CN117866053A - Polyclonal antibody for simultaneously detecting multiple porcine parvoviruses and preparation and application thereof - Google Patents
Polyclonal antibody for simultaneously detecting multiple porcine parvoviruses and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a polyclonal antibody for simultaneously detecting multiple porcine parvoviruses, and preparation and application thereof, and relates to the technical field of biological detection of parvovirus detection. The invention provides a polyclonal antibody, antigen protein for obtaining the polyclonal antibody by animal immunity, and application of the polyclonal antibody in preparing a product for detecting porcine parvovirus. The polyclonal antibody can detect eight porcine parvovirus serotypes simultaneously, has wider detection coverage to porcine parvovirus, strong specificity and high detection sensitivity; the operation is simple, the time and the labor are saved, and the sample quantity to be measured and the detection cost are saved.
Description
Technical Field
The invention relates to the technical field of biological detection of parvovirus detection, in particular to a polyclonal antibody capable of detecting multiple porcine parvoviruses simultaneously.
Background
Parvovirus is a small, highly adaptable, non-enveloped, single-stranded DNA virus with a genome of 4-6kb, and two open reading frames encoding nonstructural and capsid proteins, respectively. The capsid of porcine parvovirus is a spherical shell consisting of 60 capsid proteins, VP2 being the major capsid protein. The earliest porcine parvovirus PPV1 was discovered in 1965 in Germany, and several subtypes such as PPV2-PP8 were discovered successively in the last two decades.
Porcine parvovirus is a virus with extremely strong infectivity, mainly infections of fetuses and embryos, causes the phenomena of dead fetuses, malformed fetuses, mummy fetuses and the like, and causes great threat and loss to the breeding industry. In addition, porcine parvovirus is also a potential risk for bioproduct safety, as related raw materials of porcine origin are often used in the production process of bioproducts. Based on the detection, the detection of porcine parvovirus is one of important links in the breeding industry and the production of biological products.
The regulatory authorities in each country also make corresponding regulatory mechanisms for this, and in the general rule "animal cell matrix preparation and quality control for biological production assays" for biological production in chinese pharmacopoeias, it is pointed out that if pancreatin is used in the cell matrix in the cell bank established or passaged history before the producer, the established MCB or WCB and/or cells exceeding the production limit level should be detected at least once for exogenous viruses related to animals from which pancreatin is derived, including porcine parvovirus or bovine parvovirus. United states Pharmacopeia <1050> and <1237> also indicate that detection of corresponding viruses during manufacture of biologicals can be used to correlate detection of swine-derived viruses using infectious cell methods, blood adsorption and immunofluorescence. Federal regulation 9CFR also indicates the need for immunofluorescence detection of porcine parvovirus in biologicals.
Currently, molecular method detection is a common means for detecting porcine parvovirus, such as PCR molecular detection technology, RPA isothermal amplification technology or LAMP molecular detection technology; the method is a good detection method aiming at the detection of exogenous factors with high conservation and strong detection specificity. However, porcine parvovirus is of a large variety, with very low conservation of genes between each subtype, and existing molecular detection techniques have difficulty achieving effective coverage of all porcine parvovirus subtypes. The mutation rate of porcine parvovirus at the nucleic acid level is high, so that the detection stability of the molecular means is reduced. Therefore, it is urgently required to establish a porcine parvovirus detection method with high coverage and strong stability.
Disclosure of Invention
The invention prepares a polyclonal antibody capable of identifying a plurality of porcine parvoviruses simultaneously, and can detect all porcine parvovirus subtypes discovered at present; in theory, the research on the expression of the porcine parvovirus capsid protein can be realized by combining a western technology, and the high-sensitivity detection of the porcine parvovirus can be realized by combining an ELISA technology. Therefore, the polyclonal antibody provided by the invention can greatly promote the research and detection progress of porcine parvovirus.
In a first aspect of the present invention, there is provided an antigen protein for preparing a polyclonal antibody for simultaneously detecting a plurality of porcine parvoviruses, comprising an amino acid sequence corresponding to PPV1 as shown in SEQ ID No. 1; the amino acid sequence of the corresponding PPV2 shown as SEQ ID NO. 2; the amino acid sequence of the corresponding PPV3 shown as SEQ ID NO. 3; the amino acid sequence of the corresponding PPV4 shown as SEQ ID NO. 4; the amino acid sequence of the corresponding PPV5 shown as SEQ ID NO. 5; the amino acid sequence of the corresponding PPV6 shown in SEQ ID NO. 6; the amino acid sequence of the corresponding PPV7 shown in SEQ ID NO. 7; the amino acid sequence of the corresponding PPV8 shown as SEQ ID NO. 8; a linker sequence is also connected between two adjacent amino acid sequences.
Further, the amino acid sequence of the linker sequence is a natural amino acid sequence or an artificial synthetic sequence.
In theory, the sequence of PPV1-8 does not affect the effect of immunization of animals, and thus does not affect the acquisition of polyclonal antibodies. Therefore, the sequences of the PPV1-8 provided by the invention can be combined in sequence, and the combination sequence of the 8 sequences can be properly adjusted according to actual needs. No matter how the sequence is adjusted, the invention is within the protection scope of the invention.
It should be noted that, the linker sequence selected in the present invention can be referred to in the following documents:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514284/。
alternatively, the linker sequence selected for use in the present invention is theoretically possible as long as it is a combined sequence comprising two amino acids of GS.
Further, the amino acid sequence of the linker sequence is shown as SEQ ID NO.11 or SEQ ID NO. 12.
Further, the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO. 13; the nucleotide sequence of the amino acid sequence shown in the SEQ ID NO.2 is shown in SEQ ID NO.14, and the nucleotide sequence of the amino acid sequence shown in the SEQ ID NO.3 is shown in SEQ ID NO. 15; the nucleotide sequence of the amino acid sequence shown in the SEQ ID NO.4 is shown in SEQ ID NO. 16; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.5 is shown in SEQ ID NO. 17; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.6 is shown as SEQ ID NO. 18; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.7 is shown in SEQ ID NO. 19; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.8 is shown in SEQ ID NO. 20.
Further, the amino acid sequence of the antigen protein is shown as SEQ ID NO.9.
Further, the nucleotide sequence of the antigen protein shown in SEQ ID NO.9 is shown in SEQ ID NO. 10.
In a second aspect, the invention provides an application of the antigen protein in preparing polyclonal antibodies for simultaneously detecting a plurality of porcine parvoviruses.
In a third aspect of the invention, there is provided a product for the preparation of polyclonal antibodies for simultaneous detection of a plurality of porcine parvoviruses, the product comprising any of the antigenic proteins described above.
In a fourth aspect of the present invention, there is provided a polyclonal antibody for simultaneous detection of a plurality of porcine parvoviruses, the polyclonal antibody being obtained by immunizing an animal with any one of the antigen proteins as described above as an immunogen.
In a fifth aspect of the present invention, there is provided a method for preparing polyclonal antibodies for simultaneously detecting a plurality of porcine parvoviruses, comprising the steps of:
the method comprises the steps of synthesizing antigen protein, wherein the sequence of the antigen protein is formed by connecting amino acid sequences shown in SEQ ID NO.1-8, and a linker sequence is also connected between two adjacent amino acid sequences;
injecting the antigen protein into an immunized animal body to perform animal immunization, and obtaining serum containing polyclonal antibodies;
and carrying out affinity purification on the serum to obtain the polyclonal antibody.
Further, the method for animal immunization comprises the following steps: injecting the antigen protein with the nucleotide sequence shown as SEQ ID NO.9 into an immune animal body for immunization; uniformly mixing and emulsifying the antigen protein and Freund's complete adjuvant in equal volume during primary immunization, and uniformly mixing and emulsifying the antigen protein and Freund's incomplete adjuvant in equal volume during secondary to fourth immunization; and finally, boosting again.
Alternatively, the dose of primary immunization is 300 μg/dose, the dose of second to fourth immunization is 150 μg/dose, and the last booster immunization is 150 μg/dose; the total immune dose was 900 μg/dose.
Alternatively, the time for five immunizations was day 0, day 3, day 21, day 28 and day 35 of vaccination, respectively.
Still further, the immunized animal is a New Zealand white rabbit.
In a sixth aspect, the invention provides an application of the polyclonal antibody or the polyclonal antibody prepared by the preparation method in preparation of products for detecting porcine parvovirus.
It should be noted that, firstly, the amino acid sequences of the coat proteins of the eight subtypes of porcine parvoviruses (PPV 1-PPV 8) discovered at present are compared, and the immunogenicity, the group exposure and the hydrophilicity of peptide chains are comprehensively analyzed, and finally, a section of amino acid sequence with strong immunogenicity, good group exposure, high hydrophilicity and strong specificity, namely the amino acid sequence shown in SEQ ID NO.1-8, is selected on the eight porcine parvoviruses respectively; and a linker sequence is connected between two adjacent amino acid sequences and used as an antigen protein for animal immunization to prepare polyclonal antibodies. Finally, the polyclonal antibody is obtained based on the method, and the immunofluorescence method is used as a means to finally realize the detection of various porcine parvovirus subtypes.
The linker sequences suitable for the antigen proteins of the invention are diverse and have both synthetic and natural amino acid sequences. Generally, a sequence comprising both of G (glycine) and S (serine) can be used as the linker sequence according to the present invention.
For example, amino acid sequences GGGS, GSGSGGS, etc. can be selected as linker sequences.
It should be noted that the antigen protein provided by the invention can be used as the antigen protein claimed by the invention only by connecting amino acid sequences (shown as SEQ ID NO. 1-8) corresponding to eight subtype porcine parvoviruses according to the mode of the invention. The eight amino acid sequences can theoretically obtain corresponding antigen proteins according to any arrangement sequence, and can obtain corresponding polyclonal antibodies after animal immunization, and the eight porcine parvoviruses PPV1-8 have good detection sensitivity, accuracy and stability.
In a seventh aspect of the invention, there is provided a product for detecting porcine parvovirus, said product comprising the polyclonal antibody described above, or a polyclonal antibody prepared by the preparation method described above.
Furthermore, the product provided by the invention is a detection reagent or a kit, and the process of detecting the porcine parvovirus is performed based on an ELISA test strip or a protein chip or based on a colloidal gold test strip or a protein chip.
In an eighth aspect of the invention, a method for detecting pig serum titers is provided, which is carried out by using the polyclonal antibody or the polyclonal antibody prepared by the preparation method.
In a ninth aspect of the present invention, there is provided a method for detecting porcine parvovirus not for disease diagnosis and treatment, using the polyclonal antibody described above, or using the polyclonal antibody prepared by the preparation method described above.
Compared with the prior art, the invention has the following advantages: compared with the conventional immunofluorescence detection, the polyclonal antibody prepared by utilizing the antigen protein provided by the invention can detect eight porcine parvovirus serotypes simultaneously, and has the advantages of wider detection coverage for the porcine parvovirus, strong specificity and high detection sensitivity; the operation is simple, the time and the labor are saved, and the sample quantity to be measured and the detection cost are saved.
Drawings
FIG. 1 is an analysis of immunogenicity, group exposure and hydrophilicity of PPV1-8 capsid proteins. Eight rows from top to bottom in the figure correspond to PPV1-8 respectively, and each row from left to right corresponds to the analysis results of immunogenicity, radical exposure and hydrophilicity of each PPV respectively;
FIG. 2 shows the protein expression electropherograms of PPV1-PPV8 tandem antigens;
FIG. 3 shows the results of detection of the serial antigen immune Elisa titers of PPV1-PPV 8;
FIG. 4 is a graph showing the effect of 7 lentiviral packaging systems on PK-15 infection;
FIG. 5 shows the effect of polyclonal antibodies on the immunofluorescence detection of PPV1 virus, packaged lentiviruses and negatives of capsid proteins of the corresponding porcine parvoviruses PPV2-PPV 8;
FIG. 6 shows the immunofluorescence detection effect of the polyclonal antibody and the single PPV1 antibody on PPV1 virus, wherein the single PPV1 antibody is used on the left, and the PPV1-8 antigen is used on the right to prepare the antibody in series.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some of experimental examples of the present invention, not all experimental examples. All other examples obtained by a person of ordinary skill in the art without making any inventive effort are within the scope of the present invention based on the examples herein.
The following embodiments provided by the invention include polyclonal antibody synthesis and detection of porcine parvovirus using polyclonal antibody-bound cellular immunofluorescence.
1. Synthesis of target Gene
The amino acid sequences of the eight selected porcine parvoviruses PPV1-8 (shown in Table 1 below) were spliced and joined between adjacent amino acid sequences using linker sequences. FIG. 1 shows the results of analysis of immunogenicity, group exposure and hydrophilicity of PPV1-8 capsid proteins. Eight rows from top to bottom in the figure correspond to PPV1-8, respectively, and each row from left to right corresponds to the results of the analysis of immunogenicity, group exposure and hydrophilicity of each PPV, respectively.
Alternatively, the linker sequence of the invention may be GGGS (shown as SEQ ID NO. 11) or GSGSGGS (shown as SEQ ID NO. 12); other linker sequences disclosed in the following documents may also be used. The literature sources are: https:// www.ncbi.nlm.nih.gov/PMC/optics/PMC 4514284/.
Specifically, the following specific examples select GGGS as linker sequences.
The amino acid sequence of the finally completed splice is shown as SEQ ID NO.9.
Correspondingly, the sequence shown in SEQ ID NO.9 is encoded, and the gene sequence is subjected to codon optimization, as shown in SEQ ID NO. 10.
The gene sequence shown in SEQ ID NO.10 is synthesized by Tianyihui remote company to obtain the target gene sequence, i.e. the nucleotide sequence of the coding antigen protein.
TABLE 1 amino acid sequences of eight porcine parvoviruses
TABLE 2 Gene sequences encoding the eight amino acid sequences of TABLE 1
| Serotypes | Gene sequence encoding amino acid sequence | Sequence length |
| PPV1 | As shown in SEQ ID NO.13 | 342 |
| PPV2 | As shown in SEQ ID NO.14 | 249 |
| PPV3 | As shown in SEQ ID NO.15 | 270 |
| PPV4 | As shown in SEQ ID NO.16 | 240 |
| PPV5 | As shown in SEQ ID NO.17 | 240 |
| PPV6 | As shown in SEQ ID NO.18 | 237 |
| PPV7 | As shown in SEQ ID NO.19 | 240 |
| PPV8 | As shown in SEQ ID NO.20 | 180 |
2. Construction of prokaryotic expression plasmid
(1) The target gene was synthesized by PCR amplification using Pfu DNA polymerase (Pfu DNA polymerase, life technology, 12344024) kit using the target gene as a template, and the specific reaction system is shown in Table 1 below.
TABLE 1PCR amplification reaction System
| Composition of the components | Volume of |
| 10×pfu buffer | 5μl |
| Target gene template | 1μl(20-50ng) |
| F(10μM) | 1μl |
| R(10μM) | 1μl |
| Pfu | 1μl(5U) |
| Nuclease-free water | 41μl |
| Total | 50μl |
(2) The above-prepared reaction system was operated under the reaction conditions shown in the following table 2.
TABLE 2 amplification reaction System
Rapidly cooling to 4 ℃ for standby after the reaction is finished; and then the PCR amplification product obtained above is recovered by using an OMEGA gel recovery kit.
(3) The Pet-32a base vector and the PCR amplification product (gene fragment of interest) were digested with EcoRI (NEB, R0101V) and NotI (NEB, R0189V) endonucleases, respectively. Specific reaction systems are shown in Table 3 below.
TABLE 3 cleavage reaction System
| Composition of the components | Volume of |
| Basic vector/Gene fragment of interest (500 ng/. Mu.l) | 2μl |
| 10×cutsmart buffer | 5μl |
| EcoRI | 1μl |
| NotI | 1μl |
| Nuclease-free water | 41μl |
| Total | 50μl |
Placing the prepared system in a thermostat to react for 3 hours at 37 ℃; and then the enzyme digestion products obtained above are recovered by using an OMEGA gel recovery kit.
(4) The gene fragment of interest was recombinantly ligated onto the base vector using T4 ligase (Roche, 10481220001). The specific reaction system is shown in Table 4 below.
TABLE 4 Gene ligation reaction System
Placing the prepared system in a thermostat to react for 1h at 37 ℃; the ligation product described above (i.e., prokaryotic expression plasmid) was then transformed into TOP10 competence; finally, selecting positive clones and sequencing; two positive clones sequenced correctly were selected for transformation into DE3 competence and designated clone 1 and clone 2, respectively.
3. Protein expression and purification
The selected DE3 positive clone containing the target gene fragment is cultivated overnight, and then inoculated into 500ml of liquid LB culture solution (commercially available and prepared by a conventional formula) of 100mg/ml Amp according to the proportion of 1:100 for cultivation at 37 ℃; the OD600 value of the bacterial liquid is measured every 1 hour.
When the OD600 value reached 0.6, isopropyl β -D-1-thiogalactopyranoside (IPTG) was added and the final concentration was 0.5mM, and the culture was continued at 18℃for about 16 hours; centrifuging to remove supernatant, adding 15ml of cell lysate (commercially available, prepared by conventional formulation; containing 20mM HEPES, pH=7.5, 100mM KCl, 10mM imidazole and protease inhibitor), and crushing cells with ultrasound after ice bath for 30 min; 10000g, centrifuging at 4deg.C for 10min, collecting supernatant, filtering the supernatant into a new sterile tube with 0.22 μm filter membrane, combining the supernatant with Ni2+ resin to purify target protein, and washing the resin for 3 times; finally, elution was performed with eluent (PBS, ph=7.4, 300mM imidazole) and overnight dialysis treatment was performed with dialysate to obtain purified antigen protein. As shown in FIG. 2, M in FIG. 2 represents a protein marker,1 represents DE3 competent total cell lysate without the gene of interest, 2 and 3 represent inclusion bodies of clones 1 and 2, and 4 and 5 represent supernatant samples of clones 1 and 2.
4. Immunization of animals
Healthy female white rabbits (about 4 months old, 2 kg) were selected for animal immunization experiments. Mixing the antigen protein of the primary immunization with the Freund's complete adjuvant with the same volume, emulsifying, mixing the antigen protein of the second, third and fourth times with the Freund's incomplete adjuvant with the same volume, emulsifying; the dose of the primary immunization is 300 mug/dose, the dose of the second to fourth immunization is 150 mug/dose, and the dose of the final boost is 150 mug/dose; the total immune dose was 900 μg/dose. The time of the five immunizations was 0 day, 3 day, 21 day, 28 day and 35 day of inoculation, respectively.
5. Purification of antibodies
Carrying out ear vein blood collection on the white rabbits 7 days after the immunization is finished, centrifuging the obtained rabbit serum for 20min at a low temperature of 3400 r at 4 ℃, removing the lipid on the upper layer by using a pipette, and carrying out overnight incubation at 4 ℃ together with the beads in the proA column; serum was drawn off from the large column, washed three times with PBS and eluted with pre-chilled eluent (0.1 m glycine, ph=3.0); completion of elutionNeutralizing with antibody neutralizing solution (1M Tris-Cl, ph=8.5); after collection, the mixture was mixed with an equal volume of glycerol and then 2.5% NaN was added 3 Solution to a final concentration of two parts per million; mixing, and storing at-80deg.C.
6. ELISA determination of antibody titers
The antigen protein was diluted to 6. Mu.g/ml with 0.05mol/L carbonate (pH=9.6), 100. Mu.l/well and incubated overnight at 4 ℃;
taking out, washing with washing solution (PBST containing 0.05% Tween-20) three times for 3 min/time, adding 150 μl of blocking solution (5% skimmed milk powder+PBST) into each well, and blocking at 37deg.C for 60min;
taking out, washing with washing solution (PBST containing 0.05% Tween-20) three times for 3 min/time; antisera (containing polyclonal antibodies) were diluted 1:1000 respectively, and then twice the ratio, i.e. 1:2000, 1:4000, 1:8000 up to 1 (2) 11 *1000 Dilution (100. Mu.l of antiserum was added to the first well, then 100. Mu.l of the first well was taken into the second well, followed by sequential dilution backwards); incubating for 1h at 37 ℃;
taking out, washing with washing solution (PBST containing 0.05% Tween-20) three times for 3 min/time; horseradish peroxidase-labeled goat anti-rabbit IgG (ackson, 116154J) was diluted 1:8000, 100 μl/well and incubated at 37 ℃ for 45min;
taking out, and washing with washing solution (PBST containing 0.05% Tween-20) for five times for 3 min/time; adding 100 μl/well of substrate solution (TMB), and reacting for 5-10min; finally, 100. Mu.l of a 2mol/L stop solution (sulfuric acid) was added to terminate the reaction.
OD determination with an ELISA apparatus (Kehua ST-360) at a wavelength of 450nm 450 Values were calculated and the dilution factor of serum was calculated.
As shown in FIG. 3, in the figure, the number 1 represents a blank sample, and 2-12 represent 2 which is an antibody 0 *1000-2 11 * ELISA results at 1000-fold dilution.
7. Construction of lentiviral packaging systems
The nucleotide sequences corresponding to the selected PPV2-8 serotype antigen sequences are respectively constructed into pHAGE-CMV-MCS-IZsGreen vectors by using a homologous recombination kit (Nuozan, C115-01), then are respectively co-transfected into 293T cells along with the vectors PMD2.G and the vector psPAX2, the supernatant of the 293T cells is collected after 72h transfection, 500g centrifugation is carried out at a low temperature of 4 ℃ for 5min, and the supernatant is collected to obtain the PPV2-8 packaging virus (i.e. a lentivirus packaging system), and is stored to-80 ℃ for standby after subpackaging. FIG. 4 is a graph showing the effect of 7 lentiviral packaging systems on PK-15 infection, and shows that the lentiviruses packaged in the patent have strong infectivity on PK-15 cells, can simulate the infection of viruses to a certain extent, and can reflect the availability of the polyclonal antibody from the side by using the viruses to verify the polyclonal antibody.
8. Cell immunofluorescence method for detecting porcine parvovirus
PK-15 cells (pig kidney cells, which are independently preserved by the company) with good growth state are taken and inoculated into a 12-well plate; when cells grow to an appropriate (e.g., 80%) cell density, the culture broth is discarded and washed 1-2 times with serum-free DMEM medium.
Then, a proper amount (1000 FAID 50) of PPV1 virus and PPV2-PPV8 packaging virus are respectively inoculated, the mixture is adsorbed for 1h at 37 ℃, the adsorption liquid is discarded, the mixture is washed for 1 to 2 times (the washing times can be increased or decreased according to the situation), 5% FBSDMEM cell culture liquid is added, and a serum-free culture medium is used as a negative control.
Removing cell supernatant after 1 week, and washing with PBS solution for 1-3 times; 1ml of 4% paraformaldehyde is added into each hole, after the holes are fixed for 30min at room temperature, the fixed liquid is discarded, and the holes are washed for 1 to 3 times by PBS solution; 1ml of 1% Triton X-100 is added into each well, after 30min permeabilization at room temperature, the permeabilization solution is discarded, and the solution is washed for 1 to 3 times by PBS solution; 1ml of 3% BSA was added to each well, blocking was performed for 30min at room temperature, 0.5ml of positive serum of the corresponding virus was added to each well, after 2-8deg.C overnight (or 37℃incubation for 1-2 h), positive serum was discarded, washed 6 times with 1% PBST solution, 0.5ml of the corresponding fluorescent secondary antibody was added to each well, and incubated at room temperature or 37℃in the absence of light for 1-2h. The fluorescent antibody was discarded, washed 3 times with PBS solution, and photographed.
Each row of fig. 5 shows the effect of the polyclonal antibodies in this patent on the PPV1 virus, the lentiviruses of the capsid proteins of the seven corresponding porcine parvoviruses, and negative immunofluorescence detection, respectively. From the figure, the detection effect of the virus is better whether the virus corresponds to the lentivirus containing PPV2-PPV7 capsid protein or PPV1 virus.
In fig. 6, the left graph of the graph shows the immunofluorescence detection effect of a single PPV1 antibody, and the right graph shows the immunofluorescence detection effect of the polyclonal antibody prepared by the present invention, and it can be seen from the graph that the polyclonal antibody prepared by the present invention has a detection effect similar to that of the single PPV1 antibody.
The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.
Claims (10)
1. An antigen protein for preparing polyclonal antibodies for simultaneously detecting a plurality of porcine parvoviruses, which is characterized by comprising an amino acid sequence corresponding to PPV1 as shown in SEQ ID NO. 1; the amino acid sequence of the corresponding PPV2 shown as SEQ ID NO. 2; the amino acid sequence of the corresponding PPV3 shown as SEQ ID NO. 3; the amino acid sequence of the corresponding PPV4 shown as SEQ ID NO. 4; the amino acid sequence of the corresponding PPV5 shown as SEQ ID NO. 5; the amino acid sequence of the corresponding PPV6 shown in SEQ ID NO. 6; the amino acid sequence of the corresponding PPV7 shown in SEQ ID NO. 7; the amino acid sequence of the corresponding PPV8 shown as SEQ ID NO. 8; a linker sequence is also connected between two adjacent amino acid sequences.
2. The antigenic protein of claim 1 wherein the linker sequence has the amino acid sequence shown in SEQ ID No.11 or SEQ ID No. 12.
3. The antigenic protein of claim 1 wherein the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.1 is shown in SEQ ID No. 13; the nucleotide sequence of the amino acid sequence shown in the SEQ ID NO.2 is shown in SEQ ID NO.14, and the nucleotide sequence of the amino acid sequence shown in the SEQ ID NO.3 is shown in SEQ ID NO. 15; the nucleotide sequence of the amino acid sequence shown in the SEQ ID NO.4 is shown in SEQ ID NO. 16; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.5 is shown in SEQ ID NO. 17; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.6 is shown as SEQ ID NO. 18; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.7 is shown in SEQ ID NO. 19; the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.8 is shown in SEQ ID NO. 20.
4. An antigenic protein as claimed in claim 3 wherein the amino acid sequence of the antigenic protein is as shown in SEQ ID No.9.
5. The antigenic protein of claim 4 which encodes the antigenic protein of SEQ ID No.9 having the nucleotide sequence of SEQ ID No. 10.
6. Use of an antigenic protein as claimed in any one of claims 1 to 5 in the preparation of a polyclonal antibody for simultaneous detection of a plurality of porcine parvoviruses.
7. A product for preparing polyclonal antibodies for simultaneous detection of a plurality of porcine parvoviruses, characterized in that the product comprises an antigenic protein according to any one of claims 1-5.
8. A polyclonal antibody for simultaneous detection of a plurality of porcine parvoviruses, wherein the polyclonal antibody is obtained by immunizing an animal with the antigen protein according to any one of claims 1 to 5 as an immunogen.
9. Use of a polyclonal antibody according to claim 8 for the preparation of a product for the detection of a plurality of porcine parvoviruses, or for the detection of porcine serum titers, or for the preparation of a product for the detection of porcine serum titers, or for the detection of porcine parvoviruses not for the purpose of disease diagnosis and treatment.
10. An assay product comprising the polyclonal antibody of claim 8; the detection product is used for detecting various porcine parvoviruses without the aim of disease diagnosis and treatment or is used for detecting porcine serum titers.
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