CN115947794A - PCV2ORF3 specific polypeptide and application thereof - Google Patents
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Abstract
The invention discloses a PCV2ORF3 specific polypeptide and application thereof, relating to the technical field of biology. The invention discloses a PCV2ORF3 specific polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1. The invention also discloses application of the specific polypeptide in preparing an ELISA kit for detecting PCV2 antibody. According to the invention, the ORF3 protein is selected as a target antigen, the epitope of the antigen is predicted and analyzed by a molecular biology method, and the specific polypeptide is used as a coating antigen to establish an ELISA method for detecting the PCV2 antibody, so that the method has good repeatability, sensitivity and specificity, PCV2 wild virus infected pigs can be identified from PCV2 subunit vaccine immune pig groups, and a powerful technical support is provided for PCV2 purification in a pig farm.
Description
Technical Field
The invention relates to the technical field of biology, in particular to PCV2ORF3 specific polypeptide and application thereof.
Background
Porcine circovirus type 2 (PCV 2) is associated with many disease syndromes in pigs, the most common of which is postweaning multisystemic wasting syndrome, which is collectively referred to as porcine circovirus-associated disease (PCVAD), which is a major hazard to the swine industry (Jeong, et al., 2018). PCV2 propagates both horizontally and vertically (Dvorak, et al, 2013 eddicks, et al, 2019), and therefore rapidly. Since the discovery in 1998, it has become one of the most important viral diseases in the swine industry (Zhai, et al, 2014). Serological positive rates of 20-80% and incidence rates of 60% with 3-10% lethality in endemic pig farms were investigated (Afolabi, et al, 2017).
After PCV2 infects pig bodies, the progressive accumulation of virus occurs mainly in follicular dendritic cells, histiocytes and macrophages of pig body lymphoid tissues and other parts, which causes the significant apoptosis and defect of lymphocytes, and a large amount of typical virus inclusion bodies are distributed in macrophages and multinucleated cytoblast cytoplasm, thus seriously damaging the immune system of pigs, causing low immune function and weak body resistance, and easily inducing other diseases (Richmond, et al, 2015, du Q, et al, 2018. It has been found to be an important cause of diseases such as blueear virus, parvovirus, pseudorabies virus, eperythrozoon, etc. (Fan, et al, 2013, du q, et al, 2018). Therefore, it is important to control the infection of circovirus type 2.
In addition to taking comprehensive control measures, vaccination has become the primary measure for the prevention and control of Porcine circovirus type 2 (PCV-2) infection in severe viral-contaminated pig farms. The PCV2 vaccines commonly used at present are subunit vaccines and whole virus inactivated vaccines. Because the production cost of the whole virus inactivated vaccine is high, a genetic engineering vaccine (Blanchard, et al, 2003) based on the Cap protein encoded by ORF2 becomes the most widely used vaccine at present. However, recent studies found that vaccination may affect the PCV2 genotype circulating in the herd, PCV2d is often found in vaccinated herds after 2010 (Hennig-Pauka, 2019). PCV2d has now become the most prevalent strain and even in vaccinated herds, the evolutionary advantage of this genotype being able to replicate in vaccinated pigs cannot be ruled out (Xiao, et al, 2016). Therefore, in an immune pig farm, the evaluation and detection of PCV2 infection are important for the prevention and control of the disease. Although PCR can be used for detection (Lyoo, et al, 2008), the operation is complex and difficult to perform in batches, and a method for detecting wild-infected pigs in batches in immune pig herds is needed in actual production, and the ELISA method is very suitable for detection.
PCV2 belongs to the genus circovirus of the family circoviridae and is a single stranded circular DNA virus (Ren, et al, 2016). The PCV2 genome has 3 major Open Reading Frames (ORFs): ORF1, ORF2 and ORF3.ORF1 encodes the Rep protein, essential for viral DNA replication (Cheung, 2004), which has high homology with PCV 1; ORF2 encodes a structural capsid protein (Cap protein), which is the major antigenic material that elicits the host immune response, and is also important for viral replication (Lv, et al, 2014); the protein encoded by ORF3, now called ORF3 protein, is involved in the pathogenic process of PCV2 by inducing apoptosis of host cells (Liu, et al, 2006, 2007), being the main pathogenic protein.
Disclosure of Invention
The invention aims to provide a PCV2ORF3 specific polypeptide and application thereof, so as to solve the problems in the prior art, the invention selects ORF3 protein as a target antigen, predicts and analyzes the epitope of the target antigen by a molecular biological method, establishes an ELISA detection method for detecting PCV2 antibody by using the specific polypeptide as a coating antigen, can identify PCV2 wild virus infected pigs from PCV2 subunit vaccine immune pig groups, and provides powerful technical support for PCV2 purification in pig farms.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a PCV2ORF3 specific polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.
The invention also provides application of the specific polypeptide in preparation of an ELISA kit for detecting PCV2 antibody.
The invention also provides an ELISA kit for detecting PCV2 antibody, which comprises an ELISA plate coated with the specific polypeptide.
Further, the coating concentration of the specific polypeptide is 10 mug/mL.
Further, the ELISA kit also comprises standard negative and positive serum, rabbit anti-pig IgG marked by HRP, a washing solution, a developing solution and a stop solution.
Further, the washing solution is a TBST solution.
Further, the color developing solution is a TMB solution.
Further, the stop solution is a sulfuric acid solution.
The invention discloses the following technical effects:
the invention selects ORF3 protein as target antigen, predictively analyzes the epitope by a molecular biology method, establishes an ELISA detection method for detecting PCV2 antibody by using the specific polypeptide as coating antigen, has good repeatability, sensitivity and specificity, can identify PCV2 wild virus infected pigs from PCV2 subunit vaccine immune pig groups, and provides powerful technical support for PCV2 purification in pig farms.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the results of Dotblot detection of a specific PCV2 polypeptide sequence of the present invention; wherein A is the detection result of a positive serum sample, and B is the detection result of a negative serum sample;
FIG. 2 is a ROC curve of the kit of the present invention, based on a detection sample of the PCV2 antibody detection kit of Bionote;
FIG. 3 shows the detection of positive and negative samples by the kit of the present invention, based on the detection sample of the PCV2 antibody detection kit of Bionote.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Discovery of specific polypeptides: the protein sequences of ORF3 of PCV1 (GenBank No. AY660574.1) and PCV2 (GenBank No. KU 981062.1) were downloaded from NCBI, and the differences of the epitopes thereof were compared by analyzing using the online analysis program Bepipred 2.0, and a specific PCV2 polypeptide sequence VCKISSPFAFTTPRWPHNDV (SEQ ID No. 1) was found.
Dot blot validation of specific polypeptides: to determine the reactivity of the ORF3 polypeptide and PCV2 positive sera, 2. Mu.L of 10. Mu.g/mL polypeptide solution (SEQ ID NO. 1) was spotted onto Nitrocellulose (NC) membranes, dried and then blocked in 1% (W/V) BSA-containing PBS (pH 7.4) at 37 ℃ for 1 hour, washed and then incubated with 1. The results showed that PCV2 positive sera showed clear immunospot (fig. 1A) while negative sera showed no pot (fig. 1B).
Example 2
Establishment of a polypeptide ELISA antibody detection method:
using 10 mu g/mL polypeptide to coat an ELISA plate, sequentially incubating serum to be detected and rabbit anti-pig IgG marked by HRP for 30 minutes, washing with 300 mu L TBST solution after each incubation step, washing for 5 times in each step, then developing for 15 minutes in a developing solution (100 mu LTMB solution) in a dark place, adding 50 mu L10 wt% sulfuric acid solution to stop reaction, and finally reading OD (origin-to-destination) with an ELISA reader 450 The results were evaluated and the threshold for evaluation was 0.264 (by ROC analysis, it was found that, at 0.264, the ELISA method and ELISA kit of the present applicationCommercial kits have the highest identity, so this value was chosen as the threshold value) when OD was used 450 If the value is more than 0.264, the result is judged to be positive, otherwise, the result is negative.
Wherein the polypeptide is coated overnight at 4 ℃ and then blocked with 0.5% (W/V) BSA for 1 hour; the standard negative and positive serum is diluted 2000 times, and the horse radish peroxidase labeled rabbit anti-pig IgG antibody is diluted 5000 times.
The above-described polypeptide ELISA antibody detection method had a sensitivity of 95.4% (83/87) (95% ci of 88.64% to 98.73%) and a specificity of 100%% (6/6) (95% ci of 54.07% to 100%) compared to a commercial PCV2 antibody ELISA kit (purchased from Bionote corporation, korea), and an area under the ROC curve of 0.9789, which had a very high degree of agreement (fig. 2-3).
Specific experiments:
according to the ELISA conditions established, standard positive sera of swine fever, porcine reproductive and respiratory syndrome, porcine foot and mouth disease, porcine pseudorabies, porcine parvovirus disease and porcine epidemic encephalitis B are respectively used as primary antibodies for ELISA detection, the results are shown in Table 1, the OD values of the detection are all less than 0.264 and are negative, the detection shows that the antibodies do not react with the coating polypeptide, and the kit established by the invention has good specificity.
TABLE 1 results of specificity experiments
Example 3 detection of PCV 2-infected pigs in an Immunity pig farm
(1) In a pig farm for PCV2 subunit vaccine immunization, blood of pigs is collected and serum is centrifugally separated.
(2) 93 serum samples are detected by the method established in the example 2, 5 replicates are set for each sample, PCV2 negative serum is used as a control, and the result shows that the CV value is 9.7%, which indicates that the ELISA method established in the example 2 has good repeatability. The method can identify PCV2 wild virus infected pigs from PCV2 subunit vaccine immune swinery, and provides powerful support for PCV2 purification in a pig farm.
Example 4 detection of PCV2 infected pigs in non-immune pig farm
(1) In a pig farm not immunized by the PCV2 subunit vaccine, blood of pigs is collected and serum is separated by centrifugation.
(2) 93 serum samples are detected by the method established in the example 2, 5 replicates are set for each sample, PCV2 negative serum is used as a control, and the result shows that the CV value is 9.2%, which indicates that the ELISA method established in the example 2 has good repeatability.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (8)
1. A PCV2ORF3 specific polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. Use of a specific polypeptide according to claim 1 for the preparation of an ELISA kit for the detection of PCV2 antibodies.
3. An ELISA kit for detecting PCV2 antibody, which is characterized by comprising an ELISA plate coated with the specific polypeptide of claim 1.
4. The ELISA kit of claim 3 wherein the specific polypeptide is coated at a concentration of 10 μ g/mL.
5. The ELISA kit of claim 3 further comprising a standard negative and positive serum, an HRP-labeled rabbit anti-porcine IgG, a wash solution, a color developing solution, and a stop solution.
6. The ELISA kit of claim 5, wherein the wash solution is a TBST solution.
7. The ELISA kit of claim 5 wherein the color-developing solution is a TMB solution.
8. The ELISA kit of claim 5 wherein the stop solution is a sulfuric acid solution.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101112621A (en) * | 2006-07-26 | 2008-01-30 | 金宁一 | PRRSV and PCV-2bivalent recombinant fowl pox virus disease live vector vaccines |
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