An Indirect ELISA Kit for Detecting Feline Coronavirus Antibodies and Its Application

Abstract

This invention discloses an indirect ELISA kit for detecting feline coronavirus antibodies and its application. The kit contains a recombinant protein coated with antigen M; the nucleotides encoding the recombinant protein M are shown in SEQ ID NO:1. The nucleic acid homology rate among the truncated M protein strains is 92.6%–100%, and the corresponding amino acid homology rate is 98.35%–100%. The homology rate between the two serotypes of FCoV is 85.89%–87.77%. Therefore, the indirect ELISA method established in this invention can simultaneously detect two serotypes of FCoV. This kit uses indirect ELISA to detect antibodies against feline coronavirus infection and can be used for prophylactic detection of FCoV in pet cats, preventing FIPV at its source and reducing the mortality rate of pet cats. Simultaneously, cats are important experimental animals, such as experimental animal infection models for SARS-CoV2, and FCoV infection must be excluded.

Description

Technical Field

[0001] This invention relates to the technical field of kits for detecting feline coronavirus antibodies, and more specifically to an indirect ELISA kit for detecting feline coronavirus antibodies and its application. Background Technology

[0002] Although previous coronavirus epidemics such as SARS-CoV and MERS-CoV have raised awareness of the need for clinically available treatments or preventative interventions, no proven effective treatments have yet been established. The development of effective treatments depends on understanding the molecular and cellular mechanisms of coronavirus infection, highlighting the importance of coronavirus-experimental host interaction studies to identify targets for antiviral interventions. Therefore, there is a need to establish representative animal models for SARS-CoV-2 infection and pathogenicity studies. Studies have shown that cats are susceptible to SARS-CoV-2 and can serve as preclinical animal models for SARS-CoV-2 disease development, vaccine development, antiviral drugs, and treatments. As feline coronavirus is a close relative of SARS-CoV-2, cats need to be tested for FCoV (feline enteric coronavirus) before establishing experimental animal disease models to rule out the influence of feline coronavirus on model establishment.

[0003] Based on the in vitro viability of feline coronavirus, its antigenic relevance to canine coronavirus (cCoV), its neutralization reaction with monoclonal antibodies against the S protein, and the gene sequence of the S protein, FCoV is classified into two serotypes: serotype I FCoV and serotype II FCoV. Research results show that serotype II FCoV is formed by recombinant synthesis of serotype I FCoV and cCoV. Serotype II FCoV has been reported to be more pathogenic.

[0004] Existing indirect ELISA kits for detecting feline coronavirus antibodies are usually designed based on the feline coronavirus FCoV-S protein, but their detection range is narrow, the extracted FCoV-S protein portion is not highly conserved, and they cannot detect two serotypes of feline coronavirus simultaneously. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, one objective of this invention is to provide an indirect ELISA kit for detecting feline coronavirus antibodies. The kit uses a truncated M protein as the antigen, with nucleic acid homology of 92.6-100% and corresponding amino acid homology of 98.35-100% among different strains. Homology between the two FCoV serotypes is 85.89%-87.77%. Therefore, the indirect ELISA method established by this invention can simultaneously detect different strains of different FCoV serotypes. Another objective of this invention is to provide an application of the indirect ELISA kit for detecting feline coronavirus antibodies. This kit uses indirect ELISA to detect antibodies against feline coronavirus, and can be used for preventative detection of FCoV in pet cats, thus preventing FIPV (feline infectious peritonitis coronavirus) at its source and reducing the mortality rate of pet cats.

[0006] One of the objectives of this invention is achieved through the following technical solution:

[0007] An indirect ELISA kit for detecting feline coronavirus antibodies, the kit containing a recombinant protein coated with antigen M; the nucleotide encoding the recombinant protein M is shown in SEQ ID NO:1.

[0008] Specifically, the preparation method of the recombinant M protein includes the following steps:

[0009] (a) Based on the FCoV M gene, amino acids 124-263 of the M protein were selected as the prokaryotic expression fragment, named M124-263aa, and 6×His-tag was fused to the NH2 terminus to obtain the plasmid of the gene as shown in SEQ ID NO:1. Then, the gene was amplified by PCR using primers.

[0010] (b) The PCR-amplified plasmid was separated by electrophoresis, purified and ligated into a vector, and transformed into competent cells to obtain the first recombinant plasmid; the first recombinant plasmid was identified by double enzyme digestion electrophoresis, and the first recombinant plasmid that was correctly identified by electrophoresis was then sequenced.

[0011] (c) Ligate the first recombinant plasmid with the correct sequence to the prokaryotic expression vector to obtain the second recombinant plasmid;

[0012] (d) The second recombinant plasmid was transformed into competent E. coli cells for prokaryotic expression to obtain the recombinant protein, namely the M recombinant protein.

[0013] Furthermore, the preparation method of the M recombinant protein also includes step (e), which involves purifying the recombinant protein using a His-tagged protein purification kit, then mixing the recombinant protein with lysis buffer, centrifuging, discarding the supernatant, resuspending, and collecting the protein.

[0014] Further, in step (a), PCR amplification is performed using the upstream primer sequence shown in SEQ ID NO:2 and the downstream primer sequence shown in SEQ ID NO:3.

[0015] Further, in step (a), the PCR amplification: the PCR amplification reaction system includes: DNA polymerase, cDNA template, upstream primer, downstream primer and water; the PCR amplification reaction conditions are: pre-denaturation at 92~95℃ for 2~5 min followed by cycling, denaturation at 92~95℃ for 20~40 s; annealing at 50~65℃ for 20~40 s, extension at 70~75℃ for 10~25 s, 28~32 cycles; final extension at 70~75℃ for 5~15 min.

[0016] Furthermore, in step (b), the sites of double enzyme digestion in the double enzyme digestion electrophoresis detection are NdeⅠ and XhoⅠ, respectively; the vector is pET30a; and the competent cells are Escherichia coli competent cells DH5α.

[0017] Furthermore, in step (d), an inducer is added to the prokaryotic expression for induction optimization; the inducer is IPTG; for every 1 mL of the second recombinant protein particle bacterial culture added, (0.1~1.25) mmol·L⁻¹ is added. -1 Inducing agent.

[0018] Furthermore, the kit also includes an ELISA plate, positive control serum, negative control serum, washing buffer, serum sample diluent, enzyme-labeled antibody working solution, chromogenic solution, and stop solution; the coating concentration of the coating antigen is 1.25~40 μg / mL; the enzyme-labeled antibody working solution is HRP-labeled rabbit anti-cat IgG; the serum dilution in the serum sample diluent is 1: (50~6400).

[0019] The second objective of this invention is achieved by the following technical solution:

[0020] The above-described indirect ELISA kit for detecting feline coronavirus antibodies uses an indirect ELISA method to detect antibodies against feline coronavirus infection. This kit can also be used in conjunction with other diagnostic methods to diagnose non-exudative FIP (feline infectious peritonitis).

[0021] Furthermore, the steps and positive criteria for detecting feline coronavirus antibodies using the kit are as follows: Dissolve recombinant M protein in coating buffer, add to an ELISA plate for coating, and wash; dilute positive and negative control sera and add to the ELISA plate respectively; add serum sample dilution buffer to the remaining wells for reaction; after washing, add working concentration of enzyme-labeled antibody working solution and react; after washing, add chromogenic solution for color development, then add stop solution to stop the display, and read the OD value. 450nmAbsorbance, OD 450nm A hole larger than 0.315 is considered positive.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The M recombinant protein is selected as the coating antigen in the kit of the present invention. The M gene is a highly conserved gene. After testing, the truncated M recombinant protein is selected as the antigen. The nucleic acid homology rate between the strains is 92.6-100%, and the corresponding amino acid homology rate is 98.35-100%. The homology between the two serotypes of FCoV is 85.89%-87.77%. Therefore, the kit containing this protein can detect different strains of the two serotypes of feline coronavirus FCoV at the same time. Combined with the FCoV-ELISA method, it can specifically detect FCoV virus. It can be used for preventive detection of FCoV in pet cats and laboratory cats. Combined with other diagnostic methods, it can also prevent and control FIPV (feline infectious peritonitis coronavirus) from the root and reduce the mortality rate of pet cats.

[0024] (2) In the kit of the present invention, the M recombinant protein is dissolved in the coating solution and added to the ELISA plate. If the FCoV protein is directly coated onto the ELISA plate, there may be a risk of viral shedding. Attached Figure Description

[0025] Figure 1 This is a diagram illustrating the antigenicity analysis of the M recombinant protein.

[0026] Figure 2 The image shows the amino acid sequence alignment results of the truncated M protein.

[0027] Figure 3 Homology comparison of the two serotypes of the truncated M protein

[0028] Figure 4 The image shows an electrophoresis diagram of the FCoV-M gene after PCR amplification, where M is DNA Marker DL2000; 1 and 2 are the FCoV-M gene amplification products.

[0029] Figure 5 This is the electrophoresis result of double enzyme digestion of the first recombinant plasmid; where M1 is DNA Marker DL2000; 1: FCoV-M double enzyme digestion product;

[0030] Figure 6 Electrophoresis images of recombinant proteins under different induction expression conditions; where M: protein molecular weight standard; 1: pET30a induction (empty vector); 2: uninduced; 3: whole bacteria after induction; 4: supernatant after induction and lysis; 5: precipitate after induction and lysis.

[0031] Figure 7 Electrophoresis images of recombinant protein M at different inducing agent concentrations; where M: protein marker; 1: pET30 empty vector; 2-7: IPTG induced at 0.10 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, and 1.25 mM; 8: uninduced pET30a-M;

[0032] Figure 8 This is a purification diagram of recombinant protein M, where M: protein molecular weight standard; 1: post-lysis sample; 2: eluent; 3-4: elution buffer.

[0033] Figure 9 This is a Western blotting analysis diagram of recombinant protein, where M: protein molecular weight standard; 1: sample. Detailed Implementation

[0034] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0035] An indirect ELISA kit for detecting feline coronavirus antibodies, the kit containing a recombinant protein coated with antigen M; the nucleotide encoding the recombinant protein M is shown in SEQ ID NO:1.

[0036] Specifically, the preparation method of the recombinant M protein includes the following steps:

[0037] (a) Synthesis and optimization of pET30a-M recombinant plasmid

[0038] Based on bioinformatics analysis of the FCoV M gene (KX722531.1) published in GenBank, and due to previous failures in expressing the full-length M protein, we selected amino acids 124-263 of the M protein as the prokaryotic expression fragment in this experiment, named M... 124-263aa .like Figure 1 As shown, the M recombinant protein contains nine groups of antigenic determinants. Figure 2 As shown, the truncated M protein has removed the transmembrane region, making it easier to express in E. coli. Furthermore, the truncated M protein shows higher homology and better conservation among different strains. Based on GC content and codon bias, the gene sequence of this fragment was optimized for the E. coli expression system. To improve the ease of purification, a 6×His-tag was fused to the NH2 terminus to obtain the plasmid of the gene shown in SEQ ID NO:1, which is the pET30a-M recombinant plasmid.

[0039] like Figure 3 As shown, the gene sequence of the present invention, as shown in SEQ ID NO:1, has 83.97~97.92% homology with the full-length sequence of the M protein. Therefore, the kit containing this protein can simultaneously detect two serotypes of two feline coronaviruses of FCoV.

[0040] To facilitate gene cloning, restriction enzyme sites Nde I and Xho I were added to the 3' and 5' ends of the sequence, respectively. The pET30a-M recombinant plasmid was synthesized by Nanjing GenScript Biotech Co., Ltd. PCR amplification was then performed using the upstream primer sequence FCoV MF (SEQ ID NO:2) and the downstream primer sequence FCoV MR (SEQ ID NO:3). Primers were designed using Premier 5.0 software, as shown in Table 1, where the protective bases of the restriction enzyme sites Nde I and Xho I are underlined.

[0041] Table 1 Primers for target gene amplification

[0042]

[0043] After culturing the strains containing the pET30a-M recombinant plasmid, the plasmid was extracted and PCR amplified. Tables 2-3 show the PCR reaction system and procedure, respectively. 5 μL of the PCR product was extracted for electrophoresis, and the amplification results were detected. Figure 4 It can be seen that a 417bp target fragment was obtained by amplification, and then the target fragment was recovered by gel extraction.

[0044] Table 2 PCR reaction system

[0045]

[0046] Table 3 PCR reaction procedure for target fragment amplification

[0047]

[0048] (b) Enzyme digestion and recovery of pET30a-M plasmid

[0049] The PCR-amplified plasmid was separated by electrophoresis, purified, ligated into a vector, and transformed into competent cells to obtain the first recombinant plasmid. The first recombinant plasmid was identified by double enzyme digestion electrophoresis. The enzyme digestion reaction system is shown in Table 4. The electrophoresis results are as follows: Figure 5 The target protein fragment with a molecular weight of 16.6 kDa was obtained. The first recombinant plasmid, which was correctly identified by electrophoresis, was then sequenced.

[0050] Table 4 Enzyme digestion reaction system

[0051]

[0052] (c) The first recombinant plasmid with correct sequencing and the prokaryotic expression vector pET-30a were ligated to obtain the second recombinant plasmid pET30a-FCoV-M;

[0053] (d) The second recombinant plasmid pET30a-FCoV-M was transformed into Escherichia coli competent cells BL21 for prokaryotic expression to obtain FCoV-M recombinant protein, i.e. M recombinant protein; the FCoV-M recombinant protein is an inclusion body.

[0054] In step (c), an inducer was added to the prokaryotic expression for induction optimization. Experimental groups were set up including empty vector induction, no induction, whole bacteria after induction, supernatant collection after induction and lysis, and precipitate collection after induction and lysis. Figure 6 As shown, the target protein was present in both the whole bacteria and the precipitate after induction.

[0055] Optimization of recombinant protein IPTG-induced

[0056] IPTG was used as the inducer. Different concentrations of IPTG (0.1 mmol / L, 0.25 mmol / L, 0.5 mmol / L, 0.75 mmol / L, 1.0 mmol / L, 1.25 mmol / L) were added to the M recombinant protein in step (d) and induced for 16 h. Afterward, 1 mL of the bacterial culture was subjected to SDS-PAGE gel electrophoresis. Figure 7 As shown in the figure, the experiment indicates that when the final IPTG concentration is 0.75 mmol / L, corresponding to lane 5 in the diagram, the band is the largest and widest, indicating the highest expression level of the target protein. Based on the expression levels in lane 5, the expression level of protein M accounts for 16290.51 / 195886.7 = 83.16% of the total protein expression in lane 5. Therefore, 0.75 mmol / L was chosen as the optimal IPTG concentration.

[0057] (e) After extracting the FCoV-M recombinant protein on ice, the recombinant protein was purified using a His-tagged protein purification kit (inclusion body protein). Because the FCoV-M recombinant protein is an inclusion body, 0.1 g of bacterial cells were added to 2 mL of lysis buffer. The resuspended cells were sonicated, centrifuged at 12000 r / min for 15 min, the supernatant was discarded, and the protein was resuspended in Binding Buffer containing 8 M urea. The protein was collected as shown below. Figure 8 The purified FCoV-M recombinant protein shown is as follows: Figure 9 As shown, the purified FCoV-M recombinant protein was analyzed for immunogenicity by Western blotting, demonstrating its good antigenicity and suitability for use as an antigen in the kit.

[0058] Explore optimal antigen coating concentration and serum dilution

[0059] Checkerboard titration was performed to determine the optimal operating conditions for the indirect ELISA reagent. Purified FCoV-M protein diluted in 1×PBS was coated onto 96-well ELISA plates at concentrations of 40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL in 100 μL volumes and incubated overnight at 4 °C. The plates were blocked with 10% skim milk powder at 37 °C for 1 h and washed three times with 1×PBST. Subsequently, 100 μL of FCoV standard positive and negative sera were added at dilutions of 1:50, 1:100, 1:2000, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, and incubated at 37 °C for 1 h. The plate was washed three times, then incubated with 100 μL of HRP-labeled rabbit anti-cat IgG (1:4000 dilution) at 37 °C for 1 h. After washing three times, 100 μL of TMB was added and the plate was incubated at 37 °C for 15 min. The reaction was terminated using stop solution. Finally, the plate was analyzed by microplate reader at OD... 450nm Measurements were taken at each location. Data for each group are shown in Table 5.

[0060] Table 5 Determination of Optimal Antigen Coating Concentration and Optimal Serum Dilution

[0061]

[0062] Table 5 shows that the optimal antigen concentration and serum dilution were determined based on the matrix titration experiment, and the maximum P / N value was found according to the principle that positive OD450nm ≥ 1.0 and negative OD450nm ≤ 0.2. As shown in Table 3-1, when the protein concentration was 20 μg / mL and the serum dilution was 1:200, the OD450nm values ​​for positive and negative serum were 1.62 and 0.12, respectively, and the P / N value was 13.49. Therefore, the optimal coating protein concentration was selected as 20 μg / mL, and the optimal serum dilution as 1:200.

[0063] Determining the optimal wrapping conditions.

[0064] Five different coating conditions (4℃ overnight followed by 37℃ for 1 h, 4℃ overnight, 4℃ overnight followed by 37℃ for 2 h, 37℃ for 1 h, and 37℃ for 2 h) were designed to optimize the working conditions of indirect ELISA. Finally, the OD of each optimized condition was measured. 450nm The values ​​are shown in Table 6.

[0065] Table 6 Determination of Optimal Antigen Coating Conditions

[0066]

[0067] As shown in Table 6, the optimal result is selected according to the P / N value, so coating at 37 ℃ for 2 h is the best coating condition for the antigen.

[0068] Exploring optimal sealing fluid conditions

[0069] Five different blocking solutions (1% BSA, 2% BSA, 5% skim milk, 2% skim milk, and 1% gelatin) were designed, along with four different blocking times (37 ℃ 0.5 h, 37 ℃ 1 h, 37 ℃ 1.5 h, and 37 ℃ 2 h) to optimize the working conditions of indirect ELISA. Finally, the OD of each optimized condition was measured. 450nm The values ​​are shown in Tables 7 and 8.

[0070] Table 7 Determination of Optimal Sealing Solution

[0071]

[0072] Table 8 Determination of Optimal Closure Time

[0073]

[0074] As shown in Tables 7 and 8, the optimal result is selected according to the P / N value. Therefore, 5% skim milk is the optimal blocking solution. When the blocking time is 37 ℃ for 0.5 h, the P / N value is the largest. Therefore, blocking at 37 ℃ for 0.5 h is the optimal blocking time.

[0075] Exploring the optimal serum binding time

[0076] Four different serum binding times (30 min, 60 min, 90 min, and 120 min) were designed to optimize the working conditions of indirect ELISA, as shown in Table 9. The optimal serum binding time was determined to be 37 °C for 1.5 h.

[0077] Table 9 Determination of Optimal Serum Binding Time

[0078]

[0079] Explore the dilution of enzyme-labeled secondary antibodies

[0080] Five different enzyme-labeled secondary antibody concentrations and times (1:2500, 1:5000, 1:7500, 1:10000) were designed to optimize the working conditions of indirect ELISA, as shown in Table 10. The results showed that the optimal enzyme-labeled secondary antibody dilution was 1:10000.

[0081] Table 10 Determination of the optimal dilution of enzyme-labeled secondary antibodies

[0082]

[0083] Exploring the optimal incubation time for enzyme-labeled secondary antibodies

[0084] Four different incubation times for enzyme-labeled secondary antibodies (30 min, 60 min, 90 min, and 120 min) were designed to optimize the working conditions of indirect ELISA. The details are shown in Table 11. The optimal incubation time was determined to be 1.5 h.

[0085] Table 11 Determination of the optimal incubation time for enzyme-labeled secondary antibodies

[0086]

[0087] Exploring the optimal action time of the substrate

[0088] Four different incubation times for secondary antibodies (5 min, 10 min, 15 min, and 20 min) were designed to optimize the working conditions of indirect ELISA. The details are shown in Table 12. 10 min was determined to be the optimal reaction time for the substrate.

[0089] Table 12 Determination of Optimal Substrate Development Time

[0090]

[0091] Determination of positive and negative cutoff values ​​for indirect ELISA

[0092] Ten FCoV-negative serum samples were tested under the above optimal conditions, as detailed in Table 13. The critical value was then calculated to be 0.315. +3SD=0.315), therefore OD 450nm A value ≥0.315 is considered positive; OD 450nm A value <0.315 is considered negative.

[0093] Table 13 Determination of Critical Values ​​for Indirect ELISA

[0094]

[0095] The reagent kit components are shown in Table 14.

[0096] Table 14 Components of the Reagent Kit

[0097]

[0098] Specific experiments

[0099] Positive serum samples infected with feline herpesvirus (FHV), feline calicivirus (FCV), and feline parvovirus (FPV) were detected using the optimal FCoV-ELISA method and the kits listed in Table 14, and OD was measured.450nm The values ​​are calculated, and then the specificity of the method is determined. See Table 15 for details.

[0100] Table 15 Results of Indirect ELISA Specificity Assay

[0101]

[0102] As shown in Table 15, the kit of the present invention can specifically detect FCoV.

[0103] Indirect ELISA repeatability

[0104] Five feline serum samples with varying FCoV antibody levels were selected for repeated experiments (see Table 16). The intra-assay coefficient of variation (CV) ranged from 1.40% to 3.78%. Five different batches of purified FCoV-M protein were coated onto ELISA plates, with inter-assay coefficients of variation (CV) ranging from 4.59% to 9.40%, all less than 10%. This indicates that the reproducibility of the indirect ELISA method is quite significant.

[0105] Table 16 Results of Intra-Batch and Batch Repeatability Tests

[0106]

[0107] In summary, the kit of this invention uses a specific recombinant M protein as the antigen and employs an indirect ELISA method to detect FCoV. It can specifically detect FCoV and is convenient to operate with high detection efficiency (results are available in 4-5 hours). No other special equipment is required. The reagent, designed based on enzyme-linked immunosorbent assay (ELISA), exhibits excellent performance in terms of stability, specificity, and sensitivity. Cats, as susceptible animals to SARS-CoV-2, can serve as preclinical animal models for SARS-CoV-2 disease research, vaccine development, antiviral drugs, and treatment. Establishing animal models requires FCoV detection in cats, and this experimentally established method combines indirect ELISA detection of antibodies with RT-PCR detection of antigens to screen for FCoV-free cats.

[0108] Compared to FCoV (feline enteric coronavirus), FIPV (feline infectious peritonitis coronavirus) is more difficult and complex to detect. The lack of specific clinical symptoms and pathological biochemical abnormalities in FIP is the main reason for the difficulty in diagnosing FIP, making premortem diagnosis challenging. A definitive diagnosis of FIP is usually only possible after the death of an FIP-positive cat; histological examination and viral antigen detection are the primary methods for postmortem confirmation. In contrast, non-invasive premortem diagnosis of FIP-positive cats remains a significant challenge. Diagnosing non-exudative FIP is even more difficult. To date, the best diagnostic criteria for non-exudative FIP have been a comprehensive assessment combining serological testing with histopathological and biochemical diagnostic methods, as well as a review of medical history and clinical symptoms. Therefore, the FCoV-M indirect ELISA detection method established in this invention can be used in conjunction with other diagnostic methods to diagnose non-exudative FIP.

[0109] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. An indirect ELISA kit for detecting feline coronavirus antibodies, characterized in that, The kit contains a recombinant protein coated with antigen M; the nucleotide sequence encoding the recombinant protein M is shown in SEQ ID NO:

1.

2. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 1, characterized in that, The method for preparing the recombinant M protein includes the following steps: (a) The gene shown in SEQ ID NO:1 was ligated into a vector to obtain a plasmid, and then the gene was amplified by PCR using primers; (b) The PCR-amplified plasmid was separated by electrophoresis, purified and ligated into a vector, and transformed into competent cells to obtain the first recombinant plasmid; the first recombinant plasmid was identified by double enzyme digestion electrophoresis, and the first recombinant plasmid that was correctly identified by electrophoresis was then sequenced. (c) Ligate the first recombinant plasmid with the correct sequence to the prokaryotic expression vector to obtain the second recombinant plasmid; (d) The second recombinant plasmid was transformed into competent E. coli cells for prokaryotic expression to obtain the recombinant protein, namely the M recombinant protein.

3. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 2, characterized in that, The preparation method of the M recombinant protein further includes step (e): after purifying the recombinant protein using a His-tagged protein purification kit, the recombinant protein is mixed with lysis buffer, lysed and centrifuged, the supernatant is discarded, the protein is resuspended, and the protein is collected.

4. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 2, characterized in that, In step (a), PCR amplification is performed using the upstream primer sequence shown in SEQ ID NO:2 and the downstream primer sequence shown in SEQ ID NO:

3.

5. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 2 or 4, characterized in that, In step (a), the PCR amplification reaction system includes: DNA polymerase, cDNA template, upstream primer, downstream primer and water; the PCR amplification reaction conditions are: pre-denaturation at 92~95℃ for 2~5 min followed by cycling, denaturation at 92~95℃ for 20~40 s; annealing at 50~65℃ for 20~40 s, extension at 70~75℃ for 10~25 s, 28~32 cycles; final extension at 70~75℃ for 5~15 min.

6. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 2, characterized in that, In step (b), the double enzyme digestion sites in the double enzyme digestion electrophoresis detection are NdeⅠ and XhoⅠ, respectively; the vector is pET30a; and the competent cells are Escherichia coli competent cells BL21.

7. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 2, characterized in that, In step (d), an inducer is added to the prokaryotic expression for induction optimization; the inducer is IPTG; for every 1 mL of the second recombinant protein particle bacterial culture added, (0.1~1.25) mmol·L⁻¹ is added. -1 Inducing agent.

8. The indirect ELISA kit for detecting feline coronavirus antibodies as described in claim 1, characterized in that, The kit also includes an ELISA plate, positive control serum, negative control serum, washing buffer, serum sample diluent, enzyme-labeled antibody working solution, chromogenic solution, and stop solution; the coating concentration of the coating antigen is 1.25~40μg / mL; the enzyme-labeled antibody working solution is HRP-labeled rabbit anti-cat IgG; the serum dilution in the serum sample diluent is 1:(50~6400).

Images