An antigen for detecting type i and type ii feline coronavirus antibodies, and a kit and application thereof

Abstract

The application belongs to the technical field of antibody detection, and particularly relates to an antigen for detecting type I and type II cat coronavirus antibodies, a kit and application thereof. The antigen for detecting type I and type II cat coronavirus antibodies is recombinant N protein, IRBD protein (type I) and IIRBD (type II) protein of purified cat coronavirus, and an indirect ELISA detection kit is composed of an enzyme-labeled plate of the purified recombinant N protein and RBD protein of cat coronavirus. The results of the embodiments of the application show that the ELISA kit for cat coronavirus antibodies has the advantages of low cost, simple and convenient use, high sensitivity and strong specificity, does not produce cross-reactions with other common pathogenic agents in a cat population, and can effectively distinguish type I and type II cat coronaviruses.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibody detection, and particularly relates to an antigen for detecting type I and type II feline coronavirus antibodies and a kit and application thereof. BACKGROUND

[0002] Feline coronavirus (FCOV) is a single-stranded positive-sense RNA virus with a capsid envelope, and its genome is relatively large, containing about 29,000 nucleotides. The genome structure of feline coronavirus has typical characteristics of coronaviruses, consisting of 11 open reading frames (ORFs) that can encode 11 different proteins, including structural proteins, non-structural proteins and auxiliary proteins. The structural proteins include spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). In the field of coronavirus research, the receptor binding domain (RBD) at the C-terminal of spike protein S1 not only contains the main neutralizing epitope, but also has strong immunogenicity, and is a key target for vaccine and antiviral drug design, and has important value for the development of vaccines and diagnostic reagents.

[0003] Feline coronavirus is prone to variation during replication, which may promote its transformation from feline enteric coronavirus (FECV) to feline infectious peritonitis virus (FIPV), causing fatal feline infectious peritonitis (FIP) and becoming one of the main causes of death of domestic and wild cats. FCOV exists in two serotypes: type I and type II, of which type I can persist in the cat body for a long time, while type II may have a closer association with the lethality of feline infectious peritonitis. Therefore, distinguishing the two serotypes is of great significance for the treatment of FCOV-infected cat groups in the clinic.

[0004] The early symptoms of feline infectious peritonitis are not specific, showing decreased appetite and listlessness, which are difficult to distinguish from enteritis or other diseases, and therefore laboratory detection is needed for diagnosis, which increases the difficulty of on-site diagnosis. There is no good treatment and prevention method for FIP at present, resulting in a very high infection rate of FCOV in cat groups.

[0005] The in vitro isolation and culture of FCOV are extremely sensitive to the environment, affected by sample quality, experimental operation and culture conditions and other factors, and the detection rate is usually low. Polymerase chain reaction (PCR) and immunohistochemical techniques are the main methods for detecting FCOV. PCR technology has strict requirements for experimental conditions, and improper operation may lead to false positive results, while variation of viral sequences may lead to mismatch with primers, resulting in false negative results. Real-time quantitative PCR (RT-PCR) technology monitors the initial amount of DNA through a fluorescent probe, which shows higher sensitivity in clinical practice than traditional PCR, but has relatively high requirements for operation and instrument facilities, long detection time, which limits its application in the differential diagnosis of early FCOV infection. Immunohistochemical detection usually requires invasive operation, which causes certain physical harm to cats and is mainly used for postmortem organ sample detection.

[0006] In contrast, ELISA technology is widely used in clinical diagnosis due to its simple operation, rapidness, no need for special equipment, and advantages such as stability, specificity and sensitivity of reagents. Indirect ELISA can provide results within 4-5 hours, suitable for detection of various antigens or antibodies. However, there is no effective commercial cat coronavirus antibody ELISA detection kit on the market, and the serological diagnosis of FCOV has the limitation of sensitivity and specificity. The indirect ELISA method for detecting cat coronavirus antibodies is usually designed with a single protein (S protein, M protein or N protein) of cat coronavirus, which has a narrow detection range and cannot detect two serotypes of cat coronavirus at the same time, and cannot realize the differentiation of serotypes.

[0007] Therefore, there is an urgent need for a substance that can effectively distinguish the serotypes of FCOV. SUMMARY

[0008] The purpose of the present application is to provide an antigen for detecting type I and type II cat coronavirus antibodies, which can not only realize the differentiation of cat serotypes, but also detect quickly and accurately.

[0009] In order to achieve the above purpose, the present application provides the following technical solutions:

[0010] The present application provides an antigen for detecting type I and type II cat coronavirus antibodies, which is one or more groups of purified recombinant type I N protein, type I IRBD protein and type II IIRBD protein of cat coronavirus; the amino acid sequences of the recombinant type I N protein, type I IRBD protein and type II IIRBD protein are shown in SEQ ID NO. 1-3.

[0011] The nucleotide sequences encoding the above-mentioned recombinant type I N protein, type I IRBD protein and type II IIRBD protein are shown in SEQ ID NO. 4-6.

[0012] The application also provides an indirect ELISA detection kit for detecting type I and type II feline coronavirus antibodies, wherein an enzyme-labeled plate of the kit is coated with the above-mentioned antigen.

[0013] Preferably, the kit further comprises a positive control sample, a negative control sample, a sample diluent, a washing solution, an HRG-labeled rabbit anti-feline IgG antibody, a color developing solution and a termination solution.

[0014] More preferably, the positive control sample is FCOV positive cat serum, the negative control sample is FCOV negative cat serum, the sample diluent is PBS, the washing solution is PBST containing 0.05% Tween-20, the color developing solution is a TMB solution and the termination solution is a 2M sulfuric acid solution.

[0015] The application also provides an indirect ELISA detection method for type I and type II feline coronavirus antibodies, which specifically comprises the following steps:

[0016] S1, serum incubation: after dilution, the sample serum is added to an enzyme-labeled plate coated with protein recombinant N protein, IRBD protein (type I) and IIRBD protein (type II), and then incubated at 37℃ for 1h, washed with PBST for 5 times and dried;

[0017] S2, enzyme-labeled antibody incubation: the enzyme-labeled plate obtained in step S1 is added with 4000-fold diluted HRP-labeled rabbit anti-feline IgG antibody, and then incubated at 37℃ for 1h, the solution is discarded, washed with PBST for 5 times and dried;

[0018] S3, color development: TMB single-component color developing solution is added to each well, and color development is performed in the dark for 4min;

[0019] S4, termination: termination solution is added to each well to terminate the reaction, and the OD450 nm value of the sample to be detected is determined.

[0020] Preferably, the method further comprises determining whether the sample to be detected contains feline coronavirus antibodies.

[0021] Preferably, the standard for determining whether the sample to be detected contains feline coronavirus antibodies is that the critical value of recombinant N protein is 0.264, that is, the OD450 nm of the detected serum is greater than or equal to 0.264, which is positive; the critical value of recombinant IRBD protein is 0.254, that is, the OD450 nm of the detected serum is greater than or equal to 0.254, which is positive; the critical value of recombinant IIRBD protein is 0.222, that is, the OD450 nm of the detected serum is greater than or equal to 0.222, which is positive.

[0022] The application also provides the above-mentioned antigen or the above-mentioned kit for detecting type I and type II feline coronavirus antibodies.

[0023] The beneficial effects of the present application are:

[0024] (1) The present application uses N recombinant protein as a coating antigen, and N gene is a highly conservative gene, so the kit containing the protein can detect different strains of FCOV, and can be used for preventive detection of pet cats and experimental cats FCOV.

[0025] (2) The RBD protein on the S protein of the type I feline coronavirus and the RBD protein on the S protein of the type II feline coronavirus are selected as antigens in the kit of the present application, and there is no cross reaction between the RBD proteins of the type I and type II viruses, therefore, the indirect ELISA method established by the present application can identify the type I FCOV and the type II FCOV.

[0026] (3) The combined detection of N antigen and RBD antigen can improve the sensitivity of serological detection, especially in the early stage of the disease, the combined diagnosis of N protein and RBD protein antibody can improve the accuracy of FCoV detection.

[0027] (4) The indirect ELISA detection method established by the present application has good sensitivity, specificity and repeatability, and the preliminary clinical application also shows that the kit can detect a large number of FCOV infected cats, and the effect of identifying serotypes is good.

[0028] (5) The kit provided by the present application provides an enzyme-labeled plate coated with recombinant N protein, IRBD protein and IIRBD protein, which can be detected on site without special detection personnel, and the detection is fast and accurate, and good application effect is obtained in preliminary application, which provides an effective tool for early diagnosis of FCoV infection. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 The electrophoresis map for verifying the recombinant plasmid by PCR of the bacterial liquid in Example 1 is shown in the figure, wherein lane M is 5000 DNA marker, lane 1 is recombinant plasmid pET-28a-N, lane 2 is recombinant plasmid pET-28a-IRBD, and lane 3 is recombinant plasmid pET-32a-IIRBD.

[0031] Figure 2Figure 1 is a photograph of a gel showing the results of the induction expression and purification of the recombinant proteins in Example 1, in which lane M is a protein marker; lane 1 is supernatant of recombinant protein pET-28a-N; lane 2 is precipitate of recombinant protein pET-28a-N; lane 3 is precipitate of recombinant protein pET-28a-IRBD; lane 4 is supernatant of recombinant protein pET-28a-IRBD; lane 5 is supernatant of recombinant protein pET-32a-IIRBD; lane 6 is precipitate of recombinant protein pET-32a-IIRBD;

[0032] Figure 3 Figure 2 is a photograph of a gel showing the detection of the product during the purification of the recombinant protein pET-28a-N in Example 1, in which lane M is a protein marker; lane 1 is a flow-through; lanes 2-4 are washes; lane 5 is 200 mM imidazole eluate; lanes 6-9 are 500 mM imidazole eluate;

[0033] Figure 4 Figure 3 is a photograph of a gel showing the detection of the product during the purification of the recombinant proteins pET-28a-IRBD and pET-32a-IIRBD in Example 1, in which lane M is a protein marker; lane 1 is 1 M urea solubilized inclusion body solution of the lysis precipitate of pET-28a-IRBD; lane 2 is 3 M urea solubilized inclusion body solution of the lysis precipitate of pET-28a-IRBD; lane 3 is 6 M urea solubilized inclusion body solution of the lysis precipitate of pET-28a-IRBD; lane 4 is 8 M urea solubilized inclusion body solution of the lysis precipitate of pET-28a-IRBD; lane 5 is 8 M urea solubilized inclusion body solution of the lysis precipitate of pET-32a-IIRBD; lane 6 is 6 M urea solubilized inclusion body solution of the lysis precipitate of pET-32a-IIRBD; lane 7 is 3 M urea solubilized inclusion body solution of the lysis precipitate of pET-32a-IIRBD; lane 8 is 1 M urea solubilized inclusion body solution of the lysis precipitate of pET-32a-IIRBD;

[0034] Figure 5 Figure 4 is a graph showing the results of the reactivity of the recombinant proteins after purification using ELISA in Example 1, in which A is the ELISA results of FCOV positive and negative sera using recombinant N protein as the coating antigen; B is the ELISA results of FCOV positive and negative sera using recombinant IRBD protein as the coating antigen; C is the ELISA results of FCOV positive and negative sera using recombinant IIRBD protein as the coating antigen;

[0035] Figure 6 Figure 5 is a graph showing the results of the optimization of the antigen coating amount in Example 2;

[0036] Figure 7 Figure 6 is a graph showing the results of the optimization of the antigen coating conditions in Example 2;

[0037] Figure 8 Optimization results for the blocking solution screening in Example 2;

[0038] Figure 9 Optimization results for the blocking condition in Example 2;

[0039] Figure 10 Optimization results for the serum dilution in Example 2;

[0040] Figure 11 Optimization results for the serum incubation condition in Example 2;

[0041] Figure 12 Optimization results for the enzyme-labeled antibody dilution in Example 2;

[0042] Figure 13 Optimization results for the enzyme-labeled antibody incubation condition in Example 2;

[0043] Figure 14 Optimization results for the TMB color developing time in Example 2

[0044] Figure 15 Calculation results of the cut-off value in Example 2;

[0045] Figure 16 Specificity test results in Example 3;

[0046] Figure 17 Batch repeatability test results in Example 3;

[0047] Figure 18 Batch repeatability test results in Example 3;

[0048] Figure 19 Sensitivity test results in Example 3;

[0049] Figure 20 Statistical results of the positive detection rate of the antibodies in clinical samples in Example 4;

[0050] Figure 21 Statistical analysis results of the combined detection of the antibodies in Example 4;

[0051] Figure 22 Correlation analysis graph of the antibodies and the occurrence of FCOV symptoms in Example 4;

[0052] Figure 23 Correlation analysis graph of the antibody levels and the occurrence of FCOV symptoms in Example 4. DETAILED DESCRIPTION

[0053] The application provides an antigen for cat coronavirus antibody detection, which is purified recombinant N protein, IRBD protein (type I) and IIRBD protein (type II) of cat coronavirus; the amino acid sequence of the recombinant N protein is shown as SEQ ID NO. 1, the amino acid sequence of the recombinant IRBD protein (type I) is shown as SEQ ID NO. 2, and the amino acid sequence of the recombinant IIRBD protein (type II) is shown as SEQ ID NO. 3; the genes encoding the above proteins are shown as nucleotide sequences in SEQ ID NO. 4-6.

[0054] The recombinant N protein, IRBD protein (type I) and IIRBD protein (type II) are obtained by connecting the coding gene of the cat coronavirus N gene shown as SEQ ID NO. 4 with the prokaryotic expression vector pet-28a, connecting the coding gene of the cat coronavirus IRBD gene (type I) shown as SEQ ID NO. 5 with the prokaryotic expression vector pet-28a, connecting the coding gene of the cat coronavirus IIRBD gene (type II) shown as SEQ ID NO. 6 with the prokaryotic expression vector pet-32a, respectively, transforming into BL21 E. coli competent cells, inducing expression, obtaining bacterial liquid, performing ultrasonic crushing and purification, and obtaining the above antigen.

[0055] The amino acid sequences of the N protein, IRBD protein (type I) and IIRBD protein (type II) in the embodiment are shown as SEQ ID NO. 1-3.

[0056] N-AA: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELRR QAYMATQGQRVNWGDEPSKRRGRSNSRGRKNNIIPLSFYNPLTLEQGSKFWNVCPRDFVPKGIGNKDQQIGYWNRQERYRIVKGQRKELPERWFFYFLGTGPHADAKFKDKIDGVFWVARDGAMNKPTTLGTRGTNNESKPLKFDGKIPPQFQLEVNRSRNNSRSGSQSRSVSRNRSQSRGRQQSNNQNNNVEDTIVAVLQKLGVTDKQRSRSKSKDRSESKPRDTTPKNANKHTWKKTAGKGDVTNFYGARSASANFGDSDLVANGNAAKCYPQIAECVPSVSSMLFGSQWSAEDDGDQVKVTLTHTYYLPKDDAKTSQFLEQIDAYKRPSQVAKDQRQRKSRSKSADKKPEELSVTLVEAYTDVFDDTQVEMIDEVTNAAALEHHHHHH (SEQ ID NO. 1);

[0057] IRBD-AA: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFEL RRQAYYCDSPLNRIKCQQLKHELPDGFYSAIMLVKKDLPKTFVTMPQFYNLMNVTLHVVLNDTEKGADIILAKAPELASLADIHFEIVQANGSVTNVTSLCVQARQLALFYKYTSLQGLYTYSNLVELQNYDCPFSPQQFNNHLQFETLCFYVSPAVAGCKWSLVHYNRWRTQFATITVSYKDGTMITAAALEHHHHHH (SEQ ID NO. 2);

[0058] IIRBD-AA: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMI APILDEIADE YQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD ANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMA DIGSYCNSHINNIKCSQLTANLNNGFYPVVSSEVGFVNKSVVLLPSFFTHTAVNI TIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVYCIRSDQFSVYVHSTCKSSLWD NIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAA RTTRTNEQVVRSLYVIYEEGDNIV (SEQ ID NO. 3).

[0059] The sequences encoding the N segment, the IRBD (type I) segment, and the IIRBD (type II) segment of the gene of interest are as follows:

[0060] IRBD: ATTCGAGCTCCGTCGACAAGCTTATTATTGTGACTCACCCCT CAATAGAATTAAGTGTCAGCAATTGAAGCATGAGTTACCGGACGGTTTTTATTCTGCTATCATGCTCGTCAAAAAGGATTTACCTAAGACATTTGTAACTATGCCACAATTTTACAATTTGATGAATGTCACACTACACGTCGTGTTGAACGATACTGAAAAGGGGGCTGACATCATCTTGGCTAAGGCACCAGAGCTAGCATCACTTGCTGACATACATTTTGAAATAGTTCAGGCGAATGGAAGTGTAACTAATGTCACCAGCCTGTGTGTGCAAGCAAGACAATTGGCTTTATTCTACAAGTACACTAGCCTACAAGGCTTGTACACTTATTCAAATTTGGTTGAGTTACAAAATTACGACTGCCCTTTTTCACCACAGCAGTTTAACAATCATCTGCAGTTTGAAACTTTGTGTTTTTATGTGAGCCCAGCTGTGGCAGGTTGTAAGTGGTCGTTAGTTCATTACAACAGGTGGCGTACCCAGTTTGCCACTATCACTGTTTCTTACAAGGATGGTACTATGATCACTGCGGCCGCACTCGAGCA (SEQ ID NO. 5);

[0061] IIRBD: ACAAGGCCATGGCTGATATCGGATCCTATTGTAACAGTCAC ATTAATAACATTAAATGTTCTCAACTTACTGCTAATTTGAATAATGGATTTTATCCTGTTGTTTCAAGTGAAGTAGGTTTCGTTAATAAGAGTGTTGTGTTATTACCTAGCTTTTTCACACACACCGCTGTCAATATAACCATTGATCTTGGTATGAAGCTTAGTGGTTATGGTCAACCCATAGCCTCGACACTAAGTAACATCACACTACCAATGCAGGATAACAATACTGATGTGTACTGTATTCGTTCTGACCAATTCTCAGTTTATGTTCATTCCACTTGCAAAAGTTCTTTATGGGACAATATTTTTAATCAAGACTGCACGGATGTTTTAGAGGCTACAGCTGTTATAAAAACTGGTACTTGTCCTTTCTCATTTGATAAATTGAACAATTACTTGACTTTTAACAAGTTCTGTTTGTCGTTGAGTCCTGTTGGTGCTAATTGCAAGTTTGATGTTGCTGCACGTACAAGAACCAATGAGCAGGTTGTTAGAAGTCTATATGTAATATATGAAGAAGGAGACAACATAGTGTAAGAATTCGAGCTCCGTCGACA (SEQ ID NO. 6);

[0062]

[0063] The application also provides an indirect ELISA kit for detecting feline coronavirus antibodies and distinguishing serotypes, which uses the high-conservation N protein as an antigen, so that the kit can detect different strains of FCOV, and further includes RBD protein on S protein of type I feline coronavirus (IRBD) and RBD protein on S protein of type II feline coronavirus (IIRBD) as antigens, and there is no cross-reaction between RBD proteins of type I and type II viruses, so that the indirect ELISA method established by the application can identify type I FCOV and type II FCOV.

[0064] The application also provides an application of the indirect ELISA kit for detecting feline coronavirus antibodies, which uses indirect ELISA to detect feline coronavirus antibodies and distinguish serotypes, and can be used for preventive detection of FCOV.

[0065] The results of the embodiments of the application show that the indirect ELISA method for detecting FCOV antibodies established by the application has a sensitivity as high as 1:64000, and has no cross-reaction with common pathogens (FHV, FCV, FPV) of cats; the repeatability test shows that the variation coefficient of the detected samples in the same batch is between 3.220% and 8.413%, and the variation coefficient of the detected samples in different batches is between 1.802% and 8.948%, both of which are less than 10%, indicating that the method has good repeatability in batches and between batches; 123 clinical samples of cats are detected, and the positive rate of feline coronavirus antibodies is 73.17%, among which type I FCOV infection accounts for 91.11%, type II FCOV infection accounts for 1.11%, mixed infection accounts for 2.22%, and in addition, 5.56% of the samples are detected to be positive for feline coronavirus N antibodies but not for RBD antibodies.

[0066] The recombinant N protein, IRBD protein (type I) and IIRBD protein (type II) prepared by the application have good reactivity, the ELISA method for detecting feline coronavirus antibodies established by the application has the characteristics of sensitivity, specificity and high efficiency, is simple to operate, short in time consumption and low in cost, can detect a large number of cats infected with FCOV, and has good effect in distinguishing serotypes, thereby providing a simple and effective diagnostic tool for veterinary clinical diagnosis.

[0067] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and embodiments, but they should not be understood as limiting the protection scope of the application.

[0068] Feline panleukopenia virus (FPV), feline herpesvirus (FHV), feline calicivirus (FCV) antibody positive serum, feline coronavirus antibody positive serum and negative serum were preserved by the laboratory. 123 clinical serum samples of cats were collected from several pet hospitals in Guangzhou.

[0069] HIS protein purification kit was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.; Phanta Max Super-Fidelity DNA Polymerase, DNA maker and BL21 E. coli competent cells were purchased from Nanjing Novozyme Bio-tech Co., Ltd.; HRP labeled rabbit anti-cat IgG enzyme labeled antibody was purchased from Beijing Boao Sun Biotechnology Co., Ltd.; TMB color developing liquid and ELISA termination liquid were purchased from Beijing Solabio Technology Co., Ltd.

[0070] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special instructions, and the name and / or abbreviation thereof all belong to the conventional name in the field, which is very clear and explicit in the related application field. The skilled person in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and carry out the implementation according to the conventional conditions or the conditions recommended by the manufacturer. The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional method well known to those skilled in the art.

[0071] Example 1 Preparation of recombinant protein

[0072] 1.1 Construction of recombinant plasmid

[0073] (1) Primer design: Selecting feline coronavirus N protein, IRBD protein (type I) and IIRBD protein (type II) as the target antigen protein, designing specific primers, the upstream and downstream primers of N gene and IRBD gene (type I) contain HindIII and NotI enzyme cutting sites (indicated by underlined), and the homologous sequence of PET-28a vector is added after the enzyme cutting site. The upstream and downstream primers of IIRBD gene (type II) contain BamHI and EcoRI enzyme cutting sites (indicated by underlined), and the homologous sequence of PET-32a vector is added after the enzyme cutting site. The primer sequence is submitted to Shanghai Shengong Biotechnology Co., Ltd. for synthesis, and the detailed information of the sequence is as follows:

[0074] Table 1 primer sequence table

[0075]

[0076] (2) Amplification of the target gene: The FCOV strain cDNA preserved in the laboratory was used as the template for PCR amplification. The PCR reaction system is shown in Table 2, and the reaction conditions are as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 sec, 55°C annealing for 15 sec, 72°C extension for 15 sec, for a total of 35 cycles; and 72°C extension for 5 min. After the reaction, the target fragment was recovered by gel.

[0077] Table 2 PCR reaction system

[0078]

[0079] (3) Enzymatic digestion of the vector: The pET-28a plasmid was double-digested with HindIII and NotI restriction enzymes, and the pET-32a plasmid was double-digested with BamHI and EcoRI restriction enzymes.

[0080] (4) Plasmid construction and identification: The amplified N gene and IRBD gene (type I) were ligated to the pET-28a digested vector through homologous recombination, and the amplified IIRBD gene (type II) was ligated to the pET-32a digested vector through homologous recombination. Then, the positive bacterial liquid was inoculated into the BL21 E. coli competent cells, and the bacterial liquid PCR identification and sequencing identification were performed.

[0081] The bacterial liquid PCR test results are shown in Table 3. Figure 1 After the bacterial liquid PCR identification of N, IRBD (type I), and IIRBD (type II), the target bands with sizes of 1173 bp, 591 bp, and 601 bp were obtained by agarose gel electrophoresis, which were consistent with the expected sizes.

[0082] 1.2 Induction and expression of recombinant protein

[0083] The positive bacterial liquid with correct bacterial liquid PCR identification and sequencing was inoculated into the corresponding liquid medium with corresponding resistance, and was cultured at 37°C, 180 rpm on a shaking bed for 3-4 h. The absorbance of the bacterial liquid was measured by a spectrophotometer. When the OD600 nm value was 0.6-0.8, the bacterial liquid was taken out, 1 mM concentration of isopropyl-β-D-thiogalactoside (IPTG) was added, and the bacterial liquid was cultured at 16°C, 120 rpm for 24 h. Then, the bacterial liquid was ultrasonically lysed (the parameters were set as power 300 W, working 5 s, pausing 5 s, and time 30 min). After lysis, the supernatant and the precipitate were separated by centrifugation at 10000 rpm, 4°C for 30 min, and the supernatant and the precipitate were collected.

[0084] 1.3 Identification of the expression of recombinant protein

[0085] The expression of the target protein in the supernatant and the precipitate was analyzed by SDS-PAGE, and the specific steps were as follows: the prepared PAGE gel was placed in the electrophoresis tank, 1x SDS-PAGE electrophoresis solution was poured, the supernatant and the precipitate collected in step 1.2 were spotted into the wells, and a protein marker well was set. The electrophoresis tank device was assembled, 80V voltage for 30min, 120V voltage for 60min after the sample entered the separation gel layer from the concentrated gel layer, and the electrophoresis was stopped when the sample approached the bottom of the gel. After completing the electrophoresis separation, the gel was cut to an appropriate size, soaked in pure water for several minutes to wash away the electrophoresis solution, and then discarded, and a suitable amount of coomassie brilliant blue staining solution was added and shaken for 30min until the staining had bands. Discard the coomassie brilliant blue staining solution, and add an appropriate amount of pure water to wash away the staining solution.

[0086] The experimental results showed that the N, IRBD, and IIRBD proteins were successfully expressed, as shown in Figure 2 . Among them, the recombinant N protein was expressed in both inclusion bodies and supernatant, with a molecular weight of about 49kDa, which was consistent with the expected size and had good soluble expression. The supernatant was used for subsequent protein purification; the recombinant proteins IRBD (type I) and IIRBD (type II) were expressed in inclusion bodies, with a molecular weight of about 27kDa and 38kDa, which was consistent with the expected size. The precipitate was used for subsequent protein purification.

[0087] 1.4 Purification of soluble protein pet-28a-N

[0088] A large amount of soluble protein N expressed in the supernatant was obtained after ultrasonication, and the target protein was purified by affinity chromatography using the Biyun Tian HIS protein purification kit, and the specific steps were as follows:

[0089] (1) First, add about 4mL of 50% BeyoGold TM His-tag Purification Resin (reduction-resistant chelating type) mixed evenly to the nickel column, stand for 10min to separate the filler and the protection solution, open the lower plug of the nickel column, and then release the upper protection solution. Then wash the column with five times the column volume of ultrapure water, and then equilibrate the nickel column with ten times the column volume of non-denaturing lysis buffer. After a few minutes, open the plug and slowly release it.

[0090] (2) Put the supernatant of the pet-28a-N bacterial lysate and the equilibrated nickel into a 50ml centrifuge tube, and slowly shake at 4°C for 120min on a shaker to allow the protein to fully bind to the nickel.

[0091] (3) Then perform protein column chromatography, and the bacterial lysate and BeyoGold TMHis-tag Purification Resin (Ni-NTA) was packed into an empty affinity column tube, and after 5 minutes, the lower plug of the column was removed and the flow-through was collected. The flow-through was re-applied to the column and the process was repeated 4 times to ensure that the recombinant protein with His-tag was bound to the column and to reduce protein loss.

[0092] (4) The column was washed with 15 column volumes of Solution Binding Buffer (see Table 3 for the formulation) to remove impurities, and 100 μl of the wash was collected.

[0093] (5) The target protein was eluted with 5 column volumes of Solution Elution Buffer (see Table 3 for the formulation) at a slower flow rate to ensure complete elution of the protein. Each 1 ml of eluate was collected in a separate centrifuge tube. The samples were identified by SDS-PAGE, and the eluate with the best purification results was collected, the protein concentration was determined by BCA, and the sample was stored at -80°C.

[0094] Table 3 Formulation of the buffer for soluble protein purification

[0095]

[0096] The results of the SDS-PAGE identification are shown in Table 6. After purification of the N protein, the band was clear and the impurity band was less. Figure 3

[0097] 1.5 Renaturation and dialysis purification of inclusion body proteins pET-28a-IRBD and pET-32a-IIRBD

[0098] A large amount of inclusion body proteins pET-28a-IRBD and pET-32a-IIRBD expressed in the precipitate were obtained after ultrasonication. After the precipitate was washed with different concentrations of urea solution (1M-8M, prepared using the protein wash solution as the mother liquor, see Table 4 for the specific components), the inclusion body proteins were dissolved in different gradient urea solutions, and SDS-PAGE analysis was performed. The target protein wash solution was collected at the appropriate urea concentration, and then several gradient urea-containing target protein wash solutions were added to the dialysis bag for gradient dialysis (the dialysis solution was TGE solution, see Table 5 for the specific components, the initial concentration of dialysis was 1M lower than the urea concentration of the collected target protein wash solution, and finally dialysis was performed in TGE solution without urea). The specific steps are as follows:

[0099] (1) The protein solution after ultrasonication was centrifuged at 8000 rpm at 4°C for 20 minutes, and the supernatant and precipitate were collected separately. The precipitate was dissolved with 10 ml of 1M urea solution.

[0100] ​(2) 8000rpm 4℃ centrifuge 10 minutes, collect supernatant and precipitate respectively, dissolve precipitate with 10ml 3M urea;

[0101] (3) 8000rpm 4℃ centrifuge 10 minutes, collect supernatant and precipitate respectively, dissolve precipitate with 10ml 6M urea;

[0102] (4) 8000rpm 4℃ centrifuge 10 minutes, collect supernatant and precipitate respectively, dissolve precipitate with 10ml 8M urea;

[0103] (5) 8000rpm 4℃ centrifuge 10 minutes, discard precipitate and collect supernatant;

[0104] (6) Analyze supernatant of gradient dissolved inclusion body protein by SDS-PAGE, then verify expression by Coomassie brilliant blue staining.

[0105] (7) Take out dialysis bag soaked with 70% EtOH, boil in pure water for 10 minutes for high temperature sterilization;

[0106] (8) If the band is correct, mix (expressed obviously) inclusion body solution dissolved by gradient urea in 50ml centrifuge tube, add into dialysis bag, seal both ends;

[0107] (9) Place dialysis bag in TGE solution containing urea with gradient, finally dialyze in TGE solution without urea, TGE solution formula is shown in Table 5, generally 12h for one gradient;

[0108] (10) After dialysis, concentrate by sucrose, according to BCA kit instruction, measure protein concentration, then store at -80℃.

[0109] Table 4 Protein washing solution formula

[0110] Component Amount used Tris-HCl pH=8.0 6.5g Sodium chloride 5.8g EDTA 3.7g Triton-x-100 5 mL Single distilled water Diluted to 1 L

[0111] Table 5 TGE solution formula (pH = 7.9)

[0112] Component Amount used Tris 6g Sodium chloride 2.9g EDTA 0.186g Glycerol 5％ Single distilled water Diluted to 1 L

[0113] SDS-PAGE analysis results are shown in Figure 4 , recombinant IRBD protein inclusion body is dissolved at 6, 8M, and recombinant IIRBD protein inclusion body is dissolved at 6, 8M.

[0114] 1.6 Reactogenicity identification of recombinant protein

[0115] For the purpose of verifying whether the target protein reacts with FIPV positive cat serum, ELISA test is carried out, and the specific steps are as follows: the purified pET-28a-N, pET-28a-IRBD, pET-32a-IIRBD protein (10 μg / mL) is coated with carbonate buffer, and the coated target protein is added to a 96-well plate, 100 μL / well, and coated at 4°C overnight.

[0116] The next morning, the target protein coating solution in the 96-well plate is discarded, and the prepared PBST is washed 3 times, and 5% skimmed milk powder (50 g / L, prepared with PBST) is blocked for 2 h; the 5% skimmed milk powder is shaken off, and the PBST is washed 4 times, and finally dried to ensure that there is no PBST residue in the well; then the FIPV positive cat serum is diluted with PBS at 1:500, 100 μL of the diluted sample is added to each well, the control group is FIPV negative cat serum, and incubated at 37°C for 1 h; the diluted serum sample is shaken off, and the PBST is washed 5 times, and finally dried to ensure that there is no PBST residue in the well; then the rabbit anti-cat IgG-HRP is diluted at 1:2000 (diluent is PBST), 100 μL is added to each well, and incubated at 37°C for 1 h; the liquid in the well is shaken off, and the PBST is washed 5 times, and finally dried to ensure that there is no PBST residue in the well; 100 μL of TMB color developing liquid is added to each well at room temperature, and the reaction is avoided for 10 min; finally, the reaction is terminated by adding 50 μL of termination solution to each well. The absorbance value of each well sample at OD450 nm is detected by using an enzyme label instrument.

[0117] The results are shown in Figure 5 The reactionogenicity analysis of the purified N, IRBD, and IIRBD recombinant proteins by ELISA proves that they have good reactionogenicity and can be used as antigens for the kit.

[0118] Example 2 Establishment and optimization of indirect ELISA antibody detection method

[0119] 2.1 Establishment of indirect ELISA antibody detection method

[0120] (1) Dilute the purified recombinant protein in ELISA 1x coating solution to a final concentration of 5 μg / mL, 100 μL per well, add to a 96-well ELISA plate, 3 repeats, and coat at 4°C overnight.

[0121] (2) Shake off the coating solution, wash with PBST (PBS containing 0.05% Tween 20) three times, and dry on absorbent paper.

[0122] (3) Block with 5% skimmed milk powder in PBST, 100 μL / well, at 37°C for 2 h.

[0123] (4) Discard the blocking solution, wash 4 times with PBST, then pat dry, dilute the cat FCOV positive serum and negative serum with PBST at a ratio of 1:500, add the diluted serum to the enzyme-labeled plate, 100 μL / well, incubate at 37°C for 2 h.

[0124] (5) Discard the cat serum, wash 5 times with PBST, then pat dry, add 1:2000 diluted HRP-labeled rabbit anti-cat IgG enzyme-labeled antibody, 100 μL / well, incubate at 37°C for 1 h.

[0125] (6) Discard the enzyme-labeled antibody, wash 5 times with PBST, then pat dry, add TMB color developing solution, 100 μL / well, incubate at 37°C for a period of time in the dark, then add 50 μL ELISA stop solution to each well.

[0126] (7) Read the OD450 nm value with an enzyme-labeled instrument, and record the results.

[0127] (8) Determine the results: the antigen coating concentration corresponding to the maximum ratio of positive serum to negative serum (P / N) and the lower negative value is the optimal antigen coating concentration.

[0128] 2.2 Optimization of antigen coating concentration

[0129] Dilute the purified recombinant protein in ELISA 1x coating solution to a final concentration of 1, 2, 3, 4, 5 ug / mL, and perform detection according to the basic operation procedure of indirect ELISA described in 2.1, read OD450 nm, and select the optimal antigen coating concentration by comparing the P / N value of the positive serum to the negative serum.

[0130] The results are shown in Table 2.2. Figure 6 Based on the detection of the optimal antigen coating concentration of each protein and to ensure uniform conditions and ease of operation during detection, 3 ug / mL was selected as the coating concentration for the three proteins.

[0131] 2.3 Optimization of antigen coating conditions.

[0132] Select 4°C overnight coating, 37°C coating for 2 h, 37°C coating for 1 h, and 37°C coating for 0.5 h as the four coating conditions, and perform detection according to the basic operation procedure of indirect ELISA.

[0133] The results are shown in Table 2.3. Figure 7 Based on the detection of the optimal antigen coating conditions of each protein and to ensure uniform conditions and ease of operation during detection, 37°C for 2 h was selected as the coating condition for the three proteins.

[0134] 2.4 Selection of blocking solution

[0135] Select 2% skim milk, 5% skim milk, 2% BSA and 5% BSA as blocking solution of ELISA respectively, and detect according to the basic operation process of indirect ELISA.

[0136] As shown in the results Figure 8 , in view of the detection of the screening results of the optimal blocking solution of each protein, and in order to ensure the uniformity of the conditions and the convenience of the operation during detection, 2% skim milk is selected as the blocking solution of the three proteins.

[0137] 2.5 Optimization of blocking conditions

[0138] The blocking time is set to 37℃ for 0.5h, 37℃ for 1h, 37℃ for 2h and 37℃ for 3h, four different blocking time conditions, and detect according to the basic operation process of indirect ELISA.

[0139] As shown in the results Figure 9 , in view of the detection of the optimal blocking conditions of each protein, and in order to ensure the uniformity of the conditions and the convenience of the operation during detection, 37℃ for 3h is selected as the blocking condition of the three proteins.

[0140] 2.6 Optimization of serum dilution

[0141] The cat positive serum and cat negative serum are diluted with PBS solution, and the dilution multiples are 1:250, 1:500, 1:1000 and 1:2000, and detect according to the basic operation process of indirect ELISA.

[0142] As shown in the results Figure 10 , in view of the detection of the optimal serum dilution of each protein, and in order to ensure the uniformity of the conditions and the convenience of the operation during detection, 1:1000 is selected as the serum dilution of the three proteins.

[0143] 2.7 Optimization of serum incubation conditions

[0144] The incubation time of cat positive and negative serum is set to 30min, 1h, 2h and 4h, four time conditions, and detect according to the basic operation process of indirect ELISA.

[0145] As shown in the results Figure 11 , in view of the detection of the optimal serum incubation conditions of each protein, and in order to ensure the uniformity of the conditions and the convenience of the operation during detection, 37℃ for 1h is selected as the serum incubation condition of the three proteins.

[0146] 2.8 Optimization of enzyme-labeled antibody dilution

[0147] The rabbit anti-cat IgG enzyme-labeled antibody is diluted with PBST solution to four different dilutions of 2000 times, 4000 times, 6000 times and 8000 times, and detect according to the basic operation process of indirect ELISA.

[0148] The results are shown in Figure 12 In view of the detection of the optimal enzyme-labeled antibody dilution of each protein and in order to ensure uniform conditions and ease of operation during detection, 4000-fold dilution was selected as the dilution fold of the enzyme-labeled antibodies of the three proteins.

[0149] 2.9 Optimization of enzyme-labeled antibody incubation conditions

[0150] The incubation time of rabbit anti-cat IgG enzyme-labeled antibody was set to 15 min, 30 min, 1 h, and 1.5 h, and the detection was performed according to the basic operation procedure of indirect ELISA.

[0151] The results are shown in Figure 13 In view of the detection of the optimal enzyme-labeled antibody incubation conditions of each protein and in order to ensure uniform conditions and ease of operation during detection, 37°C for 1 h was selected as the incubation condition of the enzyme-labeled antibodies of the three proteins.

[0152] 2.10 Optimization of color development time

[0153] The TMB color developing liquid action time was set to 2 min, 4 min, 6 min, and 8 min, and the detection was performed according to the basic operation procedure of indirect ELISA.

[0154] The results are shown in Figure 14 In view of the detection of the optimal color development time of each protein and in order to ensure uniform conditions and ease of operation during detection, 4 min was selected as the TMB color developing liquid action time of the three proteins.

[0155] 2.11 Determination of the critical value

[0156] Using the above established and optimized ELISA method, 24 cat negative sera stored in the laboratory were detected, the OD450 nm was measured, the average value (X) and the standard deviation (SD) were calculated, according to the statistical principle, the value of X+3SD was taken as the critical value (cutoff value), when the sample OD450 nm≥X+3SD was determined as positive; OD450 nmX+3SD was determined as negative.

[0157] The results are shown in Figure 15 The critical value of the recombinant N protein was 0.264, i.e. the OD450 nm of the detected serum≥0.264 was positive; the critical value of the recombinant IRBD protein was 0.254, i.e. the OD450 nm of the detected serum≥0.254 was positive; the critical value of the recombinant IIRBD protein was 0.222, i.e. the OD450 nm of the detected serum≥0.222 was positive.

[0158] Example 3 Performance evaluation based on the indirect ELISA antibody detection method

[0159] 3.1 Specificity test

[0160] We selected confirmed positive sera for feline calicivirus, feline herpesvirus, and feline panleukopenia virus (FPV) stored in our laboratory, using FCOV-positive serum as a positive control, to specifically analyze the results of the established ELISA method. Figure 16 As shown, the established ELISA method has no specific response to these three clinically common feline viral infectious diseases, and only shows specificity to FCOV positive serum.

[0161] 3.2 Repeatability Experiment

[0162] Four confirmed FCOV-positive and four confirmed FCOV-negative cat serum samples were randomly selected and subjected to intra-batch reproducibility experiments using ELISA plates coated from the same batch. Six replicates were set for each sample, and OD450 nm was measured. The coefficient of variation for the same sample was calculated to verify the intra-batch reproducibility of this method. Four confirmed FCOV-positive and four confirmed FCOV-negative cat serum samples were randomly selected and subjected to inter-batch reproducibility experiments using ELISA plates coated from different batches. Six replicates were set for each sample, and OD450 nm was measured. The coefficient of variation for the same sample was calculated to verify the inter-batch reproducibility of this method.

[0163] The coefficient of variation is calculated by taking the mean and standard deviation of the same serum test values ​​and using the formula CV (coefficient of variation) = (standard deviation SD / mean X) × 100%.

[0164] The results are as follows Figure 17 and Figure 18 As shown, the intra-assay coefficient of variation (CIV) for the N recombinant protein ELISA coated plate ranged from 3.436% to 7.319%, and the inter-assay CIV ranged from 3.220% to 8.264%, all less than 10%. The intra-assay CIV for the IRBD recombinant protein ELISA coated plate ranged from 4.986% to 8.413%, and the inter-assay CIV ranged from 5.260% to 8.501%, all less than 10%. The intra-assay CIV for the IIRBD recombinant protein ELISA coated plate ranged from 1.802% to 6.121%, and the inter-assay CIV ranged from 3.346% to 8.948%, all less than 10%, indicating good reproducibility of this ELISA method.

[0165] 3.3 Sensitivity Test

[0166] FCOV positive serum was serially diluted (1000, 4000, 16,000, 32,000, 64,000, 128,000, 256,000, and 512,000 times with PBS) and tested under the optimized conditions described above. Cat negative serum was used as a corresponding negative control. The sensitivity of the method was analyzed using the cutoff value as the standard.

[0167] Results as shown in Figure 19 N, IRBD, IIRBD protein, respectively, in the positive serum dilution of 1:64,000, 1:64,000, 1:32,000, the detection result value is still greater than the critical value, and P / N≥2.

[0168] Example 4 Clinical application of indirect ELISA antibody detection method based on N, IRBD, IIRBD protein

[0169] The N, IRBD and IIRBD proteins purified in this study were used as coating antigens, and the established indirect ELISA method was used to detect antibodies in 123 cat sera collected in Guangzhou. The detection of each antibody was counted, the serotyping analysis was performed, and the correlation between antibody level and FCOV symptom occurrence was observed.

[0170] 4.1 N, IRBD and IIRBD protein antibody positive detection rate statistics

[0171] Results as shown in Figure 20 Among the 123 samples, 83 samples were detected to be positive for N antibody, and the FCOV-N antibody positive rate was 67.5%; 84 samples were positive for IRBD antibody, and the FCOV-IRBD antibody positive rate was 68.3%; 3 samples were positive for IIRBD antibody, and the FCOV-IIRBD antibody positive rate was 2.4%.

[0172] 4.2 Statistical analysis of combined antibody detection

[0173] Statistical analysis was performed on the detection results of three antibodies (N, IRBD and IIRBD) of individual individuals, and the results are shown in Figure 21 Among the 123 samples, 33 samples were not detected for any of the N, IRBD and IIRBD antibodies, indicating that 26.83% of the samples were FCOV antibody negative; 90 samples were detected for one or more of the N, IRBD and IIRBD antibodies, indicating that 73.17% of the samples were FCOV antibody positive, among which type 1 infection accounted for 91.11%, type 2 infection accounted for 1.11%, and mixed infection rate accounted for 2.22%.

[0174] 4.3 Correlation between antibody and FCOV symptom occurrence

[0175] According to whether there is FCOV symptom occurrence, 105 cats with healthy clinical manifestations and 18 cats diagnosed with FIP were divided, and statistical analysis was performed on the antibody detection results, and the results are shown in Figure 22As shown, the detection rate of N antibody in cats without FCOV symptoms was 61.9%, the detection rate of IRBD antibody was 62.86%, and the detection rate of IIRBD antibody was 2.86%. The positive rates of N antibody and IRBD antibody in cats clinically diagnosed with FIP were 100%.

[0176] 4.4 Association between antibody levels and the occurrence of FCOV symptoms

[0177] Based on the OD450 nm measured in the samples, antibody levels were categorized into high (OD450 nm > 0.8), medium (0.8 > OD450 nm > 0.5), low (0.5 > OD450 nm > critical value), and negative (OD450 nm < critical value). Statistical analysis was then performed on the antibody level detection results.

[0178] The results show that... Figure 23 As shown, statistical analysis of antibody levels in 65 out of 105 clinically healthy cats with detectable N antibodies revealed that 50.77% had high levels of N antibodies, 16.92% had intermediate levels, and 32.31% had low levels. Statistical analysis of antibody levels in 66 out of 105 clinically healthy cats with detectable IRBD antibodies revealed that 36.36% had high levels of IRBD antibodies, 27.27% had intermediate levels, and 36.36% had low levels. Statistical analysis of antibody levels in 3 out of 105 clinically healthy cats with detectable IIRBD antibodies showed that only low levels of IIRBD antibodies were detected in all three.

[0179] All 18 cats clinically diagnosed with FIP had detectable high levels of N antibodies. Additionally, 61.11% of the cats had high levels of IRBD antibodies, 16.67% had moderate levels, and 22.22% had low levels. No IIRBD antibodies were detected in any of the 18 cats with FIP.

[0180] The results show that the method established in this study has good sensitivity, specificity and repeatability, and has achieved good results in preliminary application. It demonstrates that the kit can detect large numbers of cats infected with FCOV and has a good effect on identifying serotypes.

[0181] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antigen for detecting antibodies to feline coronavirus type I and type II, characterized in that, The antigen is a purified recombinant N protein, a type I IRBD protein and a type II IIRBD protein of feline coronavirus; the amino acid sequences of the recombinant N protein, the type I IRBD protein and the type II IIRBD protein are shown in SEQ ID NO. 1-3.

2. The nucleotide sequences encoding the recombinant N protein, the type I IRBD protein and the type II IIRBD protein of claim 1 are shown in SEQ ID NO. 4-6.

3. An indirect ELISA test kit for detecting type I and type II feline coronavirus antibodies, characterized by, The kit is coated with the antigen of claim 1 on an enzyme-labeled plate.

4. The kit of claim 3, wherein The kit further comprises a positive control sample, a negative control sample, a sample diluent, a washing solution, an HRG-labeled rabbit anti-feline IgG antibody, a color developing solution and a termination solution.

5. The kit of claim 4, wherein The positive control sample is FCOV positive cat serum; the negative control sample is FCOV negative cat serum; the sample diluent is PBS; the washing solution is PBST containing 0.05% Tween-20; the color developing solution is a TMB solution; and the termination solution is a 2M sulfuric acid solution.

6. An indirect ELISA method for the detection of antibodies to feline coronavirus types I and II for non-diagnostic purposes, characterized in that, The method comprises the following steps: S1, serum incubation: after dilution, the sample serum is added to an enzyme-labeled plate coated with a recombinant N protein, a type I IRBD protein and a type II IIRBD protein, and incubated at 37℃ for 1h, then washed with PBST for 5 times and dried; the amino acid sequences of the recombinant N protein, the type I IRBD protein and the type II IIRBD protein are shown in SEQ ID NO. 1-3; S2, enzyme-labeled antibody incubation: 4000-fold diluted HRP-labeled rabbit anti-feline IgG antibody is added to the enzyme-labeled plate obtained in step S1, and incubated at 37℃ for 1h, then the solution is discarded, washed with PBST for 5 times and dried; S3, color development: TMB single-component color developing solution is added to each well, and color developed in the dark for 4min; S4, termination: termination solution is added to each well to terminate the reaction, and the OD450 nm value of the sample to be tested is determined.

7. The method of claim 6, wherein, The method further comprises determining whether the sample to be tested contains feline coronavirus antibodies.

8. The method of claim 7, wherein, The standard for determining whether the sample to be tested contains feline coronavirus antibodies is that the critical value of the recombinant N protein is 0.264, i.e. the OD450 nm of the tested serum ≥0.264 is positive; the critical value of the recombinant IRBD protein is 0.254, i.e. the OD450 nm of the tested serum ≥0.254 is positive; and the critical value of the recombinant IIRBD protein is 0.222, i.e. the OD450 nm of the tested serum ≥0.222 is positive.

9. The antigen of claim 1 or the kit of claim 3 for detecting type I and type II feline coronavirus antibodies for non-diagnostic purposes.

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