KR100354971B1 - A GST-FMDV 3D fusion protein expression vector, Transformant thereof and a method for detection of FMD antibody using GST-FMDV 3D fusion protein and anti-GST antibody - Google Patents

Abstract

The present invention relates to a method of detecting foot-and-mouth disease antibodies using a GST antibody, which is a fusion protein of the expression vector GST and the foot-and-mouth virus gene expression protein 3D as a GST-foot-and-mouth virus 3D gene fusion protein.

Accordingly, an object of the present invention is to provide a new diagnostic method that can safely and easily and quickly detect foot-and-mouth disease antibodies by simultaneously detecting a novel GST-foot-and-mouth virus 3D gene fusion protein expression vector and foot-and-mouth serotype antibodies.

The diagnostic method according to the present invention can quickly search a large amount of samples by using the principle of enzyme immunoassay, and can secure the safety of the diagnostic antigen by using an artificially made protein. Since it can be detected at the same time has the advantage of eliminating the hassle of having to perform the serotype antibody test.

Description

Expression of a foot-and-mouth disease virus 3D gene, a transformant and a method for detecting foot-and-mouth antibody using a fusion protein produced from the transformant {A GST-FMDV 3D fusion protein expression vector, Transformant Y and a method for detection of FMD antibody using GST-FMDV 3D fusion protein and anti-GST antibody}

The present invention relates to a fusion protein expression vector of a glutathione-S-transferase (hereinafter abbreviated as GST) and a foot-and-mouth virus 3D gene product and a method for detecting foot-and-mouth antibody using a GST- and foot-and-mouth virus fusion protein and a GST antibody. Specifically, by using artificially synthesized gene expression protein as a detection antigen, it is possible to remove the risk of disease transmission due to incomplete treatment of virus and to safely and easily and promptly remove foot-and-mouth antibody. It relates to a detection method.

Foot and mouth disease (FMD) is a disease that infects cows and forms blisters in the mucous membranes of the mouth, face, and hoof, and causes massive animal industry damage once caused by rapid spread and high mortality. (Office Internationale des Epizooties, OIE) A grade A malignant livestock epidemic.

Currently widely used as an antibody detection method for the diagnosis of the disease is the liquid phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) (Hamblin et al, J. Immunol. Methods 1986: 93: 115- 121).

However, the method uses inactivated foot-and-mouth virus as a detection antigen, and detects serotype-specific antibodies using respective antiserum against foot-and-mouth virus serotypes. Due to the hassle of having to carry out antibody tests for each type, a diagnostic method capable of detecting all serotype antibodies of foot-and-mouth disease at the same time is required, rather than the serotype antibody screening test for foot-and-mouth disease.

The present inventors have studied a diagnostic method that can simultaneously detect four foot-and-mouth disease virus serotypes in order to alleviate the need to perform serotype-specific antibody tests, resulting in artificially synthesized foot-and-mouth virus 3D protein and GST protein in E. coli. Using the fusion protein of the present invention was found to be able to detect foot-and-mouth virus serotypes at the same time to complete the present invention.

Accordingly, an object of the present invention is to provide a novel GST- foot and mouth virus 3D gene fusion protein expression vector.

Another object of the present invention is to provide a new diagnostic method that can detect foot-and-mouth antibody in a safe, simple and rapid manner by detecting the foot-and-mouth serotype antibody at the same time.

1 is a production process of the GST- foot and mouth virus 3D gene fusion protein expression vector pG3D-2.

2 is a block diagram of an expression vector pG3D-2.

3 is a principle of a diagnostic method for detecting foot-and-mouth disease virus,

The present invention includes a GST- foot and mouth virus (FMDV) 3D gene fusion protein expression vector pG3D-2. The GST- foot and mouth virus 3D gene fusion protein expression vector (hereinafter, abbreviated as pG3D-2) of the present invention is preferably present in a fused form of the 3D gene and GST gene.

The 3D gene contained in the pG3D-2 of the present invention is foot-and-mouth disease virus A₂₄cruzeiro (FMDV A₂₄cruzeiro_,Plum island Animal Disease Center, USA), the 3D gene is as shown in the gene sequence of SEQ ID NO: It consists of 1410 bases and 470 amino acids. The 3D gene encodes a polymerase involved in nucleic acid synthesis and has a high concordance rate between serotypes for foot-and-mouth virus. Therefore, the 3D gene can be used as a diagnostic antigen. It is characterized by the ability to detect antibodies at the same time.

In addition, the foot-and-mouth virus antibody diagnostic antigen of the present invention includes a fusion protein form of the above-mentioned 3D gene and GST. The GST functions as an antigen of the GST antibody immobilized on the bottom of a plate well when performing an enzyme immunoassay (ELISA) for diagnosing foot-and-mouth virus virus. Therefore, the preparation of an antibody for diagnosing an antibody having a function similar to that of the fusion protein disclosed in the present invention using a protein different from the GST can be easily made by those of ordinary skill in the art. It is possible to use by modifying.

As a 3D gene for producing the foot-and-mouth virus antibody diagnostic antigen of the present invention, a foot-and-mouth virus A ₂₄ synthesizes a cDNA using a random primer and reverse transcriptase, and the cDNA for the production of the antigen for antibody diagnosis It is preferable to use it as a template. The random primer and reverse transcriptase are generally commercially available and do not require any particular limitation, and the cDNA obtained at this time corresponds to the entire genome of foot-and-mouth virus containing 3D genes.

Separation and amplification of the 3D gene can be performed using a known sequence SEQ ID NO: 2 and SEQ ID NO: 3 as a forward primer and a reverse primer, respectively, by amplification through a polymerase chain reaction. The 3D cDNA obtained at this time is used for antibody diagnosis. Preference is given to using as a template for antigen production.

In addition, a Bgl II cleavage site is inserted at the 5 'end, and an EcoR 1 cleavage site is inserted at the reverse primer, and Taq polymerase ( Amplitaq, Perkin Elmer) is preferred in the practice of the present invention.

Next, the manufacturing process of the pG3D-2 recombination vector of the present invention will be described.

Figure 2 shows the pG3D-2 recombination vector of the present invention, Figure 1 shows the manufacturing process of the recombinant vector Referring to the manufacturing process with reference to Figure 1, first, foot and mouth virus gene using a random primer and reverse transcriptase CDNA was synthesized for the whole, and the cDNA was selectively amplified by 3D gene by polymerase chain reaction, and then the amplified product was inserted into pGEM-T vector having EcoR I and Bgl II cleavage sites to insert 3D gene. Synthesized pGEM-T / 3D and the pGEM-T / 3D is cloned into DH5α cells (not shown);

The cloned pGEM-T / 3D was further treated with EcoR I and Bgl II to isolate 3D genes, and then inserted the 3D gene into pGEX-3X having EcoR I and BamH I cut sites as E. coli expression vectors. Synthesizing -3X / 3D (hereinafter referred to as pG3D-2) and recloning it into BL21.

As shown in FIG. 2, the pGEX-3X vector has an ampicillin resistance gene and lac Iq as a selection marker, and has a pBR322origin as a replication start point. In addition, the GST gene is located upstream of the BamH I cleavage site where the 3D gene is to be inserted, so that the pG3D-2 recombination vector obtained when the 3D gene is inserted includes a fusion gene in which GST and 3D are fused.

The recombinant vector pG3D-2 containing the gene of GST and foot-and-mouth virus 3D is recloned into BL21, an E. coli competent cell, and used to produce a fusion protein in which GST and foot-and-mouth virus 3D are fused.

The present invention also includes a transformant transformed with the recombinant vector pG3D-2. Cells used for transformation are not particularly limited, but E. coli BL21 is preferred in the practice of the present invention. The transformation process is disclosed in Preferred Example 2 of the present invention, which is by a known method and detailed description thereof will be omitted here. The transformant obtained by the transformation process produces a GST- foot-and-mouth virus 3D fusion protein, the fusion protein produced at this time will include a bovine factor Xa between GST and 3D as shown in FIG. Those skilled in the art can be used to delete the site in the vector production step irrelevant to.

PG3D-2 / BL21 transformed with the recombinant vector pG3D-2 was deposited on February 25, 2000 with the microbial accession number KFCC-11150 to the Korean spawn association.

The present invention also includes a method for detecting foot-and-mouth antibody using a GST- and foot-and-mouth virus 3D fusion protein and a GST antibody. The foot-and-mouth antibody detection method of this invention shown in FIG. 3 uses the ELISA method generally used for antibody detection. In this case, a GST antibody is used as a primary antibody fixed to the plate, and preferably, the antibody is a goat anti-GST antibody, and specifically, a GST-foot-and-mouth virus 3D fusion protein. The secondary antibody which specifically binds to the reacting anti-FMDV 3D antibody and generates an enzymatic reaction is not particularly limited but is preferably a secondary antibody against the animal antibody of interest. When the test serum is bovine serum, it is preferable to use an anti-bovine IgG-HRP conjugate [Horserishish peroxidase conjugate], and 0-phenylenediamine as a substrate of Horseradish peroxidase linked to the secondary antibody. By using dihydrochloride, foot-and-mouth virus antibodies can be detected by enzymatic reaction.

The specific antibodies and substrates disclosed in the method for detecting foot-and-mouth disease using the GST- and foot-and-mouth virus 3D fusion proteins and GST antibodies are merely examples for explaining the present invention and are not intended to limit the scope of the present invention. Therefore, some modifications by those skilled in the art do not depart from the scope of the present invention, and as long as the detection antigen and the antibody apply the GST-foot-and-mouth virus 3D fusion protein and GST antibody according to the present invention, they are within the scope of the present invention. Belong.

Hereinafter, the present invention will be described in detail through Examples and Test Examples. However, these examples and test examples are provided only to explain the present invention in detail, and the scope of the present invention is not limited thereto.

Example 1 Amplification of Foot-and-mouth Virus 3D Gene

Nucleic acid was extracted from the culture of foot-and-mouth virus A ₂₄ (FMDV A ₂₄ ) culture and synthesized cDNA using a random primer and reverse transcriptase was used as a template for 3D gene amplification.

The foot-and-mouth virus 3D gene was amplified by a polymerase chain reaction (PCR) using a nucleic acid amplifier (Perkin Elmer). For the reaction for gene amplification, the reaction solution was prepared as shown in Table 1 below. After reacting for 5 minutes at 94 ° C. in advance, the procedure of 51 ° C. for 1 minute, 72 ° C. for 2 minutes, and 94 ° C. for 1 minute was repeated 30 times. The reaction was carried out at 72 ° C. for 5 minutes and then terminated.

TABLE 1 Reaction solution composition (100 μl).

Furtherance content Forward primer 1 μl Reverse primer 1 μl Genomic cDNA 20 μl 25 mM MgCl ₂ 16 μl Amplitaq DNA polymerase ^* 1 μl 10 × buffer 8 μl Nucleic acid free distilled water 53 μl

* Amplitaq DNA polymerase: manufactured by Perkin Elmer.

The amplified foot-and-mouth disease 3D gene was identified by two methods. First, the amplified gene product was subjected to electrophoresis using agarose gel to which ethidium bromide was added, thereby confirming that the nucleic acid size was 1.4k base pairs. The second amplified gene product was cloned in a pGEM-T vector (Promega) and analyzed by nucleotide sequence analysis.

Example 2 Preparation of pG3D-2 Vector Inserted with 3D Gene

The amplified 3D gene was inserted into the pGEM-T vector by overnight reaction at 4 ° C. using a pGEM-T vector (Promega) and a T4 DNA ligase. The pGEM-T vector into which the 3D gene was inserted was inserted into competent cells by mixing with DH5α cells, which are competent cells, and treating them at 42 ° C. for 90 seconds, and then inserting the cells into X-gal (5-bromo-4-chloro). 3D genes were grown by growing at 37 ° C. in LB (Lauria-Bertani) medium with 3-indolyl-β-D-galactopyranoside and IPTG (isopropyl-β-thiogalactopyranoside). Cloned.

Then, 3D gene was extracted by processing EcoR 1 and Bgl lI enzyme into 3D gene inserted pGEM-T vector, and extracted gene was pGEX-3X (Pharmacia) vector and T4 DNA ligase. ) Was reacted overnight at 4 ° C. and inserted into BamH 1 and EcoR 1 sites in the pGEX-3X vector to prepare a pG3D-2 vector as shown in FIG. 2.

Example 3 Preparation of Transgenic Cell pG3D-2 / BL21

The prepared pG3D-2 vector was mixed with E. coli competent cells (BL21), treated at 42 ° C. for 90 seconds, and then plated onto LB (Lauria-Bertani) agar medium containing X-gal and IPTG. E. coli competent cells (pG3D-2 / BL21, microbial accession no. KFCC-11150) transformed by proliferation at 37 ° C were prepared. The 3D protein was expressed by growing the prepared cells (pG3D-2 / BL21) by shaking culture at 37 ° C. in an LB (Lauria-Bertani) liquid medium to which ampicillin was added.

Example 4 Production of Foot-and-mouth Virus Proteins

The foot-and-mouth virus 3D protein produced in transformed Escherichia coli cells (pG3D-2 / BL21) prepared in Example 3 was fused with GST (glutathion-S-transferase) protein (GST-FMDV 3D fusion protein). ).

Escherichia coli cells producing fusion protein were proliferated in shaken culture in LB liquid medium and IPTG was added to induce the expression of fusion protein when absorbance of 0.6 to 1.0 was observed at 290 nm. Then, E. coli was harvested, sonicated for several seconds, and Triton X-100 (final concentration 1%) was added to elute the fusion protein expressed in E. coli and centrifuged at high speed (10,000xg, 5 min) to remove E. coli cell byproducts. By fusion protein was extracted.

The expressed fusion protein was purified by adsorption and release to glutathione sepharose 4B beads (Phamarcia) having GST receptor attached to its surface.

Example 5 Detection of Foot and Mouth Antibodies

Anti-GST antibody (Pharmacia, 5mg / ml) was diluted 1: 1,000 with 0.1M carbonate buffer (pH 9.6) or phosphate buffer (0.01M phosphate buffered saline, pH7.2). ELISA plate 50 μl (final concentration 0.25 μg / well) per well was adsorbed at 37 ° C. for 1 hour and then washed three times with [0.002M phosphate buffered saline (PBS), pH 7.2, 0.02% tween 20]. Washed.

Blocking buffer [0.01M PBS, pH7.2, 3.0% (w / v) Skimmed milk] was added to 100μl per well and blocking was performed at 37 ° C for 1 hour.

Thereafter, 50 µl of the expressed fusion protein (5 µg / ml) diluted with the reaction solution (0.01 M PBS, pH 7.2, 1.5% (w / v) skimmed milk) was added to each well for 1 hour at 37 ° C. I was. After washing the plate three times with washing buffer, 50 μl of control serum and test serum diluted to 1:50 were added to each well, followed by reaction at 37 ° C. for 1 hour, followed by washing with washing buffer three times. It was. Subsequently, 50 μl of anti-species IgG-Horseradish Peroxidase conjugate (2.5 μg / ml) diluted with the reaction solution was added thereto, followed by reaction at 37 ° C. for 1 hour. Wash three times.

Citrate phosphate buffer solution (pH 5.2) containing substrate solution (0.04% o-phenylenediamine dihydrochloride) added with 0.01% hydrogen peroxide was immediately added to 100 μl of each well and allowed to react at room temperature for 10 minutes.

Thereafter, 50 µl of 2.5 M sulfuric acid (H ₂ SO ₄ ) solution was added per well to stop the reaction, and then the absorbance was measured at 490 nm. As a result of the enzymatic reaction, in the case of the sample containing foot-and-mouth antibody, the final reaction solution of the tested well is colored brown.

The result was determined by obtaining a T / P value (test serum absorbance value / positive serum absorbance value) to be 0.4 or more antibody positive and less than antibody negative.

<Test Example 1> Reactivity to foot-and-mouth virus serotypes and other virus-positive serum

By using the diagnostic method according to Example 5 above, each antiserum, foot-and-mouth disease and clinical symptom for foot-and-mouth virus (FMDV) serotypes A, O, C, and Asia 1 are similar to clinical symptoms. responsiveness of antiserum of rinderpest virus (RPV), infectious bovine rhinotracheitis virus (IBRV), bluetongue virus (BTV), and bovine viral diarrhea virus BVDV The results are shown in Table 2, and it was confirmed that only specifically responded to the positive serum for foot-and-mouth virus four serotype viruses.

Table 2: Responsiveness to foot-and-mouth virus serotypes and other virus-positive sera

FMDV Other Viruses * A ₂₂ O ₁ Asia 1 C ₃ RPV IBRV BTV BVDV T / P value 0.5 0.81 1.0 0.48 0.06 0.02 0.04 0.00 result positivity positivity positivity positivity voice voice voice voice

RPV; rinderpest virus, IBRV; infectious bovine rhinotracheitis virus, BTV; bluetongue virus, BVDV; bovine viral diarrhea virus

<Test Example 2> Application to Outdoor Bovine Serum Samples with Negative Foot-and-mouth Antibody

The results of the ELISA assay using the same diagnostic method as in Example 5 on 219 domestic serum of the domestic broilers determined to be foot-and-mouth antibody negative by the foot-and-mouth antibody diagnostic kit of the British foot and mouth standard laboratory. As shown in Table 3, it was confirmed that it can be applied to foot-and-mouth antibody diagnosis by showing a specificity of 94.5%.

<Table 3> Results of the application of this diagnostic method to the outdoor bovine serum sample of foot-and-mouth antibody negative

This diagnosis method Voice (T / P value 0.0 to 0.39) 207 heads Positive (T / P value 0.40 or more) 12 heads Sum 219 heads

* Specificity = (number of voice result / total number of negative) x 100 = (207/219) x100 = 94.5%

The diagnostic method according to the present invention can quickly search a large amount of samples by using the principle of enzyme immunoassay, and the safety of the diagnostic antigen can be secured by using an artificially made protein.

In addition, the diagnostic method according to the present invention is capable of simultaneously detecting antibodies of the four kinds of foot-and-mouth virus serotypes (A, O, C, Asia 1), thereby eliminating the hassle of performing the antibody test for each serotype. There is this.

Claims (3)

A vector having a cleavage map as described in FIG. 2 as an expression vector pG3D-2 containing a GST-foot and mouth virus 3D gene E. coli KFCC-11150 transformed with the expression vector pG3D-2 containing the GST-foot and mouth virus 3D gene In the foot-and-mouth virus detection method using ELISA, A method for detecting foot-and-mouth virus, characterized by using a fusion protein expressed in E. coli KFCC-11150 transformed with the expression vector pG3D-2 as an antigen for foot-and-mouth virus detection.