Porcine circovirus type 2 antibody detection kit

Abstract

The present invention relates to a porcine circovirus type 2 antibody detection kit. The porcine circovirus type 2 antibody detection kit of the present invention comprises an antibody detection chip, wherein the antibody detection chip comprises a solid phase carrier and polypeptides represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively immobilized on the solid phase carrier. The porcine circovirus type 2 antibody detection kit of the present invention can be used to distinguish pigs immunized with porcine circovirus type 2 inactivated vaccine from pigs infected with porcine circovirus type 2 wild-type virus.

Description

Technical Field

[0001] The present invention relates generally to the field of biological detection technology. Specifically, the present invention relates to a porcine circovirus type 2 antibody detection kit, which is used to distinguish pigs immunized with porcine circovirus type 2 inactivated vaccine from pigs infected with porcine circovirus type 2 wild virus. Background Art

[0002] Porcine circovirus disease is a serious infectious disease of pigs caused by porcine circovirus (PCV).

[0003] Porcine circovirus (PCV) is the smallest animal virus discovered to date. Its genome consists of a single-stranded, negative-strand circular DNA molecule. Two serotypes are known: PCV1 and PCV2. PCV1 is non-pathogenic, while PCV2 is pathogenic. PCV2 infection of pigs can cause serious diseases such as post-weaning multisystemic wasting syndrome (PMWS) in piglets, reproductive failure in sows, respiratory disease in fattening pigs, porcine dermatitis and nephrotic syndrome, and congenital tremor in pigs.

[0004] Currently, my country primarily uses various types of PCV vaccines (primarily inactivated PCV2 vaccines, PCV2 virus-like particle vaccines, and PCV2 Cap protein subunit vaccines) to prevent and control porcine circovirus disease, i.e., immune-free (no outbreaks under vaccination conditions). However, immune-free is only the primary stage of animal disease prevention and control, while the advanced stage of animal disease prevention and control is non-immune-free (no outbreaks under non-vaccination conditions). Achieving non-immune-free requires the absence of pathogenic pathogens within a designated area (including host animals and the environment), which can be achieved through animal disease purification.

[0005] The so-called animal disease purification refers to the planned elimination of animals carrying a certain animal disease pathogen in a certain area, thereby eliminating the animal disease pathogen and achieving a state in which individual animals in the area are disease-free and infection-free.

[0006] A key step in animal disease purification is to distinguish wild-type virus-infected individuals from the immunized population through DIVA (Differentiating Infected from Vaccinated Animals) and kill them, thereby eliminating the pathogenic pathogens from the population.

[0007] Generally, DIVA can be performed through antibody testing. However, for porcine circovirus, an animal disease that is prevented and controlled by inactivated vaccines, the structural differences between inactivated vaccines and wild-type live viruses are small. Although theoretically there will be differences in the antibodies they induce in animals, these differences are difficult to detect using traditional biological probe molecules (such as the Cap protein of PCV2).

[0008] Therefore, how to develop an antibody detection kit for DIVA of porcine circovirus disease (PCV2) that can also be used for immunization with inactivated vaccines is a difficult problem faced in the purification of porcine circovirus disease. Summary of the Invention

[0009] An epitope is a region of a protein antigen that binds to an antibody. Multiple epitopes may exist on a single protein antigen. Epitope peptides are polypeptides containing epitopes and can be synthesized artificially. Epitope peptides can also be used to detect antibodies that bind to them.

[0010] After in-depth research, the inventors found a group of antigenic epitope peptides from the Capsid Protein of Porcine Circovirus Type 2 (PCV2). These peptides do not respond to biological samples of pigs immunized with the inactivated PCV2 vaccine, but respond to biological samples of pigs infected with wild-type PCV2 (including biological samples of pigs infected with wild-type PCV2 after immunization with the inactivated PCV2 vaccine). Therefore, they can be used for DIVA in pig herds immunized with the inactivated PCV2 vaccine.

[0011] That is, the present invention includes:

[0012] 1. A porcine circovirus type 2 antibody detection kit, comprising an antibody detection chip,

[0013] The antibody detection chip comprises a solid phase carrier, and polypeptides shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, which are fixed on the solid phase carrier.

[0014] SEQ ID NO: 1 (ORF2-8, MMRFNINDFLPPGGGSNPRS);

[0015] SEQ ID NO: 2 (ORF2-14, TKATALTYDPYVNYSSRHTI);

[0016] SEQ ID NO: 3(ORF2-17, FTPKPVLDSTIDYFQPNNKR);

[0017] SEQ ID NO: 4 (ORF2-20, GNVDHVGLGTAFENSIYDQE);

[0018] SEQ ID NO: 5 (ORF2-23, FREFNLKDPPLNP).

[0019] 2. The porcine circovirus type 2 antibody detection kit according to item 1, which is used to detect porcine circovirus type 2 antibodies in a biological sample derived from pigs. Here, the biological sample only needs to contain porcine circovirus type 2 antibodies, and can be, for example, whole blood or serum; serum is generally used.

[0020] 3. The porcine circovirus type 2 antibody detection kit according to item 1 is used to distinguish pigs immunized with porcine circovirus type 2 inactivated vaccine and pigs infected with porcine circovirus type 2 wild virus.

[0021] 4. The porcine circovirus type 2 antibody detection kit according to item 1 is used to distinguish pigs infected with porcine circovirus type 2 wild virus from pigs immunized with porcine circovirus type 2 inactivated vaccine. Here, the porcine circovirus type 2 wild virus-infected pigs include pigs that failed to be immunized with porcine circovirus type 2 inactivated vaccine and were infected with porcine circovirus type 2 wild virus, and also include pigs that were successfully immunized with porcine circovirus type 2 inactivated vaccine but were still infected with porcine circovirus type 2 wild virus.

[0022] 5. The porcine circovirus type 2 antibody detection kit according to item 1, wherein the porcine circovirus type 2 Cap protein is also immobilized on the solid phase carrier.

[0023] 6. Use of antibody detection chip in the preparation of porcine circovirus type 2 antibody detection kit,

[0024] The antibody detection chip comprises a solid phase carrier, and polypeptides shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, which are fixed on the solid phase carrier.

[0025] 7. The use according to item 6, wherein the porcine circovirus type 2 antibody detection kit is used to detect porcine circovirus type 2 antibodies in biological samples of pig origin.

[0026] 8. The use according to item 6, wherein the porcine circovirus type 2 antibody detection kit is used to distinguish pigs immunized with porcine circovirus type 2 inactivated vaccine and pigs infected with porcine circovirus type 2 wild virus.

[0027] 9. The use according to item 6, wherein the porcine circovirus type 2 antibody detection kit is used to distinguish pigs infected with wild porcine circovirus type 2 from a pig herd immunized with porcine circovirus type 2 inactivated vaccine.

[0028] 10. The use according to item 6, wherein the Cap protein of porcine circovirus type 2 is also immobilized on the solid phase carrier. DETAILED DESCRIPTION

[0029] The present invention is described in detail below. Although specific embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0030] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. For example, "including" or "comprising" mentioned throughout the specification and claims are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the present invention shall be as defined in the attached claims.

[0031] The full-length PCV2 Cap protein is currently the most commonly used antigen for detecting PCV2 antibodies. Existing studies have shown that it responds to antibodies elicited by both wild-type PCV2 infection and immunization with various PCV2 vaccines, with essentially identical response patterns. Therefore, the full-length PCV2 Cap protein cannot be used for PCV2 DIVA.

[0032] After extensive research, the inventors discovered that a group of epitope peptides derived from the PCV2 Cap protein exhibit distinct response patterns to antibodies elicited by wild-type PCV2 infection and antibodies induced by inactivated PCV2 vaccines. Specifically, this group of epitope peptides only responds to antibodies elicited by wild-type PCV2 infection, but not by antibodies induced by inactivated PCV2 vaccines. Therefore, this group of epitope peptides can be used as a tool to distinguish between wild-type PCV2 infection and inactivated PCV2 vaccine immunization.

[0033] In this specification, "response" means that the SNR is greater than or equal to 2, and the SNR (signal-to-noise ratio) = (antigen spot signal value - negative control spot signal value) / negative control spot signal value.

[0034] Example 1. Preparation and confirmation of polypeptide

[0035] The polypeptides of SEQ ID NOs: 1 to 5 were synthesized by GenScript and confirmed by mass spectrometry.

[0036] SEQ ID NO: 1 (ORF2-8, MMRFNINDFLPPGGGSNPRS);

[0037] SEQ ID NO: 2 (ORF2-14, TKATALTYDPYVNYSSRHTI);

[0038] SEQ ID NO: 3(ORF2-17, FTPKPVLDSTIDYFQPNNKR);

[0039] SEQ ID NO: 4 (ORF2-20, GNVDHVGLGTAFENSIYDQE);

[0040] SEQ ID NO: 5 (ORF2-23, FREFNLKDPPLNP)

[0041] Example 2. Preparation of antibody detection chip (kit)

[0042] An antibody detection chip was prepared by spotting solutions of the polypeptides shown in SEQ ID NOs: 1 to 5 on a conventional ELISA solid phase carrier using conventional methods, and also spotting a pig IgG spot as a positive quality control spot and a PB buffer spot as a negative quality control spot.

[0043] Accordingly, a porcine circovirus type 2 antibody detection kit was prepared, which, in addition to the antibody detection chip, also contained concentrated cleaning solution, sample diluent, enzyme-labeled antibody solution, luminescent substrate solution and other reagents for antibody detection.

[0044] Example 3. Antibody detection using a kit

[0045] Detection steps

[0046] (1) Dilute 20× concentrated cleaning solution (TBST: 0.4 M Tris-HCl, 2.74 M NaCl, 2% Tween 20, pH 7.2 ± 0.2) at a ratio of 1:20 with purified water to prepare the cleaning solution. To completely wet the detection chip surface, pipette approximately 200 μL of the cleaning solution onto the detection chip surface and allow the chip to soak for a specified period of time.

[0047] (2) Dilute the serum sample to be tested at 1:100 with sample diluent (0.05M PBS, 1% BSA, 0.2% PVP, 0.5% Tween 20, pH 7.2±0.2).

[0048] (3) For the detection chip with the cleaning solution discarded, when the surface is completely wet, 200 μL of the diluted serum sample is drawn and added to the detection chip.

[0049] (4) Incubate the detection chip in a constant temperature shaker at 500 rpm for 30 minutes at 37 degrees Celsius.

[0050] (5) Discard the serum sample and clean the surface of the detection chip with cleaning solution.

[0051] (6) After washing, 100 μL of enzyme-labeled antibody solution (rabbit anti-swine IgG-HRP, Sigma) was added to the detection chip and incubated in a constant temperature shaker at 500 rpm for 30 min at 37 °C.

[0052] (7) Discard the enzyme-labeled antibody solution and clean the surface of the detection chip with cleaning solution.

[0053] (8) After washing, 20 μL of luminescent substrate solution (Thermo, Prod#37074) was evenly spread on the surface of the detection chip.

[0054] (9) Use a gel imager to perform chemiluminescence imaging on the detection chip and determine the results.

[0055] (10) Result determination:

[0056] For each serum, when the SNR of the antigen epitope polypeptide spot on the antibody detection chip is greater than or equal to 2, it is considered a "response"; otherwise, it is considered no response.

[0057] When two or more of the antigenic epitope polypeptide sites of SEQ ID NOs: 1 to 5 respond, it is determined that PCV2 antibodies are detected; otherwise, it is determined that PCV2 antibodies are not detected.

[0058] Example 4. Antibody detection results

[0059] Twenty pigs that were determined to be PCV2 antibody negative by other methods were evenly divided into a challenge group and a control group. Both groups were immunized with an inactivated PCV2 vaccine. On the 21st day after immunization, both groups were determined to be PCV2 antibody positive by other methods. However, no PCV2 antibodies were detected in either group using the above-mentioned porcine circovirus type 2 antibody detection kit.

[0060] After completing the above tests, the challenge group was challenged with wild-type PCV2 virus on day 21 after immunization. Antibody testing was then performed every two days using the porcine circovirus type 2 antibody detection kit. PCV2 antibodies were detected in all participants on day 14 after challenge. Clinical observations several days before and after the detection of PCV2 antibodies confirmed that all participants in the challenge group had been infected with wild-type PCV2 virus. Antibody testing was performed every two days using the porcine circovirus type 2 antibody detection kit in the control group, and no PCV2 antibodies were detected.

[0061] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. An antibody detection kit for distinguishing pigs immunized with porcine circovirus type 2 inactivated vaccine and pigs infected with porcine circovirus type 2 wild virus, characterized in that: The kit comprises an antibody detection chip, The antibody detection chip comprises a solid phase carrier, and polypeptides shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, which are fixed on the solid phase carrier.

2. The kit according to claim 1, wherein The biological samples tested include whole blood or serum of porcine origin.

3. The kit according to claim 1, wherein The kit is used to distinguish pigs infected with wild porcine circovirus type 2 from a pig group immunized with an inactivated porcine circovirus type 2 vaccine; the pigs infected with wild porcine circovirus type 2 include pigs that failed to be immunized with an inactivated porcine circovirus type 2 vaccine and were infected with wild porcine circovirus type 2, or pigs that were successfully immunized with an inactivated porcine circovirus type 2 vaccine but were still infected with wild porcine circovirus type 2.

4. The kit according to claim 1, wherein The Cap protein of porcine circovirus type 2 is also fixed on the solid phase carrier.

5. Use of an antibody detection chip in the preparation of an antibody detection kit for distinguishing pigs immunized with porcine circovirus type 2 inactivated vaccine and pigs infected with porcine circovirus type 2 wild virus, characterized in that: The antibody detection chip comprises a solid phase carrier, and polypeptides shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, which are fixed on the solid phase carrier.

6. The use according to claim 5, characterized in that The samples tested are biological samples of pig origin.

7. The use according to claim 5, characterized in that The antibody detection kit is used to distinguish pigs infected with wild porcine circovirus type 2 from a pig group immunized with porcine circovirus type 2 inactivated vaccine.

8. The use according to claim 5, characterized in that The Cap protein of porcine circovirus type 2 is also fixed on the solid phase carrier.