A nanobody specifically binding eimeria subspores and uses thereof

Abstract

The application belongs to the technical field of biotechnology and veterinary medicine, and particularly relates to a nano-antibody specifically combined with Eimeria stiedai sporozoite and application thereof. The nano-antibody comprises an amino acid sequence shown in SEQ ID NO:1. Experiments show that the nano-antibody can be efficiently combined with Eimeria stiedai sporozoite, and significantly inhibit invasion of the sporozoite into rabbit intrahepatic bile duct epithelial cells. The nano-antibody provides a new type of efficient biological preparation candidate for preventing and controlling rabbit Eimeria stiedai disease, and has important diagnostic application value.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and veterinary medicine, specifically relating to a nanobody that specifically binds to sporozoites of Eimeria stearothermiae and its application. Background Technology

[0002] Rabbit coccidiosis is a serious parasitic disease caused by Eimeria species, specifically Eimeria spp., which severely damages the rabbit industry. Eimeria spp. primarily parasitizes the hepatobiliary epithelial cells of rabbits, causing severe hepatic coccidiosis, leading to high mortality rates in young rabbits and significant economic losses to the rabbit farming industry. Currently, control of this disease mainly relies on the long-term addition of chemical anticoccidial drugs to feed, such as sulfonamides and polyether ionotropic agents. However, the long-term and widespread use of chemical drugs has led to increasingly serious drug resistance in coccidia, reducing or even eliminating the effectiveness of many drugs. Furthermore, the use and residues of chemical drugs also pose potential food safety and public health risks.

[0003] Although a trivalent live rabbit vaccine against intestinal coccidia has been commercialized, its primary target is intestinal coccidia. Its effectiveness against hepatic coccidia caused by *Eimeria skrvii* has not been clearly reported, and live vaccines themselves suffer from issues such as virulence reversion and stringent transportation and storage conditions. Therefore, developing novel, efficient, and safe *Eimeria skrvii* control technologies, especially biological agents capable of blocking key stages of its invasion, is of significant practical importance.

[0004] Nanobodies (Nb) are variable region fragments of heavy chain antibodies lacking the light chain, derived from camelids, and are the smallest natural antigen-binding units. Compared to traditional antibodies, nanobodies have significant advantages, including small molecular weight, structural stability, high affinity, easy tissue penetration, ability to recognize cryptic epitopes, relatively low production cost, and ease of genetic engineering. In recent years, nanobodies have been widely used in infectious disease diagnosis, targeted tumor therapy, and pathogen function research. For example, studies have reported specific nanobodies against pathogens such as Brucella, porcine epidemic diarrhea virus, and Newcastle disease virus, which have been successfully used for diagnosis or have neutralizing activity.

[0005] In the field of parasitic diseases, some studies have attempted to utilize nanobodies as targeting carriers or diagnostic tools. For example, some researchers have combined nanobodies that specifically recognize the surface antigens of *Trypanosoma africanum* with drug-loaded nanoparticles to achieve targeted drug delivery. However, for *Eimeria*, especially the tissue-specific (hepatobiliary) *Eimeria stearothermiae*, there are currently no reports of functional nanobodies that can directly block the invasion of its sporozoites into host cells. Summary of the Invention

[0006] To address the shortcomings of existing technologies for the prevention and control of Eimeria schlegelii in rabbits, which rely on chemical drugs and lack highly effective targeted biological agents, this invention aims to provide a nanobody that can specifically bind to and inhibit the invasion of Eimeria schlegelii sporozoites to overcome drug resistance problems, reduce drug residues, and provide a novel approach for the prevention, control, and diagnosis of coccidiosis.

[0007] In one aspect, the present invention provides a nanobody, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] In one aspect, the present invention provides a polynucleotide encoding the aforementioned nanobody.

[0009] In one aspect, the present invention provides a recombinant vector comprising the aforementioned polynucleotide.

[0010] In one aspect, the present invention provides a host cell comprising the recombinant vector described above.

[0011] In one aspect, the present invention provides the use of the nanobodies, polynucleotides, recombinant vectors or host cells in the preparation of medicaments for the prevention and / or treatment of Eimeria tenella squarrosa disease in rabbits.

[0012] In one aspect, the present invention provides the use of the nanobody, polynucleotide, recombinant vector or host cell in the preparation of diagnostic reagents or kits for rabbit Eimeria stearothermia.

[0013] In one aspect, the present invention provides a pharmaceutical composition comprising the aforementioned nanobody.

[0014] In one aspect, the present invention provides a diagnostic reagent or kit comprising the nanobody, polynucleotide, recombinant vector or host cell.

[0015] In one aspect, the present invention provides a feed additive comprising the aforementioned nanobody.

[0016] The beneficial effects of this invention are as follows: The nanobody provided by this invention has the advantages of small molecular weight, good stability and strong tissue permeability, and can effectively inhibit the invasion of cells by Eimeria stearothermiae sporozoites; This antibody can be used in the development of drugs or feed additives for the prevention or treatment of rabbit coccidiosis, reducing the use of chemical drugs, and can also be used as the core recognition element of diagnostic kits. It has the characteristics of safety, high efficiency and versatility, and provides a new strategy for the green prevention and control of coccidiosis. Attached Figure Description

[0017] Figure 1 Electrophoresis diagram of total RNA from PBMCs;

[0018] Figure 2 Electrophoresis image of the first round of VHH-PCR amplification;

[0019] Figure 3 Electrophoresis image of the second round of VHH-PCR amplification;

[0020] Figure 4 This is a PCR electrophoresis image of the vector.

[0021] Figure 5 The results are for the determination of the library capacity of the electroporation conversion library;

[0022] Figure 6 The positive rate was determined by agarose gel electrophoresis results.

[0023] Figure 7 Immunofluorescence image of B2C8 nanobody binding to sporozoites of Eimeria stearothermiae (A: B2C8 nanobody group; B: PBS blank control group).

[0024] Figure 8 Flow cytometry analysis of the B2C8 nanobody inhibiting the invasion of cells by sporozoites of Eimeria stearothermiae. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All features disclosed in this specification, or steps in all disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0026] In this invention, unless otherwise stated, the following terms have the following meanings:

[0027] "Nucleic acid" or "polynucleotide": refers to the nucleotide sequence encoding the nanobody (with the amino acid sequence shown in SEQ ID NO:1) that specifically targets the sporozoites of *Eimeria stearothermiae* as described in this invention. This sequence can be obtained by reverse translating the amino acid sequence of the nanobody according to the genetic codon, or by codon optimization (e.g., optimization to preferred codons suitable for *E. coli* or mammalian cell expression systems) or other modifications known to those skilled in the art to improve its expression efficiency or stability. The nucleic acid includes DNA, RNA, and their analogues or derivatives.

[0028] "Vector" or "expression vector": refers to a recombinant DNA construct containing the aforementioned "nucleic acid" molecule and capable of guiding the expression of the nanobody in a suitable host cell. The vector may be selected from various vectors known in the art, including but not limited to plasmids, phage particles, and viral vectors (such as lentiviral vectors and adeno-associated virus vectors). The vector typically contains elements operatively linked to the nucleic acid and essential for controlling expression, such as promoters (e.g., CMV promoters, T7 promoters), enhancers, terminators, polyadenylation signals, and selection marker genes (e.g., antibiotic resistance genes).

[0029] "Host cell": refers to a cell that has been introduced with the above-mentioned "expression vector" through conventional molecular biology methods such as transformation, transfection, and electroporation, and can be used to produce the nanobody described in this invention. The host cell can be a prokaryotic cell (such as Escherichia coli) or a eukaryotic cell (such as yeast cells, insect cells, mammalian cells, such as HEK293 cells and CHO cells).

[0030] "Expression": In this invention, "expression" refers to the behavior of the host cell, under suitable conditions, utilizing its expression system to perform a series of processes such as transcription and translation, ultimately producing a biologically active nanobody as shown in SEQ ID NO:1. The expression product can be located intracellularly or secreted into the culture supernatant.

[0031] Example 1: Screening, expression, and purification of anti-Eimeria stearothermiae sporozoite nanobodies

[0032] 1 Experimental Methods

[0033] 1.1 Preparation of Immunogen

[0034] (1) Preparation of rSAG recombinant protein

[0035] The gene sequence encoding the sporozoite surface antigen (rSAG) of *Eimeria steggii* was codon-optimized and synthesized. The target fragment was subcloned into the pET-28a vector, fused with a His tag, and the target protein was expressed using an *E. coli* expression system. Expression and purification tests were then performed. The construction scheme is as follows: pET-28a + NcoI + 6×His + target protein + stop codon + XhoI.

[0036] (2) Preparation of inactivated sporozoites

[0037] The sporozoites of Eimeria stearothermiae were inactivated by treatment at 75°C for 8 minutes and then set aside for later use.

[0038] 1.2 Immunization regimen and potency testing

[0039] (1) Immunization regimen

[0040] Camels were immunized five times with sporozoites of Eimeria stearothermiae and recombinant sporozoite surface antigen (rSAG). The immunization schedule is as follows:

[0041] Day 1 (first immunization): 1000 μg rSAG antigen (1000 μL) and 1000 μL of sporozoites were injected subcutaneously at multiple sites, respectively, after being emulsified with an equal volume of complete Freund's adjuvant.

[0042] Day 15 (second immunization): 1500 μg rSAG antigen (1000 μL) was mixed with an equal volume of incomplete Freund's adjuvant and emulsified, then injected subcutaneously at multiple sites; 5 mL of peripheral blood was collected to detect serum titer.

[0043] Day 22: Collect 5 mL of peripheral blood to test serum titer.

[0044] Day 27 (Third Immunization): 1000 μL of sporozoites was emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple points.

[0045] Day 37: Collect 5 mL of peripheral blood to test serum titer.

[0046] Day 43 (Fourth Immunization): 1000 μg rSAG antigen (1000 μL) and 1000 μL of spirochetes were injected subcutaneously at multiple sites, respectively, and emulsified with an equal volume of incomplete Freund's adjuvant.

[0047] Day 53: Collect 5 mL of peripheral blood to test serum titer; at the same time, collect 200 mL of peripheral blood for PBMC isolation.

[0048] Day 58 (Five Immunizations): 500 μg rSAG antigen (1000 μL) and 1000 μL of spirochetes were injected subcutaneously at multiple sites, respectively, and emulsified with an equal volume of incomplete Freund's adjuvant.

[0049] Day 65: 200 mL of peripheral blood was collected, and PBMCs were isolated for library construction.

[0050] (2) Valence testing

[0051] Serum titers were determined using ELISA. rSAG antigen was dissolved in CBS coating buffer (pH 9.6) at a concentration of 2 μg / mL and coated overnight at 4°C. After blocking, serially diluted camel serum (1:10 to 1:32000) was added, followed by incubation and then secondary antibody. OD450 values ​​were read after TMB colorimetry.

[0052] 1.3 Total RNA extraction and cDNA synthesis

[0053] 200 mL of peripheral blood was collected from immunized camels, and PBMCs were isolated. Total RNA was extracted from the PBMCs, and the first strand of cDNA was synthesized using Oligo dT primers and reverse transcriptase as a template.

[0054] 1.4 First round of PCR amplification of VHH

[0055] Using cDNA as a template, amplification was performed using primers CAL001F and CAL002R. PCR reaction conditions: 94℃ for 3 min; 30 cycles (94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s); 72℃ for 7 min. The product was subjected to 2% agarose gel electrophoresis, and the target band of approximately 600 bp was excised and recovered.

[0056] 1.5 Second round of nested PCR amplification of VHH

[0057] Using the recovered product from the first round as a template, amplification was performed using primers F2 and R2. Reaction conditions: 95℃ for 3 min; 13 cycles (98℃ for 10 s, 55℃ for 10 s, 72℃ for 30 s); 72℃ for 7 min. The product was subjected to 1% agarose gel electrophoresis, and the target band of approximately 600 bp was excised and recovered.

[0058] 1.6 Preparation of phage vectors

[0059] Using pComb3x as a template, amplification was performed using primers p-SFI-F and p-SFI-R. Reaction conditions: 95℃ for 3 min; 30 cycles (98℃ for 10 s, 55℃ for 10 s, 72℃ for 60 s); 72℃ for 7 min. The product was subjected to 2% agarose gel electrophoresis, and the target band of approximately 3000 bp was excised and recovered. The recovered product was digested with SfiI enzyme overnight, and the linearized vector was purified and recovered.

[0060] 1.7 Enzyme digestion of the VHH fragment

[0061] The VHH fragment recovered in the second round was digested with SfiI overnight, and the product was recovered for later use. 1.8 Library Preparation

[0062] The enzyme-digested VHH fragment was mixed with the linearized pComb3x vector at a molar ratio of 3:1, and T4 DNA ligase was added. The mixture was ligated overnight at 16°C. After purification, the ligation product was transformed into TG1 competent cells by electroporation, and the cells were revived in 2YT medium. The library capacity was determined by plate-spreading serially diluted solutions, and the remainder was plated on large plates.

[0063] 1.8 Document Quality Inspection

[0064] Twenty-four single colonies were randomly selected for bacterial PCR, and the insertion rate was detected by agarose gel electrophoresis. 10 μL of a phage library was serially diluted and used to infect TG1 bacteria, then plated on Amp antibody plates, and the titer was calculated.

[0065] 1.9 Packaging of Phage Antibody Libraries

[0066] The library was inoculated into TG1 bacterial culture and cultured to the logarithmic phase. M13KO7 helper phage (helper phage: TG1 ≥ 10: 1) was added for superinfection. After overnight culture at 30°C, the phage supernatant was collected.

[0067] 1.10 Affinity Screening

[0068] Three rounds of screening were conducted, targeting recombinant rSAG protein and inactivated sporozoites, respectively.

[0069] (1) rSAG solid phase screening:

[0070] R1: 20 μg rSAG, washed 8 times with 0.1% PBST, phage loading 1×10¹².

[0071] R2: 20 μg rSAG, washed 12 times with 0.3% PBST, phage loading amount 7.6×10¹¹.

[0072] R3: 20 μg rSAG, washed 15 times with 0.5% PBST, phage loading amount 6.6×10¹¹.

[0073] (2) Solid-phase screening of sporozoites:

[0074] R1: Inactivated sporozoites, washed 8 times with 0.1% PBST, phage loading 1×10¹².

[0075] R2: Inactivated sporozoites, washed 12 times with 0.3% PBST, phage loading 3×10¹¹.

[0076] R3: Inactivated sporozoites, washed 15 times with 0.5% PBST, phage input amount 2.9×10¹¹.

[0077] Each round of screening steps: After coating with antigen and blocking, incubate with the blocked phage library, wash with PBST, elute with glycine-hydrochloric acid (pH 2.2), and neutralize with Tris-HCl (pH 8.0). The elution product is used to infect TG1 bacteria for amplification and is used for the next round of screening.

[0078] 1.11 Monoclonal ELISA Identification

[0079] After the third round of screening, 96 monoclonal phages were randomly selected and placed in 96-well deep-well plates to prepare monoclonal phage supernatants.

[0080] ELISA plates were coated with rSAG protein and inactivated sporozoites, respectively: rSAG protein was diluted to 0.1 μg / mL in pH 9.0 PBS, with 80 μL / well for coating; inactivated sporozoites were treated at 75℃ for 8 min and then diluted to 10 mL PBS, with 100 μL / well for coating. After blocking, monoclonal phage supernatant (mixed with 2% skim milk powder 1:1) was added, followed by incubation. M13 secondary antibody (1:10000 dilution) was then added, and OD450 values ​​were read after TMB color development. Positive wells were identified based on OD450 values.

[0081] 1.12 Sequencing of positive clones

[0082] Positive clones were sequenced to obtain the target antibody sequence.

[0083] 1.13 Expression and purification of B2C8 nanobodies

[0084] The obtained B2C8 VHH gene was cloned into the expression vector PCDNA3.4-hIgG1-Fc to construct a nanobody expression plasmid. The expression plasmid was transfected into EXPI293 cells for expression. The cell culture supernatant was collected, and the expressed nanobody-Fc fusion protein was purified to obtain a high-purity B2C8 nanobody.

[0085] 2. Experimental Results

[0086] 2.1 Serum titer test results

[0087] The results are shown in Table 1. Serum titers gradually increased with the number of immunizations. After the first immunization, the serum titer was approximately 1:10; after the second immunization, it rose to approximately 1:100; after the third immunization, it remained at approximately 1:100; and after the fourth immunization, it reached approximately 1:1000. These results indicate that the camels produced a highly efficient anti-rSAG immune response after four immunizations, which can be used for subsequent library construction.

[0088] Table 1. Serum titer ELISA results (OD450 values) at different immune stages

[0089]

[0090] 2.2 Library Construction Results

[0091] Electrophoresis analysis of extracted PBMC total RNA revealed clear 28S, 18S, and 5S rRNA bands with no obvious degradation. Figure 2 The first round of PCR amplification yielded clear electrophoretic bands for the VH and VHH chains, approximately 800 bp and 500-600 bp in size, respectively. Figure 3The second round of nested PCR amplification yielded a clear and bright VHH heavy chain target band of approximately 300 bp. Figure 4 The library volume after electroporation of the linker product was determined to be 6.07 × 10⁻⁶. 9 CFU ( Figure 5 Twenty-four single clones were randomly selected for bacterial culture PCR identification. All clones showed a target band of approximately 400 bp, with an insertion rate of 100%, and the antibody library positivity rate was satisfactory. Figure 6 The packaged phage antibody library was serially diluted, and the titer was 2.92 × 10¹³ pfu / mL.

[0092] 2.3 Affinity Screening Results

[0093] The results of each round of screening are as follows:

[0094] rSAG screening: R1 output 3.8×10 5 CFU, R2 output 4.9×10 7 CFU, R3 output 1.5×10 8 CFU.

[0095] Sporozoite screening: R1 yield 1.9 × 10 7 CFU, R2 output 3.6×10 8 CFU, R3 output 1.3×10 9 CFU.

[0096] 2.4 Identification and Sequence Analysis of Positive Clones

[0097] After screening with monoclonal phage ELISA, 23 positive clones binding to *Spirometra* were obtained. Sequencing yielded 12 unique VHH sequences, named B2A3, B2A8, B2B4, B2C2, B2C5, B2C8, B2D11, B2G7, B2H1, SA4, SB2, and SD2 (Table 2). Among them, one nanobody with strong binding ability to *Spirometra* and a unique sequence was B2C8, whose amino acid sequence is shown in SEQ ID NO:1.

[0098] SEQ ID NO: 1:

[0099] QVKLEESGGGSVQAGGPLRLSCASSGYTSTLKYMGWFRQAPGKEREGVAAISTAAGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAAKADSGFFWHLLKPTEYNYWGQGTQVTVAS

[0100] Table 2. Amino acid sequences of the obtained nanobodies VHH

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] 2.5 Results of B2C8 nanobody expression and purification

[0108] The expressed nanobody-Fc fusion protein was purified to obtain a high-purity B2C8 nanobody, which can be used for subsequent functional verification experiments.

[0109] Example 2: Verification of the binding and inhibition functions of nanobody B2C8

[0110] 1 Experimental Methods

[0111] 1.1 Immunofluorescence verification of nanobody binding to sporozoites

[0112] Purified *Eimeria stearothermiae* sporozoites were incubated with B2C8 nanobodies at room temperature for 1 h. After washing, Fluorescein (FITC)-conjugated Goat Anti-Human IgG was added, and the mixture was incubated at room temperature for 1 h. After washing, the samples were observed under a fluorescence microscope. PBS was used as a control instead of the nanobodies.

[0113] 1.2 Experiment on the inhibition of sporozoite invasion of rabbit hepatic and bile duct epithelial cells by nanobodies

[0114] (1) Cell and parasite markers

[0115] Fluorescent labeling of *Eimeria stearothermiae* sporozoites was performed using the CFDA-SE cell proliferation and tracing assay kit. Simultaneously, rabbit hepatic bile duct epithelial cells in the logarithmic growth phase were collected, washed with PBS, digested with trypsin, centrifuged at 250 g for 5 min, the supernatant was removed, and an appropriate amount of culture medium was added to form a cell suspension.

[0116] (2) Experimental grouping and treatment

[0117] After cell counting, adjust the density to 3×10⁻⁶. 5Cells were seeded at a concentration of 2 mL / well in 6-well plates. A control group and a nanobody treatment group were set up, with 3 replicates per group. After cell attachment, nanobody at a final concentration of 0.03 mg / mL was added to the treatment group, while an equal volume of PBS was added to the control group. Both groups were incubated for 22 h.

[0118] (3) Detection and analysis

[0119] After culture, cells were collected and CFDA-SE fluorescence signals were detected using flow cytometry.

[0120] Calculate the intrusion rate using the following formula:

[0121] Invasion rate (%) = number of sporozoites of invading cells / (number of sporozoites of invading cells + number of cells not invaded by sporozoites) × 100%.

[0122] 1.3 Reproducible Validation of the Inhibitory Function of B2C8 Nanobody

[0123] Based on the preliminary screening results, the nanobody with the best inhibitory effect was selected for repeated validation experiments. The experimental method was the same as in 1.2, with a total incubation time of 22 h, 3 replicates per group, and the experiment was repeated 3 times.

[0124] 2. Experimental Results

[0125] 2.1 Immunofluorescence results of B2C8 nanobody binding to sporozoites

[0126] Immunofluorescence detection results as follows Figure 7 As shown in the figure. In the B2C8 nanobody treatment group, after the sporozoites bind to the nanobody, they can bind to FITC-labeled goat anti-human IgG and be stained with green fluorescence ( ). Figure 7 A); No green fluorescence was observed in the control group ( Figure 7 B). The results showed that the B2C8 nanobody could specifically bind to Eimeria stearothermiae sporozoites.

[0127] 2.2 Sources and Preliminary Screening Results of Candidate Nanobodies

[0128] Following screening and sequencing as described in Example 1, 12 unique VHH sequences were obtained. Five representative nanobodies (B2C8, B2D11, B2H1, SA4, and SB2) were selected for preliminary screening of their inhibitory functions. The results are shown in Table 3. After 7 hours of co-incubation, the invasion rates of the different antibody treatment groups showed no significant difference. After 22 hours of co-incubation, the invasion rate of the B2C8 treatment group was the lowest (9.92%), significantly lower than the control group (25.97%), while the invasion rates of the other antibody treatment groups ranged from 20.35% to 25.60%. These results indicate that among the five candidate nanobodies, B2C8 exhibits the best inhibitory effect.

[0129] Table 3. Inhibitory effects of different nanobodies on sporozoan invasion

[0130]

[0131] 2.3 Reproducible validation results of B2C8 nanobody inhibiting sporozoite invasion

[0132] In vitro cell experiments showed that the B2C8 nanobody significantly inhibited the invasion of rabbit hepatic and bile duct epithelial cells by *Eimeria stearothermiae* sporozoites. Combining the data in Table 4 and the flow cytometry scatter plot in Figure 8, the average invasion rate in the control group was 24.48%, with the proportion of the H1-UR quadrant (invaded fluorescently positive cells) remaining stable at 19.69%-20.37% in the corresponding flow cytometry scatter plot. In contrast, the B2C8 treatment group with a final concentration of 0.03 mg / mL showed an average invasion rate of 13.68%, approximately 44.1% lower than the control group, and the proportion of the H1-UR quadrant simultaneously decreased to 12.21%-12.38%. Furthermore, no significant fluctuations were observed in the three biological replicates for both groups, indicating good experimental repeatability.

[0133] Table 4. Inhibitory effect of nanobody B2C8 on sporozoan invasion

[0134]

[0135] In summary, B2C8 can block the infection process by specifically binding to sporozoite invasion-related targets, providing reliable in vitro experimental evidence for the subsequent development of biological agents against coccidia infection.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanobody, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that, It encodes the nanobody as described in claim 1.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It contains the recombinant vector as described in claim 3.

5. The use of the nanobody of claim 1, the polynucleotide of claim 2, the recombinant vector of claim 3, or the host cell of claim 4 in the preparation of a medicament for the prevention and / or treatment of Eimeria tenella in rabbits.

6. The use of the nanobody of claim 1, the polynucleotide of claim 2, the recombinant vector of claim 3, or the host cell of claim 4 in the preparation of diagnostic reagents or kits for rabbit Eimeria stearothermia.

7. A pharmaceutical composition, characterized in that, It includes the nanobody as described in claim 1.

8. A diagnostic reagent or kit, characterized in that, It comprises the nanobody of claim 1, the polynucleotide of claim 2, the recombinant vector of claim 3, or the host cell of claim 4.

9. A feed additive, characterized in that, It includes the nanobody as described in claim 1.

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