PCV3 rep protein, expression vector, monoclonal antibody, detection kit and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a PCV3 rep protein, an expression vector, a monoclonal antibody, a detection kit and application. The amino acid sequence of the PCV3 rep protein is as shown in SEQ ID NO. 1. An insect cell baculovirus expression vector system is used for expressing the PCV3 Rep protein, and the protein expressed by the vector system, the monoclonal antibody combined with the protein and a kit containing the Rep protein and / or the monoclonal antibody can be widely applied to related research of PCV3. After a pig is immunized by the PCV3 Cap subunit vaccine, an antibody combined with the PCV3 Cap protein exists in the serum of the pig, and an antibody combined with the PCV3 Rep protein does not exist in the serum of the pig. When a pig is infected by the whole virus of the PCV3 or immunized by an inactivated vaccine of the whole virus, an antibody capable of being combined with the Rep protein of the PCV3 exists in serum of the pig. Therefore, whether the pig immunized by the PCV3 Cap subunit vaccine is infected by the PCV3 virus or not is judged by utilizing the difference between the PCV3 Cap subunit vaccine and the PCV3 Cap subunit vaccine, and the method is convenient and quick.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a PCV3 rep protein, an expression vector, a monoclonal antibody, a detection kit and application. BACKGROUND

[0002] Porcine circovirus disease (PCV) is an infectious disease that destroys the immune system of pigs caused by a circular virus. The pathogenic virus is mild and can coexist with pigs. After the pigs are infected with the virus, the immune system is often damaged, and other diseases are triggered to cause multisystemic diseases, thereby causing a large number of deaths in the pig population. So far, four circular viruses have been found and identified: PCV1, PCV2, PCV3 and PCV4. Among them, PCV2, PCV3 and PCV4 are pathogenic viruses, and PCV2 and PCV3 have strong infectivity. In particular, PCV3 is currently widely prevalent and transmitted in China, and has caused typical clinical symptoms in pig farms, resulting in huge economic losses and posing a huge challenge to the prevention and control of PCV3 diseases.

[0003] At present, PCV2 and PCV3 subunit vaccines on the market all express Cap protein as antigen protein and play a good role in disease prevention and control, while the rep protein of PCV3 is not expressed as antigen protein. After being immunized with PCV3 subunit vaccine, the corresponding rep protein is not produced, but wild virus infection can produce corresponding rep antibodies. Enzyme-linked immunosorbent assay (ELISA) has become an effective technical means for PCV3 detection due to its simple operation and high sensitivity, but there is no rep protein antibody diagnostic kit for PCV3.

[0004] Therefore, the application establishes a chemiluminescence method, which can monitor the immune PCV3 efficacy evaluation of the pig population and the PCV3 viral load of the pig population under the condition of immunizing PCV3 subunit vaccine. SUMMARY

[0005] In view of the deficiencies of the prior art, the first purpose of the application is to provide a PCV3 rep protein.

[0006] The second purpose of the application is to provide an expression vector for expressing the PCV3 rep protein, which alleviates the problem of lack of a vector and system capable of expressing the PCV3 Rep protein in the prior art.

[0007] The third purpose of the application is to provide a monoclonal antibody combined with the PCV3 rep protein, which can be widely used in PCV3 related research and lays a material foundation for PCV3 related molecular biology experiments.

[0008] A fourth object of the present application is to provide a chemiluminescence detection kit, which has a sensitivity of 10-100 times that of Elisa, can directly obtain RLU values, realizes semi-quantitative analysis of antibody titers, is compatible with automatic plate washing machines and detection equipment, is suitable for large-scale detection, does not require termination of the reaction, reduces human operation errors, and can be applied to various molecular biology experiments for studying PCV3.

[0009] A fifth object of the present application is to provide the use of the chemiluminescence detection kit described above.

[0010] A sixth object of the present application is to provide a chemiluminescence method for detecting PCV3.

[0011] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0012] A PCV3 rep protein, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene of which is shown as SEQ ID NO. 2.

[0013] The PCV3 rep protein is one of the important non-structural proteins in PCV3 and can induce the body to produce corresponding antibodies. When pigs are infected with PCV3 or immunized with PCV3 whole-virus inactivated vaccine, antibodies against Cap and Rep proteins can be produced, and subunit vaccines can produce immunizing efficacy through Cap, but not Rep protein. Therefore, the protein has great significance in the research of PCV3 subunit vaccine differential diagnosis.

[0014] An expression vector, which is a baculovirus, expresses the encoding gene of the PCV3 rep protein described above.

[0015] The baculovirus is an insect cell baculovirus.

[0016] Specifically, in some embodiments of the present application, the insect cell is an Sf-9 cell.

[0017] The insect cell baculovirus expression vector system (IC-BEVS) has the traditional advantages of eukaryotic expression systems, and at the same time has the characteristics of high expression of recombinant proteins of prokaryotic expression systems. Specifically, in some embodiments of the present application, the PCV3 Rep protein is expressed by using the insect cell baculovirus expression vector system.

[0018] A monoclonal antibody which binds to the PCV3 rep protein described above. This monoclonal antibody can be widely used in PCV3 related research, laying a material foundation for PCV3 related molecular biology experiments, for example, it can be used in immunoblotting, immunohistochemistry, enzyme-linked immunoassay or immunofluorescence techniques.

[0019] A chemiluminescence detection kit, which comprises the monoclonal antibody described above, or the horseradish peroxidase-labeled monoclonal antibody described above.

[0020] The chemiluminescence detection kit further comprises an antigen-coated plate, negative serum, positive serum, sample diluent, washing solution, and chemiluminescent substrate solution.

[0021] Specifically, the antigen is PCV3 Rep protein; the negative serum is PCV3 Rep negative serum; the positive serum is PCV3 antibody negative pig serum, and the serum after immunization with PCV3 Cap subunit vaccine.

[0022] The chemiluminescence detection kit described above is also within the scope of the present application.

[0023] The application of the chemiluminescence detection kit in detecting PCV3 includes detecting whether the sample to be tested is infected with PCV3; whether the sample to be tested produces anti-PCV3 antibodies after immunization with PCV3 whole virus (tissue) inactivated vaccine; and whether the sample to be tested contains PCV3 related antibodies.

[0024] A method for screening pigs infected with PCV3 wild strain, by detecting whether the serum of the pig immunized with PCV3 Cap subunit vaccine contains antibodies that bind to the PCV3 Rep protein, to determine whether the pig immunized with PCV3 Cap subunit vaccine is infected with PCV3 wild strain.

[0025] The detection method is a chemiluminescence method for PCV3, specifically, PCV3 Rep protein is used as an antigen to coat an ELISA enzyme-labeled plate, the sample to be tested is incubated in the ELISA enzyme-labeled plate, horseradish peroxidase-labeled monoclonal antibody is added, and chemiluminescent substrate solution is added to read the RLU value (relative light unit).

[0026] Then the blocking rate of the sample is calculated according to the value of chemiluminescence; when the blocking rate of the sample is > 35%, it is determined to be positive, ≤ 30% is determined to be negative, and 30% < blocking rate ≤ 35% is a suspicious sample.

[0027] Further, the blocking rate is calculated as follows: blocking rate = (RLU average of negative control - RLU average of sample) / RLU average of negative control * 100%.

[0028] Specifically, the conditions of the chemiluminescence method for the PCV3 include: 0.1-1 µg / mL antigen coating concentration, antigen coating conditions are divided into 37℃, 2-12 h; the blocking solution is 2-5% BSA; the blocking condition is 37℃, 1-3 h; the dilution ratio of negative serum and positive serum is 1:1-50; the serum incubation condition is 37℃, 0.5-2.5 h; the dilution ratio of enzyme-labeled monoclonal antibody is 1:1000-3000; the enzyme-labeled antibody incubation condition is 37℃, 0.5-2 h; after adding 0.4-1 mmol / L chemiluminescent substrate liquid Luminol, the RLU value (relative light unit) is read in a chemiluminescence detector.

[0029] Preferably, the conditions of the chemiluminescence method include: 0.5 µg / mL antigen coating concentration, antigen coating conditions are divided into 37℃, 2 h; the blocking solution is 2% BSA; the blocking condition is 37℃, 2 h; the dilution ratio of negative serum and positive serum is 1:50; the serum incubation condition is 37℃, 1 h; the dilution ratio of enzyme-labeled monoclonal antibody is 1:1000; the enzyme-labeled antibody incubation condition is 37℃, 1 h; after adding 0.6 mmol / L chemiluminescent substrate liquid Luminol, the RLU value (relative light unit) is read in a chemiluminescence detector.

[0030] Beneficial effects:

[0031] The expression vector for expressing the PCV3 Rep protein provided by the application is expressed by using an insect cell baculovirus expression vector system, and has the advantages of high safety, short vector construction cycle and high protein expression level.

[0032] The Rep protein expressed by the above-mentioned expression vector provided by the application can produce antibodies against the Rep protein when pigs are infected with PCV3 or immunized with a PCV3 whole virus inactivated vaccine. Therefore, the protein has very important significance in the research of PCV3 subunit vaccine differential diagnosis.

[0033] The monoclonal antibody provided by the application and combined with the above-mentioned PCV3 Rep protein can be widely applied to the research of PCV3, and lays a material foundation for PCV3 related molecular biology experiments.

[0034] The kit provided by this invention contains PCV3 Rep protein and / or monoclonal antibodies that bind to PCV3 Rep protein, and can be used in a variety of molecular biology experiments for studying PCV3, such as immunoblotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), or immunofluorescence techniques.

[0035] The PCV3 Rep protein, monoclonal antibody or kit bound to the PCV3 Rep protein provided by the present invention are widely applicable to the detection of PCV3 and the proteins contained in PCV3 or antibodies against PCV3.

[0036] The method for screening pigs infected with wild-type PCV3 provided by this invention can identify pigs infected with wild-type PCV3 from those immunized with a PCV3 Cap subunit vaccine. After immunization with a PCV3 Cap subunit vaccine, the pig's serum contains antibodies that bind to the PCV3 Cap protein but not antibodies that bind to the PCV3 Rep protein. However, after infection with whole PCV3 virus or immunization with an inactivated whole-virus vaccine, the pig's serum contains antibodies that bind to the PCV3 Rep protein. Therefore, this invention utilizes the difference between these two scenarios—the presence of antibodies that bind to the PCV3 Rep protein in the serum of the test sample—to determine whether pigs immunized with a PCV3 Cap subunit vaccine have been reinfected with PCV3 virus. This method uses molecular biology techniques; only pig serum needs to be collected, and the specific reaction between antigen and antibody can be used to detect whether the sample providing the serum is infected with PCV3 virus. Therefore, this method has the advantages of simple operation and high throughput. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 A flowchart illustrating the construction of the expression vector provided in Embodiment 1 of the present invention.

[0039] Figure 2 This is an electrophoresis image of the PCV3 Rep gene amplification. M: DL2000 Marker; 1-3: positive samples; 4: negative control.

[0040] Figure 3This image shows the identification of the PCV3 Rep gene after transfection into Sf-9 cells. In the image, M: DL2000 Marker, 1: Sf-9 cell sample infected, 2: Sf-9 cell sample infected, and 3: negative control.

[0041] Figure 4 This is a diagram identifying PCV3 Rep protein expression after transfection into Sf-9 cells. M: Protein Marker; 1-3: Positive samples; 4: Negative control.

[0042] Figure 5 A graph used to identify whether the prepared monoclonal antibody reacts with PCV3 Rep recombinant baculovirus.

[0043] Figure 6 This serves as a negative control to identify whether the prepared monoclonal antibody reacts with PCV3 Rep recombinant baculovirus. Detailed Implementation

[0044] The technical solutions of the present invention will now be clearly and completely described in conjunction with the embodiments and accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0045] In the following examples, the Sf-9 insect cells were purchased from Invitrogen Life Sciences, Inc., USA; the horseradish peroxidase-labeled rabbit anti-pig secondary antibody was purchased from Sigma-Aldrich; the FITC-labeled rabbit anti-pig IgG secondary antibody was purchased from Sigma-Aldrich; and the 6-8 week old female Balb / c mice were purchased from Xi'an Yifengda Biotechnology Co., Ltd.

[0046] Example 1: Amplification of the PCV3 rep gene

[0047] The PCV3 Rep gene was codon-optimized to obtain the optimized PCV3 Rep gene (amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as shown in SEQ ID NO:2). Based on the optimized PCV3 Rep gene's corresponding nucleotide sequence, upstream primer BacREP-F1 and downstream primer BacREP-R1 were designed and synthesized. Using the optimized PCV3 Rep gene fragment as a template, PCR amplification was performed using high-fidelity Pfu DNA polymerase to obtain the PCR product. Specifically, the primers introduced TOPO at the 5' end of the PCR product. ® Cloningsite (CACC) and Kozak transcription start sequence containing ATG (ACCATGG).

[0048] Among them, BacREP-F1: 5'-CACCATGGTTGGAACCATGGTTAGAAGGGAA-3';

[0049] BacREP-R1: 5'-CACCATAATTGATCGGGTACAAGGT-3'.

[0050] The PCR reaction system consisted of the optimized PCV3 Rep gene fragment as a template, with the following added: 1 μL 10 μM BacREP-F1 primer, 1 μL 10 μM BacREP-R1 primer, 5 μL 2.5 mM dNTPs, 5 μL 10×PCR buffer, 1 μL Pfu DNA polymerase, and 36 μL ddH2O. The PCR reaction parameters were: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 57℃ annealing for 30 seconds, and 72℃ extension for 1 minute, for 35 cycles; and a final extension at 72℃ for 10 minutes.

[0051] After the reaction, the PCR amplification products were analyzed by 1% agarose gel electrophoresis at 100 volts. The electrophoresis results are as follows: Figure 2 As shown in the figure, the target gene is approximately 909 bp in size. The purified target gene product was then recovered using the QIAquick PCR purification kit according to the instruction manual. After purification, the product was confirmed by 1.0% agarose gel electrophoresis, and the RLU value was measured using a chemiluminescence detector to calculate the recovered DNA concentration. The DNA was then stored at -20°C for later use.

[0052] Example 2: Construction of a baculovirus transfer vector

[0053] (1) TOPO ® Cloning reaction: TOPO was performed using the Invitrogen TOPO cloning kit. ® Cloning reaction. The reaction system is as follows: 0.5–4 μL of purified blunt-end PCR product (containing 5–10 ng DNA), 1 μL of salt solution (1.2 mol / L NaCl, 0.06 mol / L MgCl2), 1 μL of pENTR / D-TOPO ® Add ddH2O up to 6 μL to the carrier. After mixing, place the reaction tube at room temperature for 15 minutes, then place it on ice to obtain TOPO. ® Cloning reaction solution, ready for use.

[0054] (2) Plasmid transformation: Take 2 μL of TOPO obtained in step (1) ® cloning reaction solution added to One Shot® TOP10 Chemically Competent E. coli (Invitrogen) was placed in a reaction tube, mixed well, and placed on ice for 30 minutes. Then, it was heat-shocked in a 42°C water bath for 30 seconds. The tube was then quickly returned to ice to cool for 2 minutes. Next, 250 μL of pre-warmed SOC culture medium was added to the reaction tube, and the tube was placed in a 37°C constant temperature shaking incubator with shaking at 200 r / min for 60 minutes. 50 μL of the above bacterial suspension was evenly spread onto an LB-Kanamycin selection plate containing 50 μg / mL kanamycin. The plate was placed face up for 30 minutes until the bacterial suspension was completely absorbed by the culture medium. The plate was then inverted and incubated overnight at 37°C.

[0055] (3) Screening of PCV3 Rep recombinant transfer vectors: PCV3 Rep recombinant transfer vectors were initially screened by PCR amplification. Twenty-four single clones were picked from the LB-kana screening plate in step (2) and inoculated into 1 mL of LB-kana culture medium. The clones were then cultured overnight in a constant temperature shaking incubator at 37℃ with a shaking speed of 200 r / min to obtain 24 groups of bacterial cultures.

[0056] The PCR reaction system was as follows: 2 μL of bacterial culture obtained overnight was used as PCR templates for 24 groups of bacteria, and 0.5 μL of 10 μM BacREP-F1 primer, 0.5 μL of 10 μM BacREP-R1 primer, 2.5 μL of 2.5 mM dNTPs, 2.5 μL of 10×PCR buffer, 0.5 μL of Pfu DNA polymerase, and 16.5 μL of ddH2O were added respectively. The PCR reaction parameters were: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 57℃ annealing for 30 seconds, 72℃ extension for 1 minute, 35 cycles; and 72℃ extension for 10 minutes.

[0057] After the reaction, the PCR amplification products were analyzed by electrophoresis on 1.0% agar gel at 100 volts. If the expected products appeared, 4-5 corresponding bacterial cultures were selected and inoculated into 3 mL of LB-Kana culture medium at a ratio of 1‰. The cultures were then incubated overnight in a constant temperature shaking incubator at 37℃ with a shaking speed of 200 r / min.

[0058] (4) Extraction of PCV3 Rep recombinant transfer vector: using QIAprep TMThe recombinant vector was extracted using the Spin Plasmid Kit. Specifically, 3 mL of bacterial culture was centrifuged at 10000 r / min for 4 min to remove the supernatant. Then, 250 μL of buffer P1 (containing 100 mg / mL RNase A) was added to resuspend the bacterial cells. The resuspended mixture was transferred to a 1.5 mL centrifuge tube, 250 μL of buffer P2 was added, and the mixture was inverted several times until the liquid became transparent and viscous. Then, 350 μL of buffer N3 was added, and the mixture was slowly shaken several times. The mixture was then centrifuged at 12000 r / min for 10 min. Aspirate the supernatant and add it to a QIAprep spin column. Centrifuge at 12000 rpm for 1 min, discard the filtrate, add 750 μL of buffer PE to the column to wash away excess salts, centrifuge at 12000 rpm for 1 min, discard the filtrate, and centrifuge again for 2 min to avoid liquid residue. Finally, transfer the column to a new centrifuge tube, add 20 μL of double-distilled water, let stand for 1 min to allow the vector DNA to fully dissolve, centrifuge at 12000 rpm for 1 min, collect the filtrate, and store below -20℃. After digestion with the restriction endonuclease NotI, the recombinant vectors are analyzed by electrophoresis on a 1.0% agarose gel at 100 volts. If the size matches the expected product, select two extracted recombinant vectors for analysis using pENTR / D-TOPO. ® The vector itself was sequenced and verified.

[0059] Among them, M13F: 5'-GTAAAACGACGGCCAG-3'

[0060] M13R: 5'-CAGGAAACAGCTATGAC-3'

[0061] (5) Preservation of PCV3 Rep recombinant transfer vector: E. coli strain containing PCV3 Rep recombinant transfer vector was inoculated into 5 mL of LB-kana culture medium containing 50 μg / mL kanamycin resistance, placed in a constant temperature shaking incubator at 37℃, and shaken at 200 r / min for 16 h. 20% glycerol was added, aliquoted, and stored at -70℃ or below.

[0062] Example 3: Harvesting and Identification of PCV3 Rep Recombinant Baculovirus

[0063] (1) Obtaining baculovirus using LR recombination reaction: The PCV3 Rep recombinant transfer vector, verified by sequencing in Example 2, was extracted, and the nucleic acid concentration of the recombinant transfer vector was measured at a wavelength of 260 nm using a spectrophotometer. Then, the nucleic acid concentration was determined using the LR recombination reaction. TMThe Baculovirμs Expression System was used for LR recombination. The specific steps are as follows: 1 μL of PCV3 Rep recombinant transfer vector (100-300 ng) and 4 μL of 5×LR Clonase were added. ® 10 μL of BaculRLUirect was added to the reaction buffer and 1 μL of ddH2O, respectively. TM Linear DNA (300 ng). LR Clonase taken from a -70°C freezer. ® After warming the enzyme mix on ice for 2 minutes, shake twice for 2 seconds each time. Take 4 μL of LR Clonase. ® Add the enzyme mix to the above mixture and mix gently. After incubating at 25°C for 18 h, add 2 μL of Proteinase Ksolution and incubate at 37°C for 10 min to interrupt the LR recombination reaction and obtain the LR recombination reaction solution containing recombinant baculovirus DNA.

[0064] (2) Transfection: Take a 6-well cell culture plate and seed 1.5 × 10⁶ cells per well. 6 Two replicates were performed on each well containing 10 viable Sf-9 cells (cell viability > 95%). The Sf-9 cells were incubated at 26–28°C for 1 hour to allow complete cell attachment to the bottom of the culture plate. Transfection mixture A (20 μL LR recombinant reaction solution containing recombinant baculovirus DNA + 200 μL SF900II serum-free insect cell culture medium) and transfection mixture B (12 μL Cellfection...) were prepared. ® The reagent and 200 μL of SF900II serum-free insect cell culture medium were gently mixed and then incubated at room temperature for 45 minutes to obtain mixture A and B.

[0065] First, carefully wash the Sf-9 cells attached to the bottom of the culture plate with SF900II serum-free insect cell culture medium. Then, divide the mixture of A and B into two tubes, add 800 μL of SF900II serum-free insect cell culture medium to each tube, and invert the tubes several times. Finally, drain as much culture medium as possible from the 6-well plate, and slowly add the mixture of A and B along the tube wall into the two wells. Seal the cell culture plate and incubate at 26-28℃ for 5 h. Remove the mixture, add 3 mL of Sf-900II SFM containing antibiotics and 100 μM ganciclovir to each well, seal with transparent tape, and incubate at 26-28℃ for 5 days to obtain the virus solution.

[0066] (3) Harvesting PCV3 Rep recombinant baculovirus: Collect the virus solution into a 50 mL centrifuge tube, centrifuge at 3000 r / min for 5 min to remove cell debris, then add 10% fetal porcine serum to the virus solution, store in the dark at 4℃ or below for later use, or store for long-term use at -70℃ or below. For primary isolation after transfection, filter through a 0.45 μm filter membrane, then aliquot into 1.5 mL centrifuge tubes to harvest PCV3 Rep recombinant baculovirus.

[0067] (4) Identification of PCV3 Rep recombinant baculovirus:

[0068] Gene identification: DNA was extracted from the supernatant of Sf-9 cells infected for 4 days. PCR amplification of the DNA in the supernatant was performed using primers BacREP-F1 and BacREP-R1. The results are as follows: Figure 3 As shown in the figure, the amplified target gene fragments are of similar size, which preliminarily indicates the presence of the target gene in the infected Sf-9 cells.

[0069] Rep protein expression analysis: The expression and reactivity of PCV3 Rep protein were verified by Western blot. First, SDS-PAGE was performed. Then, the protein bands on the SDS-PAGE gel were transferred to a PVDF membrane, placed in PBST containing 5% skim milk powder, blocked at room temperature for 30 min, washed 5 times, and then primary antibody PCV3 Rep positive serum was added. After incubation at room temperature for 1 h, washed 5 times, and then secondary antibody containing horseradish peroxidase-conjugated rabbit anti-pig antibody was added. After incubation at room temperature for 1 h, washed 5 times, and finally, DAB staining was performed in a dark room. The results are as follows: Figure 4 As shown in the figure, Western blot analysis of PCV3 Rep protein expression revealed a band of approximately 35 kDa, indicating that the expressed protein is reactive.

[0070] Immunofluorescence: The constructed PCV3 Rep recombinant baculovirus was inoculated into Sf-9 cells and cultured in a constant temperature incubator at 26~28℃ for 7 days. After 7 days, each well was washed 6 times with PBST, then 100 μL of formaldehyde was added for fixation for 10 min. Primary antibody PCV3Rep positive serum (1:1000) was added, and the cells were incubated for 1 h. FITC-labeled rabbit anti-pig IgG secondary antibody was added, and the presence or absence of fluorescence signal was observed under an inverted fluorescence microscope.

[0071] Example 4: Preparation of Monoclonal Antibodies

[0072] Six- to eight-week-old female Balb / c mice were immunized with 50 μg / mouse of purified PCV3 Rep recombinant protein, followed by a final pulse immunization. Spleen cells from the immunized mice were fused with SP2 / 0 myeloma cells, and subcloning was performed four times. Indirect ELISA was used for screening to obtain positive hybridoma cell lines that secrete antibodies against PCV3 Rep protein. Hybridoma cells from six- to eight-week-old female Balb / c mice were expanded using Gibco CD hybridoma medium, with 1 × 10⁻⁶ cells per mouse. 6 Ascites fluid was prepared by intraperitoneal injection of cells, and the collected ascites fluid was purified and HRP-labeled. Sf9 cells were infected with PCV3 Rep baculovirus solution, and indirect immunofluorescence detection was performed using purified monoclonal antibody as the primary antibody and FITC-labeled rabbit anti-mouse IgG as the secondary antibody to identify the specificity of the monoclonal antibody. Results are as follows: Figure 5 , 6 As shown in the figure, Sf9 cells infected with PCV3 Rep baculovirus exhibit specific fluorescence, while those infected with negative baculovirus show no fluorescence.

[0073] Example 5: A method for screening pigs infected with wild-type PCV3 strains using chemiluminescence

[0074] (1) Establishment of chemiluminescence method and optimization of reaction conditions

[0075] PCV3 Rep protein was coated onto ELISA plates at concentrations of 1 µg / mL, 0.5 µg / mL, 0.25 µg / mL, and 0.1 µg / mL (50 µL / well). The antigen coating conditions were 37℃ for 2 h, 37℃ for 4 h, and 37℃ for 12 h. After washing three times with PBST, 200 µL of 2% BSA was added to each well for blocking at 37℃ for 1 h, 2 h, and 3 h. Positive and negative sera were diluted 1:1, 1:5, 1:10, and 1:50, and added to each well at 100 μL. The plates were then incubated at 37℃ for 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h. Finally, after washing three times with PBST, 100 μL of enzyme-labeled monoclonal antibody was diluted 1:1000 and added to each well. The plates were then incubated at 37℃ for 1 hour. h; wash 6 times with PBST, then add 0.4~1mmol / L chemiluminescent substrate solution Luminol to 100 μL / well, and take readings after 2~5 min at room temperature.

[0076] The reaction plate was placed in a chemiluminescence analyzer to detect the chemiluminescence value of each reaction well, and the blocking rate was calculated. The blocking rate was calculated according to the following formula: Blocking rate = (average RLU of negative control - average RLU of sample) / average RLU of negative control × 100%. A sample was considered positive if the blocking rate was >35%, negative if ≤30%, and suspicious if 30% < blocking rate ≤35%.

[0077] By optimizing the reaction conditions in the chemiluminescence method and calculating the blocking rate, the optimal reaction conditions were determined to be: protein coating concentration of 0.5 µg / mL, 50 µL / well; coating time of 37℃ for 2 h; blocking buffer of 2% BSA, 200 µL / well, at 37℃ for 2 h; positive and negative sera diluted 1:50, 100 μL / well, incubated at 37℃ for 1 h; enzyme-labeled monoclonal antibody diluted 1:1000, 100 μL / well, incubated at 37℃ for 1 h; and finally, 100 μL / well of 0.6 mmol / L chemiluminescent substrate solution luminol was added as the optimal reaction conditions.

[0078] (2) Sensitivity verification: Sensitivity verification was performed using each test sample, which included the following samples: swine serum immunized with PCV3 tissue inactivated vaccine; swine serum infected with whole PCV3 virus; swine serum immunized with Cap subunit vaccine; and PCV3 Rep antibody positive serum.

[0079] The results were as follows: the positive rate of Rep antibody in the serum of pigs immunized with PCV3 tissue inactivated vaccine was 96.2%; the positive rate of Rep antibody in the serum of pigs infected with whole PCV3 virus (PCR detection of viremia positive) was 100%; the positive rate of Rep antibody in the serum of pigs immunized with Cap subunit vaccine was 0%; and the positive rate of Rep antibody in PCV3 Rep antibody-positive serum was 0%.

[0080] (3) Specificity verification: Specificity verification was performed using each sample to be tested. The chemiluminescence method with optimized conditions described above was used to verify the specificity of positive sera infected with porcine circovirus type 3 (PCV3), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), and classical swine fever virus (CSFV). The results confirmed that the chemiluminescence kit did not react with positive sera from other pathogens, but only with positive sera infected with porcine circovirus type 3, and showed a positive result.

[0081] Example 6: Application of the method for screening pigs infected with wild-type PCV3 strains

[0082] Before immunization, 200 pig serum samples were collected as test serums and measured using the chemiluminescence method optimized in Example 5. The results showed that the Rep antibody positivity rate was 12.5%. 170 pigs were randomly selected from Rep antibody-negative pigs for immunization challenge experiments.

[0083] Pigs that were negative for Rep antibodies were isolated and fed a variety of vaccines. Eighty pigs were immunized with porcine circovirus type 3 (PCV3) tissue-inactivated vaccine, 80 pigs were immunized with PCV3 type 3 Cap subunit vaccine, and the remaining 10 pigs were not immunized. Blood samples were collected after 35 days to test for Rep antibodies. The detection method described above showed that the positive rate of Rep antibodies in the group immunized with the PCV3 tissue-inactivated vaccine was 96.2%, and the detection rate after challenge was 100%. The positive rate of Rep antibodies in the group immunized with the PCV3 type 3 Cap subunit vaccine and the non-immunized group was 0%, and the detection rate after challenge was 100%. This demonstrates that the detection method has good sensitivity and specificity for detecting infection with tissue-inactivated vaccines or wild-type viruses. It can be used to detect PCV3 type 3 Rep antibodies and can be used for screening pigs infected with wild-type PCV3 strains, clinical eradication, or evaluating vaccine immunization efficacy.

[0084] This invention provides a concept and method for PCV3 rep protein, expression vector, monoclonal antibody, detection kit, and applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A PCV3 rep protein, characterized in that, The amino acid sequence of the PCV3 rep protein is shown in SEQ ID NO.

1.

2. The PCV3 rep protein according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the PCV3 rep protein is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, The expression vector is a baculovirus that expresses the gene encoding the PCV3rep protein as described in claim 2.

4. A monoclonal antibody, characterized in that, The monoclonal antibody binds to the PCV3 rep protein of claim 1.

5. A chemiluminescence immunoassay kit, characterized in that, The chemiluminescence detection kit includes the monoclonal antibody of claim 4, or the monoclonal antibody of claim 4 labeled with horseradish peroxidase.

6. The chemiluminescence detection kit according to claim 5, characterized in that, The chemiluminescence detection kit includes an antigen-coated plate, negative serum, positive serum, sample diluent, washing solution, and chemiluminescence substrate solution.

7. The chemiluminescence detection kit according to claim 6, characterized in that, The antigen is PCV3 Rep protein; the negative serum is PCV3 Rep negative serum; and the positive serum is serum from pigs immunized with PCV3 Cap subunit vaccine that is PCV3 antibody negative.

8. The application of the chemiluminescence detection kit according to any one of claims 5 to 7 in the detection of PCV3.

9. A method for screening pigs infected with wild-type PCV3 strains, characterized in that, The presence of antibodies binding to the PCV3 Rep protein as described in claim 1 in the serum of pigs immunized with the PCV3 Cap subunit vaccine is used to determine whether the pigs immunized with the PCV3 Cap subunit vaccine are infected with the wild-type PCV3 strain. The detection method is a chemiluminescence method for PCV3. Specifically, PCV3 Rep protein is used as an antigen to coat the ELISA plate. After incubating the sample in the ELISA plate, horseradish peroxidase-labeled monoclonal antibody is added, and the chemiluminescence value is detected after incubation.

10. The method according to claim 9, characterized in that, The conditions for the chemiluminescence method of PCV3 include: an antigen coating concentration of 0.1–1 µg / mL; antigen coating conditions of 37°C for 2–12 h; blocking buffer of 2–5% BSA; blocking conditions of 37°C for 1–3 h; a negative serum to positive serum dilution ratio of 1:1–50; serum incubation conditions of 37°C for 0.5–2.5 h; enzyme-labeled monoclonal antibody dilution ratio of 1:1000–3000; enzyme-labeled antibody incubation conditions of 37°C for 0.5–2 h; addition of 0.4–1 mmol / L chemiluminescence substrate solution luminol; and then reading the RLU value using a chemiluminescence detector.

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