JP3681537B2 - Monoclonal antibody recognizing pathogenic E. coli, hybridoma producing said monoclonal antibody, and method for detecting pathogenic E. coli using said monoclonal antibody - Google Patents

Description

【００２８】
（２）ウエスタンブロットによる病原性大腸菌Ｏ−１５７、Ｏ−２６、Ｏ−１１１に対する反応性の確認
病原性大腸菌Ｏ−１５７、Ｏ−２６、Ｏ−１１１及び遺伝子工学の分野で用いられている大腸菌ＤＨ５α、ＢＬ２１（ＤＥ３）をＬＢ培地で３７℃一晩培養した。１ｍｌ量の菌体培養液を５０００ｒｐｍで２分間遠心分離して菌体を集め、１５０μｌのポリアクリルアミドゲル緩衝液（２０％グリセリン、１％ＳＤＳ、１０ｍＭトリス−塩酸、ｐＨ６．８、１％ ２−メルカプトエタノール、０．０１％ＢＰＢ）に懸濁し、１５分間の超音波破砕により完全に菌体を破砕した。沸騰水中で加熱した後、ＳＤＳ−ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）を行い、更にニトロセルロース膜に転写した。転写膜を１％スキムミルクを含むＰＢＳでブロッキングした後、１％ＢＳＡ−ＰＢＳで２μｇ／ｍｌに希釈した抗インティミンモノクローナル抗体ｉｎｔｎ２０２、ｉｎｔｎ２０３、ｉｎｔｎ２１５、ｉｎｔｎ２１６及びｉｎｔｎ２２８を室温、１時間反応させた。ＰＢＳＴによる洗浄後、３００倍希釈したＰＯＤ標識抗マウス免疫グロブリン抗体（ダコ社製）と室温、１時間反応させた。更にＰＢＳＴによる洗浄を行った後、ＰＯＤ不溶性基質（４クロロナフトール−過酸化水素系）を加えて、発色させた。結果を図２に示す。病原性大腸菌Ｏ−１５７、Ｏ−２６、Ｏ−１１１では分子量９４ｋＤａ付近にバンドを生じたが、大腸菌ＤＨ５α、ＢＬ２１（ＤＥ３）では同様のバンドが認められなかった。抗インティミンモノクローナル抗体ｉｎｔｎ２０２、ｉｎｔｎ２０３、ｉｎｔｎ２１５、ｉｎｔｎ２１６及びｉｎｔｎ２２８は病原性大腸菌Ｏ−１５７、Ｏ−２６、Ｏ−１１１のインティミンに反応することが確認できた。
【００２９】
実施例３ 抗インティミンモノクローナル抗体のエピトープ解析
（１）エピトープ解析用のペプチド作製
エピトープ解析用のペプチドとして、免疫原として用いたインティミンＮｐの部分ペプチドを作製した。まず、参考例１で作製したｐＷ６ＡｅａｅＡＮｐを鋳型として用いてＰＣＲ法にて特定の部分ペプチド領域をコードする遺伝子領域を増幅した。増幅した断片は、それぞれ制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで水解した後、図２に示すチオレドキシン融合発現プラスミドｐＷＴ８ＡのＥｃｏＲＩ−ＨｉｎｄＩＩＩ部位に挿入した。次いで、それぞれの組み換えプラスミドを用いて、大腸菌ＢＬ２１（ＤＥ３）（Brookhaven National Laboratoryより入手）を形質転換した。形質転換体は、参考例２と同様の方法でチオレドキシン融合蛋白として発現させた。発現させた特定の部分ペプチド領域は、配列表１に示すインティミンのアミノ酸番号で、３８〜２３５、２３７〜５２２、１５８〜３５１、３８〜１５７、１５８〜２３６、３８〜９０、９１〜１５７、８２〜９９の計８種類である。これらの部分ペプチドは、ＮｐＡ、ＮｐＢ、ＮｐＣ、ＮｐＤ、ＮｐＦ、ＮｐＨ、ＮｐＩ、ＮｐＮと命名した。
【００３０】
（２）実施例３−（１）で作製した部分ペプチド８種類を用いて、ウエスタンブロットにより、実施例１で作製したハイブリドーマが産生するモノクローナル抗体のエピトープ解析を行った。ウエスタンブロット法は、実施例３−（１）で作製した部分ペプチド８種をＳＤＳ−ＰＡＧＥした他は、実施例２−（２）に記載の方法にて行った。結果を表２に示す。表２に示すように、モノクローナル抗体ｉｎｔｎ２０２、ｉｎｔｎ２０３、ｉｎｔｎ２１５、ｉｎｔｎ２１６及びｉｎｔｎ２２８はいずれも、インティミンの３８〜１５７アミノ酸領域に結合することが明らかとなった。
【００３１】
【表２】

【００３２】
【発明の効果】
本発明により、病原性大腸菌に特異的なモノクローナル抗体、該モノクローナル抗体を産生するハイブリドーマ及び該モノクローナル抗体を用いた病原性大腸菌の免疫測定方法を提供し、病原性大腸菌を簡便に測定することができる。
【００３３】
【配列表】
配列番号：１
配列の長さ：２７１２
配列の型：核酸
トポロジー：二本鎖
配列の種類：核酸
配列

【図面の簡単な説明】
【図１】発現用プラスミドｐＷ６Ａの制限酵素地図である。
【図２】発現用プラスミドｐＷＴ８Ａの制限酵素地図である。
【図３】ウエスタンブロットによる病原性大腸菌Ｏ−１５７、Ｏ−２６、Ｏ−１１１の測定結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a monoclonal antibody specific for pathogenic E. coli, a hybridoma producing the monoclonal antibody, and a method for detecting pathogenic E. coli using the monoclonal antibody.
[0002]
[Prior art]
In 1996, food poisoning due to pathogenic Escherichia coli O-157 occurred sporadically in Japan, and the number of patients including deaths increased to 9000. Pathogenic Escherichia coli O-157 is a type of enterohemorrhagic Escherichia coli that produces verotoxin similar to Shigella toxin and causes hemorrhagic colitis, hemolytic uremic syndrome, and the like. In severe cases, the mortality rate is high, so definitive diagnosis and early treatment at the early stage of infection are essential.
[0003]
However, as a means of detecting pathogenic Escherichia coli, particularly O-157, a classic is that a patient specimen is cultured in a selective medium supplemented with sorbitol utilizing the property that O-157 is non-degradable or slow degrading. The method is generally taken and requires time and effort, so it may not be in time for urgent inspection. In addition, in clinical isolates from patients, it has been reported that not only O-157 but also O-26 and O-111 are sporadic as verotoxin producing strains (Acta Paediatr. Jpn .: 37, 469-473). , (1995), Nihon Lin: 55, 651-654, (1997)), there is a concern that the culture using an O-157 selective medium for E. coli culture may cause problems in the detection of other strains.
[0004]
On the other hand, in order to eliminate the problem of the selective medium in the culture method, a method of discriminating the O antigen type of E. coli by the sugar chain on the surface of the E. coli membrane has been taken. That is, the O antigen type is identified based on the presence or absence of reaction with E. coli grown by culture using antiserum against sugar chains present on the membrane surface. According to this method, although it can be identified more accurately than the method using a selective medium, among O antigen type classifications of which 170 types or more are known, O antigen type antiserum commercially available in Japan. There are about 40 types, and it is problematic to have antiserum corresponding to a very large number of strains in terms of specificity, reproducibility, cost, etc. of the antiserum. In addition, even when this method is used, since E. coli culture is required in terms of sensitivity, it is unsuitable for identification of toxin-producing pathogenic E. coli that requires rapid response.
[0005]
In recent years, in order to detect the presence or absence of verotoxin production, a gene amplification method by the PCR method of verotoxin gene is also used. Although gene amplification by the PCR method has excellent detection sensitivity, in order to prevent false positives due to amplification of pseudogenes and the like, sample pretreatment is required, and therefore it is undeniable when measuring a large number of samples.
In addition, a method has been established in which only E. coli O-157 is collected from a specimen using magnetic particles in which antiserum against O-157 is immobilized after pre-culture, and O-157 is selectively detected (J. Med. Microbiol .: 40, 424-427, (1994), J. Med. Microbiol .: 44, 267-271, (1996), Appl. Environ. Microbiol .: 62, 587-592, (1996)). However, as described above, only Escherichia coli O-157 is not a verotoxin-producing strain, and there is also a problem with the antiserum specificity. Therefore, a broader method for detecting pathogenic Escherichia coli having a reactivity that is not overlooked is desired. It was rare (Japan Lin: 55, 651-654 (1997)).
[0006]
These methods detect pathogenic E. coli by type classification by O antigen, but on the other hand, antibodies that recognize structures common to pathogenic E. coli are produced, and there is a high risk of producing verotoxin. Attempts have also been made to collectively detect pathogenic E. coli, and among them, intimine, an outer membrane protein of enterohemorrhagic Escherichia coli, has attracted attention. Intimin is known as an adhesive factor that exhibits strong adhesion to the intestinal mucosal epithelial cells of the large intestine mucosa, and enterohemorrhagic Escherichia coli attaches to the intestinal mucosal epithelial cells via this intimin and is called AE disorder (Attaching & Effacing) Causes a series of cytoskeletal structural disturbances to establish infection.
[0007]
However, intimin, which acts as an adhesion factor to the site of infection, has not yet been fully clarified at the stage where the structure of each O antigen type is being elucidated. M. Louie et al. Expressed O-5, O-26 using an antiserum prepared by expressing a protein in which the C-terminal 266 amino acid residues of intimine of O-157 was fused with GST and using this as an immunogen. , O-55, O-111, O-127, O-157, and the like (Infect. Immun., 61, 4085-4092 (1993)).
[0008]
On the other hand, when comparing intimin of O-157 (EHEC), which is feared as an infectious strain to humans, with intimine of O-127 (EPEC), which is best elucidated as an infectious strain of sputum, C It is presumed that the terminal side is very mutated and has a strain-specific sequence, and the N-terminal side has a region with relatively high homology. A group such as TSAgin has also tried to analyze a strain-specific C-terminal region in the gene analysis of O-26 and O-111 intimin, and has not been analyzed for the N-terminal region (Infect. Immun., 65, 320-326 (1997)).
[0009]
In these reports, the common recognition site of pathogenic Escherichia coli using intimine was not clear, and the amount of antiserum was limited, so it was not sufficient as research material.
Therefore, it has been desired to produce a monoclonal antibody that widely recognizes pathogenic E. coli and can be supplied with sufficient reproducibility even in the examination of mass infection.
[0010]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a monoclonal antibody capable of directly recognizing pathogenic E. coli, a hybridoma producing the monoclonal antibody, and a method for immunoassay of pathogenic E. coli using the monoclonal antibody.
[0011]
[Means for Solving the Problems]
As a result of repeated research on pathogenic E. coli in order to solve the conventional problems, the present inventors created a monoclonal antibody that commonly recognizes intimin of the main pathogenic E. coli that produces verotoxin, The present invention was completed by succeeding in immunoassay of pathogenic Escherichia coli by an immunoassay method using the above.
That is, the present invention provides a monoclonal antibody that recognizes pathogenic E. coli, a hybridoma that produces the monoclonal antibody, and a method for immunoassay of pathogenic E. coli using the monoclonal antibody.
[0012]
By using the monoclonal antibody of the present invention, pathogenic E. coli can be detected directly in a short time, and it can be widely applied to definite diagnosis of pathogenic E. coli infection, elucidation of the cause of food poisoning, etc. The present invention can also be applied to countermeasures after infection with pathogenic E. coli, such as collection of enterohemorrhagic Escherichia coli using immobilized monoclonal antibodies or elimination from the body.
[0013]
Hereinafter, the present invention will be described in detail.
In the present invention, pathogenic E. coli refers to enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), intestinal invasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), intestinal aggregating E. coli (EAggEC). E. coli causing disease such as E. coli, preferably enterohemorrhagic E. coli, more preferably enterohemorrhagic E. coli producing verotoxin, particularly preferably E. coli having intimin as an outer membrane protein.
[0014]
Examples of E. coli producing verotoxin include O-2, O-5, O-8, O-22, O-26, O-38, O-69, O-84, O-98, O-103, O -111, O-113, O-116, O-132, O-145, O-153, O-156, O-157 and the like, E. coli having intimin includes O-5, O-26, O-69. , O-84, O-98, O-103, O-111, O-145, O-156, O-157, and the like. Although these pathogenic Escherichia coli are thought to infect humans using bovine as a medium, there is a view that most pathogenic E. coli strains clinically isolated from humans have intimin.
[0015]
The molecular weight of intimine in E. coli O-157 is 94 to 97 kDa, and its nucleotide sequence has been clarified by G. Beebakhee, J. Yue & JBKaoer et al. (FEMS Microbiol. Lett .: 91, 63-68, (1992 ), Mol. Microbiol .: 6, 411-417, (1992)). There have also been reports on the presence or absence of eaeA gene, an intimin gene, in the enterohemorrhagic Escherichia coli strains isolated from cattle (Epidemiol. Infect., 116, 1-7 (1996)). .
[0016]
In order to obtain a monoclonal antibody that recognizes pathogenic Escherichia coli, the pathogenic Escherichia coli itself may be used as an immunogen, but it is desirable to use Intimine, which is a specific outer membrane protein of pathogenic Escherichia coli. Intimin as an immunogen may be used in whole or in part, but whatever immunogen is used, a monoclonal antibody specific for intimin should be selected when establishing a monoclonal antibody. The immunogen is not limited to these.
[0017]
An immunized animal such as a mouse, rat, hamster or the like is immunized with the immunogen as described above. Immunization can be performed according to a conventional method, and the immunogen is administered to the immunized animal alone or together with adjuvant. After immunization, serum may be collected to confirm an increase in antibody titer, and boosted as necessary. After confirming the increase in the antibody titer, antibody-producing cells such as spleen cells and tumor cells such as myeloma cells are fused with a fusion agent such as polyethylene glycol to produce a hybridoma. Subsequently, the hybridoma is selected using a selective medium such as HAT medium, monoclonalized and cultured by an appropriate method such as a limiting dilution method. The culture supernatant is analyzed by an appropriate immunoassay such as an enzyme immunoassay, and a clone producing a monoclonal antibody specific for the target pathogenic E. coli is selected. In the selection, it is preferable to select based on the presence or absence of reactivity of intimin, pathogenic E. coli, E. coli not showing pathogenicity, or the like.
[0018]
These monoclonal antibody production methods can be performed by known methods, for example, Köhler and Milstein (Nature 256 495 1975), Scherrer (Nature 285 446 1980) and the like. Further, the monoclonal antibody prepared by the above-described technique is obtained from the ascites obtained by administering a hybridoma producing the monoclonal antibody to a pristane-treated mouse, or from the culture supernatant of the hybridoma producing the monoclonal antibody, It can be recovered by analysis / purification means such as ion exchange chromatography or affinity chromatography with protein A immobilized.
[0019]
The monoclonal antibody of the present invention can be used in various ways for detecting pathogenic E. coli. For example, E. coli collection, immunoassay method and the like.
When the detection method is collection of bacteria, for example, the monoclonal antibody of the present invention can be immobilized on an insoluble carrier or the like, and pathogenic E. coli insolubilized via the monoclonal antibody can be collected together with the insoluble carrier. If this is done in the body, only pathogenic E. coli can be excluded from the infected body.
[0020]
When the detection method is an immunoassay method, it can be used for a known immunoassay method. For example, tissue immunostaining method in which the monoclonal antibody of the present invention is fluorescently labeled, Western blot method, sandwich immunoassay method using two types of monoclonal antibodies of the present invention, sandwich immunization using the monoclonal antibody of the present invention and an anti-E. Coli antibody A measurement method, a competitive immunoassay method using labeled intimine, and the like can be mentioned, but the immunoassay method of the present invention is not limited to these.
The sample to be measured is not limited, and can be applied to the measurement of pathogenic E. coli such as stool, tissue extract, tissue, food, and serum.
[0021]
【Example】
The present invention will be described in further detail with reference to the following reference examples and examples.
Reference Example 1 Isolation of intimin gene from pathogenic Escherichia coli O-157 and analysis of the reported base sequence of the intimin gene (FEMS Microbiol. Lett .: 91, 63-68, (1992)) The intimin gene 32aa-935aa region was amplified by PCR using O-157 genomic DNA as a template. The amplified fragment was hydrolyzed with restriction enzymes EcoRI and HindIII, and then inserted into the EcoRI-HindIII site of the expression plasmid pW6A shown in FIG. 1 to prepare plasmid pW6AaeAA900. When the nucleotide sequence of the inserted DNA fragment was examined, it had the DNA sequence shown in SEQ ID NO: 1, and the DNA sequence encoded the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the 38aa-522aa region of the intimin gene was amplified by PCR using plasmid pW6AaeA900 as a template. The amplified fragment was hydrolyzed with restriction enzymes EcoRI and BamHI, and then inserted into the EcoRI-BamHI site of the expression plasmid pW6A to prepare plasmid pW6AaeANP. PW6AeaA900 and pW6AaeANp are then used to transform E. coli BL21 (DE3) (obtained from Brookhaven National Laboratory), and ampicillin resistant transformant E. coli BL21 (DE3 ) / PW6AaeA900 and BL21 (DE3) / pW6AaeANP.
[0022]
Reference Example 2 Expression and Analysis of Recombinant Protein The transformant prepared in Reference Example 1 was cultured at 37 ° C. in 2 ml of LB medium containing 50 μg / ml ampicillin. After OD was adjusted to 0.6 to 0.8 at 600 nm in the preliminary culture, 0.5 mM IPTG was added to induce expression, and further cultured for 2 hours. A 1.5 ml amount of the bacterial cell culture solution is centrifuged at 5000 rpm for 2 minutes to collect the bacterial cells, suspended in 100 μl of a buffer solution (10 mM Tris-HCl pH 8.0, 0.1 M sodium chloride, 1 mM EDTA), 15 The cells were completely disrupted by ultrasonic disruption for 1 minute. This is used as a cell sample.
[0023]
4 μl of 3-fold polyacrylamide gel buffer solution (0.1 M Tris-HCl pH 6.8, 6% SDS, 24% glycerin, 6 mM EDTA, 3% 2-mercaptoethanol, 0.01% BPB) is added to 8 μl of the bacterial cell sample. In addition, after sufficient stirring, SDS polyacrylamide gel electrophoresis was performed. After electrophoresis, polyacrylamide gel was stained with protein, and the developed image of the protein contained in the bacterial cell sample was observed. In the transformants BL21 (DE3) / pW6AaeA900 and BL21 (DE3) / pW6AaeANp samples, there were polypeptides having a molecular weight of about 94-97 kDa and about 55 kDa that were not observed in the non-transformant BL21 (DE3). This is considered an expression product from plasmids pW6AaeAA900 and pW6AaeANP. The expression products were named Intimin 900 and Intimin Np, respectively.
[0024]
Reference Example 3 Purified Intimin Np Escherichia coli BL21 (DE3) / pW6AaeANP was cultured under LB medium at 37 ° C. After preliminarily culturing the OD at a concentration of about 0.7 at 600 nm, 0.5 mM IPTG was added to induce expression. After culturing for 4 hours, centrifugation was performed to recover E. coli. 200 ml of 50 mM Tris-hydrochloric acid buffer pH 8.0, 2M urea was added to the recovered E. coli and subjected to ultrasonic crushing treatment under ice cooling. The expressed intimin Np was recovered in the insoluble fraction after centrifugation. The insoluble fraction was solubilized with 6 M urea and 20 mM sodium hydroxide solution pH 12.0, and 1.0 M Tris-HCl buffer solution pH 8.0 was immediately added to pH 9.0, followed by centrifugation to collect the supernatant fraction. The supernatant was subjected to ion exchange purification using a QFF anion exchange column (Pharmacia) equilibrated with 6M urea and 50 mM Tris-HCl buffer pH 9.0. When eluted with a column equilibration buffer containing sodium chloride, intimin Np was recovered in a fraction eluted with about 0.3 M sodium chloride. Superdex 200 gel filtration column (manufactured by Pharmacia) equilibrated with 6M urea, 0.5M sodium chloride, 50mM Tris-HCl buffer pH 9.6 from the QFF elution fraction concentrated with Centriprep 10 (Amicon). Intimine Np having a molecular weight of about 55 kDa was obtained.
[0025]
Example 1 Preparation of Anti-Intimin Monoclonal Antibody-Producing Hybridoma Intimin Np prepared in Reference Example 3 was mixed with an equal volume of Freund's complete adjuvant and administered into the peritoneal cavity of BALB / c mice at 200 μl (about 40 μg / mouse). Intimin Np was mixed twice with Freund's incomplete adjuvant and administered intraperitoneally twice more at intervals of about 2 weeks. After confirming the increase in antibody titer, about 40 μg of Intimin Np was intravenously administered as the final immunization, and the spleen was removed 3 days later. The isolated spleen cells and mouse myeloma cell line P3-x63-Ag8-U1 (purchased from Dainippon Pharmaceutical Co., Ltd.) were mixed at a cell number ratio of 3: 1, and the cells were mixed with 50% polyethylene glycol 1500. Fusion was performed. The cells were suspended in RPMI 1640 medium (hereinafter referred to as HAT medium) supplemented with HAT (1 × 10 ⁻⁴ M hypoxanthine, ⁴ × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymine) and 10% fetal calf serum. It became turbid and dispensed into a 96-well microculture plate and cultured. The culture supernatant of the well in which the hybridoma had grown was examined by the following ELISA method, and a hybridoma producing a monoclonal antibody that reacts with intimin was selected. That is, intimin Np prepared in Reference Example 3 was diluted with a phosphate buffer (hereinafter referred to as PBS in the present specification), dispensed into a 96-well ELISA plate, and allowed to stand at 4 ° C. overnight. Bound to. Next, after washing with PBS containing 0.05% Tween 20 (hereinafter referred to as PBST in this specification), PBS containing 1% skim milk is dispensed and allowed to stand at 37 ° C. for 1 hour for blocking. did. After washing with PBST, the culture supernatant of the hybridoma was dispensed and left at 37 ° C. for 1 hour. Subsequently, after washing in the same manner, a 1000-fold diluted peroxidase (hereinafter referred to as POD) labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was dispensed and allowed to stand at 37 ° C. for 1 hour. . After the same washing, a POD substrate (ABTS-hydrogen peroxide system) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, a reaction stop solution was added, and absorption at 405 nm was measured. Cells in positive wells were cloned by limiting dilution. The antibody activity of the cell supernatant of wells containing a single colony was examined by the above-mentioned method, selected and cultured, and hybridomas INTN202, INTN203, INTN215, INTN216 and INTN228 producing monoclonal antibodies that react with intimin were established. These hybridomas were cultured in large quantities and each was intraperitoneally administered to the mouse, and ascites was collected. Furthermore, the antibody was purified from ascites using Affi-prep protein A maps II kit (manufactured by Biorad) to obtain a monoclonal antibody. These antibodies were named monoclonal antibodies intn202, intn203, intn215, intn216 and intn228.
Of the established hybridomas, INTN215 and INTN228 are deposited with the Biotechnology Institute of Technology, and their deposit numbers are FERM P-16729 and FERM P-16730.
[0026]
Example 2 Reactivity of Anti-Intimin Monoclonal Antibody (1) Reactivity to E. coli BL21 (DE3) / pW6AaeA900 Induced in Expression E. coli BL21 (DE3) / pW6AaeA900 and E. coli BL21 (induced in the same manner as in Reference Example 2) After DE3) was washed 3 times with PBS, it was diluted with PBS to an OD of 1.0 at 660 nm. This was dispensed into 96-well ELISA plates and allowed to dry overnight at 37 ° C. Next, after washing with PBST, PBS containing 1% skim milk was added and blocked at 37 ° C. for 1 hour. After washing with PBST, anti-intimin monoclonal antibodies intn202, intn203, intn215, intn216, intn228 diluted with 1% BSA-PBS to 2 μg / ml and a monoclonal antibody that does not react with intimin as negative control antibodies were added, respectively, at 37 ° C. Reacted for hours. Subsequently, after washing in the same manner, 1000-fold diluted POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was added and reacted at 37 ° C. for 1 hour. After the same washing, a POD substrate (ABTS-hydrogen peroxide system) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, a reaction stop solution was added, and absorption at 405 nm was measured. . The results are shown in Table 1. Anti-intimin monoclonal antibodies intn202, intn203, intn215, intn216 and intn228 did not react with normal E. coli BL21 (DE3), but could react with E. coli BL21 (DE3) / pW6AaeA900 expressing intimin 900 .
[0027]
[Table 1]

[0028]
(2) Confirmation of reactivity to pathogenic E. coli O-157, O-26 and O-111 by Western blotting Pathogenic E. coli O-157, O-26, O-111 and E. coli used in the field of genetic engineering DH5α and BL21 (DE3) were cultured overnight at 37 ° C. in LB medium. A 1 ml amount of the bacterial cell culture solution is centrifuged at 5000 rpm for 2 minutes to collect the bacterial cells, and 150 μl of polyacrylamide gel buffer solution (20% glycerin, 1% SDS, 10 mM Tris-HCl, pH 6.8, 1% 2- The suspension was suspended in mercaptoethanol (0.01% BPB), and the cells were completely disrupted by ultrasonic disruption for 15 minutes. After heating in boiling water, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and further transferred to a nitrocellulose membrane. After blocking the transfer membrane with PBS containing 1% skim milk, anti-intimin monoclonal antibodies intn202, intn203, intn215, intn216 and intn228 diluted to 2 μg / ml with 1% BSA-PBS were reacted at room temperature for 1 hour. After washing with PBST, the mixture was reacted with a POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) diluted 300 times for 1 hour at room temperature. After further washing with PBST, a POD insoluble substrate (4-chloronaphthol-hydrogen peroxide system) was added to cause color development. The results are shown in FIG. In the pathogenic E. coli O-157, O-26, and O-111, a band was generated in the vicinity of a molecular weight of 94 kDa, but a similar band was not observed in E. coli DH5α and BL21 (DE3). It was confirmed that the anti-intimin monoclonal antibodies intn202, intn203, intn215, intn216 and intn228 reacted with intimin of pathogenic E. coli O-157, O-26 and O-111.
[0029]
Example 3 Epitope Analysis of Anti-Intimin Monoclonal Antibody (1) Preparation of Peptide for Epitope Analysis A partial peptide of Intimin Np used as an immunogen was prepared as a peptide for epitope analysis. First, a gene region encoding a specific partial peptide region was amplified by PCR using the pW6AeaANp prepared in Reference Example 1 as a template. The amplified fragments were hydrolyzed with restriction enzymes EcoRI and HindIII, respectively, and then inserted into the EcoRI-HindIII site of the thioredoxin fusion expression plasmid pWT8A shown in FIG. Subsequently, E. coli BL21 (DE3) (obtained from Brookhaven National Laboratory) was transformed with each recombinant plasmid. The transformant was expressed as a thioredoxin fusion protein in the same manner as in Reference Example 2. The specific partial peptide region expressed is the amino acid number of Intimin shown in Sequence Listing 1, 38-235, 237-522, 158-351, 38-157, 158-236, 38-90, 91-157, 82. There are a total of 8 types of ~ 99. These partial peptides were named NpA, NpB, NpC, NpD, NpF, NpH, NpI, and NpN.
[0030]
(2) Epitope analysis of the monoclonal antibody produced by the hybridoma prepared in Example 1 was performed by Western blot using 8 types of partial peptides prepared in Example 3- (1). The Western blotting method was performed by the method described in Example 2- (2) except that SDS-PAGE of the eight partial peptides prepared in Example 3- (1) was performed. The results are shown in Table 2. As shown in Table 2, it was revealed that the monoclonal antibodies intn202, intn203, intn215, intn216 and intn228 all bind to the 38 to 157 amino acid region of intimin.
[0031]
[Table 2]

[0032]
【The invention's effect】
INDUSTRIAL APPLICABILITY According to the present invention, a monoclonal antibody specific for pathogenic E. coli, a hybridoma producing the monoclonal antibody, and an immunoassay method for pathogenic E. coli using the monoclonal antibody can be provided, and pathogenic E. coli can be easily measured. .
[0033]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 2712
Sequence type: Nucleic acid Topology: Double-stranded sequence type: Nucleic acid sequence

[Brief description of the drawings]
FIG. 1 is a restriction map of an expression plasmid pW6A.
FIG. 2 is a restriction enzyme map of expression plasmid pWT8A.
FIG. 3 is a view showing measurement results of pathogenic E. coli O-157, O-26, and O-111 by Western blot.

Claims (5)

  A monoclonal antibody which recognizes a peptide consisting of positions 38 to 157 or 38 to 235 of the amino acid sequence shown in SEQ ID NO: 1, which is a partial sequence of intimin contained in an outer membrane protein of enterohemorrhagic Escherichia coli. Monoclonal antibodies, hybridoma INTN215 (Accession No. FERM P-16729) monoclonal antibody intn215 produced by, or hybridoma INTN228 (Accession No. FERM P-16730) The monoclonal antibody of claim 1 which is a monoclonal antibody intn228 produced by .   A hybridoma producing the monoclonal antibody according to claim 1 or 2.   A method for detecting enterohemorrhagic Escherichia coli using the monoclonal antibody according to claim 1 or 2.   The detection method according to claim 4, wherein the detection method is an immunoassay method.

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