Application of recombinant rabbit coccidia protein in the prevention and control of rabbit coccidiosis

Abstract

This invention relates to the field of biotechnology, specifically to the application of recombinant rabbit coccidia proteins in the prevention and control of rabbit coccidiosis. In this invention, the EmGAM56 and EmROP17 genes were amplified from the first-ever determined transcriptome data of *Eimeria macrocarpa* in rabbits. These genes were cloned into the prokaryotic vector pET32a(+) and expressed in *E. coli* competent cells to obtain recombinant proteins GAM56 (rEmGAM56) and ROP17 (rEmROP17). Subsequently, a vaccine trial was conducted using saponin Quil-A as an adjuvant to preliminarily evaluate the immunoprotective effects of these proteins. Simultaneously, the stimulatory effects of recombinant proteins rEmGAM56 and rEmROP17 on humoral and cellular immune responses were determined. This invention lays the foundation for the research of recombinant subunit vaccines against *Eimeria macrocarpa* in rabbits.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of recombinant rabbit coccidia protein in the prevention and control of rabbit coccidiosis. Background Technology

[0002] Rabbit coccidiosis is a protozoan disease caused by various coccidia of the genus Eimeria parasitizing the intestines and liver of domestic rabbits. It is a common and frequently occurring disease in domestic rabbits.

[0003] Eimeria magna is one of the most common rabbit coccidia, with moderate pathogenicity. Epidemiological surveys have found that it has a high detection rate and a large amount of oocysts excreted in rabbit farms. Eimeria magna parasitizes the epithelial cells of the ileum and jejunum in rabbits, and infection can cause symptoms such as growth retardation and reduced feed conversion rate, thus impacting the economic benefits of rabbit farming. Furthermore, in subclinical conditions, intestinal coccidia infection can reduce the rabbit's immunity, leading to secondary diseases. Traditional coccidia control heavily relies on the addition of chemical drugs, resulting in problems such as drug resistance and drug residues. Therefore, vaccination has become the preferred solution for preventing rabbit coccidiosis.

[0004] However, research on rabbit coccidiosis vaccines is currently limited, and live vaccines are expensive and carry the risk of relapse. Therefore, developing novel control strategies such as subunit vaccines is of great significance. Summary of the Invention

[0005] In view of this, the present invention provides the application of recombinant rabbit coccidia protein in the prevention and control of rabbit coccidiosis.

[0006] This invention provides the application of recombinant rabbit coccidia proteins in the prevention and control of rabbit coccidiosis. rEmGAM56 and rEmROP17 can stimulate significant cellular and humoral immunity in rabbits and exhibit good anti-Eimeria macrocarcinoma infection effects, with rEmROP17 showing better performance in reducing oocyst excretion. Both can serve as candidate antigens for recombinant subunit vaccines against Eimeria macrocarcinoma. This invention lays the foundation for the research of recombinant subunit vaccines against Eimeria macrocarcinoma in rabbits.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of recombinant Eimeria macrocarpa protein in the preparation of any of the following:

[0009] (I) A recombinant subunit vaccine for the prevention and / or treatment of rabbit coccidiosis; and / or

[0010] (II) Vaccine antigens for the prevention and / or treatment of rabbit coccidiosis;

[0011] The recombinant proteins from *Eimeria macrocarpa* include rEmGAM56 and / or rEmROP17.

[0012] In some specific embodiments of the present invention, the prevention and / or treatment of rabbit coccidiosis includes inducing cellular immunity and / or humoral immunity.

[0013] In some specific embodiments of the present invention, the prevention and / or treatment of rabbit coccidiosis includes reducing oocyst expulsion.

[0014] In some specific embodiments of the present invention, the prevention and / or treatment of rabbit coccidiosis includes improving weight gain rate.

[0015] In some specific embodiments of the present invention, the prevention and / or treatment of rabbit coccidiosis includes reducing the feed conversion ratio.

[0016] In some specific embodiments of the present invention, the prevention and / or treatment of rabbit coccidiosis includes increasing antibody levels.

[0017] In some specific embodiments of the present invention, the antibody includes a specific antibody; the specific antibody includes IgG.

[0018] In some specific embodiments of the present invention, the prevention and / or treatment of rabbit coccidiosis includes increasing cytokine levels.

[0019] In some specific embodiments of the present invention, the cytokines include one or more of IL-2, IFN-γ, or IL-17.

[0020] The present invention also provides a recombinant subunit vaccine whose antigenic proteins include rEmGAM56 and / or rEmROP17.

[0021] In some specific embodiments of the present invention, the working concentration of rEmGAM56 includes 0.94 μg / well;

[0022] The working concentration of rEmROP17 is 1.13 μg / well.

[0023] In some specific embodiments of the present invention, the recombinant subunit vaccine further includes other antigenic components.

[0024] In some specific embodiments of the present invention, the recombinant subunit vaccine further includes an acceptable adjuvant.

[0025] In some specific embodiments of the present invention, the adjuvant of the recombinant subunit vaccine includes saponin Quil-A.

[0026] In some specific embodiments of the present invention, the method for detecting rabbit serum-specific antibody levels based on rEmGAM56 and / or rEmROP17 includes indirect ELISA.

[0027] In some specific embodiments of the present invention, the optimal dilution of the serum is 1:160.

[0028] In the above experiments, the present invention also provides primer sets, which include: primer set 1 for amplifying EmGAM56 and / or primer set 2 for amplifying EmROP17:

[0029] The primer set 1 includes:

[0030] (I) The upstream primer has the nucleotide sequence shown in SEQ NO:1; and

[0031] (II) The downstream primer has the nucleotide sequence shown in SEQ NO:2; or

[0032] (III) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) or (II), but differs from the nucleotide sequence shown in (I) or (II) due to the degeneracy of the genetic code; or

[0033] (IV) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to any of the nucleotide sequences shown in (I) to (III), and which has the same or similar function to any of the nucleotide sequences shown in (I) to (III); or

[0034] (V) A nucleotide sequence having at least 89.44% sequence homology with any of the nucleotide sequences described in (I) to (IV);

[0035] Primer set 2 includes:

[0036] (VI) The upstream primer has the nucleotide sequence shown in SEQ NO:3; and

[0037] (VII) The downstream primer has the nucleotide sequence shown in SEQ NO:4; or

[0038] (VIII) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (VI) or (VII), but is different from the nucleotide sequence shown in (VI) or (VII) due to the degeneracy of the genetic code; or

[0039] (IX) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to any of the nucleotide sequences shown in (VI) to (VIII), and which has the same or similar function to any of the nucleotide sequences shown in (VI) to (VIII); or

[0040] Nucleotide sequences having at least 66.96% sequence homology with any of (X) and (VI) to (IX).

[0041] In some specific embodiments of the present invention, the present invention also provides the application of the primer set in the preparation of kits for amplifying and / or detecting EmGAM56 and / or EmROP17.

[0042] Based on the above research, the present invention also provides a kit comprising the primer set and acceptable adjuvants or vectors.

[0043] Furthermore, the present invention also provides a method for constructing recombinant plasmids for obtaining the recombinant proteins rEmGAM56 and / or rEmROP17 of *Eimeria macrocarpa*, comprising the following steps:

[0044] (I) Using cDNA from Eimeria macrocarpa as a template;

[0045] (II) Amplify using the primer set or the kit.

[0046] This invention provides the application of recombinant rabbit coccidia proteins in the prevention and control of rabbit coccidiosis. rEmGAM56 and rEmROP17 can stimulate significant cellular and humoral immunity in rabbits and exhibit good anti-Eimeria macrocarcinoma infection effects, with rEmROP17 showing better performance in reducing oocyst excretion. Both can serve as candidate antigens for recombinant subunit vaccines against Eimeria macrocarcinoma. This invention lays the foundation for the research of recombinant subunit vaccines against Eimeria macrocarcinoma in rabbits. Attached Figure Description

[0047] 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.

[0048] Figure 1Multiple sequence alignments of EmGAM56 (a) and EmROP17 (b) from different species are shown in the examples. (a) shows multiple sequence alignments of the amino acid sequence of EmGAM56 with homologous sequences of other parasites GAM56: Eimeria stiedae (GenBank accession number: OL622034), Cyclospora cayetanensis (UniProt: A0A1D3D9G4), Eimeria tenella (UniProt: U6KUA4), Eimeria maxima (UniProt: U6M5G7), Eimeria acervulina (UniProt: U6GGM3); (b) shows multiple sequence alignments of the amino acid sequence of EmROP17 with homologous sequences of other parasites AMA1: Eimeria stiedae (GenBank accession number: OM451231), Eimeria mitis (UniProt: U6KAV7), Eimeria brunetti (UniProt:U6LD89), Eimeria acervulina (UniProt:U6GVC3), Eimeria tenella (UniProt:U6KG78); conserved residues are highlighted with a dark blue background; B cell antigenic epitopes are marked with dashed red boxes; signal peptides are marked with solid blue boxes.

[0049] Figure 2 SDS-PAGE and Western blot analyses of EmGAM56(a) and EmROP17(b) in the illustrated examples are shown. Lane M: protein molecular weight standard marker; Lane 1: crude extract of recombinant protein expressed by *E. coli* BL21(DE3) induced by IPTG; Lane 2: recombinant protein purified using a HisTrap HP pre-packed column; Lane 3: reaction of purified recombinant protein with rabbit *Eimeria tenella* positive serum; Lane 4: reaction of purified recombinant protein with coccidia-free rabbit serum; (arrows indicate the location of the bands).

[0050] Figure 3 The changes in specific antibody IgG in the serum of experimental rabbits after initial immunization with rEmGAM56(a) and rEmROP17(b) (week 0, indicated by arrows), booster immunization (week 2, indicated by arrows), and challenge with the parasite (week 4, indicated by arrows) are illustrated in the examples.

[0051] Figure 4The changes in serum cytokines IL-2 (a), IL-4 (b), IL-10 (c), IL-17 (d), IFN-γ (e), and TGF-β1 (f) in experimental rabbits two weeks after booster immunization in the illustrated examples; different superscript lowercase letters (a, b, c) indicate significant differences in cytokine levels among the experimental rabbits before challenge (P<0.05); the concentrations (mean±SD) of IL-2, IL-4, IL-10, IL-17, and IFN-γ are in pg / mL, and the concentration of TGF-β1 (mean±SD) is in ng / mL. Detailed Implementation

[0052] This invention discloses the application of recombinant rabbit coccidia protein in the prevention and control of rabbit coccidiosis. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted 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 obviously make modifications or appropriate alterations and combinations to 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.

[0053] In this invention, homologous genes GAM56 and ROP17 were identified in the transcriptome data of *Eimeria macrocarpa* in rabbits. Sequence analysis and prokaryotic expression were performed to obtain recombinant proteins rEmGAM56 and rEmROP17. The reactivity of the recombinant proteins was then detected by Western blotting. Subsequently, the efficacy of these two proteins against *Eimeria macrocarpa* infection was evaluated. Sixty healthy, coccidia-free New Zealand rabbits were randomly divided into six groups. The rEmGAM56 and rEmROP17 immunization groups were subcutaneously inoculated with 100 μg of recombinant protein rEmGAM56 or rEmROP17, respectively, in the neck. The PBS-uninfected and PBS-infected control groups were inoculated with an equal volume of sterile PBS. The Quil-A saponin control group was inoculated with an equal volume of Quil-A saponin solution. The Trx-His-S tag control group was inoculated with 100 μg of pET-32a(+) empty vector protein Trx-His-S tag. Except for the PBS-uninfected control group, the other experimental rabbits were orally vaccinated with 1×10⁻⁶ PBS two weeks after the booster immunization. 5 Large Eimeria coccidia sporulated oocysts were collected. Serum specific antibody levels were monitored weekly; simultaneously, ELISA was used to detect various cytokine levels in the serum of experimental rabbits two weeks after booster immunization. Clinical symptoms of the experimental rabbits were observed and recorded after infection. Oocyst excretion, relative weight gain, and feed conversion ratio were recorded for each group 14 days after challenge.

[0054] The results showed that recombinant proteins rEmGAM56 and rEmROP17 exhibited good reactivity. Immunoprotection assays indicated that, compared to the control group, immunization with recombinant proteins rEmGAM56 and rEmROP17 resulted in 63.85% and 80.10% reduction in oocysts, respectively, and 81.35% and 79.03% relative weight gain, respectively, with lower feed conversion ratios of 3.27:1 and 3.37:1, respectively. After immunization, the clinical symptoms in the protein-immunized groups were milder than in the control group, with only a few rabbits experiencing softened and unformed feces. The level of specific antibody IgG in the serum of rabbits in the protein-immunized groups increased rapidly after immunization. Significantly elevated levels of IL-2, IFN-γ, and IL-17 were detected in the serum of rabbits in both recombinant protein-immunized groups (P<0.05).

[0055] rEmGAM56 and rEmROP17 can stimulate significant cellular and humoral immunity in rabbits and exhibit good anti-Eimeria macrocephala infection effects, with rEmROP17 showing better performance in reducing oocyst excretion. Both can be considered as candidate antigens for recombinant subunit vaccines against Eimeria macrocephala.

[0056] Gametophyte antigen 56 (GAM56) is an antigen produced during the gametophyte stage of coccidia and is involved in the formation of the oocyst wall. In this invention, the number of oocysts expelled after immunization with recombinant protein rEmGAM56 in experimental rabbits was significantly reduced compared with the positive control group (P<0.05), with an oocyst reduction rate of 63.85% and a relative weight gain rate of 81.35%.

[0057] Rhoptry bodies are secretory organelles unique to Apicocomplex protozoa. The rhoptry proteins (ROPs) they secrete participate in processes such as parasite invasion of host cells and vacuole formation. Some ROPs, such as ROP16, ROP17, and ROP18, possess serine / threonine kinase domains and active sites, also known as ROP kinases (ROPK), and can function as virulence factors. In this invention, the amino acid sequence of EmROP17 was predicted, revealing the presence of a serine / threonine kinase and a catalytic domain. Immunization of experimental rabbits with recombinant protein rEmROP17 reduced weight gain loss, with a relative weight gain rate of 79.03%, while simultaneously reducing fecal oocysts by 80.10%. In summary, the results showed that immunization with recombinant proteins rEmGAM56 and rEmROP17 in rabbits demonstrated good anti-Eimeria macrocarcinoma infection effects, achieving worm reduction rates of 63.85% and 80.10%, and relative weight gain rates of 81.35% and 79.03%, respectively. Overall, rEmROP17 exhibited better immunoprotective efficacy against Eimeria macrocarcinoma infection.

[0058] In the detection results of this invention, two inoculations of experimental rabbits with rEmGAM56 and rEmROP17 induced significant humoral immunity, with a significant increase in the level of specific antibody IgG. Current research has demonstrated that antibodies play a role in combating coccidia infection; antigen-specific antibodies can inhibit the adhesion of parasites to host cells. Cytokines play an important role in combating coccidia infection, with IFN-γ, as a marker of Th1-type immune responses, playing a dominant role. Chicken IFN-γ not only inhibits the development of coccidia sporozoites in vitro, but also reduces weight loss and oocyst expulsion caused by coccidia infection after immunizing chicken flocks with its recombinant protein, thus exerting an anti-coccidia effect. IL-2 exerts its anti-coccidia effect by inducing T cell proliferation and increasing the proportion of CD8+ and CD4+ T cells in peripheral blood. Simultaneously, these cytokines can also serve as adjuvants for coccidia vaccines, enhancing the immune response to vaccine antigens and thereby improving vaccine efficacy. When researchers combined vaccine antigens with IFN-γ or IL-2, they found that compared with using vaccine antigens alone, chicken flocks showed enhanced resistance to coccidia, further reducing oocyst excretion and improving weight gain. This invention found that the recombinant immune proteins rEmGAM56 and rEmROP17 significantly increased serum IL-2 and IFN-γ levels in experimental rabbits (P<0.05), indicating that they can stimulate Th1-type immune responses. A significant increase in serum IL-17 levels was also observed in both protein immunization groups (P<0.05). IL-17 plays an important role in protective immunity against parasitic infections. Studies have found that Toxoplasma gondii infection of IL-17RA knockout mice and wild-type mice treated with IL-17 neutralizing antibodies resulted in prolonged survival time compared to the control group. Ding et al. co-immunized chicken embryos with recombinant Eimeria tenella 3-1E protein and an expression plasmid encoding IL-17, finding that the ability of chicken flocks to resist Eimeria tenella infection after hatching was further enhanced compared to immunization with 3-1E protein alone. Geriletu et al. obtained similar results, showing that the IL-17 gene can enhance the anticoccidial effect of vaccine antigens.

[0059] This invention amplifies the EmGAM56 and EmROP17 genes from the first-ever transcriptome analysis of *Eimeria tenella* in rabbits. These genes are then cloned into the prokaryotic vector pET32a(+) and expressed in *E. coli* competent cells to obtain recombinant proteins GAM56 (rEmGAM56) and ROP17 (rEmROP17). Subsequently, vaccine trials were conducted using Quil-A as an adjuvant to preliminarily evaluate the immunoprotective effects of these proteins. Simultaneously, the stimulatory effects of recombinant proteins rEmGAM56 and rEmROP17 on humoral and cellular immune responses were determined. This invention lays the foundation for the research of recombinant subunit vaccines against *Eimeria tenella* in rabbits.

[0060] Materials and Methods

[0061] 1. Insect strains and laboratory animals

[0062] The large Eimeria coccidia were passaged and preserved by the Parasitology Research Center of Sichuan Agricultural University. Unspecified oocysts, sporulated oocysts, schizonts, and gametophytes of the large Eimeria coccidia were provided by the Animal Parasitic Diseases Research Center of Sichuan Agricultural University.

[0063] Sixty healthy, coccidioidom-free New Zealand rabbits (35 days old, 0.84±0.108 kg) were bred at the Animal Parasitic Diseases Research Center of Sichuan Agricultural University. The rabbits were fed according to the method reported by Wei et al. After weighing and blood collection, the rabbits were randomly divided into 6 groups (n=10). The rEmGAM56 and rEmROP17 immunization groups received subcutaneous injections of recombinant proteins rEmGAM56 and rEmROP17 (100 μg + 1 mg Quil-Adilution in 1 mL PBS), respectively, into the neck. The PBS-infected, Quil-A saponin, and Trx-His-S tag control groups received subcutaneous injections of equal volumes of sterile PBS, Quil-A saponin solution, and pET-32a(+) empty vector protein Trx-His-S tag, respectively, into the neck as three positive control groups. The PBS-uninfected control group received an equal volume of sterile PBS using the same method as a blank control group. Except for the PBS-uninfected control group, the other experimental rabbits were orally vaccinated with 1×10⁻⁶ PBS two weeks after the second immunization. 5 Large Eimeria sporulated oocysts (Table 1).

[0064] 2. Data Analysis

[0065] All data are expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare differences among multiple groups, and IBM SPSS Statistics 22.0 was used for statistical analysis. A p-value less than 0.05 was considered statistically significant, and a p-value less than 0.01 was considered highly statistically significant. GraphPad Prismversion 5.0 (GraphPad Software) was used for plotting.

[0066] The raw materials and reagents used in the application of the recombinant rabbit coccidia protein provided by this invention in the prevention and control of rabbit coccidiosis are all commercially available.

[0067] The present invention will be further illustrated below with reference to the embodiments:

[0068] Example 1: Sequence Analysis of EmGAM56 and EmROP17

[0069] The open reading frames of genes were identified and their corresponding amino acid sequences were obtained using the ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ). ExPASy Proteomics Server

[0070] (http: / / web.Expasy.org / protparam / ) predicts isoelectric point and molecular weight. TMHMM Server v.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / #opennewwindow) predicts transmembrane regions of amino acid sequences; SignalP4.1 (http: / / www.cbs.dtu.dk / services / SignalP / ) predicts the presence of signal peptides. IEBD Analysis Resource (http: / / tools.immuneepitope.org / bcell / ) predicts B cell antigenic epitopes. Multiple sequence alignment was performed using Jalview version 2.11.2.0.

[0071] Example 2: Cloning, Expression, and Purification of rEmGAM56 and rEmROP17

[0072] Total RNA was extracted from the worms at four different stages according to the instructions of the total RNA extraction kit. Then, cDNA was synthesized according to the instructions of the reverse transcription kit. The resulting complementary double-stranded cDNA was stored at -80℃.

[0073] Based on transcriptome data of *Eimeria macrocarpa* obtained from the Animal Parasitic Diseases Research Center of Sichuan Agricultural University, two gene-specific primers, EmGAM56 (forward primer: 5'-CGGGATCCATGGAACCCTCTACCATTGAG-3', as shown in SEQ NO:1; reverse primer: 5'-GCGTCGACTTAGAAAGGCATGCCTGC-3', as shown in SEQ NO:2) and EmROP17 (forward primer: 5'-CGGGATCCATGTACAGCCTCTTACAAGGTCAC-3', as shown in SEQ NO:3; reverse primer: 5'-GCGTCGACCTACTCTGAGCTTTTTCCTTCACT-3', as shown in SEQ NO:4), were designed using Primer Premier 5.0 software. The upstream and downstream restriction enzyme sites BamHI and SalI (underlined, TaKaRa, Dalian, China) were added, respectively. Following PCR amplification, the PCR products were separated by 0.1% agarose gel electrophoresis, purified using a DNA purification kit, and then ligated into the expression vector pET-32a(+). The recombinant plasmids pET-32a(+)-EmGAM56 and pET-32a(+)-EmROP17 were sent to a sequencing facility (Sangon, Shanghai, China). Successfully sequenced recombinant plasmids were transformed into *E. coli* BL21(DE3) competent cells for protein expression (1 mM IPTG). The recombinant proteins were purified using a HisTrap HP pre-packed column (Cytiva, USA). Protein purification was assessed by SDS-PAGE, and the concentrations of the two purified recombinant proteins were determined using a BCA protein assay kit (Solarbio, Beijing, China). The pET32a(+) empty vector protein (without the inserted foreign fragment) was provided by the Animal Parasitic Diseases Research Center of Sichuan Agricultural University.

[0074] Example 3 Immunoblot Analysis

[0075] Positive serum from Eimeria macrocarpa was collected from artificially infected Eimeria macrocarpa sporulated oocysts (1×10⁻⁶). 5 (Number of rabbits per rabbit) New Zealand rabbits. Large Eimeria coccidia-negative serum was collected from 1-month-old coccidia-free rabbits. All serum samples were stored at -20°C.

[0076] After purification, the recombinant protein was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Boster, Wuhan, China) using a semi-dry transfer tank (Bio-Rad, USA) for 45 min. The membranes were washed with TBST for 5 min × 3 times, then blocked with 5% (w / v) skim milk powder at room temperature (23℃±2℃) for 2 h. Primary antibodies (1:200 v / v dilution in TBS) were used as both rabbit Eimeria macrocarpa positive and negative sera, and the membranes were incubated overnight at 4℃. After discarding the primary antibody, the membranes were washed with TBST for 5 min × 4 times, and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG was added as a secondary antibody (EarthOxLife Sciences, Millbrae, CA, USA, 1:2000 v / v dilution), and incubated at room temperature (23℃±2℃) for 2 h. Finally, discard the secondary antibody, wash the membrane with TBST for 5 min × 4 times, and use the DAB colorimetric kit (20×) to develop the color in the dark (Solarbio, Beijing, China). When the bands appear, add double-distilled water to stop the reaction.

[0077] Example 4 Experimental Design

[0078] The experimental animals were grouped and immunization procedures are shown in Table 1. The experimental rabbits were euthanized two weeks after the parasite attack.

[0079] Table 1. Grouping and Immunization Procedures of Experimental Animals

[0080]

[0081] Example 5: Evaluation of Immunoprotective Effect

[0082] Safety observation: The health status of all experimental rabbits was observed after immunization. The weight of each group of rabbits was recorded at the first immunization, the second immunization, and the time of challenge. The average weight gain after immunization = weight at challenge - weight before the first immunization, thus verifying whether the immunization process of this invention affects the weight gain of rabbits.

[0083] After the parasite treatment, observe the mental state of each group, whether there is a decrease in thirst and appetite, and whether there is diarrhea. After necropsy, observe whether there is congestion, hemorrhage and hyperplasia in the posterior end of the jejunum and ileum.

[0084] The effectiveness of immune protection was assessed based on survival rate, weight gain, oocyst expulsion, and feed conversion ratio. Survival rate was obtained by dividing the number of surviving rabbits in each group by the initial number of rabbits in each group. Weight gain after infection in each group was calculated as pre-necropsy weight minus pre-infection weight, with the relative weight gain rate calculated as: (Average weight gain in the experimental group after infection / Average weight gain in the PBS-uninfected group after infection) × 100%. At necropsy, 2g of feces was collected from the rectum of each rabbit. The McMaster method was used to calculate the oocyst expulsion rate (OPG) per gram of feces, and the oocyst reduction rate was calculated as: (PBS-infected control group OPG - immunized group OPG) / PBS-infected control group OPG × 100%. Feed conversion ratio was calculated as: (Total feed before feeding - Total feed remaining after necropsy) / Total weight gain after infection.

[0085] Example 6: Serum Antibody Level Measurement

[0086] To observe the specific changes in serum IgG levels in experimental rabbits after immunization with two recombinant proteins, serum samples from rabbits aged Weeks 0 to 6 were collected, and all serum samples were stored at -20°C for later use.

[0087] The levels of specific antibody IgG in the serum of rabbits in the corresponding protein immunization group and the control group were detected using an indirect ELISA method based on recombinant proteins rEmGAM56 and rEmROP17, respectively. The working concentrations of rEmGAM56 and rEmROP17 were 0.94 μg / well and 1.13 μg / well, respectively, and the optimal serum dilution was 1:160.

[0088] Example 7: Serum Cytokine Level Measurement

[0089] Rabbit IFN-γ, IL-2, IL-4, IL-10, IL-17, and TGF-β1 were purchased from CUSABIO Corporation in China. Cytokine assays were performed according to the manufacturer's instructions.

[0090] Example 1: Sequence characteristics of EmGAM56 and EmROP17

[0091] Sequence analysis revealed that the ORF of the EmGAM56 gene (GenBank accession number: OM451230) is 1371 bp long, encoding 456 amino acids, with a predicted protein molecular weight of approximately 51 kDa (PI = 4.91); the ORF of the EmROP17 gene (GenBank accession number: OM451229) is 1725 bp long, encoding 574 amino acids, with a predicted protein molecular weight of approximately 63 kDa (PI = 9.80). Neither gene contains a transmembrane region, but signal peptides were predicted at amino acids 1–20 and 1–22, respectively, with de-signal peptide fragment sizes of 1314 bp and 1662 bp, respectively.

[0092] Multiple sequence alignment results showed that while EmGAM56 exhibited high variability compared to other apical protozoa, it shared 89.44% homology with *Eimeria steudensis*, which also parasitizes rabbits. The EmROP17 sequence showed the highest homology with *Eimeria steudensis* (66.96%), while its homology with other *Eimeria* species ranged from 28.01% to 37.95%. Figure 1 ).

[0093] Example 2: Expression, purification, and immunoblotting analysis of recombinant proteins

[0094] Two successfully constructed recombinant plasmids were transformed into *E. coli* competent cells BL21(DE3). After induction at 27°C for 12 h with 1 mmol / L IPTG, the recombinant proteins rEmGAM56 (de-signal peptide, ~48 kDa) and rEmROP17 (de-signal peptide, ~54 kDa) were successfully expressed in BL21(DE3). Figure 2 Both were expressed in the supernatant (lane 1). The molecular weight of the recombinant protein included a ~20 kDa fusion peptide encoded by pET-32a(+). The recombinant protein was purified using a HisTrap HP pre-packed column, and the expression band was consistent with the size of the target gene (lane 1). Figure 2 (lane 2)

[0095] Immunoblotting results showed that both rEmGAM56 and rEmROP17 reacted with serum from rabbits infected with Eimeria macrocarpa, and a single specific band was observed on the NC membrane. Figure 2 Lane 3), while incubation with coccidia-free rabbit serum resulted in no band appearing at the corresponding position ( Figure 2 Lane 4 indicates that the two recombinant proteins have strong reactivity.

[0096] Example 3: Evaluation of the immunoprotective effects of rEmGAM56 and rEmROP17

[0097] After initial and booster immunization, there was no significant difference in average weight gain among the six groups of experimental rabbits (Table 2) (P>0.05), and no obvious adverse reactions were observed, indicating that the recombinant protein has good safety.

[0098] Oral infection 1×10 5 In the second week after the sporulated oocysts were formed, the control group rabbits showed a slight decrease in appetite, and some rabbits experienced negative weight gain; only a few rabbits showed obvious diarrhea, mostly manifested as soft and unformed feces. In contrast, the rEmGAM56 and rEmROP17 immunized groups showed no obvious clinical symptoms, and a few rabbits had unformed feces.

[0099] The immunogenicity of six recombinant proteins was preliminarily assessed using indicators such as survival rate, weight gain, oocyst expulsion, and feed conversion ratio. Results showed that, after challenge with the parasite, no deaths occurred in any of the groups except for the Quil-A saponin control group (1 / 10 of the rabbits). The average weight gain after challenge in the rEmGAM56 and rEmROP17 immunization groups was between that of the blank control group and the three challenged control groups, with no significant difference (P > 0.05). The relative weight gain rates were 81.35% and 79.03%, respectively.

[0100] Compared with the three control groups, the number of oocysts released in the rEmGAM56 and rEmROP17 immunization groups was significantly reduced (P<0.05), with oocyst reduction rates of 63.85% and 80.10%, respectively, while there was no significant difference in the number of oocysts released between the two protein immunization groups (P>0.05).

[0101] From infection to 14 days post-infection, the feed conversion ratios of both protein-immunized groups were higher than those of the parasite-challenged control group. The feed conversion ratios (FCRs) of the rEmGAM56 and rEmROP17 immunized groups were 3.27:1 and 3.37:1, respectively, while the FCRs of the PBS-infected, Quil-Asaponin, and Trx-His-S tag control groups reached 4.27:1, 4.15:1, and 3.94:1, respectively.

[0102] Table 2. The efficacy of rEmGAM56 and rEmROP17 against Eimeria macrocarcinoma infection under different evaluation indicators.

[0103]

[0104] Data are presented in the form of mean ± standard deviation (SD). In each column, there are significant differences between data with different letter labels (P < 0.05), and no significant differences between data with the same letter labels (P > 0.05).

[0105] Example 4: Changes in serum specific antibody levels

[0106] Following immunization with recombinant proteins rEmGAM56 and rEmROP17, specific antibody levels in rabbits gradually increased. The rEmGAM56 immunization group reached and maintained a relatively high level by week 4; while the rEmROP17 immunization group continued to show an increase in specific antibody levels after challenge. In addition, the Trx-His-S tag protein immunization group also showed a significant increase in antibody levels, indicating that the recombinant protein used for ELISA detection contained the tag protein from the pET32a(+) vector, but its antibody level was significantly lower than that of the two recombinant protein immunization groups. The PBS-infected, Quil-A saponin, and Trx-His-S tag control groups showed no significant changes in specific antibody levels (Tables 3 and 4). Figure 3 ).

[0107] Table 3. Determination of anti-rEmGAM56 specific IgG levels (OD) 450 )

[0108]

[0109]

[0110] Table 4. Determination of anti-rEmROP17 specific IgG levels (OD) 450 )

[0111]

[0112] Example 5: Changes in different cytokines in serum

[0113] Two weeks after booster immunization, the serum levels of IL-2, IL-4, IL-10, IL-17, IFN-γ, and TGF-β1 in experimental animals in the PBS-infected, Quil-A saponin, Trx-His-S tag control groups, and the rEmGAM56 and rEmROP17 immunization groups were measured. The results showed that the serum levels of IL-17 and IFN-γ in the rEmGAM56 immunization group were significantly higher than those in the three control groups (P<0.05); while the IL-2 level was significantly higher than that in the PBS-infected and Trx-His-S tag control groups (P<0.05), there was no significant difference compared to the Quil-A saponin control group (P>0.05). The serum levels of IL-2, IL-17, and IFN-γ in the rEmROP17 immunization group were all significantly higher than those in the three control groups (P<0.05). IL-4 and IL-10 levels were significantly higher in rabbit serum from both protein immunization groups, PBS-infected, and the Trx-His-S tag control group compared to PBS-infected (P<0.05). TGF-β1 levels showed no significant difference among the two protein immunization groups and the three control groups (P>0.05) (Table 5). Figure 4 ).

[0114] Table 5. Results of detection of different cytokine levels in each group of experimental rabbits.

[0115]

[0116]

[0117] 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. Application of recombinant Eimeria macrocarpa protein in the preparation of any of the following: (I) Recombinant subunit vaccines for the prevention and / or treatment of rabbit coccidiosis; and / or (II) Vaccine antigens for the prevention and / or treatment of rabbit coccidiosis; The recombinant protein from the large Eimeria coccidia is rEmROP17. The GenBank accession number for the EmROP17 gene is OM451229; The rEmROP17 is a recombinant protein encoded by the EmROP17 gene with the signal peptide removed. The rabbit coccidiosis is caused by Eimeria macrocarpa.

2. The application as described in claim 1, characterized in that, The prevention and / or treatment of rabbit coccidiosis includes inducing cellular and / or humoral immunity.

3. The application as described in claim 2, characterized in that, The prevention and / or treatment of rabbit coccidiosis includes reducing oocyst expulsion and / or increasing weight gain.

4. The application as described in claim 3, characterized in that, The prevention and / or treatment of rabbit coccidiosis includes reducing the feed conversion ratio.

5. The application as described in claim 4, characterized in that, The prevention and / or treatment of rabbit coccidiosis includes increasing antibody levels; The antibody includes a specific antibody; the specific antibody includes IgG.

6. The application as described in any one of claims 1 to 5, characterized in that, The prevention and / or treatment of rabbit coccidiosis includes increasing cytokine levels; The cytokines include one or more of IL-2, IFN-γ, or IL-17.

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