KR101876535B1 - Antibody for detecting of bovine viral diarrhea virus(bvdv), bvdv antigen detecting method and test kit using thereof - Google Patents

Abstract

The present invention relates to an antigenic epitope of bovine viral diarrhea virus (BVDV), a vaccine composition comprising the same, an antibody or fragment thereof using the same, an antibody or fragment thereof, A kit for detecting a diarrheal viral antigens, and a method for detecting a small viral diarrhea virus antigen using the same. According to the present invention, it is very useful not only to quickly measure the infectivity of BVDV in the blood in a cow but also to test it in the field. In particular, the detection kit for BVDV antigens can be used to prevent the spread of BVDV since it can be diagnosed at the early stage of infection rather than the antibody differentiation method by serological method, which can contribute to the economic profit of the cattle breeding farm .

Description

FIELD OF THE INVENTION [0001] The present invention relates to an antibody for detecting bovine viral diarrhea virus, an antigen detection method using the same, and a detection kit comprising the antibody,

The present invention relates to an antigenic epitope of bovine viral diarrhea virus (BVDV), a vaccine composition comprising the same, an antibody or fragment thereof using the same, an antibody or fragment thereof, A kit for detecting a diarrheal viral antigens, and a method for detecting a small viral diarrhea virus antigen using the same.

The BVDV (Bovine Viral Diarrhea Virus) belongs to the family Pestivirus with Flaviviridae, and the virus includes swine fever virus (CSFV) and positive borderline virus (BDV). BVDV can be a carrier to infect other cows from infected cattle, or to infect other cows by continuously infecting calves born from infected birds according to the time of infection during pregnancy (Nettleton et al., 1995 [JoC]; Bri Vet J 151: 615-642). BVDV consists of positive-sense, single-stranded RNA with about 12,500 nucleotides and has a single open reading frame (ORF) (Collett et al., 1988. Virology 63: 191-199, Deng et al., 1992. Virology 191: 867-879). BVDV is divided into genotypes of BVDV1 and BVDV2 according to the result of gene analysis of 5'-UTR (untranslated region), and BVDV1 is divided into BVDV1a and BVDV1b. And cytopathic BVDV (cp BVDV) and non-cytopathic BVDV (ncp BVDV) that do not show CPE, both showing cytopathic effect (CPE) in infected cells, and most (Ridpath et al., 1998. Mol Cell Probes 12: 101-106, Van et al., 1997. Virology 142: 2049-2057).

BVDV is a causative agent of bovine viral diarrhea that is spread all over the world. In addition to diarrheal acute death, leukopenia, congestion of digestive mucosa, ulceration, respiratory lesion, pregnancy abortion or stillbirth, And it is causing great economic damage to the breeding farm at the onset.

Because BVDV has a variety of symptoms, complete diagnosis is difficult only with clinical symptoms. Laboratory diagnostics include serological diagnostic methods such as neutralization test (VN), serum enzyme immunoassay (Ab-ELISA), and polymerase chain reaction (PCR). In order to test persistent infections during the diagnosis, BVDV antigen and antibody (antibody to the maternal antibody) are detected at intervals of at least 3 weeks with serum and ear skin patches. Antigen is detected twice and antibody is persistent It can be judged as a continuous infectious disease due to infectious disease, or immunohistological staining of pieces of ear skin to judge whether or not the infection is persistent due to dyke dyeing. However, these methods are troublesome to perform tissue culture, take a long period of inspection, require expert knowledge for analysis, and require expensive equipment.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made in order to solve the problems of the prior art, and it is an object of the present invention to provide an anti-viral anti-viral diarrhea virus (BVDV) It is an object of the present invention to provide a BVDV antigen detection kit capable of rapidly and easily diagnosing BVDV in a field by using an amino acid sequence having a particularly high immunogenicity and thereby producing an antibody against the peptide, .

In order to solve the above problems, the inventors of the present invention have proposed a method for specifically diagnosing three types of bovine viral diarrhea virus (BVDV) viruses (BVDV1a, BVDV1b, BVDV2a) Specifically, a nucleotide sequence for a gp53 / E2 (hereinafter referred to as "E2") protein contained in the above three types of BVDVs was obtained, and an effective antigen common to all these types of E2 sequences The epitope sequences were searched. Thereafter, a peptide having high immunogenicity was synthesized by the above sequence and an antibody against the peptide was synthesized. Finally, a BVDV rapid diagnostic kit was developed to complete the present invention. Accordingly, a method of detecting or diagnosing bovine BVDV in the field can be quickly and conveniently replaced with a conventional serotyping method or PCR.

Hereinafter, the present invention will be described in more detail.

As used herein, an 'epitope polypeptide molecule' refers to a molecule of the polypeptide of the present invention, which is referred to as BVDV (hereinafter referred to as 'BVDV') or a bovine viral diarrhea virus ) Is a physical representation of the use as an antigenic determinant of an anti-circular type 1 (type 1a or type 1b) or type 2 protein.

One preferred embodiment of the invention is a polypeptide molecule which is an epitope of the E2 protein of BVDV, in particular BVDV, comprising a polypeptide consisting of 10 to 40 consecutive amino acids, including the amino acid sequence of SEQ ID NO: to provide.

More preferably, the polypeptide molecule may comprise a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, most preferably a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 8, 8, or may further comprise 10 to 30 additional amino acid sequences at the N-terminus and / or the C-terminus of the sequence.

The polypeptide molecule, which is the antigenic determinant site, may be for a type 1 or type 2 protein, more preferably three types of bovine viral diarrhea viruses, i.e., 1a, 1b, and 2a type proteins of BVDV Lt; RTI ID = 0.0 > 1 < / RTI >

On the other hand, the polypeptide molecule is a specific active antigenic determinant commonly present in both bovine viral diarrhea viruses, preferably bovine viral diarrhea virus antigens 1a, 1b, and 2a, and the three BVDVs The antigenic determinant site is useful as a vaccine against any one or more of the three types of BVDV, since it effectively produces an antibody specific for the anti-circular type.

Accordingly, another preferred embodiment of the present invention relates to a method for producing a polypeptide comprising the antigenic region polypeptide molecule, a DNA molecule encoding the polypeptide molecule, an expression vector comprising the DNA molecule, and a transformed cell transformed with the expression vector (BVDV, Bovine Viral Diarrhea Virus) comprising at least one member selected from the group consisting of the active ingredients of the present invention.

Another preferred embodiment of the present invention provides an antibody or fragment thereof that specifically recognizes the polypeptide molecule.

The antibody may be prepared by a conventional method using the polypeptide molecule as described above as an antigen. For example, the antibody may be prepared by injection into an animal commonly used in the art, such as a rabbit or a mouse. Or may be produced by a known method well known in the art.

The antibody may be a polyclonal antibody or a monoclonal antibody, or a fragment thereof. The prepared monoclonal and polyclonal antibodies can be used as a tool to quickly and accurately identify three types of BVDV, and thus have important functions that can be applied to BVDV diagnosis and the like.

The antibody may be, for example, a monoclonal antibody produced by a conventional method by a hybridoma cell obtained by a conventional method from a mouse injected with the above-mentioned polypeptide molecule. (A) immunizing a cell such as a mouse or a rabbit by immunizing a cell expressing the antigenic determinant site polypeptide molecule or the antigenic determinant site, (b) splenocytes producing an antibody specific to the antigenic determinant site And (c) fusing the spleen cells with the myeloma cells to select hybridoma cells that produce the specific antibody.

The antibody production strain may be cultured in vitro or in vivo to isolate the antibody. For example, the antibody producing strain can be inserted into the abdominal cavity of a mouse, and the antibody produced can be isolated and purified from a plurality. Separation and purification of the antibody can be carried out using affinity chromatography such as ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column.

Alternatively, the antibody may be a polyclonal antibody produced by immunizing an experimental animal with the above-mentioned polypeptide molecule as an antigen by a conventional method.

Accordingly, another preferred embodiment of the present invention provides a composition for preventing or treating bovine viral diarrhea virus (BVDV) infection comprising the antibody or fragment thereof as an active ingredient.

As used herein, the term " treatment " refers to a reduction or amelioration of symptoms, a reduction in the extent of disease, a delay or alleviation of disease progression, an improvement, alleviation or stabilization of a disease state, partial or complete recovery, Is used to mean both.

The composition for preventing and / or treating BVDV infection and the vaccine composition may be administered by a conventional route such as intravenous (intravenous), subcutaneous, peritoneal administration, but not limited thereto. Usually, It is possible. The compositions may further comprise additives such as conventionally used excipients. The route of administration, dosage, additives, etc. of such compositions can be easily determined by those skilled in the art.

On the other hand, the antibody thus obtained recognizes a specific antigenic determinant site commonly present in BVDV antigens 1a, 1b, and 2a, and thus can detect BVDV antigen.

Accordingly, another preferred embodiment of the present invention provides a kit for detecting bovine viral diarrhea virus (BVDV) antigen comprising the antibody or fragment thereof. The kit may preferably comprise a membrane on which the antibody or fragment thereof is adsorbed as a test line.

Another preferred embodiment of the present invention provides a method for detecting bovine viral diarrhea virus (BVDV) antigen using the kit.

Antibody detection is a detection method that overcomes the limitations of molecular biology and requires quick, easy and relatively low level of quality control. Sensitive and specific monoclonal and polyclonal antibodies are indispensable and necessary to develop new BVDV diagnostic techniques using antibodies. Therefore, developing antibodies that react with various types of BVDV is at the heart of immunological assays. Furthermore, the development of specific monoclonal and polyclonal antibodies that react with BVDV can be used not only as a diagnostic method, but also as an important research material that can be used for virus transcription, replication, and the like.

The kit may preferably be an immunity kit that detects BVDV using the antibody or fragment thereof contained therein and an antigen-antibody reaction with an antigen (antigenic determinant site) in the sample to be applied thereto, and preferably, an enzyme immunoassay A kit (ELISA), a blotting kit, an immunoprecipitation kit, an immunofluorescence kit, or an immunological strip.

The kit may further comprise a detection reagent for detecting a hybridization reaction between the antibody and an antigen in the sample, and the detection reagent may detect the presence of a target analyte to be examined externally through the naked eye or other instrument (Labeled reagent), an auxiliary specific binding member, and / or a component of the signal generation system to allow for the production of a signal.

The labeled reagent, that is, the labeled detection reagent may be those well known to those skilled in the art, and the label may be, for example, a catalyst, enzyme (for example, phosphatase, peroxidase, etc.) , And more specific examples include basic phosphatase and amphoteric peroxidase used together in combination with an enzyme substrate), an enzyme substrate (e.g., nitro blue tetrazolium, 3,5 ', 5,5'-tetranitro Bromo-4-chloro-3-indolephosphate, chemiluminescent enzyme substrates such as dioxetane, and induction peptides and their analogues), fluorescent compounds ( For example, fluorescein, phycobiliprotein, rhodamine, and derivatives and analogs thereof), chemiluminescent compounds, metal sols, dye sols, particulate latexes, (Particulate latex), color indicator (Col or coloring matter contained in liposomes, chemiluminescent compounds, metal sols, dye sols, particulate latex, color indicators, and liposomes. Color matter, non-metallic sol such as carbon sol and selenium, and the like.

The labeling reagent that can be included in the control reagent can be applied to the labeling reagent in the same way.

Examples of the auxiliary specific binding member include, but are not limited to, avidin, biotin, FITC, anti-FITC antibody, mouse immunoglobulin, and anti-mouse immunoglobulin antibody.

In the present invention, the specimen may be a body fluid or secretion of an animal to be diagnosed, and may be urine, tears, saliva, milk, vomit, or the like secreted from the blood, serum, plasma, lymph, And feces, and preferably blood, but is not limited thereto.

As an example of a detection kit according to a preferred embodiment of the present invention, an immunoassay apparatus 1 of FIG. 1, which is an exploded perspective view of a diagnostic kit incorporating one assay strip, The structure of the diagnostic kit and the analysis strip according to the present invention will be described in more detail. In addition to the matters described below, reference can also be made to the present invention's registered patent No. 10-1069818 (immunoassay apparatus).

The detection kit of the present invention preferably includes a sample pad 5 for dropping and developing a sample; A conjugation pad (6) conjugated to gold particles and adsorbed with a BVDV antibody-gold conjugate for carrying out a primary antigen-antibody reaction with a BVDV antigen present in said sample; The E2 antigen that produces the highest level of neutralizing antibody in the BVDV infection, preferably the E2 antigenic determinant of the present invention, as a part for carrying out the secondary antigen-antibody reaction and showing the detection result, A membrane 9 having an antibody against the peptide molecule, preferably a test line 7 to which the polyclonal antibody is immobilized; And an analytical strip 2 comprising at least one analytical strip 2 comprising an absorbent pad 10 for absorbing the sample elutriation fluid, the analytical strip preferably having a width in the short side and a length in the long side Lt; / RTI >

The BVDV antibody for performing the primary antigen-antibody reaction is not particularly limited and various known antibodies can be used. Preferably, monoclonal antibodies against any one of the three BVDV antigens can be used.

In addition to the inspection line, the membrane may also include a control line 8 for determining whether the kit is operating normally and comparing it with the inspection line to become a standard for disease analysis. As the control line, there is no particular limitation and can be used as long as it does not cross-react with BVDV. For example, an anti-mouse IgG antibody may be additionally contained as a control line.

The membrane that can be used for the preparation of the assay strip may be one that is used as a material of a conventional diagnostic strip and may be one selected from various synthetic polymers such as nitrocellulose, cellulose, cellulose acetate, polyethylene, and the like .

Also preferably, the kit comprises: a lower case (3) supporting one side of the analysis strip; And a sample inlet (11) coupled to the lower case and covering the other surface of the analyte strip, the sample inlet being formed in correspondence with the sample pad, and a test result formed to correspond to the membrane, And an upper case 4 including a display window 12 and a character display corresponding to the absorbent pad.

On the other hand, the analysis strip 2 has a form in which the sample pad 5, the conjugation pad (gold bonding pad) 6, the membrane 9, and the absorption pad 10 are superposed and joined together, And a sample pad on the upper portion of the conjugation pad, covering the upper part of one end of the membrane in the form of a strip, covering the absorption pad on the opposite end, and attaching a backing plate for support on the lower part of the membrane . The conjugation pad 6 may further comprise gold particles to which mouse IgG has been adsorbed to exhibit a reaction in the control tank.

A BVDV antigen detection method according to another preferred embodiment of the present invention is a method for detecting a BVDV antigen in a sample, that is, a blood sample of a bovine, plasma, serum, lymph, tissue fluid, urine, tear, saliva, milk, vomit, May be one or more selected from the group consisting of the BVDV antigens 1a, 1b, and 2a.

The method includes both a sandwich assay or a competition assay, preferably immobilizing a BVDV antibody in a conjugation pad, preferably a monoclonal antibody-gold conjugate, and isolating a sample obtained from an animal serum And then performing a first antigen-antibody reaction with the antibody in the conjugation pad, followed by a second antigen-antibody reaction with the antibody according to an embodiment of the present invention.

Preferably, the method further comprises the steps of: (1) injecting a predetermined amount of the sample into the drip region of the assay strip; (2) forming a complex by an antibody-gold conjugate having a predetermined label and a first antigen-antibody reaction with the analyte in the specimen; (3) developing the complex on a membrane; And (4) performing a secondary antigen-antibody reaction with at least one antibody or hapten selected from an antibody or a hapten which can be obtained from the polypeptide molecule according to an embodiment of the present invention through a specific immune reaction in a predetermined region on the membrane And determining the presence or absence of the BVDV antigen in the specimen by observing the change in the appearance.

The principle of detecting the BVDV antigen using the above kit will be schematically described with reference to FIGS. 1, 7, and 8. FIG.

An appropriate amount (for example, 40 ((1 drop)) of a specimen (for example, blood such as whole blood, serum, plasma, etc.) obtained from the animal to be diagnosed is placed in the specimen input port 11 of the immunoassay apparatus 1, (40 [mu] l (1 drop)).

After the spotting, the sample is passed through the nitrocellulose membrane (9) by capillary action after the primary antigen-antibody reaction in the conjugation pad (6) on which the BVDV antibody (E2 antibody) Gold conjugate adsorbed to the conjugation pad 6 in the same manner is also transferred to the absorption pad 10 through the nitrocellulose membrane 9. The BVDV antibody 7, Direction.

In the presence of the BVDV antigen in the sample, the antibody according to one embodiment of the present invention binds to the nitrocellulose membrane (9) after the primary antibody-antibody reaction with the E2 antibody-gold conjugate on the conjugation pad (6) (7), which in turn exhibits a unique red band due to the gold particles sensitized to the primary antibody (E2 antibody-gold conjugate). Thereby, it is possible to judge positive (antigenic) or negative (absence of antigen) according to presence or absence of an antigen in the bovine blood. On the other hand, anti-mouse IgG is adsorbed at the position of the control line (8), so that the gold particles adsorbed by mouse IgG always react to the red band regardless of the presence or absence of the viral antigen in the sample.

The developing solution is not particularly limited and may be any of those known in the art. Preferably, a buffer solution having a pH of 7 to 8 may be used. For example, a phosphate buffer solution (PBS) at pH 7.4, 0.5% TW- KCl, 300 mM NaCl and 0.1% NaN ₃ may be used.

As described above, the use of the detection kit according to the present invention makes it possible to rapidly measure the presence or absence of BVDV infection in the cow, and it is very useful because it can be tested in the field. In particular, the detection kit for BVDV antigens can be used to prevent the spread of BVDV since it can be diagnosed at the early stage of infection rather than the antibody differentiation method by serological method. This also contributes to increase the economic profit of the cattle breeding farm .

1 is an exploded perspective view of a kit for detecting BVDV according to an embodiment of the present invention.
FIG. 2 to FIG. 4 show the results of ELISA for detecting the antibody production according to Example 2 of the present invention (FIG. 2: primary immunity, FIG. 3: secondary immunization, and FIG. 4: tertiary immunization).
FIG. 5 shows the specificity of the antibody of Example 2 of the present invention against BVDV in comparison with BVDV E2 monoclonal antibody B20.24 (Jenobiotech, Korea).
FIG. 6 shows the results of analysis of the antibody of Example 2 of the present invention and the antigen-antibody reaction of BVDV E2 monoclonal antibody B20.24 through Western blotting.
Figure 7 shows an example of the preparation of a BVDV antigen diagnostic strip according to an embodiment of the invention.
Figure 8 shows the results of testing the efficacy of BVDV antigen diagnostic strips prepared according to Example 8 of the present invention.
Figure 9 shows the results of multiple alignments of amino acid sequences of three types of E2 proteins in order to determine the antigenic determinants common to the three types of BVDV antigens obtained according to Example 1 of the present invention.

Hereinafter, the BVDV antigen diagnostic system according to the present invention will be described in more detail with reference to the following examples. It is to be understood, however, that these embodiments are provided to understand the scope of the present invention, and the scope of the present invention is not limited to these embodiments.

Example  One: BVDV E2  ≪ RTI ID = 0.0 > Peptides  order

In order to establish a method for diagnosing the above three types of BVDV by predicting the epitope common to each amino acid sequence of three types of BVDV antigens (BVDV1a, BVDV1b, and BVDV2a) isolated from domestic cattle, The nucleotide sequences of E2 structural proteins present in each type of BVDV antigen were provided by the Ministry of Agriculture, Forestry and Fisheries Quarantine and Inspection, and the nucleotide sequences and amino acid sequences of the respective types were as shown in SEQ ID NOS: 1 to 3 and SEQ ID NOS: 4 to 6, respectively.

Specifically, in order to predict the epitope common to each type of E2 amino acid sequence, multi-alignment was performed using a bioinformatics program (DNAMAN version 6 (Lynnon Corporation), followed by domain exclusion analysis Transmembrane domain searches were used to analyze regions of the transmembrane domain that are likely to form other structures by post-translational modification and are shown in Figure 9. As a result of analysis, A peptide sequence having a low hydrophobicity and high antigenicity could be obtained while being located close to the C-terminal of the sequence (SEQ ID NO: 8).

Example  2: BVDV E2 To peptides  About Polyclone  Antibody ( poly E2 - peptiede  antibody, pE2 - pep Ab ) Produce

The peptide obtained from Example 1 was conjugated with preactivated BSA (pre-activated Bovine Serum Albumin) or similarly preactivated KLH (pre-activated Keyhole Limpet Hemocyanin) Zealand White Rabbits), 2.5-3.0 kg, 10-16 weeks of age, male), and the serum was separated and the antibody against the peptide was prepared by ELISA (Enzyme Linked Immuno-Sorbent Assay) (1986) Diana, M., et al Analytical Biochemistry 166: 224-229 (1987) Halliday, M., et al. LC, et al. Contemporary Topice, 43 (1): 8-13 (2004)).

Specifically, the antigen used in the ELISA was coated on the plate at a concentration of 100 ng / well in which the peptide obtained from Example 1 was conjugated with BSA or KLH. Serum from immunized rabbits was diluted 1 / 1,000 to 1 / 100,000 by volume in PBS and reacted at 100 μl / well at room temperature for 2 hours. After washing 5 times with PBST (PBS + 0.1% Tween-20) / 5000 diluted Rabbit-IgG HRP (Goat anti-Rabbit IgG (H + L) -HRP, SURMODICS, USA) as a secondary antibody for 1 hour. After washing PBST 7 times, the plate was reacted with TMB substrate (BioFX, SurModics, USA) for 10 minutes at room temperature to detect the antibody bound to the antigen. The reaction was terminated by adding 20 μl of 5N HCl per well The reactivity was measured by measuring with an ELISA reader (Sunrise, TECAN) at 450 nm. As a result of observation through ELISA, as shown in Figs. 2 to 4, when sera were collected from peptide-immunized rabbits and non-immunized rabbits respectively and subjected to an ELISA reaction with peptides used as immunogens, (OD) of the antibody was found to be 0.5-1.5 fold depending on the degree of dilution of the serum. Thus, it was confirmed that an antibody showing reactivity to the peptide antigen was generated. After confirming antibody production, rabbits were anesthetized and whole blood was collected and allowed to stand at 37 ° C for 30 minutes to 60 minutes. When blood cells were clotted to form blood clots, serum was obtained after they were removed. The prepared serum was centrifuged at 10,000 rpm for 10 minutes at 4 ° C, and the supernatant was taken and the antibody was purified from the supernatant using the immunoaffinity chromatogarphy method. That is, the supernatant was loaded onto an agarose bead column (empty column, Pierce) conjugated Protein A equilibrated with 5 times the volume of beads of phosphate (PBS) After washing the column with phosphate buffered saline (PBS) in bed volumn, the polyclonal antibody was eluted with a buffer solution of 0.1 M Glycine, pH 3.0. The eluted polyclonal antibody was further added with 1/10 volume of 1M Tris, pH 8.0 for neutralization. Finally, the pure polyclonal antibody was isolated and dialyzed against phosphate buffer (PBS) to isolate and purify the antibody. Purified antibody (pE2-pep Ab) was dialyzed into PBS buffer, and 0.02% NaN ₃ was added and stored in the refrigerator.

Example  3: IFA  And western blot through E2 Polyclone  Antibody BVDV Specific reaction confirmation

The antibody (pE2-pep Ab) prepared from Example 2 was identified by indirect immunofluorescent antibody technique (IFA) to confirm that it specifically reacted with BVDV. BVDV domestic isolate type 1a (agriculture, forestry and fisheries quarantine inspection headquarters) was inoculated into a MDBK (Madin-Darby Kindney: ATCC CCL22) cell line with a degree of cell saturation of 70%, cultured in a CO ₂ incubator (37 ° C) for 3 days, Cells were washed twice with buffer (pH 7.2).

The washed cells were fixed with acetone / PBS (90: 10) (v / v) for 10 minutes at room temperature. (PE2-pep Ab) prepared in Example 2 and BVDV E2 (gp53) monoclonal antibody B20.24 (Jenobiotech, Korea) were added to the fixed cells, respectively. After the reaction time and PBS was washed four times, anti-rabbit IgG FITC (fluorosane isothiocyanate, surmodics) was diluted 1: 1000 by volume on the basis of the supplied manufacturer's instructions and reacted at room temperature for 1 hour.

After the reaction, the cells were washed three times with PBS buffer for 5 minutes each. Afterwards, it was confirmed by using a fluorescence microscope (IX51 Inverted microscope, OLYMPUS) that the BVDV-infected cells and the antibodies produced in the cells that did not react specifically with the viruses Reference). As shown in FIG. 5, in the FA (florescent antibody) test, both of the antibodies of the examples and the comparative examples showed fluorescence reaction in cells infected with the BVDV Type 1a domestic isolate. It was confirmed that both antibodies react with antibody specific to BVDV.

In addition, western blots were performed with BVDV-infected cells and total cell lysates obtained from uninfected cells. The anti-mouse HRP (Goat anti-Mouse IgG, (H +) mouse anti-mouse HRP was used for the monoclonal antibody (the antibody of the comparative example) when diluted with 1: 5000 (v / v) Rabbit IgG (H + L) -HRP, SURMODICS, LR-HRP, SURMODICS, USA) at a concentration of 1: 1000 (v / v) USA) was diluted 1: 2000 (v / v). DAB (Diaminobenzidine tetrahydrochloride) was used as a substrate for color development. Refer to the manufacturer's instructions for use (D5905, Sigma).

As a result, as shown in FIG. 6, in the case of the antibody of Experimental Example 2, it was confirmed that a band having a reactivity between 48 kDa and 63 kDa was clearly observed for the total cell lysate infected with the domestic isolate BVDV Type 1a, whereas BVDV E2 gp53) Monoclonal Antibody B20.24 (Jenobiotech, Korea) was found to be less sensitive than polyclonal antibodies. On the other hand, in the total cell lysate in which the virus used as a control was not infected, neither band showed any specific band seen in the experimental group.

As a result, it was confirmed through IFA and Western blot that both the polyclonal antibody of Example 2 and the B20.24 monoclonal antibody were specific antibodies to BVDV.

Example  4: BVDV E2 Polyclone  Antibody is stuck Nitrocellulose Membrane  Strip manufacturing

The BVDV antibody prepared according to Example 2 was mixed with buffer (0.1% trehalose, 10 mM PB) adjusted to a concentration of 1 to 4 mg / ml and used as a test line. The control line was a goat anti-mouse immunoglobulin G mouse IgG) (Arista Biologicals Inc.) was used. The control solution of the recombinant antibody and the control line of the test line was injected into a nitrocellulose membrane (MDI, India) using a dispensing device (KINAMETICS, USA). It was then dried in a low humidity laboratory overnight or in a fan for at least 5 hours. The plates of the prepared membrane were stored in a container sealed with a desiccant or in a laboratory of low humidity.

Example  5: BVDV E2 Monoclonal  Antibody ( Monoclonal antibody )-gold( Gold ) Conjugate  Produce

BVDV E2 monoclonal antibody (anti-BVDV E2 Monoclonal Antibody, Genobiotech, Korea) was added dropwise to the gold solution with stirring to a final concentration of 4 to 20 ug / ml, and the solution was further stirred for 15 minutes. A 10% BSA solution (Sigma) was then added to the gold particle suspension. After stirring again for 15 minutes, the bound gold solution was centrifuged (6000 g, 15 minutes) to discard the supernatant to remove the unbound E2 mAb. To the bound gold solution pellet was added 3 times 1 After addition of 5 mM Sodium Tetraborate (pH 7.2) (Sigma) to which% BSA had been added, the pellet was resuspended. The suspension was centrifuged again (6000 g, 15 minutes), and the final pellet was prepared by adjusting the absorbance to 5 +/- 1 at 530 nm in 5 mM sodium tetraborate (Sigma) to which 1-fold 1% BSA had been added .

Mouse IgG purchased from Arista (USA) was added to the gold solution with stirring to a final concentration of 4 to 20 ug / ml to prepare a gold conjugate for the control, Lt; / RTI > A 10% BSA solution was then added to the gold particle suspension. After stirring again for 15 minutes, the bound gold solution was centrifuged (6000 g, 15 minutes), and the supernatant was discarded to remove unbound mouse IgG. To the bound gold solution pellet, 1% After addition of 5 mM sodium borate (pH 7.2) supplemented with BSA, the pellet was resuspended. The suspension was centrifuged again (6000 g, 15 minutes), and the final pellet was prepared by adjusting the absorbance to 5 +/- 1 at 530 nm in 5 mM sodium borate (pH 7.2) to which 1-fold 1% BSA had been added. Respectively.

Example  6: Preparation of gold bonding pad

The gold conjugate prepared in Example 5 was prepared as follows by adding 1 to 5% trehalose. The BVDV E2 mAb-gold conjugate was adjusted to a final concentration of 0.5 to 2.5 OD (Optical Density) on 0.5 x 20 cm glass fibers, and the mouse IgG-gold conjugate was mixed to a final concentration of 0.5 OD and adsorbed evenly. It was then dried in a low humidity laboratory overnight or in a fan for at least 5 hours. The prepared conjugate pad was stored in a container sealed with a desiccant or in a low humidity laboratory.

The other hygroscopic pad and sample pad were dried using a method known in the art so that the reaction solution could be well absorbed.

Example  7: device  Assembly

Each of the membranes and pads prepared above was superimposed on the sample pad, the gold pad (gold-bonded body treatment pad), the nitrocellulose membrane, and finally the moisture-absorbing pad in the overlapping manner from the right side, . The cut strips were finally placed in the bottom plate of the immunoassay device and covered with the top plate to prepare a diagnostic kit (Figs. 1 and 7). The immunoassay and sample solution (PBS / 0.5%, Tween-20, 50 mM KCl, 300 mM NaCl, 0.1% NaN ₃ ) was made up as a final product.

Example  8: BVDV  Efficacy testing of antigen diagnostic strips

The kit was tested according to the method of Example 7. The samples used for the efficacy test of strips were reacted with the kit prepared as inactivated BVDV domestic isolate type 1a (agriculture, forestry and fisheries quarantine inspection headquarters), and the reactivity was visually confirmed after 10 minutes. As a result, there was no reactivity in the test line in the negative specimen, but the test line was reactive to the positive specimen BVDV. Therefore, it can be confirmed that the BVDV antigen detection method is possible using the kit according to the present invention.

Description of Reference Numerals for Main Parts of FIG.
1. Immunoassay 2. Analytical strip
3. Lower Case 4. Upper Case
5. Sample pad 6. Conjugation pad
7. Inspection line
9. Membrane 10. Absorbent pad
11. Sample inlet 12. Results display window

<110> VETALL CO., LTD. <120> ANTIBODY FOR DETECTING OF BOVINE VIRAL DIARRHEA VIRUS (BVDV), BVDV          ANTIGEN DETECTING METHOD AND TEST KIT USING THEREOF <130> DPP20122575 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 1122 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of BVDV 1a E2 <400> 1 cacctggaat gcaaacctga atactcatat gccatagcta agagtgacag aattggccca 60 ctaggagctg aaggccttac cactgtttgg aaggattact cacatgaaat gacattggaa 120 gacacaatgg ttatagcttg gtgtaaagat ggtaagtttg tacaccttgc aaggtgcacc 180 agagaaacta gatatcttgc agttttgcat tcaagagcct tgccgaccag tgtagtattt 240 gagaaacttt tcgaggggct aaggcaagag gaaacaattg aaatggatga caactttgaa 300 tttgcgctct gcccatgcga tgccaaaccc atagtaagag ggaagttcaa tacaacactg 360 ctgaacggac cagccttcca ggtggtatgt cccataggat ggactgggac tgtgagctgt 420 atgatagcta atagagatac cctagacaca gtagtagtac ggacatatag gaggtccaga 480 ccattccctt ataggcaagg ctgtattacc caaaaaactt tgggagagga tctctatgac 540 tgtattcttg gaggaaactg gacttgtgtg actggggatc aactaaaata cacaggaggc 600 cttattgaat cctgcaagtg gtgtggtttc aaattccaaa aagatgaggg actaccacac 660 taccccatcg gcaagtgtag gttaaaaaac gagactggct acagacttgt agatgacacc 720 tcttgcaaca gagaaggtgt ggccattgtg ccacacgggc aggtaaagtg taagatagga 780 gacacaattg tacaggttat agctcttgac accagacttg ggcctatgcc ttgcaagcca 840 tatgagatca tatcaagtga gggacctgtt gaaaagacgg cctgcacctt caactacacg 900 aaaacattga aaaataaata ttttgagccc agagatagtt acttccagca atacatgtta 960 aaaggagaat atcaatactg gtttgacctg gaggtcactg accatcatcg ggattacttc 1020 gt; ctggtcacat acatggtttt atcagaacaa aaggccttag gg 1122 <210> 2 <211> 1122 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of BVDV 1b E2 <400> 2 tacccagact gcaaacccga cttttcatac gccatagcca aaaatgatga gattggccca 60 cttggagcta caggcctcac cactcagtgg tacgaatact cagatggaat gcggctgcag 120 gactcaacag ttgaagtttg gtgtaaagat ggaaagttca gatatctaac caaatgtgag 180 agggaagcca gatatctggc tattctacac acgagagcct tgccaacatc tgtagaattt 240 aagaaaattt taaatggaaa agaacaagag gacatagtag aaatggaaga caactttgaa 300 ttcggtcttt gcccatgtga tgctagaccc ttggtaaagg gaaaattcaa tacaacactt 360 ctaaatgggc cggccttcca gatggtttgt cctataggat ggacagggac tgtaagctgc 420 atactggcca ataaggatac gttagccaca atcgttgtgc gaacgtataa gagggtcagg 480 cctttcccat ataggcagta ctgcgttacc cagaagacca ttggggagga ccttcacgac 540 tgtgcgtgag gagggaattg gacttgtgtg ccgggggatc aactacgata tgtggctggg 600 cccgtcgagt cttgtaagtg gtgtggttac aagtttttta aaagtgagga tctgccgcat 660 ttcccaattg gtaaatgcag gcttaagaat gagagtggct ataggctggt ggatgagact 720 ccttgcaata gagacggcgt ggctatagta ccatctggcc tggtcaaatg caagataggg 780 gacacagtag tgcaagtcat cgcaatggat agtaaactag ggcctatgcc ttgcaaacca 840 tatgaaatca tacccagtga ggggccagtg gaaaagacgg catgcacctt caactacaca 900 agaacattag aaaacaagta ctttgagcct agggacaact atttccaaca atacatgttg 960 aagggggggt accaatattg gtttgatcta gagatcactg accaccaccg ggattacttc 1020 gctgagtccc tactggtgat agtagttgca ctcctgggtg gtaggtacgt gctttggttg 1080 ctggtcacat atatggtcct atcagaacaa atggcctcgg gt 1122 <210> 3 <211> 1116 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of BVDV 2a E2 <400> 3 ttccctgaat gcaaaggggg cttccaatat gccatatcga aagacagaaa aatggggtta 60 ttgggaccag agagcttgac tacaacatgg caccttccca cacaaaaaat agtggactcc 120 atggtacaag tatggtgtga aggaaaaaac ttgaaaatat taaaaacgtg cacaaaggaa 180 gagaggtacc tagtggctgt gcacgagaga gccttatcga ccagtgcaga gtttatgcag 240 atcagtgaag ggacgagagg cctagaagtg atagatatgc ctgatgactt tgagtttgga 300 ctctgccctt gtgactcaaa accagtgata aagggcaaat tcaatgccag cttactgaat 360 ggaccagctt tccagatggt atgcccacag gggtggactg gtagaataga atgcacccta 420 gtgaaccaag acaccttgga cacaactgtc attaggacat atataagaac tactccattc 480 cagcggagaa aatggtgcac ctatgaaaaa ataatagggg aagatatcca tgaatgcgtt 540 ctaggtggaa actggacatg cataactggt gaccatagca ggttgaaaga cgggcctatc 600 aagaagtgta agtggtgtgg ctatgacttc ttcaactcag agggactgcc acactaccca 660 ataggtaagt gcatgctcat caatgagggt gggtacaggt atgtagatga cacctcttgc 720 gataggggtg gtgtagccat agttccaaca ggcactgtaa agtgtagaat aggtaacgtc 780 acggtgcagg ttatcgctac taacaataat ctgggaccca tgccctgcag cccagctgaa 840 gtgatagcaa gtgaaggacc agtagaaaag actgcatgca catttaacta ttcaaggaca 900 ctacctaata agtactatga gccaagggac cggtacttcc aacaatacat gttaaaaggg 960 gagtggcaat attggttcga cctggattct gtagaccacc acaaagacta cttctcagag 1020 ttcataatca tagcagtggt cgccttgttg ggtggtaagt acgtactgtg gctcttgata 1080 acatacacaa tattgtctga gcagatggct atgggt 1116 <210> 4 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of BVDV 1a E2 <400> 4 His Leu Glu Cys Lys Pro Glu Tyr Ser Tyr Ala Ile Ala Lys Ser Asp   1 5 10 15 Arg Ile Gly Pro Leu Gly Ala Glu Gly Leu Thr Thr Val Trp Lys Asp              20 25 30 Tyr Ser His Glu Met Thr Leu Glu Asp Thr Met Val Ile Ala Trp Cys          35 40 45 Lys Asp Gly Lys Phe Val His Leu Ala Arg Cys Thr Arg Glu Thr Arg      50 55 60 Tyr Leu Ala Val Leu His Ser Arg Ala Leu Pro Thr Ser Val Val Phe  65 70 75 80 Glu Lys Leu Phe Glu Gly Leu Arg Gln Glu Glu Thr Ile Glu Met Asp                  85 90 95 Asp Asn Phe Glu Phe Ala Leu Cys Pro Cys Asp Ala Lys Pro Ile Val             100 105 110 Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro Ala Phe Gln Val         115 120 125 Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys Met Leu Ala Asn     130 135 140 Arg Asp Thr Leu Asp Thr Val Val Val Arg Thr Tyr Arg Arg Ser Arg 145 150 155 160 Pro Phe Pro Tyr Arg Gln Gly Cys Ile Thr Gln Lys Thr Leu Gly Glu                 165 170 175 Asp Leu Tyr Asp Cys Ile Leu Gly Gly Asn Trp Thr Cys Val Thr Gly             180 185 190 Asp Gln Leu Lys Tyr Thr Gly Gly Leu Ile Glu Ser Cys Lys Trp Cys         195 200 205 Gly Phe Lys Phe Gln Lys Asp Glu Gly Leu Pro His Tyr Pro Ile Gly     210 215 220 Lys Cys Arg Leu Lys Asn Glu Thr Gly Tyr Arg Leu Val Asp Asp Thr 225 230 235 240 Ser Cys Asn Arg Glu Gly Val Ala Ile Val Pro His Gly Gln Val Lys                 245 250 255 Cys Lys Ile Gly Asp Thr Ile Val Gln Val Ile Ala Leu Asp Thr Arg             260 265 270 Leu Gly Pro Met Pro Cys Lys Pro Tyr Glu Ile Ile Ser Ser Glu Gly         275 280 285 Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys     290 295 300 Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu 305 310 315 320 Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu Val Thr Asp His His                 325 330 335 Arg Asp Tyr Phe Ala Glu Ser Ile Leu Val Val Val Ala Leu Leu             340 345 350 Gly Gly Arg Tyr Val Leu Trp Leu Leu Val Thr Tyr Met Val Leu Ser         355 360 365 Glu Gln Lys Ala Leu Gly     370 <210> 5 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of BVDV 1b E2 <400> 5 Tyr Pro Asp Cys Lys Pro Asp Phe Ser Tyr Ala Ile Ala Lys Asn Asp   1 5 10 15 Glu Ile Gly Pro Leu Gly Ala Thr Gly Leu Thr Thr Gln Trp Tyr Glu              20 25 30 Tyr Ser Asp Gly Met Arg Leu Gln Asp Ser Thr Val Glu Val Trp Cys          35 40 45 Lys Asp Gly Lys Phe Arg Tyr Leu Thr Lys Cys Glu Arg Glu Ala Arg      50 55 60 Tyr Leu Ala Ile Leu His Thr Arg Ala Leu Pro Thr Ser Val Glu Phe  65 70 75 80 Lys Lys Ile Leu Asn Gly Lys Glu Glu Glu Asp Ile Val Glu Met Glu                  85 90 95 Asp Asn Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala Arg Pro Leu Val             100 105 110 Lys Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro Ala Phe Gln Met         115 120 125 Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys Ile Leu Ala Asn     130 135 140 Lys Asp Thr Leu Ala Thr Ile Val Val Arg Thr Tyr Lys Arg Val Arg 145 150 155 160 Pro Phe Pro Tyr Arg Gln Tyr Cys Val Thr Gln Lys Thr Ile Gly Glu                 165 170 175 Asp Leu His Asp Cys Ala Leu Gly Gly Asn Trp Thr Cys Val Pro Gly             180 185 190 Asp Gln Leu Arg Tyr Val Ala Gly Pro Val Glu Ser Cys Lys Trp Cys         195 200 205 Gly Tyr Lys Phe Phe Lys Ser Glu Asp Leu Pro His Phe Pro Ile Gly     210 215 220 Lys Cys Arg Leu Lys Asn Glu Ser Gly Tyr Arg Leu Val Asp Glu Thr 225 230 235 240 Pro Cys Asn Arg Asp Gly Val Ala Val Val Ser Gly Leu Val Lys                 245 250 255 Cys Lys Ile Gly Asp Thr Val Val Gln Val Ile Ala Met Asp Ser Lys             260 265 270 Leu Gly Pro Met Pro Cys Lys Pro Tyr Glu Ile Ile Pro Ser Glu Gly         275 280 285 Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr Arg Thr Leu Glu     290 295 300 Asn Lys Tyr Phe Glu Pro Arg Asp Asn Tyr Phe Gln Gln Tyr Met Leu 305 310 315 320 Lys Gly Gly Tyr Gln Tyr Trp Phe Asp Leu Glu Ile Thr Asp His His                 325 330 335 Arg Asp Tyr Phe Ala Glu Ser Leu Leu Val Ile Val Val Ala Leu Leu             340 345 350 Gly Gly Arg Tyr Val Leu Trp Leu Leu Val Thr Tyr Met Val Leu Ser         355 360 365 Glu Gln Met Ala Ser Gly     370 <210> 6 <211> 372 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of BVDV 2a E2 <400> 6 Phe Pro Glu Cys Lys Gly Gly Phe Gln Tyr Ala Ile Ser Lys Asp Arg   1 5 10 15 Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu              20 25 30 Pro Thr Gln Lys Ile Val Asp Ser Met Val Gln Val Trp Cys Glu Gly          35 40 45 Lys Asn Leu Lys Ile Leu Lys Thr Cys Thr Lys Glu Glu Arg Tyr Leu      50 55 60 Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu Phe Met Gln  65 70 75 80 Ile Ser Glu Gly Thr Arg Gly Leu Glu Val Ile Asp Met Pro Asp Asp                  85 90 95 Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly             100 105 110 Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys         115 120 125 Pro Gln Gly Trp Thr Gly Arg Ile Glu Cys Thr Leu Val Asn Gln Asp     130 135 140 Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Ile Arg Thr Thr Pro Phe 145 150 155 160 Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile                 165 170 175 His Glu Cys Val Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His             180 185 190 Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys Trp Cys Gly Tyr         195 200 205 Asp Phe Phe Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys     210 215 220 Met Leu Ile Asn Glu Gly Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys 225 230 235 240 Asp Arg Gly Gly Val Ala Ile Val Pro Thr Gly Thr Val Lys Cys Arg                 245 250 255 Ile Gly Asn Val Thr Val Gly Val Ile Ala Thr Asn Asn Asn Leu Gly             260 265 270 Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val         275 280 285 Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys     290 295 300 Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr Met Leu Lys Gly 305 310 315 320 Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val Asp His His Lys Asp                 325 330 335 Tyr Phe Ser Glu Phe Ile Ile Ala Val Val Ala Leu Leu Gly Gly             340 345 350 Lys Tyr Val Leu Trp Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln         355 360 365 Met Ala Met Gly     370 <210> 7 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> The common epitope sequence of E2 <400> 7 Met Pro Cys Lys Pro Tyr Glu Ile Ile Ser Ser Glu Gly Pro Val Glu   1 5 10 15 Lys Thr Ala Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys Asn Lys Tyr              20 25 30 Phe Glu Pro Arg Asp Ser Tyr Phe          35 40 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> The common epitope sequence of E2 <400> 8 Ser Glu Gly Pro Val Glu Lys Thr Ala Cys   1 5 10

Claims (11)

Comprising the amino acid sequence of SEQ ID NO:
Bovine Viral Diarrhea Virus (BVDV) Polypeptide molecule that is an epitope of E2 protein.
2. The polypeptide molecule of claim 1, wherein the antibody produced by the polypeptide molecule has cross-neutralizing ability to E2 proteins of BVDV antigens 1a, 1b and 2a.
The polypeptide molecule according to claim 1, wherein the polypeptide molecule is at least one antigenic determinant selected from the group consisting of anti-viral diarrhea virus antigens 1a, 1b and 2a.
At least one selected from the group consisting of an antigenic determinant site polypeptide molecule of claim 1, a DNA molecule encoding the polypeptide molecule, an expression vector comprising the DNA molecule, and a transformant cell transformed with the expression vector Vaccine compositions for bovine viral diarrhea virus (BVDV).
An antibody or fragment thereof, which specifically recognizes the polypeptide molecule of claim 1 and has cross-neutralizing ability to the E2 protein of the 1a type, 1b type and 2a type antiviral of bovine viral diarrhea virus (BVDV) .
6. The antibody or fragment thereof of claim 5, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
A composition for the prophylaxis or treatment of bovine viral diarrhea virus (BVDV) infection comprising the antibody of claim 5 or a fragment thereof as an active ingredient.
A kit for detecting bovine viral diarrhea virus (BVDV) antigen comprising the antibody of claim 5 or a fragment thereof.
9. The kit according to claim 8, wherein the kit comprises a membrane on which the antibody or fragment thereof is adsorbed as a test line.
A method for detecting bovine viral diarrhea virus (BVDV) antigen using the kit of claim 8.
11. The method of claim 10,
(Bovine viral diarrhea virus) antigens of type 1a, type 1b, and type 2a in human blood, plasma, serum, lymph, liquid, urine, tears, saliva, milk, vomit or feces Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

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