantly after challenge. Different from the inhibition in the control group, the early immune response in this group was not inhibited. At the same time, different from the control group, the level of IL-10 increased after challenge, while it decreased in the control group. IL-5 showed a significant decrease at the 4th week after challenge.( Figure 11 )

[0163] Effect Example 5 Immunoprotection Test

[0164] 1. Immunization grouping

[0165] Twelve beagle dogs were randomly divided into 2 groups, with 6 dogs in each group, half male and half female, for immunization experiments.

[0166] 2. Immunization procedure

[0167] Each dog in each experimental group was subcutaneously injected with 1 mL of the immunization mixture in the neck:

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

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

[0170] A total of two immunizations were performed, and the second immunization was carried out two weeks after the first immunization.

[0171] 3. The remaining treatments were carried out in the same manner as shown in 3 - 8 of Effect Example 3.

[0172] 4. Challenge protection results

[0173] Four weeks after oral infection with protoscoleces, all beagle dogs in the immunization group and the control group were dissected, and the infection conditions of each group are shown in Table 5. The average number of worms in the control group was 100,852, the average length of the worms was 1.199 mm, and the average width was 0.199 mm; the average number of worms in the rEgENO(150 μg)&rEgSev(150 μg)&rEgCyc(150 μg) immunization group was 19,368, the average length of the worms was 0.901 mm, and the average width was 0.155 mm. The worm reduction rate was 80.8% (P<0.05), and the length and width of the worms decreased by 24.85% and 22.1% respectively (P<0.05).

[0174] Table 5 Post-challenge autopsy results of beagle dogs

[0175]

[0176]

[0177] 5. Serum specific IgG detection

[0178] Compared with the control group, the specific IgG of rEgENO&rEgSev&rEgCyc in the serum of dogs in the immunization group reached a relatively high level one week after the second immunization; the antibody level remained at a relatively high level within 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 increased significantly; IL-4 decreased significantly, showing a Th1-biased immunity. IL-10 decreased one week after challenge and returned to the normal level at the 4th week after challenge. Its inhibition may be one of the reasons for the occurrence of intestinal hemorrhagic injury. IL-4 increased to the normal level after challenge, and the colonization of worms induced the body to produce IL-4-type immune protection. INF-γ was significantly inhibited one week after challenge, which could hinder the early immune response, and the host immunity returned to a balanced state at the 4th week after challenge.

[0181] One week after the second immunization with rEgENO&rEgSev&rEgCyc, there were no significant changes in cytokines. The participation of these antigens enabled the host to maintain an immune balance state. Compared with the control group, IL-2 and IL-4 showed insignificant increases. One week after challenge, IL-2 decreased, while IL-4 and IL-10 increased significantly, turning to a Th2-biased type. It is worth noting that INF-γ increased significantly after challenge. Different from the inhibition in the control group, the early immune response in this group was not inhibited. At the same time, different from the control group, the level of IL-10 increased after challenge, while it decreased in the control group. IL-5 showed a significant decrease at the 4th week after challenge.(Figure 13 )

[0182] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A candidate antigen combination of Echinococcus granulosus, characterized in that: The candidate antigen combination comprises EgEno protein, EgSev protein and EgCyc protein; The EgEno protein, EgSev protein and EgCyc protein have the following characteristics: (I), the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; or (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in (I); or (III) A sequence having a homology of 90% or more to the amino acid sequence shown in (I) or (II).

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

3. A recombinant vector, characterized in that: Comprising the nucleic acid molecule as described in claim 2.

4. A host, characterized in that Comprising the recombinant vector as described in claim 3.

5. A primer pair for amplifying a nucleic acid molecule according to claim 2, characterized in that: include: A primer pair for amplifying the EgENO gene, wherein the upstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 1, and the downstream primer sequence has the nucleotide sequence shown in SEQ ID NO: 2; A primer pair for amplifying the EgSev gene, wherein the upstream primer sequence has the nucleotide sequence shown in SEQ ID NO:3, and the downstream primer sequence has the nucleotide sequence shown in SEQ ID NO:4; The primer pair used to amplify the EgCyc gene, the upstream primer sequence has the nucleotide sequence shown in SEQ ID NO:5, and the downstream primer sequence has the nucleotide sequence shown in SEQ ID NO:

6.

6. Use of any of the following in the preparation of Echinococcus granulosus vaccines: (I), the candidate antigen combination as claimed in claim 1; and / or (II), the nucleic acid molecule according to claim 2; and / or (III), the recombinant vector according to claim 3; and / or (IV), the host according to claim 4; and / or (V) The primer pair according to claim 5.

7. Use of any of the following in the preparation of a test kit for detecting Echinococcus granulosus: (I), the candidate antigen combination as claimed in claim 1; and / or (II), the nucleic acid molecule according to claim 2; and / or (III), the recombinant vector according to claim 3; and / or (IV), the host according to claim 4; and / or (V) The primer pair according to claim 5.

8. A method for producing an Echinococcus granulosus vaccine, characterized in that: The method comprises the steps of: using the primer pair as claimed in claim 5 to amplify the nucleic acid molecule as claimed in claim 2, obtaining rEgENO, rEgSev and rEgCyc after expression and purification, and mixing them to obtain the Echinococcus granulosus vaccine.

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

1.

10. An Echinococcus granulosus vaccine, characterized in that: The method comprises preparing the product by the method as claimed in claim 8 or 9.

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