Indirect ELISA detection kit based on recombinant Et CDPK4 protein and application

Abstract

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection kit based on recombinant Et CDPK4 protein and application, and belongs to the technical field of biology. The invention discloses an indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit based on a recombinant Et CDPK4 protein. The recombinant Et CDPK4 protein is used as a coating antigen. The indirect ELISA detection method established on the basis of the recombinant Et CDPK4 protein is high in specificity, high in sensitivity, good in repeatability, convenient and rapid in detection and suitable for large-scale application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an indirect ELISA detection kit based on recombinant Et CDPK4 protein and its application. Background Art

[0002] Healthy chicken farming is vital to global food supply. Chicken coccidiosis is a protozoan disease caused by Eimeria parasitizing the intestinal epithelial cells of chickens, causing at least £10.6 billion in losses to the global poultry industry each year. Eimeria tenella is the most pathogenic, harmful and widely distributed of the seven species of chicken coccidia discovered.

[0003] A preliminary diagnosis of chicken coccidiosis can be made based on clinical symptoms, epidemiology, pathological anatomy and other diagnostic methods, and the diagnosis is generally confirmed by saturated salt water flotation method to check whether there are coccidia oocysts in feces or intestinal contents. The development of coccidia in chickens mainly occurs in the intestines through schizogony and gametogenesis, and finally develops into coccidia and is excreted from the body. Existing methods cannot detect coccidia oocysts in the early and middle stages of coccidia infection. Moreover, there is currently no technology for effectively detecting the immune effect of coccidia vaccines after immunization, except for the immune effect test at the factory.

[0004] Therefore, providing an indirect ELISA detection kit based on recombinant Et CDPK4 protein and its application is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an indirect ELISA detection kit based on recombinant Et CDPK4 protein and its application.

[0006] Eimeria tenella calcium-dependent protein kinase (Et CDPK4) plays an important role in coccidia invasion. Since Et CDPK4 is not found in chickens, Et CDPK4 can be used as a candidate molecule for diagnostic antigens and a coating protein in serological diagnosis for diagnosis of E. tenella disease and vaccine immune effect detection. Therefore, the present invention uses recombinant Et CDPK4 protein as an antigen to establish an easy-to-operate, rapid and specific chicken coccidiosis ELISA antibody detection kit.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The invention relates to the use of a recombinant Et CDPK4 protein in the preparation of a preparation for diagnosing Eimeria tenella infection. The amino acid sequence of the Et CDPK4 protein is shown in SEQ ID NO.2.

[0009] Furthermore, the recombinant Et CDPK4 protein is used in the preparation of a reagent for monitoring the antibody level of a live chicken E. tenella vaccine, wherein the amino acid sequence of the Et CDPK4 protein is shown in SEQ ID NO.2.

[0010] Furthermore, the preparation method of the recombinant Et CDPK4 protein is as follows: using E. tenella cDNA as a template, specific primers are designed according to the CDPK4 gene sequence, the target fragment is amplified by PCR and cloned into the expression vector pET28a, the successfully constructed recombinant expression plasmid pET28a-Et CDPK4 is transformed into Escherichia coli E. coli BL21, IPTG is induced for expression, and the recombinant CDPK4 protein is obtained after high purification by affinity chromatography and molecular sieve chromatography; the CDPK4 gene sequence is shown in SEQ ID NO.1.

[0011] Furthermore, an indirect ELISA detection kit based on recombinant Et CDPK4 protein uses the recombinant Et CDPK4 protein as the coating antigen.

[0012] Furthermore, the kit also includes enzyme-labeled secondary antibody, blocking solution, positive serum, negative serum, and color developing solution;

[0013] The enzyme-labeled secondary antibody is HRP-labeled goat anti-chicken IgY;

[0014] The blocking solution is 3% skimmed milk powder;

[0015] The color developing solution is TMB color developing solution.

[0016] Furthermore, the kit is used in detecting antibodies against Eimeria tenella.

[0017] The method for detecting chicken E.tenella antibodies using the ELISA kit comprises the following steps:

[0018] a Preparation of solid-phase antigen: connect the antibody capture agent to the solid-phase support, block with blocking solution, wash to remove unbound antigen and impurities, and obtain solid-phase antigen;

[0019] b. Primary antibody binding: The sample to be tested is allowed to bind to the solid phase antigen through an incubation reaction to form a solid phase antigen-antibody complex, and then washed to remove impurities on the solid phase carrier;

[0020] c. Secondary antibody binding: combining the enzyme-labeled secondary antibody dilution with the solid phase antigen-antibody complex to obtain an antigen-antibody-secondary antibody complex;

[0021] d. Color development: After the antigen-antibody-secondary antibody complex obtained in step c is added to the chromogenic substrate for color development in the dark, a stop solution is added to terminate the reaction to obtain a color development sample solution;

[0022] e. Detection and determination: The color-developed sample solution in step d is measured with an enzyme marker to measure OD 450 nm value, judgment result.

[0023] Furthermore, the method also includes a blank control experiment and a negative control experiment.

[0024] Furthermore, an indirect ELISA detection method based on recombinant Et CDPK4 protein, the specific steps are as follows:

[0025] ① Preparation of solid-phase antigen: Coat the recombinant Et CDPK4 protein at a concentration of 0.625 μg / mL in an ELISA plate, add 100 μL / well, incubate at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry; add 200 μL / well blocking solution, block at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry; the blocking solution is 3% skim milk powder;

[0026] ② Primary antibody binding: dilute the serum to be tested at a volume dilution ratio of 1:80 and add it to the ELISA plate, 100 μL / well, incubate at 37°C for 2 hours, wash the plate 3 times, 5 minutes / time, and pat dry;

[0027] ③ Secondary antibody binding: Dilute the enzyme-labeled secondary antibody with PBS at a volume dilution of 1:15000 and add it, 100 μL / well, react at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry; the enzyme-labeled secondary antibody is goat anti-chicken IgY-HRP;

[0028] ④ Color development: Add 100 μL / well of TMB color development solution, color for 10 min in the dark, and then add 50 μL / well of 2 mol / L sulfuric acid to terminate the reaction;

[0029] ⑤ Detection and judgment: Measure OD with an enzyme marker 450 nm value, when OD 450 A nm value greater than 0.452 is positive, and a value less than or equal to 0.452 is negative.

[0030] Solid phase carrier: The solid phase carrier acts as an adsorbent and container during the ELISA assay and does not participate in chemical reactions. There are many materials that can be used as solid phase carriers in ELISA, such as polystyrene. It must have strong protein adsorption properties. After the antibody or protein antigen is adsorbed on it, it still retains the original immunological activity and can be made into various forms. There are three main shapes of ELISA carriers: microtiter plates, beads and small test tubes. Microtiter plates are the most commonly used. Products dedicated to ELISA are called ELISA plates. The international standard microtiter plate is 8×12 96-well type. In order to facilitate the detection of a small amount of specimens, 8-well strips or 12-well strips are made. After being placed in the stand, the size is the same as the standard ELISA plate. The characteristic of ELISA plates is that a large number of specimens can be detected at the same time, and the results can be quickly read on a special colorimeter. There are now a variety of automated instruments for microtiter plate-type ELISA detection, including sample addition, washing, insulation, colorimetry and other steps, which are extremely beneficial to the standardization of operations.

[0031] Antibody capture agent: The antibody capture agent used in the present invention is recombinant EtCDPK4 protein.

[0032] Enzyme-labeled secondary antibody: The enzyme-labeled secondary antibody used in the present invention is goat anti-chicken IgY-HRP (goat anti-chicken IgY labeled with horseradish peroxidase). In addition to being labeled with horseradish peroxidase, other enzyme labels, such as phosphatase, can also be used.

[0033] Substrate: Tetramethylbenzidine (TMB), o-phenylenediamine, 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and other HRP colored conjugates can be used as substrates. When other enzymes are used for labeling, their colored conjugates can be used as substrates.

[0034] The TMB colorimetric solution was SureBlue TMB Microwell Substrate from KPL, and the substrate was 3,3',5,5'-tetramethylbenzidine.

[0035] Blocking solution: Blocking is the process of re-coating with a high concentration of irrelevant protein solution after coating. The concentration used for antigen or antibody coating is relatively low, and there are still unoccupied gaps on the surface of the solid phase carrier after absorption. Blocking is to allow a large amount of irrelevant proteins to fill these gaps, thereby excluding the re-adsorption of interfering substances in the subsequent steps of ELISA. The blocking procedure is similar to coating. Among them, the common ones are 0.05%-0.5% bovine serum albumin, 10% calf serum, 1% gelatin, and skimmed milk powder with a volume fraction of 0.05%-0.5%.

[0036] The present invention discloses a series of related applications such as using Et CDPK4 protein as a diagnostic antigen for chicken coccidiosis. The relevant experimental results show that Et CDPK4 protein can be recognized by E.tenella positive serum and has good immunogenicity and reactivity. At the same time, the indirect ELISA method for detecting chicken E.tenella antibodies established with recombinant CDPK4 protein as a coating antigen has strong specificity, high sensitivity, good repeatability, good safety, convenient and fast detection, and is suitable for large-scale application. Moreover, the recombinant CDPK4 protein expressed by Escherichia coli is easier to obtain, which greatly reduces the production cost and operation difficulty of the indirect ELISA detection method. It is shown that Et CDPK4 protein can be used as a diagnostic antigen for chicken coccidiosis and applied to related vaccines and detection kits.

[0037] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses an indirect ELISA detection kit based on recombinant EtCDPK4 protein and its application. The indirect ELISA detection method established based on the purified recombinant Et CDPK4 protein has good specificity. The positive sera of Eimeria tenella, Eimeria acervulina, Eimeria giant and Eimeria precocious were tested, and the results were all negative. The method showed that the coefficient of variation of repeated tests within or between batches was less than 10%, and the positive serum of E. tenella diluted 1:300 times could be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0039] Figure 1 The accompanying drawing shows the PCR amplification of the Et CDPK4 gene of the present invention;

[0040] Wherein, M is the DNA relative molecular mass standard D2000; 1 is the PCR amplification product using E. tenella genomic DNA as template (molecular weight, about 1803 bp);

[0041] Figure 2 The accompanying drawing shows the double enzyme digestion result of the recombinant plasmid pET28a-Et CDPK4 of the present invention;

[0042] Wherein, M is the DNA relative molecular mass standard D5000; 1 is the two fragments obtained by double digestion of the recombinant plasmid pET28a-Et CDPK4 with NdeI and XhoI;

[0043] Figure 3 The accompanying drawing shows the PCR identification of the pET28a-Et CDPK4 recombinant plasmid bacterial solution of the present invention;

[0044] Among them, M is the protein relative molecular mass standard; bacterial solutions 1, 2, and 3 are selected positive colonies; "-" is a negative control (template is ultrapure water); "+" is a positive control (template is a recombinant plasmid) (the molecular weight of Et CDPK4 is about 1803bp);

[0045] Figure 4 The accompanying drawings are diagrams showing the test expression results of the recombinant Et CDPK4 protein of the present invention;

[0046] Among them, M is the protein relative molecular mass standard; 1 and 2 are the induced expression results of colonies 1 and 2 respectively; 3 is the blank control;

[0047] Figure 5 The accompanying drawing shows the purification of the recombinant Et CDPK4 protein of the present invention;

[0048] Wherein, M is the protein relative molecular mass standard; 1 is the protein after bacterial disruption; 2 is the protein in the supernatant after centrifugation; 3 is the protein contained in the flow-through; 4 is the protein contained in the washing liquid after washing; 5 is the protein in the eluate (i.e., the target protein) (the molecular weight of Et CDPK4 is about 1803bp);

[0049] Figure 6 The accompanying figure shows the molecular screening results of the recombinant Et CDPK4 protein of the present invention;

[0050] Among them, M is the protein relative molecular mass standard; 1-14 are the screened samples; finally, the samples in tubes 2-14 are selected for concentration;

[0051] Figure 7 The accompanying drawing shows the reactivity detection (Western blotting analysis) of the recombinant Et CDPK4 protein of the present invention;

[0052] Among them, M is the relative molecular mass standard of pre-stained protein; 1 is the detection of recombinant Et CDPK4 protein (about 68KD) using E. tenella positive serum as the primary antibody; 2 is the detection of recombinant Et CDPK4 protein using negative serum as the primary antibody, with no specific band. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] The preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The experimental methods in the preferred embodiments where specific conditions are not specified are usually carried out according to conventional conditions, such as the conditions described in Molecular Cloning Laboratory Manual (3rd edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002), or according to the conditions recommended by the manufacturer.

[0055] Agarose (Takara Biotechnology Co., Ltd.), 2×Taq PCR MasterMix (Takara Biotechnology Co., Ltd.), restriction endonucleases Nde I and Xho I (Takara Biotechnology Co., Ltd.), DNA ligase Mix (BioLabs Co., Ltd.), plasmid DNA minipreparation kit (Tiangen Biotechnology Co., Ltd.) and universal DNA purification and recovery kit (Tiangen Biotechnology Co., Ltd.), PrimeScript TM FAST RT reagent Kit with gDNA Erase (Takara Biotechnology Co., Ltd.).

[0056] E. tenella strong strain (Tsx) see the literature: Zhang Yu. Preliminary study on the mechanism of Eimeria tenella-induced autophagy in chicken embryo cecal epithelial cells [D]. Shanxi Agricultural University, 2023).

[0057] E.tenella positive serum: SPF chickens inoculated with 2×10 virulent strains of E.tenella 4 The serum was obtained by separating blood from oocysts 7 days after inoculation.

[0058] Negative serum: serum separated from SPF chicken blood.

[0059] The CDS region of Et CDPK4 is shown in SEQ ID NO.1.

[0060] atggagcgaggccacg acatgaaactcaactga ; SEQ ID NO.1.

[0061] The amino acid sequence of EtCDPK4 is shown in SEQ ID NO.2.

[0062] ; SEQ ID NO.2.

[0063] Example 1 Preparation of Antibody Capture Agent

[0064] 1) Cloning of EtCDPK4 gene

[0065] (1) Primer design

[0066] Primers were designed using SanpGene software with reference to the Et CDPK4 gene sequence (GenBank accession number: KU925778.1) and synthesized by Sangon Biotechnology Co., Ltd.

[0067] The primer sequences are as follows:

[0068] EtCDPK4 F:

[0069] 5'-GCCGCGCGGCAGC CATATG ATGGAGCGAGGCCACG-3'; SEQ ID NO.3; the underlined part is the Nde I restriction site;

[0070] EtCDPK4 R:

[0071] 5'-TGGTGGTGGTGGTG CTCGAG TCAGTTGAGTTTCATGTA-3'; SEQ ID NO.4; the underlined part is the Xho I restriction enzyme cleavage site.

[0072] After synthesis, it was dissolved in an appropriate amount of sterile deionized water to a final concentration of 10 μmol / L and stored at -20°C for later use.

[0073] (2) Extraction of total RNA

[0074] Take the sporozoite precipitate (extracted according to the method of "Zhang Yu. Preliminary study on the mechanism of autophagy in chicken embryo cecal epithelial cells induced by Eimeria tenella [D]. Shanxi Agricultural University, 2023"), add 1mL Trizol, aspirate and mix, and let stand at room temperature for 10 minutes; add 200μL chloroform and mix, the solution emulsifies and becomes milky white, and let stand for 10 minutes; 12000r / min, 4℃, centrifuge for 15min, the liquid is divided into three layers (ie: colorless supernatant (containing RNA), the middle white protein layer (mostly DNA) and the colored lower organic phase), aspirate the supernatant and transfer it to another new centrifuge tube. Add 400 μL of isopropanol, mix thoroughly by inverting the centrifuge tube, and let it stand for 10 minutes; centrifuge at 12000 r / min, 4°C for 10 minutes, and carefully discard the supernatant; add 1 mL of 75% ice ethanol, wash the precipitate, centrifuge at 7500 r / min, 4°C for 10 minutes, and discard the supernatant; open the centrifuge tube cover and dry the precipitate in a clean hood for 5-10 minutes; after the precipitate is dry, add an appropriate amount of RNase-free water (DEPC water is also acceptable) to dissolve the precipitate; determine the RNA concentration.

[0075] (3) Determination of total RNA concentration and purity

[0076] Use 2 μL DEPC-treated water as blank to adjust to zero, take 2 μL RNA sample and add it to the sample well of Eppendorf μCuvette to detect the concentration and purity of the sample RNA. A260 / A280 ratio of 1.8-2.0 indicates a high-purity RNA sample that is conducive to PCR amplification.

[0077] (4) cDNA synthesis: using PrimeScript TMFAST RT reagent Kitwith gDNA Erase (Takara Biotechnology Co., Ltd.)

[0078] ① Removal of genomic DNA

[0079] Prepare the reaction mixture on ice according to the following ingredients:

[0080] Reagent name Formula volume (μL) 8×gDNAEraserPremix 2 Total RNA (sample) 12 <![CDATA[RNaseFreeH2O]]> 2 Total 16

[0081] First, add 8×g DNA Eraser Premix to the reaction tube, then add RNase Free H2O and RNA sample in sequence. Centrifuge to sink the liquid to the bottom of the tube, and place it in the PCR instrument and select the following programs: 42°C, 2min; 4°C, ∞ to remove genomic DNA.

[0082] ②Reverse transcription reaction

[0083] Add 4 μL of 5×RT Premix to the reaction solution (1) on ice, mix gently and then immediately perform reverse transcription reaction. The reaction system is as follows:

[0084] Reagent name Formula volume (μL) Total RNA (genomic DNA-free reaction product) 16 5×RTPremix 4 Total 20

[0085] After the system is prepared, centrifuge it to sink the liquid to the bottom of the tube, place it in the PCR instrument and select the following programs: 37℃, 10min; 85℃, 5s; 4℃, ∞. The cDNA product can be stored at 4℃ for 1-2 days and at -20℃ for a long time.

[0086] (5) PCR amplification of EtCDPK4 gene

[0087] The PCR reaction system is:

[0088] TaKaRaExPremierDNAPolymerase(2X) 12.5μL EtCDPK4F 1.0μL EtCDPK4R 1.0μL cDNA 2.0μL Ultrapure water 8.5μL Total volume 25μL

[0089] Mix gently, centrifuge briefly at 2000r / min, and then perform PCR.

[0090] Reaction parameters: 94℃ pre-denaturation for 1min; 98℃ denaturation for 10s, 63℃ annealing for 15s, 68℃ extension for 1min, 30 cycles; stored at 4℃ for later use. Take 5μL of PCR product and run it on 1% agarose gel to observe the length of the amplified fragment, and use gel recovery kit to recover the target fragment.

[0091] The E. tenella virulent strain genomic DNA was used as a template to amplify the Et CDPK4 gene by PCR. The product was subjected to 1.0% agarose gel electrophoresis, and a specific DNA band of about 1803 bp was obtained, which was consistent with the expected result ( Figure 1 ).

[0092] 2) Construction and identification of prokaryotic expression plasmid pET28a-Et CDPK4

[0093] (1) Enzyme digestion and ligation of target fragments

[0094] The prokaryotic expression vector pET28a(+) was double-digested with restriction endonucleases Nde I and Xho I;

[0095] The enzyme digestion system is:

[0096] Prokaryotic expression vector pET28a(+) 34μL N i 1.0μL XOt 1.0μL 10×Mbuffer 4.0μL Ultrapure water 10μL

[0097] Digest at 37℃ for 1h. Recover the target fragment using a DNA recovery kit. Ligate at 50℃ for 1h according to the following ligation system.

[0098] Connection system:

[0099] Purpose fragment 4.5μL pET28a(+) double restriction enzyme digestion and recovery fragment 0.5μL 2×DNA Ligase Mixture 5.0μL

[0100] (2) Transformation of recombinant plasmid

[0101] Pipette 1-2 μL of the extracted plasmid, add it to BL21 competent cells, place on ice for 30 minutes, then heat shock at 42°C for 90 seconds, place on ice, add 500 μL of antibiotic-free LB medium, shake for 30-60 minutes, then centrifuge at 5000 rpm for 3-4 minutes, discard part of the supernatant (or add 200 μL of antibiotic-free medium, shake for one hour and directly pipette 50 μL to plate without centrifugation), mix the precipitate and spread on a plate (with 100 μg / mL kanamycin resistance), add glass beads and shake well, and culture overnight.

[0102] (3) Identification of recombinant plasmid by bacterial culture PCR and double enzyme digestion

[0103] Three colonies of the above-preserved clones were selected and inoculated into 5 mL of LB liquid culture medium (containing 100 μg / mL of kanamycin), cultured overnight in a 37°C water bath with shaking, and 1 μL was measured for bacterial solution PCR. The recombinant plasmid was extracted according to the conventional method, and then the recombinant plasmid was identified by double restriction digestion with Nde I and Xho I, and the results were observed by 1% gel electrophoresis. At the same time, the primer Et CDPK4 F / R was used for PCR reaction, and the method and amplification conditions were the same as above, and the PCR product was taken for electrophoresis detection on 1% agarose gel. After restriction digestion and PCR identification, the recombinant prokaryotic expression plasmid pET28a-Et CDPK4 was obtained.

[0104] The enzyme digestion system is as follows:

[0105] pET28a-EtCDPK4 plasmid 10μL N i 1.0μL XOt 1.0μL 10×Mbuffer 2.0μL Ultrapure water 15μL

[0106] The sizes of the two fragments obtained after double digestion with Nde I and Xho I were approximately 5289 bp and 1083 bp, respectively, which were consistent with the theoretical values, indicating that the prokaryotic expression vector was successfully constructed ( Figure 2 ). PCR detection using bacterial solution as template showed that all three colonies were positive ( Figure 3 ).

[0107] (4) Experimental expression of recombinant plasmid pET28a-Et CDPK4

[0108] Pick 3 single colonies, add 500μL culture medium (100μg / mL kanamycin resistance), shake for 4h, add 1μL IPTG to 2 of them for induction, and the other one as negative control, shake for another 3 hours, centrifuge at 12000r / min for 3min, take 5μL bacterial solution, add 20μL loading buffer, place in 100℃ metal bath for 15min, and then stain with Coomassie brilliant blue. Results are shown in Figure 4 .

[0109] Depend on Figure 4 It can be seen that after adding inducers, the strains expressing the recombinant plasmid pET28a-Et CDPK4 in 1 and 2 showed a specific protein band at 68KD, while 3 showed no band at 68KD without adding inducers, indicating successful induction. The results showed that bacterial solution 1 had the best induced expression, and bacterial solution 1 was selected for induced expression purification.

[0110] (5) Large-scale expression and purification of recombinant plasmid pET28a-Et CDPK4

[0111] Large-scale expression of recombinant plasmid pET28a-Et CDPK4: Add the bacterial solution to 25 mL of culture medium for overnight expansion, then pour the bacterial solution into 1 L of LB medium containing 100 μg / mL kanamycin resistance, shake the bacteria at 37°C and 220 rpm. When OD 600 When the value reaches 1.8-2.0, cool down to 16℃, add 200μL IPTG to each 1L bacterial solution, and induce overnight. After shaking, centrifuge at 4200r / min for 15min, add 20mL A buffer (25mM Tris-HCl, 500mM NaCl, 5mM imidazole) to resuspend (this process is carried out on ice), add lysozyme at 1mg / mL, and store at -20℃ for later use.

[0112] (6) Purification and concentration of recombinant plasmid pET28a-Et CDPK4

[0113] ① Wash the crusher twice with water, covering the joint, then wash it with A buffer, pour in about 150mL of bacterial solution each time, pressurize to 700-800bar, and crush for 5 minutes. Take 10μL of sample (bacterial lysis solution) after crushing.

[0114] ② The broken bacterial liquid was divided into 50mL centrifuge tubes, centrifuged at 10000r / min at 4℃ for 45min, and the supernatant was filtered on ice using a 0.45μm filter membrane. 10μL of the supernatant was sampled (supernatant).

[0115] ③ Pass the supernatant through the nickel column. Before using the nickel column, treat it first and elute it with B buffer (25mM Tris-HCl, 500mM NaCl, 250mM imidazole). Then change to Abuffer (25mM Tris-HCl, 500mM NaCl, 5mMimidazole) for balancing once. Then pass the supernatant through the bulk nickel column in batches, repeat twice (flow-through sampling), and then wash with Abuffer (wash sampling) to remove impurities. When eluting the target protein with B buffer, blow up the Niresin with a gun tip, and elute with 5mL B buffer in each tube (elution sampling).

[0116] ④ Take samples of bacterial lysate, supernatant, flow-through, wash and eluate and use SDS-PAGE gel for identification ( Figure 5 ).

[0117] ⑤ Concentrate the eluate using a 30KD concentrator, and add 1mM DTT to the protein concentrate (the cutoff value of the concentrator and the addition of DTT are determined according to the properties of the target protein) to prevent the protein from forming aggregates.

[0118] ⑥ Use superdex gel chromatography column for purification, use molecular sieve to further purify the protein and replace the protein buffer with PBS, number 1-14 from the 14 tubes of protein selected after the molecular sieve, then take 5μL sample from each tube, add 20μL Loading buffer to the corresponding number and mix well, add 10μL of the mixture to the SDS-PAGE gel loading wells in sequence according to the number, perform electrophoresis at a voltage of 240v after adding the sample, and stop electrophoresis when the bromophenol blue indicator migrates to 1-2cm from the front. The observation results show that the protein purity of tubes 2-14 is high, so the protein of tubes 2-14 is selected for concentration ( Figure 6 ). Then the protein concentration of each tube was detected, and the aliquots were stored at -80℃ for later use.

[0119] (7) Reactogenicity detection of recombinant Et CDPK4 protein

[0120] The purified recombinant Et CDPK4 protein was analyzed by SDS-PAGE, and detected by Western blotting using E. tenella positive and negative sera as primary antibodies and HRP-labeled goat anti-chicken IgY as secondary antibodies.

[0121] Western blotting analysis showed that the recombinant Et CDPK4 protein could strongly react with anti-E. tenella positive serum ( Figure 7 ), indicating that the recombinant Et CDPK4 protein has a high reactivity with E. tenella positive serum and can be used as a detection antigen, i.e., an antibody capture agent, for clinical detection of chicken E. tenella antibodies.

[0122] Example 2 Method for detecting chicken E. tenella antibodies using ELISA kit

[0123] Purified recombinant Et CDPK4 protein, E. tenella positive serum, E. necatrix positive serum (SPF chickens infected with E. necatrix virulent strain 5×10 4 oocysts, blood was collected 7 days later to separate serum), positive serum for Eimeria acervulina (SPF chickens were infected with a strong strain of E. acervulina 10×10 4 oocysts, blood was collected 7 days later to separate serum), Eimeria maxima positive serum (SPF chickens were infected with 10×10 4 oocysts, blood was collected 7 days later to separate serum), Eimeria praecox positive serum (SPF chickens were infected with 20×10 4 oocysts, and blood was collected 7 days later to separate serum), SPF chicken negative serum; goat anti-chicken IgY-HRP (goat anti-chicken IgY labeled with horseradish peroxidase) was purchased from Thermo Fisher Scientific. Goat anti-chicken IgY-HRP was a lyophilized agent and packaged as 0.1 mg / bottle. When used, it was dissolved into 1 mL according to the instructions; ELISA coating solution was purchased from Solebao Biotechnology Co., Ltd.; color development solution (TMB) was purchased from Solebao Biotechnology Co., Ltd.; skimmed milk powder was purchased from Yamei Biotechnology Co., Ltd.

[0124] 1) Establishment of Et CDPK4-ELISA method for detecting chicken E. tenella antibodies

[0125] (1) Determination of the optimal coating concentration and optimal serum dilution of recombinant Et CDPK4 protein

[0126] The optimal antigen coating concentration and serum dilution concentration were determined by the chessboard method. The recombinant Et CDPK4 protein with a concentration of 1.6 mg / mL was diluted with the coating solution (1:320, 1:640, 1:1280, 1:2560, 1:5120) and coated on the enzyme-labeled reaction plate. Each well was coated with 100 μL. Chicken serum (E. tenella positive serum or negative serum) was serially diluted at a dilution of 1:40, 1:80, 1:160, and 1:320. Sheep anti-chicken IgY-HRP was diluted 1:20000 and determined by the indirect ELISA method. The OD of the positive serum was selected. 450 The nm value is close to 1.0, and the OD of negative serum 450 The dilution of CDPK4 protein and chicken serum with a smaller nm value and a larger P / N value is the optimal working concentration for the antigen-antibody reaction of this ELISA.

[0127] After the recombinant Et CDPK4 protein antigen was serially diluted with the coating solution, the positive serum or negative serum diluted 1:40-1:320 was added; the sheep anti-chicken IgY-HRP enzyme-labeled secondary antibody diluted 1:20000 was added, and the color developing solution (TMB) was added, and the OD was measured with an automatic microplate reader. 450 When the antigen coating concentration is 0.625 μg / mL and the dilution of the positive serum is 1:80, the positive value is close to 1 and the maximum P / N value is 2.356. 450 The nm values ​​are shown in Table 1; the P / N values ​​of each antigen-antibody concentration are shown in Table 2.

[0128] Table 1 OD of square array titration 450 nm Results

[0129]

[0130] Note: “P” stands for positive serum; “N” stands for negative serum.

[0131] Table 2 Determination of the optimal coating concentration and serum dilution (P / N value) of recombinant Et CDPK4 protein

[0132]

[0133] (2) Determination of the optimal antigen coating time

[0134] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The coating conditions were: 4°C overnight, 37°C 1h, 37°C 1.5h, 37°C 2h, 37°C 3h, and the secondary antibody was diluted 1:20000 for ELISA determination. The coating time corresponding to the maximum P / N was selected.

[0135] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then the positive serum or negative serum diluted 1:80 was added. When the antigen was coated at 37°C for 1 hour, the P / N value was the largest, so the coating time was 1 hour, see Table 3.

[0136] Table 3 Determination of the optimal coating time for antigens

[0137] Antigen coating time 4℃ overnight 37℃3h 37℃2h 37℃1h 37℃0.5h Positive serum (P) 1.182 1.285 1.295 1.226 1.083 Negative serum (N) 0.453 0.518 0.486 0.452 0.407 P / N 2.612 2.481 2.665 2.712 2.663

[0138] (3) Determination of the optimal closure time

[0139] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, and the secondary antibody was diluted 1:20000. The screening blocking conditions were: 37°C for 0.5 hours, 37°C for 1 hour, 37°C for 1.5 hours, and 37°C for 2 hours, and the ELISA was determined. The blocking time corresponding to the maximum P / N was selected.

[0140] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL. For every 100 μL, a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, and the secondary antibody was diluted 1:20000. When the blocking time was 1 hour, the P / N value was the largest, so the blocking time was 1 hour, see Table 4.

[0141] Table 4 Determination of the optimal sealing time

[0142]

[0143]

[0144] (4) Determination of the optimal blocking solution

[0145] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the secondary antibody was diluted 1:20000, and the screening blocking solution was 1% skim milk powder, 3% skim milk powder, 5% skim milk powder, and 10% skim milk powder for ELISA determination. The blocking solution corresponding to the maximum P / N was selected.

[0146] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 h, the blocking time was 37°C for 1 h, and the secondary antibody was diluted 1:20000. When the blocking solution was 3% skim milk powder, the P / N value was the largest, so 3% skim milk powder was selected, see Table 5.

[0147] Table 5 Determination of the best blocking solution

[0148]

[0149] (5) Determination of the optimal serum incubation time

[0150] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the blocking solution was 3% skim milk powder, the serum incubation time was 0.5h, 1h, 1.5h, 2h, and the secondary antibody was diluted 1:20000 for ELISA determination. The serum incubation time corresponding to the maximum P / N was selected.

[0151] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 diluted positive serum or negative serum was added. The antigen was coated at 37°C for 1 h, the blocking time was 37°C for 1 h, the blocking solution was 3% skim milk powder, and the secondary antibody was diluted 1:20000. When the serum was incubated for 2 h, the P / N value was the largest, so 2 h was selected, see Table 6.

[0152] Table 6 Determination of the optimal incubation time for serum

[0153]

[0154]

[0155] (6) Determination of the optimal secondary antibody dilution

[0156] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the blocking solution was 3% skim milk powder, the serum was incubated for 2 hours, and the secondary antibody was diluted 1:20000. The secondary antibody dilutions were PBS, 5% skim milk powder, and 3% skim milk powder. ELISA was performed. The secondary antibody dilution corresponding to the maximum P / N was selected.

[0157] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 diluted positive serum or negative serum was added. The antigen was coated at 37°C for 1 h, the blocking time was 37°C for 1 h, 3% skim milk powder was selected as the blocking solution, the serum was incubated for 2 h, and the secondary antibody was diluted 1:20000. When the secondary antibody diluent was PBS, the P / N value was the largest, so PBS was selected, see Table 7.

[0158] Table 7 Determination of the optimal secondary antibody dilution

[0159] Secondary Antibody Dilution PBS 5% skim milk powder 3% skim milk powder Positive serum (P) 0.810 1.005 0.862 Negative serum (N) 0.337 0.420 0.421 P / N 2.404 2.394 2.046

[0160] (7) Determination of the optimal secondary antibody dilution ratio

[0161] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the blocking solution was 3% skim milk powder, the serum was incubated for 2 hours, the secondary antibody dilution solution was PBS dilution, and the secondary antibody was diluted at 1:5000, 1:10000, 1:15000, 1:20000, and 1:25000. ELISA was performed. The secondary antibody dilution corresponding to the maximum P / N was selected.

[0162] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 diluted positive serum or negative serum was added. The antigen was coated at 37°C for 1 h, the blocking time was 37°C for 1 h, 3% skim milk powder was selected as the blocking solution, the serum was incubated for 2 h, and PBS was selected as the secondary antibody dilution solution. When the secondary antibody dilution was 1:15000, the P / N value was the largest, so a 1:15000 dilution was selected, see Table 8.

[0163] Table 8 Determination of the optimal secondary antibody dilution ratio

[0164]

[0165] (8) Determination of the optimal secondary antibody incubation time

[0166] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the blocking solution was 3% skim milk powder, the serum was incubated for 2 hours, the secondary antibody dilution solution was PBS dilution, the secondary antibody was diluted 1:15000, and the secondary antibody incubation time was 0.5h, 1h, 1.5h, 2h, and the ELISA determination was performed. The secondary antibody incubation time corresponding to the maximum P / N was selected.

[0167] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 diluted positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, 3% skimmed milk powder was selected as the blocking solution, the serum was incubated for 2 hours, PBS was selected as the secondary antibody dilution solution, and the secondary antibody was diluted 1:15000. When the secondary antibody incubation time was 1 hour, the P / N value was the largest, so the secondary antibody was selected for 1 hour, see Table 9.

[0168] Table 9 Determination of optimal secondary antibody incubation time

[0169] Secondary antibody incubation time 0.5h 1h 1.5h 2h Positive serum (P) 0.564 0.777 0.992 1.015 Negative serum (N) 0.234 0.310 0.404 0.527 P / N 2.409 2.505 2.455 1.927

[0170] (9) Determination of the optimal color development time

[0171] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the blocking solution was 3% skim milk powder, the serum was incubated for 2 hours, the secondary antibody dilution solution was PBS dilution, the secondary antibody was diluted 1:15000, the secondary antibody action time was 1 hour, and the color development conditions were: 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 25 minutes, and the ELISA determination was performed. The color development time corresponding to the maximum P / N was selected.

[0172] The purified recombinant Et CDPK4 protein antigen was coated on the ELISA reaction plate at a concentration of 0.625 μg / mL, 100 μL per well, and then a 1:80 dilution of positive serum or negative serum was added. The antigen was coated at 37°C for 1 hour, the blocking time was 37°C for 1 hour, the blocking solution was 3% skim milk powder, the serum was incubated for 2 hours, the secondary antibody dilution solution was PBS, the secondary antibody was diluted 1:15000, and the secondary antibody was incubated for 1 hour. When the color development time was 10 minutes, the P / N value was the largest, so 10 minutes was selected, see Table 10.

[0173] Table 10 Determination of the optimal color development time

[0174]

[0175]

[0176] (10) Determination of positive and negative cutoff value of Et CDPK4-ELISA

[0177] Take 30 E. tenella negative serum samples. 450 The mean value (X) of the nm value and the sum of three times the standard deviation (SD) are used as the upper limit of negative serum, that is, when the OD450nm value of the serum sample to be tested is greater than X+3SD, it is judged as positive; otherwise, it is negative. By testing 30 negative sera, the results showed that the X value was 0.338, the SD value was 0.038, and the critical value (X+3SD) was 0.452. That is, the OD value of the sample to be tested is greater than 0.452 for positive, and less than or equal to 0.452 for negative, see Table 11.

[0178] Table 1130 E.tenella negative serum OD 450 nm value

[0179] serial number 1 2 3 4 5 6 7 8 9 10 <![CDATA[OD 450 nm]]> 0.358 0.324 0.362 0.355 0.306 0.338 0.347 0.38 0.4 0.339 serial number 11 12 13 14 15 16 17 18 19 20 <![CDATA[OD 450 nm]]> 0.326 0.359 0.375 0.371 0.288 0.277 0.385 0.292 0.28 0.376 serial number 21 22 23 24 25 26 27 28 29 30 <![CDATA[OD 450 nm]]> 0.343 0.37 0.301 0.255 0.316 0.378 0.342 0.309 0.296 0.385

[0180] (11) Detection procedure of recombinant EtCDPK4-ELISA method

[0181] According to the above optimization results, the detection procedure of the indirect ELISA method is as follows:

[0182] ① Preparation of solid-phase antigen: Coat the recombinant Et CDPK4 protein at a concentration of 0.625 μg / mL in an ELISA plate, add 100 μL / well, incubate at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry; add 200 μL / well blocking solution (3% skim milk powder), block at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry;

[0183] ② Primary antibody binding: dilute the serum to be tested at a volume dilution ratio of 1:80 and add it to the ELISA plate, 100 μL / well, incubate at 37°C for 2 hours, wash the plate 3 times, 5 minutes / time, and pat dry;

[0184] ③ Secondary antibody binding: Dilute the enzyme-labeled secondary antibody (sheep anti-chicken IgY-HRP) with PBS at a volume dilution of 1:15000, add 100 μL / well, react at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry;

[0185] ④ Color development: Add 100 μL / well of TMB color development solution (chromogenic substrate tetramethylbenzidine), color development for 10 min in the dark, and then add 50 μL / well of 2 mol / L sulfuric acid to terminate the reaction;

[0186] ⑤ Detection and judgment: Measure OD with an enzyme marker 450 nm value, when OD 450 A nm value greater than 0.452 is positive, and a value less than or equal to 0.452 is negative.

[0187] 2) Sensitivity test

[0188] The recombinant Et CDPK4 protein was coated at the optimal coating concentration, and the E. tenella positive serum was diluted to 5 gradients of 1:100, 1:200, 1:300, 1:400, and 1:500, and the other conditions were the same as those of this embodiment for the Et CDPK4-ELISA test. The result of the positive serum diluted 1:300 was higher than the critical value of 0.452, indicating that this method can detect the E. tenella positive serum diluted 1:300, as shown in Table 12.

[0189] Table 12 Sensitivity test results

[0190] Positive serum dilution multiple 1：100 1：200 1：300 1：400 1：500 <![CDATA[OD 450 nm]]> 0.925 0.553 0.482 0.383 0.275 PorN P P P N N

[0191] 3) Repeatability experiment

[0192] The recombinant Et CDPK4 protein was coated at the optimal coating concentration, 3 test sera and standard negative sera were diluted at the optimal dilution, added to the antigen-coated wells, 4 wells per portion, and ELISA was performed. The average OD value and standard deviation (SD) were calculated from the OD value of each serum, and then the intra-batch coefficient of variation of each serum was calculated. The results are shown in Table 13. In addition, four batches of recombinant Et CDPK4 protein were coated, 3 test sera and standard negative sera were diluted at the optimal dilution, diluted at the optimal dilution, added to the antigen-coated wells, 4 wells per portion, and ELISA was performed. The average OD value and standard deviation (SD) were calculated from the OD value of each serum, and then the inter-batch coefficient of variation of each serum was calculated. The results are shown in Table 14.

[0193] The coefficients of variation were all less than 10%. The variation of the same sample in the same batch of tests was very small, and the variation of the same sample in different batches of antigen tests was also small. The established Et CDPK4-ELISA method had good repeatability.

[0194] Table 13 Intra-batch repeatability test results

[0195]

[0196] Table 14 Results of inter-batch repeatability test

[0197]

[0198]

[0199] 4) Specificity experiments

[0200] Using the established ELISA conditions, the purified Et CDPK4 protein was coated on the ELISA plate, and the Et CDPK4-ELISA specificity test was performed using E. necatrix, E. maxima, E. praecox, E. acervulina, and E. tenella positive sera, respectively. The results are shown in Table 15. According to the critical value judgment standard, the OD 450 nm was 1.059, much larger than 0.452, and was judged as positive; E.necatrix, E.maxima, E.praecox, E.acervulina and negative serum OD 450 If the nm values ​​are all less than the critical value, they are all negative, indicating that the Et CDPK4-ELISA method established for detecting E. tenella antibodies has good specificity.

[0201] Table 15 Specificity test results

[0202] serum E. necatrix E.maxima E. praecox E. acervulina Negative serum E. tenella <![CDATA[OD 450 nm]]> 0.371 0.407 0.375 0.214 0.289 1.059 PorN N N N N N P

[0203] The Et CDPK4 gene prokaryotic expression antigen provided by the present invention is cloned on the basis of chicken Eimeria tenella virus CDPK4 gene by genetic engineering technology, connected with the prokaryotic expression vector pET28a (+), and transformed into an Escherichia coli expression system for induced expression. The expression form of the protein is His-CDPK4 fusion protein, and the molecular weight of the expressed fusion protein is about 68kDa. The product is designed as fusion expression for the convenience of purification and the increase of the immunogenicity of the expressed protein. The product expressed by the prokaryotic expression system is shown to have an immune response activity similar to that of the natural protein after Western blot identification. The product expressed by the prokaryotic expression system is an Et CDPK4-ELISA method for detecting E.tenella antibodies established by the coated antigen, which has strong specificity, good repeatability and high sensitivity.

[0204] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of recombinant Et CDPK4 protein in the preparation of a preparation for diagnosing Eimeria tenella, characterized in that: The amino acid sequence of the EtCDPK4 protein is shown in SEQ ID NO.

2.

2. The use of recombinant EtCDPK4 protein in the preparation of a reagent for monitoring the antibody level of a live chicken E. tenella vaccine, characterized in that: The amino acid sequence of the Et CDPK4 protein is shown in SEQ ID NO.

2.

3. The use according to claim 1 or 2, characterized in that: The preparation method of the recombinant Et CDPK4 protein is as follows: using E. tenella cDNA as a template, designing specific primers according to the CDPK4 gene sequence, amplifying the target fragment by PCR and cloning it into the expression vector pET28a, transforming the successfully constructed recombinant expression plasmid pET28a-EtCDPK4 into Escherichia coli E. coli BL21, inducing expression by IPTG, and obtaining the recombinant CDPK4 protein after high purification by affinity chromatography and molecular sieve chromatography; the CDPK4 gene sequence is shown in SEQ ID NO.

1.

4. An indirect ELISA detection kit based on recombinant Et CDPK4 protein, characterized in that: Recombinant Et CDPK4 protein was used as coating antigen.

5. An indirect ELISA detection kit based on recombinant Et CDPK4 protein according to claim 4, characterized in that: The kit also includes enzyme-labeled secondary antibody, blocking solution, positive serum, negative serum, and color developing solution; The enzyme-labeled secondary antibody is HRP-labeled goat anti-chicken IgY; The blocking solution is 3% skimmed milk powder; The color developing solution is TMB color developing solution.

6. An indirect ELISA detection method based on recombinant Et CDPK4 protein, characterized in that: The specific steps are as follows: ① Preparation of solid-phase antigen: Coat the recombinant Et CDPK4 protein at a concentration of 0.625 μg / mL in an ELISA plate, 100 μL / well, incubate at 37°C for 1 h, wash the plate 5 times, 3 min / time, and pat dry; Add 200 μL / well blocking solution, block at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry; the blocking solution is 3% skimmed milk powder; ② Primary antibody binding: dilute the serum to be tested at a volume dilution ratio of 1:80 and add it to the ELISA plate, 100 μL / well, incubate at 37°C for 2 hours, wash the plate 3 times, 5 minutes / time, and pat dry; ③ Secondary antibody binding: Dilute the enzyme-labeled secondary antibody with PBS at a volume dilution of 1:15000 and add it, 100 μL / well, react at 37°C for 1 hour, wash the plate 5 times, 3 minutes / time, and pat dry; the enzyme-labeled secondary antibody is goat anti-chicken IgY-HRP; ④ Color development: Add 100 μL / well of TMB color development solution, color for 10 min in the dark, and then add 50 μL / well of 2 mol / L sulfuric acid to terminate the reaction; ⑤ Detection and judgment: Measure OD with an enzyme marker 450 nm value, when OD 450 A nm value greater than 0.452 is positive, and a value less than or equal to 0.452 is negative.

7. Use of the kit according to claim 4 or 5 in detecting antibodies against Eimeria tenella.

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