CN110794134B - Application of Eimeria siei SAG4 protein - Google Patents
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Abstract
The invention relates to the technical field of biology, and discloses a series of related applications of Eimeria sieboldii SAG4 protein as rabbit coccidiosis diagnosis antigen, and the like, wherein related experimental results show that the Eimeria sieboldii SAG4 protein can be identified by Eimeria sieboldii positive serum, and has good immunogenicity and reactogenicity; meanwhile, the protein shows extremely high sensitivity and specificity in an indirect ELISA method, and various results prove that the Eimeria sieboldii SAG4 protein can be used as a diagnostic antigen of rabbit coccidiosis and applied to related vaccines and detection kits.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of Eimeria sieboldii SAG4 protein.
Background
Rabbit coccidiosis (rabbitcoccidiosis) is a protozoal disease caused by various coccidia of eimeria parasitizing in the intestinal tract and liver of rabbits, is a common and multiple disease of rabbits, and is one of the main diseases seriously harming the rabbits. Eimeria stiedai (Eimeria stiedai), a rabbit coccidium most virulent, attacks primarily the liver and bile duct epithelial cells of rabbits, causing cirrhosis and cholestasis, causing severe hepatic rabbit coccidiosis. The diseased rabbits are mainly characterized clinically by diarrhea, growth and development retardation, weakness and emaciation, and can die when seriously infected. Because liver type rabbit coccidiosis has high morbidity and mortality, eimeria sieboldii is regarded as one of the most harmful pathogens in domestic rabbit raising industry, and the development of domestic rabbit raising industry is severely restricted.
The rabbit coccidiosis is distributed worldwide, causing serious economic loss to rabbit industry in many countries, and the liver type rabbit coccidiosis is the most pathogenic and prevalent. At present, the diagnosis of the disease needs to be performed by a cesarean section, and an effective prenatal diagnosis method is still lacked; eimeria sieboldii oocysts have been studied and used as natural diagnostic antigens for diagnosis, however, the oocysts as the diagnostic antigens have a series of problems that antigen standard products are difficult to collect and prepare, sources and dosage are difficult to determine, and popularization and application are difficult.
In view of the strong pathogenicity of Eimeria stipitis and the severity of its harm to the rabbit industry, the diagnosis of diseased rabbits using reliable diagnostic techniques is the basis for effective prevention and control. However, the current methods for prenatal diagnosis of the disease are still lack of reports, so that the establishment of a serological diagnosis method for accurately diagnosing the hepatic rabbit coccidiosis has important significance for preventing and treating the disease.
Disclosure of Invention
In view of the above, the invention aims to provide Eimeria sieboldii SAG4 protein (ESAG 4) as a diagnostic antigen of rabbit coccidiosis and an application thereof in preparation of the diagnostic antigen of rabbit coccidiosis, so that the ESAG 4 has higher specificity and sensitivity, and good immunogenicity and reactogenicity;
the invention also aims to provide the application of ESAG 4 in the preparation of a kit for detecting rabbit coccidiosis, so that an ELISA method established by the ESAG 4 shows higher specificity and sensitivity and can be used for ELISA detection;
another object of the invention is to provide the application of ESAG 4 in preparing rabbit coccidiosis vaccine.
In this context, the eimeria stuartii SAG4 protein (EsSAG 4) may be non-natural, e.g., synthetic or expressed from an artificial vector (often referred to in the art as recombinant protein rsesag 4). The term "non-natural" means that the target substance is not naturally occurring in nature, which does not preclude the non-natural substance from having the same structure and/or composition as the naturally occurring substance.
SAGs are located on the surface of cell membranes of protozoa and are a type of surface protein related to Glycosyl Phosphatidylinositol (GPI), and related researches on SAGs in Toxoplasma gondii (Toxoplasma gondii), plasmodium (Plasmodium), neospora caninum (Neosporium caninum) and Sarcocystis neurona (Sarcocystis neurona) are reported in the literature, and no related researches on the SAG gene family of Eimeria grisea are found at present.
The invention clones and expresses the EsSAG4 in a prokaryotic way, verifies the reactionogenicity of the recombinant protein rEsSAG4 by utilizing immunoblotting, and establishes an indirect ELISA method to evaluate the diagnostic value of the recombinant protein rEsSAG4 as a recombinant antigen to the liver type rabbit coccidiosis. The result showed that the protein encoded by the ORF of the ESAG 4 (full length 444bp, SEQ ID NO: 1) gene had a molecular weight of about 16.17kDa (SEQ ID NO: 2). Immunoblotting showed that the recombinant protein rEsSAG4 could be recognized by Eimeria sieversii positive serum, indicating that it has good reactogenicity. The indirect ELISA method established based on rESAG 4 had a sensitivity of 97.92% (47/48) and a specificity of 100% (48/48). Therefore, ESAG 4 can be used as a candidate diagnostic antigen for rabbit coccidiosis caused by Eimeria siei.
Based on the content, the invention provides the application of the Eimeria sieboldii SAG4 protein as a diagnosis antigen of rabbit coccidiosis and the application in preparing the rabbit coccidiosis diagnosis antigen. Meanwhile, the invention also provides application of the Eimeria sieboldii SAG4 protein in preparing a kit for diagnosing rabbit coccidiosis; among them, the kit is preferably an ELISA kit, and more specifically, the ELISA kit is a kit based on an ELISA indirect method. In addition, the invention also provides application of the Eimeria swirskii SAG4 protein in preparation of a rabbit coccidiosis vaccine.
According to the application of the Eimeria sieboldii SAG4 protein in the preparation of the kit, the invention provides an ELISA kit for diagnosing rabbit coccidiosis, which comprises a solid phase carrier coated with the Eimeria sieboldii SAG4 protein (ESAG 4). In a specific embodiment of the invention, the solid phase carrier can be selected from a 96-well culture plate or a solid phase carrier similar to the 96-well culture plate, the ESAG 4 coating concentration is 1.45 mug/well, and the carrier can be coated by a coating solution, wherein the coating solution is 0.39g of Na 2 CO 3 ，35mM NaHCO 3 And adjusting the pH value to 9.6 to obtain the product, wherein the concentration of the NaCl is 0.2M.
After the core components of the kit are determined, the ELISA kit further comprises one or more than two of enzyme-labeled secondary antibody, washing solution, developing solution, confining solution, diluent and stop solution.
The enzyme-labeled secondary antibody is preferably goat anti-rabbit IgG labeled with HRP, in the specific embodiment of the invention, the enzyme-labeled secondary antibody is a product purchased from biological engineering Limited company of Bausch & Wuhan, and the dilution ratio of the enzyme-labeled secondary antibody is 1: 3000A;
the wash solution is preferably PBS-T wash solution, which in a specific embodiment of the present invention consists of 0.01M PBS +0.05% Tween-20; the color development liquid is preferably TMB color development liquid;
the blocking fluid is preferably skim milk, which in a specific embodiment of the invention is 5% diluted in 0.01M PBS solution.
The stop solution is preferably a sulfuric acid solution, and the concentration is preferably 2mol/L; the preparation method comprises slowly dripping 21.7mL of 98% concentrated sulfuric acid into 178mL of deionized water, cooling to room temperature, and storing at 4 ℃;
the diluent is preferably 0.01M PBS; the preparation method comprises 8g NaCl,0.2g KCl,1.42g Na 2 HPO 4 ，0.27gKH 2 PO 4 Dissolving in 800mL deionized water, dissolving to 1L, sterilizing, and storing at room temperature.
According to the technical scheme, the Eimeria sieboldii SAG4 protein provided by the invention is used as a series of relevant applications such as rabbit coccidiosis diagnosis antigen, and relevant experimental results show that the Eimeria sieboldii SAG4 protein can be identified by Eimeria sieboldii positive serum, and has good immunogenicity and reactogenicity; meanwhile, the protein shows extremely high sensitivity and specificity in an indirect ELISA method, and various results prove that the Eimeria sieboldii SAG4 protein can be used as a diagnostic antigen of rabbit coccidiosis and applied to related vaccines and detection kits.
Drawings
FIG. 1 shows the expression purification of rEsSAG4 and immunoblotting results; wherein, lane M: a protein standard substance marker; lane 1: induction of rESAG 4 expressed from E.coli BL21 (DE 3) with IPTG (unpurified); lane 2: purified rEsSAG4 (8. Mu.g); lane 3: eressag 4 recognized by eimeria siella positive sera; lane 5: rEsSAG4 recognized by Eimeria sienna negative sera; lanes 5-7: rESAG 4 recognized by rabbit coccidiosis positive serum; lanes 8-10: rESAG 4 recognized by rabbit sarcoptidosis positive serum;
FIG. 2 shows the detection of E.stuartii positive and negative serum samples using an established indirect ELISA method; where the horizontal lines represent the Cut-Off values for the ELISA method (Cut-Off = 0.447), 48 eimeria siella positive and negative serum samples were detected, respectively, with significant variability between the data (P < 0.01).
Detailed Description
The invention discloses application of Eimeria siei SAG4 protein, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the application. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the embodiments described herein without departing from the spirit and scope of the invention.
The invention carries out reverse transcription to synthesize cDNA by extracting Eimeria sieboldii sporulated oocyst total RNA and taking OligodT (18) as a reverse transcription primer, and amplifies an Eimeria sieboldii SAG4 protein coding sequence from the cDNA. After T cloning, the amplified product is introduced into an expression vector in a mode of enzyme digestion connection, and prokaryotic expression is carried out by using escherichia coli to obtain recombinant rEsSAG4.
In experiments of a specific embodiment, all experimental animals were treated strictly according to the "animal protection law of the people's republic of china" (draft published on 9/18 th in 2009). All procedures were performed in accordance with the rules of animal care and use of the animal ethics committee for animal ethics of the university of Sichuan agriculture (China, yaan) (approval No.: 2015-028).
Eimeria sieboldii species used in the present invention were isolated from the liver and gall bladder of naturally infected New Zealand rabbits, sporulated in 2.5% potassium dichromate solution at 28 ℃ and then stored at 4 ℃;48 30-day-old young non-coccidiosis rabbits are bred by animal parasitic disease research center of Sichuan agriculture university and strictly raised in a non-coccidiosis environment, boiled drinking water and 80 ℃ roasted rabbit feed are provided for feeding, and anti-coccidiosis drugs diclazuril and decoquinate are alternately used for cross-rotation, and meanwhile, rabbit cages are periodically sprayed and burnt to prevent coccidiosis pollution.
48 parts of Eimeria sieboldii negative rabbit serum in the test is collected from 48 coccidian-free young rabbits, the coccidian-free young rabbits check excrement by a saturated saline floating method every day in the period of 30-35 days of age, and the coccidian oocysts are negative after 5 days of continuous excrement check; negative sera were used to determine Cut-Off values and specificity of indirect ELISA. 48 parts of Eimeria sieboldii positive rabbit serum were collected from artificially infected coccidian sporulated oocysts (8X 10) 4 One/one) to 30 days and the liver was confirmed by necropsy to show significant symptoms in 48 experimental rabbits; positive sera were used to determine the sensitivity of the indirect ELISA method. 3 parts of rabbit sarcoptidosis scabies (sarcoptitis scabies) positive serum was collected from a New Zealand rabbit artificially infected with rabbit sarcoptidosis scabies, 3 parts of rabbit coccidiosis positive serum was collected from a New Zealand rabbit naturally infected with and etiologically detected for coccidiosis; rabbit sarcoptidosis and rabbit enterococcidia positive sera were used for immunoblotting to verify the cross-reactivity of the recombinant protein with the rest of the rabbit parasites. All serum samples were stored at-20 ℃ until use.
All data are expressed as mean ± standard deviation (s.d.) and all correlation analyses were performed using GraphPad Prism version 5.0 (GraphPad Software). The P value of less than 0.05 is judged to be significant by using IBM SPSS statistics 22.0 (SPSS software) to analyze the difference between batches.
The application of the Eimeria swirskii SAG4 protein provided by the present invention is further described below.
Example 1: bioinformatics analysis and clonal sequencing of Essag4
1. cDNA amplification of Essag4
Determining the open reading frame and the deduced amino acid sequence of ESAG 4 by using an open reading frame Finder ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf. Html); signalP4.1 is used for predicting whether a signal peptide and a transmembrane region exist in the protein at a biological sequence analysis center (http:// www.cbs.dtu.dk/services/SignalP /); the ExPASy website (http:// web. ExPASy. Org /) was used to predict Molecular Weight (MW), isoelectric point (pI), conserved domain and protein properties of proteins.
Eimeria sieboldii stored at 4 ℃ in 2.5% potassium dichromateSporulated oocysts were washed repeatedly 5 times with physiological saline, RNA was extracted using a total RNA Extraction Kit (MiniBEST Universal RNA Extraction Kit, taKaRa, japan), and then extracted Eimeria spergualis total RNA was used as a template and a reverse transcription Kit (PrimeScript) was used TM RT reagent kit with gDNA Eraser, taKaRa, japan) and the complementary double-stranded cDNA obtained is preserved at-80 ℃.
Eimeria siei cDNA was used as a template to amplify the ESAG 4 gene. Designing a primer according to Eimeria sieboldii transcriptome data measured by the animal parasitic disease research center of Sichuan university: SAG4-F:5' -CCAAGCTTATTCAACTTGGGGGGATGGT-3 'and SAG4-R:5' -CCTCGAGTTACACTGTGACCATTGCCAACG-3'. The primers add restriction sites HindIII and XhoI (the restriction sites are underlined). The EsSAG4 was amplified by the following PCR program: pre-denaturation at 95 ℃ for 4min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 60s, extension at 72 ℃ for 1min, and repeating the denaturation step for 35 cycles; final extension at 72 ℃ for 10min. After separation by agarose gel electrophoresis and purification using a DNA purification kit (Novagen, germany), the amplified product of the gene was cloned into the vector pMD19-T (TaKaRa, dalian, china), transformed into e.coli DH5 α (Tiangen, beijing, china) and subsequently sent to the company for sequencing (Invitrogen, shanghai, china).
The ORF total length of the EsSAG4 gene is 444bp, and 147 amino acids are coded; the protein has a molecular weight of about 16.17kDa, no signal peptide, and no transmembrane region. Is that
2. Expression and purification of recombinant protein rEsSAG4
And recovering and purifying a PCR product of ESAG 4, connecting the PCR product to a pMD19-TVector vector, performing double enzyme digestion by using HindIII and XhoI (TaKaRa, dalian and China), connecting the enzyme digestion fragment to a pET32a (+) expression vector, transferring the enzyme digestion fragment into escherichia coli BL21 (DE 3), and sequencing after identification. And (3) inoculating the recombinant expression strain with correct sequencing into LB culture solution containing 50 mu g/mLAmp, culturing at 37 ℃ for 4h until the OD value of the bacterial solution reaches 0.6, and adding 1mMIPTG to induce and express for 6h. Subsequent use of NGC TM Purifying the expressed recombinant protein with high pressure chromatography system (Bio-RadUSA) in 10, and collecting the proteinWas ultrafiltered and characterized by SDS-PAGE.
The gene encoding SAG4 was successfully ligated to expression vector pET-32a (+), and the plasmid was transformed into E.coli BL21 to induce protein expression. After 12-% SDS-PAGE separation, a single band of approximately 34.17kDa was observed (FIG. 1, lane 1 and lane 2), which was consistent with the expected size (including approximately 18kDa from the pET-32a (+) vector tag).
Example 2: immunoblot analysis
Preparing 12% SDS-PAGE gel, and performing SDS-PAGE separation on the purified rESAG 4 protein obtained by the method in example 1; after electrophoresis, cutting the part containing the protein sample on the gel, and adopting a semi-dry type membrane transfer device at 0.8mA/cm 2 Transferring for 40min under constant current; after the end, the film is taken out and washed with TBST for 3 times, 5min each time; blocking for 2h at room temperature by using 5% skim milk powder PBS solution; washing the membrane with TBST for 5min for 3 times; rabbit positive serum infected with eimeria stuartii (diluted at 1; the liquid was decanted and washed 3 times with TBST, 5min each time; then, a rabbit anti-goat IgG secondary antibody with HRP label diluted with PBS (dilution ratio 1.
Immunoblot analysis showed that rESAG 4 was recognized by Eimeria sieboldii positive serum and presented a single band, indicating that the recombinant protein has good reactogenicity and antigenicity (FIG. 1, lane 3); while no band was observed when the rESAG 4 was recognized using Eimeria siei-negative serum (FIG. 1, lane 4). No band was observed with both sarcoptic mite and rabbit E.coli positive sera for rESAG 4, indicating that there was no cross-reaction of the recombinant protein with sarcoptic mite with E.coli (FIG. 1, lanes 5-10).
Example 3: establishment of indirect ELISA method and detection verification
1. Establishment of Indirect ELISA method
Indirect ELISA was established according to previous reports (CROWTHER J. The ELISA Guideboost [ J ]. Methods in Molecular Biology,2000,149 (149): III-IV, 1-413.). Purified rEsSAG4 protein was diluted in 0.1M carbonate buffer (pH 9.6) by standard checkerboard titration at 6 different concentrations in two to be used as antigen in an indirect ELISA (1. After 3 washes with PBST (0.01M PBS +0.05% Tween-20), the coated ELISA plates were blocked with 5% skim milk (diluted in 0.01M PBS solution) and incubated at 37 ℃ for 2h at 300. Mu.L per well. The eimeria siedii negative and positive sera were again washed 3 times with PBST while being diluted in 0.01M PBS solution in 6 different concentrations of a double dilution (1. Washing with PBST was continued 3 times, HRP-labeled goat anti-rabbit IgG (Boster, wuhan, china) was diluted with 0.01M PBS solution at the recommended working concentration (1 3000) and added to the plate at 100 μ Ι _ per well for 1h at 37 ℃. After washing thoroughly 4 times with PBST, 100. Mu.L of tetramethylbenzidine color developing solution (TIANGEN, beijing, china) was added to each well and allowed to act at room temperature for 20min. And finally, adding 100 mu L of 2mol/L concentrated sulfuric acid into each hole of the ELISA plate to terminate the reaction, and determining the optimal working concentration of the antigen and the serum according to the P/N value after determining the OD450 value by using an ELISA reader (Thermo, USA). Meanwhile, the selection of the optimal blocking solution, the optimal blocking time, the optimal time of serum reaction, the optimal dilution of the HRP-labeled goat anti-rabbit IgG and the action time are optimized according to the previous reports.
The optimal antigen coating concentration of rESAG 4 is 1.45 mug/hole, the optimal serum dilution ratio is 1.
Under the optimal conditions of indirect ELISA, 48 negative serum samples from coccidiless young rabbits are used to determine a Cut-Off value (Cut-Off) of the indirect ELISA, the calculation method is to add three times of standard deviation (S.D.) to the average value of the OD450 of the 48 negative sera, and the detection sample is determined to be positive if the OD450nm value of the detection sample is greater than or equal to the Cut-Off value, and is determined to be negative if the OD450nm value of the detection sample is not less than the Cut-Off value.
Under optimal reaction conditions, 48 E.siemens negative serum samples were examined to determine the Cut-Off value (Cut-Off) of the indirect ELISA method established by the recombinant protein. The cutoff value of the indirect ELISA method established by rsesag 4 was calculated to be 0.447 according to the cutoff value calculation formula (Cut-Off = negative sample OD450 mean +3 × s.d.). It can be concluded that when rabbit serum samples are detected by using the indirect ELISA method established by rEsAG 4, eimeria spongiella spongiensis is determined to be positive when the OD450 value is more than or equal to 0.447; conversely, eimeria siei-negative was determined when the OD450 value was < 0.447.
2. Intra-and inter-batch reproducibility of indirect ELISA detection methods
6 E.stuartii positive sera were selected to determine the intra-and inter-batch reproducibility of the Essag4 indirect ELISA. Respectively detecting 6 positive serum samples in the ELISA plates coated in the same batch and 5 different batches, making 3 repeated holes on the same ELISA plate for each sample, determining OD450 values, respectively calculating the OD450 average value and Standard Deviation (SD) of each serum sample, and determining the in-batch and inter-batch repeatability of the established indirect ELISA method by calculating the coefficient of variation.
The result shows that the intra-batch variation coefficient of the indirect ELISA method established based on the rEsSAG4 is between 0.29 and 4.57 percent, the inter-batch variation coefficient is between 0.98 and 8.23 percent, and all the variation coefficients are within 10 percent, which indicates that the rEsSAG4 indirect ELISA method has good repeatability and reliable result.
3. Sensitivity and specificity of Indirect ELISA detection method
48 parts E.stuartii positive rabbit serum and 48 parts E.stuartii negative rabbit serum were tested according to the established ESAG 4 indirect ELISA method to determine the sensitivity and specificity of the indirect ELISA method. And (3) carrying out indirect ELISA detection on all the sera under the optimal working condition, measuring the OD450 value of the sera, and judging the negativity and the positivity of each serum sample according to the critical value of ESAG 4. And evaluating the sensitivity and specificity of the indirect ELISA method according to a calculation formula.
The calculation formula is as follows: sensitivity = (number of ELISA-detected positive samples/number of eimeria stuartii positive serum samples) × 100%; specificity = (number of negative samples detected by ELISA/number of eimeria siella negative serum samples) × 100%.
The results show that, for the indirect ELISA method established on the basis of rEsSAG4, the OD450 values of only 1 sample of 48 positive sera were lower than their cut-off value of 0.447, so that their sensitivity was 97.92% (47/48) (FIG. 2); meanwhile, 48 parts of Eimeria sieversii negative rabbit serum were respectively detected by an indirect ELISA method established based on rESAG 4 to determine the specificity of the detection method. The results showed that the OD450 values of 48 negative sera were all lower than their cut-off value of 0.447 (48/48), so that their specificity was 100% (48/48) (FIG. 2).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Sichuan university of agriculture
Application of <120> Eimeria siei SAG4 protein
<130> MP1930886
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 444
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggtattaa cctacgagga tacgagcgga agtaccccca attacaagaa ccttgtccaa 60
acggtcttga ccaagggtct cggtgtactc gcgcaacagt cgtcatcagg gaacacaggt 120
tggactcaag gattctggag ccaaaatgcc gatgcagcta atttgggcta cttgatgtgg 180
agcggttcgg tatcggtcgg ctgcgccgtc accgacggat gtgaagataa taaattcctc 240
atcctttgcc tattcaatcc aacagctgcc ggagacgtcg cgcctttcga cgaaaacgta 300
tacaaagccc tcgtggcaag aacaacccta ctgacccaaa tgacagagag tgatcttcag 360
gccgcaggca gcggtgccgt catgggactt ccttccctcc tccttgccgg tttcgttgcc 420
gcgttggcaa tggtcacagt gtaa 444
<210> 2
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Val Leu Thr Tyr Glu Asp Thr Ser Gly Ser Thr Pro Asn Tyr Lys
1 5 10 15
Asn Leu Val Gln Thr Val Leu Thr Lys Gly Leu Gly Val Leu Ala Gln
20 25 30
Gln Ser Ser Ser Gly Asn Thr Gly Trp Thr Gln Gly Phe Trp Ser Gln
35 40 45
Asn Ala Asp Ala Ala Asn Leu Gly Tyr Leu Met Trp Ser Gly Ser Val
50 55 60
Ser Val Gly Cys Ala Val Thr Asp Gly Cys Glu Asp Asn Lys Phe Leu
65 70 75 80
Ile Leu Cys Leu Phe Asn Pro Thr Ala Ala Gly Asp Val Ala Pro Phe
85 90 95
Asp Glu Asn Val Tyr Lys Ala Leu Val Ala Arg Thr Thr Leu Leu Thr
100 105 110
Gln Met Thr Glu Ser Asp Leu Gln Ala Ala Gly Ser Gly Ala Val Met
115 120 125
Gly Leu Pro Ser Leu Leu Leu Ala Gly Phe Val Ala Ala Leu Ala Met
130 135 140
Val Thr Val
145
Claims (5)
1. The application of Eimeria sieboldii SAG4 protein in the preparation of a kit for diagnosing rabbit coccidiosis;
the amino acid sequence of the Eimeria sieboldii SAG4 protein is shown in SEQ ID NO. 2.
2. The use according to claim 1, wherein the kit is an ELISA kit.
3. The use according to claim 2, wherein the ELISA kit is a kit based on an ELISA indirect method.
4. An ELISA kit for diagnosing rabbit coccidiosis is characterized by comprising a solid phase carrier coated with Eimeria sieboldii SAG4 protein;
the amino acid sequence of the Eimeria spergualis SAG4 protein is shown in SEQ ID NO. 2.
5. The ELISA kit of claim 4, further comprising one or more of an enzyme-labeled secondary antibody, a washing solution, a developing solution, a blocking solution, a diluting solution and a stop solution.
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