CN114814210A - Fluorescence immunoassay kit for detecting PCV3 - Google Patents

Abstract

The invention discloses a fluorescence immunoassay kit for detecting PCV3, which mainly comprises the following components: PCV3Cap single factor serum antibody, by the amino acid sequence as SEQ ID NO 1 antigen peptide immune mice, separate immune serum preparation; and a fluorescent secondary antibody: FITC-labeled goat anti-mouse IgG. The primary antibody is prepared by blood sampling and serum precipitation after a specific PCV3Cap protein immunized mouse, the specificity and the sensitivity are very high, the repeatability is good, the PCV3 virus infection condition can be visually seen under a fluorescence microscope by directly processing pathogenic tissues, adding the primary antibody and then carrying out fluorescence labeling on the secondary antibody, the detection is convenient, and the detection result is accurate.

Description

Fluorescence immunoassay kit for detecting PCV3

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.

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 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).

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; 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 animal individuals.

Disclosure of Invention

The invention aims to provide a fluorescence immunoassay kit for detecting PCV3, 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:

a fluorescence immunoassay kit for detecting PCV3, comprising:

a first antibody: PCV3Cap single factor serum antibody, prepared by separating immune serum from antigen peptide immune mouse whose amino acid sequence is shown in SEQ ID NO. 1;

fluorescent secondary antibody: FITC-labeled goat anti-mouse IgG;

optionally or preferably, the kit further comprises:

fixing liquid: the formaldehyde-free polyurethane adhesive is prepared by mixing paraformaldehyde and PBS, wherein the volume percentage of the paraformaldehyde is 4%;

sealing liquid: normal sheep serum 8% by volume;

washing liquid: PBS.

Optionally or preferably, the kit further comprises a positive control, wherein the positive control is PCV3 virus tissue.

Alternatively or preferably, in the kit, the PBS is composed of 8.0g of NaCl, 0.2g of KCl and Na ₂ HPO ₄ 3.58g、KH ₂ PO ₄ 0.24g of water is added to the mixture until the volume is 1L.

Alternatively or preferably, in the kit, the PCV3Cap single factor serum antibody is prepared by the following method:

(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.

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.

Escherichia 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 chemical formula C9H18O5S, is capable of causing the transcription process of the lactose operon and thus of inducing the expression of the protein corresponding to the gene downstream 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.

FITC: fluorescein isothiocyanate can be combined with goat anti-mouse IgG to form a fluorescent secondary antibody, and has strong green fluorescence in an alkaline solution.

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

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

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

the fluorescence immunoassay kit for detecting PCV3 provided by the invention takes PCV3Cap single factor serum antibody as primary antibody, has good specificity, takes FITC-labeled goat anti-mouse IgG as fluorescent secondary antibody, and observes a tissue specimen incubated by the primary antibody and the secondary antibody by using a fluorescence microscope when detecting PCV3 infected pig tissue, namely, virus display and positioning can be carried out through a fluorescence position, and the fluorescence immunoassay kit has strong specificity, high sensitivity, good repeatability and high detection speed.

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 430072, China;

the preservation number is: CCTCC No. v202208;

preservation time: 3 months and 10 days in 2022.

Drawings

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

FIG. 2 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 specific descriptions of the embodiments. 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. 1.

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 XPS Buffer according to the ratio of 1:1, boiling for 10min, centrifuging to take supernatant, identifying and analyzing the supernatant by 12% SDS-PAGE electrophoresis to obtain target protein of about 27-28 kDa, and purifying the target protein by using a cation affinity purification kit according to the specified operation as shown in figure 2.

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. After 2 weeks, the mice were immunized a second time, 0.2 mL/mouse, subcutaneously in the back 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 embedded and preserved by OCT in laboratory (PCV3 virus nucleic acid load is 3.65 multiplied by 10) ^6.0 Clinical, no morbid symptoms are yet manifested), the method is carried out according to the operating procedure of the frozen section immunofluorescence method:

(1) frozen sections of at least 10 μm thickness were air dried for 2 minutes and stored at-20 ℃;

(2) taking out the slices from-20 ℃, then placing the slices in a fume hood for 10 minutes to remove water vapor, and fixing the slices by paraformaldehyde with the volume concentration of 4% for 15 minutes;

(3) washing the slide with 1 × PBS for 3 times, and keeping the slide wet;

(4) 8% normal sheep serum was incubated for 30 minutes at room temperature;

(5) PCV3 single factor serum antibody (control replaced PBS) was incubated overnight at 4 ℃;

(6) washing with 1 × PBS for 3 times, 2 min/time;

(7) FITC-labeled goat anti-mouse IgG secondary antibody is incubated for 1 hour at 37 ℃;

(8) washing with 1 × PBS for 3 times, 2 min/time;

(9) sealing the anti-quenching agent;

(10) dried and stored at 4 ℃.

The PCV3 single-factor serum antibody serum in the step (5) is diluted by 1 XPBS according to the ratio of 1:50, 1:100, 1:200 and 1:500 respectively in four dilutions, and the experimental results are observed under a microscope, and the identification results are shown in the following steps: the effect is better when the dilution is 200 degrees, and the cytoplasm of the positive porcine inguinal lymph node tissue cells is dyed into green fluorescence; negative porcine tissue cells are not stained with green fluorescence; PBS was used as a control instead of primary antibody.

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

Example 2 preparation of fluorescent immunoassay kit and verification of sensitivity, specificity and repeatability thereof

1. PCV3 fluorescence immunoassay kit construction

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

TABLE 1 immunofluorescence assay kit for porcine circovirus 3

Preferably, the kit further comprises an anti-quencher, which is commercially available directly and is used for reducing photobleaching of the test sample to improve the photostability of the fluorescent dye in the fixed cell sample.

2. Sensitivity, specificity and repeatability tests of the kit

(1) IFA and PCR comparative detection

IFA (immunofluorescence assay, kit with components in table 1) and PCR (polymerase chain reaction) methods are respectively applied to carry out comparative detection on 134 tissue samples which are clinically diagnosed to be infected by PCV3 and are sent to pig farms in North river, Liaoning, Henan, Shandong, Sichuan and the like. The results of IFA 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

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

(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, and the PCR method has more positive rates than the IFA method, wherein the PCR positive detection rate of the Hebei sample is 2 more than the IFA, the PCR positive detection rate of the Liaoning sample is 3 more than the IFA, and the PCR positive detection rate of the Henan sample is 1 more than the IFA.

And performing repeated detection twice on the samples of Hebei, Liaoning and Henan, wherein the second and third detection results of the IFA method are the same as the first result of the IFA. The second and third detection results of the PCR method are consistent with the detection result of the IFA and different from the first detection result 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 repeated measurements are shown in Table 3 below.

Table 3 comparison of the results of the repeatability tests of the IFA and PCR methods on 134 samples

(3) Crossover experiment

Randomly drawing a batch of PCV3 fluorescence immunoassay kit, and detecting porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine encephalitis B virus, classical swine fever virus and porcine pseudorabies virus on some pathological tissue samples stored in a laboratory, wherein the result is negative result without green fluorescence when observed under a microscope. Further indicates that the porcine circovirus 3 immunohistochemical detection kit does not have false negative and has good specificity.

In conclusion, the PCV3 fluorescence immunoassay 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.

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

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<120> a fluorescence immunoassay kit for detecting PCV3

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Claims (5)

1. A fluoroimmunoassay kit for detecting PCV3, comprising:

a first antibody: PCV3Cap single factor serum antibody, prepared by separating immune serum from antigen peptide immune mouse whose amino acid sequence is shown in SEQ ID NO. 1;

fluorescent secondary antibody: FITC-labeled goat anti-mouse IgG.

2. The kit of claim 1, further comprising:

fixing liquid: the formaldehyde-free polyurethane adhesive is prepared by mixing paraformaldehyde and PBS, wherein the volume percentage of the paraformaldehyde is 4%;

sealing liquid: normal sheep serum 8% by volume;

washing liquid: PBS.

3. The kit of claim 1, further comprising a positive control, wherein the positive control is PCV3 viral tissue.

4. The kit of claim 1, wherein the PBS is composed of 8.0g NaCl, 0.2g KCl Na ₂ HPO ₄ 3.58g、KH ₂ PO ₄ 0.24g of water is added to the mixture until the volume is 1L.

5. The kit according to claim 1, wherein the PCV3Cap single factor serum antibody is prepared by the following method:

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

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