CN115028688A - PCV3Cap protein antigen peptide, antibody and immunohistochemical kit for PCV3 detection - Google Patents

Abstract

The invention discloses an antigen peptide and an antibody of PCV3Cap protein and an immunohistochemical kit for PCV3 detection. The amino acid sequence of the antigen peptide is shown as SEQ ID NO. 1; the antibody is prepared by separating immune serum after immunizing a mouse by the antigen peptide; the kit comprises the antibody, goat anti-mouse IgG marked by HRP, SABC, DAB, confining liquid and PBS. The antigen peptide has high specificity, and the antibody and the kit can detect the antigen in the clinical PCV3 infected pig tissue pathological material and judge whether to be infected with PCV3, and have strong specificity, high sensitivity, good repeatability and high detection speed.

Description

PCV3Cap protein antigen peptide, antibody and PCV3 detection immunohistochemical kit

Technical Field

The invention relates to the technical field of livestock pathogenic virus diagnosis, in particular to a Cap protein antigen peptide and antibody of porcine circovirus 3 and an immunohistochemical kit for PCV3 detection.

Background

Porcine Circovirus (PCV) currently has four established genotypes: PCV1, PCV2, PCV3 and PCV 4. PCV1 is a non-pathogenic virus, the latter three being all pathogenic viruses.

PCV3 is an ancient virus, but its presence has not been discovered and confirmed by humans. Until 2016, the American scholars Palinski et al found and reported the porcine circovirus type 3 (PCV3) for the first time by means of metagenomic sequencing from a dead fetus sample of a certain pig farm sow with unexplained dermatitis syndrome and abortion. Subsequently, most swine-raising countries in the world report the prevalence of PCV3 in pig farms (Faccini et al, 2017; Stadejek et al, 2017; Bedolla et al, 2018; Hayashi et al, 2018; Kim et al, 2018; Ye et al, 2018; Yuzhakov et al, 2018; Arruda et al, 2019; Savic et al, 2020). The laboratories which began PCV3 epidemiological investigation and virus isolation research in China were respectively a university of Huazhong agriculture who inspires professor team and university of Huanan agriculture who has a long lead, and the results showed that the sample positive rate of PCV3 in each province of south China is (313/1078) 29.04%, the pig farm positive rate is (79/177) 44.63%, and the prevalence of PCV3 in domestic pig farms and the pathogenicity to pig herds are confirmed (Shen et al, 2018). Subsequently, a large number of domestic scholars reported the prevalence of PCV3 in various regions of the country (Ha et al, 2018; Sun et al, 2018; Wen et al, 2018; Xu et al, 2018). Sundobo et al collected 2015-2017 616 suspected porcine circovirus infected samples from 21 provinces in China. The test results showed that the positive rate of PCV3 in these samples with respiratory tract disease was 26.6% (Qi et al, 2019); xupengli et al collected 170 relevant samples of 14 cities in the central region suspected of porcine circovirus infection with a positive rate of 31.18% PCV3 and a positive rate of 48.78% (20/41) in the pig farm (Xu et al, 2018). From these data, it can be found that PCV3 is already widely present and prevalent in swine farms in China, and also has a cross-infection phenomenon with other diseases (such as PCV2, PEDV, PRRSV, etc.) (Chen et al, 2019). The professor group in zingiberine detected PCV3(Zhang et al, 2018) from samples collected from dogs, and also showed a high percentage of PCV3 nucleic acid positivity among the piggery mosquitoes. These epidemiological studies provide important guidance for the prevention and control of PCV 3.

Since PCV3 was discovered in the united states, china, korea, polish, brazil and italy have also reported the presence of the virus in succession. Pigs infected with PCV3 show Porcine Dermatitis and Nephrotic Syndrome (PDNS), reproductive disorders, respiratory, gastrointestinal and nervous system diseases, multiple system inflammation, myocarditis, etc. PCV3 genome has been detected by PCR methods in oral fluid and nasal swabs as well as feces, semen and colostrum, horizontal transmission by direct contact may be the main transmission pathway. The pathogenicity and immunological mechanisms of PCV3 are currently unknown, its high incidence may pose a potential threat to the swine industry worldwide, and the widespread presence of PCV3 has raised a high level of concern in the swine industry of countries worldwide. PCV3 is harmful in that it can impair the immune function of infected pigs, leading to a decline in the body resistance, and often presents as a subclinical infection, which is easily overlooked. Since PCV3 infection damages the immune system, it is prone to secondary or concurrent infections with other pathogens, exacerbating the disease and causing greater harm.

PCV3 is a small, non-enveloped virus particle, approximately 15-20nm in diameter, with icosahedral symmetry. The whole genome is a single strand circular DNA, and the genome is 2000 bp. Mainly comprises two main Open Reading Frames (ORFs): ORF1 encoding a viral replication protein (Rep) and ORF2 encoding a viral capsid protein (Cap). Rep and Cap genes are encoded in opposite directions, resulting in an ambisense strand structure of the genome. The 5' spacer between Cap and Rep has a stem-like characteristic (stem-loop) viral replication initiation site (Ori).

Since the strain PCV3 is difficult to isolate on cells, the development of in vitro differential diagnostic methods against PCV3 is greatly limited. At present, methods such as Polymerase Chain Reaction (PCR) and animal regression are mainly adopted for the differential diagnosis of PCV 3. Wherein, PCR requires known, definite, universal and specific nucleic acid sequence to design and identify primers, has high requirement on the operation technique of experimenters and is easy to generate false positive or false negative; however, animal regression experiments require raising experimental animals to combat toxicity and observe clinical symptoms for identification, and are long in time consumption, high in cost and greatly influenced by individual animals.

Disclosure of Invention

The invention aims to provide an antigenic peptide of PCV3Cap protein capable of specifically labeling PCV3, an antibody prepared according to the antigenic peptide, and an immunohistochemical kit for PCV3 detection, which can sensitively and specifically detect PCV3 virus, thereby making up the defects in the prior art.

The technical scheme of the invention is detailed as follows:

in a first aspect, the invention provides PCV3Cap protein antigen peptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

In a second aspect, the invention provides a coding gene of the above antigen peptide, and the nucleotide sequence is shown in SEQ ID NO. 2.

In a third aspect, the present invention provides an expression vector comprising the above-described encoding gene.

In a fourth aspect, the present invention provides a host bacterium comprising the above expression vector.

In a fifth aspect, the present invention provides the use of the above antigenic peptide in the preparation of an antibody for detecting PCV 3.

In a sixth aspect, the invention provides a PCV3Cap single factor serum antibody, which is prepared by immunizing a mouse with the antigen peptide and separating immune serum.

Alternatively or preferably, the above antibody is prepared by a method comprising the following steps:

(1) extracting DNA from tissue virus liquid of PCV3 LY strain of porcine circovirus as a template, and performing PCR amplification by using nucleotide sequences shown in SEQ ID NO. 3-4 as primers to obtain a target gene;

(2) respectively carrying out double enzyme digestion on the target gene and a vector PET-28a (+) and then connecting to obtain a PET-Cap recombinant plasmid;

(3) transferring the PET-Cap recombinant plasmid into host bacterium Escherichia coli BL21, culturing, and identifying positive clone;

(4) after the identification is correct, inoculating the host bacteria into an LB culture medium containing ampicillin, and performing shake culture at 37 ℃;

(5) culturing to logarithmic phase, cooling the culture temperature to 30 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) to the final concentration of 1.0mmol/L, and carrying out induced culture for 4 hours;

(6) taking out the bacteria liquid for induction culture expression, centrifuging to remove supernatant, adding PBS to resuspend the bacteria, cracking and crushing, adding 2 times of sample buffer according to the volume ratio of 1:1, boiling for 10min, centrifuging and taking supernatant;

(7) purifying the target protein by using a His label purification kit to obtain PCV3Cap protein antigen peptide;

(8) diluting PCV3Cap protein antigen peptide to 1.05mg/mL, adding equivalent volume Freund's complete adjuvant, carrying out first immunization injection on BALB/C mice, wherein the injection part is neck subcutaneous, the injection dose is 100-110 mu g/mouse, and feeding for 2 weeks;

(9) diluting PCV3Cap protein antigen peptide to 1.05mg/mL, adding equivalent Freund incomplete adjuvant, performing second immunization injection, wherein the injection part is subcutaneous on the back, the injection dose is 100-110 mu g/mouse, and feeding for 2 weeks;

(10) performing third immunization injection according to the method in the step (9), and feeding for 10 days;

(11) and (3) collecting blood from the heart of the mice which are raised for 10 days after the third immunization, and separating immune serum, namely PCV3Cap single factor serum antibody.

In a seventh aspect, the invention provides an immunohistochemical kit for detecting PCV3, comprising the PCV3Cap single factor serum antibody.

Alternatively or preferably, the kit further comprises HRP-labeled goat anti-mouse IgG, SABC, DAB, blocking solution and PBS.

Description of the drawings:

PET-28a (+): an expression carrier contains kanamycin-resistant gene, the corresponding expression host bacterium is colibacillus for expressing target gene, the genome contains protein labels N-His, N-T7 or C-His, and IPTG or lactose and their analogues can be used for induction expression.

Coli BL 21: a host bacterium for broad protein expression, also known as Escherichia coli BL21(DE3) bacterium. The chromosome carries a T7 RNA polymerase gene controlled by a lacUV5 promoter, and the exogenous target gene driven by a T7 promoter can be efficiently expressed under IPTG induction.

IPTG: isopropyl-beta-D-thiogalactoside of the formula C ₉ H18O ₅ S, can cause the transcription process of the lactose operon, and therefore can induce the expression of the protein corresponding to the downstream gene of the lactose operon.

Loading buffer: the loading buffer in step (6) is protein loading buffer, and mainly adopts Tris-HCl (Tris-hydroxymethyl aminomethane hydrochloride) or Tris glycine, and EDTA salt (disodium ethylenediaminetetraacetate), and if necessary, SDS (sodium dodecyl sulfate), DTT (dithiothreitol), bromophenol blue, glycerol, and the like.

HRP: horse Radish Peroxidase, horseradish Peroxidase, when incubated with a substrate, can produce colored, fluorescent or luminescent derivatives of the labeled molecules, thereby permitting quantitation. .

SABC: StreptAvidin-Biotin Complex, StreptAvidin-Biotin-peroxidase Complex.

DAB: 3, 3' -diaminobenzidine, the most commonly used chromogenic substrate for HRP, exhibit a color change and accumulate in the presence of hydrogen peroxide upon electron loss, forming a light brown insoluble product.

Sealing liquid: for preventing non-specific binding of antibodies to tissues or Fc receptors to reduce background signal and reduce false positives.

PBS: phosphate buffer saline, generally a solvent, acts to solubilize the protective agent.

The "single factor" in PCV3Cap single factor serum antibodies refers to a single antigen.

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

the invention provides a section of antigen peptide capable of specifically labeling PCV3 virus, and a single-factor serum antibody, namely a polyclonal antibody, is prepared by the antigen peptide, so that an Immunohistochemical (IHC) detection kit is formed, the antigen in a pig tissue disease material infected with PCV3 clinically can be detected, whether PCV3 is infected or not is judged, and the kit has the advantages of strong specificity, high sensitivity, good repeatability and high detection speed; compared with the traditional PCR, ELISA, ISH, IHC and animal regression test diagnosis methods, the kit is more convenient and more accurate to detect by an indirect immunofluorescence assay (IFA) technology.

Preservation information:

name: porcine circovirus type 3 PCV3 LY strain (PCV3 LY strain);

the preservation unit is as follows: china center for type culture Collection;

and (4) storage address: wuhan, Wuhan university, post code 430072;

the preservation number is as follows: CCTCC No. v202208;

preservation time: 3 months and 10 days in 2022.

Drawings

FIG. 1 is a graph showing the following conditions in 1: positive porcine inguinal lymph node tissue cells are detected by PCV3Cap single factor serum antibody at 200 dilution for immunohistochemical staining, wherein cytoplasm is stained, A is the condition that cell nucleus is stained to be brown yellow, B is negative porcine tissue cells which do not show brown yellow, and C is a condition that PBS is used for replacing primary antibody as a control and does not show brown yellow.

FIG. 2 shows the nucleic acid electrophoresis of PCV3 ORF2 gene, M:5000 Marker; 1. a gene of interest; 2. negative water control.

FIG. 3 is SDS-PAGE electrophoresis of expression PCV3Cap protein, 1. expression protein induced by whole engineering bacteria (unpurified); 2. the engineering bacteria do not induce to express protein; 3. and (5) purifying the target protein.

Detailed Description

The present invention will be described in detail with reference to the preferred embodiments, and the chemicals or reagents used in the present invention may be selected from other existing products according to their functions, and are not limited to the embodiments specifically described. For experiments where no specific conditions are noted in the examples, it is generally possible to operate under conventional conditions, such as those described in the molecular cloning instructions written by J.Sambruke (Sambrook), et al, or according to the manufacturer's recommendations.

EXAMPLE 1 preparation of PCV3Cap Single factor serum antibodies

1. Preparation of Cap protein antigen of PCV3

According to a sequence at two ends of PCV3 ORF2 gene and a polyclonal enzyme cutting site of an expression vector PET-28a (+), the following specific primers aiming at PCV3 ORF2 are designed, and the primers are synthesized by Shanghai biological engineering Co., Ltd.

PCV3 ORF 2-F: 5'-GGG GGA TCC ATG AGA CAC AGA GCT ATA TTC-3' (BamHI sites added),

PCV3 ORF 2-R: 5'-GGGAAG CTT TTAGAGAAC GGACTT GTAACG-3' (HindIII sites added).

PCR amplification is carried out by taking DNA extracted from virus culture solution of a strain PCV3 LY (preservation number CCTCC NO: V202208) of the porcine circovirus type 3 screened in the early stage of the inventor as a template. The target gene with the amplification product size of 645bp is shown in detail in FIG. 2.

The amplified target gene is recovered by using a DNA purification recovery kit, and simultaneously the target gene is connected with a carrier PET-28a (+) double enzyme digestion and ligase to obtain a PET-ORF2 recombinant plasmid, the recombinant plasmid is transferred into an expressive escherichia coli BL21 host bacterium according to a conventional method, the recombinant plasmid is cultured, a single clone is selected, a positive clone is identified by an enzyme digestion method, the bacterium is shaken, the sequence is determined, and the result of the sequence determination shows that the nucleotide sequence is shown as SEQ ID NO. 2, and the amino acid sequence of the translated protein is shown as SEQ ID NO. 1. The sequence is found to be a new gene sequence of PCV3 through BLAST tool analysis of NCBI website, the homology with the gene sequence of the known PCV3 strain is about 87.08 to 87.85 percent, the homology with the amino acid sequence of the known PCV3 strain is 90.70 to 93.49 percent, and the homology with the genome of other PCV (PCV2/PCV4) is 43.2 to 51.5 percent.

In order to exclude the gene from being caused by errors generated in the amplification process, multiple amplifications and sequencing are carried out, and the Cap protein gene expressing PCV3 amplified in PCV3 LY strain is proved to be a new gene and can express a novel Cap protein.

2. Preparation of PCV3Cap Single factor serum antibody (Primary antibody) with Cap protein as antigen

Inoculating expressive BL21 host bacteria transformed by single clone recombinant plasmid PET-ORF2 with correct sequencing result into 5mL LB bacterial culture medium containing ampicillin (100mg/L), shaking and culturing overnight at 37 ℃, taking 1mL culture out the next day, inoculating the culture into 120mL LB culture medium, shaking and culturing for 2.5h at 37 ℃, shaking the bacterial liquid to be in a semitransparent and semi-turbid state, measuring OD600 to be 0.6-0.8 by an absorptiometry, taking 10mL before induction as a control before induction, changing the shaking temperature to 30 ℃, adding IPTG (final concentration is 1.0mmol/L) for induction, collecting 10mL bacterial liquid after 4h induction, taking the bacterial liquid inducing non-transformed PET vector as a blank control, and taking the bacterial liquid inducing transformed PET vector as an empty vector control.

Taking out the bacteria liquid for induction expression, centrifuging to remove supernatant, then resuspending the bacteria by 0.5mL of 1 XPBS, ultrasonically cracking and crushing the bacteria, adding 2 Xsample Buffer according to the ratio of 1:1, boiling for 10min, centrifuging to take supernatant, and carrying out electrophoretic identification and analysis on the supernatant by 12% SDS-PAGE to obtain the target protein of about 27-28 kDa, which is detailed in figure 3. The target protein is purified by using a cation affinity purification kit according to the specified operation, and the electrophoretogram of the purified protein is shown in detail in figure 3.

The purified Cap protein of interest was quantified spectrophotometrically, diluted to (1.0mg/mL) with an equal amount of Freund's complete adjuvant, and injected subcutaneously into the neck of 20 BALB/C mice, 0.2mL (100. mu.g/mouse) each. The second, 0.2 ml/mouse, was immunized subcutaneously on the back 2 weeks later with Cap protein antigen containing Freund's incomplete adjuvant. A third immunization (the same procedure as the second) was carried out after 2 weeks, in which 10 BALB/C mice without the immunizing antigen served as a negative control group. And (3) collecting blood from the heart to kill on the 10 th day after the third immunization, and storing the separated immune serum at the temperature of-20 ℃ to obtain the PCV3Cap single-factor serum antibody.

3. Sensitivity and specificity identification of monoclonal antibody of single factor serum

Inguinal lymph node tissue (PCV3 virus nucleic acid load of 3.65 × 10) preserved in the laboratory ^6.0 Clinical, no morbid symptoms present), according to immunohistochemical ABC method staining procedure:

(1) slicing paraffin and pasting;

(2) washing the slices in PBS buffer for 10 min;

(3) incubating with 0.6% hydrogen peroxide at room temperature for 30 min;

(4) washing in 0.4% triton-100PBS for 3 times (5 min/time);

(5) 8% normal sheep serum is sealed for 30 minutes at room temperature;

(6) PCV3Cap single factor serum antibody (control replaced PBS) was incubated overnight at 4 ℃;

(7) washing with 0.2% triton-100PBS for 3 times (5 min/time);

(8) incubating the goat anti-mouse IgG secondary antibody marked by HRP for 1 hour at 37 ℃;

(9) washing with 0.2% triton-100PBS for 4 times (5 min/time);

(10) incubation of the SABC complex for 1 hour at 37 ℃;

(11) washing with 0.2% triton-100PBS for 4 times (5 min/time);

(12) Tris-HCl (0.05M pH 7.6) for 15 min;

(13) DAB color development is carried out for about 3-10 minutes;

(14) washing with PBS 2 times, and washing with deionized water 1 time;

(15) counter-staining with hematoxylin for 30 seconds, and washing with deionized water for 10 min;

(16) drying, and sealing with neutral gum.

The PCV3Cap single factor serum antibody in the step (6) was diluted with 1 XPBS at four dilutions of 1:50, 1:100, 1:200, and 1:500, respectively, and the experimental results were observed under a microscope. The identification results are shown in 1: the effect is better when the dilution is 200 degrees, as shown in figure 1, cytoplasm of positive porcine inguinal lymph node tissue cells is dyed into brown yellow, and nucleus is dyed into blue after being counterstained by hematoxylin, see A; negative porcine tissue cells do not show brown yellow, see B; PBS was used as a control instead of primary antibody, see C.

The result proves that PCV3Cap single factor serum antibody (primary antibody) has high sensitivity and specificity, and particularly can be related to the variation of the amino acid sequence of a new strain Cap protein which is optimally expressed, so that the immunized single factor serum generates the antibody aiming at PCV3 specific epitope.

Example 2 immunohistochemical kit preparation and verification of sensitivity, specificity, reproducibility thereof

1. Construction of porcine circovirus 3 immunohistochemical detection kit

The kit comprises the following components listed in the following table 1:

TABLE 1 immunohistochemical detection kit for porcine circovirus 3

Item	Composition (I)
		A primary antibody	PCV3Cap single factor serum antibody
Enzyme-labeled secondary antibody	HRP-labeled goat anti-mouse IgG
		Substrate	SABC (streptavidin-biotin-peroxidase complex)
Sealing liquid	8% Normal sheep serum
		Color developing liquid	DAB (diaminobenzidine)
Buffer system	PBS powder (1 package can be constant volume to 1L with deionized water as buffer)

2. Sensitivity, specificity and repeatability tests of the kit

(1) IHC and PCR comparative detection

134 clinically diagnosed suspected PCV3 infected tissue samples were examined from pig farms in Hebei, Liaoning, Henan, Shandong, and Sichuan provinces using IHC (kit of Table 1) and PCR method, respectively. The results of IHC and PCR detection of tissue samples from different regions are shown in Table 2 below.

TABLE 2 comparison of the positive detection rates of the two methods for 134 samples

(2) Repeatability test

134 parts of clinical disease materials are detected by using the kit with the components in the table 1, and meanwhile, sample DNA is extracted and amplified by using a PCR method to verify the detection result of the kit, wherein except that the positive numbers of Shandong and Sichuan tissue samples are consistent: the other detection results are inconsistent, the PCR method has more positive rates than the IHC method, wherein the PCR positive detection rate of the Hebei sample is 2 more than that of the IHC, the PCR positive detection rate of the Liaoning sample is 3 more than that of the IHC, and the PCR positive detection rate of the Henan sample is 1 more than that of the IHC.

And performing repeated detection twice on the samples of Hebei, Liaoning and Henan, wherein the second and third detection results of the IHC method are the same as the first detection results of the IHC. The second and third detection results of the PCR method are consistent with the detection results of IHC, and are different from the first detection results of the PCR method. The PCR detection result is easy to generate false positive result, and the specificity, sensitivity and repeatability of the detection result of the kit with the components in the table 1 are also shown to be very good. The results of the reproducibility tests are shown in Table 3 below.

Table 3 comparison of results of reproducibility tests on 134 samples by IHC and PCR methods

(3) Crossover experiment

Randomly drawing a batch of porcine circovirus 3 immunohistochemical detection kits, detecting porcine reproductive and respiratory syndrome viruses, porcine parvoviruses, porcine encephalitis B viruses, classical swine fever viruses and porcine pseudorabies viruses of some pathological tissue samples stored in a laboratory, and observing the result under a microscope to obtain a negative result without brown yellow cells. Further indicates that the porcine circovirus 3 immunohistochemical detection kit does not have false negative and has good specificity.

In conclusion, the porcine circovirus 3 immunohistochemical detection kit has the following advantages:

the detection result A has high accuracy, and false positive and false negative results cannot be generated.

B sensitivity and specificity are high: PCV3Cap single factor serum antibody working concentration 1: the 200-time dilution effect is good, the PCV3 virus can be accurately and sensitively detected and diagnosed, and other viruses are negative.

C, the clinical application effect is better: the kit and the PCR method with higher sensitivity are used for simultaneously detecting 134 clinical samples of pig tissues and the like caused by suspected PCV3, 23 infections containing PCV3 are detected by results, and the detection results of the kit are consistent with the detection results of the second PCR method and the third PCR method, so that the kit is proved to have higher sensitivity, specificity and repeatability of the detection results and better clinical application effect.

The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.

Sequence listing

<110> Shandong Xin De science and technology Co., Ltd

<120> PCV3Cap protein antigen peptide, antibody and immunohistochemical kit for PCV3 detection

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Claims (9)

1. An antigen peptide of PCV3Cap protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The gene encoding the antigenic peptide of claim 1, wherein the nucleotide sequence is represented by SEQ ID NO. 2.

3. An expression vector comprising the coding gene according to claim 2.

4. A host bacterium comprising the expression vector of claim 3.

5. Use of the antigenic peptide of claim 1 in the preparation of an antibody for the detection of PCV 3.

6. A PCV3Cap single factor serum antibody, which is prepared by immunizing a mouse with the antigenic peptide of claim 1 and isolating the immune serum.

7. The antibody of claim 6, wherein the method of preparation comprises the steps of:

(1) extracting DNA from tissue virus liquid of PCV3 LY strain as a template, and carrying out PCR amplification by using nucleotide sequences shown in SEQ ID NO. 3-4 as primers to obtain a target gene;

(2) respectively carrying out double enzyme digestion on the target gene and a vector PET-28a (+) and then connecting to obtain a PET-Cap recombinant plasmid;

(3) transferring the PET-Cap recombinant plasmid into host bacterium Escherichia coli BL21, culturing, and identifying positive clone;

(4) after the identification is correct, inoculating the host bacteria into an LB culture medium containing ampicillin, and performing shake culture at 37 ℃;

(5) culturing to logarithmic phase, cooling the culture temperature to 30 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) to the final concentration of 1.0mmol/L, and carrying out induced culture for 4 hours;

(6) taking out the bacteria liquid for induction culture expression, centrifuging to remove supernatant, adding PBS to resuspend the bacteria, cracking and crushing, adding 2 times of sample buffer according to the volume ratio of 1:1, boiling for 10min, centrifuging and taking supernatant;

(7) purifying the target protein by using a His label purification kit to obtain PCV3Cap protein antigen peptide;

(8) diluting PCV3Cap protein antigen peptide to 1.05mg/mL, adding equivalent volume Freund's complete adjuvant, carrying out first immunization injection on BALB/C mice, wherein the injection part is neck subcutaneous, the injection dose is 100-110 mu g/mouse, and feeding for 2 weeks;

(9) diluting PCV3Cap protein antigen peptide to 1.05mg/mL, adding equivalent Freund incomplete adjuvant, performing second immunization injection, wherein the injection part is subcutaneous on the back, the injection dose is 100-110 mu g/mouse, and feeding for 2 weeks;

(10) performing third immunization injection according to the method in the step (9), and feeding for 10 days;

(11) and (3) collecting blood from the heart of the mice which are raised for 10 days after the third immunization, and separating immune serum, namely PCV3Cap single factor serum antibody.

8. An immunohistochemical kit for detecting PCV3, comprising the PCV3Cap single factor serum antibody of claim 6 or 7.

9. The kit of claim 8, further comprising HRP-labeled goat anti-mouse IgG, SABC, DAB, blocking solution, and PBS.

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