KR101101386B1 - Immunogenic peptide specific for a north American type PRRSV and use thereof - Google Patents

Abstract

One or more exemplary embodiments provide immunogenic peptides specific for North American PRRSV, methods for diagnosing infection with PRRSV in pigs using the same, and kits for the same.

PRRSV, North American, Peptide

Description

Immunogenic peptide specific for a north American type PRRSV and use etc}

This study was conducted as part of the research project for agricultural and forestry technology development of the Department of Agriculture, Forestry and Technology Center (ARPC) [107086-03-2-CG000, Development of PRRSV vaccine and differential diagnosis using heterologous antigen expressing live bacterial vaccine system].
The present invention relates to a method for diagnosing porcine respiratory syndrome virus infection using the PRRSV GP5 peptide antigen. The present invention relates to a method for diagnosing PRRSV infection using a peptide antigen of a high immunogenic part in antigenic glycoprotein 5 (GP5).

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, which causes sows miscarriage, stillbirth, and pneumonia and dysplasia in fetuses, low birth rate, low growth rate, other secondary bacterial infections, and respiratory disease in pigs of all ages, both domestically and internationally. Is a contagious disease that has seriously affected pig industry. PRRSV, which was first isolated and reported in 1993 in Korea, preferentially proliferates in macrophages. Its onset forms are genital, respiratory, and mixed, primarily from pigs to pigs, and colostrum antibodies can be transmitted to pigs without lactation, through feces, urine, milk, It can also be transmitted by needles, insects and air.

PRRSV has a wide variety of genetically diverse strains, depending on the geopolitical location at the same time. The two main genotypes are European (genotype 1) and North American (genotype 2), which show 40% nucleotide differences in terms of genome level. The strain that occurs mainly in Korea has been found to be VR2332. According to the study, North American PRRSV has been evolving since it spread to Korean piglets, and it is predicted that genomic alteration of PRRSV gene is caused by geopolitical influence.

The overall genome size of PRRSV is 15 kb and has 9 open reading frames (ORFs) (ORFs 1a, 1b, 2a, 2b, 3-7) with the lower genome mRNA overlapping each other. ORF 1a and 1b are replication related nonstructural proteins and ORF 2-7 constitute structural proteins as GP2a, GP2b, GP3, GP4, GP5, membrane / matrix (M) protein and nucleocapsid (N) protein. The major structural proteins are N protein, M protein and GP5, the primary envelope glycoprotein. Among them, GP5 (ORF 5) protein is the most abundant protein known to be able to induce neutralizing antibodies in the host during infection (Domestic Patent Publication No. 2008-0064813).

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, with anti-GP5 neutralizing antibodies lasting much longer than anti-N protein antibodies in infected pigs. 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.

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, it is not easy to prepare the diagnosis because the antigen must be prepared through two tissue cultures, and the cultured virus must be purified and used. As a recently developed diagnostic method, Korean Patent Application Publication No. 2008-0055792 discloses a diagnostic method for detection of an antibody that recognizes a PRRSV virus. In addition, International Application WO 2006/091824 and Korean Patent Application Publication No. 2002-0015919 disclose a method for detecting an antibody against PRRSV using a protein or peptide based on PRRSV, but it is highly sensitive and effective and can be performed quickly in the field. There is still a need for a method of diagnosing possible PRRSV infection.

The inventors have surprisingly found a method for diagnosing PRRSV infection by selecting a neutralizing region that is highly antigenic and specific to North American strains from the GP5 protein of the PRRS virus, and thus in a sample such as pig serum. By determining the presence of antibodies to the PRRSV GP5 protein antigen, we tried to diagnose PRRSV more accurately.

One exemplary embodiment provides a method for efficiently diagnosing the infection of PRRSV in pigs.

Another exemplary embodiment provides a kit for efficiently detecting the presence of PRRSV antibodies in a sample.

Another exemplary embodiment provides an isolated peptide having the amino acid sequence of SEQ ID NO: 1.

One exemplary embodiment of the present invention comprises the steps of contacting a sample comprising an anti-Swine Respiratory Syndrome Virus (PRRSV) antibody to a peptide of SEQ ID NO: 1 immobilized on a solid substrate; Contacting the contact result with an anti-pig antibody labeled with a detectable label; And it provides a method for diagnosing the infection of PRRSV in pigs, comprising the step of measuring the signal from the label.

The method comprises contacting a sample comprising an anti-Swine Respiratory Syndrome Virus (PRRSV) antibody to a peptide of SEQ ID NO: 1 immobilized on a solid substrate. The peptide of SEQ ID NO: 1 is a group consisting of amino acid sequences 145-175 of glycoprotein 5 (GP5) of PRRSV, and is an immunogenic antigen specific for North American type PRRSV. Since the peptide of SEQ ID NO: 1 is composed of 31 amino acids and is an immunogenic antigen specific for North American type PRRSV, and its length is short, it can be usefully used for detection of North American type PRRSV in a sample. Therefore, the PRRSV may be a North American type PRRSV.

The peptide of SEQ ID NO: 1 was selected by a method comprising analyzing the antigenic and hydrophilic regions of the European PRRSV and North American PRRSV GP5 proteins, and selecting regions having different sequences from each other. Analysis of antigenicity and hydrophilicity can be carried out using known methods, preferably antigenicity Hopp-Woods method (Hopp, TP and Woods, KR 1981, Proc. Natl. Acad. Sci., USA, 78, 3824-3828), and hydrophilicity can be analyzed according to the Kyte-Doolittle method (Kyte, J. and Doolittle, R. 1982, J. Mol. Bio. 157, 105-132). The peptide of SEQ ID NO: 1 may be carried out using a known method, preferably using a peptide synthesizer.

The solid substrate can be any substrate known in the art. For example, the substrate may be selected from the group consisting of silicon, glass, or plastic. The solid substrate may also be in any shape, for example in the form of a plate, well, bead, spherical, or porous material. In addition, the substrate may be to form the inner surface of the microchannel substrate or the microchannel or chamber of the microfluidic device.

The contact can be made by adding the sample, for example a liquid sample, to the substrate on which the peptide is immobilized. The liquid medium in which the contact is made may be made in a suitable buffer, for example a buffer such as PBS. Such contact may be appropriately selected by those skilled in the art. The contact temperature may be, for example, room temperature to 45 ° C., but is not limited thereto. The contact results in the anti-PRRSV antibody in the sample binding to the peptide to form an anti-PRRSV antibody-peptide complex. The PRRSV antibody may be an anti-GP5 antibody, eg, an antibody that binds to the peptide of SEQ ID NO: 1. The sample may be selected from whole blood, serum and plasma.

The method may further include washing after the contacting step. The washing step may be performed using a washing solution which removes a substance in a sample that does not bind the peptide or a substance that is not specifically bound to the peptide. For example, the wash may be a buffer containing a suitable surfactant, eg TWEEN ^™ , for example PBS buffer.

The method also includes contacting the contact result with an anti-pig antibody labeled with a detectable label. The contact can be made by adding and culturing a liquid comprising a labeled anti-pig antibody to the contact result. The contact conditions and time can be appropriately selected by those skilled in the art.

Such detectable labels are known in the art. For example, the detectable label can be an enzyme that catalyzes the reaction of converting the fluorescent material, radioactive material, and substrate into a light emitting material. The enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP) or beta-galactosidase. The substrate can be TMB, ABTS or X-gal. The fluorescent material may be Cy3 or Cy5. The Hyogo conjugated anti-pig antibody can also be purchased commercially. The anti-pig antibody may be anti-pig IgG, anti-pig IgM, anti-pig IgE, anti-pig IgA or anti-pig IgE.

After contacting the anti-pig antibody labeled with the label, the method may further include washing. The washing can be accomplished using a wash solution which removes unbound material or nonspecifically bound material to the peptide and anti-PRRSV antibody complex in the anti-pig antibody solution. For example, the wash may be a buffer containing a suitable surfactant, eg TWEEN ^™ , for example PBS buffer.

The method also includes measuring a signal from the label. The signal may be an optical signal, for example absorbance, transmittance, or fluorescence. The wavelength of the optical signal can be appropriately selected by those skilled in the art according to the label selected.

As a result of the signal measurement, when the optical signal measured in the sample is significantly higher than the negative control, it can be determined that the anti-PRRSV antibody is present in the sample. The significant level can be appropriately set with reference to the signal from the positive control.

Another embodiment of the present invention provides a kit for detecting the presence of a PRRSV antibody in a sample comprising the peptide of SEQ ID NO: 1 and an anti-pig antibody labeled with a detectable label.

The peptide of SEQ ID NO: 1, the detectable label, the anti-swine antibody, and the PRRSV are as described above.

Another exemplary embodiment of the invention provides an isolated peptide having the amino acid sequence of SEQ ID NO: 1. The peptide is specifically present in North American PRRSV and has immunogenicity, and thus can be used to specifically detect North American PRRSV.

Another exemplary embodiment of the present invention provides a microarray or microplate having a substrate to which an isolated peptide having the amino acid sequence of SEQ ID NO: 1 is immobilized.

According to one exemplary embodiment, a method for efficiently diagnosing the infection of PRRSV in a pig is provided.

According to another exemplary embodiment, a kit is provided for efficiently detecting the presence of a PRRSV antibody in a sample.

According to another exemplary embodiment, an isolated peptide having the amino acid sequence of SEQ ID NO: 1 can be used to efficiently detect the presence of a PRRSV antibody.

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

Example 1: GP5 Antigenicity and Hydrophobicity Analysis of PRRSV

For North American (VR2332) and European PRRSV GP5 proteins, antigenicity was analyzed using Hopp-Woods method and hydrophobicity was analyzed using Kyte-Doolittle method. The results are shown in FIG. Based on the results, two antigenic and hydrophilic patterns were compared. According to Figure 1, the amino acid sequence of the 145th to 175th amino acid portion is different between the North American type and the European type, the hydrophilicity and antigenicity is high, this part is selected as the peptide antigen of the present invention, synthesized as shown in Table 1 below Prepared peptides (Anygen, Korea). In Figure 1, regions 148 to 184 represent the high antigenic-hydrophilic sites where the peptide antigens of the present application are selected.

Table 1. Selected PRRSV GP5 high antigenic-hydrophilic sites

Amino acid sequence Amino acid position LLDTKGGLYRWRSPVIIEKRGKVEVEGHLID 145-175

Example 2 Neutralizing Site Peptide Antigen Preparation for Comparative Experiments

Previous studies have reported that neutralizing antibody sites exist in PRRSV GP5. This site is called the ectodomain or neutralization site. The neutralizing antibody site was prepared as shown in Table 2 below as a control of the present invention.

Table 2. PRRSV GP5 neutralizing antibody sites used as controls in the present invention

Amino acid sequence Amino acid position SHLQLIYNL 37-45th

Example 3: Experiment with HerdCheck PRRS Virus Antibody Test Kit as Positive Control

Serum tests using an ELISA kit (Herdcheck® PRRSV Antibody Kit, IDEXX Laboratories, USA) as a control for this experiment were performed according to the manufacturer's instructions. When the S / P (Sample / Positive Control mean) value was less than 0.4 for the OD value measured after the experiment, the serum was negative. This value was used to calculate sensitivity and specificity using the ELISA results obtained from the peptide antigens of the present application. The result of ELISA showed that the antibody titer of the experimental group was 0.853 ± 0.519 and 1.633 ± 1.15 by S / P ratio. According to the above results, 209 positive individuals out of a total of 258 samples showed a natural infection rate of 81%.

Example 4: Serum Diagnosis with GP5 Peptide Antigen of PRRSV (ELISA)

ELISA analysis was performed using the peptide antigen of the present invention prepared in Example 1. That is, the peptide antigen was diluted to 10 µg / ml with PBS and coated at a concentration of 100 µl / well in 96-well plates (Maxisorp ™, Nunc, Denmark). The plate was blocked for 1 hour with PBS with 1% skim milk and washed 3-4 times with PBST (PBS + 0.05% Tween-20, pH 7.4).

As porcine samples (primary antibody solution) for diagnosis, 258 pig serum randomly selected from 21 farms without PRRSV vaccine were used. As a positive control, 5 pigs whose vaccine had been identified as having antibodies to PRRSV were used. As a negative control, 10 colostrums born from PRRSV-free sows were used for the previous piglets. Serum was used.

The primary antibody solution obtained from the pigs was diluted with PBS and a total of 90 μl was added to the wells, followed by reaction at 37 ° C. for 1 hour, washed three times with PBST, and further diluted 1: 500 with HRP-conjugated goat antiserum. Pig IgG (Serotec, UK) was used as the secondary antibody and reacted. The secondary antibody was also reacted at 37 ° C. for 1 hour and then washed three times with PBST. Detection of bound secondary antibody was performed using a substrate solution [100 mM citric acid buffer, pH 4.0, 10 ml, 250 µl of ABTS stock solution (ABTS 100 mg in 4.5 ml distilled water), 50 µl of H ₂ O ₂ ]. The reaction with the substrate solution was carried out in the dark for 10 minutes and the OD value was measured using an ELISA reader (Multiskan EX, Thermo LabSystems, Beverly, MA) at absorbance 405 nm. The measured OD values were expressed as mean ± standard deviation (SD) and the values were statistically tested by Student's t-test.

result

ELISA was performed on 260 pigs randomly selected from 21 farms. As a result, ELISA using the neutralizing antibody peptide (amino acids 37-45: SEQ ID NO: 2) as the control group was performed. Antibody titers were found not to be high or induced (Table 3). However, the peptide of SEQ ID NO: 1 according to the present invention showed very high sensitivity (86.6%), which was concluded to be very suitable as an antigen used for serum diagnosis. Sensitivity is statistically represented as compared to the control group means true positive / (true positive + negative). True positive indicates positive in both experiments and false negative indicates negative on only one side.

Table 3. Sensitivity and Specificity Analysis by Serum Diagnosis (ELISA) Results Using the Peptide Antigens of the Application and Comparison with ELISA Kits

antigen ELISA kit Chinese Antibody Site
(37-45 SEQ ID NO: 2) Peptide Antigens herein
Example 1 positivity 209 83 208 voice 49 175 50 Jinyangseong 74 181 False positive 9 27 False negative 135 28 True voice 40 22 responsiveness 35.41 86.60 Specificity 81.63 44.90

Specificity is expressed as true negative / (responsive + positive). Since looking at the example results in the embodiment 175 in the experiment using a neutralizing antibody region that is, almost always appeared negative, the negative overlap with the sample found in the ELISA kit specific lot be as both widening the negative jineung property also (Please confirm) Is higher. In addition, the ELISA kits may differ in specificity because the antigen is GP7, ie the "N" protein and the antigen is GP5. However, what is important in the diagnosis is the sensitivity regarding the efficiency of detecting true positives.

Through the above results, the method for diagnosing PRRSV infection using a peptide comprising a PRRSV GP5 high antigenic site according to the present invention can accurately and easily detect infection with a genotype-specific North American virus compared to the prior art. Turned out.

1 shows antigenicity (Hopp-Woods method) and hydrophobicity analysis (Kyte-Doolittle method) of North American type (A) and European type (B) PRRSV GP5.

<110> Industry Foundation of Chonnam National University <120> Immunogenic peptide specific for a north American type PRRSV and use <130> PN081134 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 31 <212> PRT <213> Arteriviridae arterivirus <220> <221> PEPTIDE <222> (1) <223> PRRSV GP5 145-175 <400> 1 Leu Leu Asp Thr Lys Gly Gly Leu Tyr Arg Trp Arg Ser Pro Val Ile   1 5 10 15 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp              20 25 30 <210> 2 <211> 9 <212> PRT <213> Arteriviridae arterivirus <220> <221> PEPTIDE <222> (2) <223> PRRSV GP5 37-45 <400> 2 Ser His Leu Gln Leu Ile Tyr Asn Leu   1 5  

Claims (12)

Contacting a sample comprising a North American anti-swine respiratory syndrome virus (PRRSV) antibody to a peptide of SEQ ID NO: 1 immobilized on a solid substrate; Contacting the contact result with an anti-pig antibody labeled with a detectable label; And A method for diagnosing the infection of a North American PRRSV in a pig, comprising measuring a signal from the label. The method of claim 1, wherein the sample is selected from whole blood, serum, and plasma. The method of claim 1, wherein the detectable label is an enzyme that catalyzes the reaction to convert the fluorescent material, radioactive material, and substrate into an optical signal generating material. The method of claim 3, wherein the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (AP). The method of claim 1, wherein the anti-pig antibody is anti-pig IgG, anti-pig IgM, anti-pig IgE, anti-pig IgA or anti-pig IgE. delete A kit for detecting the presence of a North American PRRSV antibody in a sample comprising the peptide of SEQ ID NO: 1 and an anti-pig antibody labeled with a detectable label. 8. The kit of claim 7, wherein the detectable label is an enzyme that catalyzes the reaction to convert the fluorescent material, radioactive material, and substrate into an optical signal generating material. The kit of claim 8, wherein the enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (AP). The method of claim 8, wherein the anti-pig antibody is anti-pig IgG, anti-pig IgM, anti-pig IgE, anti-pig IgA or anti-pig IgE. delete delete

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