CN117288955A - Porcine circovirus 4-type Cap protein indirect ELISA detection kit, detection method and application - Google Patents

Abstract

The invention discloses a porcine circovirus 4-type Cap protein indirect ELISA detection kit, a detection method and application. The kit comprises a coated ELISA plate taking porcine circovirus type 4 recombinant Cap protein as a coating antigen, and also comprises negative serum, positive serum, HRP-labeled goat anti-pig IgG, a washing liquid, a diluent, a coating liquid, a sealing liquid, a color development liquid and a termination liquid. The kit has the advantages of strong specificity, high sensitivity, simplicity, convenience and practicability, and lays a foundation for clinical detection and pathogenicity research of porcine circovirus type 4.

Description

Porcine circovirus 4-type Cap protein indirect ELISA detection kit, detection method and application

Technical Field

The invention relates to the technical field of biology, in particular to a porcine circovirus 4 type Cap protein indirect ELISA detection kit, a detection method and application.

Background

Porcine Circovirus (Porcine Circovirus, PCV) is the smallest DNA virus known to date, belonging to one of the members of the genus Circoviridae, the family Circoviridae, single stranded circular, icosahedral symmetric, non-capsular, genome sizes between 1.7 and 2.1 kb. Based on differences in PCV gene sequence homology and antigenicity, 4 types of porcine circovirus have been identified in swine herds: PCV1, PCV2, PCV3 and PCV4. Studies have shown that PCV1 is not pathogenic to herds; porcine circovirus-related diseases (Porcine Circovirus Associated Disease, PCVAD) can occur after PCV2 infection of pigs, and are still the most dominant immunosuppressive pathogen in the pig industry at present; there is a controversy over the direct association between PCV3 and disease, and both sick and healthy pigs are infected with PCV 3.

PCV4 is a novel circular ring virus with low homology with other PCV nucleic acids, which is first identified by Chinese scholars in 2019 in pigs suffering from respiratory diseases, diarrhea, dermatitis nephrotic syndrome and the like in Hunan province. Studies show that the genome of the virus has a total length of 1700bp and mainly comprises two open reading frames (open reading frame, ORF): ORF1 encodes a replicase-related protein on the positive strand (Replicase protein, rep) and ORF2 encodes a Capsid protein on the negative strand (Capsed protein, cap). Cap is the only structural protein in PCV4, contains a plurality of B cell and T cell epitopes, has good immunogenicity, and can be used as a candidate protein for developing PCV4 vaccines and related diagnostic kits.

PCV4 was found to exist in 2 modes of infection, alone and in combination. Individual infection of pigs with PCV4 causes significant pathological changes in organ tissues (liver, lymph nodes, lung, etc.), but clinical symptoms are not apparent; the PCV4 and other porcine viruses (PRRS, PCV2 and PEDV) are mixed to infect pigs, so that the diagnosis of swine diseases is more difficult, and the health of the pig industry is seriously threatened. Because PCV4 viruses cannot be stably subjected to in vitro subculture at present, the infectivity, pathogenicity, host range, existence time of the viruses, economic losses brought to pig farming in China and the like are not clear, an effective method for antigen and antibody detection is established, PCV4 epidemiological investigation is conducted, and the method has important significance for enriching PCV4 epidemiological data and early warning, prevention and control of PCV4.

At present, ELISA is still the main method for detecting serum antibodies, and the method has the advantages of simple operation, strong specificity, high sensitivity and the like, and is suitable for detecting a large number of clinical samples. Studies on PCV2 and PCV3 have found that Cap proteins are the major immune-related proteins of PCV2 and PCV3, and ELISA methods established based on Cap proteins have been widely used for serodiagnosis of PCV2 and PCV 3. However, ELISA detection methods for PCV4 Cap proteins are rarely reported. In addition, no ELISA commercial antibody detection kit for PCV4 Cap protein has been developed on the market, which causes inconvenience to the immunogenicity evaluation of early PCV4 vaccine research. Therefore, the PCV4 Cap protein is expressed by adopting an escherichia coli expression system, and the purified recombinant Cap protein is used as a coating antigen, so that a PCV4 indirect ELISA method which is strong in specificity, high in sensitivity, convenient, practical and used for clinical detection is established, and a scientific and effective method is provided for evaluating the PCV4 infection condition of the pig farm in Fujian province, developing a PCV4 antibody diagnostic kit, developing PCV4 pathogenicity research and the like.

Disclosure of Invention

The invention aims to provide a porcine circovirus 4-type Cap protein indirect ELISA detection kit, a detection method and application.

The aim of the invention is achieved by the following technical scheme:

an indirect ELISA detection kit for porcine circovirus type 4 Cap protein comprises a coated ELISA plate taking porcine circovirus type 4 recombinant Cap protein as a coating antigen;

the preparation method of the porcine circovirus 4-type recombinant Cap protein comprises the following steps: cloning a porcine circovirus 4-type Cap gene with a sequence shown in SEQ ID NO.1 onto a prokaryotic expression vector through two enzyme cutting sites of NdeI and HindIII to construct a recombinant plasmid; transforming the recombinant plasmid into escherichia coli DH5 alpha to obtain a recombinant expression strain; inducing recombinant expression strain by IPTG, ultrasonic crushing, centrifuging, and collecting supernatant; purifying the supernatant by Ni-NTA affinity chromatography to obtain porcine circovirus 4-type recombinant Cap protein; the prokaryotic expression vector is a pET-30a (+) vector.

The porcine circovirus 4 Cap protein indirect ELISA detection kit according to claim 2, characterized in that: the IPTG induction is to add IPTG to a final concentration of 0.5mM, and the induction of expression is carried out for 16h at a temperature of 15 ℃.

The indirect ELISA detection kit for the 4-type Cap protein of the porcine circovirus also comprises negative serum, positive serum, enzyme-labeled antibodies, washing liquid, diluent, coating liquid, sealing liquid, color development liquid and stop liquid;

the enzyme-labeled antibody is HRP-labeled goat anti-pig IgG, the washing solution is 0.01mol/L PBST of pH7.4, the diluent is 0.01mol/L PBST of pH7.4 and 2% skim milk powder, the coating solution is 0.05mol/L phosphate buffer solution of pH9.6, the blocking solution is 5% skim milk powder, the color development solution is TMB, and the stopping solution is 0.5M H ₂ SO ₄ 。

The preparation of the porcine circovirus 4-type Cap protein indirect ELISA detection kit is carried out according to the following operations: coating antigen: diluting the porcine circovirus 4-type recombinant Cap protein with coating liquid to 1.25 mug/mL, adding the diluted porcine circovirus 4-type recombinant Cap protein into an ELISA plate, coating overnight at 4 ℃ and 100 mug/hole to obtain a coated ELISA plate;

washing: discarding the coating liquid and washing with a washing liquid;

closing: adding 200 mu L of sealing liquid into each hole, sealing for 1h at 37 ℃, discarding the sealing liquid and washing with a washing liquid;

incubating primary antibodies: pig serum was mixed with dilution, dilution 1:200 100. Mu.L/well, incubated at 37℃for 60min, discarded and washed with wash solution;

incubating a secondary antibody: mixing the enzyme-labeled antibody with a diluent, wherein the dilution is 1:10000 100. Mu.L/well, incubated at 37℃for 30min, discarded and washed with wash solution;

color reading: the reaction was stopped by adding 100. Mu.L/well of a color developing solution, adding 50. Mu.L/well of a stop solution after 10min of a color developing reaction at 37℃and measuring the OD value at a wavelength of 450nm on a microplate reader.

The invention has the remarkable advantages that:

ELISA detection methods for PCV4 Cap proteins are reported rarely, and ELISA commercial antibody detection kits for PCV4 Cap proteins are not developed on the market at present, which cause inconvenience to immunogenicity evaluation of early research of PCV4 vaccines. Therefore, the PCV4 Cap protein is expressed by adopting an escherichia coli expression system, and the purified recombinant Cap protein is used as a coating antigen, so that a PCV4 indirect ELISA method which is strong in specificity, high in sensitivity, convenient, practical and used for clinical detection is established, and a scientific and effective method is provided for evaluating the PCV4 infection condition of the pig farm in Fujian province, developing a PCV4 antibody diagnostic kit, developing PCV4 pathogenicity research and the like.

Drawings

Fig. 1: and (3) carrying out double enzyme digestion verification on the recombinant plasmid pET-30a (+) -Cap. Lane 1, recombinant plasmid pET-30a (+) -Cap; lane 2, pET-30a (+) empty vector; lane M, DNAmarker.

Fig. 2: SDS-PAGE analysis of recombinant Cap proteins. Lane M, protein molecular mass standard; lane 1, uninduced thallus; lane 2, post-induction whole mycoprotein; lane 3, cell lysis supernatant after induction; lane 4, post-induction cell lysis pellet.

Fig. 3: SDS-PAGE analysis of purified recombinant Cap proteins. Lane M, protein molecular mass standard; lane 1, bovine serum albumin; lane 2, purified recombinant Cap protein.

Fig. 4: and (5) purifying recombinant Cap protein Western blotting identification. Lane M, protein molecular mass standard; lane 1, porcine PCV4 positive serum; lane 2, swine PCV4 negative serum.

Detailed Description

In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.

Example 1

1 materials and methods

1.1 disease agent, serum and Main reagent

Both PCV4 positive disease and swine PCV4 positive serum were collected from pig farms identified by qPCR as strong PCV4 positive. PCV4 negative serum was collected from SPF pigs and identified as PCV4 negative by qPCR. Pig breeding and respiratory syndrome virus (PRRSV), porcine Epidemic Diarrhea Virus (PEDV), porcine pseudorabies virus (PRV), 3 porcine circovirus (PCV 1, PCV2, PCV 3) positive sera were all maintained in the laboratory for swine disease at the institute of livestock and veterinary, the national academy of agricultural sciences, fowls. pET-30a (+) vector, restriction enzymes (EcoR I and HindIII), competent cells of E.coli BL21 (DE 3), E.coli DH 5. Alpha,T1 Cloning Kit, protein Marker, pre-dye protein Marker, -/-> Ni-NTA Resin was purchased from full gold Biotechnology (Beijing) Inc. PCR amplification reagents 2X Taq Plus PCR MasterMix, T4 DNA ligase, DNA extraction, rapid plasmid Small mention DNA purification gel recovery kits were purchased from Tiangen Biochemical technologies (Beijing) Inc. Sheep anti-pig IgG-HRP, PVDF membrane, 1% TMB solution were purchased from Beijing Soy Corp technology Co. The ELISA plate was purchased from corning (Co.) of America. Other reagents were all analytically pure products purchased by commercial companies.

1.2 construction of prokaryotic expression-Positive recombinant plasmid pET-30a (+) -Cap

PCR amplification primers for Cap gene were designed based on PCV4 FJ2020001 strain complete gene sequence (accession number: MW 238796.1) provided in NCBI using oligo 7.0 software; the 5' -ends of the upstream and downstream primers are respectively inserted with NdeI and HindIII enzyme cutting sites, and the primers are pre-mixedThe phase amplified fragment is 720bp (the nucleotide sequence is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2); an upstream primer: 5' -AATAATCATATGCATGCCCATAAGGTCAAGATATA-3' (Nde I cleavage site underlined); a downstream primer: 5' -CCCAATAAGCTTTCATTAGCCTTGCTTCGGATAATTCACC-3' (HindIII cleavage site underlined) the primers were synthesized by the company HindIII, inc. of Biotechnology (Shanghai).

And (3) performing PCR amplification by using the designed primer and taking genomic DNA of PCV4 FJ2020001 strain as a template, purifying the PCR product, and connecting the PCR product to a pEASY-T1 vector to construct a T-Cap cloning plasmid. E.coli DH5 alpha is transformed by the T-Cap cloning plasmid, monoclonal colony is selected for culture, plasmid is extracted, and the bacterial liquid PCR and double enzyme digestion identification are carried out correctly and then sequencing is carried out. And (3) carrying out double enzyme digestion on the T-Cap cloning plasmid and a prokaryotic expression vector pET-30a (+) respectively by NdeI and HindIII, recovering enzyme digestion products by cutting gel, connecting the recovered products by using T4 DNA ligase overnight, transforming the connected products into escherichia coli DH5 alpha, picking up a monoclonal colony for culture, extracting the plasmid, and carrying out PCR, double enzyme digestion and sequencing identification respectively. The correct recombinant plasmid was verified to be designated pET-30a (+) -Cap.

As shown in FIG. 1, after the recombinant plasmid pET-30a (+) -Cap is subjected to NdeI and HindIII, 2 bands (FIG. 1) with the sizes of about 5400bp and 720bp appear and the sizes of the bands are consistent with those of the pET-30a (+) vector and the target gene. The plasmid sequencing result shows that the sequence is consistent with the target gene sequence, which shows that the recombinant plasmid pET-30a (+) -Cap is successfully constructed.

1.3 preparation and identification of recombinant Cap proteins

Transforming recombinant plasmid pET-30a (+) -Cap into escherichia coli BL21 (DE 3), inoculating positive monoclonal colonies into LB liquid medium containing 50 mug/mL kanamycin, culturing by shaking at 37 ℃ and 200r/min until the OD600nm value is 0.6-0.8, adding IPTG with the final concentration of 0.5mM, inducing expression for 16h at 15 ℃, centrifuging for 4000r/min for 10min, collecting bacterial liquid precipitate, re-suspending bacterial liquid precipitate by using 1 XPBS (pH=7.2-7.4), performing ultrasonic disruption for 1min, centrifuging, collecting supernatant and precipitate respectively, and analyzing protein expression form by SDS-PAGE; purifying Cap protein from supernatant by using a Ni-NTA affinity chromatography column purification kit, carrying out SDS-PAGE electrophoresis, transferring to a PVDF membrane, wherein the primary antibody is clinically collected pig source PCV4 positive serum (1:400), incubating for 2 hours at room temperature, and washing 3 times by using PBST for 5 minutes each time; HRP-labeled rabbit anti-pig IgG (1:5000) was added, incubated at room temperature for 1h, PBST was washed 3 times, developed with TMB, and the purified Cap protein concentration was measured by the Bradford method and stored for later use.

SDS-PAGE analysis shows that: recombinant Cap proteins were mainly present in the supernatant, belonging to soluble expression, with a size of about 28.3ku, consistent with the theoretical value of recombinant proteins (FIG. 2); the supernatant was purified by Ni-NTA affinity chromatography to give a single target band at 28.3ku (FIG. 3), which was over 90% pure and had a protein concentration of 0.26mg/ml (Bradford method).

Western blotting identification of purified recombinant Cap protein was performed with HRP-labeled rabbit anti-pig IgG, and the results showed that: the presence of a specific band at about 28.3ku on the PVDF membrane (FIG. 4), consistent with the expected band size, shows that the purified recombinant Cap protein can be specifically recognized by porcine PCV4 positive serum and has good immunogenicity.

1.4 optimization and establishment of the reaction conditions of the indirect ELISA of the recombinant Cap protein

The purified recombinant Cap protein was diluted at 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL and 0.625. Mu.g/mL and coated overnight at 4℃using matrix titration (ref: ZHANG H.H, HU W.Q, et al Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4,Hunan province,China[J ]. Transboundary and Emerging Diseases,2020,67 (3): 1057-1061.); diluting pig source PCV4 positive serum (primary antibody) according to 1:50, 1:100, 1:200, 1:400 and 1:800, performing other steps according to conventional ELISA, and taking the antigen concentration and serum dilution of a hole with the OD450nm value of about 1.0 and the maximum P/N value in the positive serum as the optimal antigen coating concentration and serum dilution; after the antigen coating concentration and the serum dilution are determined, the blocking solution, the blocking time, the primary antibody incubation time, the enzyme-labeled secondary antibody (HRP-labeled goat anti-pig) dilution, the secondary antibody incubation time and the TMB color development time are optimized, and the optimal reaction condition of the indirect ELISA is determined.

The conditions after the final determination of the indirect ELISA optimization of the recombinant Cap protein were (table 1):

coating antigen: diluting the porcine circovirus 4-type recombinant Cap protein to 1.25 mug/mL with a coating solution (0.05 mol/L phosphate buffer solution with pH of 9.6), and then adding the diluted solution into an ELISA plate for coating at 4 ℃ overnight, wherein 100 mug/hole is formed to obtain a coated ELISA plate;

washing: the coating solution was discarded and washed 3 times with washing solution (0.01 mol/LPBST pH 7.4) for 5min each;

closing: adding 200 μl of sealing solution (skimmed milk powder with mass fraction of 5%) into each hole, sealing at 37deg.C for 1 hr, discarding sealing solution, and washing with washing solution for 3 times each for 5min;

incubating primary antibodies: pig serum was mixed with diluent (0.01 mol/L PBST pH7.4 plus 2% skimmed milk powder), dilution 1:200 100. Mu.L/well, incubated at 37℃for 60min, discarded and washed 3 times with wash solution for 5min each;

incubating a secondary antibody: HRP-labeled goat anti-pig IgG was mixed with diluent (0.01 mol/L PBST pH7.4 plus 2% nonfat dry milk), dilution 1:10000 100. Mu.L/well, incubated at 37℃for 30min, discarded and washed 3 times with wash solution for 5min each;

color reading: adding TMB color development solution at 100 μl/well, shading at 37deg.C for 10min, adding stop solution at 50 μl/well (0.5M H) ₂ SO ₄ ) The reaction was terminated and the OD was measured on a microplate reader at a wavelength of 450 nm.

TABLE 1 optimization of the reaction conditions for indirect ELISA

1.5 Positive and negative Critical determination

Under the optimal working concentration condition, 30 PCV4 negative serum samples are detected by adopting an established indirect ELISA method, and negative control and positive control are set. The average value (X) and Standard Deviation (SD) of S/P were calculated by converting the antibody titer (S/P) of OD450nm of each serum by (sample OD450 nm-negative control OD450 nm)/(positive control OD450 nm-negative control OD450 nm). According to the statistical principle, when the S/P value of the sample is less than or equal to (X+2SD), the sample is judged as negative; the S/P value of the sample is not less than (X+3SD), and the sample is judged to be positive; if the S/P value is still smaller than (X+3SD), the test is negative.

Under the optimized indirect ELISA reaction condition, 30 parts of PCV4 antibody negative serum collected in clinic are detected, and the S/P average value (X) and Standard Deviation (SD) are respectively 0.117 and 0.01. According to the statistical principle, when the S/P value is more than or equal to X+3SD (0.147), judging that the sample is positive; when the S/P value is less than or equal to X+2SD (0.137), the result is judged as negative; between them (0.147 < S/P < 0.137) and is determined as suspicious, and after repeated measurement, the interval is still negative.

1.6 specificity test

Under the condition of optimal working concentration, positive serum of porcine pseudorabies virus (PRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine Epidemic Diarrhea Virus (PEDV) and 3 porcine circovirus (PCV 1, PCV2 and PCV 3) is detected by adopting an established indirect ELISA method, meanwhile, porcine PCV4 positive serum is established as a control, 3 repeats are established for each serum, and the specificity of the established indirect ELISA method is evaluated.

Under the optimized indirect ELISA reaction conditions, PCV4, PRV, PRRSV, PEDV, PCV1, PCV2, PCV3 positive serum and pig source negative control serum are detected, and the result shows that: except that the average value of S/P of PCV4 positive serum was 1.071±0.028, the average value of S/P of other porcine common pathogenic positive serum (PRV, PRRSV, PEDV, PCV, PCV2, PCV 3) and negative control serum was lower than 0.137 (see table 2), indicating that the indirect ELISA method based on PCV4 recombinant Cap protein was highly specific and did not have serological cross-reaction with PCV1, PCV2 and PCV 3.

TABLE 2 specificity detection results of indirect ELISA method

1.7 sensitivity test

Under the condition of optimal working concentration, continuous multiple ratio dilution (1:100-1:6400) is carried out on the PCV4 positive serum of the swine source, PCV4 negative serum is set as negative control, the established indirect ELISA method is adopted for detection, and different dilutions of the PCV4 positive serum of the swine source and OD450nm values corresponding to the PCV4 negative serum are calculated.

The established indirect ELISA method is used for detecting the pig source PCV4 positive serum and PCV4 negative serum which are serially diluted in multiple ratios, and the result shows that: the result of the pig source PCV4 positive serum after 3200 times dilution is still positive, the OD450nm is 0.158, the result after 6400 times dilution is negative, which shows that the sensitivity of the method is better, and the detection lower limit can reach 1:3200 (see Table 3).

TABLE 3 sensitivity detection results of indirect ELISA method

1.8 repeatability test

Taking the same batch of coated ELISA plates according to an established Cap protein indirect ELISA method, randomly extracting 4 parts of pig source PCV4 positive serum, repeating 6 holes for each part of serum, and carrying out a batch repeatability test; and taking enzyme-labeled plates coated in different batches, carrying out batch-to-batch repeatability test on the 4 pig source PCV4 positive serum, calculating intra-batch and inter-batch variation Coefficients (CV), and evaluating the repeatability of the established indirect ELISA method.

Under the optimized indirect ELISA reaction conditions, the 4 pig source PCV4 positive serum is subjected to the repeated test in the group and between groups respectively. The results show that: the intra-group variation coefficient was 1.91-6.22, the inter-group variation coefficient was 2.91-8.59, both of which were less than 10%, indicating that the established indirect ELISA method was excellent in reproducibility (see Table 4).

TABLE 4 results of intra-and inter-group reproducibility test of indirect ELISA method

1.9 detection of clinical samples

PCV4 antibody detection is carried out on 715 parts of serum samples collected from a pig farm of the Fujian province scale in 2020-2022 by using an indirect ELISA method based on PCV4 Cap protein established by the research, and the epidemic situation of PCV4 of pig herds in Fujian province in different years is evaluated;

the PCV3 and PCV2 antibody detection was performed on the above detected PCV4 antibody positive samples using the previously established ELISA method based on PCV3 Cap protein (ref: chen Rujing, wu Xuemin, chen Qiuyong, etc.. Cap protein prokaryotic expression of porcine circovirus type 3 nuclear localization signal [ J ]. Animal medicine progression, 2020,41 (11): 24-28.), PCV2-ELISA commercialized kit (instruction manual operation), and the mixed infection between PCV4 and PCV2, PCV3 was evaluated in the pig farm of Fujian province.

PCV4 antibody detection is carried out on 715 parts of serum samples collected from pig farms in different areas of Fujian province in 2020-2022 by using an optimized indirect ELISA method, and the results show (table 5): the positive part of PCV4 antibody detected in 715 serum samples is 47 parts, and the total positive rate is 6.57%; PCV4 antibody positive rates in 2020, 2021 and 2022 were 4.26% (10/235), 5% (14/280) and 11.5% (23/200), respectively. PCV3 and PCV2 antibody assays were performed on 47 PCV4 antibody positive serum samples, respectively, and showed that: the positive fraction of PCV3 antibody in the 47 serum samples was 6 parts, the positive fraction of PCV2 antibody was 18 parts, the mixed infection rate of PCV4 and PCV2 was 25.53% (12/47), and the mixed infection rate of PCV4, PCV3 and PCV2 was 12.77% (6/47).

TABLE 5 detection results of antibodies in clinical serum samples

In conclusion, the research successfully expresses the soluble PCV4 Cap protein, establishes a detection method of PCV4 serology indirect ELISA, can be applied to the antibody detection of clinical PCV4, can effectively supplement PCV4 serum epidemiological investigation data, and provides a scientific basis for prevention and control of PCV4 and development of a commercial PCV4 serum diagnosis kit.

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (7)

1. An indirect ELISA detection kit for 4-type Cap proteins of porcine circovirus is characterized in that: comprises a coated ELISA plate taking porcine circovirus 4-type recombinant Cap protein as a coating antigen.

2. The porcine circovirus 4 Cap protein indirect ELISA detection kit according to claim 1, characterized in that: the preparation method of the porcine circovirus 4-type recombinant Cap protein comprises the following steps: by passing throughNdeI andHindIII two enzyme cutting sites, the porcine circovirus type 4 with the sequence shown in SEQ ID NO.1CapCloning genes onto a prokaryotic expression vector to construct a recombinant plasmid; transforming the recombinant plasmid into escherichia coli DH5 alpha to obtain a recombinant expression strain; inducing recombinant expression strain by IPTG, ultrasonic crushing, centrifuging, and collecting supernatant; and (3) performing affinity chromatography purification on the supernatant Ni-NTA to obtain the porcine circovirus 4-type recombinant Cap protein.

3. The porcine circovirus 4 Cap protein indirect ELISA detection kit according to claim 2, characterized in that: the prokaryotic expression vector is a pET-30a (+) vector.

4. The porcine circovirus 4 Cap protein indirect ELISA detection kit according to claim 2, characterized in that: the IPTG induction is to add IPTG to a final concentration of 0.5. 0.5mM, and the induction expression is 16h under the condition of 15 ℃.

5. The porcine circovirus 4 Cap protein indirect ELISA detection kit according to claim 2, characterized in that: the kit also comprises negative serum, positive serum, enzyme-labeled antibodies, washing liquid, diluent, coating liquid, sealing liquid, chromogenic liquid and stop liquid.

6. The porcine circovirus 4-type Cap protein indirect ELISA detection kit according to claim 5, characterized in that: the enzyme-labeled antibody is HRP-labeled goat anti-pig IgG, the washing solution is 0.01mol/L PBST of pH7.4, the diluent is 0.01mol/L PBST of pH7.4 and 2% skim milk powder, the coating solution is 0.05mol/L phosphate buffer solution of pH9.6, the blocking solution is 5% skim milk powder, the color development solution is TMB, and the stopping solution is 0.5M H ₂ SO ₄ 。

7. The porcine circovirus 4 Cap protein indirect ELISA detection kit according to claim 6, characterized in that: the preparation of the porcine circovirus 4-type Cap protein indirect ELISA detection kit is carried out according to the following operations:

coating antigen: diluting the porcine circovirus 4-type recombinant Cap protein with coating liquid to 1.25 mug/mL, adding the diluted porcine circovirus 4-type recombinant Cap protein into an ELISA plate, coating overnight at 4 ℃ and 100 mug/hole to obtain a coated ELISA plate;

washing: discarding the coating liquid and washing with a washing liquid;

closing: adding 200 mu L of sealing liquid into each hole, sealing 1h at 37 ℃, discarding the sealing liquid and washing with a washing liquid;

incubating primary antibodies: pig serum was mixed with dilution, dilution 1:200 100. Mu.L/well, incubated at 37℃for 60min, discarded and washed with wash solution;

incubating a secondary antibody: mixing the enzyme-labeled antibody with a diluent, wherein the dilution is 1:10000 100. Mu.L/well, incubated at 37℃for 30min, discarded and washed with wash solution;

color reading: the reaction was stopped by adding 100. Mu.L/well of a color developing solution, adding 50. Mu.L/well of a stop solution after 10min of a color developing reaction at 37℃and measuring the OD value at a wavelength of 450nm on a microplate reader.