KR101149402B1 - Monoclonal antibody for discriminating and detecting abnormal prion causing bovine spongiform encephalopathy - Google Patents

Abstract

The present invention is a fusion cell line produced by fusing a specific portion of the bovine PrP peptide, inoculated into a gene-targeting mouse and immunized and then fused with immune B cells isolated from mouse myeloma-derived cells; Monoclonal antibody produced from the fusion cell line and detecting modified prion (PrP ^sc ) of bovine spongiform encephalopathy with other priming of bovine normal prion and other infectious cavernous encephalopathy (Scrapie) and its use It is about.

Bovine spongiform encephalopathy, modified prion, monoclonal antibody, fusion cell line, PrP peptide, infectious cavernous encephalopathy

Description

Monoclonal antibody capable of discriminating and detecting modified prion of bovine spongiform encephalopathy, a fusion cell line producing the same and its use {Monoclonal antibody for discriminating and detecting abnormal prion causing bovine spongiform encephalopathy}

The present invention is a fusion cell line produced by fusing a specific portion of the bovine PrP peptide, inoculated into a gene-targeting mouse and immunized and then fused with immune B cells isolated from mouse myeloma-derived cells; Monoclonal antibody produced from the fusion cell line and detecting the modified prion of bovine spongiform encephalopathy (PrP ^sc ) by differentiating prion of bovine normal prion and other infectious cavernous encephalopathy (Scrapie) and its use It is about.

Transmissible Spongiform Encephalopathies (TSE) include scrapie in sheep and goats, infectious mink encephalopathies in mink (TME), and chronic chronic congenital disease in deer and elk in North America (CWD). wasting disease). TSE in humans includes sporadic Creutzfeldt-Jakob disease (sCJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Stroisler-Shinker syndrome (GSS: Gertsmann-). Straussler-Scheinker Syndrome (Kuru), Kuru and Fatal Familial Insomnia (FFI) are known. In 1986, the first clinical findings in the UK, similar to the neurological symptoms of scrapie in goats and goats, occurred in cattle and were termed Bovine Spongiform Encephalopathy (BSE). In 1996, a link between BSE and vCJD was presented to classify BSE as a common infectious disease.

Normal prion (PrP ^c ), a protein present in cells, is a sialolycoprotein with a molecular weight of 33-35 kDa, and glycosyl phosphatidyl inositol anchors are attached to mammalian cell membranes. . The primary structure of normal prion and modified prion is the same, but the modified prion PrP ^sc is structurally more β-sheet and partially resistant to proteinase K than PrP ^c . .

Until now, TSE has been diagnosed using immunological detection of modified prions mainly resistant to protease K, using specific antibodies of prions in TSE-infected brains. Recently, many monoclonal antibodies produced using knock-out mice (transgenic mice without prion protein) have been used to diagnose prion diseases. However, most of these conventional monoclonal antibodies have a limitation in distinguishing between normal prion and modified prion or detecting species-specific modified prion, and in particular, it was difficult to selectively discriminate only BSE, which is a common infectious disease. Therefore, it is necessary to develop monoclonal antibodies that specifically react to the species of infectious spongiform encephalopathy for differential diagnosis of various infectious spongiform encephalopathies.

Therefore, the present inventors selected a specific site of bovine PrP peptide, immunized it by inoculating it in a gene-targeting mouse, and then monoclonal antibody produced from a fusion cell prepared by fusing the isolated immune B cells and mouse myeloma-derived cells. The present invention was completed by specifically reacting only with modified prions and distinguishing them from modified prions such as normal normal prion of bovine or other TSE diseases such as CWD and scrapie.

Accordingly, an object of the present invention is to provide a monoclonal antibody capable of discriminating and detecting only BSE modified prion and its use.

It is also an object of the present invention to provide a fusion cell line for producing monoclonal antibodies capable of detecting BSE modified prion.

In order to achieve the above object, the present invention provides a fusion cell line of Accession No. KCLRF-BP-00213 for producing a monoclonal antibody for detecting the modified prion of bovine spongiform encephalopathy.

In addition, in the present invention 1) inoculating the mouse of the gene of SEQ ID NO: 1 into the genetically targeted mouse; 2) separating immune B cells from the mouse of 1); And 3) fusing the immune B cells isolated in 2) and mouse myeloma-derived cells to provide a method for producing a fusion cell line of Accession No. KCLRF-BP-00213.

In another aspect, the present invention provides a monoclonal antibody for detecting modified prion of bovine spongiform encephalopathy, and a method for detecting modified prion of bovine spongiform encephalopathy through western blotting using the monoclonal antibody.

The monoclonal antibody produced by the fusion cell line of the present invention is modified prion of bovine spongiform encephalopathy by discriminating it from normal prion (PrP ^c ) of bovine, and other modified prion (PrP ^sc ) of infectious spongiform encephalopathy such as deer chronic congestive disease and scrapie. Only PrP ^sc ) was specifically detected.

The present invention relates to a monoclonal antibody capable of distinguishing a modified prion of bovine spongiform encephalopathy from a modified prion of bovine normal prion and other infectious cavernous encephalopathy, such as deer chronic congestive disease and scrapie, and a fusion cell line producing the same.

The process for preparing a fusion cell line according to the present invention comprises the following steps:

1) immunizing the mouse with the peptide of SEQ ID NO: 1 by gene targeting;

2) separating immune B cells from the mouse of 1); And

3) fusing the immune B cells isolated from 2) and mouse myeloma-derived cells.

In step 1), a peptide of SEQ ID NO: 1 corresponding to the 150-169th amino acid sequence (IHFGSDYEDRYYRENMHRYP) of the bovine PrP gene is used. In the present invention, the cysteine is added to the N-terminus of the peptide and then selectively polymerized with a carrier such as Keyhole Limpet Hemocyanin (KLH) and / or Ovalbumin to increase immunogenicity. It can be used for enzyme-linked immunoassay (ELISA) for testing a sample of or screening a manufactured fusion cell line.

Synthetic techniques for the preparation of the peptides can be carried out by those skilled in the art can be easily applied to conventional techniques well known in the art. For example, the peptide of SEQ ID NO: 1 may be synthesized using a Fmoc Soiled phase synthesis procedure, and then purified by HPLC using a Waters C18 column. It is not limited.

A monoclonal antibody that specifically reacts with a modified prion of BSE by inoculating the synthetic peptide into a genetically targeted mouse (transgenic mouse without prion protein) and then fusion between immune B cells and myeloma-derived cells of the mouse isolated therefrom. The fusion cell line (hybridoma cell) which produced the was produced. The fusion cell line according to the present invention was deposited with the Bank of Korea Cell Line on July 13, 2009 and was given accession number KCLRF-BP-00213.

The monoclonal antibody produced from the fusion cell line was named "N18-5 monoclonal antibody".

In addition, in the present invention, after preparing a peptide spot membrane (Pepspot membrane) to analyze the epitope through the reactivity of the monoclonal antibody and bovine prion peptide, using this N18-5 monoclonal antibody as a diagnostic antibody showing a response to the BSE modified prion As a result of investigating the difference in reactivity between the N18-5 monoclonal antibody and modified prion of BSE, CWD and scrapie by Western blotting, the antibody binds only to the modified prion of bovine spongiform encephalopathy and did not react with other antigens. Therefore, when using the monoclonal antibody according to the present invention was confirmed that can be distinguished from normal prion of bovine and modified prion of CWD and scrapie. That is, the present invention provides a new differential diagnosis method that distinguishes infectious spongiform encephalopathy other than BSE, such as CWD and scrapie, by using N18-5 monoclonal antibody in western blotting, one of the BSE definite diagnosis methods.

Hereinafter, the content of the present invention will be described in more detail through examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1 Preparation of Bovine PrP Synthetic Peptide

Peptide of SEQ ID NO: 1 comprising epitope “YYR” capable of differentiating between modified prions and normal prions while having hydrophilicity based on the amino acids of the small normal prions shown in Table 1 was commissioned to Peptron Co., Ltd. .

Bovine PrP amino acid sequence used to prepare synthetic peptides Species (Accession Number) location Synthetic peptide sequences Cattle (P10279) 150-169 IHFGSDYEDRYYRENMHRYP (SEQ ID NO: 1)

Peptide synthesis method is as follows. Peptron Co., Ltd. synthesized the peptide of SEQ ID NO: 1 using Fmoc Soiled phase synthesis procedure, purified by HPLC using Waters C18 column, and then purified by 0.1% (v / v) tree. It was eluted with an acetonitrile linear gradient using acetonitrile 5 to 75% (v / v) solution containing fluoroacetic acid (trifluoroacrtic acid).

After cysteine was added to the peptide N-terminus of SEQ ID NO: 1, it was polymerized with keyhole-limpet hemocyanin (KLH) to increase the immunogenicity of the peptide fragment. In addition, the peptide fragment was polymerized with Ovalbumin and used for enzyme-linked immunoassay (ELISA) for screening a large number of samples or for screening fusion cell lines.

Example 2 Preparation of Fusion Cell Line Producing Monoclonal Antibody

Synthetic peptide-kiholimpethemocyanin (KLH) polymer prepared in Example 1 was mixed in the same amount with complete Freund's adjuvant and then inoculated subcutaneously by 100 μg per 8-week-old mouse. Three weeks after the first immunization, the same antigen and incomplete Freund's adjuvant were inoculated three times by the same immunization method. Three days before cell fusion, mice were inoculated with 100 μg peptide in phosphate buffer (PBS) without adjuvant and injected into the abdominal cavity to induce secondary immunity.

To prepare a fusion cell line producing monoclonal antibodies, B lymphocytes were collected from spleens of immunized gene-targeted mice and used for cell fusion. Cell fusion was carried out as follows using a general method using polyethylene glycol. The collected lymphocytes were washed with serum-free medium, and then fused by adding 1 ml of mouse myeloma-derived cell line (Sp 2 / 0-Ag14) and polyethylene glycol 1500. After completion of the fusion, the cells were appropriately diluted in proliferation medium (DMEM, HAT (Hypoxanthine Aminopterine Thymidine) and 10% Fetal Bovine Serum), and then 100 μl of a 96-well microplate in which mouse peritoneal cells were incubated in advance. Incubated for 7-14 days in% carbon dioxide incubator.

In order to select and isolate the fusion cell line, the supernatant obtained above was taken, and Western blotting using peptide, prion recombinant protein, and BSE infected brain tissue in which Ovalbumin was polymerized to the peptide fragment specified above by indirect ELISA method was performed. After selecting the positive clones for the bovine modified prion used, the fusion cells of the wells showing positive reaction were added to include 96 cells per well in 96 well plates in which mouse peritoneal cells were cultured and cultured until monoclonal formation. The fusion cell line was deposited with the Bank of Korea Cell Line on July 13, 2009 and was given accession number KCLRF-BP-00213.

Example 3 Monoclonal Antibody Production and Isolation

In order to prepare a mouse intraperitoneal culture solution using the positive clone obtained in Example 2, the positive clone fusion cells producing monoclonal antibodies identified by ELISA method were cultured and 5 × 10 ⁶ cells were incomplete Freund's adjuvant one week ago. Was injected into the abdominal cavity of pretreated BLB / C mice. After 10-15 days, 5-10 ml of ascites were extracted.

Antibodies were purified from the intraperitoneal culture solution containing monoclonal antibodies in the following manner. The obtained ascites was purified using an IgG purification kit (Immunopure (A / G) IgG purification kit; Pierce, USA) containing an Immobilized protein A / G column (Pierce, USA). , Its isotype was determined as shown in Table 2 using an isotyping test strip (Serotec, UK). The concentration of the purified antibody was quantified by BCA (bicinchoninic acid; Pierce, U.S.A) method, and the concentration was diluted to 0.5 mg / ml with phosphate buffer solution.

Monoclonal antibody  N18-5 Isotype  IgG1, kappa

Example 4 Epitope Analysis of Monoclonal Antibody (N18-5)

In order to analyze specific epitopes of the produced monoclonal antibodies, a peptide spot membrane was prepared and its reactivity was investigated. In other words, 11 peptides per spot were synthesized from bovine PrP 139-180th amino acids, one amino acid was moved to the side, and 32 peptide spot membranes were customized (Sigma Genosys) to determine the antigenic determinant against monoclonal antibody by immunoblot method. Was analyzed. More specifically, after washing the membrane with TBST (Tris-buffered saline (0.05% Tween 20)), 5 g blocking powder and 5 g sucrose were added to 100 ml TBST to prepare a 5% blocking buffer. It was used after melting. After reacting with 20 ml of blocking buffer for 1 hour at room temperature, the mixture was washed once with TBST, and then prepared into 2 ㎍ / ml of monoclonal antibody prepared in 20 ml of antibody diluent. . After washing 6 times with TBST for 5 minutes, 2 μl of secondary antibody (anti-mouse HRP, 1: 10,000) was added to 20 ml of antibody dilution, and the membrane was washed for 30 minutes at room temperature. In order to develop color, ECL western blotting detection reagent (Amersham RPN2106) was immersed in the membrane using a chromophore, and confirmed by LAS 4000 (Kodak). The results are shown in FIG. 1.

From the results of FIG. 1, it was confirmed that FGSD, the 144th to 147th amino acids, which are overlapping amino acids, was an epitope of N18-5 monoclonal antibody, in view of the positive reaction of the 7th to 14th spots. .

Example 5 Extraction of Normal Prion and Modified Prion from Brain Tissue

In order to extract prion from the normal brains of sheep, cattle and elk for SDS-PAGE and Western blotting, 100 mg of brain tissue was taken, a 10% cerebral emulsion was prepared using a homogenized buffer, and then 10,000 g. Centrifuge for 5 minutes at. 100 μl of the supernatant was collected, and the same amount of 2 × sample loading buffer was added and heated at 100 ° C. for 5 minutes. In addition, in order to extract modified prion from infected brains, brain emulsions of sheep, elk and cows infected with each type of TSE were prepared in the same manner as described above. After addition, it was made to react at 37 degreeC for 2 hours from 4 hours. 40 μg of proteinase K per 100 mg tissue was added and reacted at 37 ° C. for 1 hour. Then, 10 μl of 0.1 M Pepablock was added, followed by centrifugation at 15,000 rpm for 15 minutes, and then the supernatant was removed. 100 μl of 5% SDS (sodium dodecyl sulfate) was added to the pellet and boiled at 100 ° C. for 5 minutes, and 1 ml of cold methanol was added thereto and left in a -20 ° C. freezer for at least 30 minutes. In this state, it is possible to overnight, and then centrifuged at 15,000 rpm at 4 ° C. for 10 minutes to discard the methanol, and then allowed to stand for 5 minutes to evaporate the methanol. It was dissolved in 100 μl of 1 × sample loading buffer and boiled at 100 ° C. for 5 minutes.

Example 6 SDS-PAGE and Western Blotting

Normal prion and modified prion extracted in Example 5 were loaded on a 12% Bis-tris polyacrylamide gel (Invitrogen). Electrophoresis was performed at 200 V for 35 minutes, and protein samples were electrically transferred to 150 V for 1 hour using polyvinylidene difluoride (PVDF) membrane. After blocking the membrane for 30 minutes, the N18-5 monoclonal antibody was reacted at room temperature for 2 µg / ml for 1 hour. After washing 3 times with TBST for 5 minutes, the secondary antibody (alkaline phosphatase-conjugated goat antibody to mouse IgG) was diluted 1: 3000 in TBST for 1 hour at room temperature. Reacted for a while. After washing 3 times with TBST for 5 minutes, it was confirmed as LAS 4000 (Kodak, Japan) by using a CDP star of the chemiluminescence (chemiluminescence) as a coloring agent, the results are shown in FIG.

In the results of Figure 2, the N18-5 monoclonal antibody according to the present invention did not react with sheep, elk and bovine normal prion, and did not react with sheep scrapie, scrapie and elk CWD among modified prions, It was confirmed that only reacted with the modified prion.

The monoclonal antibody produced by the fusion cell line of the present invention has excellent specificity and can be used as a diagnostic reagent for bovine bovine spongiform encephalopathy infection.These results are specific diagnostic methods for selectively detecting only modified prions of bovine spongiform encephalopathy. It can be used as valuable data for the prevention and analysis of the causes of bovine spongiform encephalopathy.

FIG. 1 shows 11 peptides arranged per spot for amino acid sequences 139 to 180 of a bovine prion protein and binds N18-5 monoclonal antibody to a peptide membrane prepared by overlapping one amino acid. It is a diagram analyzing the prion protein epitope.

2 is a result of Western blotting using the monoclonal antibody according to the present invention.

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries.National Veterinary Research and Quarantine Service (NVRQS)) <120> Monoclonal antibody for discriminating and detecting abnormal          prion causing bovine spongiform encephalopathy <130> YPD / 200906-0015 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met   1 5 10 15 His Arg Tyr Pro              20  

Claims (5)

Fusion cell line of Accession No. KCLRF-BP-00213, which produces a monoclonal antibody for detecting modified prions of bovine spongiform encephalopathy. 1) immunizing the peptide of SEQ ID NO: 1 by inoculating a knock-out mouse; 2) separating immune B cells from the mouse of 1); And 3) fusion of immune B cells isolated from 2) with mouse myeloma-derived cells; Method for preparing a fusion cell line of Accession No. KCLRF-BP-00213 via. Monoclonal antibody produced from the fusion cell line according to claim 1 for detecting the modified prion of bovine spongiform encephalopathy. 4. The monoclonal antibody according to claim 3, wherein the monoclonal antibody is an antigenic determinant of the "FGSD" site, which is an amino acid of the 144th to 147th amino acids. Method for detecting the modified prion of bovine spongiform encephalopathy by western blotting using the monoclonal antibody according to claim 3.

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