PCV4-c157 recombinant antigen synthesized based on pcv4 specific protein sequence and application thereof in preparation of anti-porcine pcv4 specific antibody

Abstract

The application discloses a kind of anti-swine PCV4 monoclonal antibodies, it is characterized in that, based on PCV4 specific protein sequence synthesis sequence PCV4-C157, wherein the PCV4-C157 sequence is KPQDPSGETHTLHFQP;Synthetic sequence is coupled with inert protein hemocyanin after injection immunization is carried out to animal, after immunization, serum is collected, antibody in serum is extracted, and specific antibody is prepared, and the monoclonal antibody prepared based on the specific polypeptide fragment in PCV4 without cross reaction with PCV1, PCV2, PCV3 is used as detection approach, PCV4 virus can be quickly identified, simple operation, short detection time and low cost, the provided detection pig PCV4 antigen colloidal gold test strip is strong in specificity, high in sensitivity, accurate in result, does not need any instrument and equipment, can be operated on site, and does not need to be operated by professional personnel.

Description

[0001] This application is a divisional application of patent application 202210990670.7

[0002] Filing date: August 18, 2022

[0003] Application number: 202210990670.7

[0004] Invention name: Anti-swine PCV4 specific antibody, detection of swine PCV4 antigen colloidal gold test strip and preparation method TECHNICAL FIELD

[0005] The present application relates to the field of novel porcine circovirus detection technology, specifically an anti-swine PCV4 specific antibody, a detection of swine PCV4 antigen colloidal gold test strip and a preparation method. BACKGROUND

[0006] In 2019, the Biological College Veterinary and Poultry Virus Research Group discovered a new type of porcine circovirus (tentative PCV4). PCV4 is a new type of circovirus, with a genome size of 1770 nucleotides. Its homology with other PCV genomes is only 43.2%-51.5%. Porcine circovirus belongs to the Circoviridae family and the Circovirus genus, and is a icosahedral, non-enveloped, single-stranded, closed, circular DNA virus. PCV2 was first discovered by ELLIS et al. in Canada in 1991. It has strong pathogenicity to pig populations and is the main pathogen causing post-weaning multisystemic syndrome. PCV3 was identified by American researchers in 2016 through metagenomic sequencing technology from sows and aborted fetuses with dermatitis and kidney disease syndrome and reproductive disorders.

[0007] At present, PCV4 has been detected in Hunan, Henan and Shanxi provinces in China, but it has not been detected in Italy and Spain. As a newly discovered circovirus, the existence time, infection rate, prevalence and pathogenicity of PCV4 virus are not clear due to the short time of onset. Swine fever, pseudorabies and porcine circovirus disease are known as the world's three major swine diseases, causing huge economic losses to the pig industry every year. As the first detection site of PCV4 in China, we are facing even more severe challenges. Therefore, establishing a specific, sensitive, inexpensive and simple detection method is an urgent requirement for the prevention and control of the disease in our country, and there is currently no rapid detection test paper product in China.

[0008] Therefore, the prior art still needs to be further improved and developed. SUMMARY

[0009] In order to solve the above problems, an anti-swine PCV4 specific antibody, a detection of swine PCV4 antigen colloidal gold test strip and a preparation method are proposed:

[0010] An anti-swine PCV4 specific antibody, characterized in that a sequence PCV4-C157 is synthesized based on a PCV4 specific protein sequence, wherein the PCV4-C157 sequence is KPQDPSGETHTLHFQP; after coupling the synthetic sequence with an inert protein hemocyanin, a serum of a SPF mouse is collected, antibodies in the serum are extracted, and the specific antibodies are prepared.

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

[0012] Further, after coupling the synthetic sequence with an inert protein hemocyanin, animal injection immunization is performed, specifically:

[0013] The synthetic sequence PCV4-C157 is coupled with an inert protein hemocyanin to prepare C157-KLH, and the C157-KLH is used as a mouse immunization antigen for immunization and booster immunization.

[0014] Further, 8-10 week old mice are used, each of which is subcutaneously injected with 50 μg / each of C157-KLH once a week, for a total of 3 inoculations, wherein the first 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, the antibody titer in the serum is detected by ELISA indirect method, the mouse with the highest titer is selected, the serum is collected, and the antibodies are purified by protein G.

[0015] Further, the serum is obtained by blood collection from the enucleated eyes of the immunized mice.

[0016] Preferably, a preparation method of a colloidal gold test strip for detecting a swine PCV4 antigen is provided, and the specific steps are as follows: after C157-KLH specific antibodies (final concentration 20 ug / ml) and goat anti-rabbit IgG (final concentration 40 ug / ml) are dialyzed in 0.005 Mol / L pH 7.0 NaCl solution at 4°C overnight, the dialyzed solution is mixed with a colloidal gold solution with PH = 8.2 and stirred for 30 min, then a solution with a final concentration of 0.1% PEG-20000 and 0.5% BSA is added to the mixed solution, stirred for 10 min, and then centrifuged at 9000g for 30 min, the supernatant is discarded, and the colloidal gold is resuspended in PBS with an original volume of 1 / 10 to obtain a mixed solution of C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label.

[0017] Further, the colloidal gold label pad is treated, and the specific steps are as follows: the mixed solution of C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label is diluted with a point membrane diluent, and is sprayed on the colloidal gold label pad at a rate of 2 μL / cm.

[0018] Further, the nitrocellulose membrane treatment, the specific steps are as follows: the PCV4-C157 sequence is coupled with bovine serum albumin, and a polypeptide C157-BSA is prepared, after being diluted with a dot membrane diluent, 2 μL / cm is sprayed on the detection line, and the rabbit IgG antibody is diluted with a dot membrane diluent, and 2 μL / cm is sprayed on the control line, wherein the concentration of the capture antigen dot membrane liquid is 3 μg / mL, and the concentration of the quality control antibody dot membrane liquid is 2 μg / L, wherein the dot membrane diluent comprises 0.15M sodium chloride, 10mM ethylenediaminetetraacetic acid, 0.1% proclin300 and 20g / L methanol in 0.02M phosphate buffer, and the rest is water.

[0019] Further, a colloidal gold test strip for detecting a pig PCV4 antigen, wherein a sample pad, a colloidal gold labeled pad, a nitrocellulose membrane and a water absorption paper are sequentially fixed on a substrate, the sample pad, the colloidal gold labeled pad, the nitrocellulose membrane and the water absorption paper partially overlap, and a part of the water absorption paper is overlapped with the nitrocellulose membrane.

[0020] Beneficial effects:

[0021] 1、The colloidal gold test strip for detecting a pig PCV4 antigen provided by the application, based on the specific polypeptide fragment prepared from PCV4 which has no cross reaction with PCV1, PCV2 and PCV3, uses the antibody as a detection approach, can quickly identify the PCV4 virus, is simple to operate, short in detection time and low in cost.

[0022] 2、The colloidal gold test strip for detecting a pig PCV4 antigen provided by the application is high in specificity, high in sensitivity, accurate in result, does not need any instrument and equipment, can be operated on site, and does not need to be operated by professional personnel. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is an SDS-PAGE electrophoretogram of the PCV4 specific antibody purified by Protein G;

[0024] Figure 2 It is a front view of the colloidal gold test strip for detecting a pig PCV4 antigen;

[0025] Marked for explanation:

[0026] 1, substrate, 2, sample pad, 3, colloidal gold labeled pad, 4, nitrocellulose membrane, 5, water absorption pad, 6, detection line, 7, control line. DETAILED DESCRIPTION

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

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

[0029] 1. Peptide design and synthesis

[0030] By comparing the homology of PCV4 with PCV1, PCV2, and PCV3 sequences, differential fragments were screened out. The sequence 157-172 (KPQDPSGETHTLHFQP) of the PCV4-cap protein sequence was designated as PCV4-C157. After synthesis, it was conjugated with the inert protein hemocyanin to prepare C157-KLH as an antigen for SPF mouse immunization. In addition, PCV4-C157 was conjugated with bovine serum albumin to prepare the polypeptide C157-BSA for detection line antibody spraying.

[0031] 2. Animal immunization

[0032] Six 8-10 week old mice were subcutaneously injected with 50 μg of C157-KLH per mouse once a week for a total of three times. The initial immunization was emulsified with Freund's complete adjuvant, and the booster immunization was emulsified with Freund's incomplete adjuvant. Seven days after the third immunization, the antibody titer in the serum was detected by indirect ELISA, and the mice with the highest titer were selected.

[0033] 3. PCV4-specific antibody purification:

[0034] Combination Figure 1 After obtaining serum by enucleating the eyes of immunized mice, Protein G agarose gel medium was loaded into an affinity purification chromatography column. The immunized serum was slowly loaded onto the column, and after antibody binding, it was eluted with glycine elution buffer to obtain the desired purified antibody. Figure 1 As shown in Figure a, serum before purification is displayed.

[0035] The sample contained a large amount of extraneous proteins. Figure b shows the purified serum sample. The image shows that protein bands are only present at 25 kDa (antibody light chain) and 55 kDa (antibody heavy chain), indicating that the antibody has been well purified.

[0036] Specific antibodies prepared based on specific polypeptide fragments in PCV4 which have no cross-reaction with PCV1, PCV2 and PCV3 can be used as a detection approach to quickly identify PCV4 virus, and the operation is simple, the detection time is short and the cost is low.

[0037] Example 2 Preparation method of a colloidal gold test strip for detecting porcine PCV4 antigen

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

[0039] After dialysis of C157-KLH specific antibody (final concentration 20 ug / ml) and goat anti-rabbit IgG (final concentration 40 ug / ml) in 0.005Mol / L pH7.0 NaCl solution at 4℃ overnight, mix with PH = 8.2

[0040] colloidal gold solution, stir for 30 min, then add 0.1% PEG-20000 and 0.5% BSA solution with a final concentration to the mixture, stir for 10 min, then centrifuge at 9000g for 30 min, discard the supernatant, resuspend the colloidal gold in PBS at 1 / 10 of the original volume, and obtain the C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label mixture.

[0041] 2. Treatment of colloidal gold label 3

[0042] Dilute the C157-KLH specific antibody colloidal gold label and goat anti-rabbit IgG colloidal gold label mixture with dot membrane diluent, and spray 2μL / cm on the colloidal gold label pad 3.

[0043] 3. Nitrocellulose membrane treatment

[0044] Dilute the C157-BSA protein with dot membrane diluent, and spray 2μL / cm on the detection line 6. Dilute the rabbit IgG antibody with dot membrane diluent, and spray 2μL / cm on the control line 7. The concentration of the capture antigen dot membrane solution is 3μg / mL, and the concentration of the quality control antibody dot membrane solution is 2μg / L. The dot membrane diluent contains 0.15M sodium chloride, 10mM ethylenediaminetetraacetic acid, 0.1% proclin300 and 20g / L methanol in 0.02M phosphate buffer, and the rest is water.

[0045] 4. Assembly

[0046] Fix the sample pad 2, colloidal gold label pad 3, nitrocellulose membrane 4 and water absorption pad 5 on the base plate 1 in sequence, with some overlap between the sample pad 2, colloidal gold label 3, nitrocellulose membrane 4 and water absorption pad 5, and part of the water absorption pad 5 overlaps with the nitrocellulose membrane 4. The assembled test strip structure is shown in Figure 2 .

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

[0048] Take the sample such as peripheral blood or tissue eluate, add 50ul sample diluent (0.01mol / L Tris-HCL pH7.6; 0.01mol / L NaCl; 0.005mol / L MgCl2) in it, mix and stir, then drop on the sample pad of the sample area, react for 5 minutes at room temperature, if the detection line T and the quality control line C both show color, then the sample has no PCV4 antigen; if the detection line does not show color and the quality control line shows color, the sample contains PCV4 antigen.

[0049] Example 4 Conformity test

[0050] 30 serum samples were detected by the colloidal gold test strip established by the present application and the ELISA blocking method technology at the same time, and the results are shown in Table 1:

[0051] Table 1: 30 serum samples are detected by ELISA and colloidal gold test strip and the results are compared.

[0052] Detection sample ELISA detection Test paper detection Number Blocking rate Judgment result 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 blocking rate of the elisa detection result is ≥25% for positive, and <25% for negative.

[0054] The results show that the colloidal gold detection method of the present application has a positive coincidence rate of 100%, a negative coincidence rate of 100%, and a total coincidence rate of 100% with the ELISA blocking method, which is consistent with the detection results of the ELISA blocking method and can be used for clinical monitoring.

[0055] The colloidal gold test strip for detecting porcine PCV4 antigen provided by the present application has high specificity, high sensitivity, accurate results, does not require any instrument and equipment, can be operated on site, does not require operation by professional personnel, and is simple and convenient.

[0056] It is obvious to those skilled in the art that the present application 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 essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application.

[0057] Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes every feature or implementation described herein. The specification can include implicit combinations of explicitly mentioned features and / or implicit combinations of implicitly mentioned features. Such combinations are also expressly included within the scope of the specification and an embodiment.

Claims

1. A PCV4-C157 recombinant antigen synthesized based on PCV4-specific protein sequences, characterized in that: The PCV4-C157 sequence is coupled with inert protein hemocyanin to obtain an antigen C157-KLH for preparing an antibody specific to swine PCV4, The PCV4-C157 sequence is KPQDPSGETHTLHFQP.

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

3. The PCV4-C157 recombinant antigen synthesized based on the PCV4-specific protein sequence according to claim 1, for use in the preparation of an anti-swine PCV4-specific antibody. The antigen C157-KLH is used to immunize SPF mice, serum is collected and antibodies in the serum are extracted to prepare a specific antibody. 8-10 week old mice are used, each of which is subcutaneously injected with 50 μg of C157-KLH at a time, once a week, for a total of 3 times, wherein the first immunization is emulsified with Freund's complete adjuvant, the reinforcement immunization is emulsified with Freund's incomplete adjuvant, and 7 days after the third inoculation, the antibody titer in the serum is detected by an indirect ELISA method, the mouse with the highest titer is selected, serum is collected, and the antibodies are purified by protein G. Protein G agarose gel medium is loaded into an affinity purification chromatography column, the post-immune serum is slowly loaded, and after the antibodies are combined, the antibodies are eluted with glycine elution buffer to obtain the desired purified antibodies.

4. The use of PCV4-C157 recombinant antigen synthesized based on PCV4 specific protein sequence according to claim 3 in the preparation of anti-porcine PCV4 specific antibody, characterized in that, The PCV4 specific protein sequence is a differential fragment obtained by comparing the homology of PCV4 with PCV1, PCV2 and PCV3 sequences.

5. The use of the PCV4-C157 recombinant antigen synthesized based on the PCV4-specific protein sequence according to claim 3 in the preparation of an antibody specific to porcine PCV4, characterized in that, Serum is obtained by blood collection from the enucleated eyes of the immunized mice.

Images