PCV4-C157 recombinant antigen synthesized based on PCV4 specific protein sequence and application of PCV4-C157 recombinant antigen in preparation of anti-porcine PCV4 specific antibody

Abstract

The anti-porcine PCV4 monoclonal antibody disclosed by the invention is characterized in that a sequence PCV4-C157 is synthesized on the basis of a PCV4 specific protein sequence, and the sequence of the PCV4-C157 is KPQDPSGETHTLHFQP; a synthetic sequence and inert protein hemocyanin are coupled and then subjected to animal injection immunization, serum is collected after immunization, an antibody in the serum is extracted, a specific antibody is prepared, the monoclonal antibody prepared on the basis of specific polypeptide fragments which have no cross reaction with PCV1, PCV2 and PCV3 in PCV4 serves as a detection way, the PCV4 virus can be rapidly identified, operation is easy, detection time is short, cost is low, and the method is suitable for large-scale popularization and application. The colloidal gold test strip for detecting the porcine PCV4 antigen is high in specificity, high in sensitivity and accurate in result, does not need any instrument and equipment, can be operated on site, and does not need to be operated by professionals.

Description

[0001] This application is a divisional application of patent application No. 202210990670.7

[0002] Application date: August 18, 2022

[0003] Application number: 202210990670.7

[0004] Invention Name: Anti-swine PCV4 specific antibodies, colloidal gold test strips for detecting swine PCV4 antigens, and preparation method Technical Field

[0005] The present invention relates to the technical field of novel porcine circovirus detection, in particular to an anti-porcine PCV4 specific antibody, a colloidal gold test strip for detecting porcine PCV4 antigen and a preparation method thereof. Background Art

[0006] In 2019, the Veterinary Virology Research Group of the College of Life Sciences discovered a new porcine circovirus (tentatively designated PCV4). PCV4 is a novel circovirus with a genome size of 1770 nucleotides, sharing only 43.2%-51.5% homology with other PCV genomes. Porcine circoviruses belong to the Circoviridae family and the genus Circovirus. They are single-stranded, closed, circular DNA viruses with icosahedral symmetry and no envelope. PCV2 was first discovered in Canada by Ellis et al. in 1991. It is highly pathogenic to pigs and is the primary cause of post-weaning multisystemic wasting syndrome (MPWS). PCV3 was identified in 2016 by US researchers using metagenomic sequencing from sows with dermatitis and nephrotic syndrome, reproductive failure, and aborted fetuses.

[0007] Currently, PCV4 has been detected in Hunan, Henan, and Shanxi provinces in China, but not in Italy or Spain. As a newly discovered circovirus, PCV4's duration of existence, infection rate, prevalence, and pathogenicity are unclear due to its relatively short onset. Classical swine fever, pseudorabies, and porcine circovirus are considered the world's three major swine diseases, each causing significant economic losses to the pig industry. As the first site of PCV4 detection, China faces even more severe challenges. Therefore, establishing specific, sensitive, inexpensive, and simple detection methods is an urgent need for my country's current prevention and control of the disease. However, my country currently lacks rapid detection test strips.

[0008] Therefore, the existing technology still needs to be further improved and developed. Summary of the Invention

[0009] In order to solve the above problems, anti-swine PCV4 specific antibodies, colloidal gold test strips for detecting swine PCV4 antigens and preparation methods are proposed:

[0010] An anti-swine PCV4 specific antibody, characterized in that the sequence PCV4-C157 is synthesized based on the PCV4 specific protein sequence, wherein the PCV4-C157 sequence is KPQDPSGETHTLHFQP; the synthesized sequence is coupled to the inert protein hemocyanin, and then SPF mice are immunized to collect serum, and antibodies in the serum are extracted to prepare the specific antibody.

[0011] Furthermore, the PCV4-specific protein sequence is a differential fragment obtained through homology comparison analysis of PCV4 and PCV1, PCV2, and PCV3 sequences.

[0012] Furthermore, the synthetic sequence is coupled to the inert protein hemocyanin and then injected into animals for immunization as follows:

[0013] The synthetic sequence PCV4-C157 was coupled with the inert protein hemocyanin to prepare C157-KLH, which was used as an immune antigen for mice to be immunized and boosted.

[0014] Furthermore, 8-10 week old mice were subcutaneously injected with 50 μg / time C157-KLH once a week for a total of 3 vaccinations. The primary immunization was emulsified with Freund's complete adjuvant, and the booster immunization was emulsified with Freund's incomplete adjuvant. Seven days after the third vaccination, the antibody titer in the serum was detected by indirect ELISA method. The mouse with the highest titer was selected, the serum was collected, and the antibody was purified by protein G.

[0015] Furthermore, serum was obtained by removing the eyes of the immunized mice and collecting blood.

[0016] Preferably, a method for preparing a colloidal gold test strip for detecting porcine PCV4 antigen comprises the following steps: dialyzing C157-KLH-specific antibody (final concentration 20 μg / ml) and goat anti-rabbit IgG (final concentration 40 μg / ml) against 0.005 Mol / L pH 7.0 NaCl solution at 4°C overnight, mixing with a pH 8.2 colloidal gold solution and stirring for 30 minutes; then adding a final concentration of 0.1% PEG-20000 and 0.5% BSA solution to the mixture, stirring for 10 minutes, centrifuging at 9000 g for 30 minutes, discarding the supernatant, and resuspending the colloidal gold in PBS at 1 / 10 of the original volume to obtain a mixed solution of C157-KLH-specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label.

[0017] Furthermore, the colloidal gold labeling pad was treated in the following specific steps: a mixed solution of C157-KLH specific antibody colloidal gold labeling substance and goat anti-rabbit IgG colloidal gold labeling substance was diluted with a dot film diluent and sprayed on the colloidal gold labeling pad at 2 μL / cm.

[0018] Furthermore, the nitrocellulose membrane is treated, specifically by coupling the PCV4-C157 sequence with bovine serum albumin to prepare the polypeptide C157-BSA, which is diluted with a spotting diluent and sprayed on the test line at 2 μL / cm, and the rabbit IgG antibody is diluted with a spotting diluent and sprayed on the control line at 2 μL / cm, wherein the concentration of the capture antigen spotting solution is 3 μg / mL, and the concentration of the quality control antibody spotting solution is 2 μg / L, wherein the spotting solution diluent contains 0.15M sodium chloride, 10mM ethylenediaminetetraacetic acid, 0.1% proclin 300 and 20g / L methanol in 0.02M phosphate buffer, and the remainder is water.

[0019] Furthermore, a colloidal gold test strip for detecting porcine PCV4 antigen is provided, wherein a sample pad, a colloidal gold labeling pad, a nitrocellulose membrane and an absorbent paper are fixed on a substrate in sequence, the sample pad, the colloidal gold labeling pad, the nitrocellulose membrane and the absorbent paper partially overlap, and a portion of the absorbent paper overlaps the nitrocellulose membrane.

[0020] Beneficial effects:

[0021] 1. The colloidal gold test strip for detecting porcine PCV4 antigen provided by the present invention uses antibodies prepared from specific polypeptide fragments of PCV4 that have no cross-reaction with PCV1, PCV2, and PCV3 as a detection method, which can quickly identify the PCV4 virus with simple operation, short detection time and low cost.

[0022] 2. The colloidal gold test strip for detecting porcine PCV4 antigen provided by the present invention has strong specificity, high sensitivity, and accurate results. It does not require any instruments or equipment and can be operated on-site without the need for professional personnel to operate it. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SDS-PAGE electrophoresis diagram after purification of PCV4-specific antibodies using Protein G;

[0024] Figure 2 Front view of the colloidal gold test strip for detecting porcine PCV4 antigen;

[0025] Description of reference numerals:

[0026] 1. Substrate, 2. Sample pad, 3. Colloidal gold labeling pad, 4. Nitrocellulose membrane, 5. Absorbent pad, 6. Test line, 7. Control line. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0028] Example 1: A method for preparing an anti-swine PCV4 specific antibody

[0029] 1. Peptide design and synthesis

[0030] The homology of PCV4 with PCV1, PCV2, and PCV3 sequences was compared and analyzed, and the differential fragment was screened out. The sequence was 157-172 (KPQDPSGETHTLHFQP) of the PCV4-cap protein sequence and was recorded as PCV4-C157. After synthesis, it was coupled with the inert protein hemocyanin to prepare C157-KLH as an antigen for immunizing SPF mice. In addition, PCV4-C157 was coupled with bovine serum albumin to prepare the polypeptide C157-BSA for detection line antibody spraying.

[0031] 2. Animal immunization

[0032] Six mice aged 8-10 weeks were subcutaneously injected with 50 μg / time / mouse C157-KLH once a week for a total of three vaccinations. The primary immunization was emulsified with Freund's complete adjuvant, and the booster immunization was emulsified with Freund's incomplete adjuvant. Seven days after the third vaccination, the antibody titer in the serum was tested by indirect ELISA method, and the mice with the highest titer were selected.

[0033] 3. PCV4-specific antibody purification:

[0034] Combine Figure 1 After collecting blood from the eyes of immunized mice, serum is obtained. Protein G agarose gel medium is loaded into affinity purification chromatography column, and the immunized serum is slowly loaded. After the antibody binds, it is eluted with glycine elution buffer to obtain the desired purified antibody. Figure 1 As shown, Figure a is the serum before purification

[0035] The sample contains a lot of impurities. Figure b is the serum sample after purification. It can be found from the picture that there are only protein bands at 25KDa (antibody light chain) and 55KDa (antibody heavy chain), and the antibody is well purified.

[0036] Specific antibodies prepared based on specific polypeptide fragments in PCV4 that have no cross-reaction with PCV1, PCV2, and PCV3 are used as a detection method to quickly identify the PCV4 virus. It is simple to operate, has a short detection time, and is low cost.

[0037] Example 2 Preparation Method of Colloidal Gold Test Strip for Detecting Swine PCV4 Antigen

[0038] 1. Preparation of colloidal gold-labeled C157-KLH-specific antibody

[0039] C157-KLH specific antibody (final concentration 20ug / ml) and goat anti-rabbit IgG (final concentration 40ug / ml) were dialyzed against 0.005Mol / L pH 7.0 NaCl solution at 4°C overnight and then diluted with pH 8.2

[0040] The colloidal gold solution was mixed and stirred for 30 minutes, and then a final concentration of 0.1% PEG-20000 and 0.5% BSA solution were added to the mixture. After stirring for 10 minutes, the mixture was centrifuged at 9000g for 30 minutes, the supernatant was discarded, and the colloidal gold was resuspended in 1 / 10 of the original volume of PBS to obtain a mixed solution of C157-KLH-specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label.

[0041] 2. Treatment of colloidal gold labeling 3

[0042] The mixed solution of C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label was diluted with membrane spotting diluent and sprayed onto the colloidal gold labeling pad 3 at 2 μL / cm.

[0043] 3. Nitrocellulose membrane treatment

[0044] The C157-BSA protein was diluted with a spotting diluent and sprayed on the test line 6 at 2 μL / cm, and the rabbit IgG antibody was diluted with a spotting diluent and sprayed on the control line 7 at 2 μL / cm, wherein the concentration of the capture antigen spotting solution was 3 μg / mL, and the concentration of the quality control antibody spotting solution was 2 μg / L, wherein the spotting solution diluent contained 0.15 M sodium chloride, 10 mM ethylenediaminetetraacetic acid, 0.1% proclin300 and 20 g / L methanol in 0.02 M phosphate buffer, and the rest was water.

[0045] 4. Assembly

[0046] The sample pad 2, colloidal gold label pad 3, nitrocellulose membrane 4 and absorbent pad 5 are fixed on the substrate 1 in sequence. The sample pad 2, colloidal gold label 3, nitrocellulose membrane 4 and absorbent pad 5 are partially overlapped, and a part of the absorbent pad 5 is overlapped with the nitrocellulose membrane 4. The assembled test strip structure is as follows Figure 2 shown.

[0047] Example 3: A method for using a colloidal gold test strip for detecting porcine PCV4 antigen

[0048] Take a sample such as peripheral blood or tissue eluate, add it to 50 μL of sample diluent (0.01 mol / L Tris-HCl, pH 7.6; 0.01 mol / L NaCl; 0.005 mol / L MgCl2), mix well, and then add it dropwise to the sample loading area of ​​the sample pad. After incubation at room temperature for 5 minutes, if both the test line T and the quality control line C show color, the sample does not contain PCV4 antigen. If the test line does not show color but the quality control line does, the sample contains PCV4 antigen.

[0049] Example 4 Compliance Test

[0050] Thirty serum samples were tested simultaneously using the colloidal gold test strips and ELISA blocking assay established by the present invention. The results are shown in Table 1:

[0051] Table 1: ELISA and colloidal gold test strip test results of 30 serum samples and controls.

[0052] Test samples ELISA test Test strips serial number Blocking rate Judgment results Result determination 1 65.3％ + + 2 72.1％ + + 3 61.0％ + + 4 121％ - - 5 33.3％ + + 6 21.9％ - - 7 16.8％ - - 8 57.2％ + + 9 24.1％ - - 10 36.4％ + + 11 26.9％ + + 12 53.20％ + + 13 9.20％ - - 14 3.30％ - - 15 69.00％ + + 16 41.60％ + + 17 36.30％ + + 18 74.60％ + + 19 77.10％ + + 20 5.50％ - - 21 6.40％ - - 22 51.30％ + + 23 22.30％ - - 24 43.20％ + + 25 66.60％ + + 26 30.10％ + + 27 34.10％ + + 28 29.30％ + + 29 44.90％ + + 30 12.20％ - -

[0053] Among them, the ELISA test result blocking rate ≥ 25% is positive, and < 25% is negative.

[0054] The results showed that the colloidal gold detection method of the present invention had a positive coincidence rate of 100% with the ELISA blocking method, a negative coincidence rate of 100%, and an overall coincidence rate of 100%; the results were consistent with the ELISA blocking method and could be used for clinical monitoring.

[0055] The colloidal gold test strip for detecting porcine PCV4 antigen provided hereby has strong specificity, high sensitivity and accurate results, does not require any instruments or equipment, can be operated on-site, does not require professional personnel to operate, and is simple and convenient.

[0056] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced thereby.

[0057] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A PCV4-C157 recombinant antigen synthesized based on a PCV4-specific protein sequence, characterized by: The PCV4-C157 sequence was coupled to the inert protein hemocyanin to obtain the antigen C157-KLH for the preparation of anti-swine PCV4 specific antibodies. The PCV4-C157 sequence is KPQDPSGETHTLHFQP.

2. The PCV4-C157 recombinant antigen synthesized based on the PCV4-specific protein sequence according to claim 1, characterized in that: The antigen C157-KLH was used to immunize SPF mice to obtain anti-swine PCV4 specific antibodies.

3. Use of the PCV4-C157 recombinant antigen synthesized based on the PCV4 specific protein sequence according to claim 1 in the preparation of anti-swine PCV4 specific antibodies, characterized in that: SPF mice were immunized with antigen C157-KLH, serum was collected and antibodies were extracted from the serum to prepare specific antibodies; 8-10 week old mice were subcutaneously injected with 50 μg / dose of C157-KLH once a week for a total of three vaccinations. The primary immunization was emulsified with Freund's complete adjuvant, and the booster immunization was emulsified with Freund's incomplete adjuvant. Seven days after the third vaccination, the antibody titer in the serum was tested by indirect ELISA. The mice with the highest titer were selected, and the serum was collected and the antibodies were purified using protein G. The Protein G agarose gel medium is loaded into the affinity purification chromatography column, and the immunized serum is slowly loaded. After the antibody binds, it is eluted with glycine elution buffer to obtain the desired purified antibody.

4. The use of the PCV4-C157 recombinant antigen synthesized based on the PCV4 specific protein sequence in the preparation of anti-swine PCV4 specific antibodies according to claim 3, characterized in that: The PCV4-specific protein sequence is a differential fragment obtained through sequence homology comparison analysis of PCV4 with PCV1, PCV2, and PCV3.

5. The use according to claim 3, characterized in that Serum was obtained by removing the eyes of immunized mice and collecting blood.

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