Porcine circovirus type 2 antibody detection kit for diva

Abstract

The present application discloses a porcine circovirus type 2 antibody detection kit which can be used for DIVA. The porcine circovirus type 2 antibody detection kit of the present application 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 and SEQ ID NO: 4 are respectively immobilized on the solid phase carrier. The porcine circovirus type 2 antibody detection kit of the present application can be used for DIVA (Differentiating Infected from Vaccinated Animals) of pigs immunized by porcine circovirus type 2 Cap protein subunit vaccine and pigs infected by porcine circovirus type 2 wild virus.

Description

TECHNICAL FIELD

[0001] The present application mainly relates to the field of biological detection technology. Specifically, the present application relates to a porcine circovirus type 2 antibody detection kit for distinguishing porcine circovirus type 2 Cap protein subunit vaccine immunized pigs and porcine circovirus type 2 wild virus infected pigs. BACKGROUND

[0002] Porcine circovirus disease is a serious infectious disease of pigs caused by porcine circovirus (PCV), which is a category II animal disease specified in the "List of Category I, II and III Animal Disease Species" issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

[0003] Porcine circovirus PCV is the smallest animal virus discovered so far, and its genome is a single-stranded negative-strand circular DNA molecule. It is known to have two serotypes, PCV1 and PCV2. PCV1 is a non-pathogenic virus, and PCV2 is a pathogenic virus. PCV2 infection in pigs can cause post-weaning multisystemic wasting syndrome (PMWS), sow reproductive disorders, respiratory disease in growing pigs, pig dermatitis and nephropathy syndrome, and congenital tremor in pigs.

[0004] Currently, China mainly uses various types of PCV vaccines (mainly PCV2 inactivated vaccine, PCV2 virus-like particle vaccine, PCV2 Cap protein subunit vaccine) to prevent and control porcine circovirus disease, i.e. immune no epidemic (no epidemic under the condition of vaccination). However, immune no epidemic is the primary stage of animal disease prevention and control, and the advanced stage of animal disease prevention and control is non-immune no epidemic (no epidemic under the condition of no vaccination). The realization of non-immune no epidemic requires the absence of pathogenic agents in a designated area (including host animals and environment), which can be achieved through animal disease purification.

[0005] Animal disease purification refers to the planned eradication of animals carrying certain animal disease agents in a certain area, thereby eliminating the animal disease agents, so as to achieve the state of no disease and no infection of individual animals in the area.

[0006] In October 2021, the Ministry of Agriculture and Rural Affairs of the People's Republic of China issued "Opinions on Promoting Animal Disease Purification Work", which clearly states that China will carry out purification of a series of major animal diseases in the current and future period.

[0007] A key step in animal disease purification is to distinguish wild virus infected individuals from immune populations through DIVA (Differentiating Infected from Vaccinated Animals), and to cull them, thereby eliminating pathogenic agents from the population.

[0008] Generally, DIVA can be performed by antibody detection. The Cap protein of PCV2 is the main immunogen of PCV2 and is also the commonly used biological probe (antigen) for detecting PCV2 antibodies. However, for the animal disease of porcine circovirus disease immunized by Cap protein subunit vaccine, because the Cap protein subunit vaccine and the live virus both contain Cap protein in structure and Cap protein is the main immunogen, the antibody patterns induced in the animal body are similar, and the Cap protein of PCV2 cannot be used as a biological probe for differentiation.

[0009] Therefore, how to develop an antibody detection kit for DIVA of porcine circovirus disease (PCV2) in the case of immunization with Cap protein subunit vaccine is an important issue in the porcine circovirus disease eradication work. SUMMARY

[0010] Antigen epitope is the region of a protein antigen that binds to an antibody. There can be multiple antigen epitopes in a protein antigen. Antigen epitope peptide is a polypeptide containing an antigen epitope, which can be obtained by artificial synthesis. Antigen epitope peptide can also be used to detect antibodies that can bind thereto.

[0011] The inventors have found a group of antigen epitope peptides from porcine circovirus type 2 (PCV2) Cap protein (Capsid Protein) which respond to biological samples of pigs immunized with PCV2 Cap protein subunit vaccine and disappear around 73 days after immunization, and then reappear if the pigs are infected with PCV2 wild virus, and thus can be used for DIVA of pigs immunized with PCV2 Cap protein subunit vaccine.

[0012] That is, the present application comprises:

[0013] 1. A porcine circovirus type 2 antibody detection kit comprising an antibody detection chip,

[0014] 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 and SEQ ID NO: 4 respectively fixed on the solid phase carrier.

[0015] SEQ ID NO: 1 (MMRFNINDFLPPGGGSNP);

[0016] SEQ ID NO: 2 (TKATALTYDPYVNYSSRH);

[0017] SEQ ID NO: 3 (FTPKPVLDSTIDYFQPNNK);

[0018] SEQ ID NO: 4 (REFNLKDPPLNP).

[0019] 2. The porcine circovirus type 2 antibody detection kit according to claim 1, which is used for detecting porcine circovirus type 2 antibody in a biological sample of porcine origin. Here, the biological sample can be, for example, whole blood, plasma, serum, as long as it contains porcine circovirus type 2 antibody.

[0020] 3. The porcine circovirus type 2 antibody detection kit according to claim 1, which is used for differentiating a porcine circovirus type 2 Cap protein subunit vaccine-immunized pig from a porcine circovirus type 2 wild virus-infected pig.

[0021] 4. The porcine circovirus type 2 antibody detection kit according to claim 1, which is used for differentiating a porcine circovirus type 2 wild virus-infected pig from a porcine circovirus type 2 Cap protein subunit vaccine-immunized pig in a porcine circovirus type 2 Cap protein subunit vaccine-immunized pig group. Here, the porcine circovirus type 2 wild virus-infected pig includes a porcine circovirus type 2 wild virus-infected pig that has failed porcine circovirus type 2 Cap protein subunit vaccine immunization and a porcine circovirus type 2 wild virus-infected pig that has succeeded porcine circovirus type 2 Cap protein subunit vaccine immunization.

[0022] 5. The porcine circovirus type 2 antibody detection kit according to claim 1, wherein a Cap protein of porcine circovirus type 2 is further immobilized on the solid support.

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

[0024] The antibody detection chip comprises a solid support, and polypeptides represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, immobilized on the solid support.

[0025] 7. The use according to claim 6, wherein the porcine circovirus type 2 antibody detection kit is used for detecting porcine circovirus type 2 antibody in a biological sample of porcine origin.

[0026] 8. The use according to claim 6, wherein the porcine circovirus type 2 antibody detection kit is used for differentiating a porcine circovirus type 2 Cap protein subunit vaccine-immunized pig from a porcine circovirus type 2 wild virus-infected pig.

[0027] 9. The use according to claim 6, wherein the porcine circovirus type 2 antibody detection kit is used to distinguish the porcine circovirus type 2 wild virus infected pigs from the porcine circovirus type 2 Cap protein subunit vaccine immunized pig herd.

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

[0029] The full-length Cap protein of PCV2 is the antigen commonly used for detecting PCV2 antibody at present, and the existing researches show that it has the same response mode to the antibodies caused by PCV2 wild virus infection and various types of PCV2 vaccine immunization. Therefore, the full-length Cap protein of PCV2 cannot be used for DIVA of PCV2.

[0030] The inventors have found, through in-depth research, that a group of antigen epitope peptides from the Cap protein of PCV2 have a significantly different response mode to the antibodies caused by PCV2 Cap protein subunit vaccine immunization than the full-length Cap protein of PCV2. Specifically, the full-length Cap protein of PCV2 has a sustained response to the antibodies caused by PCV2 Cap protein subunit vaccine immunization (the response lasts for at least 100 days or more after immunization), while the response of the group of antigen epitope peptides to the antibodies caused by PCV2 Cap protein subunit vaccine immunization disappears around 73 days after immunization. Therefore, the group of antigen epitope peptides can be used as a tool for distinguishing PCV2 wild virus infection and PCV2 Cap protein subunit vaccine immunization. That is, for the pig herd immunized with PCV2 Cap protein subunit vaccine, the antibody monitoring can be continuously performed with the porcine circovirus type 2 antibody detection kit of the present application after immunization, and if the porcine circovirus type 2 antibody is detected at any time point after about 73 days of immunization, it indicates that the pig has been infected with porcine circovirus type 2 wild virus.

[0031] In the present specification, "response" refers to that the SNR (signal-to-noise ratio) = (antigen spot signal value - negative control spot signal value) / negative control spot signal value is greater than or equal to 2. When two or more of the antigen epitope polypeptide spots of SEQ ID NO: 1-4 have a response, it is determined that PCV2 antibody is detected; otherwise, it is determined that PCV2 antibody is not detected.

[0032] Example 1. Preparation and confirmation of polypeptides

[0033] The polypeptides of SEQ ID NO: 1-4 are synthesized by Kings River Biotechnology, and confirmed by mass spectrometry. Among them,

[0034] SEQ ID NO: 1 (MMRFNINDFLPPGGGSNP);

[0035] SEQ ID NO: 2 (TKATALTYDPYVNYSSRH);

[0036] SEQ ID NO: 3 (FTPKPVLDSTIDYFQPNNK);

[0037] SEQ ID NO: 4 (REFNLKDPPLNP).

[0038] Example 2. Antibody detection chip Preparation of kit Example 3. Antibody detection using the kit

[0039] The solutions of the polypeptides shown in SEQ ID NO: 1-4 above were spotted on a conventional solid carrier for ELISA by a conventional method, and a pig IgG was spotted as a positive quality control point and a PB buffer solution was spotted as a negative quality control point to prepare an antibody detection chip.

[0040] Correspondingly, a porcine circovirus type 2 antibody detection kit was prepared, which contains the antibody detection chip and reagents for antibody detection such as concentrated washing solution, sample diluent, enzyme-labeled antibody solution, and luminescent substrate solution in addition to the antibody detection chip.

[0041] Example 4. Antibody detection results

[0042] Detection step

[0043] (1) The 20x concentrated washing solution (TBST: 0.4M Tris-HCl, 2.74M NaCl, 2% Tween 20, pH 7.2±0.2) was diluted 1:20 with purified water to obtain a washing solution. To completely soak the surface of the detection chip, about 100 μL of the washing solution was added to the surface of the detection chip using a pipette, and the detection chip was soaked for a certain period of time.

[0044] (2) The serum sample to be tested was diluted 1:1000 with a sample diluent (0.05M PBS, 1% BSA, 0.2% PVP, 0.5% Tween 20, pH 7.2±0.2).

[0045] (3) For the detection chip from which the washing solution was discarded, 100 μL of the diluted serum sample was added to the detection chip while the surface was completely wet.

[0046] (4) The detection chip was incubated in a constant-temperature shaker at 500 rpm for 30 minutes at a temperature of 37 degrees Celsius.

[0047] (5) The serum sample was discarded, and the surface of the detection chip was washed with the washing solution.

[0048] ​​(6) After washing, 100 μL of enzyme-labeled antibody solution (rabbit anti-pig IgG-HRP, Sigma, A5420) was added to the detection chip, and the detection chip was incubated in a constant-temperature shaker at 500 rpm for 30 minutes at 37 degrees Celsius.

[0049] (7) Discard the enzyme-labeled antibody solution and clean the surface of the detection chip with washing solution.

[0050] (8) After washing, 20 μL of luminescent substrate solution (Thermo, Prod#37074) was evenly spread on the surface of the detection chip.

[0051] (9) The detection chip was chemiluminescence imaged using a gel imager, and the results were determined.

[0052] (10) Result determination:

[0053] For each serum, if the SNR of the antigen epitope polypeptide spot on the antibody detection chip is greater than or equal to 2, it is "responsive"; otherwise, it is not responsive.

[0054] When two or more of the antigen epitope polypeptide spots of SEQ ID NO: 1-4 are responsive, it is determined that PCV2 antibody is detected; otherwise, it is determined that PCV2 antibody is not detected.

[0055] ​

[0056] Ten pigs determined to be PCV2 antibody negative by other methods were divided into an attack group and a control group, and both groups were immunized with a PCV2 Cap protein subunit vaccine. On the 85th day after immunization, both groups were determined to be PCV2 antibody positive by other methods, but no PCV2 antibody was detected using the porcine circovirus type 2 antibody detection kit described above.

[0057] After the above detection was completed, the attack group was strongly attacked with PCV2 wild virus on the 85th day after immunization, and then the antibody detection was performed every 2 days using the porcine circovirus type 2 antibody detection kit described above. On the 14th day after the attack, all the PCV2 antibodies were detected, and through clinical observation it was confirmed that all the pigs in the attack group were infected with PCV2 wild virus before and after the detection of PCV2 antibody. The control group was detected for PCV2 antibody every 2 days using the porcine circovirus type 2 antibody detection kit described above, and no PCV2 antibody was detected. SEQUENCE LISTING <110> Institute of Suzhou Nanotechnology and Nanobionics, Chinese Academy of Sciences <120> Porcine circovirus type 2 antibody detection kit for DIVA <130> 2022-03-28 <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <400> 1 Met Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser 1 5 10 15 Asn Pro <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <400> 2 Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser 1 5 10 15 Arg His <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <400> 3 Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 1 5 10 15 Asn Asn Lys <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <400> 4 Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro 1 5 10

Claims

1. An antibody detection kit for distinguishing porcine circovirus type 2 Cap protein subunit vaccine immunized pigs from porcine circovirus type 2 wild virus infected pigs, comprising an antibody detection chip, wherein the antibody detection chip comprises a solid phase carrier, and polypeptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are respectively immobilized on the solid phase carrier; and the biological sample detected by the kit comprises whole blood, plasma or serum of porcine origin. 2.The kit of claim 1, which is used for distinguishing porcine circovirus type 2 wild virus infected pigs from a porcine circovirus type 2 Cap protein subunit vaccine immunized pig herd; the porcine circovirus type 2 wild virus infected pigs include porcine circovirus type 2 Cap protein subunit vaccine immunized pigs that fail to be immunized by the porcine circovirus type 2 Cap protein subunit vaccine and are infected by the porcine circovirus type 2 wild virus, or porcine circovirus type 2 Cap protein subunit vaccine immunized pigs that are successfully immunized by the porcine circovirus type 2 Cap protein subunit vaccine but are still infected by the porcine circovirus type 2 wild virus.

3. The kit of claim 1, wherein, Cap protein of porcine circovirus type 2 is also immobilized on the solid phase carrier. 4.Use of an antibody detection chip in the preparation of an antibody detection kit for distinguishing porcine circovirus type 2 Cap protein subunit vaccine immunized pigs from porcine circovirus type 2 wild virus infected pigs, wherein the antibody detection chip comprises a solid phase carrier, and polypeptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are respectively immobilized on the solid phase carrier.

5. Use according to claim 4, wherein, The biological sample detected comprises whole blood, plasma or serum of porcine origin.

6. The use according to claim 4, wherein, The kit is used for distinguishing porcine circovirus type 2 wild virus infected pigs from a porcine circovirus type 2 Cap protein subunit vaccine immunized pig herd.

7. The use according to claim 4, wherein, Cap protein of porcine circovirus type 2 is also immobilized on the solid phase carrier.