KR20080087317A - Protein chip for diagnosing porcine circovirus and method of diagnosing porcine circovirus using the chip - Google Patents

Abstract

A protein chip is provided to diagnose infection of porcine circovirus(PCV) by identifying a concentration of a PCV type 2(PCV2) through an SPR imaging diagnosis system. A protein chip is characterized in that PCV2 capsid antigen is supported on a metal thin film substrate, wherein the metal is gold, silver, copper or aluminum. A method for measuring a concentration of the PCV antigen comprises the steps of: (a) preparing the protein chip by supporting the PCV2 capsid antigen on the metal thin film substrate; (b) reacting the protein chip with a serum sample; and (c) detecting an SPR signal strength of an antigen-antibody complex formed on the surface of the SPR protein chip.

Description

Protein chip for diagnosing porcine circovirus and method for diagnosing porcine circovirus using same {{Protein chip for diagnosing porcine circovirus and method of diagnosing porcine circovirus using the chip}

1 is a photograph of cloning the capsid gene, which is the ORF2 portion of PCV2, to a pRSET vector and performing PCR, followed by 1.0% agarose gel electrophoresis of the PCR product.

Figure 2 is a Western blotting whether the protein expressed from the pLysS (pRSET-circo-cap) plasmid hourly (1-4 hours).

3 is a graph showing signal intensity according to control antibody concentration in an SPR imaging protein chip in which PCV2 capsid antigen is immobilized on a substrate via carboxymethylated dextran matrix.

FIG. 4 shows the results of measuring antibody concentrations in 70 pig serum using an SPR imaging protein chip in which PCV2 capsid antigen was immobilized on a substrate via carboxymethylated dextran matrix.

Figure 5 is a result of comparing the antibody concentration in pig serum using SPR imaging and ELISA.

The present invention relates to a protein chip immobilized with a pig circovirus type 2 (PCV2) capsid antigen on a metal thin film substrate, a method for producing the protein chip, and a method for measuring the concentration of the pig circovirus antibody in serum samples using the chip. will be.

One pathogen that is a serious problem in the swine industry at home and abroad is Porcine circovirus type 2 (PCV2). The pathogen was first discovered in weaned pigs in 1991, with weight loss, dyspnea, and jaundice, with gross interstitial pneumonia, systemic lymph nodes, hepatitis, and nephritis. The pathogen also increases in pathogenicity when mixed with other pathogens, including the swine parvovirus and the swine genital respiratory syndrome virus.

Porcine circovirus type 2 (PCV2) is a small nonenveloped virus with a single circular circular DNA genome and belongs to Circoviridae . PCV has two open reading frames (ORFs), where ORF1 is an integral part of the virus's replication, while ORF2 encodes the capsid protein. ORF2 protein is an antigenic site that distinguishes between type 1 and type 2, which is an important site used to produce diseases and to produce vaccines.

The method of testing the antibody concentration of PCV2 has been used to confirm that the virus has been previously infected or has been infected until now, when no vaccine against this virus was developed. Neutralizing antibody assay and enzyme-linked immunosorbent assay (ELISA) have been used for this method. However, the neutralizing antibody test is stable but time-consuming and costly. Although the ELISA method has been used as an effective method for measuring trace antigen proteins, the analysis range of antigen proteins is 10 pg / ml ~ 1 It is about ng / ml, and one antigen protein can be analyzed at a time, so that a large amount of sample is required for analyzing various antigen proteins. Therefore, a more effective diagnostic method is required.

Protein chip measurement technology has been developed and utilized as a technique for analyzing a large number of antigenic proteins at once. A protein chip is a variety of antibody or receptor (receptor) protein is arranged on a single substrate, by reacting a sample on the protein chip to measure the amount of protein bound to the chip. In order to measure the amount of various sample proteins bound on a protein chip, analytical methods capable of converting signals by isotopes, fluorescence, enzymatic reactions, etc. have been introduced. However, isotope measurement has a problem in safety, and enzyme reaction measurement has a narrow analysis range, making it difficult to analyze samples in various concentrations, and fluorescence measurement has a problem of using expensive fluorescent materials (Schweitzer, B. and Kingsmore, SF, Curr. Opin. Biotechnol., 2002, 13, 14-19; Schweitzer, B. et al., Nature Biotechnol., 2002, 20, 359-366). Thus, there has been a continuing need for a method for solving the problems of the above assays.

Surface plasmons are collective vibrations of electrons occurring on the surface of the metal thin film as an activating material, and surface plasmon waves generated by them are surface electromagnetic waves propagating along the interface between the metal and the adjacent dielectric material. When an electric field is applied to the interface of two media having different dielectric functions from the outside, surface charges are induced due to the discontinuity of the vertical component of the electric field at the interface, and the surface charge oscillates as surface plasmon waves. Surface plasmon resonance (SPR) occurs under conditions where the wavelength and propagation constants of the applied electromagnetic waves coincide with the surface plasmons occurring on the metal thin film surface. The propagation constant is increased by using an evanescent wave generated when light is totally reflected through a medium having a high refractive index such as glass, and is incident when it is coincided with the propagation constant of the surface plasmon wave at a specific angle of incidence of light. The energy of light is transmitted, causing resonance. These surface plasmon resonance phenomena are used to analyze biological changes such as antigenic antibody responses in protein chips.

Therefore, the present inventors fabricated a protein chip using recombinant PCV2 ORF2 protein (capsid), and a pig serum using surface plasmon resonance (SPR) imaging diagnostic method that can effectively detect antigen-antibody reactions in a short time as a non-labeling method. We have developed a system for measuring PCV2 antibody concentration, and completed the present invention.

It is an object of the present invention to provide a protein chip in which a pig circovirus type 2 (PCV2) capsid antigen is immobilized on a thin metal substrate which can reduce the diagnosis time and cost of the pig circovirus.

Another object of the present invention is to provide a method for measuring the pig circovirus antibody concentration in the serum sample which can reduce the diagnostic time and cost of the pig circovirus.

Another object of the present invention is to provide a method for preparing a protein chip to which the pig circovirus type 2 (PCV2) capsid antigen is immobilized.

In order to achieve the object of the present invention, the present invention provides a protein chip immobilized porcine circovirus type 2 (PCV2) capsid antigen on a metal thin film substrate.

Porcine circoviruses (PCVs) have two open reading frames (ORFs), where ORF1 is an integral part of the replication of the virus, while ORF2 encodes a capsid protein. ORF2 protein is an antigenic site that distinguishes between type 1 and type 2, which is an important site used to produce diseases and to produce vaccines. In the protein chip of the present invention, a capsid antigen corresponding to type 2 is accumulated among ORF2 of PCV. Preferably, the capsid antigen of PCV2 ORF2 may have an amino acid sequence of SEQ ID NO: 1.

The metal thin film forming the protein chip of the present invention may be gold, silver, copper or aluminum, but may be made of a metal which is not limited thereto, and preferably, may be made of gold. In the present invention, it is preferable to use gold (Au, purity of 99.9% or more) as a metal capable of producing surface plasmon waves and measuring surface plasmon resonance angles. This is because gold thin films are easy to form self-assembled monolayers for organic functional groups that are selective for the detection of contaminants and are advantageous metals for generating surface plasmon waves. Metal thin films can be prepared using techniques generally known in the art.

Protein chip according to an embodiment of the present invention can be used for the measurement of porcine circovirus antibody, preferably can be used for the measurement of porcine circovirus type 2 antibody. The protein chip of the present invention is produced by cloning a capsid protein of porcine circovirus type 2 and dripping it onto an SPR gold thin film chip. The protein chip thus prepared can react with pig serum and check the results with SPR imaging equipment to evaluate antibody concentration.

The protein chip according to one embodiment of the present invention may be for surface plasmon resonance (SPR) imaging measurement. SPR is a method of sensitively measuring the change in refractive index on a metal surface, and when a biochemical sample such as a protein is bound to the metal surface, it can detect its amount (Nice, EC and Catimel, B., BioEssays, 1999, 21, 339-352). SPR has the advantage of directly measuring the binding of the biological sample without a separate signal amplification.

Protein chip according to an embodiment of the present invention is a transparent support layer, a gold thin film layer coated with gold (Au) on one side on the transparent support layer and an alkane comprising a thiol group (SH) on the gold thin film layer It may include a self-assembled monolayer formed of a compound.

The transparent support layer is preferably a silica material, in particular glass of SF10 or BK7 material when used in surface plasmon resonance (SPR) devices. The reason is that the material is the same as the prism of the surface plasmon device to be used in the present invention. When the material is used, the surface plasmon (SPR) resonator can be used in various media (air, aqueous solution, organic solvent, etc.). This is because the plasmon resonance (SPR) angle can be measured.

On the other hand, it is preferable to further layer a chromium (Cr) layer between the transparent support layer and the gold thin film layer. In this case, the chromium layer serves as an adhesive between the transparent support layer and the gold thin film layer, and when used as a sensor chip, prevents detachment of the chip surface and enables reuse.

The thickness of this chromium layer is preferably 3 to 10 nanometers. This is because it is an appropriate thickness for forming the surface plasmon wave of the metal while serving as an adhesion with the gold thin film layer.

Herein, the gold thin film layer may be directly deposited on the transparent support or, if desired, on the chromium layer, and the thickness thereof is preferably 10 to 100 nanometers. If the thickness is smaller than 10 nanometers, the thickness of the thin film is difficult to form, and if larger than 100 nanometers, there is a problem that is not economical in the absence of an improved effect. For this reason, the thickness of the gold thin film layer is preferably 40 to 60 nanometers.

The chromium layer and the gold thin film layer are sequentially stacked on one side of the transparent support layer, and thermal evaporation is preferable. This is because it is a suitable way to coat metal to the nano level above, which is a suitable way to measure the surface plasmon resonance angle when using a surface plasmon resonance (SPR) device.

Meanwhile, the self-assembled monomolecular layer introduced on the thus formed gold thin film layer includes a self-assembled monomolecular layer formed of an alkane compound containing a thiol group (SH). The self assembled monolayer (SAM) refers to a monomolecular layer in which the organic molecules constituting the alkane compound are regularly well aligned by spontaneous chemical bonding on the surface of the substrate.

It is preferable to use an alkane compound containing a thiol group (SH) at both ends, because one of the thiol groups (SH) exhibits a well-ordered surface structure with a crystalline metal such as gold (Au). It is because it is suitable to form an assembled monolayer. In addition, the other thiol group (SH) is for inducing adsorption bond with the antibody of interest in the present invention.

This self-assembled monolayer is formed by preparing the appropriate alkane compound in an aqueous solution or an ethanol solution and soaking the glass coated with the gold thin film layer for several hours. Specifically, the concentration of the aqueous solution and the ethanol solution is preferably 1 to 10 mM, and the production time is preferably 5 hours to 20 hours.

This is because the stability of the self-assembled monolayer in the above time range is the best. This manufacturing method is a chemical bond is formed by the self-assembly of the alkane compound, and does not require an additional chemical process, and is formed at room temperature and normal pressure, and thus undergoes a very simple process compared to the conventional technology.

In order to achieve another object of the present invention, the present invention

i) immobilizing porcine circovirus type 2 (PCV2) capsid antigen protein on a metal thin film substrate to prepare a protein chip for SPR;

ii) reacting a serum sample to the protein chip; And

iii) detecting the SPR signal intensity of the antigen-antibody complex formed on the surface of the SPR protein chip.

The method of the present invention comprises the step of immobilizing porcine circovirus type 2 (PCV2) capsid antigen protein on a metal thin film substrate to prepare a protein chip for SPR. The present invention relates to a method for measuring the antibody concentration in serum samples using the SPR phenomenon. At the interface of two transparent media with different refractive indices, a portion of the light coming from the medium with the high refractive index is reflected and partially refracted. However, above a certain angle of incidence (determined by the refractive index ratio of the two media), all the light is totally reflected rather than refracted by the other media. Although all light is reflected, an electromagnetic wave called an evanescent wave penetrates a very short distance of about one wavelength into a medium with low refractive index. If the two media are coated with a very thin metal, and the incident light is polarized and monochromatic light, the reflected light at a particular incident angle will cause the light density to decrease significantly. This phenomenon is called SPR and the incident angle is called SPR angle. The SPR angle depends on the refractive index of the solution in which the electromagnetic wave is penetrated. The optical device for detecting the SPR phenomenon can continuously observe the refractive index change of the surface plasmon layer. The refractive index changes as the buffer changes, but also changes in the mass change of the surface plasmon layer.

After preparing an antigen that can detect the target protein as an antibody, the antigen is immobilized on the surface plasmon layer, and then reacted with the sample to induce an immune response. The concentration of the antibody in the sample is measured by measuring the refractive index change (ΔRefractive index unit; ΔRIU) by irradiating the polarized light to the sensor. The antibody may be a monoclonal antibody or a polyclonal antibody, with polyclonal antibodies being more preferred.

An antigen to which a particle made of a metal or an organic or inorganic material is attached is attached to a particle made of a metal or an organic material to the antigen. The particles are 1-200 nm in diameter, preferably 1-50 nm. In the case of metal particles, gold, silver, copper, aluminum or alloys thereof are preferable, and oxides such as alumina, titania, and the like are also possible. Most preferably gold. In the case of an organic substance, as a polymer, they are polyethylene, polystyrene, polyethyleneglycol, polyurethane, polyacrylic acid, polymethyl methacrylate, or derivatives thereof. Inorganic materials include glass, silica, and silicone polymers. These particles can attach to the antigen using covalent bonds, hydrogen bonds, electrostatic attraction, and the like.

The method of measuring the refractive index change of the present invention can be carried out by a method using a conventional SPR, preferably a surface plasmon layer, a light emitting diode (LED) in a portable device. )), Reflective mirror and photodiode array. The light source is irradiated with a wavelength of 800 to 850 nm, but the wavelength range can be adjusted. The LED and the detector are in one semiconductor, and the information in the semiconductor can be output as a refractive index change to a computer through a digital signal processor. Since the refractive index varies with temperature, it is preferable to further include a temperature sensor on the semiconductor to correct the refractive index according to the temperature. Representative SPR devices include Spreeta ^™ (US 6191847, US 611652, US 611248, US 5946083, US 5912456, US 5898503) developed by Texas Machinery. The Spreeta ^™ is a type of semiconductor (semiconductor based optoelectronic device) that has an optical function, uses LED as light, and is made of a photodiode array, a temperature sensor, and an epoxy composition inside.

The method of immobilizing the antigen for the target protein on the surface plasmon layer (a) reacts with a compound capable of forming a self-assembled monolayer (SAM) on the gold surface of the surface plasmon layer. Forming and (b) immobilizing the antigen to the SAM.

The SAM acts as a linker for densely accumulating antigens on the surface plasmon layer, and has a three-dimensional structure, thereby widening the binding surface area of the antibody. The compound forming the SAM may be used a conventional material, and may be selected from the group consisting of a compound containing a thiol group and a carboimide, a compound containing a thiol group and an acetic acid group, and a calixarene derivative. .

Calixarene derivatives have a thiol group as a functional group capable of forming SAM on the gold surface and include a crown ring that can specifically recognize cations of proteins or amino acids, in particular amine groups (-NH3 +). Unlike the conventional method of fixing the biomaterial by chemical bonding, the crown ring immobilizes the cationic material on the surface plasmon layer by using polyion recognition, which is one of molecular recognition. Therefore, it is possible to fix the biomaterial without chemical treatment or change, and most proteins, such as antigens, antibodies and enzymes, can solve the orientation problem because cations such as amine groups are distributed on the opposite side of the protein active site.

Representative SAM-forming compounds include mercaptohexadecanoic acid, mercaptoundecanoic acid, and mercaptohexadecylamine (1,6-mercaptohexadecylamine).

The method of the present invention comprises the step of reacting a serum sample to the SPR protein chip. When a serum sample is reacted with a protein chip to which the PCV2 capsid antigen protein is immobilized, an antigen-antibody binding reaction will occur if an antibody to the PCV2 capsid antigen is present in the serum sample.

The method of the present invention comprises detecting the SPR signal intensity of the antigen-antibody complex formed on the surface of the SPR protein chip. As described above, SPR is a method for sensitively measuring the change in refractive index on the metal surface. When the biochemical sample such as protein is bound to the metal surface, the SPR is able to detect the amount thereof. Is formed, the SPR signal strength can be detected using a device such as an SPR imaging system.

In the method according to the embodiment of the present invention, the protein chip for SPR is a transparent support layer, a gold thin film layer coated with gold (Au) on one side on the transparent support layer and a thiol group on the gold thin film layer ( It may include a self-assembled single molecule layer formed of an alkane compound containing SH). Details of each component of the SPR protein chip are as described above.

In the method according to an embodiment of the present invention, the protein chip may be made of a metal thin film chip, the metal thin film may be made of a metal, but is not limited to gold, silver, copper or aluminum, Preferably it may be made of gold. In the present invention, it is preferable to use gold (Au, purity of 99.9% or more) as a metal capable of producing surface plasmon waves and measuring surface plasmon resonance angles. This is because gold thin films are easy to form self-assembled monolayers for organic functional groups that are selective for the detection of contaminants and are advantageous metals for generating surface plasmon waves. Metal thin films can be prepared using techniques generally known in the art.

In a method according to one embodiment of the invention, the antibody concentration can be measured quantitatively. It is also possible to qualitatively analyze the presence or absence of antibodies in serum samples using this method, but it is also possible to measure them quantitatively. That is, as shown in FIG. 3 below, after preparing a standard curve of SPR imaging signal intensity for antibody concentration using a PCV2 capsid antibody having a known concentration, the SPR imaging signal intensity of an unknown serum sample is measured, and the standard By substituting the curve, the antibody concentration can be quantitatively measured.

The method according to an embodiment of the present invention is characterized in that the steps ii) and iii) are completed within 1 hour. That is, the time required to detect the SPR signal intensity by reacting the serum sample to the protein chip immobilized with the PCV2 capsid antigen may be within 1 hour. Therefore, by using the method of the present invention, by measuring the antibody concentration of the pig circovirus in a short time, it is possible to reduce the time and manpower to determine whether the pig circovirus infection.

The method according to one embodiment of the present invention can preferably measure the pig circovirus type 2 antibody concentration.

The method according to an embodiment of the present invention can diagnose swine circovirus infection through the measurement of the pig circovirus antibody concentration. In other words, the pig circovirus antibody concentration is measured by the method of the present invention, and it is possible to diagnose whether the pig circovirus infection according to the measurement result. For example, if the measured pig circovirus antibody concentration is high, the probability of infection of the pig circovirus is high. If the measured pig circovirus antibody concentration is low, the probability of infection of the pig circovirus is low. It is.

In the method according to an embodiment of the present invention, the volume of the serum sample may be 1-2 μl. Whereas conventional ELISA assays require at least 20-100 μl of serum, the protein chip of the present invention requires a relatively small amount of serum samples as described above, thus reducing the burden on animals by collecting small amounts of serum samples. And a variety of additional tests.

In a method according to an embodiment of the present invention, the SPR signal intensity of the antigen-antibody complex may be measured by an SPR sensor or a surface plasmon resonance imaging (SPRI) sensor. The SPR sensor is used to measure the thickness of a sample, which is a thin film, from quantitative analysis, qualitative analysis, and a thin film of a material close to a metal thin film, that is, a change in refractive index of the sample. The SPR sensor can be referred to Korean Patent Publication No. 2004-102847. Which is incorporated herein by reference in its entirety. SPR sensors typically provide the lowest reflectivity, ie the amount of biomaterial combined with the surface of the SPR chip, by showing one or more angles of light at a given SPR chip point. Proportional to the change in refractive index of the chip surface). SPRI sensors, on the other hand, use a principle to measure the difference in intensity of reflected light by projecting a balanced light with a constant angle over a larger area. This principle allows the SPR sensor to be more sensitive while limiting the amount of sample that can be analyzed at one time, while the SPRI sensor is capable of microarray analysis while the sensitivity is reduced and the reflection intensity is saturated with changes in the refractive index of the chip surface. It is difficult to measure biomaterials of various concentrations.

To achieve another object of the present invention, the present invention provides an SPR immobilized with a pig circovirus type 2 (PCV2) capsid antigen, comprising immobilizing a pig circovirus type 2 (PCV2) capsid antigen protein on a metal thin film substrate. Provided are a method for preparing a protein chip for a dragon. The method of immobilizing the protein on the metal thin film substrate is as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example

Example  1: pig Circovirus  Type 2 Capsid  Preparation of the antigen

Disclosure strains, virus strains and plasmids

E. coli strains JM109, BL21 (DE3) pLysS and pRSET vectors were purchased from Invitrogen (Carlsbad, CA, USA). The published virus was used for lymph node samples of pigs diagnosed with porcine circovirus type 2 infection.

gene Cloning

Viral DNA was extracted from porcine lymph nodes. Primers for amplifying the capsid gene include 5'-primer (5'-ATAT GGATCC ATG ACG TAT CCA AGG AGG C-3 '; SEQ ID NO: 2) (underlined Bam HI restriction site), 3'-primer (5'- TGTG AAGCTT TTA GGG TT T AAG TGG GGG G-3 '; SEQ ID NO: 3) (underlined Hind III restriction enzyme site).

PCR conditions consisted of 2 μl (50 ng / μl) DNA and 1 μl of each primer (50 pM), 5 μl 10 mM dNTP (2.5 mM each) and 1 μl 5U Ex TaqTM DNA polymerase (5 μl 10 × reaction buffer). TaKaRa, Shiga, Japan) was performed in a thermal cycler (PTC-100TM, MJ Research, Waltham, Mass., USA) to a final volume of 50 μl. PCR conditions were last extended at 72 ° C. for 7 minutes after 95 cycles / 5 minutes followed by 30 cycles (95 ° C./30 sec, 55 ° C./30 sec, and 72 ° C./30 sec) PCR. The amplified PCR product was 1.0% (w / v) agarose gel electrophoresis to confirm the product of 0.7kb size (Fig. 1). As can be seen in Figure 1, the ORF2 coding gene sequence was digested with Bam HI and Hind III restriction enzymes and observed in two bands of 3 kb (pRSET vector) and 0.7 kb (PCV2 ORF2).

The PCR product was purified using a PCR purification kit (Qiagen, Hilden, Germany), digested with two restriction enzymes ( Bam HI and Hind III), and then placed in Bam HI + Hind III-cut pRSET to prepare pRSET-circo-cap. It was. The recombinant DNA was transformed into chemically qualified Escherichia coli JM109 cells. Transformed cells were selected in Luria-Bertani (LB) plates containing 100 μg / ml ampicillin. The plasmid was then sequenced (ABI Prism 3100 Genetic Analyzer, Applied Biosystems, Foster City, Calif., USA) to confirm the sequence alignment through the BLAST method.

Capsid  Protein expression

For capsid protein expression, BL21 (DE3) pLysS (pRSET-circo-cap) was prepared by transforming BL21 (DE3) pLysS host cell (Invitrogen) with sequencing completed pRSET-circo-cap. Transformed cells were incubated for 24 hours at 37 ° C. in LB plates containing 100 μg / ml ampicillin and 35 μg / ml chloramphenicol. Protein expression was confirmed by SDS-PAGE and Western blot analysis at 1 hour intervals over 4 hours. As a result, capsid protein expression of 27 kDa was confirmed (FIG. 2). In Figure 2, lane 1 is the result of expression in untransformed cells, lane 2 is BL21 (DE3) pLysS (pRSET-circo-cap) before induction of isopropyl-β-D-thiogalactopyranoside (IPTG) ) Expression results, lanes 3 to 6 represent expression for 1 hour, 2 hours, 3 hours and 4 hours, respectively. In FIG. 2, the recombinant PCV2 capsid protein corresponds to 27 kDa.

Recombinant capsid antigen was purified through 6X His affinity column (Probond, Invitrogen) and used for SPR imaging and ELISA experiments.

Example  2: ELISA test

Porcine circovirus antibody assays were performed using ELISA (Frey, A. et al., J. Immunol. Methods, 1998, 221, 35-41). Briefly, 96-well plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with recombinant capsid protein (1 g / well / 90 μl) and washed with PBST (PBS + 0.05% Tween-20, pH 7.4). 90 μl of the serum sample was reacted at 37 ° C. for 1 hour, and then washed three times with PBST. The goat anti-pig horseradish peroxidase-conjugated IgG (Serotec, USA) was diluted 1: 500 and reacted at 37 ° C. for 1 hour and washed three times with PBST. Substrate solution [0.1 M 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 ] was reacted at room temperature for 15 minutes, and the absorbance of 405 nm was confirmed by ELISA reader (Multiskan EX, Thermo Lab Systems, Beverly, MA, USA).

Example  3: Fabrication of Gold Thin Film Chips

Gold thin film chips were immersed in a solution of 30% hydrogen peroxide solution and 95% sulfuric acid in a volume ratio of 1: 3 at 60 ° C. for 30 minutes, and the gold thin film chips were washed with distilled water and ethanol in that order. An ethanol solution in which 10 mM mercaptoundecanol was dissolved in a gold thin film chip was supported at room temperature for about 20 hours to form a self-assembled molecular film. In order to activate the chip after the gold thin film chip was washed with ethanol and distilled water, epichlorohydrin was 0.6 M in a mixed solution of 0.4 M sodium hydroxide solution and 2-methoxyethyl ether in a volume ratio of 1: 1. The solution made to the concentration was put in a gold thin film chip and reacted at room temperature for 4 hours. The reaction solution was discarded and the chips were washed in the order of ethanol and distilled water. In order to coat dextran on the chip surface, a solution in which dextran was dissolved in 0.1 M sodium hydroxide solution at a concentration of 0.3 g / ml was supported on the gold thin film chip and reacted for 20 hours. The reaction solution was discarded and the chips were washed with distilled water, 0.1 M sodium hydroxide solution, and distilled water in that order. 0.4 M sodium hydroxide solution and 2-mercaptoethyl ether solution were added to the gold thin film chip, and the solution of epichlorohydrin was dissolved in a mixture of volume ratio 1: 1 at a concentration of 0.6 M, and reacted at room temperature for 4 hours. The reaction solution was discarded and the chips were washed in the order of ethanol and distilled water. In order to introduce iminodiacetic acid (IDA) into the gold thin film chip, an aqueous solution of 1.7 M iminodiacetic acid (disodium salt) and 2 M sodium carbonate was placed in a container with a chip and reacted at 60 ° C. for 20 hours. I was. The reaction solution was discarded and the chips were washed with distilled water. In order to introduce nickel into the chip prepared above, 50 mM nickel chloride (Nichel (II) chloride) solution was placed in a vessel containing chips and reacted at room temperature for 3 to 4 hours. The reaction solution was discarded and washed with distilled water.

Example  4: pig Circovirus Capsid  Antigenic SPR Imaging  Diagnostic system

<1> Signal intensity analysis according to antibody concentration in serum

Purified capsid protein (15 ng / ml) was spotted by dropping 1 µl into a 1 mm diameter microwell prepared on a gold thin film. After spotting, the chip was reacted at 37 ° C. for 20 minutes while maintaining 80% humidity, and then washed three times with PBST and then once with distilled water. The surface of the chip was blocked by reacting PBS with 1% BSA for 10 minutes, and the sample was diluted from 1-fold to 1/5000 with positive control serum, reacted for 20 minutes, washed, and dried for SPR imaging system (SPRi LAB). , K-MAC, Daejeon, Korea). As a result, as shown in FIG.

<2> SPR imaging diagnostic system

The chip prepared by the above method was reacted with PBS containing 1% BSA for 10 minutes to block the surface of the chip, and then reacted for 20 minutes by adding serum and positive serum, followed by washing and drying, followed by SPR imaging system (SPRi LAB, K- MAC, Daejeon, Korea). Serum obtained from six farms 70 pigs is shown in FIG. In Figure 4, numbers 1 to 70 represent serum sample numbers, P represents a positive control, PCV2 capsid antibody, N represents a negative control, reacted with pig serum not infected with PCV2. As can be seen in Figure 4, 1-70 serum samples show various positive reaction results according to various antibody concentrations.

The test results were examined for correlation between SPR imaging and ELISA through Pearson's correlation coefficient (SPSS Base 12.0, SPSS, Chicago, IL). As a result, as shown in FIG. 5, high correlation (n = 70, r = 0.911, p <0.01) was confirmed.

According to the present invention, since the antibody concentration of the pig circovirus type 2 in the pig can be confirmed through the SPR imaging diagnostic system, it is possible to diagnose whether the pig circovirus is infected. In addition, it is possible to quickly and accurately diagnose swine circovirus type 2 infection within 1 hour, which can save time and manpower, and has higher sensitivity and specificity than the conventional ELISA. In addition, while the existing ELISA assay requires a minimum of 20-100 μl of serum, the protein chip of the present invention requires significantly less serum samples, so a small amount of serum samples can be used to reduce the burden on animals and add a variety of additions. Can be used for inspection.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Protein chip for diagnosing porcine circovirus and method of          diagnosing porcine circovirus using the chip <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 233 <212> PRT <213> Porcine circoviurs type 2 <400> 1 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg   1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro              20 25 30 Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg          35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Arg Thr Thr Val Thr Thr      50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Lys Leu Asp Asp Phe Val  65 70 75 80 Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr                  85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr             100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn         115 120 125 Phe Val Pro Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr     130 135 140 Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr 145 150 155 160 Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro                 165 170 175 Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ser Arg Asn             180 185 190 Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Asn Lys Tyr Asp         195 200 205 Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe     210 215 220 Asn Leu Lys Asp Pro Pro Leu Lys Pro 225 230 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR forward primer <400> 2 atatggatcc atgacgtatc caaggaggc 29 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR reverse primer <400> 3 tgtgaagctt ttagggttta agtgggggg 29  

Claims (12)

A protein chip immobilized with porcine circovirus type 2 (PCV2) capsid antigen on a metal thin film substrate. The protein chip of claim 1, wherein the metal is gold, silver, copper, or aluminum. 2. The protein chip of claim 1, wherein the protein chip is for surface plasmon resonance (SPR) imaging measurement. The alkane compound according to claim 3, wherein the protein chip comprises a transparent support layer, a gold thin film layer coated with gold (Au) on one side of the transparent support layer, and a thiol group (SH) on the gold thin film layer. Protein chip comprising a self-assembled monolayer formed by. i) immobilizing porcine circovirus type 2 (PCV2) capsid antigen protein on a metal thin film substrate to prepare a protein chip for SPR; ii) reacting a serum sample to the protein chip; And iii) detecting the SPR signal intensity of the antigen-antibody complex formed on the surface of the SPR protein chip. The protein chip for SPR comprises a transparent support layer, a gold thin film layer coated with gold (Au) on one side of the transparent support layer, and a thiol group (SH) on the gold thin film layer. And a self-assembled monolayer formed of an alkane compound. 6. The method of claim 5 wherein the metal is gold, silver, copper or aluminum. The method of claim 5, wherein the antibody concentration is measured quantitatively. 6. The method of claim 5, wherein steps ii) and iii) are completed within 1 hour. The method of claim 5, wherein the porcine circovirus infection is diagnosed by measuring the pig circovirus antibody concentration. The method of claim 5, wherein the SPR signal intensity of the antigen-antibody complex is measured by an SPR sensor or a surface plasmon resonance imaging (SPRI) sensor. A method for producing a protein chip for SPR, wherein the pig circovirus type 2 (PCV2) capsid antigen is immobilized on a metal thin film substrate.

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