KR20120131531A - Peptide for detecting Porcine reproductive and respiratory syndrome virus and application thereof - Google Patents

Abstract

PURPOSE: A peptide for diagnosing porcine reproductive and respiratory syndrome virus(PRRSV) is provided to effectively diagnose PRRSV infection and to detect the presence of PRRSV antibody. CONSTITUTION: A peptide for diagnosing porcine reproductive and respiratory syndrome(PRRS) virus is a peptide of sequence number 1 or 2. A composition for diagnosing PRRSV contains the peptide of sequence number 1 or 2. A kit for diagnosing PRRSV contains the peptide of sequence number 1 or 2. The kit contains an antibody for the peptide of sequence number 1 or 2. A method for detecting a PRRSV antibody in a biological sample comprises: a step of providing the biological sample; a step of reacting the biological sample with the peptide; and a step of detecting the presence of the antibody.

Description

Peptide for detecting Porcine reproductive and respiratory syndrome virus and application approximately

The present invention relates to a peptide for diagnosing porcine respiratory syndrome virus (hereinafter referred to as 'PRRSV') and its application. More specifically, a PRRSV GP3 peptide antigen and a kit for diagnosing porcine respiratory syndrome virus infection using the same And a diagnostic method thereof.

Porcine reproductive and respiratory syndrome (PRRS) was first discovered in North America about 10 years ago (Keffaber KK, American Association Swine Practitioners Newsletter 1: 1-9, 1989) and soon after Europe (Paton DJ et al., Vet. Rec. 128: 617, 1991) and Asia (Shimizu et al., J. Vet. Med. Sci. 56: 389-391, 1994). Since then, PRRS has become one of the most common and economically important infectious diseases in pig breeding worldwide (Albina E., Vet. Microbiol. 55: 309-316, 1997; Dee et al., Vet. Rec. 140: 498-). 500, 1997), characterized by mild or severe fertility decline in sows and respiratory failure in piglets (Rossow KD Vet Pathol. 35: 1-20, 1998; Done SHand Paton D. J, Vet. Rec. 136 : 32-35, 1995; Rossow KD et al., Vet. Pathol. 32: 361-373, 1995; Christianson WT and Joo HS, Swine Health Production 2: 10-28, 1994).

The first PRRS virus (PRRSV) isolated from Europe and North America at about the same time was Lelystad (Wensvoort GC et al., Vet. Q. 13: 121-130, 1991) and VR-2332 (Benfield DA et al., J. Vet, respectively). Diagn. Invest. 4: 127-133, 1992; Collins JE et al., J. Vet. Diagn. Invest. 4: 117-126, 1992).

Although these species cause phenotypically indistinguishable conditions (Halbur et al., Vet. Pathol. 32: 649-660, 1995), genetically (Allende RT et al. J. Gen. Virol. 80: 307-315, 1999; Nelsen CJ et al., J. Virol. 73: 270-280, 1999; Meng XJ et al., Arch. Virol. 140: 745-755, 1995) and serologically (Wootton SK et al., Clin. Diagn. Lab. Immunol. 5: 773-779, 1998; Nelson EA et al., J. Clin.Microbiol. 31: 3184-3189, 1993; Wesvoort GE et al., J. Vet. Diagn. Invest. 4: 134-138, 1992) Clearly distinguished PRRSV viruses are currently divided into two different genotypes.

The virus belongs to Arteriviridae and has a positive strand RNA genotype. Viruses belonging to the arterviridae family include Equine arteritis virus, Lactate dehydrogenase elavating virus, Simian hemorrhagic fever virus, and PRRSV. PRRSV causes respiratory disease in young pigs, which has a significant economic impact. A virus that occurs mainly in Korea has been reported as VR 2332 strain.

Pigs have a rapid antibody response during PRRSV infection, but the synthesis of neutralizing antibodies is slow and their titers are low. This may vary from pig to pig. Attenuated vaccines and the like have been developed to prevent PRRSV infection, but the effect is rather low, and PRRSV infection has not been successfully controlled. The main cause of infection prevention failure in pig farms is the lack of accurate and sensitive diagnosis of PRRSV infection, while being relatively simple to use.

PRRSV is an enveloped virus, with a gene size of approximately 15 kb, with nine opening reading frames, ORF 1a, 1b, 2a, 2b, and 3-7. ORFs 1a, 1b are nonstructural proteins and ORFs 2-7 constitute composition proteins as GP2a, GP2b, GP3, GP4, GP5, membrane / matrix (M) proteins and nucleocapsid (N) proteins. The size of the virus has a diameter of 40-60 nm (Fig. 1).

Current diagnostic methods for identifying PRRSV infection include, in addition to conventional virus isolation, reverse transcription polymerase chain reaction (RT-PCR), a gene detection method, serum neutralization assay (SNA), and immunoenzyme tomography (IPMA). ), Indirect fluorescent antibody method (IFA) and enzyme immunosorbent method (ELISA). Of these, the most commonly used is IFA, which is known as a very sensitive diagnostic method along with IPMA. However, the antigen must be prepared through two tissue cultures, and the cultured virus must be purified and used, so it is not easy to prepare a diagnosis. Therefore, a high sensitivity and a diagnostic method that can be used in the field are required.

The present invention solves the above problems and the object of the present invention is to provide a kit for efficiently diagnosing the infection of PRRSV.

Another object of the present invention is to provide a method for efficiently diagnosing infection with PRRSV.

In order to achieve the above object, the present invention provides a peptide for diagnosing porcine respiratory syndrome virus selected from the group consisting of the peptides of SEQ ID NO: 1 and 2.

The present invention also provides an antibody that specifically recognizes the peptide of the present invention.

In another aspect, the present invention is a composition for diagnosing swine genital respiratory syndrome virus comprising the peptide of the present invention.

The present invention also provides a kit for diagnosing swine genital respiratory syndrome virus comprising the peptide of the present invention.

In another aspect, the present invention provides a kit for diagnosing swine genital respiratory syndrome virus comprising the antibody of the present invention.

The present invention also provides a method for (a) providing a biological sample; and (b) binding to said peptides of the invention of the swine genital respiratory syndrome virus when the swine genital respiratory syndrome virus is present in said biological sample to form an antibody / antigen complex. Under conditions which permit the biological sample to react with the peptides of the invention;

(c) detecting the presence of the complex to detect the presence of the antibody of swine genital respiratory syndrome virus in the sample, thereby providing a method for detecting the swine genital respiratory syndrome virus antigen in a biological sample.

In another aspect, the present invention is a diagnosis of swine genital respiratory syndrome comprising reacting the antibody according to the present invention to a sample containing the pig respiratory syndrome virus, and judging a sample showing a positive response to the antibody as swine genital respiratory syndrome Provide a method.

Hereinafter, the present invention will be described.

The present invention includes analyzing the antigenic and hydrophilic regions of the North American type PRRSV GP3 protein and selecting regions with different sequences. The peptide of SEQ ID NO: 1 is composed of amino acid sequence 83-89 of glycoprotein 3 (GP3) of PRRSV and the peptide of SEQ ID NO: 14 is amino acid sequence of 144-158, which is an immunogenic antigen specific for North American type PRRSV . The SEQ ID Nos. 1 and 2 are immunogenic antigens specific for North American PRRSV, and because of their short length, they can be usefully used for the detection of North American PRRSV in a sample.

The peptides of SEQ ID NOS: 1 and 2 were selected by analyzing the antigenic and hydrophilic regions of the North American PRRSV GP3 protein. Antigenicity and hydrophilicity analysis can be carried out using known methods, preferably antigenicity is Hopp-Woods method (Hopp, TP and Woods, KR 1981, Proc. Natl. Acad. Sci., USA, 78, 3824). Hydrophilicity can be analyzed according to the Kyte-Doolittle method (Kyte, J. and Doolittle, R. 1982, J. Mol. Bio. 157, 105-132). The peptides of SEQ ID NOs: 1 and 2 may be synthesized using known methods, preferably peptide synthesizers or obtained through genetic engineering methods.

The present inventors use a North American specific sequence having high antigenicity at the high antigenic region of PRRSV and at the Envelop GP3 portion other than ORF7 (Nuclocapsid P) or ORF5 (Envelope GP5) which are important sites for neutralizing antibody formation. A method for diagnosing PRRSV infection was found, and the presence of PRRSV GP3 antibody in porcine serum was used to determine the diagnosis of infection.

By immunogenic fragment in the present invention is meant a fragment of a biomarker protein having one or more epitopes that can be recognized by an antibody against a biomarker peptide of the invention.

In order to achieve another object of the present invention, the present invention provides a swine genre respiratory syndrome virus diagnostic agent comprising an antibody that specifically binds to the biomarker protein or immunogenic fragment thereof of the present invention.

The antibody of the present invention may be a polyclonal antibody, but is preferably a monoclonal antibody.

Polyclonal antibodies can be prepared by injecting a fragment of the biomarker protein of the invention or a peptide of the invention into an external host according to conventional methods known to those skilled in the art. Outer hosts include mammals such as mice, rats, sheep and rabbits. Immunogens are injected intramuscularly, intraperitoneally or subcutaneously, and are usually administered with an adjuvant to increase antigenicity. Blood is collected periodically from the external host to collect serum that shows improved titers and specificity for the antigen or to separate and purify the antibody therefrom.

Monoclonal antibodies can be prepared by techniques for generating immortalized cell lines by fusions known to those skilled in the art (Koeher and Milstein 1975, Nature, 256: 495). The manufacturing method is briefly described as follows. First, 20 µg of pure protein is obtained to immunize Balb / C mice or a peptide is synthesized and combined with bovine serum albumin to immunize mice. Thereafter, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and only hybridoma cells that produce the desired monoclonal antibody using ELISA method. After selection and propagation, monoclonal antibodies are isolated and purified from the culture.

In the method of the present invention, the detecting step detects the presence of the biomarker protein or immunogenic fragment thereof directly by two-dimensional (2-D) electrophoresis from the blood solution of the liver of the individual, or by contacting the blood with the antibody of the present invention. Indirectly confirming the presence of a biomarker protein or peptide of the invention through an antigen antibody reaction.

Currently known immunoassay as an antigen antibody reaction, enzyme immunoassay (ELISA, Coated tube), antibody-bound magnetic particles are bound to a tube, and then antigen-tracer and refractory contaminants are reacted competitively to induce enzymatic reaction. Magnetic particle method, latex particle method using antibody bound latex particle.

Diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins and the like. The antibody may be labeled by radionuclides, fluorescent sources, enzymes, and the like.

Monoclonal antibodies of the present invention can be used in a variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as various pig genitalia through the development of antibodies showing higher specificity and sensitivity It can also be used to develop protein chips with respiratory syndrome virus detection spectrum.

As described above, the peptides of the present invention can be used as a diagnostic agent to detect the presence of reactive antibodies of swine genital respiratory syndrome virus in a biological sample to determine the presence of porcine respiratory syndrome virus infection. For example, the presence of antibodies reactive to the peptides of the invention can be detected by using standard electrophoresis and immunodiagnostic techniques, including competition, direct response or immunoassay such as sandwich type assays. Such assays include, but are not limited to, Western blots; Coagulation test; Enzyme labeled and mediated immune assays such as ELISA; Biotin / avidin type assays; Radioimmunoassay; Immunoelectrophoresis; And immunoprecipitation. Such reactions generally involve identifying labels such as fluorescence, chemiluminescence, radioactive activity, enzymatic labels or dye molecules, or include other methods of detecting the formation of an antigen and the antibody or antibodies that react with it. .

The assay described above generally involves isolating unbound antibodies in the liquid phase from the solid phase support to which the antigen-antibody complexes are bound. Solid supports that can be used in the practice of the present invention include substrates such as nitrocellulose (eg, in the form of membranes or microtiter wells); Polyvinylchloride (eg, sheet or microtiter wells); Polystyrene latex (eg, beads or microtiter plates); Polyvinylidene fluoride; Diazotized paper; nylon membrane; And activated beads, magnetically reactive beads, and the like.

Typically, the solid support first reacts with a solid phase component (eg, one or more porcine respiratory syndrome virus peptides) under suitable binding conditions to ensure that the component is sufficiently anchored to the support. Sometimes, the immobilization of the antigen to the support can be enhanced by binding the antigen to peptides with better binding properties. Suitable binding proteins include, but are not limited to, macromolecules such as serum albumin, including bovine serum albumin (BSA), kiolimpet hemocyanin, immunoglobulin molecules, tyroglobulins, ovalbumins, and other proteins known to those skilled in the art. Included. Other molecules that can be used to bind the antigen to the support include polysaccharides, polylactic acid, polyglycolic acid, polymerizable amino acids, amino acid copolymers, and the like. Such molecules and methods of binding such molecules to antigens are known to those skilled in the art. Brinkley, M.A. Bioconjugate Chem. (1992) 3: 2-13; Hashida et al., J. Appl. Biochem. (1984) 6: 56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30: 117-124.

After reacting the solid support with the solid phase component, any unfixed solid phase component is removed from the support by washing, and the support bound component is subsequently found to contain ligand residues (eg, antibodies to immobilized antigen) under suitable binding conditions. Contact with the expected biological sample. After washing to remove any unbound ligand, a secondary binder moiety is added under suitable binding conditions, where the secondary binder can optionally be bound to the bound ligand. The presence of secondary binders can then be detected by using techniques known in the art.

More particularly, ELISA methods are used wherein the wells of the microtiter plates can be coated with the required protein. Biological samples containing or expected to contain anti-protein immunoglobulin molecules are then added to the coated wells. After sufficient incubation to allow the antibody to bind to the immobilized antigen, the plate is washed to remove unbound residues and detectably labeled secondary binding molecules added. The secondary binding molecules are allowed to react with any captured sample antibody, the plates are washed, and the presence of secondary binding molecules is detected by using methods known in the art.

Thus, in one specific embodiment, the presence of the bound anti-antigen ligand from the biological sample can be readily detected by using secondary binders that include antibodies directed against the antibody ligand. Many anti-pig immunoglobulin (Ig) molecules that can be readily conjugated to enzyme labels detectable by using methods known to those skilled in the art such as horseradish peroxidase, alkaline phosphatase or urease are known in the art. It is. Appropriate enzyme substrates are then used to generate a detectable signal. In other related embodiments, competitive type ELISA techniques can be practiced using methods known to those skilled in the art.

The assay is performed in solution such that antibodies specific for the peptides of the invention form complexes under precipitation conditions. In one particular embodiment, the peptides of the invention can be bound to solid phase particles (eg, agarose beads, etc.) by using binding techniques known in the art, such as by direct chemical bonding or indirect bonding. have. The antigen coated particles are then contacted with biological samples that are expected to contain antibodies to the protein under suitable binding conditions. Crosslinking between bound antibodies leads to the formation of particle-antigen-antibody complex aggregates that can be precipitated and separated from the sample by using washing and / or centrifugation. The reaction mixture can be analyzed to determine the presence or absence of the antibody-antigen complex by using any of a number of standard methods, such as the immunodiagnostic method described above.

In another embodiment, an immunoaffinity matrix is provided wherein a group of polyclonal antibodies from a biological sample expected to contain an antibody against a desired peptide of the invention is immobilized on a substrate. In this regard, initial affinity purification of the sample can be performed by using immobilized antigen.

Many methods are known in the art for immobilizing immunoglobulins (in gratuitous or specific fragments) at high yields and good retention levels of antibody binding activity. Although not limited to any particular method, immobilized Protein A or Protein G can be used to immobilize immunoglobulins.

Thus, when an immunoglobulin molecule is immobilized to provide an immunoaffinity matrix, the peptide is contacted with the bound antibody under suitable binding conditions. After any nonspecifically bound antigen has been washed out of the immunoaffinity support, the presence of the bound antigen can be measured by assaying the label using methods known in the art.

In addition, the antibodies to be raised against the peptides of the invention, rather than the peptides themselves, can be used in the assays described above to detect the presence of antibodies to proteins in a given sample. Such assays are basically performed as described above and are known to those skilled in the art.

As can be seen through the present invention, the peptide of the present invention was able to efficiently diagnose the infection of PRRSV in pigs, and can be used to efficiently detect the presence of PRRSV antibodies.

1 is a diagram showing the structure of the PRRSV virus model and seven ORFs.
Fig. 2 shows the antigenicity (Hopp-Woods method) and hydrophilicity analysis (Kyte-Doolittle method) of North American type PRRSV GP3.
3 is a diagram showing the antigenicity and hydrophilicity analysis (IEDB: Immune epitope database method) of the North American type PRRSV GP3.
4 is a diagram showing the results of serum diagnostics (ELISA) using the PRRSV GP3 peptide antigen.

The following examples are intended to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

Example  One: PRRSV of GP3  Antigenicity, Hydrophobicity Analysis

Antigenicity of the GP3 portion of PRRSV using the gene sequence of the North American PRRSV VR 2332 strain (Hopp, TP and Woods, KR 1981, Proc. Natl. Acad. Sci., USA, 78, 3824-3828) Hydrophilicity was analyzed according to the Kyte-Doolittle method (Kyte, J. and Doolittle, R. 1982, J. Mol. Bio. 157, 105-132) and the Immune epitope database and analysis resource of NIAID. Immune epitope database (IEDB) was used to find specific sequences of interest. High antigenic-hydrophilic sites were identified in the peptides of 83-89 and 144-158 of GP3.

Peptide Amino acid sequence Amino acid position GP3-1 eeddhde (SEQ ID NO: 1) 83-89th GP3-2 ICAEHDGQNTTLPRH ( SEQ ID NO: 2) 144-158th

Table 1 is a table showing selected PRRSV GP3 high antigenic-hydrophilic sites.

Example  2: PRRSV of GP3 peptide  Serum diagnosis using antigens ELISA )

ELISA was analyzed using two kinds of peptides prepared above (COSMO, Korea). The peptide antigen was diluted to 10 µg / ml using PBS, and two peptides were coated at 100 µl / well in each well of a 96 well plate (Maxisorp, Nunc, Denmark).

The plate was stored at 4 ° C. for one day, washed three times with PBST (1 × PBS + 0.1% Tween-20), then blocked at room temperature for 30 minutes with PBS with 5% skim milk powder, and with PBST. Washed three times.

As a pig sample for diagnosis, 10 rats not vaccinated with the PRRSV vaccine and 10 sera vaccinated were used. The serum was diluted 1: 100 with PBS and reacted at room temperature at a concentration of 100 μl / well for 2 hours, and washed 5 times with PBST. HRP-conjugated goat anti-pig IgG (Serotec, UK) diluted 1: 500 was used as a secondary antibody. The secondary antibody was also reacted at room temperature for 1 hour and then washed 7 times with PBST. The detection of the bound secondary antibody was reacted at room temperature for 10 minutes with a TMB substrate (BioFX, SurModics, USA), and then 5N HCl was administered at well / 20μl to measure 450nm after completion of the reaction to observe reactivity.

Based on the above results, it can be confirmed that PRRSV infection diagnosis can be effectively performed in porcine serum through a diagnosis method using two types of peptides of the GP3 moiety presented in the present invention.

<110> Konkuk University Industrial Cooperation Corp. <120> Peptide for detecting Porcine reproductive and respiratory          syndrome virus and application <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 7 <212> PRT <213> PRRSV <400> 1 Glu Glu Asp Asp His Asp Glu   1 5 <210> 2 <211> 15 <212> PRT <213> PRRSV <400> 2 Ile Cys Ala Glu His Asp Gly Gln Asn Thr Thr Leu Pro Arg His   1 5 10 15

Claims (7)

Peptide for diagnosing porcine respiratory syndrome virus selected from the group consisting of peptides of SEQ ID NO: 1 and 2. An antibody that specifically recognizes the peptide of claim 1. Composition for diagnosing swine genital respiratory syndrome virus comprising the peptide of claim 1. Pig genital respiratory syndrome virus diagnostic kit comprising the peptide of claim 1. Pig genital respiratory syndrome virus diagnostic kit comprising the antibody of claim 2. (a) providing a biological sample;
(b) when the swine genital respiratory syndrome virus is present in the biological sample, the biological sample is combined with the peptide of claim 1 under conditions such that the swine genital respiratory syndrome virus can bind to the peptide of claim 1 to form an antibody / antigen complex. React;
(c) detecting the presence of the antibody of swine genital respiratory syndrome virus in the sample by detecting the presence of the complex, thereby detecting the swine genital respiratory syndrome virus antibody in the biological sample. A method for diagnosing swine genital respiratory syndrome, comprising reacting an antibody according to claim 2 to a sample comprising swine genital respiratory syndrome virus and judging a sample showing a positive response to the antibody as swine genital respiratory syndrome.

Images