JP2012017334A - Antibody for detecting chlamydia pneumoniae - Google Patents
Antibody for detecting chlamydia pneumoniae Download PDFInfo
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- JP2012017334A JP2012017334A JP2011196225A JP2011196225A JP2012017334A JP 2012017334 A JP2012017334 A JP 2012017334A JP 2011196225 A JP2011196225 A JP 2011196225A JP 2011196225 A JP2011196225 A JP 2011196225A JP 2012017334 A JP2012017334 A JP 2012017334A
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- antibody
- protein
- ribosomal protein
- chlamydia pneumoniae
- microorganism
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/125—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Chlamydiales (O)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
Abstract
Description
本発明は、一般的な肺炎の原因微生物であるクラミジア・ニューモニア(Chlamydia pneumoniae)に属する微生物の検出に有用な抗体、該微生物の検出方法、該微生物の検出用試薬キット、及び該微生物検出用抗体の製造方法に関する。 The present invention, an antibody useful for detecting a microorganism belonging to Chlamydia pneumoniae , which is a general causative microorganism of pneumonia, a method for detecting the microorganism, a reagent kit for detecting the microorganism, and an antibody for detecting the microorganism. Manufacturing method.
本発明は、医療上、特にChlamydia pneumoniaeによって惹起される非定型肺炎の診断に重要である。 INDUSTRIAL APPLICABILITY The present invention is important medically, especially for diagnosing atypical pneumonia caused by Chlamydia pneumoniae .
本発明は、検体、例えば、のど綿棒、組織サンプル、および体液から採取された検体中に含まれる微生物Chlamydia pneumoniaeを検出するのに有用である。 The present invention is useful for detecting the microorganism Chlamydia pneumoniae contained in specimens such as throat swabs, tissue samples, and specimens collected from body fluids.
微生物感染症の診断は、通常感染部位などでの原因菌の検出か、血清、体液中の原因菌に対する抗体の検出により確定される。特に、この診断は原因菌の検出が患者への迅速な治療を可能にする意味で重要である。 Diagnosis of microbial infection is usually confirmed by detection of causative bacteria at the site of infection or detection of antibodies against the causative bacteria in serum or body fluids. In particular, this diagnosis is important in that the detection of the causative bacteria enables rapid treatment of the patient.
感染症原因菌の検出は、一般に、原因菌の分離培養を経て、その生理学的、生化学的あるいは構造的な特性に基づきこれを同定する培養同定法、原因菌の遺伝子をPolymerase chain reaction (PCR)法または特異的核酸ハイブリダイゼーションにより増幅させて、これを検出する遺伝子的診断法および抗体と原因菌の抗原マーカーとの特異的反応を利用して原因菌を検出する免疫学的方法に分類される。 Generally, the causative bacterium of infectious disease is detected by a culture identification method in which the causative bacterium is isolated and cultured to identify it based on its physiological, biochemical or structural characteristics. ) Method or specific nucleic acid hybridization to detect it, and a genetic diagnostic method to detect this and an immunological method to detect the causative bacterium by utilizing the specific reaction between the antibody and the antigen marker of the causative bacterium. It
しかしながら、培養同定法または遺伝子的診断法を用いる場合には、結果を得るのに長時間を要する。従って、原因菌を短時間にしかも高感度で検出することができ、迅速かつ適切な患者の治療につながる免疫学的方法による診断が汎用されている。 However, when using the culture identification method or the genetic diagnosis method, it takes a long time to obtain the result. Therefore, the diagnosis by an immunological method, which can detect the causative bacterium in a short time and with high sensitivity and leads to rapid and appropriate treatment of a patient, is widely used.
従来、免疫学的方法による感染症原因菌の検出には、菌種によって様々なマーカー抗原と抗体の組み合わせが使われている。
Chlamydia pneumoniaeは世界的に肺炎の一般的な原因菌である。小さな、非運動性のグラム陰性菌であり、選択的にヒトの体内に侵入し、病気を引き起す。病原巣となる動物は知られていない。血清陽性率は30〜40代の成人で40〜50%である(Hyman, Roblin et al. 1995)。この微生物は喉頭炎、気管支炎および軽度の肺炎を発症せしめる。
Conventionally, in order to detect infectious disease-causing bacteria by immunological methods, various combinations of marker antigens and antibodies are used depending on the bacterial species.
Chlamydia pneumoniae is a common cause of pneumonia worldwide. It is a small, non-motile, Gram-negative bacterium that selectively invades the human body and causes disease. There are no known pathogenic animals. Seropositivity is 40-50% in adults in their 30s and 40s (Hyman, Roblin et al. 1995). This organism causes laryngitis, bronchitis and mild pneumonia.
この菌は、非常に微小の偏性寄生生物であり、宿主細胞の細胞質中で成長する。組織培養液中でのChlamydia pneumoniaeの成長は非常に緩慢であり(Godzik, O'Brien et al.1995)、培養液中に菌を同定するまでに少なくとも3〜5日間を要する(Essig, Zucs et al. 1997)。従って、迅速に病原菌として検出する診断法としては、グラム染色法と培養法などを適用することができない。従って、クラミジアの迅速診断法としては、抗体を用いた免疫学的手法がしばしば用いられる。 This fungus is a very small obligate parasite that grows in the cytoplasm of host cells. Growth of Chlamydia pneumoniae in tissue culture is very slow (Godzik, O'Brien et al. 1995) and requires at least 3-5 days to identify the fungus in culture (Essig, Zucs et al. 1997). Therefore, the Gram staining method, the culture method, and the like cannot be applied as diagnostic methods for rapidly detecting pathogenic bacteria. Therefore, an immunological method using an antibody is often used as a rapid diagnostic method for chlamydia.
クラミジア（Chlamydia）属の場合、属特異的抗原であるリポ多糖(LPS)の抗原決定基としての存在が知られており(Verkooyen, Van Lent et al.1998)、様々な診断用キットにおいて特にChlamydia trachomatisの検出用試薬抗体に利用されている。
更に、Peterson ら (Peterson, Cheng et al. 1993 ; Peterson, de la Maza et al. 1998)およびBatteiger ら(Batteiger, Newhallet al. 1986)はChlamydia属の主要外膜蛋白質(MOMP)に対するモノクローナル抗体を報告している。
In the case of the genus Chlamydia , it is known that the genus-specific antigen, lipopolysaccharide (LPS), exists as an antigenic determinant (Verkooyen, Van Lent et al. 1998), and Chlamydia is particularly useful in various diagnostic kits. It is used as a reagent antibody for detection of trachomatis .
Furthermore, Peterson et al. (Peterson, Cheng et al. 1993; Peterson, de la Maza et al. 1998) and Batteiger et al. (Batteiger, Newhall et al. 1986) reported a monoclonal antibody against the major outer membrane protein (MOMP) of the genus Chlamydia . is doing.
これらの抗体がChlamydia pheunoniaeとChlamydia trachomatisを区別することに役立つことが明らかになったが、その後、これらの抗体はChlamydia pheunoniae種内の複数の抗原性の違いを明らかにすることに重要な役割をはたした。このような抗原はChlamydia pheunoniae種内の血清型分類には役立つかもしれないがChlamydia pheunoniae種の全ての菌株の検出を必要とする通常の診断には役に立つものではない。普遍的な機能を持つ共通な抗原、しかも大部分な構造は微生物種間で保存されているにもかかわらず、それを区別して検出するのに利用できる抗原はいままで知られていなかった。 Although these antibodies were shown to help distinguish Chlamydia pheunoniae from Chlamydia trachomatis , they subsequently played an important role in revealing multiple antigenic differences within Chlamydia pheunoniae species. I played. Such antigens may be useful for serotyping within the Chlamydia pheunoniae species, but not for routine diagnostics that require detection of all strains of the Chlamydia pheunoniae species. Although a common antigen with a universal function, and most of the structure is conserved among microbial species, no antigen has been known to date that can be used to detect it.
本発明は全ての微生物に同一機能の分子として普遍的に存在し、しかも抗体を取得するための蛋白質抗原として有用な蛋白質に関するものである。通常、このような分子は小規模の構造変化しか伴わないものである。このような同一機能の普遍的な分子に対する大規模な構造変化は生物の生存に対して重大な悪影響を及ぼす可能性のあるものである。 The present invention relates to a protein which is universally present in all microorganisms as a molecule having the same function and is useful as a protein antigen for obtaining an antibody. Usually such molecules are associated with only small structural changes. Such large-scale structural changes to a universal molecule of the same function can have serious adverse effects on the survival of living organisms.
クラミジア病原体を検出する商業的に利用可能なモノクローナル抗体の数はごく僅かであり、十分というには程遠い。最近までTWAR株のみが肺炎の原因菌であると信じられてきた(Thom and Grayston 1991 ; United States Patent No.5,008,186)。最近になって、いくつかのクラミジア病原体の血清型が報告された。LPSまたはMOMPは菌株によって異なり、また1つの血清型に対する抗体では全てをカバーできない状況である。 The number of commercially available monoclonal antibodies that detect Chlamydia pathogens is very small and far from sufficient. Until recently, it was believed that only the TWAR strain was the causative agent of pneumonia (Thom and Grayston 1991; United States Patent No. 5,008,186). Recently, several serotypes of Chlamydia pathogens have been reported. LPS or MOMP varies depending on the strain, and antibodies against one serotype cannot cover all.
本発明は、上記のような課題を解決するためになされたものである。すなわち、本発明は、Chlamydia pneumoniaeに属する微生物を特異的、かつ高感度に迅速に検出する方法、その検出に用いる検出用抗体、検出用試薬キットを提供することを課題とする。さらに、本発明は、その検出に用いる検出用抗体の製造方法を提供することを課題とする。 The present invention has been made to solve the above problems. That is, an object of the present invention is to provide a method for rapidly and specifically detecting a microorganism belonging to Chlamydia pneumoniae with high sensitivity, a detection antibody used for the detection, and a detection reagent kit. Another object of the present invention is to provide a method for producing a detection antibody used for the detection.
本発明者等は、全ての微生物において同一の機能が保存されている蛋白質を有用な抗原蛋白質として見出した。通常、このような蛋白質の構造変化はきわめて少ないと予想される。しかし驚くべきことに、該蛋白質に対する抗体は、微生物の種あるいは属特異的であり、該蛋白質に対する抗体は、微生物の種あるいは属特異的な識別に用いることが可能な多様性を持つとともに、対象となる微生物についてはその全ての血清型を検出しうるものであることが見出されたのである。 The present inventors have found a protein in which the same function is conserved in all microorganisms as a useful antigen protein. Usually, such structural changes in proteins are expected to be extremely small. Surprisingly, however, antibodies to the protein are species- or genus-specific for microorganisms, and antibodies to the protein have diversity that can be used for species- or genus-specific identification of microorganisms and It has been found that all the serotypes of the microorganisms to be detected can be detected.
本発明者らは全ての微生物細胞に同一機能の分子として存在し、しかもそのアミノ酸構造が微生物間である程度の相違点をもつ細胞内分子、特にリボソーム蛋白質の一種であるRibosomal Protein L7/L12蛋白質に着目した。Ribosomal Protein L7/L12蛋白質は分子量約13キロダルトンの蛋白質であり、蛋白質合成に必須のリボソーム蛋白質として存在することが知られている。特にChlamydia pneumoniaeを含むいくつかの微生物ではRibosomal Protein L7/L12蛋白質の全アミノ酸配列が解析されている。 The present inventors present an intracellular molecule that exists in all microbial cells as a molecule having the same function, and its amino acid structure has some differences between microorganisms, particularly Ribosomal Protein L7/L12 protein which is a kind of ribosomal protein. I paid attention. Ribosomal Protein L7/L12 protein is a protein having a molecular weight of about 13 kilodaltons, and is known to exist as a ribosomal protein essential for protein synthesis. Especially in some microorganisms including Chlamydia pneumoniae , the entire amino acid sequence of Ribosomal Protein L7/L12 protein has been analyzed.
本発明者らはこの分子が微生物間で類似しているにもかかわらずその一部に各微生物固有の構造部分を持つことに着目し、このChlamydia pneumoniae のRibosomal Protein L7/L12蛋白質に対する抗体を利用することで様々な微生物、細菌の種特異的でかつ全ての同一菌種内の血清型について検出が可能であることを見出した。 The present inventors have focused on the fact that this molecule has a structural part unique to each microorganism in spite of the fact that the molecule is similar among microorganisms, and an antibody against the Ribosomal Protein L7/L12 protein of Chlamydia pneumoniae is used It was found that by doing so, it is possible to detect various microorganisms and bacteria species-specifically and to detect serotypes in all the same bacterial species.
本発明者等はChlamydia pneumoniaeの該蛋白質に特異的な抗体が得られること、および該抗体を用いることによりChlamydia pneumoniaeの特異的な検出が可能であることを見出し、本発明を完成した。 The present inventors have completed the present invention by finding that an antibody specific to the protein of Chlamydia pneumoniae can be obtained and that the specific detection of Chlamydia pneumoniae can be performed by using the antibody.
本発明により、Chlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質に対して特異的なモノクローナル抗体が見出され、開発された。この抗体は新規であり、従来公知のいかなる抗体とも異なり、上記蛋白質と特異的に反応する性質を有する。 According to the present invention, a monoclonal antibody specific to the Ribosomal Protein L7/L12 protein of Chlamydia pneumoniae was found and developed. This antibody is novel, and unlike any conventionally known antibody, has the property of reacting specifically with the above protein.
配列表において配列番号１及び２はChlamydia pneumoniaeのRibosomal Protein L7/L12遺伝子のDNA配列(NCBI database accession ♯NC♯000922)及び対応するアミノ酸配列(NCBI database accession ♯AE001593.1, NCBI data base)である。なお、配列表に記載されたアミノ酸配列の左端および右端はそれぞれアミノ基末端（以下、N末端）およびカルボキシル基末端（以下、C末端）であり、また塩基配列の左端および右端はそれぞれ5'末端および3'末端である。クロスマッチテストのアミノ酸配列はアミノ酸１文字略字によって記している。また、クロスマッチテストにおける「+」の表記は、異なるアミノ酸であるが、疎水性などの性質が類縁のアミノ酸であること、「 」のブランクは性質も含めて全く異なるアミノ酸であることを示す。また、本発明で述べられる遺伝子操作の一連の分子生物学的な実験は通常の実験書の記載方法によって行うことができる。前記の通常の実験書としては、例えばMolecular Cloning, A laboratory manual, Cold Spring Harber Laboratory Press, Sambrook, J.ら(1989)を挙げることができる。
In the sequence listing, SEQ ID NOs: 1 and 2 are the DNA sequence (NCBI database accession #NC#000922) and the corresponding amino acid sequence (NCBI database accession #AE001593.1, NCBI data base) of the Ribosomal Protein L7/L12 gene of Chlamydia pneumoniae. .. The left and right ends of the amino acid sequences shown in the sequence listing are amino group terminals (hereinafter, N terminal) and carboxyl group terminals (hereinafter, C terminal), respectively, and the left and right ends of the nucleotide sequences are 5'terminals, respectively. And 3'end. The amino acid sequence of the cross-match test is indicated by the one-letter amino acid abbreviation. Further, the notation "+" in the cross-match test indicates that the amino acids are different amino acids, but the amino acids have similar properties such as hydrophobicity, and the blank "" indicates completely different amino acids including the properties. In addition, a series of molecular biology experiments of gene manipulation described in the present invention can be carried out by a method described in a usual experiment manual. Examples of the above-mentioned usual experiment manual include Molecular Cloning, A laboratory manual, Cold Spring Harber Laboratory Press, Sambrook, J. et al. (1989).
本発明において、″微生物と特異的に反応する抗体″とは、微生物の種あるいは属に特異的に反応する抗体をさすが、微生物感染症の診断においては微生物の種に特異的に反応する抗体が特に有用となる。 In the present invention, the "antibody that specifically reacts with a microorganism" refers to an antibody that specifically reacts with the species or genus of a microorganism, but in the diagnosis of microbial infection, an antibody that specifically reacts with the species of the microorganism is Especially useful.
本発明において抗体は、ポリクローナル抗体またはモノクローナル抗体を指し、Ribosomal Protein L7/L12蛋白質の全長あるいはその部分ペプチドを用いて作成することができる。抗体を作成するためのペプチドの長さは特に限定されないがRibosomal Protein L7/L12蛋白質に対する抗体の場合、この蛋白質を特徴づけられる長さがあれば良く、好ましくは5アミノ酸以上、特に好ましくは8アミノ酸以上のペプチドを用いれば良い。 In the present invention, the antibody refers to a polyclonal antibody or a monoclonal antibody, and can be prepared using the full length Ribosomal Protein L7/L12 protein or a partial peptide thereof. The length of the peptide for producing the antibody is not particularly limited, but in the case of an antibody against the Ribosomal Protein L7/L12 protein, the protein may have a length that characterizes this protein, preferably 5 amino acids or more, particularly preferably 8 amino acids. The above peptides may be used.
このペプチドあるいは全長蛋白質をそのまま、またはKLH(keyhole-limpet hemocyanin)やBSA(bovine serum albumin)といったキャリア蛋白質と架橋した後必要に応じてアジュバントとともに動物へ接種せしめ、その血清を回収することでRibosomal Protein L7/L12蛋白質を認識する抗体（ポリクローナル抗体）を含む抗血清を得ることができる。また抗血清より抗体を精製して使用することもできる。接種する動物としてはヒツジ、ウマ、ヤギ、ウサギ、マウス、ラット等であり、特にポリクローナル抗体作製にはヒツジ、ウサギなどが好ましい。また、ハイブリドーマ細胞を作製する公知の方法によりモノクローナル抗体を得ることも可能であるが、この場合はマウスが好ましい。 This peptide or full-length protein as it is, or after cross-linking with a carrier protein such as KLH (keyhole-limpet hemocyanin) or BSA (bovine serum albumin) is inoculated into an animal with an adjuvant as necessary, and the serum is collected to collect the Ribosomal Protein. An antiserum containing an antibody (polyclonal antibody) that recognizes the L7/L12 protein can be obtained. Alternatively, the antibody may be purified from the antiserum and used. The animals to be inoculated are sheep, horses, goats, rabbits, mice, rats and the like, and sheep and rabbits are particularly preferable for producing polyclonal antibodies. It is also possible to obtain a monoclonal antibody by a known method for producing hybridoma cells, but in this case, a mouse is preferable.
また該蛋白質の全長または5残基以上、望ましくは8残基以上のアミノ酸配列をグルタチオンＳ−トランスフェラーゼ（GST）などとフュージョン蛋白質としたものを精製して、または未精製のまま、抗原として用いることもできる。成書(Antibodies a laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory)に示された各種の方法ならびに遺伝子クローニング法などにより分離されたイムノグロブリン遺伝子を用いて培養した細胞に発現させた遺伝子組み換え抗体によっても作製することができる。 In addition, the full-length or 5 or more residues, preferably 8 or more residues of amino acid sequence of which is a fusion protein with glutathione S-transferase (GST) or the like is purified or used as an antigen without purification. You can also Genes expressed in cells cultured using immunoglobulin genes isolated by various methods and gene cloning methods described in the published literature (Antibodies a laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory) It can also be produced by a recombinant antibody.
本発明のマーカー抗原として用いることができるRibosomal Protein L7/L12蛋白質に対する抗体は、以下の方法あるいはその他の類似の方法によって取得することができるがこれらの方法に限定されるものではない。
ａ）Ribosomal Protein L7/L12蛋白質の遺伝子配列およびアミノ酸配列が既知の微生物については、他の微生物における該蛋白質のアミノ酸配列との類似性の少ない領域についてペプチド断片を合成し、それを免疫原としてポリクローナル抗体、あるいはモノクローナル抗体を作製することにより目的の抗体を取得することができる。
The antibody against Ribosomal Protein L7/L12 protein that can be used as the marker antigen of the present invention can be obtained by the following method or other similar methods, but is not limited to these methods.
a) For microorganisms in which the gene sequence and amino acid sequence of Ribosomal Protein L7/L12 protein are known, a peptide fragment is synthesized for a region having a low similarity to the amino acid sequence of the protein in other microorganisms, and the fragment is used as an immunogen for polyclonal production. The desired antibody can be obtained by producing an antibody or a monoclonal antibody.
また、既知の該遺伝子の両端部位におけるDNA配列をプローブとしたPCR手法による遺伝子増幅、相同部分配列を鋳型プローブとしたハイブリダイゼーション法など通常の遺伝子操作手法を用いることにより該遺伝子の全長配列を取得することができる。 In addition, the full-length sequence of the gene is obtained by using a normal gene manipulation method such as gene amplification by PCR method using a DNA sequence at both ends of the known gene as a probe, or hybridization method using a homologous partial sequence as a template probe. can do.
その後他の蛋白質遺伝子とのフュージョン遺伝子などを構築し、大腸菌等を宿主として公知の遺伝子導入手法により宿主内に該当フュージョン遺伝子を挿入し大量に発現させた後にフュージョン蛋白質として用いた蛋白質に対する抗体アフィニティカラム法などにより発現蛋白質を精製することにより目的とする蛋白質抗原を取得することができる。この場合Ribosomal Protein L7/L12の全長蛋白質が抗原となるため微生物間で保存されているアミノ酸部分に対する抗体を取得しても本発明の目的に合致しない。従って、本法によって取得した抗原に対しては公知の手法によりモノクローナル抗体を産生するハイブリドーマを取得し、該当する微生物とのみ反応する抗体を産生するクローンを選択することにより目的の抗体を取得することができる。
ｂ）Ribosomal Protein L7/L12蛋白質のアミノ酸配列が未知の微生物については１つにはRibosomal Protein L7/L12蛋白質のアミノ酸配列が菌種間で50〜60%相同であることにより、そのアミノ酸配列の相同部分の配列を基にしてPCR法による特定配列部分の遺伝子増幅や相同部分配列を鋳型プローブとしたハイブリダイゼーション法など通常の遺伝子操作手法を用いることにより該蛋白質遺伝子を容易に取得することができる。
After that, a fusion gene or the like with other protein genes is constructed, and the fusion gene is inserted into the host by a known gene introduction method using Escherichia coli or the like as a host and a large amount is expressed, and then an antibody affinity column for the protein used as the fusion protein The target protein antigen can be obtained by purifying the expressed protein by a method or the like. In this case, since the full-length protein of Ribosomal Protein L7/L12 serves as an antigen, obtaining the antibody against the amino acid portion conserved among microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, a hybridoma producing a monoclonal antibody is obtained by a known method, and a target antibody is obtained by selecting a clone producing an antibody that reacts only with the corresponding microorganism. You can
b) For microorganisms in which the amino acid sequence of the Ribosomal Protein L7/L12 protein is unknown, one is that the amino acid sequence of the Ribosomal Protein L7/L12 protein is 50-60% homologous between bacterial strains. The protein gene can be easily obtained by using an ordinary gene manipulation method such as gene amplification of a specific sequence portion by PCR method based on the partial sequence or hybridization method using a homologous partial sequence as a template probe.
その後他の蛋白質遺伝子とのフュージョン遺伝子などを構築し、大腸菌等を宿主として公知の遺伝子導入手法により宿主内に該当フュージョン遺伝子を挿入し大量に発現させた後にフュージョン蛋白質として用いた蛋白質に対する抗体アフィニティカラム法などにより発現蛋白質を精製することにより目的とする蛋白質抗原を取得することができる。この場合Ribosomal Protein L7/L12の全長蛋白質が抗原となるため微生物間で保存されているアミノ酸部分に対する抗体を取得しても本発明の目的に合致しない。従って、本法によって取得した抗原に対しては公知の手法によりモノクローナル抗体を産生するハイブリドーマを取得し、該当する微生物とのみ反応する抗体を産生するクローンを選択することにより目的の抗体を取得することができる。
ｃ）あるいはRibosomal Protein L7/L12蛋白質のアミノ酸配列が未知な場合の別な方法として、既知のRibosomal Protein L7/L12蛋白質のアミノ酸配列のうち微生物間で保存されている共通配列部分に相当する5〜30アミノ酸の合成ペプチドを作製し、そのペプチド配列に対し公知の方法でポリクローナル抗体あるいはモノクローナル抗体を作製する。該抗体を用いたアフィニティカラムクロマトによって目的の微生物細胞破砕液を精製することにより高度に精製されたRibosomal Protein L7/L12蛋白質を取得することができる。
After that, a fusion gene or the like with other protein genes is constructed, and the fusion gene is inserted into the host by a known gene introduction method using Escherichia coli or the like as a host and a large amount is expressed, and then an antibody affinity column for the protein used as the fusion protein The target protein antigen can be obtained by purifying the expressed protein by a method or the like. In this case, since the full-length protein of Ribosomal Protein L7/L12 serves as an antigen, obtaining the antibody against the amino acid portion conserved among microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, a hybridoma producing a monoclonal antibody is obtained by a known method, and a target antibody is obtained by selecting a clone producing an antibody that reacts only with the corresponding microorganism. You can
c) Alternatively, as another method when the amino acid sequence of Ribosomal Protein L7/L12 protein is unknown, 5 ~ corresponding to the common sequence portion conserved among microorganisms among known amino acid sequences of Ribosomal Protein L7/L12 protein A synthetic peptide of 30 amino acids is prepared, and a polyclonal antibody or a monoclonal antibody is prepared for the peptide sequence by a known method. A highly purified Ribosomal Protein L7/L12 protein can be obtained by purifying the target microbial cell disruption solution by affinity column chromatography using the antibody.
蛋白質の精製度が不足している場合は公知の精製手法であるイオン交換クロマトグラフィー、疎水クロマトグラフィー、ゲル濾過などの手法により精製したのち作製した抗体によるウェスタンブロットなどの方法によりRibosomal Protein L7/L12蛋白質の溶出画分を同定し精製蛋白質を得ることができる。得られた精製Ribosomal Protein L7/L12蛋白質抗原を基にして公知の方法によりハイブリドーマを取得し、目的の微生物に特異的に反応するハイブリドーマを選択することにより目的の抗体を取得することができる。 If the degree of purification of the protein is insufficient, Ribosomal Protein L7/L12 can be purified by a known purification method such as ion exchange chromatography, hydrophobic chromatography, gel filtration, etc., followed by Western blotting with the produced antibody. The elution fraction of the protein can be identified to obtain a purified protein. Based on the obtained purified Ribosomal Protein L7/L12 protein antigen, a hybridoma can be obtained by a known method, and a target antibody can be obtained by selecting a hybridoma that specifically reacts with a target microorganism.
上記の方法ａ）、ｂ）およびｃ）によって得られた、種々の微生物に特異的な本発明の抗体は、微生物に特異的な各種の診断試薬およびキットを利用する、種々の免疫学的分析法に用いることができる。例えば、この抗体は、公知の測定手法であるポリスチレンラテックス粒子上に該抗体を吸着させた凝集反応、マイクロタイタープレート中で行う公知技術であるELISA法、既存のイムノクロマト法、着色粒子もしくは発色能を有する粒子、または酵素もしくは蛍光体でラベルされた該抗体とともに捕捉(capture)抗体で被覆した磁気微粒子などを用いるサンドイッチアッセイなど既知の全ての免疫測定手法に利用できる。 The antibodies of the present invention specific to various microorganisms obtained by the above-mentioned methods a), b) and c) can be used in various immunological analyzes utilizing various diagnostic reagents and kits specific to microorganisms. Can be used in law. For example, this antibody is an agglutination reaction in which the antibody is adsorbed on polystyrene latex particles which is a known measurement method, an ELISA method which is a known technique performed in a microtiter plate, an existing immunochromatography method, colored particles or a coloring ability. It can be used for all known immunoassay techniques such as a sandwich assay using particles having the same or a magnetic fine particle coated with a capture antibody together with the antibody labeled with an enzyme or a fluorophore.
抗体を用いる微生物診断方法とは、ポリスチレンラテックス粒子上に該抗体を吸着させた凝集反応、マイクロタイタープレート中で行う公知技術であるELISA法、既存のイムノクロマト法、着色粒子もしくは発色能を有する粒子、または酵素もしくは蛍光体でラベルされた該抗体とともに捕捉抗体で被覆した磁気微粒子などを用いるサンドイッチアッセイなど既知の全ての免疫測定手法を利用する診断方法を意味する。 Microbial diagnostic method using an antibody is an agglutination reaction in which the antibody is adsorbed on polystyrene latex particles, an ELISA method which is a known technique performed in a microtiter plate, an existing immunochromatographic method, a particle having a coloring particle or a coloring ability, Alternatively, it means a diagnostic method utilizing all known immunoassay techniques such as a sandwich assay using magnetic fine particles coated with a capture antibody together with the antibody labeled with an enzyme or a fluorescent substance.
また、特に抗体を用いる有用な微生物診断方法として特表平７−５０９５６５号公報に記載されているシリコン、窒化珪素などにより形成された光学薄膜上で抗体反応をおこない光干渉原理等により検出するいわゆるオプティカルイムノアッセイ(OIA, Optical Immunoassay)などが高感度な診断方法として有用である。 In addition, as a useful method for diagnosing microorganisms particularly using an antibody, so-called detection by the principle of light interference by causing an antibody reaction on an optical thin film formed of silicon, silicon nitride or the like described in JP-A-7-509565. Optical immunoassays (OIA, Optical Immunoassay) and the like are useful as highly sensitive diagnostic methods.
また該検出方法において必要となる微生物からの細胞内マーカー抗原の抽出方法としては、TritonX-100, Tween-20をはじめとする種々の界面活性剤を用いた抽出試薬による処理法、適当なプロテアーゼなどの酵素を用いる酵素処理法、物理的方法による微生物細胞の破砕をはじめ既知の細胞構造の破砕手法が用いられる。界面活性剤等の組み合わせにより微生物ごとに試薬による最適な抽出条件を設定することが望ましい。 Further, as a method for extracting an intracellular marker antigen from a microorganism required in the detection method, a treatment method with an extraction reagent using various surfactants such as TritonX-100 and Tween-20, an appropriate protease, etc. Known cell disruption techniques, including disruption of microbial cells by an enzymatic treatment method using an enzyme and a physical method, are used. It is desirable to set the optimum extraction conditions with reagents for each microorganism by combining surfactants and the like.
また、本発明における、抗体を用いる微生物検出用試薬キットとは、当該検出方法を用いた検出用試薬キットに相当する。 The reagent kit for detecting a microorganism using an antibody in the present invention corresponds to a reagent kit for detection using the detection method.
Chlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質のアミノ酸配列及びDNA配列を配列表配列番号：１及び２に示す。従って、この微生物の場合はRibosomal Protein L7/L12蛋白質のアミノ酸配列を、配列表に「クロスマッチ」と記した類似した微生物の同種の蛋白質と比較することが可能である。相同性の低いセグメントのペプチドを合成し、これに対するポリクローナルあるいはモノクローナル抗体を作成することは、微生物に対する特異性を有するものの選択を省略することを可能とする。 The amino acid sequence and DNA sequence of the Ribosomal Protein L7/L12 protein of Chlamydia pneumoniae are shown in SEQ ID NOs: 1 and 2 in the Sequence Listing. Therefore, in the case of this microorganism, it is possible to compare the amino acid sequence of the Ribosomal Protein L7/L12 protein with a homologous protein of a similar microorganism described as "cross match" in the sequence listing. By synthesizing a peptide of a segment with low homology and producing a polyclonal or monoclonal antibody against it, it becomes possible to omit selection of those having specificity to microorganisms.
特にポリクローナル抗体の場合、免疫した動物の抗血清をProtein Aカラム等で精製しIgG画分を取得したのち、さらに動物の免疫に用いた合成ペプチドによるアフィニティ精製を実施することが望ましい。 In the case of a polyclonal antibody in particular, it is desirable to purify the antiserum of the immunized animal using a Protein A column or the like to obtain an IgG fraction, and then further perform affinity purification using the synthetic peptide used for immunizing the animal.
更に、当該微生物のRibosomal Protein L7/L12蛋白質のDNA配列からN末端とC末端の配列に基づいてPCRプライマーが作成された。このPCRプライマーの相同性を利用して、ゲノムDNAを用いてPCR法によりDNA断片を増幅させ、これを抽出し、常法によりChlamydia pneumoniaeのRibosomal Protein L7/L12遺伝子の断片を取得することができる。Chlamydia pneumoniaeのRibosomal Protein L7/L12遺伝子の全長はこれらの断片のDNA配列情報を分析することにより知ることができる。 Further, PCR primers were prepared from the DNA sequence of Ribosomal Protein L7/L12 protein of the microorganism based on the N-terminal and C-terminal sequences. By utilizing the homology of this PCR primer, a DNA fragment is amplified by a PCR method using genomic DNA, this is extracted, and a Ribosomal Protein L7/L12 gene fragment of Chlamydia pneumoniae can be obtained by a conventional method. .. The full length of the Ribosomal Protein L7/L12 gene of Chlamydia pneumoniae can be known by analyzing the DNA sequence information of these fragments.
取得したChlamydia pneumoniaeのRibosomal Protein L7/L12遺伝子は、例えばGSTなどとフュージョン蛋白質遺伝子を構成し、適当な発現用プラスミドを用いて発現ベクターを構築後、大腸菌等を形質転換して該蛋白質を大量発現させうる。形質転換した大腸菌を適当量培養し、菌体破砕液をGSTを用いたアフィニティカラムで精製することにより、Chlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質とGSTのフュージョン蛋白質が得られる。 The obtained Ribosomal Protein L7/L12 gene of Chlamydia pneumoniae constitutes a fusion protein gene with, for example, GST and the like, and after constructing an expression vector using an appropriate expression plasmid, transforming E. coli etc. and expressing the protein in large quantities. Can be done. By culturing an appropriate amount of transformed Escherichia coli and purifying the disrupted cell suspension with an affinity column using GST, the Ribosomal Protein L7/L12 protein of Chlamydia pneumoniae and the fusion protein of GST can be obtained.
この蛋白質をそのまま、あるいはGST部分を切断後、抗原蛋白質として公知の手法により、複数のハイブリドーマクローンを確立し、Chlamydia pneumoniae菌体あるいは菌体破砕液またはChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質に特異的な反応を示す抗体を選択することにより目的の特異的モノクローナル抗体を取得することも可能である。 This protein as it is, or after cleaving the GST portion, by a method known as an antigen protein, a plurality of hybridoma clones were established, and specific to the Chlamydia pneumoniae bacterial cells or cell disruption solution or Chlamydia pneumoniae Ribosomal Protein L7/L12 protein. It is also possible to obtain the target specific monoclonal antibody by selecting an antibody that exhibits various reactions.
本発明に基づき作製された抗体は、公知の測定手法であるポリスチレンラテックス粒子上に該抗体を吸着させた凝集反応、マイクロタイタープレート中で行う公知技術であるELISA法、既存のイムノクロマト法、着色粒子もしくは発色能を有する粒子、または酵素もしくは蛍光体でラベルされた該抗体とともに捕捉抗体で被覆した磁気微粒子などを用いるサンドイッチアッセイなど既知の全ての免疫測定手法に利用できる。 The antibody produced according to the present invention is an agglutination reaction in which the antibody is adsorbed on polystyrene latex particles which is a known measurement method, an ELISA method which is a known technique performed in a microtiter plate, an existing immunochromatography method, and colored particles. Alternatively, it can be used for all known immunoassay techniques such as a sandwich assay using particles having a coloring ability, or magnetic fine particles coated with a capture antibody together with the antibody labeled with an enzyme or a fluorescent substance.
また、本発明に基づき作製された抗体は全ての免疫測定手法において当該抗原蛋白質を固相あるいは液相中で捕獲するいわゆる捕捉抗体として機能しうると同時にパーオキシダーゼやアルカリフォスファターゼなどの酵素を公知の方法により修飾していわゆる酵素標識抗体とすることにより、検出用抗体としても機能しうる。 Further, the antibody produced according to the present invention can function as a so-called capture antibody that captures the antigen protein in a solid phase or a liquid phase in all immunoassay methods, and at the same time, known enzymes such as peroxidase and alkaline phosphatase are known. When modified by a method to obtain a so-called enzyme-labeled antibody, it can also function as a detection antibody.
以下の例は本発明を具体的に説明するためのものであって本発明について何らその範囲を限定するものではない。 The following examples are for specifically explaining the present invention and are not intended to limit the scope of the present invention.
Chlamydia pneumoniaeからのRibosomal Protein L7/L12遺伝子のクローニング
Chlamydia pneumoniae(ATCC VR-1310、ATCCから分譲、購入)をHL細胞のモノレイヤー上で培養した。Chlamydia pneumoniaeの詳細な培養方法はKuo らなど(Cles and Stamm 1990 ; Kuo and Grayston 1990 ; Yoshizawa, Dairiki et al. 1992)の記載に従った。CO2インキュベータ内にて37℃、5% CO2の条件下で微生物を5日間培養した。感染した細胞を遠心分離により集め、最終的に5×107個/ml前後の濃度となるようにTE緩衝液（和光純薬工業製）に懸濁した。この懸濁液約1.5mlを微量遠心チューブに移し取り10,000rpmで2分間遠心した。
Cloning of Ribosomal Protein L7/L12 gene from Chlamydia pneumoniae
Chlamydia pneumoniae (ATCC VR-1310, purchased from ATCC, purchased) was cultured on a monolayer of HL cells. The detailed culturing method of Chlamydia pneumoniae was as described by Kuo et al. (Cles and Stamm 1990; Kuo and Grayston 1990; Yoshizawa, Dairiki et al. 1992). The microorganism was cultured for 5 days in a CO 2 incubator under the conditions of 37° C. and 5% CO 2 . The infected cells were collected by centrifugation and suspended in TE buffer (Wako Pure Chemical Industries, Ltd.) so that the final concentration was about 5×10 7 cells/ml. About 1.5 ml of this suspension was transferred to a microcentrifuge tube and centrifuged at 10,000 rpm for 2 minutes.
上澄み液を棄てた。沈殿部分を567μlのTE緩衝液に再懸濁した。さらに30μlの10%SDSと3μlの20mg/mlのProteinaseK溶液を加えて良く混合し、37℃で1時間インキュベートした。懸濁液を56℃で更に1時間インキュベートした。次に10%のセチルトリメチルアンモニウムブロマイド/0.7M NaCl溶液を80μl加え、よく混合したのち65℃で10分間インキュベートした。同一体積の24:1のクロロホルム−イソアミルアルコール混合液を700μl加えよく攪拌した。 The supernatant was discarded. The precipitated portion was resuspended in 567 μl of TE buffer. Further, 30 μl of 10% SDS and 3 μl of 20 mg/ml Proteinase K solution were added, mixed well, and incubated at 37° C. for 1 hour. The suspension was incubated at 56°C for another hour. Next, 80 μl of 10% cetyltrimethylammonium bromide/0.7 M NaCl solution was added, mixed well, and then incubated at 65° C. for 10 minutes. The same volume of 24:1 chloroform-isoamyl alcohol mixed solution (700 μl) was added and stirred well.
この溶液を微量遠心機で12,000rpm、5分間、4℃で遠心処理したのち、水層画分を新しい微量遠心管に移した。そこに0.6倍量のイソプロパノールを加えチューブをよく振ってDNAの沈殿を形成した。白いDNA沈殿をガラス棒ですくって1mlの70%エタノール（-20℃に冷却したもの）が入った別の微量遠心管に移した。その後チューブを10,000rpmで5分間遠心し、上清を静かに除去した。1mlの70%エタノールを追加し、混合物を更に5分間遠心した。
再び上澄みを除去したのち沈殿を100μlのTE緩衝液に溶解しDNA溶液を得た。このゲノムDNA溶液の濃度をMolecular Cloning, A laboratory manual, 1989, Eds. Sambrook, J., Fritsch, E. F., and Maniatis, T., Cold Spring harbor Laboratory PressのE5, Spectrophotometric Determination of the Amount of DNA or RNAに従って定量した。
このゲノムDNAのうち10ngを用いてPCR(polymerase chain reaction)を行った。PCRはTaqポリメラーゼ（宝酒造社製、コードR001A）を用いた。酵素に添付の緩衝液5μl、酵素に添付のdNTP混合物4μlおよび各200pmolのオリゴヌクレオチド（配列表配列番号：３および４に示すもの）を酵素に加えた。全体の容量が50μlとなるように精製水を加えた。
This solution was centrifuged at 12,000 rpm for 5 minutes at 4°C in a microcentrifuge, and then the aqueous layer fraction was transferred to a new microcentrifuge tube. A 0.6-fold amount of isopropanol was added thereto, and the tube was shaken well to form a DNA precipitate. The white DNA precipitate was scooped with a glass rod and transferred to another microcentrifuge tube containing 1 ml of 70% ethanol (cooled to -20°C). The tube was then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was gently removed. 1 ml 70% ethanol was added and the mixture was centrifuged for a further 5 minutes.
After removing the supernatant again, the precipitate was dissolved in 100 μl of TE buffer to obtain a DNA solution. The concentration of this genomic DNA solution was changed to Molecular Cloning, A laboratory manual, 1989, Eds. Sambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring harbor Laboratory Press E5, Spectrophotometric Determination of the Amount of DNA or RNA. Quantified according to.
PCR (polymerase chain reaction) was performed using 10 ng of this genomic DNA. For PCR, Taq polymerase (Takara Shuzo, code R001A) was used. 5 μl of the buffer solution attached to the enzyme, 4 μl of the dNTP mixture attached to the enzyme and 200 pmol of each oligonucleotide (shown in SEQ ID NOs: 3 and 4 in the Sequence Listing) were added to the enzyme. Purified water was added so that the total volume was 50 μl.
この混合物を、TaKaRa PCR Thermal Cycler 480を用いて、95℃1分、50℃2分、72℃3分を5サイクル行ったのち、95℃1分、60℃2分、72℃3分を25サイクル行った。このPCR生成物の一部を用いて、1.5%アガロースゲル中にて電気泳動を実施した。エチジウムブロマイド（日本ジーン社製）にて染色後、紫外線下で観察し、約400bpのDNAが増幅されていることを確認した。制限酵素BamHIおよびXhoIを用いて切断後、1.5%アガロースゲル中にて電気泳動とエチジウムブロマイドによる染色を行った。ゲルから約400bpのバンドを切り取った。このバンドをSuprec01（宝酒造株式会社製）で精製し、一般的なベクターであるpGEX-6P-1(Pharmacia社製)に挿入した。同ベクターは目的の遺伝子断片を適当な制限酵素サイトに組み込むことによりGST蛋白質とのフュージョン蛋白質を発現しうる目的分子の発現ベクターとして機能することができる。 This mixture was subjected to TaKaRa PCR Thermal Cycler 480 for 5 cycles of 95°C for 1 minute, 50°C for 2 minutes, and 72°C for 3 minutes, and then 95°C for 1 minute, 60°C for 2 minutes, and 72°C for 3 minutes. Went cycle. A portion of this PCR product was used to perform electrophoresis in a 1.5% agarose gel. After staining with ethidium bromide (manufactured by Nippon Gene Co., Ltd.), it was observed under ultraviolet light to confirm that about 400 bp of DNA was amplified. After digestion with restriction enzymes BamHI and XhoI, electrophoresis was performed in 1.5% agarose gel and staining with ethidium bromide was performed. A band of approximately 400 bp was excised from the gel. This band was purified with Suprec01 (Takara Shuzo Co., Ltd.) and inserted into pGEX-6P-1 (Pharmacia), which is a general vector. The vector can function as an expression vector for a target molecule capable of expressing a fusion protein with the GST protein by incorporating the target gene fragment into an appropriate restriction enzyme site.
具体的にはベクターpGEX-6P-1と先のDNAとをそのモル比が1:3となるように混ぜ合わせて、T4 DNAリガーゼ(Invitrogen社製)にてベクターにDNAを組み込んだ。DNAを組み込んだベクターpGEX-6P-1は大腸菌のワンショットコンピテントセルに遺伝子学的手法により導入し、ついで50μg/mlのアンピシリン（シグマ社）を含む半固体状の培養プレートであるLB L-ブロス寒天（宝酒造株式会社製）に接種した。プレートを37℃で12時間インキュベートし、成長したコロニーを無差別に選択し、同じ濃度のアンピシリンを含むL-ブロス培養液に接種した。37℃で8時間振とう培養・集菌後、Wizard Miniprepを用い、添付の説明書に従ってプラスミドを分離した。プラスミドは制限酵素BamHI/XhoIにて切断処理した。約370bpのDNAを切断することによってPCR生成物の挿入を確認した。挿入されたDNAの塩基配列を上記クローンを用いて決定した。 Specifically, the vector pGEX-6P-1 and the above DNA were mixed at a molar ratio of 1:3, and the DNA was incorporated into the vector using T4 DNA ligase (Invitrogen). The vector pGEX-6P-1 incorporating the DNA was introduced into one-shot competent cells of Escherichia coli by a genetic method, and then a semisolid culture plate containing 50 μg/ml ampicillin (Sigma) LBL-. Broth agar (Takara Shuzo Co., Ltd.) was inoculated. Plates were incubated for 12 hours at 37° C., grown colonies were indiscriminately selected and inoculated into L-broth medium containing the same concentration of ampicillin. After shaking culture and collection at 37° C. for 8 hours, the plasmid was isolated using Wizard Miniprep according to the attached instruction. The plasmid was digested with the restriction enzymes BamHI/XhoI. Insertion of the PCR product was confirmed by cutting approximately 370 bp of DNA. The nucleotide sequence of the inserted DNA was determined using the above clone.
挿入DNA断片の塩基配列の決定は、Applied Biosystems社製の蛍光シークエンサーを用いて実施した。 The nucleotide sequence of the inserted DNA fragment was determined using a fluorescent sequencer manufactured by Applied Biosystems.
シークエンスサンプルの調製はPRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit(Applied Biosystems社製)を用いて行った。先ず、9.5μlの反応液、4.0μlの0.8pmol/μlのT7プロモータープライマー(Gibco BRL)、および6.5μlの0.16μg/μlテンプレートDNAを0.5mlのマイクロチューブに加え、混合した。混合物を2層の100μl鉱油で覆ったのち、25サイクルPCR増幅処理を行った。ここで、1サイクルは、96℃での30秒間の処理、55℃での15秒間の処理、および60℃での4分間の処理からなる。生成物を4℃で5分間保持した。反応終了後、80μlの無菌精製水を加え、攪拌した。生成物を遠心分離し、水層をフェノールークロロホルム混合液で３回抽出した。10μlの3M酢酸ナトリウムpH5.2と300μlのエタノールを100μlの水層に加え、攪拌した。その後14,000rpm、室温で15分間遠心し、沈殿を回収した。沈殿を75%エタノールで洗浄後、真空下に2分間静置して乾燥させ、シークエンス用サンプルとした。シークエンスサンプルは、4μlの10mMのEDTAを含むホルムアミドに溶解して90℃で2分間変性した。このものは氷中で冷却してシークエンスに供した。
無差別に選択した5個のクローンのうち2個はPCRに用いたプローブと配列上の相同性を有していた。また、Ribosomal Protein L7/L12蛋白質の遺伝子配列と一致したDNA配列が明白であった。その構造遺伝子部分の全塩基配列及び対応するアミノ酸配列は配列表配列番号：１及び２に示すような配列であった。この遺伝子断片は、明らかにChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質の遺伝子をコードするものである。
The sequence sample was prepared using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems). First, 9.5 μl reaction solution, 4.0 μl 0.8 pmol/μl T7 promoter primer (Gibco BRL), and 6.5 μl 0.16 μg/μl template DNA were added to a 0.5 ml microtube and mixed. The mixture was covered with two layers of 100 μl mineral oil before 25 cycles of PCR amplification. Here, one cycle consists of treatment at 96°C for 30 seconds, treatment at 55°C for 15 seconds, and treatment at 60°C for 4 minutes. The product was held at 4°C for 5 minutes. After the reaction was completed, 80 μl of sterile purified water was added and stirred. The product was centrifuged and the aqueous layer was extracted 3 times with a phenol-chloroform mixture. 10 μl of 3M sodium acetate pH5.2 and 300 μl of ethanol were added to 100 μl of the aqueous layer and stirred. Then, it was centrifuged at 14,000 rpm at room temperature for 15 minutes to collect the precipitate. After washing the precipitate with 75% ethanol, the precipitate was left to stand under vacuum for 2 minutes to be dried, and used as a sample for sequencing. The sequence sample was dissolved in formamide containing 4 μl of 10 mM EDTA and denatured at 90° C. for 2 minutes. This product was cooled in ice and subjected to sequencing.
Two of the five clones selected indiscriminately had sequence homology with the probe used for PCR. In addition, a DNA sequence that matched the gene sequence of Ribosomal Protein L7/L12 protein was clear. The entire base sequence of the structural gene portion and the corresponding amino acid sequence were the sequences shown in SEQ ID NOs: 1 and 2 of the Sequence Listing. This gene fragment clearly encodes the gene of Ribosomal Protein L7/L12 protein of Chlamydia pneumoniae .
Chlamydia pneumoniaeからのRibosomal Protein L7/L12蛋白質の大腸菌での大量発現と精製
発現ベクターを組み込んだ大腸菌をLB培地中で50ml、37℃、一昼夜培養した。500mlの2倍濃度のYT培地を37℃で1時間加熱した。1晩培養した大腸菌液50mlを500mlの前述の培地に入れた。一時間後、550μlの100mM イソプロピルβ-D(-)-チオガラクトピラノシド(IPTG)を導入し、4時間培養した。生成物を回収し、250mlの遠心チューブに移し、7000rpmで10分間遠心した。上澄みを棄てて50mM Tris緩衝液 pH7.4、25% Sucroseを含むLysis緩衝液25mlずつに溶解した。さらに10% NP-40 1.25ml、1M MgCl2 125μlを加えてプラスチックチューブに移した。氷冷下、1分間の超音波処理を5回行った。その後、12,000rpmで15分間遠心し、上清を回収した。
Large-scale Expression and Purification of Ribosomal Protein L7/L12 Protein from Chlamydia pneumoniae in Escherichia coli Escherichia coli containing the expression vector was cultured in LB medium at 50 ml at 37°C overnight. 500 ml of double concentration YT medium was heated at 37° C. for 1 hour. 50 ml of the E. coli solution cultured overnight was added to 500 ml of the above-mentioned medium. After 1 hour, 550 μl of 100 mM isopropyl β-D(−)-thiogalactopyranoside (IPTG) was introduced, and the cells were cultured for 4 hours. The product was collected, transferred to a 250 ml centrifuge tube and centrifuged at 7000 rpm for 10 minutes. The supernatant was discarded and dissolved in 25 ml each of Lysis buffer containing 50 mM Tris buffer pH 7.4 and 25% Sucrose. Further, 1.25 ml of 10% NP-40 and 125 μl of 1M MgCl 2 were added and transferred to a plastic tube. Ultrasonic treatment for 1 minute was performed 5 times under ice cooling. Then, the mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected.
次に、PBSでコンディショニングしたグルタチオンアガロースカラムに前記の上澄み液を吸着させた。次に、20mM Tris緩衝液pH7.4、4.2mM MgCl2、1mM ジチオスレイトール(DTT)を含む洗浄液でカラムを2ベッドボリューム分洗浄した。5mMのグルタチオンを含む50mMTris緩衝液pH9.6中で溶離処理をした。溶出画分の蛋白質含有量をピグメント結合法(Bradford法 ; BioRad Co.)で決定し、主画分を取得した。 Next, the above supernatant was adsorbed on a glutathione agarose column conditioned with PBS. Next, the column was washed for 2 bed volumes with a washing solution containing 20 mM Tris buffer pH 7.4, 4.2 mM MgCl 2 , and 1 mM dithiothreitol (DTT). Elution was performed in 50 mM Tris buffer pH 9.6 containing 5 mM glutathione. The protein content of the eluted fraction was determined by the pigment binding method (Bradford method; BioRad Co.), and the main fraction was obtained.
得られた精製GSTフュージョンRibosomal Protein L7/L12蛋白質の純度は電気泳動法により確認したところ約75%であり免疫源として充分な純度を確保できた。 The purity of the obtained purified GST fusion Ribosomal Protein L7/L12 protein was confirmed to be about 75% by electrophoresis, and a sufficient purity as an immunogen could be secured.
Chlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質に対するモノクローナル抗体の作製
まずマウスの免疫についてはChlamydia pneumoniaeのGSTフュージョンRibosomal Protein L7/L12蛋白質抗原100μgを200μlのPBSに溶解後フロイントのコンプリートアジュバントを200μl加え混合した。エマルジョン化したのち200μlを腹腔内に注射した。同じエマルジョン化抗原を2週間後、4週間後、および6週間後に腹腔内に注射した。2倍濃度のエマルジョン化抗原を10週間後および14週間後に腹腔内に注射した。最終の免疫化終了の3日後に脾臓を摘出し、細胞融合した。
Preparation of Monoclonal Antibody against Ribosomal Protein L7/L12 Protein of Chlamydia pneumoniae First, for immunization of mice, 100 μg of GST fusion Ribosomal Protein L7/L12 protein antigen of Chlamydia pneumoniae was dissolved in 200 μl of PBS, and then 200 μl of Freund's complete adjuvant was mixed. After emulsification, 200 μl was injected intraperitoneally. The same emulsified antigen was injected intraperitoneally after 2, 4, and 6 weeks. A double concentration of emulsified antigen was injected intraperitoneally after 10 and 14 weeks. Three days after the final immunization, the spleen was removed and cell fusion was performed.
無菌的に取り出したマウスの脾細胞108個に対し骨髄腫細胞2×107個をガラスチューブに取り良く混合したのち1500rpmで5分間遠心し上澄みを棄て、その後細胞をよく混合した。 After aseptically removing 10 8 mouse splenocytes, 2×10 7 myeloma cells were placed in a glass tube and mixed well, followed by centrifugation at 1500 rpm for 5 minutes, discarding the supernatant, and then thoroughly mixing the cells.
細胞融合に使用した骨髄腫細胞は、NS-1系の細胞株を用い10%の牛胎児血清を含むRPMI1640培地で培養し、細胞融合の2週間前から0.13mMのアザグアニン、0.5μg/mlのMC-210、10%の牛胎児血清を含むRPMI1640培地で1週間培養後、さらに10%の牛胎児血清を含むRPMI1640培地で1週間培養したものを用いた。 Myeloma cells used for cell fusion were cultured in RPMI1640 medium containing 10% fetal bovine serum using NS-1 cell line, and 0.13 mM azaguanine, 0.5 μg/ml from 2 weeks before cell fusion. MC-210 was cultured in RPMI1640 medium containing 10% fetal bovine serum for 1 week, and then further cultured in RPMI1640 medium containing 10% fetal bovine serum for 1 week.
37℃に保持したRPMI1640培養液50mlを混合細胞試料に加え、1,500rpmで遠心分離した。上澄み液を除去後、37℃に保持した50%ポリエチレングリコール1mlを加え、1分間攪拌した。37℃に保持したRPMI1640培養液10mlを加え、混合液を殺菌したピペットで約5分間吸引・排出することにより激しく攪拌した。 50 ml of RPMI1640 culture solution kept at 37° C. was added to the mixed cell sample and centrifuged at 1,500 rpm. After removing the supernatant, 1 ml of 50% polyethylene glycol kept at 37° C. was added and stirred for 1 minute. 10 ml of RPMI1640 culture solution kept at 37° C. was added, and the mixed solution was agitated and discharged with a sterilized pipette for about 5 minutes, thereby vigorously stirring.
5分間1,000rpmで遠心分離し、上澄み液を除去したのち、細胞濃度が5×106/mlとなるように30ml HAT培養液を加えた。この混合物を均一になるまで攪拌し、0.1mlずつを96穴の培養プレートに注ぎ、37℃、7％の炭酸ガス雰囲気下で培養した。HAT培地を、第1日、第1週、および第2週にそれぞれ0.1mlずつ加え、ELISA法により所望の抗体を産生する細胞をスクリーニングした。 After centrifugation at 1,000 rpm for 5 minutes to remove the supernatant, 30 ml HAT culture medium was added so that the cell concentration was 5×10 6 /ml. The mixture was stirred until it became homogeneous, 0.1 ml each was poured into a 96-well culture plate, and the mixture was cultured at 37° C. in a 7% carbon dioxide gas atmosphere. HAT medium was added at 0.1 ml each on the first day, the first week, and the second week, and cells producing the desired antibody were screened by the ELISA method.
0.05%のアジ化ソーダ含むPBS中に溶解したGSTフュージョンRibosomal Protein L7/L12蛋白質およびGST蛋白質をそれぞれ10μg/ml濃度で希釈した液を100μlずつ96穴プレートに別々に分注し4℃で1晩吸着させた。 GST fusion Ribosomal Protein L7/L12 protein and GST protein dissolved in PBS containing 0.05% sodium azide were diluted to a concentration of 10 μg/ml each in 100 μl aliquots and then dispensed separately into 96-well plates at 4°C overnight. Adsorbed.
上澄み除去後、1%牛血清アルブミン溶液(PBS中)200μl添加し室温で1時間反応しブロッキングした。上澄み液を除去後、生成物を洗浄液(0.02%Tween20,PBS)で洗浄した。これに融合細胞の培養液100mlを加え、室温にて2時間反応させた。上澄み液を除去し、沈殿を洗浄液で洗浄した。次いで、濃度50ng/mlのペルオキシダーゼでラベルしたgoat anti-mouse IgG抗体溶液100μlを加え、室温にて1時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。TMB溶液(KPL社製) を100μlずつ加え、混合物を室温にて20分間反応させた。着色したところで1Nの硫酸100μlを加えて反応を停止し、450nmの吸光度を測定した。 After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour and blocked. After removing the supernatant, the product was washed with a washing solution (0.02% Tween20, PBS). To this, 100 ml of the culture solution of fused cells was added, and the mixture was reacted at room temperature for 2 hours. The supernatant liquid was removed, and the precipitate was washed with a washing liquid. Then, 100 μl of a goat anti-mouse IgG antibody solution labeled with peroxidase having a concentration of 50 ng/ml was added, and the mixture was reacted at room temperature for 1 hour. The supernatant liquid was removed, and the product was washed again with the washing liquid. 100 μl of TMB solution (manufactured by KPL) was added, and the mixture was reacted at room temperature for 20 minutes. When colored, 100 μl of 1N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured.
この結果、GSTフュージョンRibosomal Protein L7/L12蛋白質にのみ反応しGST蛋白質には反応しない陽性ウェルが見いだされRibosomal Protein L7/L12蛋白質に対する抗体が含まれていることが判明した。 As a result, positive wells were found that reacted only with the GST fusion Ribosomal Protein L7/L12 protein but not with the GST protein and were found to contain antibodies against the Ribosomal Protein L7/L12 protein.
そこで陽性ウェル中の細胞をそれぞれ回収し24穴プラスティックプレート中、HAT培地で培養した。 Therefore, the cells in the positive wells were collected and cultured in a HAT medium in a 24-well plastic plate.
培養した融合培地を細胞数が約20個/mlになるようにHT培地で希釈し50μlを、HT培地に懸濁した6週齢のマウス胸腺細胞106個と96穴培養プレート中で混合した。混合後、7%CO2条件下、37℃で2週間培養した。 The cultured fusion medium was diluted with HT medium so that the cell number was about 20 cells/ml, and 50 μl was mixed with 10 6 6-week-old mouse thymocytes suspended in HT medium in a 96-well culture plate. .. After mixing, the cells were cultured at 37° C. for 2 weeks under 7% CO 2 .
培養上澄み中の抗体活性を前述のELISA法にて同様に検定し、Ribosomal Protein L7/L12蛋白質との反応陽性の細胞を回収した。さらに、同様の希釈検定、クローニング操作を繰り返し、ハイブリドーマCPRB-1〜5の計5クローンを取得した。 Similarly, the antibody activity in the culture supernatant was assayed by the above-mentioned ELISA method, and cells positive for the reaction with Ribosomal Protein L7/L12 protein were collected. Further, the same dilution assay and cloning operation were repeated to obtain a total of 5 clones of hybridoma CPRB-1-5.
Chlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質を検出するモノクローナル抗体の選択
前述のようにして取得した陽性ハイブリドーマ細胞を用いて定法にしたがってモノクローナル抗体を生産回収した。
Selection of Monoclonal Antibody for Detecting Ribosomal Protein L7/L12 Protein of Chlamydia pneumoniae Monoclonal antibody was produced and recovered by a standard method using the positive hybridoma cells obtained as described above.
具体的には、RPMI1640培地(10%FCS入り)を用いて継代培養した細胞をあらかじめ2週間前に0.5mlのプリスタンを腹腔内に注射したBalb/Cマウスの腹腔内に5×106個(PBS中)注射した。 3週間後腹水を回収し、その遠心上澄みを取得した。 Specifically, cells subcultured using RPMI1640 medium (containing 10% FCS) were pre-injected intraperitoneally in Balb/C mice with 0.5 ml of pristane 2 weeks in advance 5×10 6 cells intraperitoneally. Injection (in PBS). After 3 weeks, the ascites was collected, and the centrifugal supernatant was obtained.
得られた抗体を含む溶液をProtein Aカラム(5ml, Pharmacia製)に吸収させ、3倍量のPBSで洗浄した。次いで、クエン酸緩衝液pH3で溶出した。抗体画分を回収し、各ハイブリドーマによって産生されたモノクローナル抗体を取得した。この5株のハイブリドーマ由来のモノクローナル抗体を用いてELISA法により評価した。 The solution containing the obtained antibody was absorbed on a Protein A column (5 ml, manufactured by Pharmacia) and washed with 3 volumes of PBS. It was then eluted with citrate buffer pH 3. The antibody fraction was collected and the monoclonal antibody produced by each hybridoma was obtained. The monoclonal antibodies derived from the hybridomas of these 5 strains were used for evaluation by the ELISA method.
モノクローナル抗体の評価にはサンドウィッチ分析法を用いた。作成されたモノクローナル抗体をペルオキシダーゼに結合せしめることにより検出用の抗体として用いた。 The sandwich analysis method was used for evaluation of the monoclonal antibody. The prepared monoclonal antibody was used as an antibody for detection by binding to peroxidase.
酵素標識はホースラディッシュパーオキシダーゼ（SigmaグレードVI）を用い結合には試薬S-アセチルチオ酢酸N-ヒドロキシスクシンイミドを使用しAnalytical Bio-chemistry 132 (1983), 68-73に述べられている方法に従って行った。ELISA反応においては市販の抗Chlamydia pneumoniaeポリクローナル抗体（ウサギ）を10μg/ml濃度で希釈した液を100μlずつ96穴プレートに別々に分注し4℃で1晩吸着させた。 Enzyme labeling was performed using horseradish peroxidase (Sigma Grade VI) and conjugation was performed using the reagent S-acetylthioacetic acid N-hydroxysuccinimide according to the method described in Analytical Bio-chemistry 132 (1983), 68-73. . In the ELISA reaction, a commercially available anti- Chlamydia pneumoniae polyclonal antibody (rabbit) diluted at a concentration of 10 μg/ml was separately dispensed into a 96-well plate by 100 μl and allowed to adsorb at 4° C. overnight.
上澄み除去後、1%牛血清アルブミン溶液(PBS中)200μl添加し室温で1時間反応しブロッキングした。上澄み液を除去後、生成物を洗浄液(0.02%Tween20, PBS)で洗浄した。これに各微生物の培養液に濃度0.3%となる量のTriton X-100を加え常温で5分間抽出することにより得られた抗原溶液100μlを加え、室温で2時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。次いで、濃度5μg/mlのペルオキシダーゼ標識抗Ribosomal Protein L7/L12蛋白質抗体溶液100μlを加え、室温にて1時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。TMB溶液(KPL社製)を100μlずつ加え、混合物を室温にて20分間反応させた。着色したところで1Nの硫酸100μlを加えて反応を停止した。450nmの吸光度を測定した。 After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour and blocked. After removing the supernatant, the product was washed with a washing solution (0.02% Tween20, PBS). To this was added 100 μl of the antigen solution obtained by adding Triton X-100 in an amount of 0.3% to the culture solution of each microorganism and extracting at room temperature for 5 minutes, and reacted at room temperature for 2 hours. The supernatant liquid was removed, and the product was washed again with the washing liquid. Then, 100 μl of peroxidase-labeled anti-Ribosomal Protein L7/L12 protein antibody solution having a concentration of 5 μg/ml was added, and the mixture was reacted at room temperature for 1 hour. The supernatant liquid was removed, and the product was washed again with the washing liquid. 100 μl of TMB solution (manufactured by KPL) was added, and the mixture was reacted at room temperature for 20 minutes. When colored, 100 μl of 1N sulfuric acid was added to stop the reaction. The absorbance at 450 nm was measured.
酵素標識抗体としてハイブリドーマCPRB-1由来のモノクローナル抗体を用いた場合、試験したChlamydia pneumoniaeの全ての株を106個/mlの感度で検出すると同時に他のHaemophilus influenzae, Klebsiella pneumoniae, Mycoplasma pneumoniaeおよびNeisseria meningitides等の微生物について108個/mlの高濃度でも反応性を示さずRibosomal Protein L7/L12蛋白質に対するモノクローナル抗体を用いることでChlamydia pneumoniae特異的な反応性をもつ抗体を取得したことが明確に確認できた。この抗体はAMCP-1と名付けられた。表２はAMCP-1を用いた結果のみを示す。別の微生物と交差反応を示す他の抗体を使用した結果はここで言及しない。 When a monoclonal antibody derived from hybridoma CPRB-1 was used as the enzyme-labeled antibody, all strains of Chlamydia pneumoniae tested were detected at a sensitivity of 10 6 cells/ml, while at the same time other Haemophilus influenzae , Klebsiella pneumoniae , Mycoplasma pneumoniae and Neisseria meningitides were detected. It can be clearly confirmed that antibodies having specificity for Chlamydia pneumoniae were obtained by using a monoclonal antibody against Ribosomal Protein L7/L12 protein, which did not show reactivity with microorganisms such as 10 8 cells/ml even at high concentrations. It was This antibody was named AMCP-1. Table 2 shows only the results using AMCP-1. The results of using other antibodies that cross-react with other microorganisms are not mentioned here.
Ribosomal Protein L7/L12蛋白質固定化アフィニティカラムを用いたChlamydia pneumoniae Ribosomal Protein L7/L12蛋白質と特異的に反応するポリクローナル抗体の取得
実施例１に記載の方法により取得したChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質またはTriton X-100処理した菌体の上清を抗原として使用した。100μgの抗原を含む生理食塩水約1.2mlをフロイントアジュバント1.5mlとともに乳化した。エマルジョンをSPF日本白色ウサギに皮下注射してウサギを免疫化した。2週間おきに5〜6回免疫し、抗体価を確認した。
Ribosomal Protein L7/L12 Protein Immobilization Affinity Column for Chlamydia pneumoniae Acquisition of Ribosomal Protein L7/L12 Protein Polyclonal Antibody That Reacts Specificly Ribosomal Protein L7/L12 Protein of Chlamydia pneumoniae Obtained by the Method of Example 1 Alternatively, the supernatant of the cells treated with Triton X-100 was used as an antigen. About 1.2 ml of physiological saline containing 100 μg of antigen was emulsified with 1.5 ml of Freund's adjuvant. Rabbits were immunized by subcutaneous injection of the emulsion into SPF Japanese white rabbits. Immunization was performed 5 to 6 times every 2 weeks, and the antibody titer was confirmed.
抗体価の確認はELISA法により実施した。0.05%のアジ化ソーダ含むPBS中に溶解したChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質を10μg/ml濃度に希釈した液を100μlずつ96穴プレートに分注し4℃で1晩吸着させた。上澄み除去後、1%牛血清アルブミン溶液(PBS中)200μl添加し室温で1時間反応しブロッキングした。上澄み液を除去後、生成物を洗浄液(0.02% Tween20, PBS)で洗浄した。正常のウサギ血清および免疫化したウサギの抗血清を希釈して得られた溶液100μlを加え、室温にて2時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。次いで、濃度50ng/mlのペルオキシダーゼ標識抗ウサギIgG抗体溶液100μlを加え、室温にて1時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。OPD溶液(Sigma社製)を100μlずつ加え、混合物を室温にて20分間反応させた。着色したところで1Nの硫酸100μlを加えて反応を停止した。492nmの吸光度を測定した。 The antibody titer was confirmed by the ELISA method. A solution of Chlamydia pneumoniae Ribosomal Protein L7/L12 protein dissolved in PBS containing 0.05% sodium azide at a concentration of 10 μg/ml was dispensed in 100 μl aliquots into 96-well plates and adsorbed at 4° C. overnight. After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour and blocked. After removing the supernatant, the product was washed with a washing solution (0.02% Tween20, PBS). 100 μl of a solution obtained by diluting normal rabbit serum and immunized rabbit antiserum was added and reacted at room temperature for 2 hours. The supernatant liquid was removed, and the product was washed again with the washing liquid. Then, 100 μl of peroxidase-labeled anti-rabbit IgG antibody solution having a concentration of 50 ng/ml was added, and the reaction was carried out at room temperature for 1 hour. The supernatant liquid was removed, and the product was washed again with the washing liquid. OPD solution (manufactured by Sigma) was added in 100 μl portions, and the mixture was reacted at room temperature for 20 minutes. When colored, 100 μl of 1N sulfuric acid was added to stop the reaction. The absorbance at 492 nm was measured.
抗体価上昇を確認後、大量採血を実施した。耳動脈から血液をガラス製遠心管に採取し、37℃で1時間放置後、4℃で一晩静置した。その後3000rpm5分間遠心し、上清を回収した。得られた抗血清は4℃で保存した。 After confirming the antibody titer increase, a large amount of blood was collected. Blood was collected from the ear artery into a glass centrifuge tube, allowed to stand at 37°C for 1 hour, and then allowed to stand at 4°C overnight. Then, the mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. The obtained antiserum was stored at 4°C.
Chlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質を固定化したアフィニティカラムを調製した。HiTrap NHS活性化カラム(1ml, Pharmacia社製)を用いた。カラムを1mM HClで置換後直ちにRibosomal Protein L7/L12蛋白質のPBS溶液(1mg/ml)を加えた。カラムを30分間静置後、ブロッキング試薬を加え、PBSで平衡化した。 An affinity column on which the Ribosomal Protein L7/L12 protein of Chlamydia pneumoniae was immobilized was prepared. A HiTrap NHS activation column (1 ml, manufactured by Pharmacia) was used. Immediately after replacing the column with 1 mM HCl, a PBS solution (1 mg/ml) of Ribosomal Protein L7/L12 protein was added. After allowing the column to stand for 30 minutes, a blocking reagent was added and equilibrated with PBS.
このChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質固定化アフィニティカラムを使用して、Chlamydia pneumoniaeのTriton X-100処理した菌体の上清を抗原として得られた抗血清中のポリクローナル抗体の精製を行った。この抗血清をPBSで5倍に希釈し、0.45μmのフィルターを通した後、流速0.5ml/minでChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質固定化カラムに吸着させた。その後0.1Mグリシン緩衝液pH2.1でカラムから溶出し、直ちに1M Tris緩衝液pH9.0で中和した後、抗体価測定法と同様のELISA法により目的とする抗体の溶出画分を回収した。 Using this Chlamydia pneumoniae Ribosomal Protein L7/L12 protein-immobilized affinity column, the polyclonal antibody in the antiserum obtained by using the supernatant of the Chlamydia pneumoniae Triton X-100-treated cells as an antigen was purified. .. This antiserum was diluted 5 times with PBS, passed through a 0.45 μm filter, and then adsorbed on a Ribosomal Protein L7/L12 protein-immobilized column of Chlamydia pneumoniae at a flow rate of 0.5 ml/min. Then eluted from the column with 0.1M glycine buffer pH2.1, immediately neutralized with 1M Tris buffer pH9.0, then the elution fraction of the desired antibody was collected by the same ELISA method as the antibody titer measurement method. ..
このようにして得られたポリクローナル抗体は特表平７−５０９５６５号公報に記載されているOIA法により評価した。 The polyclonal antibody thus obtained was evaluated by the OIA method described in JP-A-7-509565.
精製した抗体はOIA法の捕捉抗体として使用した。また検出抗体としては実施例４に記載したAMCP-1モノクローナル抗体をパーオキシダーゼで酵素標識したものを使用した。酵素標識はホースラディッシュパーオキシダーゼ(SigmaグレードVI)を用い結合には試薬S-アセチルチオ酢酸N-ヒドロキシスクシンイミドを使用しAnalytical Bio-chemistry 132 (1983), 68-73に述べられている方法に従って行った。 The purified antibody was used as a capture antibody for the OIA method. The detection antibody used was the AMCP-1 monoclonal antibody described in Example 4 which was enzyme-labeled with peroxidase. Enzyme labeling was performed using horseradish peroxidase (Sigma Grade VI) and conjugation was performed using the reagent S-acetylthioacetic acid N-hydroxysuccinimide according to the method described in Analytical Bio-chemistry 132 (1983), 68-73. ..
OIA反応においては0.05%アジ化ナトリウムを含むPBS中の精製ポリクローナル抗体を10μg/ml濃度に0.1M HEPES緩衝液pH8.0で希釈した液を50μlずつシリコンウエハー上に添加し室温で30分反応させた後、蒸留水で洗浄し、サッカロース及びアルカリ処理カゼインを含むコーティング溶液でコーティング後、使用した。 In the OIA reaction, purified polyclonal antibody in PBS containing 0.05% sodium azide was diluted to a concentration of 10 μg/ml with 0.1 M HEPES buffer pH 8.0, and 50 μl of each solution was added on a silicon wafer and reacted at room temperature for 30 minutes. Then, it was washed with distilled water, coated with a coating solution containing saccharose and alkali-treated casein, and then used.
上記操作で得られた各微生物の培養液に濃度0.5%となる量のTritonX-100を加え常温で5分間抽出することにより得られた抗原溶液15μlを上記シリコンウェハー上に添加し、室温で10分間反応させた。次いで、20μg/mlペルオキシダーゼ標識化モノクローナル抗体を15μl加え、10分間反応させた。蒸留水で洗浄後、TMB溶液(KPL社製)を15μlずつ加え、混合物を室温にて5分間反応させた。生成物を蒸留水で洗浄し、酵素反応により生成した青色を肉眼で観察した。 15 μl of the antigen solution obtained by adding Triton X-100 in a concentration of 0.5% to the culture solution of each microorganism obtained by the above operation and extracting at room temperature for 5 minutes was added on the silicon wafer, and at room temperature 10 Let react for minutes. Then, 15 μl of 20 μg/ml peroxidase-labeled monoclonal antibody was added and reacted for 10 minutes. After washing with distilled water, 15 μl of TMB solution (manufactured by KPL) was added, and the mixture was reacted at room temperature for 5 minutes. The product was washed with distilled water and the blue color produced by the enzymatic reaction was visually observed.
この結果、表３に示すように、精製ポリクローナル抗体APCP-1を捕捉抗体として用いることにより、Chlamydia pneumoniaeを108個/mlの感度で検知できること、および他の微生物の反応性は検知できないことが明らかである。このようにしてChlamydia pneumoniaeのRibosomal Protein L7/L12蛋白質を固定化したアフィニティカラムにより、Chlamydia pneumoniaeに特異的に反応するポリクローナル抗体を取得したことを確認した。 As a result, as shown in Table 3, by using the purified polyclonal antibody APCP-1 as a capture antibody, Chlamydia pneumoniae can be detected with a sensitivity of 10 8 cells/ml, and the reactivity of other microorganisms cannot be detected. it is obvious. The Ribosomal Protein L7 / L12 affinity column where the protein immobilized in this way Chlamydia pneumoniae, it was confirmed that the obtained polyclonal antibodies specifically reactive with Chlamydia pneumoniae.
本発明によると微生物の進化の過程で機能的に保持された細胞内分子に対する抗体を用いて特定の種の微生物を特異的に検出できるだけでなく、同一種内における全ての血清型の微生物を精度よく検出することができる。 According to the present invention, not only can a microorganism of a specific species be specifically detected by using an antibody against an intracellular molecule that is functionally retained in the process of evolution of a microorganism, but all serotype microorganisms within the same species can be detected accurately. Can be detected well.
このような抗体として微生物のリボソーム蛋白質、Ribosomal Protein L7/L12蛋白質に対する抗体を用い、Chlamydia pneumoniaeの検出を精度良く行うことができる。 As such an antibody, an antibody against a ribosome protein of a microorganism, Ribosomal Protein L7/L12 protein can be used to detect Chlamydia pneumoniae with high accuracy.
また、このような抗体を構成要素とする微生物検出用試薬キットを用いることで、微生物の検出をより汎用的に精度良く行なうことができる。 Further, by using a microorganism detection reagent kit containing such an antibody as a component, microorganisms can be detected more generally and accurately.
Claims (3)
（１）遺伝子操作手法によりあるいはクラミジア・ニューモニア（Chlamydia pneumoniae）に属する細菌からの単離精製により得られた当該細菌の全長リボソーム蛋白質Ｌ７／Ｌ１２を免疫源として、当該細菌の全長リボソーム蛋白質Ｌ７／Ｌ１２に対するモノクローナル抗体を産生するハイブリドーマを選択する工程
（２）上記（１）で得られたハイブリドーマから、さらに、当該細菌の全長リボソーム蛋白質Ｌ７／Ｌ１２に特異的に反応し、当該細菌を他の細菌と種あるいは属で識別できるモノクローナル抗体を産生するハイブリドーマを選択する工程
（３）上記（２）で得られたハイブリドーマから抗体を取得する工程 The method for producing the antibody according to claim 1 or 2, which comprises the following steps: a method for producing a monoclonal antibody against ribosomal protein L7/L12 of a bacterium belonging to Chlamydia pneumoniae.
(1) Using the full-length ribosomal protein L7/L12 of the bacterium obtained by a gene manipulation method or by isolation and purification from a bacterium belonging to Chlamydia pneumoniae as an immunogen, the full-length ribosomal protein L7/L12 of the bacterium Step (2) of selecting a hybridoma producing a monoclonal antibody against Escherichia coli is further reacted specifically with the full-length ribosomal protein L7/L12 of the bacterium to obtain another bacterium from the hybridoma obtained in (1) above. Step of selecting hybridoma producing a monoclonal antibody which can be discriminated by species or genus (3) Step of obtaining antibody from the hybridoma obtained in (2) above
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