Anti-Porcine PCV4 Specific Antibody, Colloidal Gold Test Strip for Detecting Porcine PCV4 Antigen and Preparation Method

Abstract

An anti - porcine PCV4 specific antibody of the present invention is characterized in that sequence PCV4 - C157 is synthesized based on the PCV4 specific protein sequence, wherein the PCV4 - C157 sequence is KPQDPSGETHTLHFQP; after coupling the synthesized sequence with the inert protein hemocyanin, animal injection immunization is carried out, serum is collected after immunization, antibodies in the serum are extracted to prepare specific antibodies. Monoclonal 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, which can quickly identify PCV4 virus, with simple operation, short detection time and low cost. The provided colloidal gold test strip for detecting porcine PCV4 antigen has strong specificity, high sensitivity and accurate results, does not require any instrument and equipment, can be operated on - site, and does not require professional personnel to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of novel porcine circovirus detection, and specifically to an anti-porcine PCV4 specific antibody, a colloidal gold test strip for detecting porcine PCV4 antigen, and a preparation method thereof. Background Art

[0002] In 2019, the research group on veterinary and avian virology in the College of Biology discovered a novel porcine circovirus (tentatively named PCV4). PCV4 is a completely new circovirus with a genome size of 1,770 nucleotides, and its homology with other PCV genomes is only 43.2% - 51.5%. Porcine circovirus belongs to the family Circoviridae and the genus Circovirus, and is an icosahedral symmetric, non-enveloped single-stranded, closed, circular DNA virus. PCV2 was first discovered by ELLIS et al. in 1991 in Canada and has strong pathogenicity to pig herds, and is the main pathogen causing postweaning multisystemic wasting syndrome in piglets; PCV3 was identified by American researchers through metagenomic sequencing technology in 2016 from sows with dermatitis and nephropathy syndrome and reproductive disorders and aborted fetuses.

[0003] Currently, PCV4 has been detected in Hunan Province, Henan Province and Shanxi Province in China, but the virus has not been detected in Italy and Spain. As a newly discovered circovirus, due to the short onset time, the existence time, infection rate, epidemic situation and pathogenicity of PCV4 are not clear. Classical swine fever, pseudorabies and porcine circovirus disease are known as the world's three major swine diseases, which cause huge economic losses to the pig industry every year. As the first place where PCV4 was detected, China faces more severe challenges. Therefore, establishing a specific, sensitive, inexpensive and simple detection method is an urgent requirement for the prevention and control of this disease in China, and there is currently no rapid detection test strip product in China.

[0004] Therefore, the existing technology still needs to be further improved and developed. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides an anti-porcine PCV4 specific antibody, a colloidal gold test strip for detecting porcine PCV4 antigen, and a preparation method thereof:

[0006] An anti-porcine PCV4 specific antibody, characterized in that sequence PCV4-C157 is synthesized based on the PCV4 specific protein sequence, wherein the PCV4-C157 sequence is KPQDPSGETHTLHFQP; the synthesized sequence is coupled with the inert protein hemocyanin and then used to immunize SPF mice to collect serum, and the antibody in the serum is extracted to prepare the specific antibody.

[0007] Further, the PCV4 specific protein sequence is a differential fragment obtained by comparative analysis of the homology between PCV4 and the sequences of PCV1, PCV2, and PCV3.

[0008] Further, the specific operation of coupling the synthetic sequence with the inert protein hemocyanin and then performing animal injection immunization is as follows:

[0009] Couple the synthetic sequence PCV4-C157 with the inert protein hemocyanin to prepare C157-KLH, and use C157-KLH as the immunogen for mice for immunization and booster immunization.

[0010] Further, use 8-10-week-old mice, inject 50 μg / mouse of C157-KLH subcutaneously each time, once a week, for a total of 3 inoculations. Among them, the primary immunization is emulsified with Freund's complete adjuvant, and the booster immunization is emulsified with Freund's incomplete adjuvant. 7 days after the third inoculation, detect the antibody titer in the serum by the indirect ELISA method, select the mouse with the highest titer, collect the serum, and purify the antibody by protein G.

[0011] Further, obtain the serum by enucleating the eyes of the immunized mice for blood collection.

[0012] Preferably, a preparation method of a colloidal gold test strip for detecting porcine PCV4 antigen, the specific steps are as follows: Dialyze the C157-KLH specific antibody (final concentration 20 μg / ml) and goat anti-rabbit IgG (final concentration 40 μg / ml) overnight at 4°C in 0.005 Mol / L pH 7.0 NaCl solution, then mix and stir with the pH = 8.2 colloidal gold solution for 30 min. Then add the final concentration of 0.1% PEG-20000 and 0.5% BSA solution to the mixture, stir for 10 min, and then centrifuge at 9000 g for 30 min. Discard the supernatant, and resuspend the colloidal gold with 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.

[0013] Further, the treatment of the colloidal gold labeling pad, the specific steps are as follows: Dilute the mixed solution of C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label with a dot membrane diluent, and spray it on the colloidal gold labeling pad at 2 μL / cm.

[0014] Further, for the treatment of the nitrocellulose membrane, the specific steps are as follows: The PCV4-C157 sequence is conjugated with bovine serum albumin to prepare the polypeptide C157-BSA, which is diluted with a dotting membrane diluent and sprayed on the test line at 2 μL / cm. The rabbit IgG antibody is diluted with a dotting membrane diluent and sprayed on the control line at 2 μL / cm. Among them, the concentration of the antigen-capturing dotting membrane solution is 3 μg / mL, and the concentration of the quality control antibody dotting membrane solution is 2 μg / L. Among them, the dotting membrane diluent contains 0.02 M phosphate buffer with 0.15 M sodium chloride, 10 mM ethylenediaminetetraacetic acid, 0.1% proclin300, and 20 g / L methanol, and the rest is water.

[0015] Further, a colloidal gold test strip for detecting porcine PCV4 antigen, wherein a sample pad, a colloidal gold-labeled pad, a nitrocellulose membrane, and a blotting paper are sequentially fixed on a substrate, and a part of the sample pad, the colloidal gold-labeled pad, the nitrocellulose membrane, and the blotting paper overlap, and a part of the blotting paper overlaps with the nitrocellulose membrane.

[0016] Beneficial effects:

[0017] 1. The colloidal gold test strip for detecting porcine PCV4 antigen provided by the present invention uses an antibody prepared based on a specific polypeptide fragment in PCV4 that has no cross-reaction with PCV1, PCV2, and PCV3 as a detection method, which can quickly identify PCV4 virus, is simple to operate, has a short detection time, and is low in cost.

[0018] 2. The colloidal gold test strip for detecting porcine PCV4 antigen provided by the present invention has strong specificity, high sensitivity, accurate results, does not require any instrument and equipment, can be operated on-site, and does not require professional personnel for operation. Description of the Drawings

[0019] Figure 1 It is the SDS-PAGE electrophoresis diagram after purifying the PCV4-specific antibody using Protein G;

[0020] Figure 2 It is the front view of the colloidal gold test strip for detecting porcine PCV4 antigen;

[0021] Description of the Reference Numerals:

[0022] 1. Substrate, 2. Sample pad, 3. Colloidal gold-labeled pad, 4. Nitrocellulose membrane, 5. Blotting pad, 6. Test line, 7. Control line. Detailed Embodiments

[0023] Combined with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to 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 the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0024] Example 1 A method for preparing anti-Porcine circovirus 4 (PCV4) specific antibody

[0025] 1. Polypeptide design and synthesis

[0026] By comparing and analyzing the homology of the sequences of PCV4 with PCV1, PCV2, and PCV3, the differential fragment was screened out. The sequence was the 157-172nd position (KPQDPSGETHTLHFQP) of the PCV4-cap protein sequence, denoted as PCV4-C157. After synthesis, it was conjugated with the inert protein hemocyanin to prepare C157-KLH as the antigen for immunizing SPF mice. In addition, PCV4-C157 was conjugated with bovine serum albumin to prepare the polypeptide C157-BSA for detecting the spraying of linear antibodies.

[0027] 2. Animal immunization

[0028] Six 8-10-week-old mice were taken, and each was subcutaneously injected with 50 μg / mouse / time of C157-KLH once a week for a total of 3 times. Among them, 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 inoculation, the antibody titer in the serum was detected by the indirect ELISA method, and the mouse with the highest titer was selected.

[0029] 3. Purification of PCV4 specific antibody:

[0030] Binding Figure 1 , after obtaining the serum by enucleating the eyes of the immunized mice, the Protein G agarose gel medium was loaded into the affinity purification chromatography column, and the immunized serum was slowly loaded. After the antibody was bound, it was eluted with glycine elution buffer to obtain the required purified antibody. As Figure 1 shown, Figure a is the serum sample before purification, which contains more miscellaneous proteins. 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.

[0031] Using a specific antibody prepared from a specific polypeptide fragment in PCV4 that has no cross-reaction with PCV1, PCV2, and PCV3 as a detection approach, the PCV4 virus can be rapidly identified, with simple operation, short detection time, and low cost.

[0032] Example 2 Preparation method of a colloidal gold test strip for detecting porcine PCV4 antigen

[0033] 1. Preparation of colloidal gold-labeled C157-KLH specific antibody

[0034] The C157-KLH specific antibody (final concentration 20 μg / ml) and goat anti-rabbit IgG (final concentration 40 μg / ml) were dialyzed overnight at 4°C in 0.005 Mol / L pH 7.0 NaCl solution, then mixed and stirred with the pH = 8.2 colloidal gold solution for 30 min. Next, a solution with a final concentration of 0.1% PEG-20000 and 0.5% BSA was added to the mixture, and after stirring for 10 min, it was centrifuged at 9000 g for 30 min. The supernatant was discarded, and the colloidal gold was resuspended with PBS at 1 / 10 of the original volume to obtain a mixture of C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label.

[0035] 2. Treatment of the colloidal gold label 3

[0036] The mixture of C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label was diluted with the dot-membrane diluent and sprayed on the colloidal gold label pad 3 at 2 μL / cm.

[0037] 3. Treatment of the nitrocellulose membrane

[0038] The C157-BSA protein was diluted with the dot-membrane diluent and sprayed on the test line 6 at 2 μL / cm. The rabbit IgG antibody was diluted with the dot-membrane diluent and sprayed on the control line 7 at 2 μL / cm. Among them, the concentration of the antigen-capturing dot-membrane solution is 3 μg / mL, and the concentration of the quality control antibody dot-membrane solution is 2 μg / L. Among them, the dot-membrane diluent contains a 0.02 M phosphate buffer of 0.15 M sodium chloride, 10 mM ethylenediaminetetraacetic acid, 0.1% proclin300, and 20 g / L methanol, and the rest is water.

[0039] 4. Assembly

[0040] The sample pad 2, the colloidal gold label pad 3, the nitrocellulose membrane 4, and the absorbent pad 5 were sequentially fixed on the substrate 1. The sample pad 2, the colloidal gold label 3, the nitrocellulose membrane 4, and the absorbent pad 5 partially overlap, and a part of the absorbent pad 5 overlaps with the nitrocellulose membrane 4. The structure of the assembled test strip is as Figure 2 shown.

[0041] Example 3 Method of using a colloidal gold test strip for detecting porcine PCV4 antigen

[0042] Take samples such as peripheral blood or tissue eluate and add them to 50 μl of sample diluent (0.01 mol / L Tris-HCL pH7.6; 0.01 mol / L NaCl; 0.005 mol / L MgCl2). After mixing and stirring, drop the mixture onto the sample application area of the sample pad. After reacting at room temperature for 5 minutes, if both the test line T and the control line C are colored, then there is no PCV4 antigen in the sample; when the test line is not colored and the control line is colored, the sample contains PCV4 antigen.

[0043] Example 4 Compliance test

[0044] Thirty serum samples were detected simultaneously using the colloidal gold test strip established by the present invention and the ELISA blocking method. The results are shown in Table 1:

[0045] Table 1: Detection results and controls of 30 serum samples by ELISA and colloidal gold test strip.

[0046]

[0047] Among them, for the ELISA test results, a blocking rate of ≥ 25% is positive and < 25% is negative.

[0048] The results showed that the positive coincidence rate of the colloidal gold detection method of the present invention and the ELISA blocking method is 100%, the negative coincidence rate is 100%, and the total coincidence rate is 100%; it is consistent with the detection results of the ELISA blocking method and can be used for clinical monitoring.

[0049] The provided colloidal gold test strip for detecting porcine PCV4 antigen has strong specificity, high sensitivity, accurate results, does not require any instrument and equipment, can be operated on-site, does not require professional personnel to operate, and is simple and convenient.

[0050] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed by the present invention.

[0051] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A synthetic sequence PCV4-C157 based on the specific protein sequence of PCV4, characterized in that, The PCV4-C157 sequence is KPQDPSGETHTLHFQP.

2. Use of a synthetic sequence PCV4-C157 based on the PCV4 specific protein sequence as claimed in claim 1 in the preparation of anti-PCV4 specific antibodies, characterized in that, The synthetic sequence was conjugated with the inert protein hemocyanin and then used to immunize SPF mice. Serum was collected and antibodies in the serum were extracted to prepare specific antibodies.

3. The use according to claim 2, characterized in that, The PCV4-specific protein sequence is the differential fragment obtained by comparative analysis of the homology of the PCV4 sequence with the PCV1, PCV2, and PCV3 sequences.

4. The use according to claim 2, wherein The specific method of conjugating the synthetic sequence with the inert protein hemocyanin and then immunizing SPF mice is as follows: The synthetic sequence PCV4-C157 was conjugated with the inert protein hemocyanin to prepare C157-KLH. C157-KLH was used as the immunogen for mice for immunization and booster immunization.

5. The use according to claim 4, characterized in that, 8- to 10-week-old mice were used. Each mouse was subcutaneously injected with 50 μg / time of C157-KLH once a week for a total of 3 times. 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 inoculation, the antibody titer in the serum was detected by the indirect ELISA method. The mouse with the highest titer was selected, serum was collected, and the antibody was purified by protein G.

6. The use according to claim 5, characterized in that, Serum was obtained by enucleating the eyes of the immunized mice for blood collection.

7. Use of a synthetic sequence PCV4-C157 based on the PCV4 specific protein sequence as described in claim 1 in the preparation of a colloidal gold test strip for detecting porcine PCV4 antigen, characterized in that, The specific steps for preparing a colloidal gold test strip for detecting porcine PCV4 antigen are as follows: The specific antibody against C157-KLH and goat anti-rabbit IgG were dialyzed overnight at 4°C in 0.005 Mol / L pH 7.0 NaCl solution, then mixed and stirred with the pH = 8.2 colloidal gold solution for 30 min. Then, a solution of 0.1% PEG-20000 and 0.5% BSA was added to the mixture, and after stirring for 10 min, it was centrifuged at 9000 g for 30 min. The supernatant was discarded, and the colloidal gold was resuspended in PBS at 1 / 10 of the original volume to obtain a mixed solution of the C157-KLH-specific antibody colloidal gold label and the goat anti-rabbit IgG colloidal gold label.

8. The use according to claim 7, characterized in that, The specific steps for preparing a colloidal gold test strip for detecting porcine PCV4 antigen also include the treatment of the colloidal gold labeling pad: The specific antibody against C157-KLH and goat anti-rabbit IgG were dialyzed overnight at 4°C in 0.005 Mol / L pH 7.0 NaCl solution. The mixed solution of the C157-KLH-specific antibody colloidal gold label and the goat anti-rabbit IgG colloidal gold label was diluted with the membrane spotting diluent and sprayed on the colloidal gold labeling pad at 2 μL / cm.

9. The use according to claim 7, characterized in that, The specific steps for preparing a colloidal gold test strip for detecting porcine PCV4 antigen also include the treatment of the nitrocellulose membrane: The PCV4-C157 sequence was conjugated with bovine serum albumin to prepare the polypeptide C157-BSA. After dilution with the membrane spotting diluent, it was sprayed on the test line at 2 μL / cm. The rabbit IgG antibody was diluted with the membrane spotting diluent and sprayed on the control line at 2 μL / cm. Among them, the concentration of the antigen-capturing membrane spotting solution is 3 μg / mL, and the concentration of the quality control antibody membrane spotting solution is 2 μg / L.

10. The use according to claim 8, characterized in that, The membrane spotting diluent contains a 0.02 M phosphate buffer containing 0.15 M sodium chloride, 10 mM ethylenediaminetetraacetic acid, 0.1% proclin300, and 20 g / L methanol, and the rest is water.

11. A colloidal gold test strip for detecting porcine PCV4 antigen, characterized in that, Prepared by using the preparation method according to any one of claims 8-9, wherein the sample pad, the colloidal gold labeling pad, the nitrocellulose membrane and the absorbent paper are sequentially fixed on the substrate, and the sample pad, the colloidal gold labeling pad, the nitrocellulose membrane and the absorbent paper partially overlap, and a part of the absorbent paper overlaps with the nitrocellulose membrane.

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