CN110183527B

CN110183527B - Neosporozoan NcMIC26 antigen and application thereof

Info

Publication number: CN110183527B
Application number: CN201910422519.1A
Authority: CN
Inventors: 刘晶; 王先梅; 刘群; 许建海
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2021-03-12
Anticipated expiration: 2039-05-21
Also published as: CN110183527A

Abstract

The invention discloses a neospora NcMIC26 antigen and application thereof. The invention firstly discloses a neospora NcMIC26 antigen, and the amino acid sequence of the antigen is shown in SEQ ID NO. 1. The invention further discloses application of the recombinant protein of the antigen in constructing an indirect ELISA diagnostic kit of neospora caninum. The invention obtains a new diagnosis antigen suitable for neosporosis by screening, establishes an indirect ELISA kit by using the recombinant protein thereof as the envelope antigen, has simple operation, high speed and efficiency, good specificity, high sensitivity and good repeatability, can be used for detecting the infection condition of the bovine neosporosis, can detect a positive sample which can not be detected by the common NcSRS2 protein, can obtain more accurate detection data, and has important significance for preventing and treating the neosporosis.

Description

Neosporozoan NcMIC26 antigen and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a neospora NcMIC26 antigen and application thereof.

Background

Neosporosis (neosporiosis) is a protozoal disease caused by neosporosis (Neospora caninum) that infects a variety of animals, and the disease caused by neosporosis has a tendency to be distributed globally. The disease has the most serious harm to cattle and dogs, is one of the main causes of nervous system disorder diseases of cattle abortion, weak fetus, dead fetus, mummy fetus and dogs, and brings serious economic loss to animal husbandry. The infection intensity of the bovine neosporosis reported in different countries and regions has certain difference, and the positive rate of serum antibodies is 13.5-82%. At present, no effective drugs or vaccines exist for neospora.

The main detection methods of the neosporosis comprise etiological diagnosis, histopathological diagnosis, molecular biological diagnosis, serological diagnosis and the like. Because the neospora is an obligate intracellular parasitic protozoa, the etiology and pathology diagnosis is difficult, and the serological diagnosis method can be applied to the live animal detection, the information of the animal infection stage can be provided in the epidemiological investigation process, and the antibody level after the neospora is infected by the neospora can provide information about the risk that the animal suffers from neosporosis, ruminants and the neospora is spread through placenta, and the like, so the method has become an important means for the epidemiological investigation and research. The earliest serological technique applied to the detection of neospora antibodies was the indirect immunofluorescence assay (IFAT), which is also the "gold standard" for the current serological detection of neospora. However, reagents (such as the quality of a specific fluorescent labeled antibody) and instruments (such as a fluorescent microscope) required by the experiment and operation steps are complex, the requirement on experiment operators is high, and the judgment of the result critical value is difficult to determine, so that the method is not suitable for clinical field large-batch screening experiments. With the development of large-scale epidemiological research, the ELISA detection method is widely applied due to its advantages of simplicity, rapidness, high efficiency, no influence of subjective factors on the result judgment, and the like, and gradually becomes a main method for serological detection.

Early indirect ELISA (enzyme-Linked immunosorbent assay) is used for detecting tachyzoites, cracked neospora antigen and Immune Stimulating Complex (ISCOM) diagnosis antigen, and shows good diagnosis parameters in the neospora diagnosis process, but because the relative relationship of neospora and toxoplasma belonging to the same genus of Plasmodium, cross reaction is easy to occur, so that the specificity is not high, and the preparation cost of the antigen is high. With the development of recombinant protein expression and purification technology, the diagnosis technology of using high-purity recombinant protein to replace natural antigen as detection antigen makes up the above disadvantages. It is currently accepted that the most widely used neospora diagnostic antigen is NcSRS2, which shows good specificity and sensitivity in serological tests. However, there is some fluctuation in the infected cattle due to different periods of infection of the cattle with neospora and antibody levels. In some studies, it was found that the serology of cows with neospora positive fetuses is negative and the cause of this phenomenon may be due to fluctuations in antibody levels in the cows or to false negatives due to the lack of sensitivity of the detection method. The reported detection of antigens still has limitations in the actual diagnosis of neosporosis in cattle, causing errors in the results of epidemiological investigations.

Therefore, there is an urgent need to screen a new dominant antigen with better diagnostic effect, establish a more sensitive and specific serological diagnostic method, apply to the detection of animal population, and provide a basis for the diagnosis of neosporosis.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

It is an object of the present invention to provide a novel dominant neospora antigen which has high sensitivity and good specificity for detecting neospora.

The invention also aims to provide an indirect ELISA kit which comprises the recombinant protein of the antigen as a coating antigen, and a simple, quick and efficient serological diagnosis method is established.

In order to achieve the aim, the invention provides a neospora NcMIC26 antigen, and the amino acid sequence of the antigen is shown in SEQ ID NO. 1.

In a preferred embodiment, the screening and identification of the antigen for the preparation of neospora NcMIC26 according to the invention comprises the following steps:

culturing neospora, proliferating, collecting polypide, re-suspending, depositing, incubating, and collecting the excretory secretion antigen of tachyzoite and the whole antigen of tachyzoite;

carrying out immunoblotting on the collected tachyzoite excretory secretion antigen and tachyzoite whole worm antigen, and screening to obtain a neospora specificity protein strip which does not react with positive serum of the toxoplasma gondii;

the neospora specific protein band was collected by SDS-PAGE electrophoresis and subjected to mass spectrometry to obtain the neospora NcMIC26 antigen of the present invention.

The present invention further provides a gene encoding the aforementioned neospora NcMIC26 antigen.

In a preferred embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

The invention also provides application of the neospora NcMIC26 antigen or the gene coding the neospora NcMIC26 antigen in preparing products for treating, diagnosing or preventing neosporosis.

The invention further provides a neospora indirect ELISA diagnostic kit, which comprises a recombinant protein rNcMIC26 with an amino acid sequence shown in SEQ ID NO. 1.

In a preferred embodiment, the method for preparing the recombinant protein rNcMIC26 comprises the following steps:

1) design of primers F and R:

F：5’-AGCAAATGGGTCGCGGATCCGTTCTGGATTTCATAGACTTGG-3’；

R：5’-TCGAGTGCGGCCGCAAGCTTCGAAGTCCATTCGCCCCACGTT-3’；

2) carrying out PCR amplification by using cDNA of neospora using primers F and R as a template to obtain a target fragment;

3) connecting the target fragment with a vector pET-28a, transforming the connection product into an escherichia coli Trans1-T1 competent cell to obtain a recombinant prokaryotic expression plasmid pET-28a-MIC 26;

4) the constructed recombinant prokaryotic expression plasmid pET-28a-MIC26 is transformed into an escherichia coli Transetta competent cell, the expression of recombinant protein is induced, and the recombinant protein rNcMIC26 is obtained by collection and purification.

The kit for diagnosing the neospora indirect ELISA further comprises an HRP-labeled detection antibody, a coating solution, a confining solution, an antibody diluent, a washing solution, a substrate solution, a stop solution, neospora positive serum and negative serum.

In a preferred embodiment, the HRP-labeled detection antibody is an HRP-labeled goat anti-bovine antibody; the coating solution is 0.05M carbonate buffer solution with pH value of 9.6; the confining liquid is 5% horse serum-PBST; the antibody diluent is 2% horse serum-PBST; the wash was 0.5% PBST; the substrate solution is TMB substrate solution A and substrate solution B; the stop solution is 2M concentrated sulfuric acid.

The invention provides a using method of the neospora indirect ELISA diagnostic kit, which specifically comprises the following steps:

a) antigen coating: diluting the recombinant protein rNcMIC26 to 10 mu g/mL by using 0.05M carbonate buffer solution with the pH value of 9.6, and standing at 4 ℃ for 14-16 h after the coating condition is that the temperature is 37 ℃ for 1 h;

b) washing: discarding the liquid in the wells, adding 300. mu.L of 0.5% PBST, washing 4 times, each time with 5min intervals;

c) and (3) sealing: blocking solution using 5% horse serum-PBST, each well 100 u L, 37 degrees C blocking for 1 h;

d) washing: the operation steps are the same as those in b);

e) primary antibody incubation: 2% horse serum-PBST solution 1: diluting bovine serum to be detected by 200, wherein each hole is 100 mu L, and each plate is provided with standard bovine neospora positive serum and negative serum as positive and negative controls; incubating at 37 ℃ for 1 h;

f) washing: the operation steps are the same as those in b);

g) and (3) secondary antibody incubation: dilution of HRP-labeled goat anti-bovine antibody with 2% horse serum-PBST solution 1: 25000 dilution, 100 μ L per well, incubation at 37 ℃ for 0.5 h;

h) washing: the operation steps are the same as those in b);

i) color development: TMB substrate solution A and substrate solution B were mixed in the following ratio of 1: 1, 100 mu L of the mixture is mixed in each hole, and the mixture is developed for 10min at room temperature;

j) and (4) terminating: terminating the reaction by 2M concentrated sulfuric acid in each hole, wherein each hole is 50 mu L; after the reaction is finished, reading the OD of the double wavelengths of each hole on an enzyme-labeling instrument_450/630nmA value;

h) and (4) judging a result: OD of ELISA when serum sample to be detected_450/630nmWhen the value is not less than 0.165, a positive result can be judged at a level of 99.9%.

Compared with the prior art, the invention has the following beneficial effects:

the invention obtains a new neospora diagnosis antigen by screening, establishes an indirect ELISA kit by using the recombinant protein thereof as the envelope antigen, has simple operation, high speed and efficiency, good specificity, high sensitivity and good repeatability, can be used for detecting the infection condition of the bovine neospora, can detect a positive sample which can not be detected by the common NcSRS2 protein, can obtain more accurate detection data, and has important significance for preventing and treating the neospora.

Drawings

FIG. 1 is an SDS-PAGE pattern and a Western Blot identification pattern of a secreted protein of neospora according to an embodiment of the present invention; wherein, 1: tachyzoite excreting secretory antigen esa; 2: the tachyzoite holozoite antigen pellet.

FIG. 2 is an amplification diagram of the MIC26 gene according to one embodiment of the present invention; wherein, M: DL-2000plus DNA Marker; 1: MIC26 gene amplification product.

FIG. 3 is a diagram of prokaryotic inducible expression and purification of rNcMIC26-His protein according to one embodiment of the present invention; wherein, M: a protein Marker; 1: a non-induced bacteria liquid sample; 2: a bacterial liquid sample after induction; 3: a supernatant protein sample; 4: an inclusion body protein sample; 5: purified rNcMIC26-His protein.

FIG. 4 is a graph of rNcMIC26-His reactogenicity and specificity identification according to one embodiment of the present invention; wherein, M: pre-staining a protein Marker; 1: murine Nc negative serum; 2: positive serum of murine origin; 3: murine Tg positive serum; 4: murine His monoclonal antibody.

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

The experimental methods involved in the invention are all conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The biological material is publicly available to the applicant as Neospora Nc1(Conrad P A, Barr B C, Sverlow K W, et al. in vitro isolation and characterization of a Neospora sp. from the isolated born foods [ J ]. Parasitology,1993,106.) and is only useful for repeating the experiments related to the present invention and is not otherwise useful.

The strain is as follows: coli Transetta (DE3), Trans1-T1 competent cells were purchased from Kyoto Kogyo gold Biotech, Inc.; m5DH5a complete Cell was purchased from Convergence, Inc., Beijing.

Carrier: pET28a prokaryotic expression plasmid, kanamycin resistance, T7 promoter initiated expression fusion of His tag of the protein of interest.

cDNA reverse transcription kit: from Beijing-Okayama-Kagaku gold Biotech Ltd

2 XExTaq Mix PCR reagent, Goldview nucleic acid dye, DL2000plus DNA marker, reverse transcription kit (EasyScript One-step gDNA Removal and cDNA synthesis Supermix): purchased from Beijing Quanjin Biotechnology Ltd.

His fusion protein purification column (Ni2+), PVDF membrane: purchased from Merck Millipore Novagen, Germany.

Low background ECL luminescence color development kit: purchased from Beijing kang, a century biotechnological technology, Inc.

Single fragment ligation kit: purchased from Nanjing Novozam Biotechnology, Inc.

HRP-labeled goat anti-bovine IgG antibody: purchased from Southern Biotechnology Associates, Inc. (USA).

The following detailed description is provided to provide a better understanding of the invention; it should be understood, however, that the scope of the present invention is not limited by the particular embodiments described.

Example 1: screening of neospora diagnostic antigens

1) Collection of secreted protein of neospora

Neospora Nc1 was cultured in large quantities in Vero cells, and when the neospora propagated for about 72 hours to a state to be released, the polypide was isolated, purified and collected from the Vero cells. The count reaches 5 multiplied by 10⁷Resuspending the precipitate with 200 μ L serum-free DMEM, incubating at 37 deg.C for 3-5 hr; after the incubation is finished, the neospora secretes the protein by centrifugation, namely the extracellular tachyzoite excretory secretory antigen (esa) and the tachyzoite holozoite antigen (p) respectivelyellet); the collected secreted proteins were subjected to SDS-PAGE and silver stained according to the instructions of the silver staining kit, and the results are shown in FIG. 1.

2) Western Blot identification of neospora secretory protein

Identifying antigen reactogenicity and specificity of the secretory protein collected by the method by using a Western blot method, wherein the primary antibody is murine neospora positive serum and rabbit toxoplasma positive serum, and the secondary antibody is goat anti-mouse IgG marked by HRP and goat anti-rabbit IgG marked by HRP; as a result, as shown in FIG. 1, a protein band (50 to 70kDa) specific to neospora, which did not react with the positive serum of Toxoplasma gondii, was selected.

3) Mass spectrometric identification of neospora specific proteins

According to the result of Western blot, cutting off a corresponding specific band (50-70 kDa) in SDS-PAGE protein gel, sending the cut band to Beijing Huada protein research and development center Limited company for mass spectrometry to obtain corresponding polypeptide information, analyzing the immunogenicity of proteins in combination with bioinformatics, comprehensively analyzing the immunogenicity of each antigen gene, and determining that the selected dominant antigen of neospora is NcMIC26, such as an amino acid sequence shown in SEQ ID No. 1.

Example 2: prokaryotic induction expression and purification of recombinant protein r NcMIC26

1) Cloning of NcMIC26 Gene

According to the advantageous antigen NcMIC26 of neospora as obtained in example 1, the coding gene sequence of NcMIC26 antigen of neospora is found in ToxoDB online database (http:// www.toxodb.org), the signal peptide is removed, the part (271bp-1014bp) with lower similarity to the homologous gene of Toxoplasma is selected, primers F and R are designed, the cDNA of neospora is used as a template, the gene fragment of the MIC26 of neospora is amplified, the nucleotide sequence of the gene fragment is shown in SEQ ID NO.2, and the sequence size is 744bp as shown in FIG. 3;

wherein, the cDNA of the neospora is used for extracting RNA of the neospora and carrying out reverse transcription by a Trizol-chloroform method, and the method specifically comprises the following steps:

collecting purified neospora tachyzoites up to 3-5 x 10⁷1mL of the powder

Resuspending the RNA separating reagent evenly, and standing for 10min at room temperature; adding 200 μ L chloroform, vortex oscillating for 20s, standing at room temperature for 5min, centrifuging at 4 deg.C at 12000rpm for 15 min; transferring the sample to a new EP tube after centrifugation, adding equal volume of isopropanol precooled at-80 ℃, standing for 15min in a refrigerator at-20 ℃, and then centrifuging at 12000rpm for 15min at 4 ℃; discarding the supernatant, adding 1mL of 75% ethanol precooled at-20 ℃, slowly suspending the precipitate, and centrifuging at 12000rpm at 4 ℃ for 15 min; this step was repeated and the supernatant discarded. Adding about 15 mu L DEPC water to dissolve the precipitate after the ethanol is completely volatilized, thus obtaining the RNA after extraction.

Using cDNA reverse transcription kit, reverse transcription was performed according to the reaction system of Table 1 to obtain the cDNA of neospora.

TABLE 1 reaction System for reverse transcription of neospora RNA into cDNA

The primers are as follows:

F：5’-AGCAAATGGGTCGCGGATCCGTTCTGGATTTCATAGACTTGG-3' (shown in SEQ ID NO. 3) contains a BamHI cleavage site (underlined),

R：5’-TCGAGTGCGGCCGCAAGCTTCGAAGTCCATTCGCCCCACGTT-3' (shown in SEQ ID NO. 4) contains a HindIII cleavage site (underlined);

the reaction system is as follows: template cDNA 1. mu.L, primer F1. mu.L, primer R1. mu.L, 2 XExTaq Mix PCR polymerase 25. mu.L, DDH₂O22. mu.L, 50. mu.L total;

the PCR reaction process is as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 57 deg.C for 1min, extension at 72 deg.C for 1min, setting 35 cycles, and final extension at 72 deg.C for 10 min; and (3) after nucleic acid electrophoresis of the PCR product, recovering the target fragment obtained by PCR by using a gel recovery kit, and storing at-20 ℃.

2) Construction and protein expression of recombinant prokaryotic expression plasmid pET-28a-MIC26

The purified neospora caninum MIC26 gene fragment and pET-28a double enzyme digestion framework are subjected to single-fragment connection by using a single-fragment connection kit (the reaction system is 3 mu L of pET-28a double enzyme digestion vector framework, 4 mu L of MIC26 gene fragment, 2 mu L of 5 XCE Buffer, 1 mu L of Exnase and 10 mu L in total); transforming the ligation product into an escherichia coli Trans1-T1 competent cell, inoculating a proper amount of bacterial liquid into a kanamycin-resistant LB solid culture medium, and inverting for overnight culture; and selecting a single colony for PCR identification, selecting positive bacteria liquid for sequencing, and obtaining the recombinant prokaryotic expression plasmid pET-28a-MIC 26.

The constructed recombinant prokaryotic expression plasmid pET-28a-MIC26 is transformed into an escherichia coli Transetta competent cell, the monoclonal bacteria which are identified as positive by PCR are subjected to amplification culture in a Kanna resistant LB liquid culture medium, and the expression of the recombinant protein is induced by 0.8mM IPTG.

Collecting thalli, carrying out ice bath ultrasonic lysis, carrying out centrifugal separation on supernatant and inclusion body protein at 4 ℃, carrying out SDS-PAGE electrophoresis analysis on the expression form of the recombinant protein, and obtaining a result shown in figure 3, wherein the recombinant protein rNcMIC26 is mainly expressed in an inclusion body, and the recombinant protein is purified by a nickel affinity chromatography method after the inclusion body is washed and renatured; protein purity was analyzed by SDS-PAGE, and the results are shown in FIG. 3, and the resulting purified recombinant protein rNcMIC26-His was 35kDa in size, and the purified protein was stored at-80 ℃ after being dispensed.

3) rNcMIC26-His reactogenicity and specificity identification

Identifying the reactogenicity of recombinant protein rNcMIC26-His by using Western Blot, and specifically operating as follows: respectively taking the neospora murinus positive serum (1: 400), the neospora murinus negative serum (1: 400), the toxoplasma murinus positive serum (1: 400) and the His monoclonal antibody (1: 500 is only used for recombinant protein with His labels) as primary antibodies for incubation; the secondary antibody was subjected to immunoblotting using a corresponding HRP-labeled goat anti-mouse IgG (1: 5000) antibody; the results are shown in FIG. 4, which shows that the recombinant protein rNcMIC26-His has good reactogenicity, the recombinant protein rNcMIC26-His of the neospora is determined not to have cross reaction with the toxoplasma, and the protein is initially evaluated to have the diagnostic value of the neosporosis.

Example 3: establishment of bovine serum neospora antibody indirect ELISA detection method

1) Reaction conditions

The reaction conditions of bovine serum neospora antibody indirect ELISA are shown in Table 2:

TABLE 2 bovine serum neospora antibody Indirect ELISA reaction conditions

The method comprises the following steps:

a) antigen coating: diluting the recombinant protein rNcMIC26 to 10 mu g/mL by using 0.05M carbonate buffer solution with the pH value of 9.6, and coating the recombinant protein rNcMIC26 at 37 ℃ for 1h and at 4 ℃ overnight (14-16 h) in a 96-well enzyme-labeled microplate at each well;

d) washing: the operation steps are the same as those in b);

f) washing: the operation steps are the same as those in b);

h) washing: the operation steps are the same as those in b);

j) and (4) terminating: terminating the reaction by 2M concentrated sulfuric acid in each hole, wherein each hole is 50 mu L; after the reaction is finished, reading the OD of the double wavelengths of each hole on an enzyme-labeling instrument_450/630nmThe value is obtained.

2) Determination of the cut-off value for the ELISA detection method

rNcMIC26-ELISA detection of 30 bovine neospora antibody negative serum OD_450/630nmValue asAs shown in Table 3, average values were obtained

0.065, Standard Deviation (SD) 0.033, cut-off value

0.165, according to the statistical principle, when the OD of the ELISA is found in the serum sample to be examined_450/630nmWhen the value is not less than 0.165, a positive result can be judged at a level of 99.9%.

TABLE 3 determination of rNcMIC26-ELISA cut-off values

3) Sensitivity test

Standard positive serum samples were prepared from 1: 50, starting to dilute at a multiple ratio, performing an ELISA experiment, and judging the highest dilution of serum by using a critical value; the results are shown in table 4, and the optimized indirect ELISA method can detect the highest serum dilution of 1: 3200, indicates that the method has high detection sensitivity.

TABLE 4 rNcMIC26-ELISA sensitivity test results

4) Cross reaction experiment

The results of cross reaction tests with toxoplasma gondii are shown in Table 5, which indicates that the OD values of the positive serum of the bovine toxoplasma gondii detected by the method are all smaller than the critical value, and indicates that the created rNcMIC26-ELISA is a neospora antibody specificity indirect ELISA detection method, and bovine toxoplasma gondii infection can be distinguished in the process of diagnosing neosporosis.

TABLE 5 rNcMIC26-ELISA specificity test results

5) Repeatability test

The results of the in-batch and in-batch repeat tests are shown in tables 6 and 7, and the coefficient of variation of all the comparative samples is less than 15%, which indicates that the method has better repeatability.

TABLE 6 rNcMIC26-ELISA in-batch repeat experiment results

TABLE 7 rNcMIC26-ELISA batch-to-batch repeat results

6) Comparative experiment

Detecting 98 parts of neospora positive and 39 parts of neospora negative bovine serum by using IFAT, respectively detecting by using indirect ELISA established by two antigens of MIC26 and SRS2, and comparing specificity and sensitivity of the ELISA; as shown in Table 8, compared with IFAT, rNcMIC26-ELISA has 76.53% (75/98) higher sensitivity and 84.62% (33/39) higher specificity than SRS2, and the two proteins have similar diagnostic effects; in the process of detecting 98 parts of neospora positive serum, 75 parts of serum which is positive by rNcMIC26-ELISA and 65 parts of serum which is positive by rSRS2-ELISA are detected, and the positive results of the serum and the serum are not consistent; the tested 137 sera were statistically analyzed, and the combined results of the two diagnostic antigens showed a positive match rate of 93.88% (92/98) and a negative match rate of 84.62% (33/39) with IFAT.

TABLE 8 comparison of compliance rates of different antigen-coated indirect ELISA with IFAT

Therefore, the neospora antigen screened by the method has good diagnostic sensitivity and high specificity, can be used as a supplementary antigen for missed detection of the diagnostic antigen SRS2 at present, improves the detection sensitivity to a greater extent, can be applied to detection of bovine serum neospora antibody, and has high reliability.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Sequence listing

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Claims

1. A gene for coding neospora NcMIC26 antigen, wherein the amino acid sequence of the antigen is shown in SEQ ID NO.1, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

2. Use of the gene of claim 1 for the preparation of a product for the treatment, diagnosis or prevention of neosporosis.

3. An indirect ELISA diagnostic kit for neospora caninum is characterized in that the diagnostic kit comprises a recombinant protein rNcMIC26 with an amino acid sequence shown as SEQ ID NO. 1.

4. The kit for the indirect ELISA diagnosis of neospora according to claim 3, wherein the recombinant protein rNcMIC26 is prepared by the following steps:

1) design of primers F and R:

F：5’-AGCAAATGGGTCGCGGATCCGTTCTGGATTTCATAGACTTGG-3’；

R：5’-TCGAGTGCGGCCGCAAGCTTCGAAGTCCATTCGCCCCACGTT-3’；

5. The kit for diagnosing neospora caninum indirect ELISA according to claim 3, wherein the kit further comprises HRP-labeled detection antibody, coating solution, blocking solution, antibody diluent, washing solution, substrate solution, stop solution, neospora caninum positive serum, negative serum.

6. The indirect ELISA diagnostic kit of neospora caninum as claimed in claim 5 wherein the HRP-labeled detection antibody is an HRP-labeled goat anti-bovine antibody; the coating solution is 0.05M carbonate buffer solution with pH value of 9.6; the confining liquid is 5% horse serum-PBST; the antibody diluent is 2% horse serum-PBST; the wash was 0.5% PBST; the substrate solution is TMB substrate solution A and substrate solution B; the stop solution is 2M concentrated sulfuric acid.