CN115028688B - PCV3 Cap protein antigen peptide, antibody and PCV3 detection immunohistochemical kit - Google Patents

Abstract

The invention discloses an immunohistochemical kit for PCV3Cap protein antigen peptide, an antibody and 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 mice are immunized by the antigen peptide; the kit comprises the antibody, HRP-labeled goat anti-mouse IgG, SABC, DAB, blocking solution and PBS. The antigen peptide has high specificity, the antibody and the kit can detect the antigen in the clinical PCV3 infected pig tissue disease material and judge whether PCV3 is infected, and the antigen peptide has strong specificity, high sensitivity, good repeatability and high detection speed.

Description

PCV3 Cap 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 (porcine circovirus, PCV) has been identified to date with four genotypes: PCV1, PCV2, PCV3, PCV4.PCV1 is a non-pathogenic virus, the latter three being pathogenic viruses.

PCV3 is an ancient virus, but its presence has not been found and demonstrated. Until 2016 American scholars Palinski et al were the first report of a report by metagenomic sequencing from a sample of dead fetus from a pig farm sow suffering from the symptom of dermatitis due to unknown causes and abortion, designated porcine circovirus type 3 (PCV 3). Subsequently, most pig countries in the world report that 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 etal.,2020). China began PCV3 epidemiological investigation and virus isolation studies at the earliest in laboratories, respectively, the university of agriculture in China He Qigai professor team and the university of agriculture in China Song Changxu professor team, which showed that PCV3 had a sample positive rate of (313/1078) 29.04% in each province in south China and a pig farm positive rate of (79/177) 44.63%, and confirmed the prevalence of PCV3 in domestic pig farms and pathogenicity to swine herds (Shen et al, 2018). Numerous scholars in China subsequently reported the prevalence of PCV3 in various regions in China (Ha et al, 2018; sun et al, 2018; wen et al, 2018; xu et al, 2018). Sun Dongbo et al collected 616 samples suspected of porcine circovirus infection in 21 provinces 2015-2017 of China. The test results showed that the PCV3 positive rate was 26.6% in these samples showing respiratory diseases (Qi et al, 2019); xu Pengli et al collected 170 samples of a suspected porcine circovirus infection in 14 cities in China with a PCV3 positive rate of 31.18% and a pig farm positive rate of 48.78% (20/41) (Xu et al, 2018). From these data, PCV3 has been found to be widespread and prevalent in pig farms in China, along with cross-infection with other diseases (e.g., PCV2, PEDV, PRRSV, etc.) (Chen et al, 2019). Jiang Ping teaches that team detects PCV3 (Zhang et al, 2018) from samples taken from dogs, and also has a high PCV3 nucleic acid positive rate in pig farm mosquitoes. These epidemiological studies provide important instructive significance for the control of PCV 3.

Since PCV3 was found in the united states, china, korea, poland, brazil and italy have also reported the presence of the virus in succession. Pigs infected with PCV3 exhibit swine dermatitis and nephrotic syndrome (PDNS), reproductive disorders, respiratory, gastrointestinal and nervous system diseases, multisystem inflammation, myocarditis, and the like. PCV3 genome has been detected by PCR methods in oral fluid and nasal swabs as well as faeces, semen and colostrum, horizontal transmission by direct contact being probably the major transmission pathway. The pathogenicity and immune mechanism of PCV3 is currently unknown, its high incidence may pose a potential threat to the global pig industry, and the widespread presence of PCV3 has attracted a great deal of attention to the pig industry worldwide. PCV3 is a hazard in that it can impair the immune function of infected pigs, resulting in reduced body resistance, and is often present in the form of subclinical infections, which are easily overlooked. Since PCV3 infection damages the immune system, it is susceptible to secondary or concurrent infection with other pathogens, exacerbating the condition and causing greater damage.

PCV3 is a small, non-enveloped virus particle with a diameter of about 15-20nm and is icosahedral in symmetry. The whole genome is a single-stranded circular DNA, and the genome is 2000bp. Mainly comprises two main open reading frames (open READING FRAMS, ORFs): ORF1 encoding viral replication protein (REPLICASE PROTEIN, rep) and ORF2 encoding viral capsid protein (capsid protein, cap). Rep and Cap genes are encoded in opposite directions, resulting in a genome with a double sense strand structure. The 5' spacer between Cap and Rep has a stem-loop viral replication initiation site (Ori).

Since PCV3 strains are difficult to isolate on cells, development of differential diagnostic methods in vitro against PCV3 is greatly limited. At present, differential diagnosis of PCV3 mainly adopts methods such as Polymerase Chain Reaction (PCR), animal regression and the like. The PCR requires known definite, universal and specific nucleic acid sequences to design and identify primers, has high technical requirements for experimenters to operate and is easy to generate false positive or false negative; the animal regression experiment requires feeding experimental animals to attack toxin and observe clinical symptoms for identification, and has the defects of long time consumption, high cost and large influence by animal individuals.

Disclosure of Invention

The invention aims to provide a PCV3 Cap protein antigen peptide capable of specifically marking PCV3, an antibody prepared according to the antigen peptide and an immunohistochemical kit for PCV3 detection, which can sensitively and specifically detect PCV3 virus, thereby overcoming the defects in the prior art.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a PCV3 Cap 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 antigen peptide, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

In a third aspect, the present invention provides an expression vector comprising the above-described coding 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 the detection of PCV 3.

In a sixth aspect, the invention provides a PCV3 Cap 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 the method of:

(1) DNA is extracted from a tissue virus liquid of a PCV3 LY strain of the porcine circovirus as a template, and a nucleotide sequence shown by a PCV3 ORF2 primer is used as a primer for PCR amplification to obtain a target gene;

(2) The target gene and a vector PET-28a (+) are respectively subjected to double enzyme digestion and then connected to obtain a PET-Cap recombinant plasmid;

(3) Transferring the PET-Cap recombinant plasmid into host bacterium escherichia coli BL21, culturing, and identifying positive clones;

(4) After identification, inoculating host bacteria into LB culture medium containing ampicillin, and shake culturing at 37 ℃;

(5) Culturing to logarithmic growth phase, reducing the culture temperature to 30deg.C, adding IPTG to final concentration of 1.0mmol/L, and performing induction culture for 4 hr;

(6) Taking out the bacterial liquid of induced culture expression, centrifuging to remove the supernatant, adding PBS to resuspend the bacterial body, cracking and crushing, adding a 2X sample buffer according to the volume ratio of 1:1, boiling for 10min, centrifuging to obtain the supernatant;

(7) Purifying the supernatant by using a His tag purification kit to obtain PCV3 Cap protein antigen peptide;

(8) Diluting PCV3 Cap protein antigen peptide to 1.05mg/mL, adding Freund's complete adjuvant with equal volume, performing primary immunization injection on BALB/C mice, wherein the injection site is subcutaneous at the neck, the injection dose is 100-110 mug/mouse, and feeding for 2 weeks;

(9) Diluting PCV3 Cap protein antigen peptide to 1.05mg/mL, adding Freund's incomplete adjuvant with equal volume, performing a second immunization injection, wherein the injection site is subcutaneous on the back, and the injection dose is 100-110 mug/dose, and feeding for 2 weeks;

(10) Performing a third immunization injection according to the method of the step (9), and raising for 10 days;

(11) Taking the mice which are bred for 10 days after the third immunization, taking blood from hearts, and separating out immune serum, namely the PCV3 Cap single factor serum antibody.

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

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

Description:

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

Coli BL21: a host bacterium for widely expressing protein, which is also called as Escherichia coli BL21 (DE 3) bacterium. The chromosome carries a T7 RNA polymerase gene controlled by a lacUV5 promoter, and can efficiently express an exogenous target gene driven by the T7 promoter under the induction of IPTG.

IPTG: isopropyl-beta-D-thiogalactoside, with the chemical formula of C9H18O5S, can cause the transcription process of lactose operon, and can induce the expression of the corresponding protein of the downstream gene of lactose operon.

Loading buffer: the loading buffer solution in the step (6) is also called loadingbuffer, and the loading buffer solution mainly adopts Tris-HCl (Tris (hydroxymethyl aminomethane) hydrochloride) or Tris glycine, EDTA salt (disodium ethylenediamine tetraacetate), and if necessary, SDS (sodium dodecyl sulfate), DTT (dithiothreitol), bromophenol blue, glycerol and the like are added.

HRP: horse Radish Peroxidase, horseradish peroxidase, when incubated with a substrate, can produce colored, fluorescent or luminescent derivatives of the labeling molecules, thereby allowing quantification. .

SABC: STREPT AVIDIN-Biotin Complex, streptavidin-Biotin-peroxidase Complex.

DAB:3,3' -diaminobenzidine, the most commonly used chromogenic substrate for HRP, loses electrons in the presence of hydrogen peroxide to exhibit a color change and accumulation, forming a pale brown insoluble product.

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

PBS: phosphate buffer saline phosphate buffer salt solution, generally used as a solvent, acts as a dissolution protecting agent.

"Single factor" in PCV3 Cap single factor serum antibodies refers to a single antigen.

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

The invention provides an antigen peptide capable of specifically marking PCV3 virus, and a monoclonal antibody, namely a polyclonal antibody, is prepared by the antigen peptide, so that an Immunohistochemical (IHC) detection kit is formed, antigens in a clinical PCV3 infected pig tissue disease material can be detected, whether PCV3 is infected or not is judged, and the antigen peptide has strong specificity, high sensitivity, good repeatability and high detection speed; compared with the prior PCR, ELISA, ISH, IHC and animal regression test diagnosis method, the kit is more convenient and more accurate to detect by an indirect Immunofluorescence (IFA) technology.

Preservation information:

name: porcine circovirus type 3 (Porcine circovirus, PCV 3) LY strain (PCV 3 LY strain for short);

preservation unit: china center for type culture Collection;

preservation address: chinese university of Wuhan, post code 430072;

Preservation number: cctccc No. v202208;

preservation time: 2022, 3 and 5.

Drawings

Fig. 1 is a diagram of the process at 1: at 200 dilutions, positive pig inguinal lymph node tissue cells were detected with PCV3 Cap single factor serum antibodies for immunohistochemical staining, the cytoplasm was stained, A was brown-yellow, B was negative pig tissue cells did not show brown-yellow, C was PBS instead of primary antibody control, and no brown-yellow was shown.

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

FIG. 3 shows SDS-PAGE of expressed PCV3 Cap protein, 1. Engineering bacterium whole-cell induced expression protein (not purified); 2. the engineering bacteria do not induce the expression protein; 3. purifying target protein.

Detailed Description

The present invention will be described in detail with reference to preferred embodiments, but the chemicals or reagents used in the present invention may be selected from other existing types of products according to their functions, not limited to the specific descriptions of the embodiments. For the experimental methods in which specific conditions are not noted in the examples, it is generally possible to run the method under conventional conditions, such as those described in the molecular cloning experimental guidelines written by j.

EXAMPLE 1 preparation of PCV3 Cap single factor serum antibodies

1. Cap protein antigen preparation of PCV3

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

The DNA extracted from the virus culture solution of a strain PCV3 LY strain (preservation number CCTCCNO: V202208) of porcine circovirus type 3, which is screened by the inventor in the earlier stage, is used as a template for PCR amplification. The amplified product is 645bp target gene, which is shown in FIG. 2.

And (3) recovering the amplified target gene by using a DNA purification recovery kit, simultaneously carrying out double digestion and ligase connection on the target gene and a carrier PET-28a (+) to obtain a PET-ORF2 recombinant plasmid, transferring the recombinant plasmid into an expressive escherichia coli BL21 host bacterium according to a conventional method, culturing, selecting a single clone, identifying positive clones by using an enzyme digestion method, shaking, sequencing, and displaying the nucleotide sequence of the positive clone as shown in SEQ ID NO.2, wherein the amino acid sequence of the translated protein is shown in SEQ ID NO. 1. BLAST tool analysis of NCBI website shows that the sequence is a new gene sequence of PCV3, the homology with the gene sequence of known PCV3 strain is about 87.08-87.85%, the homology with the amino acid sequence of known PCV3 strain is 90.70-93.49%, and the homology with other PCV (PCV 2/PCV 4) genomes is 43.2% -51.5%.

In order to exclude the gene due to errors generated in the amplification process, repeated amplification and sequencing are performed, and it is proved that the amplified Cap protein gene expressing PCV3 in the PCV3 LY strain is a novel gene, and can express novel Cap protein.

2. PCV3 Cap single factor serum antibody (primary antibody) prepared by using Cap protein as antigen

The single recombinant plasmid PET-ORF2 transformed expression BL21 host bacteria with correct sequencing results are inoculated into 5mL of LB bacterial culture medium containing ampicillin (100 mg/L), shaking culture is carried out at 37 ℃ for overnight, 1mL of culture is taken out the next day and inoculated into 120mL of LB culture medium, shaking culture is carried out at 37 ℃ for 2.5 hours, bacterial liquid is shaken to a semitransparent semi-turbid state, OD600 = 0.6-0.8 is measured by a light absorption photometry, 10mL is taken as a pre-induction control before induction, the shaking temperature is changed to 30 ℃ and IPTG (with the final concentration of 1.0 mmol/L) is added for induction, 10mL of bacterial liquid is collected after induction for 4 hours, bacterial liquid of an unconverted PET carrier is used as a blank control, and bacterial liquid of the converted PET carrier is used as an empty carrier control.

Taking out the bacterial liquid for inducing expression, centrifuging, discarding the supernatant, re-suspending the bacterial body by using 0.5mL of 1 XPBS, performing ultrasonic wave pyrolysis on broken bacteria, adding 2 XP Buffer for boiling for 10min according to the ratio of 1:1, centrifuging, taking the supernatant, and performing 12% SDS-PAGE electrophoresis identification analysis on the supernatant to obtain target proteins of about 27-28 kDa, wherein the detail is shown in figure 3. Purifying the target protein by using a cation affinity purification kit according to a specified operation, and detailedly showing an electrophoresis chart of the purified protein in figure 3.

The purified target Cap protein was quantified spectrophotometrically and diluted to (1.0 mg/mL) plus an equivalent amount of Freund's complete adjuvant, and 20 BALB/C mice were injected subcutaneously on the neck, each 0.2mL (100. Mu.g/mouse). After 2 weeks, the second subcutaneous immunization was performed with Cap protein antigen containing Freund's incomplete adjuvant, 0.2 ml/mouse. A third immunization (method same as the second) was performed after 2 weeks, with 10 naive BALB/C mice as negative control. And on the 10 th day after the third immunization, heart blood is collected and killed, and the separated immune serum is preserved at the temperature of minus 20 ℃ to obtain the PCV3 Cap single factor serum antibody.

3. Sensitivity and specificity identification of monoclonal serum polyclonal antibodies

Inguinal lymph node tissue (PCV 3 virus nucleic acid load 3.65x10 ^6.0, clinical as yet not showing symptoms of disease) preserved in this laboratory was subjected to immunohistochemical ABC staining procedure:

(1) Paraffin section and patch;

(2) Washing the slices in PBS buffer solution for 10 minutes;

(3) Incubating for 30 minutes at room temperature with 0.6% hydrogen peroxide;

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

(5) 8% normal sheep serum was blocked for 30min at room temperature;

(6) PCV3 Cap single factor serum antibody (control replaced with PBS) was incubated overnight at 4deg.C;

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

(8) Incubation of HRP-labeled goat anti-mouse IgG secondary antibody for 1 hour at 37 ℃;

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

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

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

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

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

(14) Washing with PBS for 2 times and with deionized water for 1 time;

(15) Hematoxylin counterstain for 30 seconds, and deionized water is used for washing for 10 minutes;

(16) Drying, and sealing with neutral resin.

The PCV3 Cap single factor serum antibodies in step (6) were subjected to four dilutions of 1:50, 1:100, 1:200, 1:500 with 1 XPBS, respectively, and the experimental results were observed under a microscope. The identification result is shown in 1: the effect is better at 200 dilutions, as shown in figure 1, the cytoplasm of positive pig inguinal lymph node tissue cells is stained brown yellow, and the nucleus is blue after being counterstained by hematoxylin, see A; negative pig tissue cells did not appear brown yellow, see B; PBS was used as a control instead of primary antibody, see C.

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

Example 2 preparation of immunohistochemical kit and verification of sensitivity, specificity and repeatability

1. Construction of porcine circovirus 3 immunohistochemical detection kit

Included in the kit are the components listed in table 1 below:

table 1, porcine circovirus 3 immunohistochemical detection kit

Project	Composition of the components
		First antibody	PCV3 Cap single factor serum antibody
Enzyme-labeled secondary antibody	HRP-labeled goat anti-mouse IgG
		Substrate(s)	SABC (streptavidin-biotin-peroxidase complex)
Sealing liquid	8% Normal sheep serum
		Color development liquid	DAB (diaminobenzidine)
Buffer system	PBS powder (1 package of deionized water to 1L as buffer)

2. Kit sensitivity, specificity and repeatability assays

(1) IHC and PCR comparative detection

IHC (kit of Table 1 components) and PCR method were used separately to carry out comparative detection on 134 tissue samples submitted for clinical diagnosis of suspected PCV3 infection by pig farms such as Hebei, liaoning, henan, shandong and Sichuan. IHC and PCR detection results of tissue samples from different regions are shown in Table 2 below.

Table 2, comparison of the Positive detection Rate of 134 samples by two methods

Sample source	Number of samples	IHC positive number	Positive rate (%)	Number of PCR positives	Positive rate (%)
						Hebei river	35	7	20	9	25.71
Liaoning (Liaoning)	41	12	29.27	15	36.59
						Henan province	20	4	20	5	25
Shandong province	24	3	12.5	3	12.5
						Sichuan (Sichuan)	14	2	14.29	2	14.29
Totals to	134	28	20.9	34	25.37

(2) Repeatability test

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

And simultaneously, repeating the detection for the samples of Hebei, liaoning and Henan twice, 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 result of IHC, and are different from the first detection result of the PCR method. The PCR detection result is easy to generate false positive results, and the specificity, the sensitivity and the repeatability of the detection result of the kit of the components in the table 1 are very good. The results of the repeatability test are shown in table 3 below.

Table 3, IHC and PCR methods comparison of results of duplicate assays for 134 samples

(3) Cross test

Randomly extracting a batch of porcine circovirus 3 immunohistochemical detection kit, detecting porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine Japanese encephalitis virus, swine fever virus and porcine pseudorabies virus of some disease tissue samples stored in a laboratory, and observing the result under a microscope that no brown yellow cells appear, and obtaining a negative result. Further shows 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 A detection result has high accuracy and can not generate false positive and false negative results.

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

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

Specific examples are set forth herein to illustrate the invention in detail, and the description of the above examples is only for the purpose of aiding in understanding the core concept of the invention. It should be noted that any obvious modifications, equivalents, or other improvements to those skilled in the art without departing from the inventive concept are intended to be included in the scope of the present invention.

Sequence listing

<110> Shandong Xinde science and technology Co., ltd

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

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Claims (7)

1. The PCV3 Cap protein antigen peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The antigen peptide coding gene of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 2.

3. An expression vector comprising the coding gene of claim 2.

4. A host bacterium comprising the expression vector according to claim 3.

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

6. A PCV3 Cap single factor serum antibody is characterized in that the antibody is prepared by immunizing a mouse with the antigen peptide of claim 1 and separating immune serum.

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