CN114814211A - ELISA kit for detecting PCV3 - Google Patents

Abstract

The invention discloses an ELISA kit for detecting PCV3, which comprises an ELISA plate coated by PCV3Cap protein antigen peptide, wherein the amino acid sequence of the PCV3Cap protein antigen peptide is shown as SEQ ID NO: 1. The kit also comprises positive control serum, negative control serum, sample diluent, enzyme-labeled secondary antibody, washing liquid, substrate solution A, substrate solution B and stop solution. The kit is constructed on the basis of an ELISA plate coated by PCV3Cap protein expressed and purified by genetic engineering escherichia coli, can detect an antibody in clinical PCV3 infected pig serum, judges whether the pig serum is infected with PCV3, can be used for evaluating the immune efficacy of PCV3 vaccine, and has the advantages of strong specificity, high sensitivity, good repeatability and high detection speed.

Description

ELISA kit for detecting PCV3

Technical Field

The invention relates to the technical field of livestock pathogenic virus diagnosis, in particular to an ELISA kit for detecting PCV 3.

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 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 high concern to the swine industry in countries worldwide.

The PCV3 has the highest accuracy in the current detection method by using a PCR method, but the PCR method is tedious to detect, has higher technical requirements on operators and high requirements on instruments and equipment, and brings inconvenience to popularization and application.

Disclosure of Invention

The invention aims to provide an ELISA kit capable of conveniently and rapidly detecting PCV3, which has very high detection sensitivity and specificity and accurate detection result.

The technical scheme of the invention is detailed as follows:

an ELISA kit for detecting PCV3 comprises an ELISA plate coated by PCV3Cap protein antigen peptide, wherein the amino acid sequence of the PCV3Cap protein antigen peptide is shown as SEQ ID NO: 1.

Alternatively or preferably, in the kit, the nucleotide sequence of the gene encoding the antigenic peptide of the PCV3Cap protein is shown in SEQ ID NO. 2.

Alternatively or preferably, in the kit, the PCV3Cap protein antigenic peptide is prepared by the following method:

(1) extracting DNA from tissue virus liquid of PCV3 LY strain of porcine circovirus 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, wherein the nucleotide sequence is shown in SEQ ID NO. 2;

(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 a final concentration of 1.0mmol/L, and carrying out induction 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) and purifying the target protein by using a His label purification kit to obtain the PCV3Cap protein antigen peptide.

Alternatively or preferably, in the kit, the PCV3Cap protein antigen peptide is coated as follows: PCV3Cap protein antigen peptide is diluted to 12.5 mu g/mL by 0.05mol/L PBS (pH9.5), added into a coated plate, 100 mu L/hole, placed at 4 ℃ overnight, washed for 3 times by PBS, added with blocking liquid for sealing overnight, the next day, discarded, naturally dried for 48 hours under the conditions of humidity of 40 percent and room temperature, and the coating is completed.

Optionally or preferably, the kit further comprises:

positive control serum: preparing inactivated vaccine by PCV3 LY strain, collecting blood after immunizing SPF pig to obtain serum, and adding PBS according to the proportion of 1: diluting by 200 times;

negative control serum: serum obtained by blood collection of SPF pigs;

sample diluent: a sheep serum solution with a volume concentration of 3% diluted with PBS;

enzyme-labeled secondary antibody: diluting 10000 times of enzyme-labeled antibody diluent to prepare horse radish peroxidase-labeled goat anti-pig antibody;

washing liquid: PBS;

substrate solution a: the preparation is prepared by adding distilled water to 500mL according to 1.6g of citric acid, 13.6g of sodium acetate and 0.3mL of 30 volume percent hydrogen peroxide;

substrate solution B: dissolving 0.2g of disodium ethylene diamine tetraacetate (EDTA-2Na), 0.95g of citric acid and 0.15g of TMB in 50mL of DMSO (dimethyl sulfoxide), and adding distilled water to a constant volume of 500mL to obtain the product;

stopping liquid: 2mol/L sulfuric acid solution.

Alternatively or preferably, in the kit, the PBS is added according to 8.0g of NaCl, 0.2g of KCl and Na ₂ HPO ₄ 3.58g、KH ₂ PO ₄ 0.24g、NaN ₃ 1.0g and 0.5ml Triton-100, adjusting pH to 7.2, and diluting to 1L. Adding NaN into PBS ₃ And Triton-100 facilitates preservation and detergency by washing.

Description of the drawings:

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

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.

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

the ELISA kit for detecting PCV3 provided by the invention uses the unique Cap protein antigen peptide as the primary antibody to carry out ELISA plate coating, can detect whether PCV3 infection exists or not by collecting serum on site, and has the advantages of strong specificity, high sensitivity, good repeatability, high detection speed, and more convenient and accurate detection.

Preservation information:

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

the preservation unit: 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'-GGG AAG CTT TTA GAG AAC GGA CTT GTA ACG-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 87.08-87.85%, the homology with the amino acid sequence of the known PCV3 strain is 90.70-93.49%, and the homology with other PCV (PCV2/PCV4) genome is 43.2-51.5%.

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 novel Cap proteins as antigenic peptides

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 out 1mL culture in 120mL LB culture medium the next day, shaking and culturing for 2.5h at 37 ℃, shaking the bacterial liquid to be in a semitransparent semi-turbid state, measuring OD600 to be 0.6-0.8 (reaching logarithmic growth phase) 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 of 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 2. The objective protein was purified by using a His tag purification kit according to a predetermined procedure. The purity of the purified His-Cap protein is more than 95%, the protein concentration is 1.05mg/mL, and the purified His-Cap protein is Cap protein with a histidine tag (the protein sequence shown in SEQ ID NO:1 is provided with the histidine tag).

3. PCV3Cap protein antigen peptide coated ELISA plate

And (3) diluting the His-Cap protein obtained in the step (2) to 12.5 mu g/mL by using 0.05mol/L PBS (pH9.5), adding the diluted His-Cap protein into a 96-hole ELISA (enzyme-Linked immuno sorbent assay) coated plate, keeping the diluted protein at the temperature of 4 ℃ overnight, washing the plate for 3 times by using the PBS, adding bovine serum albumin sealing liquid with the mass concentration of 1% to seal the plate overnight, discarding the sealing liquid the next day, naturally drying the plate for 48 hours at room temperature and under the humidity of 40%, completing coating, and finally carrying out vacuum packaging.

Example 2ELISA kit preparation and use

1. ELISA kit construction for detecting PVC3

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

TABLE 1 immunohistochemical detection kit for porcine circovirus 3

The kit is stored in a refrigerator at 4 ℃.

2. Kit using method

(1) Taking out the kit from the refrigerator, and returning to room temperature (about 25 ℃);

(2) diluting the serum to be detected by 1:150 times with the sample diluent (for example, taking 1 muL +149 muL sample diluent of the serum to be detected, and mixing uniformly);

(3) add 100. mu.L of undiluted negative control serum to ELISA plates A1 and A2 wells;

(4) add 100. mu.L of undiluted positive control serum to ELISA plates A3 and A4 wells;

(5) add 100 μ Ι _ of sample diluent to ELISA plate a5 and a6 wells as blank background control;

(6) adding 100 mu L of diluted serum sample to be detected into other blank holes of the ELISA plate;

(7) placing the ELISA plate added with the sample at room temperature for incubating for 45 minutes or placing the ELISA plate in a constant-temperature incubator at 37 ℃ for incubating for 30 minutes;

(8) washing: adding PBS into the ELISA plate holes, adding 300 mu L of PBS into each hole, and repeatedly washing for 3-5 times;

(9) adding enzyme-labeled secondary antibody into the coated plate, wherein each well is 100 mu L, and placing the coated plate in a room temperature incubator for 45 minutes or in a constant temperature incubator at 37 ℃ for 30 minutes;

(10) washing: repeating the step 8;

(11) color development: adding 50 mu L of each of the substrate solution A and the substrate solution B, shaking up lightly, and incubating for 10 minutes in a constant-temperature incubator at 37 ℃;

(12) and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction;

(13) reading: placing the ELISA plate on an enzyme-linked immunosorbent assay (ELISA) instrument, and reading the light absorption value under the wavelength of 450 nm;

the calculation method comprises the following steps: negative control mean (a1 wells + a2 wells)/2; the positive control mean value (A3 wells + a4 wells)/2.

Positive judgment standard of sample to be detected: when the standard positive control serum OD is judged ₄₅₀ > 1.0, negative control serum OD ₄₅₀ < 0.2, blank background control OD ₄₅₀ If the number is less than 0.05, the experiment is established, otherwise, the experiment is invalid.

Calculating the formula: S/P value (OD of sample to be detected) ₄₅₀ Positive control mean OD ₄₅₀ And judging the standard: S/P is more than or equal to 0.3 and is positive; S/P < 0.3 is negative.

Example 3 kit sensitivity, specificity, reproducibility verification

(1) Experiment of specificity

A batch of ELISA kits for detecting PVC3 are randomly extracted, positive serum samples of porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine encephalitis B virus, classical swine fever virus, porcine pseudorabies virus, porcine circovirus type 2, porcine circovirus type 3 and the like stored in a laboratory are detected, except that the positive serum sample of PCV3 is positive, other detection results are negative results, and the kit for diagnosing the porcine circovirus type 3 antibody, which is detected by the invention, can not generate false negative and has good specificity.

(2) ELISA and Agar Diffusion Test (ADT) sensitivity and repeatability comparison experiment

187 serum samples of suspected PCV3 infected pigs clinically inspected in pig farms such as North Hei, Liaoning, Henan, Shandong and Sichuan were subjected to comparative detection by ELISA (kit of Table 1) and ADT methods, respectively. The results of ELISA and ADT detection of serum 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

The results of comparative detection of 187 clinical serum samples by using an ELISA detection kit and an ADT method for porcine circovirus type 3 antibodies show that the positive sera detected by the ADT are consistent with those detected by the ELISA, but the positive numbers of the ELISA detected sera are 24 more than those detected by the ADT, namely 7 in Hebei, 8 in Liaoning, 3 in Henan, 4 in Shandong and 2 in Sichuan.

Meanwhile, 24 serum samples with more positive ELISA detection results are repeatedly detected twice, the ELISA detection results are the same as the first detection results, and the second and third ADT detection results are also consistent with the first detection results, so that the repeatability of the ELISA detection results and the ADT detection results is good, but the ELISA detection results are more sensitive, and the repeated detection results are shown in the following table 3.

Table 3 comparison of the results of the repeated detection of 24 samples by ELISA and ADT methods

In conclusion, the 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: compared with the ADT detection result, the ELISA kit has more sensitive detection result, does not react with other swine pathogen positive serum, and has better specificity.

The clinical application effect is better: the kit is used for detecting 187 clinical pig serum samples initiated by suspected PCV3, an ADT method is used for verification, 97 serum PCV3 antibody positivity is detected in the result, the detection result of the kit is consistent with the detection results of the second PCR method and the third PCR method, and the sensitivity, the specificity and the repeatability of the detection result of the kit are proved to be high, and the clinical application effect is good.

Example 4 ELISA kit for detecting PVC3 an evaluation test for the immunopotency of inactivated vaccine against PCV3 LY strain

1. ELISA antibody level detection before PCV3 LY inactivated vaccine immunization

Randomly selecting 10 SPF pigs (porcine circovirus type 3, porcine circovirus type 2, classical swine fever virus, porcine pseudorabies virus, porcine reproductive failure and respiratory syndrome virus, porcine parvovirus, porcine encephalitis B virus antigen and antibody detection are all negative) with the age of 4-6 weeks, wherein the porcine circovirus type 3 antibody is detected by adopting the kit disclosed by the invention, and the detection results are all negative and are detailed in the following table 4.

2. ELISA antibody level detection after PCV3 LY inactivated vaccine immunization

10 SPF pigs were randomly divided into 2 groups of 5 pigs each. One of the 1 groups was immunized with PCV3 vaccine and the remaining 1 group was not immunized as a blank control. Blood is collected from all test pigs 28 days after immunization, serum is separated to detect the antibody level, the result shows that the antibody level is increased after the vaccine immunization, and the antibody of the non-immune control group is still negative, which indicates that the PCV3 ELISA antibody detection kit can be used for PCV3 vaccine immunity efficacy evaluation and is detailed in Table 4.

Table 4, PCV3 antibody ELISA detection kit for PCV3 LY vaccine immunity effectiveness application evaluation test results

Note: S/P is more than or equal to 0.3 and is positive; S/P < 0.3 is negative.

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> an ELISA kit for detecting PCV3

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Claims (6)

1. An ELISA kit for detecting PCV3 is characterized by comprising an ELISA plate coated by PCV3Cap protein antigen peptide, wherein the amino acid sequence of the PCV3Cap protein antigen peptide is shown as SEQ ID NO: 1.

2. The kit according to claim 1, wherein the nucleotide sequence of the gene encoding the antigenic peptide of the PCV3Cap protein is shown as SEQ ID NO. 2.

3. The kit according to claim 2, wherein the PCV3Cap protein antigenic peptide is prepared by the following method:

(1) extracting DNA from tissue virus liquid of PCV3 LY strain of porcine circovirus 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, wherein the nucleotide sequence is shown in SEQ ID NO. 2;

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

4. The kit according to claim 1, wherein the PCV3Cap protein antigen peptide is coated by the following method: PCV3Cap protein antigen peptide is diluted to 12.5 mu g/mL by 0.05mol/L PBS (pH9.5), added into a coated plate, 100 mu L/hole, placed at 4 ℃ overnight, washed for 3 times by PBS, added with blocking liquid for sealing overnight, the next day, discarded, naturally dried for 48 hours under the conditions of humidity of 40 percent and room temperature, and the coating is completed.

5. The kit according to any one of claims 1 to 4, further comprising:

positive control serum: preparing inactivated vaccine by PCV3 LY strain, collecting blood after immunizing SPF pig to obtain serum, and adding PBS according to the proportion of 1: diluting by 200 times;

negative control serum: serum obtained by blood collection of SPF pigs;

sample diluent: a sheep serum solution with a volume concentration of 3% diluted with PBS;

enzyme-labeled secondary antibody: diluting 10000 times of enzyme-labeled antibody diluent to prepare horse radish peroxidase-labeled goat anti-pig antibody;

washing liquid: PBS;

substrate solution a: the preparation is prepared by adding distilled water to 500mL according to 1.6g of citric acid, 13.6g of sodium acetate and 0.3mL of 30 volume percent hydrogen peroxide;

substrate solution B: 0.2g of ethylene diamine tetraacetic acid disodium, 0.95g of citric acid and 0.15g of TMB, dissolving in 50mL of DMSO (dimethyl sulfoxide), and adding distilled water to a constant volume of 500 mL;

stopping liquid: 2mol/L sulfuric acid solution.

6. The kit of claim 5, wherein the PBS is NaCl 8.0g, KCl 0.2g, Na ₂ HPO ₄ 3.58g、KH ₂ PO ₄ 0.24g、NaN ₃ 1.0g and 0.5ml Triton-100, adjusting pH to 7.2, and diluting to 1L.

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