KR20050074847A - Antibody detecting reagent for blv, manufacturing method of antigen for the detection, antibody detecting method thereby, and the kit - Google Patents

Abstract

The present invention relates to a small-lipopathic leukemia comprising a recombinant antigen and a p24 or NP protein produced by coat-fecting a recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of the small-lipopathic leukemia virus. Provided are reagents for detecting virus antibodies.

In addition, the present invention is a method for producing an antigen for the detection of small-lipopathic leukemia virus antibody by coating the host with a recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of small-lipopathic leukemia virus To provide.

In addition, the present invention is to coat the recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of bovine leukemia leukemia virus to prepare a P24 or NP protein of bovine leukemia virus The present invention provides a method for detecting bovine leukemia virus antibody using the antigen as a detection antigen.

In addition, the present invention is directed to a P24 or NP protein prepared by coat-fecting a recombinant vector and baculovirus containing p24 gene or p15-24-12 gene of bovine lipopathic leukemia virus on a strip with a detection antigen. Provided is a diagnostic kit for detecting an immobilized bovine leukemia virus antibody.

Description

Antibody Detecting Reagent for BLV, Manufacturing Method of Antigen for The Detection, Antibody Detecting Method and, The Kit}

The present invention relates to a reagent for detecting bovine leukemia virus antibody, and more particularly, the antibody against bovine leukemia leukemia virus can be detected accurately and quickly from a large amount of test material, so that it can be easily determined whether the disease is infected or not. The present invention provides a reagent for detecting an anti-liposomal leukemia virus, a method for producing an antigen, a method for detecting an antibody, and a diagnostic kit using the same.

Bovine leukemia is a sexually transmitted disease caused by the bovine leukemia virus belonging to the retrovirus. It can be infected in all ages of cows, but it is mostly subclinical and shows permanent lymphocytosis in cattle older than 3 years. In about 0.1% to 1% of the disease, it develops malignant tumor, which is an impulse.

Agar gel immunodiffusion test (AGID), fluorescence antibody test, complement binding reaction, virus neutralization test, and enzyme-linked immunosorbent assay (ELISA) can be used to diagnose the disease. However, there are two methods that are recognized by the Office International des Epizooties (OIE), which are AGID and ELISA. In particular, ELISA is widely used worldwide because it is more sensitive, reproducible, and time-saving than AGID. The antigen used in ELISA is purified from virus from fetal lamb kidney cell (FLK), a cell permanently infected with small-lipopathic leukemia virus, mainly gp51 protein and internal protein p24. However, if the FLK cell line is used as an antigen used in developing an ELISA, a virus, which is difficult to detect by proliferating within the cell without causing cell degeneration, may be contaminated, especially a bovine diarrhea virus. And, there is a problem that can lower the diagnostic efficiency. In addition, it is necessary to supplement the virus because many tests have to be carried out in order to extract and use only specific parts of the virus.

The present invention has been made to solve the problems of the prior art as described above, the object is to easily detect the presence or absence of a disease infection by quickly detecting antibodies to the small-fat disease leukemia virus from a large amount of test material In addition, the present invention provides a reagent for detecting small-fatty leukemia virus antibody, which can greatly contribute to the national defense project against the disease.

Another object of the present invention is to provide a method for producing an antigen for detecting bovine leukemia virus antibody.

Another object of the present invention is to provide a method for detecting bovine leukemia virus antibodies simply and accurately.

Still another object of the present invention is to provide a diagnostic kit for detecting bovine leukemia virus antibody.

As a means for achieving the above object, the present invention provides a P24 or NP protein prepared by coat-transfecting a recombinant vector and baculovirus containing p24 gene or p15-24-12 gene of bovine pathogenic leukemia virus into a host. Provided are a reagent for detecting small-lipopathic leukemia virus antibody as a detection antigen.

In addition, the present invention is a method for producing an antigen for the detection of small-lipopathic leukemia virus antibody by coating the host with a recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of small-lipopathic leukemia virus To provide.

In addition, the present invention is to coat the recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of bovine leukemia leukemia virus to prepare a P24 or NP protein of bovine leukemia virus The present invention provides a method for detecting bovine leukemia virus antibody using the antigen as a detection antigen.

In addition, the present invention is a p24 or NP protein prepared by coat-transfecting a recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of bovine leukemia leukemia virus on a plate as a detection antigen Provided is a diagnostic kit for detecting an immobilized bovine leukemia virus.

The baculovirus in the present invention is preferably selected from those belonging to the multicapsid nuclear polyhedron virus.

In the present invention, the host cell is preferably an insect cell, more preferably a Spododotera pruperfera (sf) cell, and more preferably is selected from sf9 and sf21 cells.

In the present invention, the content of antigen P24 or NP protein used in the process of detecting bovine leukemia virus antibody is preferably 0.1 to 0.15 µg / ml, more preferably 0.125 µg / ml.

Hereinafter, the content of the present invention in more detail as follows.

According to the present invention, an antibody detecting reagent, a method for producing an antibody for detecting an antibody, and a method for detecting an antibody include p24 or p15-24-12 constituting a bovine leukemia virus (hereinafter referred to as 'BLV') gene. It is characterized by using an antigen obtained from a host by coat-transfecting a recombinant vector obtained by binding to a baculovirus. In other words, the antigens for detecting antibodies thus provided are directly extracted and provided from the BLV, and the present invention can provide a basis for purely mass production of desired antigenic proteins using gene recombination technology. have.

The p24 gene is part of the proteins that make up the nucleocapsid, and the p15-24-12 gene is the gene that encodes the entire nucleocapsid protein. Proteins expressed by the p24 gene or p15-24-12 gene will be named P24 and NP below, respectively, and these proteins can act as antigens that cause antibodies in cattle.

The p24 gene or p15-24-12 gene may be obtained by extracting probiral DNA from cows infected with BLV and performing PCR amplification using a predetermined primer set. Specific conditions of the PCR used in the reaction are sufficient to follow the conditions set forth by the OIE.

Figure 1 shows the process of producing a recombinant vector p24 gene or p15-24-12 gene amplified by the PCR process. Referring to this, the products obtained by PCR amplification using a predetermined primer set from the probiral DNA of BLV are 650bp genes containing p24 and 1178bp genes containing p15-24-12 genes, respectively. Sites For example, it has restricted sites of EcoRI and KpnI.

The obtained genes were cloned by inserting into the pGEM-T vector, and then each gene was cleaved using EcoRI and KpnI, and the cleaved gene was ligated to a transition vector, for example, pAcHLT-A to transform the recombinant. Prepare the vector.

The present invention relates to a recombinant vector containing the p24 gene or the p15-24-12 gene and a baculovirus which can be safely used without an animal as a host while being an insect as a host. Purification process.

Baculoviruses are preferably selected from those belonging to the multicapsid nuclear polyhedral virus and do not require any particular limitation, but in a preferred embodiment of the present invention AcMNPV was used.

As the host cell, cells of the spordogera pruperferda, which is a kind of chestnut moth, are preferably used. More preferably, sf9 and sf21 are preferable. These cells are now widely used as a transformant for expression of specific genes. However, it has not been reported as a use for the expression of P24 or NP protein expressed by BLV so far.

Currently, the process of transfecting the ligated recombinant vector into a specific host and purifying the recombinant protein from these hosts is a known technique, and these are provided by various manufacturers (eg, PharMingen).

P24 or NP protein purified from the final transformant according to the present invention is very useful as a use of the antigen to confirm the infection of BLV in cattle. That is, since they are produced using recombinant technology, unlike conventional techniques using cell lines, the detection efficiency is very high because there is no fear of contamination by viruses that are difficult to detect, for example, novel death virus.

 The antigen according to the present invention may be provided in an immunological detection method, for example, may be variously applied to an enzyme immunoassay (ELISA), a direct test method using a diagnostic kit. In addition, one or two antigens according to the present invention may be used simultaneously in an immunological test for identifying BLV antibodies. When two species are used at the same time, the accuracy of detection can be further improved.

An example of application in the case of using an enzyme immunoassay (ELISA) as a detection method is shown in FIG. 2. Referring to this in more detail the enzyme immunoassay (ELISA) as follows.

First, the antigen, ie P24 or NP protein and normal cells are coated on the plate. At this time, the content of P24 or NP protein is preferably 0.1 to 0.15 µg / ml, more preferably 0.125 µg / ml. In this case, 0.05 M carbonate bicarbonate buffer (pH, 9.6) is used as the coating buffer. At this time, the coating reaction is preferably carried out at about 4 to 15 hours at 4 ℃.

The plate coated with the antigen is washed three times with a washing solution and then twice with PBS. PBS-0.05% Tween20 is used here as a washing buffer.

In the blocking process, 3% skim milk mixed in PBS-0.01% Tween10 is used as a blocking solution. The reaction is performed at 37 ° C. for 1 hour, and then washed 4 times with a washing solution.

The subject to be used for the serum reaction was diluted about 25-fold with 3% skim milk mixed in PBS-0.05% Tween20 as a dilution and added to the well coated with the antigen and cells, and reacted at room temperature for 1.5 hours.

The conjugate to be used for the reaction is HPL conjugated KPL chlorine antigen IgG is used, diluted about 4,000-fold diluted to each well and reacted for 30 minutes at room temperature, washed again four times with a washing solution.

ABTS (KPL) was added thereto as a substrate, and after 10 minutes, 1% SDS was added as a reaction stopper solution.

The result obtained through the above process can be measured as absorbance at 405 nm, and the intrinsic OD (TO) can be obtained from the following equation (1).

(1) intrinsic OD (TO) = added antigen coated well OD (AO)-cell coated well OD (CO)

The final result is weakly positive serum for BLV diluted 10-fold with E4, an OIE standard positive serum, and can be judged as shown in relation (2) in comparison with the result of E4 / 10, the standard serum for BLV diagnosis. have.

(2) Negative (sample TO) <E4 / 10 (TO) ≤ positive (sample TO)

As a direct test method using a diagnostic kit, a diagnostic kit currently available from various manufacturers may be used. For example, the sample according to the present invention is fixed to a specific position on a strip using nitrocellulose as a developing membrane by being spaced apart from each other together with a marker material forming a control line, and having appropriate pores and made of a material such as cellulose. While having a loading pad at the bottom of the strip, it can be implemented including a housing for covering these strips. Detection is possible with the naked eye, and after loading the sample, the antigen lines and the control lines can be identified after a predetermined time, and the BLV infection can be easily confirmed.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Cloning of Bovine leukemia virus (BLV) p24 and p15-24-12 genes

Extract BLV Probiral DNA from domestic cows infected with bovine leukemia leukemia virus (Genomic DNA Purification kit, Promega) and set primers (p24: anterior primer 5`-G GAA TTC ATG TTG CCA ATT ATA TCT GAA GGA-3) `, Rear primer 5`-GGT ACC TTA GAC GAG GAT TGC AGG CTG TTT-3`, P15-24-12: forward primer 5`-G GAA TTC ATG GGA AAT TCC CCC TCCC TAT AAC-3`, rear primer 5 DNA was amplified by PCR conditions presented by the International Water Bureau (OIE) by mixing with a PCR reaction reagent (Taq PCR Master Mix, Qiagen) using `-GGT ACC TTA GTT TTT TGA TTT GAG GGT TGG -3`. Each amplified gene was inserted into the pGEM-T vector, cloned, and then digested with EcoRI and KpnI restriction enzymes. The cleaved gene was ligated to a pAcHLT-A transfer vector and then transformed into DH5α competent cells, and recombinant DH5α bacteria were selected (see FIG. 1). Recombinant plasmids were extracted from the bacteria prior to coverage. The process from the transfection process to purification of the recombinant protein was performed according to the procedure of the manufacturer (BaculoGold 6x His Purification and Expression kit, PharMingen). In short: The extracted p24 and p15-24-12 pAcHLT-A plasmids were cotransfected into Sf9 insect cells simultaneously with linear AcMNPV DNA.

<Example 2> Small lipopathic leukemia virus recombinant BLV-P24 and BLV-NP (P15-24-12) protein expression confirmation

The presence of cell degeneration was confirmed by observing the coat-treated Sf9 cells at 27 ° C. for 7 days. Cells with confirmed cell degeneration were infected with new Sf9 cells and then cultured. After 5 days of cell degeneration, small aliquots were stored and stored at -70 ° C. Some cell culture supernatants were used to determine whether recombinant proteins were expressed. In order to determine the presence or absence, the virus was re-inoculated with HI-5 insect cells having excellent virus proliferation. Cells confirmed infection after re-inoculation were lysed using a kit (BaculoGold 6x His Purification and Expression kit, PharMingen), purified with Ni-NTA agarose, and analyzed for recombinant proteins using SDS-PAGE and Western blotting. It was.

(1) SDS-PAGE analysis

Purified recombinant protein was mixed with protein sample loading buffer (10% SDS, 10 mM Tris-HCl, pH6.8, 25% glycerol, 0.01% bromophenol blue, 10% β-mercaptoethanol) for 5 minutes at 95 ° C. Boil and then centrifuged at 12,000 rpm / 3 min. A 12.5% polyacrylamide gel and buffer strip for SDS-PAGE were used commercially (Pharmacia Biotech). 15 μl of treated sample per well of gel was loaded and then reacted at 300 V for 2 hours (GenePhor, Pharmacia Biotech). After the electrophoresis was completed, the gel was immersed in a dye solution (50% methanol, 0.2% Coomassice Brilliant Blue R-250, 7% acetic acid, 43% DW) of Zehr et al. (Anal. Biochem., 1989, 182: 157-159). It was heated for 2 minutes in a microwave oven (Daewoo, KOR-603MT) and then decolorized overnight in a decolorizing solution (15% methanol, 10% acetic acid). As a result of analyzing the bands on the gel, BLV-P24 and BLV-NP showed molecular weights of about 28 and 50 kDa, respectively (see FIG. 3).

(2) Western blotting analysis

Towbin et al. (Proc. Natl. Acad. Sci. USA., 1979, 76: 4350-4354) and Blake et al. (Anal. Biochem., 1984, 136: 175-179) were applied to the NovaBlot System using the electroblotting method ( Pharmacia Biotech). Electroblotting conditions were calculated as 0.8 mA per cm 2 and then blotted for 1.5 hours. The blotting-completed nitrocellulose paper (NC paper) was immersed in TBS (10 mM Tris (pH 8.0), 150 mM NaCl) and reacted for 10 minutes at room temperature, followed by blocking buffer (5% scheme milk, 5% lactoalbumin hydrolyzate). , TBST; 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween20)). After washing twice with TBST at 10-minute intervals, add anti-His antibody (Qiagen) diluted 1: 1000 with blocking buffer and anti-BLV bovine serum (NVSL) diluted 1: 100 in separate NC paper for 1 hour. Reacted. After the reaction, the cells were washed in the same manner, and the anti-mouse IgG HRP solution or the anti-bovine IgG HRP solution was reacted for 1 hour by diluting 1: 1000 with TBST containing 3% BSA (W / V). The wash solution was washed four times at 10 minute intervals and then developed by adding DAB substrate. As a result, as in SDS-PAGE, BLV-P24 and BLV-NP can identify specific bands at positions of about 28 and 50 kDa molecular weight, respectively, confirming that the recombinant protein for bovine leukemia virus is expressed (FIG. 3). Reference).

Example 3 Enzyme Immunoassay Using Recombinant BLV-P24 and BLV-NP Proteins

Recombinant virus-infected cells were lysed using a commercialized kit (BaculoGold 6x His Purification and Expression kit, PharMingen), purified with Ni-NTA agarose, and the content of recombinant protein was measured using Agilent Technologies 2100 Bioanalyzer. . Based on the measured content of the recombinant protein, the content of the recombinant protein suitable for the enzyme immunoassay (ELISA) was found to be 0.125 μg / ml for both the recombinant BLV-P24 and BLV-NP proteins (see FIGS. 4 and 5).

Example 4 Correlation Comparison between ELISA and Commercialized ELISA Kits According to the Present Invention

Comparison of ELISA and Bovine Leukemia Virus Antibody Test Kit (IDEXX laboratories, inc., USA) recognized by ELISA according to the present invention developed using recombinant small-lipopathic leukemia virus proteins BLV-P24 and BLV-NP proteins As a result, it was confirmed that the sensitivity and specificity showed a high correlation as shown in Table 1 below.

<Table 1> Correlation between ELISA and commercialized ELISA kit according to the present invention

division P24 NP positivity voice positivity voice IDEXX ¹⁾ positivity 165 4 165 4 voice 11 233 13 231 system 413 176 237 178 235 Sensitivity (%) ²⁾ 97.6 97.6 Specificity (%) ³⁾ 95.5 94.7

IDEXX laboratories, inc. (USA): Bovine Leukemia Virus Antibody Test Kit

2) Sensitivity (%) = positive serum count consistent with IDEXX company in development diagnosis method / positive serum number of IDEXX company X 100

3) Specificity (%) = negative serum count consistent with IDEXX company in development diagnosis / IDEXX negative serum water X 100

Referring to the above results, the sensitivity and specificity of the ELISA using the BLV-P24 recombinant protein according to the present invention as a result of comparing with the enzyme immunoassay (ELISA) kit of IDEXX, which is used as one of the existing internationally recognized standard diagnostic kit Were 97.6% and 95.5%, respectively, and the BLV-NP protein showed a high concordance rate of 97.6% and 94.7%, respectively.

According to the present invention, it is possible to easily determine the presence or absence of a disease infection by quickly detecting an antibody against bovine pathogenic leukemia virus from a large amount of test material, and greatly contribute to national defense projects for the disease.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the present invention described in the claims below. It will be appreciated that it can be changed.

Figure 1 is a schematic diagram showing the cloning process of the small-fatty leukemia virus p24 and p15-24-12 genes presented as a preferred embodiment according to the present invention, and the expression vector creation process using baculovirus.

Figure 2 is a flow chart illustrating a method for detecting antibodies against bovine leukemia leukemia virus using an ELISA presented as a preferred embodiment according to the present invention.

Figure 3 is a photograph confirming the expression of bovine leukemic virus recombinant BLV-P24 and BLV-NP (P15-24-12) protein according to the present invention.

1.coomassie brilliant blue stain,

2. Western blotting (1st antibody: anti 6xHis-Mouse IgG, 2nd antibody: anti-Mouse IgG conjugated HRP),

3. Western blotting (1st antibody: anti-BLV bovine serum, 2nd antibody: anti-bovine IgG conjugated HRP),

A: control cell, B: BLV-NP (P15-24-12) recombinant protein, C: BLV-P24 recombinant protein]

Figure 4 is a concentration diagram of the optimal BLV-P24 recombinant protein for ELISA according to the present invention.

Figure 5 is a determination of the optimal BLV-NP recombinant protein concentration for ELISA according to the present invention.

Claims (11)

Detection of small-lipopathic leukemia virus antibody using p24 or NP protein produced by coat-fecting recombinant vector and baculovirus containing p24 gene or p15-24-12 gene Reagent The reagent of claim 1, wherein the baculovirus belongs to a multicapsid nucleopolyvirus. The reagent of claim 1, wherein the host is an insect cell. 4. The reagent of claim 3, wherein the insect cell is a spordogera pruperfera cell. A method for producing an antigen for detecting the small-fatal leukemia virus antibody by coat-fecting a host with a recombinant vector and baculovirus containing the p24 gene or p15-24-12 gene of the small-fatal leukemia virus 6. The method of claim 5, wherein the baculovirus belongs to a multicapsid nucleopolyvirus. The method of claim 5, wherein the host is an insect cell. 6. The method of claim 5, wherein the insect cell is a Spododotera pruperfera cell. Recombinant vector and baculovirus containing p24 gene or p15-24-12 gene of bovine pathogenic leukemia virus were coat-fected into a host to prepare P24 or NP protein of bovine leukemia leukemia virus, To detect bovine leukemia virus antibody 10. The method according to claim 9, wherein the content of P24 or NP protein used for detection is 0.1 to 0.15 µg / ml. Small lipopathic disease in which p24 or NP protein prepared by coat-transfecting a recombinant vector and baculovirus containing p24 gene or p15-24-12 gene of small-fatty leukemia virus on a strip with a detection antigen Diagnostic kit for detecting leukemia virus antibody