KR101258611B1 - An isolated protein comprising north American type PRRSV antigen and European type PRRSV antigen and use thereof - Google Patents

Abstract

Provided are a fused PRRSV antigen comprising a North American PRRSV antigen and a European PRRSV antigen and a method of using the same.

Description

Isolated protein comprising north American type PRRSV antigen and European type PRRSV antigen and use}

The present invention relates to a PRRSV antigen and a method of using the same.

Porcine reproductive respiratory syndrome virus (PRRSV) is an enveloped, single-strand-positive sense RNA virus, from the genus Nidovirales, Arteriviridae, and Arterivirus. Belong. PRRSV is a recently developed pathogen that causes sow sowing, stillbirth and fetal pneumonia and hypoplasia, lowering of delivery rate, lowered growth rate, secondary bacterial infection and respiratory diseases in pigs of all ages, Is a contagious disease that is seriously affecting the pig industry. In Korea, the first isolated and reported PRRSV in 1993 proliferated preferentially in macrophages. The onset is genital, respiratory, and mixed, primarily transmitted from pigs to pigs, can be transmitted to piglets not fed with colostral antibodies through feces, urine, milk, It may also be transmitted by needles, insects and air.

PRRSV has a genetically diverse range of strains, depending on geographical location at the same time. The two major genotypes are the European (genotype 1) and the North American (genotype 2), which show 40% nucleotide phase heterogeneity in terms of genome level. The strain that is mainly occurring in Korea has been identified as VR2332. According to the results of the study, the North American type PRRSV has been evolving since it spread to Korean pig farmers, and it is predicted that genetic modification of PRRSV is caused by geopolitical influence.

The entire genome size of PRRSV is 15 kb and has nine open reading frames (ORFs) (ORFs 1a, 1b, 2a, 2b, 3-7) in which the lower genomic mRNAs overlap each other. ORFs 1a and 1b are replication related nonstructural proteins and ORF 2-7 constitutes structural proteins as GP2a, GP2b, GP3, GP4, GP5, membrane / matrix (M) protein and nucleocapsid (N) protein. The major structural proteins are the N protein, the M protein, and the GP5, the primary envelope glycoprotein. The GP5 (ORF 5) protein is the most abundant protein known to be capable of inducing neutralizing antibodies in the host during infection.

As a conventional method for diagnosing PRRSV infection, a method of detecting the presence of a PRRSV antigen or an antibody binding thereto in a sample isolated from pigs is known. For example, US Pat. No. 6,060,86 is a kit for detecting an antibody that specifically recognizes a Lelystad agent in a biological sample, comprising an antigen and antibody detection means selected from the group consisting of viruses, proteins, polypeptides, and peptides. The antigen is immunoreactive with a serum antibody of a pig, and the serum antibody is a) a virus substantially corresponding to a virus deposited at the Institute of Pasteur, Paris, Accession No. 1-1102, deposited on June 5, 1991. (Passage level 3, titer 10 ^4.8 TCID ₅₀ / ml) obtained by intranasal inoculation of 2 ml of specific pathogen free pigs, and b) collecting serum antibodies from inoculated pigs after 25 to 33 days Phosphorus kits are disclosed.

Therefore, even with the prior art as described above, there is a need for a method of detecting PRRSV capable of simultaneously detecting North American and European PRRSVs with high sensitivity.

One aspect is to provide isolated proteins comprising North American and European PRRSV antigens.

Another aspect is to provide a method for producing the isolated protein.

Another aspect is to provide a method for efficiently detecting anti-PRRSV antibodies in a sample.

Another aspect is to provide a method of producing an antibody that binds to said isolated protein.

Another aspect is to provide a method for efficiently detecting PRRSV antigen in a sample.

Another aspect is to provide a kit for detecting PRRSV in a sample.

One aspect of the present invention provides an isolated protein in which the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4 are fused. The amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4 may be linked by a linker amino acid sequence. The isolated protein may be one having an amino acid sequence of SEQ ID NO: 1. The linker sequence may be selected from the group consisting of GS, GSGS, and VE. The fusion may be a fusion of the C terminus of the amino acid sequence of SEQ ID NO: 2 and the N terminus of the amino acid sequence of SEQ ID NO: 4, or vice versa.

Another aspect of the invention provides an isolated nucleic acid molecule encoding said isolated protein. The isolated nucleic acid molecule may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. For example, it may have a nucleotide sequence of SEQ ID NO: 6.

Another aspect of the invention involves a method of producing the isolated protein. The method may be one or more of the methods produced by chemical synthesis, semichemical synthesis and biotechnological methods.

The method may include, for example, culturing a host cell into which the nucleic acid is introduced to express the protein; And it may be a method of producing an isolated protein, comprising the step of separating the expressed protein. The host cell may be a prokaryotic or eukaryotic cell. For example, the host cell may be Escherichia coli. In the step of culturing the host cell into which the nucleic acid is introduced to express the protein, a method known in the art may be used to introduce the nucleic acid into the host cell, to culture the host cell, and to express the protein. In addition, the step of separating the expressed protein may be performed according to methods known in the art.

Another aspect of the invention provides a method for preparing a protein comprising contacting a protein as described above with a sample suspected of containing an anti-PRRSV antibody; Detecting the presence of a complex of the protein and an anti-PRRSV antibody and if the complex is detected, the sample is determined to contain an anti-PRRSV antibody and if the complex is detected to be absent Providing a method of detecting an anti-PRRSV antibody in a sample, comprising determining that the sample does not comprise an anti-PRRSV antibody.

The method comprises contacting a protein as described above with a sample suspected of containing an anti-PRRSV antibody. The contacting may be performed by mixing and incubating the protein with the sample in a liquid medium. The liquid medium includes anything that can provide a medium for antigen-antibody reactions without destabilizing the proteins and antibodies. For example, the liquid medium includes a buffer, such as a PBS buffer. The contact can be used conditions known in the art to form a complex of antigen and antibody. For example, the contact can be carried out at 4 to 37 ℃. In addition, the contact can be carried out with or without stirring.

The protein may be fixed to a solid substrate. The solid substrate may have one or more shapes selected from well, plate and bead shapes. The well may be a micro well. The immobilization of the protein may be one in which the N and C termini are randomly immobilized on the surface of the solid substrate, such that the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 4 are randomly exposed from the outside. For example, the immobilization process is briefly described. The purified PRRSV antigen (US5C3 + EU7TC2), produced using genetic recombination technology, is diluted to an appropriate concentration with a carbonate buffer solution and dispensed into a well of a 96 microwell plate, followed by 37 ° C in a thermostat. Keep constant temperature. The antigen solution is aspirated and the blocking solution containing protein stabilizer is dispensed and kept at 25 ° C. in a thermostat. After inhalation of the blocking solution, dry it using a vacuum dryer, and keep it sealed with a silver foil. The sample may include any sample derived from pigs. For example, the sample may be pig blood, serum or plasma.

The method includes detecting the presence of a complex of the protein and anti-PRRSV antibody. The detection may be an antigen-antibody complex detection method known in the art. The detection may be made by, for example, an electrical method, a spectroscopic method, or a combination thereof. In addition, the detection may be made by a known ELISA analysis process. Anti-pig anti-PRRSV antibodies labeled with a detectable label can be made by binding to the antibody and detecting a signal from the label of the bound product. The detectable label can be a fluorescent label, an electrical label, a radioactive material, an enzyme having the activity of converting the substrate into a spectroscopically detectable material, or a combination thereof. The enzyme may include alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

The method determines that the sample contains an anti-PRRSV antibody when the complex is detected, and wherein the sample does not contain an anti-PRRSV antibody when it is detected that the complex is not present. Determining. The PRRSV may be of North American type and European type. The North American type may be a VR2332 strain and the European type may be a Lelystad strain.

Another aspect of the invention comprises the steps of administering a protein as described above to a non-human mammalian animal; And separating the antibody which binds to the protein from the animal.

The method comprises administering a protein as described above to a non-human mammalian animal. The animal may be an animal used for antibody production in the art. For example, it can be a mouse, rabbit, sheep, pig or cow. Such administration can be used administration methods known in the art for producing antibodies in an animal. For example, the animals may be administered with an amount sufficient to induce an immune response, followed by boost administration two weeks later.

The method may comprise the step of isolating an antibody that binds to the protein from the animal. The separation can be used for antibody separation methods known in the art. For example, affinity chromatography using the protein as a ligand or an antibody that specifically binds to the antibody may be used.

According to the above method, a polyclonal antibody comprising an antibody binding to the amino acid sequence of SEQ ID NO: 2 and an antibody binding to the amino acid sequence of SEQ ID NO: 4 can be obtained.

Another aspect of the invention comprises the steps of contacting a sample suspected of containing a PRRSV antigen with an antibody obtained according to the method for producing the antibody; Detecting a complex of the antibody with a PRRSV antigen; And if it is detected that the complex is present, the sample is determined to contain a PRRSV antigen, and if it is detected that the complex is not present, determining that the sample does not contain a PRRSV antigen. And a method for detecting PRRSV antigen in a sample.

The method includes contacting a sample suspected of containing a PRRSV antigen with an antibody obtained according to the method for producing the antibody. The contacting may be performed by mixing and incubating the protein with the sample in a liquid medium. The liquid medium includes anything that can provide a medium for antigen-antibody reactions without destabilizing the proteins and antibodies. For example, the liquid medium includes a buffer, such as a PBS buffer. The contact can be used conditions known in the art to form an antigen and a complex of antigens. For example, the contact can be carried out at 4 to 37 ℃. In addition, the contact can be carried out with or without stirring.

The antibody may be immobilized on a solid substrate. The solid substrate may have one or more shapes selected from well, plate and bead shapes. The well may be a micro well. The sample may include any sample derived from pigs. For example, the sample may be pig blood, serum or plasma.

The method includes detecting the presence of a complex of the antibody and a PRRSV antigen. The detection may be an antigen-antibody complex detection method known in the art. The detection may be made by, for example, an electrical method, a spectroscopic method, or a combination thereof. In addition, the detection may be made by a known ELISA analysis process. Anti-PRRSV labeled with a detectable label can be combined with the antigen and detecting a signal from the label of the bound product. The detectable label can be a fluorescent label, an electrical label, a radioactive material, an enzyme having the activity of converting the substrate into a spectroscopically detectable material, or a combination thereof. The enzyme may include alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

The method determines that the sample contains a PRRSV antigen when it is detected that the complex is present and determines that the sample does not contain a PRRSV antigen when it is detected that the complex is not present. Include. The PRRSV may be of North American type and European type. The North American type may be a VR2332 strain and the European type may be a Lelystad strain.

Another aspect of the invention provides a kit for detecting PRRSV in a sample comprising the isolated protein as described above. The kit may further comprise a reagent for detecting a complex of the isolated protein and anti-PRRSV antibody. The reagent may be an anti-pig anti-PRRSV antibody labeled with a detectable label. The detectable label can be a fluorescent label, an electrical label, a radioactive material, an enzyme having the activity of converting the substrate into a spectroscopically detectable material, or a combination thereof. The enzyme may include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. The PRRSV may be of North American type and European type. The North American type may be a VR2332 strain and the European type may be a Lelystad strain. The kit may comprise one or more selected from the group consisting of standard controls, diluents and buffers.

Another aspect of the present invention provides a kit for detecting PRRSV in a sample comprising the antibody obtained according to the above antibody production method. The antibody may be an antibody pool including both an antibody binding to the amino acid sequence of SEQ ID NO: 2 and an antibody binding to the amino acid sequence of SEQ ID NO: 4.

The isolated protein of the present invention can bind to both North American and European anti-PRRSV antibodies and can be used to detect both North American and European PRRSV in a sample.

According to the method for producing an isolated protein of the present invention, the isolated protein can be efficiently produced.

According to the method for detecting the anti-PRRSV antibody in the sample of the present invention, the anti-PRRSV antibody in the sample can be detected with high sensitivity and accuracy.

According to the method for producing the antibody of the present invention, it is possible to efficiently produce an antibody that can specifically bind to both North American and European PRRSV.

According to the method of detecting PRRSV antigen in the sample of this invention, the PRRSV antigen in a sample can be detected efficiently.

According to the kit for detecting PRRSV in a sample of the present invention, it can be used to efficiently detect PRRSV in a sample.

1 is a diagram showing a restriction map of an expression vector.
FIG. 2 shows genes of recombinant US5C3 + EU7TC2 antigens derived from vectors isolated from transformed cells. FIG.
3 shows SDS-PAGE (left) and western blotting (right) results for proteins expressed from transformed strains.
4 shows the SDS-PAGE (left) and western blot (right) results of rabbit polyclonal antibodies against recombinant US5C3 + EU7TC2 antigen.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  1: SEQ ID NO: 1 of  Preparation of Antigens Having Amino Acid Sequences

In this example, an isolated protein was prepared in which the amino acid fragment of ORF5 of North American PRRSV and the fragment of ORF7 of European PRRSV were fused. The amino acid fragment of ORF5 of North American PRRSV consists of 138-185 amino acid residues of ORF5 of North American type: CP626-2 strain and is also referred to as US5C3. A fragment of ORF7 of European PRRSV consists of 1 to 103 residues of ORF7 of European type: EU-like virus, also referred to as EU7TC2.

(1) gene amplification

First, template DNA was extracted from the virus. Specifically, a template RNA distributed from Green Cross Co., Ltd. (Yongin, Korea) was used (virus name North American Type: CP626-2 strain, EU Type: EU-like virus).

Next, PCR was performed using the template DNA as a template, and an expression vector for expression of an antigen having the amino acid sequence of SEQ ID NO: 1 was prepared using the obtained amplification product.

First, genes encoding US5C3 and EU7TC2 antigens were obtained by performing RT-PCR using PRRSV RNA as a template. The following oligonucleotide primers were used to perform PCR, which were prepared by obtaining information from the GenBank (Accession No. US: EF536003, EU: M96262) (Table 1).

Oligonucleotide PCR Primers Used  Name of the primer  The sequence (5'-3 ') SEQ ID NO: direction US5C3 F GGA TCC atgatgtcctggcgctac 7 Forward US5C3 R GTC GAC TTA cac aac tct ttt gag 8 Reverse EU7TC2 F GGA TCC atg gcc ggt aaa aac cag 9 Forward EU7TC2 R GTC GAC TTA aac ctg aaa act gac ctt 10 Reverse

Thermocycle for amplification of US5C3 and EU7TC2 antigen genes was (42 ° C., 30 minutes) × 1 time, (94 ° C., 5 minutes) × 1 time, (94 ° C., 1 minute) using a thermocycler (Mycycler: BioRad). , 45 ° C., 1 minute, 72 ° C., 1 minute) × 28 times, (94 ° C., 1 minute, 60 ° C., 1 minute, 72 ° C., 10 minutes) × 1 time, 4 ° C., 16 hours. After PCR amplification was complete, 1.5% agarose gel was prepared, and 1 µl of the PCR product was electrophoresed to determine the size of the antigen gene, and the DNA amplified using a Gene clean Kit (MP biochemical, # 1101-400, France). Was extracted. The sequence of the amplified DNA was confirmed by requesting DNA sequencing from a specialized company (Jenotech, Korea) to ensure the efficiency and accuracy of the work. As a result, the confirmed US5C3 gene and EU7TC2 gene had a nucleotide sequence of SEQ ID NO: 3 and 5, respectively, and confirmed that their coding sequence has an amino acid sequence of SEQ ID NO: 2 and 4, respectively.

PBM vector (BioNote Inc., Korea), an E. coli expression vector for fusion of USS5C3 gene and EU7TC2 gene, also contains BamH I (NEB, # R0136S, USA) and SalI (NEB, # R0138S, USA) and the product was electrophoresed on agarose gel and extracted using Gene Clean Kit (MP biochemical, # 1101-400, France).

Using T ₄ DNA ligase (Roche, 10481220001, Germany), the amplified and extracted insert and the vector were ligation at 16 ° C. overnight, using transgenic E. coli cells. (Top10F '). 1 is a diagram showing a restriction map of an expression vector.

The transformation was performed using a heat-shock method, and the transgenic cells stored at −70 ° C. were taken out and thawed on ice. Then, 5 μl of the ligation was added and mixed carefully for about 3 seconds. It was left for 30 minutes. The tube of transduced cells mixed with the reactants was placed in a constant temperature water bath at 42 ° C. for 90 seconds, and then thermally shocked and immediately placed in ice. 1 ml of LB liquid medium was incubated at 37 ° C. for 1 hour, followed by 100 μl of bacterial solution on an LB agar plate containing antibiotic ampicillin as a selection marker, followed by incubation at 37 ° C. overnight. It was.

Colonies grown on LB agar plates were inoculated into liquid LB medium and cells were grown using the Wizard Plus SV Minipreps Kit (Promega, # A1460, USA). The extracted DNA was extracted from BamHI (NEB, # R0136S, USA). And Sal I (NEB, # R0138S, USA) and electrophoresis to confirm that each antigen insert was linked to the vector. FIG. 2 shows genes of recombinant US5C3 + EU7TC2 antigens derived from vectors isolated from transformed cells. FIG. In FIG. 2, M is a size marker (Biorad 100bp ladder), and lane 1 shows the result of electrophoresis after BamHI / SalI digestion of pBM US5C3 + EU7TC2. As shown in Figure 2, 465bp US5C3 + EU7TC2 gene was confirmed.

(2) transformation

The US5C3 and EU7TC2 fusion (US5C3 + EU7TC2 antigen) genes having the nucleotide sequence of SEQ ID NO: 6 were extracted from the ligation cells, and the extracted DNA was transformed into BL21, which is an E. coli expression strain, to convert US5C3 + EU7TC2. Escherichia coli expressing the antigen was obtained.

E. coli strains expressing the recombinant US5C3 + EU7TC2 antigen were put in a Erlenmeyer flask and shaken for 18 hours or more. The cultured strains were inoculated in a fermentor and incubated at 37 ° C. for about 2 hours, and the strains were grown and cultured at 600 nm until the absorbance value was about 1.0. When the strains were propagated to absorbance of 1.0 or so, the addition of IPTG was added to 2.5mM, followed by incubation for about 5 hours, and the cultured strain solution was recovered. The cells were recovered by centrifugation, followed by SDS-PAGE and Western blot expression. Genetic recombinant antigen (US5C3 + EU7TC2: 50KDa) was identified. FIG. 3 shows SDS-PAGE and Western blotting results for proteins expressed from transformed strains. In FIG. 3, M is the size marker (Biorad protein standard), lane 1 is before pBM US5C3 + EU7TC2 induction, and lane 2 is after pBM US5C3 + EU7TC2 induction. US5C3 + EU7TC2 antigen has a molecular weight of 50KDa.

(3) Preparation of Recombinant Antigen

Cells recovered by culturing E. coli strains expressing the recombinant US5C3 + EU7TC2 antigen were crushed by treatment with an ultrasonic crusher (VCX750; Sonics & materials) and centrifuged at 8,000 rpm, 20 minutes, and 4 ° C (VS24SMTi; vision science) to precipitate. Recovered. Antigen-containing precipitate was dissolved in 6M guanidine solution. Antigens were purified by ion exchange chromatography using DEAE-Sepharose gel. That is, the dissolved inclusion body solution was reacted to the gel equilibrated with 6M guanidine solution and subjected to NaCl gradient to analyze each fraction by SDS-PAGE method, and only fractions containing the recombinant US5C3 + EU7TC2 antigen were selected. The selected antigen was quantified by BCA method (Pierce, USA) and used as an antigen to be adsorbed to microwells.

Example  2: immobilization of antigen and PRRSV Detection

1-2 μl of the purified recombinant US5C3 + EU7TC2 antigen was diluted in 50 mM carbonate buffer (pH 9.6), then 100 μl aliquoted in a 96 well ELISA plate (Costar, USA) and the antigen at 37 ° C. for 2-4 hours. Was attached. At this time, the antigen is randomly attached to the solid support by ionic bonds, hydrogen bonds and van der Waals bonds.

Next, after the antigen solution was removed, 100 μl of PBS containing 1% Scheme Milk (Sigma, USA) was again dispensed into the well of the microplate to which the antigen was attached, and incubated at 37 ° C. for 1 hour, followed by a humidity of 10 It was used after drying for 4 hours in a dryer of less than%.

Example 3 : PRRSV Detection

The test for PRRSV antibodies was carried out using enzyme-linked immunosorbent assay (ELISA) using 10 antibody positive antiserum 10 samples for European PRRSV, 10 antibody positive antiserum samples for North American PRRSV, and 10 antibody negative serum sera against PRRSV.

0.39 ml of sample dilution was dispensed into each tube in a sample dilution tube, and then three antiserum samples were diluted 40-fold by dispensing 10 μl into a tube containing the sample dilution. Subsequently, 100 μl of serum samples diluted in the plate coated with the recombinant US5C3 + EU7TC2 antigen were dispensed into each well. After 30 minutes of reaction at room temperature (18-25 ° C.), the plate was washed 5 times with PBS (pH 7.2) solution containing 0.5% Tween 20, and then goat-anti-pig antibody-HRP conjugate was added to each plate 100 Μl was added and reacted at room temperature (18-25 ° C.) for 30 minutes. After the reaction, the contents of each well were aspirated, washed 5 times with 0.5% Tween20-containing PBS (pH 7.2) solution, and 100 μl of 0.002% tetramethylbenzidine substrate solution was added to all wells. The color change was observed by reacting at room temperature (18-25 degreeC) for 15 minutes. After the reaction, 1 normal sulfuric acid was added and the absorbance of the sample was measured by using an absorbometer (sunrise basic tecan).

The enzyme immunoassay (ELISA) titer was calculated using the S / P value, and the S / P value was calculated as follows.

Positive S / P values of 0.4 or greater were considered positive. As a result, all 10 antibody positive antiserum samples for European PRRSV and 10 antibody positive antiserum samples for North American PRRSV were positive for PRRSV antibody, and all 10 antibody negative serum sera against PRRSV were antibody negative (Table 2). ).

Thus, the purified genetically modified US5C3 + EU7TC2 antigen used in the present invention could be used to efficiently detect antibody positive antiserum samples for European PRRSV and antibody positive antiserum samples for North American PRRSV.

Antibody Detection Test Results for PRRSV Specimen Absorbance value S / P value Judgment Control Voice sample 0.1000 0.0 voice Positive specimen 0.7035 1.0 positivity Antibody Positive Antiserum Against European PRRSV D8 EU27 0.9300 1.4 positivity D9 EU27 1.6190 2.5 positivity D11 EU27 2.0840 3.3 positivity D12 EU27 2.1750 3.4 positivity D10 EU30 0.4250 0.5 positivity D11 EU30 0.7900 1.1 positivity D12 EU30 0.7880 1.1 positivity D14 EU30 1.5970 2.5 positivity D8 EU No 0.4040 0.5 positivity D10 EU 1.4730 2.3 positivity Antibody Positive Antiserum Against North American PRRSV D10 NA21 0.4540 0.6 positivity D11 NA21 1.0950 1.6 positivity D12 NA21 0.8610 1.3 positivity D9 NA22 0.4940 0.7 positivity D11 NA22 0.7500 1.1 positivity D11 NA23 1.8710 2.9 positivity D14 NA23 2.1180 3.3 positivity D14 NA24 0.4780 0.6 positivity D10 NA nothing 0.9130 1.3 positivity D11 NA nothing 1.1960 1.8 positivity Antibody Negative Serum Against PRRSV PRRS Voice 1 0.0810 0.0 voice PRRS Voice 2 0.1660 0.1 voice PRRS Voice3 0.0690 -0.1 voice PRRS Voice 4 0.0900 0.0 voice PRRS Voice 5 0.0910 0.0 voice PRRS Voice 6 0.0770 0.0 voice PRRS Voice 7 0.0880 0.0 voice PRRS Voice 8 0.1070 0.0 voice PRRS Voice 9 0.0830 0.0 voice PRRS Voice 10 0.0770 0.0 voice

Example  4: PRRSV For Polyclonal  Production of antibodies

Emulsions were prepared by mixing 1 mg of the recombinant US5C3 + EU7TC2 antigen per rabbit in the same amount and complete Freund's adjuvant, and the emulsion was immunized by injection into the rabbit subcutaneously. Immunization was performed such that 1 mg of the recombinant antigen was administered once, and 5 times at weekly intervals. One week after the end of final immunization, whole blood of immunized rabbits was collected and antiserum was prepared. Polyclonal antibodies were isolated from the antisera as follows. First, the recombinant US5C3 + EU7TC2 antigen protein used for immunization of the rabbit was immobilized by covalent binding to a HiTrap ^™ NHS-activated column (Amersham Pharmacia). Next, the antiserum is loaded onto a column immobilized with the recombinant antigen to form a complex between the recombinant antigen and a polyclonal antibody, and 0.1M glycine-HCl (pH 2.7) is used as the elution solution from the complex. Polyclonal antibody was eluted to obtain a polyclonal antibody (Fig. 4). 4 shows the SDS-PAGE (left) and western blot (right) results of rabbit polyclonal antibodies against recombinant US5C3 + EU7TC2 antigen. In Figure 4, M is a marker and lanes 1 and 2 represent purified recombinant US5C3 + EU7TC2 antigens, respectively.

Example  5: PRRSV For Polyclonal  With antibody PRRSV  Antigen Detection Experiment

For detection of the PRRSV antigen, the serum of the target pig was used for the sandwich enzyme immunoassay. Conjugate fluids in which horseradish peroxidase was conjugated to European PRRSV positive serum, North American PRRSV positive serum, PRRSV antigen negative serum, and anti-PRRSV polyclonal antibody were simultaneously reacted on a plate coated with anti-PRRS polyclonal antibody. The plates were allowed to react at 37 ° C. for 60 minutes, after which the contents of each well were aspirated and washed 5 times with PBS (pH 7.2) solution containing 0.5% Tween20 and the 0.002% tetramethylbenzidine substrate solution 100 μl each well was added to block the light and reacted at room temperature (18-25 ° C.) for 15 minutes to observe color change. After the reaction, 1N sulfuric acid was added and the absorbance of the sample was measured by using an absorbometer (sunrise basic tecan).

The enzyme immunoassay (ELISA) titer was calculated using the S / P value, and the S / P value was calculated as follows.

Positive S / P values of 0.4 or greater were considered positive. As a result of the test, serum with European PRRSV and serum with North American PRRSV were positive for PRRSV and negative for PRRSV was negative (Table 3).

Therefore, the polyclonal antibodies against purified genetically recombinant US5C3 + EU7TC2 used in the present invention could be used to efficiently detect positive serum samples for European PRRSV and positive serum samples for North American PRRSV.

PRRSV detection test result Specimen Absorbance value S / P value Judgment Control Voice sample 0.0401 0 voice Positive specimen 0.4112 One positivity Positive Serum for European PRRSV VEUP1 0.5310 1.3 positivity VEUP2 0.6487 1.6 positivity VEUP3 0.5548 1.4 positivity VEUP4 0.3180 0.7 positivity VEUP5 0.2193 0.5 positivity Positive Serum for North American PRRSV VUSP1 0.8418 2.2 positivity VUSP2 0.7456 1.9 positivity VUSP3 0.5418 1.4 positivity VUSP4 0.5290 1.3 positivity VUSP5 0.2651 0.6 positivity Negative Serum for PRRSV VPN1 0.0503 0.0 voice VPN2 0.0487 0.0 voice VPN3 0.0624 0.1 voice VPN4 0.1254 0.2 voice VPN5 0.0548 0.0 voice

<110> Bionote, Inc. <120> An isolated protein comprising north Ameican type PRRSV antigen          and European thpe PRRSV antigen and use <130> PN091741 <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> Antigen derived from a fusion of US5C3 and EU7TC2 <400> 1 Met Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe Leu Leu   1 5 10 15 Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile Ile Glu              20 25 30 Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu Lys Arg          35 40 45 Val Val Gly Ser Met Ala Gly Lys Asn Gln Ser Gln Lys Lys Lys Lys      50 55 60 Ser Thr Ala Pro Met Gly Asn Gly Gln Pro Val Asn Gln Leu Cys Gln  65 70 75 80 Leu Leu Gly Ala Met Ile Lys Ser Gln Arg Gln Gln Pro Arg Gly Gly                  85 90 95 Gln Ala Lys Lys Lys Lys Pro Glu Lys Pro His Phe Pro Leu Ala Ala             100 105 110 Glu Asp Asp Ile Arg His His Leu Thr Gln Thr Glu Arg Ser Leu Cys         115 120 125 Leu Gln Ser Ile Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Ala Ser     130 135 140 Leu Ser Ser Ser Gly Lys Val Ser Phe Gln Val 145 150 155 <210> 2 <211> 50 <212> PRT <213> PRRSV VR2332 <400> 2 Met Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe Leu Leu   1 5 10 15 Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile Ile Glu              20 25 30 Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu Lys Arg          35 40 45 Val Val      50 <210> 3 <211> 150 <212> DNA <213> PRRSV VR2332 <400> 3 atgatgtcct ggcgctacgc atgtaccaga tataccaact ttcttctgga tactaagggt 60 agactctatc gttggcggtc gcctgtcatc atagagaaaa ggggcaaagt tgaggtcgaa 120 ggtcatctga tcgacctcaa aagagttgtg 150 <210> 4 <211> 103 <212> PRT <213> PRRSV Lelystad <400> 4 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             100 <210> 5 <211> 309 <212> DNA <213> PRRSV Lelystad <400> 5 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 tttcaggtt 309 <210> 6 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> Fusion gene <400> 6 atgatgtcct ggcgctacgc atgtaccaga tataccaact ttcttctgga tactaagggt 60 agactctatc gttggcggtc gcctgtcatc atagagaaaa ggggcaaagt tgaggtcgaa 120 ggtcatctga tcgacctcaa aagagttgtg ggatctatgg ccggtaaaaa ccagagccag 180 aagaaaaaga aaagtacagc tccgatgggg aatggccagc cagtcaatca actgtgccag 240 ttgctgggtg caatgataaa gtcccagcgc cagcaaccta ggggaggaca ggccaaaaag 300 aaaaagcctg agaagccaca ttttcccctg gctgctgaag atgacatccg gcaccacctc 360 acccagactg aacgctccct ctgcttgcaa tcgatccaga cggctttcaa tcaaggcgca 420 ggaactgcgt cgctttcatc cagcgggaag gtcagttttc aggtt 465 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer US5C3 F <400> 7 ggatccatga tgtcctggcg ctac 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer US5C3 R <400> 8 gtcgacttac acaactcttt tgag 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer EU7TC2 F <400> 9 ggatccatgg ccggtaaaaa ccag 24 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer EU7TC2 R <400> 10 gtcgacttaa acctgaaaac tgacctt 27

Claims (15)

An isolated protein in which the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4 are fused together. The isolated protein of claim 1, wherein the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4 are linked by a linker amino acid sequence. The isolated protein of claim 1, having the amino acid sequence of SEQ ID NO: 1. An isolated nucleic acid encoding a protein according to any one of claims 1 to 3. The isolated nucleic acid of claim 4 having the nucleotide sequence of SEQ ID NO: 6. 6. Culturing the host cell into which the nucleic acid according to claim 4 is introduced to express the protein; And
Isolating the expressed protein, the method of producing an isolated protein. Contacting the protein according to any one of claims 1 to 3 with a sample suspected of containing an anti-PRRSV antibody;
Detecting the presence of a complex of said protein with an anti-PRRSV antibody and
If it is detected that the complex is present, the sample is determined to contain an anti-PRRSV antibody, and if it is detected that the complex is not present, determining that the sample does not contain an anti-PRRSV antibody A method for detecting an anti-PRRSV antibody in a sample comprising a. 8. The method of claim 7, wherein the protein is immobilized on a solid substrate. 8. The method of claim 7, wherein the PRRSV is North American and European. 8. The method of claim 7, wherein said sample is pig blood, serum or plasma. Administering the protein according to claim 1 to a mammalian animal which is not a human; And
Separating the antibody that binds to the protein from the animal. The method of claim 11, wherein the antibody comprises an antibody binding to an amino acid sequence of SEQ ID NO: 2 and an antibody binding to an amino acid sequence of SEQ ID NO: 4. 13. Contacting the antibody obtained according to the method of claim 11 with a sample suspected of containing a PRRSV antigen;
Detecting a complex of the antibody with a PRRSV antigen; And
Determining that the sample contains a PRRSV antigen when the complex is present, and determining that the sample does not contain a PRRSV antigen when it is detected that the complex is not present. A method of detecting PRRSV antigen in a sample. A kit for detecting PRRSV in a sample comprising the isolated protein of any one of claims 1 to 3. A kit for detecting PRRSV in a sample comprising an antibody obtained according to the method of claim 11.

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