KR20170041956A - Rapid Kit for Diagnosing the specific antibodies against European type Porcine Respiratory Reproductive Syndrome Virus - Google Patents

Abstract

The present invention provides a rapid kit for diagnosing a European-type porcine reproductive and respiratory syndrome (PRRS) virus specific antibody, and to a method for diagnosing a European-type porcine reproductive and respiratory syndrome virus specific antibody by using the same. When the kit of the present invention is used, infection of a European-type porcine reproductive and respiratory syndrome virus can be conveniently and rapidly diagnosed on a site by using a rapid immune chromatography scheme without using a special inspection tool. The kit of the present invention uses a European-type porcine respiratory reproductive syndrome virus gene recombinant nucleocapsid protein and a single clone antibody in response to the same, thereby showing excellent functions in terms of sensitivity and specificity. Since results can be expressed by values by using a gold reader, the kit can conveniently and accurately make determination compared to a conventional kit confirmed by a naked eye.

Description

TECHNICAL FIELD [0001] The present invention relates to a rapid kit for diagnosing a pancreaticobiliary disease syndrome virus-specific antibody in a European type porcine reproductive reproductive syndrome virus

The present invention relates to a diagnostic kit for European type porcine respiratory syndrome virus-specific antibody and a European type porcine respiratory syndrome virus-specific antibody diagnostic use thereof.

Porcine Reproductive and Respiratory Syndrome (PRRS) was first reported in North America at the end of the 1980s.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an RNA virus belonging to Nidovirales, Arterviridae, Arterivirus. Pig reproductive and respiratory syndrome viruses are largely divided into European type 1 (European genotype) and North American type 2 (NA) according to their antigenic and genetic characteristics. Symptoms of pig genital respiratory syndrome include infertility, late pregnancy mummy fetuses, abortion, premature birth, and infected piglets born to the virus are known to die from secondary infections in the respiratory tract.

It is important to distinguish between the North American and European types in order to more effectively identify the infection status of farmed pig genital respiratory syndrome, which can lead to the selection of a vaccine for effective control.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a diagnostic kit capable of quickly and accurately diagnosing the infection of the European pig genital respiratory syndrome virus in a biological sample. As a result, when using a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and a monoclonal antibody responsive thereto, the presence of a European pig genital respiratory syndrome virus antibody in swine-derived biological samples is rapidly detected by rapid immunochromatography And thus the present invention has been completed.

Therefore, it is an object of the present invention to provide a rapid kit for diagnosing European pig genital respiratory syndrome virus-specific antibodies.

Another object of the present invention is to provide a method for diagnosing European type porcine respiratory syndrome virus specific antibody using the kit of the present invention.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a rapid kit for the diagnosis of a European type porcine respiratory syndrome virus-specific antibody comprising:

(i) biological samples; (Ii) a conjugate of a European porcine respiratory syndrome virus genetically engineered nucleocapsid protein and gold nanoparticles consisting of the amino acid sequence of Sequence Listing 1; And (iii) a sample pad to which is applied a liquid sample comprising a binding agent of a control antibody and gold nanoparticles used to confirm that the sample has been developed;

(b) a monoclonal antibody that binds to said European pig genital respiratory syndrome virus recombinant nucleocapsid protein and an antibody that specifically binds to said control antibody is immobilized; And

(c) an absorbent pad capable of absorbing a liquid sample.

The present inventors have sought to develop a diagnostic kit capable of quickly and accurately diagnosing the infection of the European pig genital respiratory syndrome virus in a biological sample. As a result, when using a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and a monoclonal antibody responsive thereto, the presence of a European pig genital respiratory syndrome virus antibody in swine-derived biological samples is rapidly detected by rapid immunochromatography And it can be detected accurately.

In the present invention, the immunochromatography method used for the diagnosis of the European pig genital respiratory syndrome is a diagnostic method which enables the diagnosis of various diseases such as diagnosis of pregnancy or hepatitis by using the antigen-antibody reaction, It is a type of immunoassay for rapid field diagnosis using a thin film as a solid phase and using colored particles such as gold colloid or colored polystyrene nanoparticles as a measurement label.

The kit of the present invention uses a monoclonal antibody reacting with a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and a high affinity to the same, thereby to produce a European type pig by competing with an antibody in the body induced by the European pig genital respiratory syndrome virus It is possible to diagnose the presence of genital respiratory syndrome virus antibody specifically.

As used herein, the term " European-type porcine respiratory syndrome virus recombinant nucleocapsid protein " refers to a polynucleotide that amplifies the ORF7 gene encoding a nucleocapsid protein from a Lelystad strain, a type of European pig genital respiratory syndrome virus, Lt; RTI ID = 0.0 > expression system. ≪ / RTI >

As used herein, the term " monoclonal antibody " refers to a single molecule composition of antibody molecules obtained in a substantially identical population of antibodies, wherein the monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope.

According to one embodiment of the present invention, the monoclonal antibody of the present invention binds to the European pig genital respiratory syndrome virus recombinant nucleocapsid protein consisting of the amino acid sequence of the first sequence of the sequence listing.

The monoclonal antibody of the present invention is an antibody produced by a hybridoma cell line deposited with the Korean Collection for Type Cultures (KCTC) on Jul. 17, 2015 under the deposit number KCTC KCTC18397P.

The kit of the present invention includes a sample pad to which a liquid sample is applied, a membrane to which the antibody is immobilized, and an absorption pad capable of absorbing the liquid sample.

According to one embodiment of the present invention, in the kit of the present invention, the membrane comprises a test line immobilized with a monoclonal antibody binding to a European pig genital respiratory syndrome virus recombinant nucleocapsid protein, and a test line And a control line for performing the control.

An antibody that specifically binds to the control antibody contained in the liquid sample is immobilized on the control panel to confirm that the sample has been developed.

According to one embodiment of the present invention, rabbit IgG, mouse IgG and the like may be used as a control antibody in the present invention, but the present invention is not limited thereto. Preferably, the control antibody contained in the liquid sample is rabbit IgG, and the antibody immobilized on the control line is anti-rabbit IgG. When the liquid sample is normally developed, the control antibody and the gold nanoparticle conjugate contained in the liquid sample react with the antibody immobilized on the control line to develop color.

Examples of the biological sample applied to the kit of the present invention include, but are not limited to, whole blood derived from pigs, serum, plasma, saliva, and other body fluids (urine, etc.).

According to one embodiment of the present invention, the biological sample applied to the kit of the present invention is whole blood, plasma or serum of a pig.

According to another embodiment of the present invention, the biological sample is mixed with a conjugate of a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and gold nanoparticles and then applied to the kit of the present invention.

More specifically, the biological sample is used as a binding agent for the European pig genital respiratory syndrome virus recombinant nucleocapsid protein and gold nanoparticles, a control antibody used to confirm that the sample is developed, and a tube in which the binding agent of gold nanoparticles is dried In a dilution buffer, and then introduced into the inlet of the sample pad. In the presence of an antibody against the European pig genital respiratory syndrome virus in the sample, it is blocked in association with the European pig genital respiratory syndrome virus recombinant nucleocapsid protein-gold nanoparticle binding agent, and is distributed on the membrane while moving the membrane by capillary force The European pig genital respiratory syndrome virus is prevented from reacting with antibodies that bind to the recombinant nucleocapsid protein, resulting in no gold color on the test line. Therefore, if there is an antibody against the European pig genital respiratory syndrome virus in the biological sample, it will only develop in the control line of the membrane. If there is no antibody against the European pig genital respiratory syndrome virus in the biological sample, ) And a reference line (see Figs. 4 to 6).

In the kit of the present invention, the biological sample is determined to have been produced by infection with European pig genital respiratory syndrome virus or European vaccine against genital respiratory syndrome virus, , The biological sample is judged to have not been infected with the European pig genital respiratory syndrome virus or that the antibody has not been produced after vaccination against the European pig genital respiratory syndrome virus.

The kit of the present invention can be used to determine whether a porcine-derived biological sample has a European pig genital respiratory syndrome virus-specific antibody. Determining whether the pig is infected with the European pig genital respiratory syndrome virus or judging whether the European pig genital respiratory syndrome virus specific antibody is present from the biological sample derived from the pig or not, Or not.

In addition, the kit of the present invention can be used to determine whether or not a European pig genital respiratory syndrome virus is infected, and then the type of vaccine to be inoculated into pigs can be determined.

According to one embodiment of the present invention, the kit of the present invention is a kit for the prevention and / or treatment of European North Pig Reproductive Respiratory Syndrome viruses, such as CSFV, BVDV, FMDV, PCV, APP5, HP, PCV2, APP2, And so on.

According to another aspect of the present invention, the present invention provides a method of diagnosing European type porcine respiratory syndrome specific antibody comprising the following steps:

(a) applying a biological sample to a kit of the present invention by mixing with a conjugate of a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and gold nanoparticle consisting of the amino acid sequence of Sequence Listing 1; And

(b) analyzing the signal generated in the kit to measure the presence of the European pig genital respiratory syndrome virus specific antibody in the biological sample.

Since the present invention is a method for diagnosing European type porcine respiratory syndrome specific antibody using the kit, common description between them is omitted in order to avoid excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a rapid kit for diagnosing European type porcine respiratory syndrome virus-specific antibody and a method for diagnosing porcine reproductive-respiratory syndrome virus-specific antibody using the same.

(Ii) Using the kit of the present invention, rapid immunochromatography can be used to easily and quickly diagnose the European poultry respiratory syndrome virus infection in the field without special test equipment.

(Iii) The kit of the present invention shows excellent performance in terms of sensitivity and specificity because it uses a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and a monoclonal antibody reactive therewith, This makes it possible to make more convenient and accurate judgments than conventional kits that are visible to the naked eye.

1 is a schematic diagram showing the production of a European pig genital respiratory syndrome virus recombinant nucleocapsid protein (ORF7).
FIG. 2 shows the results of confirming expression and purification of a European pig genital respiratory syndrome virus recombinant nucleocapsid protein (ORF7).
FIG. 3 is a result of screening for a monoclonal antibody specific to the European type porcine respiratory syndrome virus antiserum.
4 is a schematic diagram of the immunochromatography method.
5 is a schematic diagram showing the principle of positive / negative discrimination for the diagnostic kit of the present invention.
PRRSV mAb: A monoclonal antibody that binds to a European pig genital respiratory syndrome virus recombinant nucleocapsid protein immobilized on a test line and consisting of the amino acid sequence of Sequence Listing 1 sequence.
Anti-rabbit IgG: Fixed on the control line and reacted with rabbit IgG gold conjugate.
PRRSV EU recombinant antigen: European pig genital respiratory syndrome virus consisting of the amino acid sequence of the sequence of SEQ ID NO: 1 Gene Recombinant nucleocapsid protein with gold nanoparticles bound and not associated with PRRSV EU antibody .
Rabbit IgG Gold Conjugate: Rabbit IgG and gold nanoparticles combined with anti-rabbit anti-rabbit IgG immobilized in the form of color reaction.
PRRSV EU Type Antiserum: European PRRSV specific antibody.
6 is a schematic view of a strip.
FIG. 7 shows the result of measuring the detection limit for the diagnostic kit.
FIG. 8 shows the results of testing the sensitivity and specificity of the North American and European PRRSV antiserum of the rapid kit for the measurement of the European type porcine respiratory syndrome virus-specific antibody.
FIG. 9 shows the results of measurement of cross reaction with 10 kinds of antigens of porcine disease.
Fig. 10 shows the result of evaluation of reproducibility.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Production of recombinant antigens

To produce one kind of European PRRSV recombinant nucleocapsid protein (Fig. 1). The European PRRSV gene recombinant nucleocapsid protein is an E. coli recombinant protein produced by PCR cloning of the entire ORF7 gene of the European PRRSV strain Lelystad strain into the pET21a vector.

Genetic information of the European PRRSV gene recombinant nucleocapsid protein Gene Origin Recombinant protein Gene site primer size
(kDa) Fusion protein PRRSV
(Lelystad) pET21a
(BL21 DE3) 387bp
(ORF7) Forward:
5'-AATGGATCCATGGCCGGTAAAAACCAGAG-3`
Reverse:
5'-TATCTCGAGTTAACTTGCACCCTGACTGG-3` 17 C-terminus
6X histidine

PCR was performed using the primers shown in Table 1 and the PCR product (387 bp) was inserted into the pET21a vector in order to prepare the European PRRSV recombinant nucleocapsid protein. The PCR conditions were as follows: initial denaturation, 94 ° C for 3 min; 3-step cycling (35 cycles), 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute; Final extension, 72 ° C for 10 minutes. Colonies were selected for the cloned vector, followed by induction and sequencing to confirm the nucleotide sequence.

The clone identified as the nucleotide sequence was cultured in E. coli to induce induction, and SDS-PAGE was performed to confirm the size and protein expression of the expressed protein. The expression protein was purified using a 6XHis Tag and subjected to SDS-PAGE to determine the degree of purification and the amount thereof (FIG. 2). The European PRRSV gene recombinant nucleocapsid protein was expressed as a soluble protein and when purified, obtained a protein of 17 KDa.

Example 2: Preparation and screening of antibodies

The mouse PRBSV recombinant nucleocapsid protein was used for mouse immunization to develop an antibody that reacts with the European PRRSV recombinant nucleocapsid protein.

2-1: Monoclonal Antibodies to Recombinant Proteins

Step 1: Immunity

Immunization was performed on the experimental animals to prepare monoclonal antibodies against the recombinant proteins prepared above. Recombinant proteins (European PRRSV recombinant nucleocapsid protein) were prepared at 0.5 mg / ml as an antigen, and 200 μl of recombinant protein and 200 μl of Freund's Adjuvant (FA) were mixed and injected into BALB / Third immunization was performed. One week after the third immunization, indirect ELISA and IFA tests were performed using mouse serum for fusion immunization. For final boosting, 100 μl of recombinant protein was immunized peritoneally and used for cell fusion 4 days later.

Step 2: Cell fusion and hybridoma production

The spleens located on the left side of the immunized mouse body are harvested and suspended in DMEM medium. The harvested spleen was ground with a mesh to separate cells and mixed with DMEM in culture medium to make a spleen cell suspension. The suspension is centrifuged to remove the supernatant, and the mixture is mixed with the spleen cells and bone marrow cell line (SP2 / 0 Ag14 (ATCC, USA)) at a ratio of 1: 5, followed by centrifugation to remove the supernatant. Centrifuged cells are slowly dispersed, treated with 1 ml of polyethylene glycol 1500 (PEG 1500, Roche), maintained at 37 ° C for 1 min, and then 1 ml of DMEM is added. After that, 10 ml of DMEM is added for 1 minute, reacted in water at 37 ° C for 5 minutes, and then centrifuged again to 50 ml. The cell pellet was resuspended in a separate medium (HAT medium) at a concentration of 1 to 2 × 10 ⁵ cells / ml, and 0.1 ml each was dispensed into a 96-well plate and cultured in a 37 ° C. carbon dioxide incubator.

Step 3: Screening of hybridoma cells

Among the hybridoma cell lines prepared in step 2, IFA and ELISA analysis were used to select hybridoma cells that specifically react with the European PRRSV recombinant nucleocapsid protein.

In the IFA assay, MA-104 cells were cultured in 96-well microplates, PRRSV LV and Lelystad were infected, and cells were fixed with methanol to prepare PRBS-infected cell plates. Untreated plates were prepared Lt; / RTI > Hybridoma cells were incubated at 37 ° C for 1 hour with 100 μl each of the culture medium, and the cells were washed three times with phosphate buffered saline-Tween 20 (PBS-T) to remove unreacted culture medium. Mouse IgG-fluorescein isothiocyanate (Goat anti-mouse IgG-FITC) was added thereto, followed by reaction at 37 ° C for 1 hour, followed by washing three times with PBS-T. The washed plate was subjected to fluorescence microscopy to select a hybridoma cell line that was reactive to PRRSV infected plates and not reactive to normal cells.

The ELISA test method was performed by adding 100 μl (0.5 μg / ml) of the antigen-European type PRRSV recombinant nucleocapsid protein and the comparative originating normal cell lysate to the plate surface in a 96-well microplate, Antigen not removed was washed away. Hybridoma cells were incubated at room temperature for 1 hour with 100 μl of the culture solution, and then washed three times with phosphate buffer-Tween 20 (PBS-T) to remove unreacted culture medium. To this, a goat anti-mouse IgG-horseradish peroxidase (Goat anti-mouse IgG-HRP) was added and reacted at room temperature for 1 hour and then washed three times with PBS-T. After washing, the peroxidase substrate solution (TMB) was added and reacted. 50 μl of 0.5 M sulfuric acid was added to each well of the plate, and the absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan) Hybridoma cell lines secreting antibodies that are highly reactive to the antigen and not reactive to the control antigen were screened. The selected hybridoma cell line was restrictedly diluted and cloned into monoclonal. After that, a hybridoma cell line secreting the desired antibody was selected by the above method.

Six types of monoclonal antibodies reactive with the European PRRSV recombinant nucleocapsid protein were selected by the above method.

The 6 selected antibodies are spotted on nitrocellulose membrane at a concentration of 2 mg / ml and then dried at 37 ° C for 30 minutes.

Antibodies were selected by reacting the European PRRSV recombinant nucleocapsid protein-gold conjugate (under the preparation method) with each of six specimens, one buffer, one negative type, two NA type positive and one EU type positive specimen . The 11D56 antibody showed excellent sensitivity to the buffer and the negative specimen (excellent reactivity with the EU recombinant antigen to which the antibody was immobilized), and 11D56 antibody (No. 7) was selected to specifically block only the EU type positive specimen (Figs. 3A and 3B).

Example 3: Rapid diagnostic kit manufacture

3-1: Manufacture of gold conjugate

The gold conjugate is a conjugate between the antigen and gold particles (40 nm). The conjugate was prepared as follows using an adsorption method.

Manufacturing method

1) Preparation of antigen: Prepare the concentration of antigen / gold solution to be 5 μg / mL.

2) Preparation of gold solution: Prepare 40 nm colloidal gold to a OD value of 1 at 525 nm wavelength using a spectrophotometer, and adjust to pH 9.

3) Preparation of conjugate: 5 μg / ml of antigen and gold prepared above are mixed and reacted at room temperature for 1 hour.

4) Blocking and washing: 0.3% casein was added to block the portion of the gold solution that did not react with the antigen, and the reaction was allowed to proceed at room temperature for 1 hour. To remove unbound unbound antigens and blocking solution, the supernatant was discarded using centrifugation, 1% BSA solution was added, and the supernatant was removed. And the OD value is measured at a wavelength of 525 nm using a spectrophotometer.

5) Drying to maintain gold conjugate stability

Gold conjugates have the disadvantage that their activity decreases over time. To overcome these drawbacks and to ensure stability, a method was used in which three chemicals were added to a 1.5 ml tube and dried. Add 5% sucrose, 0.1% casein, 0.1% NaN ₃ , and add rabbit IgG gold conjugate to the spectrophotometer OD value of 0.5. The European PRRSV recombinant nucleocapsid protein-gold conjugate should be OD 3 It is dried in a dryer for 10 minutes.

3-2: Fabrication of Strip

The strip has a form in which a sample pad (Ahlstrom), a membrane (Millipore), and an absorbent pad (Ahlstrom) are superimposed to react with capturing while flowing in a certain direction (FIG.

Manufacturing method

1) Preparation of antibody: Prepare by adding 0.5% sucrose (Sigma) and 0.1% NaN ₃ (Sigma) to 1 mg / ml of anti-rabbit IgG (Arista) and 2 mg / ml of PRRSV mAb 11D56.

2) Antibody Dissociation and Immobilization: Membrane was attached to a backing card (PJ Co.). Anti-rabbit IgG was added to the control line and 11D56 antibody was added to the test line at a bed speed of 10 cm / sec and a dispensing volume of 0.9 μl / cm It is busy. It is then dried in a dehumidifying cabinet for more than 24 hours to fix the antibody to the membrane.

3) Pad attaching and cutting: When the antibody is fixed, attach the sample pad and the absorbing pad to the specified positions and cut them at 4 mm intervals using a rotary slitter.

4) Assembly and Packing: Assemble the completed strip into the housing and seal it with silica gel in an aluminum pouch.

3-3: Detection solution preparation

Add 0.2% Tween 20 (Sigma) and 0.01% NaN ₃ to 50 mM Boolean buffer.

3-4: Method of use and judgment of result

1) Take the serum sample to be tested to the titration line of the dropper for sample, place it in the tube where the gold conjugate is dried, add 3 drops of the dilution buffer using the dropper and react well for 5 minutes.

2) Remove the device, place it on a flat surface, and add 3 drops of the mixed solution from sample No. 1 to the injection port.

3) Load at room temperature for 10 minutes and measure the numerical value using a gold reader.

4) If the numerical value is 600 or more, it is negative. If it is less than 600, it is judged to be positive.

3-5: Analysis of Results

Detection limit measurement

This kit was found to be about 4 times lower than the detection limit of the conventional ELISA kit (IDEXX PRRS X3 Ab Test). However, the kit was designed to measure without discrimination between North American or European PRRSV antibodies (plate cross reactivity between North American and European PRRSV antibodies Present), it may be difficult to compare the exact detection limits (Fig. 7).

Sensitivity measurement

We confirmed the antitumor activity using the ELISA method (North American type & European type PRRSV antibody detection method) which is the highest method to measure the 4 kinds of NA antiserum and 2 kinds of EU antiserum received from the Agriculture, Forestry and Livestock Quarantine Headquarters. As a result, it was confirmed that only EU antiserum specimens were specifically blocked (FIG. 8).

IDEXX ELISA results of antiserum Sample OD SP (0.4 < positive) NA: 21 (VR2332) 0.35 0.91 (weakly positive) NA: 22 (VR2332) 0.55 1.53 (weakly positive) EU: 23 (LV) 0.90 2.61 (moderately positive) NA: 36 (LMY) 0.68 1.92 (weakly positive) NA: 37 (PL97) 1.07 3.14 EU: 39 (3E8) 1.13 3.33 (strong)

Specificity measurement

Sensitivity specificity was measured in comparison with the ELISA method, which is a higher method of PRRSV antibody measurement. 40 specimens tested by the IDEXX ELISA test were tested and all of them were negative. The specificity was 100%.

Example 4: Cross-reactivity evaluation

To confirm cross-reactivity with other antisera, tests were conducted with porcine antiserum confirmed to have antibodies to several diseases as a result of an NA type positive antiserum and ELISA. It was confirmed that there was no cross reaction with four kinds of NA antiserum provided by the Agriculture, Forestry and Livestock Quarantine Headquarters, and it was confirmed that there was no cross reaction with 10 other antiretroviral agents (FIGS. 9 and 10).

Example 5: Reproducibility test

In order to confirm the reproducibility of the rapid test, reproducibility tests were carried out using three strips, two positive specimens and one negative specimen. All of the gold readers showed less than 10% of the numerical values, and the three of the weakly positive ones showed a cut-off value of less than 600 (FIG. 11).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC18397P 20150717

<110> MEDIAN Diagnostics Inc. <120> Rapid Kit for Diagnosing the specific antibodies against European          type Porcine Respiratory Reproductive Syndrome Virus <130> PN150228 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 128 <212> PRT <213> PRRSV LV ORF7 Amino acid <400> 1 Met Ala Gly Lys Asn Gln Ser Gln Lys Lys Lys Lys Ser Thr Ala Pro   1 5 10 15 Met Gly Asn Gly Gln Pro Val Asn Gln Leu Cys Gln Leu Leu Gly Ala              20 25 30 Met Ile Lys Ser Gln Arg Gln Gln Pro Arg Gly Gly Gln Ala Lys Lys          35 40 45 Lys Lys Pro Glu Lys Pro His Phe Pro Leu Ala Ala Glu Asp Asp Ile      50 55 60 Arg His His Leu Thr Gln Thr Glu Arg Ser Leu Cys Leu Gln Ser Ile  65 70 75 80 Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Ala Ser Leu Ser Ser Ser                  85 90 95 Gly Lys Val Ser Phe Gln Val Glu Phe Met Leu Pro Val Ala His Thr             100 105 110 Val Arg Leu Ile Arg Val Thr Ser Thr Ser Ala Ser Gln Gly Ala Ser         115 120 125 <210> 2 <211> 387 <212> DNA <213> PRRSV LV ORF7 Nucleotide <400> 2 atggccggta aaaaccagag ccagaagaaa aagaaaagta cagctccgat ggggaatggc 60 cagccagtca atcaactgtg ccagttgctg ggtgcaatga taaagtccca gcgccagcaa 120 cctaggggag gacaggccaa aaagaaaaag cctgagaagc cacattttcc cctggctgct 180 gaagatgaca tccggcacca cctcacccag actgaacgct ccctctgctt gcaatcgatc 240 cagacggctt tcaatcaagg cgcaggaact gcgtcgcttt catccagcgg gaaggtcagt 300 tttcaggttg agtttatgct gccggttgct catacagtgc gcctgattcg cgtgacttct 360 acatccgcca gtcagggtgc aagttaa 387

Claims (5)

European type Porcine Respiratory Reproductive Syndrome Virus including: Rapid kit for the diagnosis of specific antibodies:
(a) (i) a biological sample; (Ii) a conjugate of a European porcine respiratory syndrome virus genetically engineered nucleocapsid protein and gold nanoparticles consisting of the amino acid sequence of Sequence Listing 1; And (iii) a sample pad to which is applied a liquid sample comprising a binding agent of a control antibody and gold nanoparticles used to confirm that the sample has been developed;
(b) a monoclonal antibody that binds to the European pig genital respiratory syndrome virus recombinant nucleocapsid protein and an antibody to which the antibody binding to the control antibody is immobilized; And
(c) an absorbent pad capable of absorbing a liquid sample.
2. The kit according to claim 1, wherein said monoclonal antibody is a monoclonal antibody produced by hybridoma cells having accession number KCTC 18397P.
The kit according to claim 1, wherein the biological sample is whole blood, plasma or serum of a pig.
A method for detecting a European type porcine respiratory syndrome virus-specific antibody comprising the steps of:
(a) mixing a biological sample with a conjugate of a European pig genital respiratory syndrome virus recombinant nucleocapsid protein and gold nanoparticles consisting of the amino acid sequence of Sequence Listing 1, Applying to the protest kit; And
(b) analyzing the signal generated in the kit to measure the presence of the European pig genital respiratory syndrome virus specific antibody in the biological sample.
5. The method according to claim 4, wherein the biological sample is whole blood, plasma or serum of a pig.

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