JP2017032588A - Biomarkers for tuberculosis testing - Google Patents
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- JP2017032588A JP2017032588A JP2016214285A JP2016214285A JP2017032588A JP 2017032588 A JP2017032588 A JP 2017032588A JP 2016214285 A JP2016214285 A JP 2016214285A JP 2016214285 A JP2016214285 A JP 2016214285A JP 2017032588 A JP2017032588 A JP 2017032588A
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Abstract
Description
本発明は、結核検査用バイオマーカーに関する。より詳しくは、ロイシンリッチα２グリコプロテイン（ｌｅｕｃｉｎｅ ｒｉｃｈ ａｌｐｈａ ２ ｇｌｙｃｏｐｒｏｔｅｉｎ：ＬＲＧ）からなる結核検査用バイオマーカー、疾患活動性を判断しうるバイオマーカー、さらに詳しくは治療薬の有効性を判断しうるバイオマーカーに関し、さらに該バイオマーカーを検出又は定量することによる結核の検査方法に関する。 The present invention relates to a biomarker for tuberculosis examination. More specifically, a biomarker for tuberculosis testing comprising leucine rich alpha 2 glycoprotein (LRG), a biomarker capable of judging disease activity, and more specifically a biomarker capable of judging the effectiveness of a therapeutic agent Further, the present invention relates to a method for examining tuberculosis by detecting or quantifying the biomarker.
結核とは、結核菌（Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｔｕｂｅｒｃｕｌｏｓｉｓ）によって引き起こされる感染症であり、一度発病すると、再発する可能性も高い。国際的にも、三大感染症の一つとされ、ＨＩＶや後天性免疫不全症候群との合併結核のまん延や、多剤耐性菌結核の伝播が大きな問題になっている。日本においても、結核患者数は減少傾向にあるものの、２００９年には約２４，０００人の新規患者が発生するなど、依然として主要な感染症である。
結核の診断方法としては、結核が疑われる患者の痰を採取、培養し、結核菌の有無を調べる喀痰培養検査、痰から結核菌のＤＮＡあるいはＲＮＡがあるかどうかをＰＣＲ法により調べる検査等が挙げられる。
喀痰培養検査は、検査結果が出るまでに数週間の時間が必要であり、より迅速な検査方法が求められていた。また、ＰＣＲ法による検査では、検査結果はすぐ得られるものの、死んだ結核菌が含まれている場合でも陽性となることが問題であった。
Tuberculosis is an infection caused by Mycobacterium tuberculosis, and once it occurs, there is a high possibility of recurrence. Internationally, it is regarded as one of the three major infectious diseases, and the spread of tuberculosis associated with HIV and acquired immune deficiency syndrome and the transmission of multidrug-resistant bacterial tuberculosis are major problems. In Japan, the number of tuberculosis patients is decreasing, but it is still a major infectious disease, with approximately 24,000 new patients occurring in 2009.
Methods for diagnosing tuberculosis include collecting sputum from patients suspected of having tuberculosis, culturing them, and examining sputum culture tests for the presence or absence of M. tuberculosis, tests for examining the presence of M. tuberculosis DNA or RNA from sputum, etc. Can be mentioned.
The sputum culture test requires several weeks before the test result is obtained, and a more rapid test method is required. Moreover, in the test by PCR method, although the test result can be obtained immediately, even when dead tuberculosis bacteria are included, there is a problem that it becomes positive.
ベーチェット病、関節リウマチ、クローン病、キャッスルマン氏病などの自己免疫疾患の炎症マーカーとして、Ｃ−ｒｅａｃｔｉｖｅ ｐｒｏｔｅｉｎ（ＣＲＰ）が臨床上よく用いられる。ＣＲＰは、炎症に伴って上昇するインターロイキン−６（ＩＬ−６）などの炎症性サイトカインによって肝臓で合成される急性期タンパク質のひとつである。急性期タンパク質には、ＣＲＰの他にα１−アシドグリコプロテイン、ハプトグロビン、シアル酸などがあり、血清中のこれらの物質の濃度増加は、炎症性疾患の診断や経過の推移の判定に用いられる。これらの炎症マーカーは、体内に炎症があればその程度に応じて異常値を示すが、炎症性疾患のみならず、炎症マーカーによっては、組織破壊・細胞壊死を伴う悪性腫瘍や、急性心筋梗塞、貧血、溶血、妊娠、外傷など様々な病態で異常値を示すことがあり、各マーカーにより各疾患に対する特異度や感度が異なる場合がある。そこで、自己免疫疾患において疾患活動性を判断しうる新規なマーカー、さらには治療薬の有効性を判断しうる新規なバイオマーカーが求められており、検討したところ、ロイシンリッチα２グリコプロテイン（以下、単に「ＬＲＧ」という）が、関節リウマチやクローン病、ベーチェット病、潰瘍性大腸炎などの自己免疫疾患の活動性と相関することを本発明者らが初めて見出し、報告した（非特許文献１、２）。 C-reactive protein (CRP) is frequently used clinically as an inflammatory marker for autoimmune diseases such as Behcet's disease, rheumatoid arthritis, Crohn's disease, and Castleman's disease. CRP is one of the acute phase proteins synthesized in the liver by inflammatory cytokines such as interleukin-6 (IL-6) that increases with inflammation. Acute phase proteins include α1-acid glycoprotein, haptoglobin, sialic acid and the like in addition to CRP, and the increase in the concentration of these substances in serum is used for diagnosis of inflammatory diseases and determination of the course of the course. These inflammation markers show abnormal values depending on the degree of inflammation in the body, but depending not only on inflammatory diseases, depending on the inflammation marker, malignant tumors with tissue destruction / cell necrosis, acute myocardial infarction, Abnormal values may be shown in various pathologies such as anemia, hemolysis, pregnancy, trauma, and the specificity and sensitivity to each disease may differ depending on each marker. Thus, there is a need for a novel marker that can determine disease activity in autoimmune diseases, and further, a novel biomarker that can determine the effectiveness of therapeutic agents. The present inventors have found and reported for the first time that the activity of autoimmune diseases such as rheumatoid arthritis, Crohn's disease, Behcet's disease, and ulcerative colitis is simply referred to as “LRG” (Non-patent Document 1, 2).
ＬＲＧは血清タンパク質のひとつで、約５０ｋＤａの糖タンパク質であり、好中球から分泌されることが報告されている（非特許文献３）。上述の如く、ＬＲＧが自己免疫疾患のバイオマーカーとなりうることは非特許文献１、２に開示されているものの、ＬＲＧと結核の関係についての報告はされていない。 LRG is one of the serum proteins, is a glycoprotein of about 50 kDa, and has been reported to be secreted from neutrophils (Non-patent Document 3). As described above, although it has been disclosed in Non-Patent Documents 1 and 2 that LRG can be a biomarker of autoimmune disease, there has been no report on the relationship between LRG and tuberculosis.
本発明は、結核を迅速に精度よく診断するバイオマーカー、疾患活動性を判断しうる結核検査用バイオマーカー、さらに詳しくは治療薬の有効性を判断しうる検査方法を提供することを課題とする。 It is an object of the present invention to provide a biomarker for quickly and accurately diagnosing tuberculosis, a biomarker for tuberculosis testing that can determine disease activity, and more specifically, a testing method that can determine the effectiveness of therapeutic agents. .
本発明者らは、上記課題を解決するために、検討を重ねた結果、結核患者の血清中のＬＲＧ濃度が健常者と比較して有意に高いことを見出した。また、結核患者に結核治療剤を投与した場合に、治療後の血清中のＬＲＧ濃度が、治療前に比べて低かったことから、ＬＲＧは結核のバイオマーカー及び薬剤投与に伴う疾患活動性の指標マーカーとなりうることを確認し、本発明を完成した。 As a result of repeated studies to solve the above problems, the present inventors have found that the LRG concentration in the serum of tuberculosis patients is significantly higher than that of healthy individuals. In addition, when tuberculosis patients were administered a tuberculosis therapeutic agent, the LRG concentration in the serum after treatment was lower than before treatment, so LRG is an indicator of tuberculosis biomarkers and disease activity associated with drug administration After confirming that it could be a marker, the present invention was completed.
すなわち、本発明は以下よりなる。
［１］ロイシンリッチα２グリコプロテインからなる、結核検査用バイオマーカー。
［２］生体から分離した生体検体について、前記［１］に記載のバイオマーカーを検出または定量することを特徴とする結核検査方法。
［３］生体検体が細胞、細胞を含有する組織、血液、尿、髄液、体腔穿刺液またはリンパ液である、前記［２］に記載の結核検査方法。
［４］下記（ａ）または（ｂ）：
（ａ）ロイシンリッチα２グリコプロテイン遺伝子の転写産物を特異的に検出し得る核酸プローブまたは核酸プライマー
（ｂ）ロイシンリッチα２グリコプロテインを特異的に認識する抗体
を用いて検出または定量することを特徴とする、前記［２］又は［３］に記載の結核検査方法。
［５］生体から分離した生体検体について、前記［１］に記載のバイオマーカーを検出または定量することを特徴とする、結核に対する抗結核薬による治療奏功性の予測方法。
［６］抗結核薬がイソニアジド（ＩＮＨ）、リファンピシン（ＲＩＦ）、ピラジナミド（ＰＺＡ）、エタンブトール（ＥＭＢ）またはストレプトマイシン（ＳＭ）である、前記［５］記載の治療奏功性の予測方法。
［７］生体から分離した生体検体について、前記［１］に記載のバイオマーカーを検出または定量することを特徴とする、結核の疾患活動性判定方法。
［８］下記（ａ）または（ｂ）：
（ａ）ロイシンリッチα２グリコプロテイン遺伝子の転写産物を特異的に検出し得る核酸プローブまたは核酸プライマー
（ｂ）ロイシンリッチα２グリコプロテインを特異的に認識する抗体
を含有してなる、前記［２］〜［４］のいずれか１つに記載の方法を用いる結核検査用キット。
That is, this invention consists of the following.
[1] A biomarker for tuberculosis testing comprising leucine-rich α2 glycoprotein.
[2] A method for detecting tuberculosis, comprising detecting or quantifying the biomarker according to [1] above from a biological specimen separated from a living body.
[3] The tuberculosis examination method according to the above [2], wherein the biological specimen is a cell, a tissue containing the cell, blood, urine, spinal fluid, body cavity puncture fluid or lymph fluid.
[4] The following (a) or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of a leucine-rich α2 glycoprotein gene; and (b) a detection or quantification using an antibody that specifically recognizes leucine-rich α2 glycoprotein. The tuberculosis test method according to [2] or [3].
[5] A method for predicting the efficacy of treatment with an anti-tuberculosis drug for tuberculosis, comprising detecting or quantifying the biomarker according to [1] for a biological specimen separated from a living body.
[6] The method for predicting therapeutic efficacy according to the above [5], wherein the antituberculosis drug is isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (EMB), or streptomycin (SM).
[7] A method for determining disease activity of tuberculosis, comprising detecting or quantifying the biomarker according to [1] above from a biological specimen separated from a living body.
[8] The following (a) or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of a leucine-rich α2 glycoprotein gene; and (b) an antibody that specifically recognizes leucine-rich α2 glycoprotein. A tuberculosis test kit using the method according to any one of [4].
本発明のＬＲＧは、結核の検査用バイオマーカーとなりうる。ＬＲＧをバイオマーカーとして検出又は定量することにより、結核の検査を行うことができ、また疾患活動性を評価することができる。また、結核治療薬を投与したときの治療効果を評価しうるバイオマーカーともなる。具体的には、イソニアジド（ＩＮＨ）、リファンピシン（ＲＩＦ）、ピラジナミド（ＰＺＡ）、エタンブトール（ＥＭＢ）またはストレプトマイシン（ＳＭ）などの抗結核薬による治療を行った際の疾患活動性を本発明のバイオマーカーにより評価することができる。その結果、例えば薬剤投与終了時の判断や投与薬剤や治療方針の変更などに対して、最も効果的な方法を選択することができる。 The LRG of the present invention can be a biomarker for testing tuberculosis. By detecting or quantifying LRG as a biomarker, tuberculosis can be examined and disease activity can be evaluated. It is also a biomarker that can evaluate the therapeutic effect when a tuberculosis therapeutic agent is administered. Specifically, the biomarker of the present invention indicates the disease activity when treated with an antituberculosis drug such as isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (EMB) or streptomycin (SM). Can be evaluated. As a result, for example, the most effective method can be selected with respect to the determination at the end of drug administration, the change of the administered drug and the treatment policy, and the like.
本発明の結核検査用バイオマーカーは、ＬＲＧからなる。ここでＬＲＧとは、約５０ｋＤａの糖タンパク質であり、健常人血清では約３．０μｇ／ｍＬ濃度含まれていることが知られている。また、ＬＲＧは好中球から分泌されることが報告されている（非特許文献３）。また、ＬＲＧは顆粒球で発現することも報告されているが、ＬＲＧの発現はＩＬ−６の刺激により誘導されるものではない。かかるＬＲＧは、ベーチェット病、キャッスルマン氏病又は関節リウマチ等の自己免疫疾患、潰瘍性大腸炎のバイオマーカーとなりうることが、本発明者らにより報告されている（非特許文献１、２）。自己免疫疾患のマーカーとして汎用されていたＣＲＰは、炎症に伴って上昇するＩＬ−６などの炎症性サイトカインによって肝臓で合成される急性期タンパク質のひとつであるのに対し、ＬＲＧはＩＬ−６の影響を受けにくいことが確認されている（非特許文献１、２）。一方、血中ＬＲＧは、炎症バイオマーカーであるのみならず、上記の疾患における疾患活動性とも相関する有用な分子であり、ＬＲＧはＣＲＰとは異なるメカニズムにより発現するバイオマーカーである。 The biomarker for tuberculosis examination of the present invention consists of LRG. Here, LRG is a glycoprotein of about 50 kDa, and is known to contain about 3.0 μg / mL concentration in healthy human serum. In addition, it has been reported that LRG is secreted from neutrophils (Non-patent Document 3). Although it has been reported that LRG is expressed in granulocytes, the expression of LRG is not induced by stimulation with IL-6. It has been reported by the present inventors that such LRG can be a biomarker for autoimmune diseases such as Behcet's disease, Castleman's disease or rheumatoid arthritis, and ulcerative colitis (Non-patent Documents 1 and 2). CRP, which has been widely used as a marker for autoimmune diseases, is one of the acute phase proteins synthesized in the liver by inflammatory cytokines such as IL-6 that increases with inflammation, whereas LRG is an IL-6 protein. It has been confirmed that it is not easily affected (Non-Patent Documents 1 and 2). On the other hand, blood LRG is not only an inflammation biomarker, but also a useful molecule that correlates with disease activity in the above-mentioned diseases, and LRG is a biomarker expressed by a mechanism different from CRP.
また、本発明は、生体から分離した生体検体について、ＬＲＧを検出または定量することを特徴とする結核検査方法を提供する。結核は、Ｍ．ｔｕｂｅｒｃｕｌｏｓｉｓの感染であれば、特に限定はされないが、例として、肺結核、結核性髄膜炎または結核性リンパ節炎が挙げられる。 The present invention also provides a tuberculosis examination method characterized by detecting or quantifying LRG in a biological specimen separated from a living body. Tuberculosis is Although it will not specifically limit if it is a tuberculosis infection, For example, pulmonary tuberculosis, tuberculous meningitis, or tuberculous lymphadenitis will be mentioned.
本発明の検査方法において検出または定量されるＬＲＧタンパク質のアミノ酸配列は公知であり、当該タンパク質をコードするＤＮＡ配列も公知である（ＧｅｎＢａｎｋ Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＭ＿０５２９７２．２）。本発明の検査方法において検出または定量されるＬＲＧは、タンパク質を構成するアミノ酸配列が、上記で特定されるアミノ酸配列であってもよいし、当該アミノ酸配列とは１〜複数個のアミノ酸が置換、欠失、付加、導入されていても良い。さらには、ＬＲＧタンパク質全体であってもよいし、部分タンパク質であってもよい。以下、本明細書において「ＬＲＧタンパク質」とは、上述のように、ＧｅｎＢａｎｋ Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＰ＿４４３２０４．１で特定されるアミノ酸配列、又は前記特定されるアミノ酸配列から１〜複数個のアミノ酸が置換、欠失、付加、導入されているアミノ酸配列から構成されるタンパク質や、ＬＲＧの部分タンパク質も含む意味で用いられる。また、本発明の検査方法において検出または定量されるＬＲＧ遺伝子の転写産物は、上記ＬＲＧをコードする塩基配列と同一または実質的に同一な塩基配列を含有するＤＮＡなどが挙げられる。ＬＲＧをコードする塩基配列と実質的に同一なＤＮＡとしては、例えば、該塩基配列と約５０％以上、好ましくは約６０％以上、さらに好ましくは約７０％以上、特に好ましくは約８０％以上、最も好ましくは約９０％以上の相同性を有する塩基配列を含有するＤＮＡなどが挙げられる。本明細書における塩基配列の相同性は、相同性計算アルゴリズムＮＣＢＩ ＢＬＡＳＴ（Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ Ｂａｓｉｃ Ｌｏｃａｌ Ａｌｉｇｎｍｅｎｔ Ｓｅａｒｃｈ Ｔｏｏｌ）を用い、以下の条件（期待値＝１０；ギャップを許す；フィルタリング＝ＯＮ；マッチスコア＝１；ミスマッチスコア＝−３）にて計算することができる。 The amino acid sequence of the LRG protein to be detected or quantified in the test method of the present invention is known, and the DNA sequence encoding the protein is also known (GenBank Accession No. NM_052972.2). In the LRG detected or quantified in the test method of the present invention, the amino acid sequence constituting the protein may be the amino acid sequence specified above, or the amino acid sequence is substituted with one or more amino acids, It may be deleted, added or introduced. Furthermore, the whole LRG protein may be sufficient and a partial protein may be sufficient. Hereinafter, as used herein, “LRG protein” refers to GenBank Accession No. A protein composed of an amino acid sequence specified by NP_443204.1, or an amino acid sequence in which one to a plurality of amino acids are substituted, deleted, added or introduced from the specified amino acid sequence, or a partial protein of LRG Used to mean including. Examples of the transcription product of the LRG gene detected or quantified by the test method of the present invention include DNA containing the same or substantially the same base sequence as that encoding the LRG. Examples of DNA substantially the same as the base sequence encoding LRG include, for example, about 50% or more, preferably about 60% or more, more preferably about 70% or more, particularly preferably about 80% or more. Most preferably, DNA containing a base sequence having a homology of about 90% or more can be mentioned. The homology of the base sequences in this specification is determined by using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Alignment Search Tool) and the following conditions (expectation value = 10; allow gap; filtering = ON; match Score = 1; mismatch score = -3).
本発明の検査方法が適用できる生体は、特に制限されないが、例えば、結核に罹患しているおそれがある生体、もしくは罹患していることが疑われる生体、あるいは現に結核に罹患している生体であって、例えば、ヒト、サル、ウシ、ウマ、ブタ、マウス、ラット、モルモット、ハムスター、イヌ、ネコ、ウサギ、ヒツジ、ヤギ等が挙げられる。好ましくは、ヒトである。 The living body to which the test method of the present invention can be applied is not particularly limited. For example, in a living body that may be affected by or suspected of having tuberculosis, or a living body that is actually suffering from tuberculosis. Examples thereof include humans, monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats and the like. Preferably, it is a human.
本発明の検査方法に用いられる生体検体としては、検査対象である上記生体から分離されるものであって、検出または定量する対象であるＬＲＧ遺伝子産物（例、ＲＮＡ、タンパク質、その分解産物など）を含有し得る細胞、細胞を含有する組織、血液（血清、血漿等）、尿、髄液、体腔穿刺液（胸水、腹水）またはリンパ液等であれば特に制限されない。例えば、脳、髄膜、脊髄、下垂体、胃、膵臓、腎臓、肝臓、生殖腺、甲状腺、胆嚢、骨髄、副腎、皮膚、肺、消化管（例、大腸、小腸）、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子宮、骨、関節、脂肪組織、骨格筋、リンパ節、血液（血清、血漿等）、尿、髄液、体腔穿刺液（胸水、腹水）またはリンパ液などが挙げられるが、好ましくは肺、髄膜、リンパ節、血液（血清、血漿等）、尿、髄液、体腔穿刺液（胸水、腹水）またはリンパ液、より好ましくは血液（血清、血漿等）またはリンパ液、特に好ましくは血清である。生体検体が血清の場合、ＬＲＧの検出を容易にするために、予め発現量の高い血清タンパク質、例えばアルブミン、免疫グロブリンＧ（ＩｇＧ）、トランスフェリン、免疫グロブリンＡ（ＩｇＡ）、ハプトグロビン、α１アンチトリプシン、フィブリノゲン、α２マクログロブリン、免疫グロブリンＭ（ＩｇＭ）、α１−酸性糖タンパク質、補体Ｃ３、アポリポタンパク質ＡＩ、アポリポタンパク質ＡＩＩ、トランスサイレチンなどを除去するための前処理を行っても良い。 The biological specimen used in the examination method of the present invention is an LRG gene product (eg, RNA, protein, degradation product thereof, etc.) that is separated from the organism to be examined and is to be detected or quantified. Cell, tissue containing cells, blood (serum, plasma, etc.), urine, spinal fluid, body cavity puncture fluid (pleural effusion, ascites) or lymph, etc. are not particularly limited. For example, brain, meninges, spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, Spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue, skeletal muscle, lymph node, blood (serum, plasma, etc.), urine, cerebrospinal fluid, body cavity puncture fluid (pleural effusion) , Ascites) or lymph fluid, preferably lung, meninges, lymph nodes, blood (serum, plasma, etc.), urine, spinal fluid, puncture fluid (pleural fluid, ascites) or lymph fluid, more preferably blood ( Serum, plasma, etc.) or lymph, particularly preferably serum. When the biological specimen is serum, in order to facilitate the detection of LRG, serum proteins with high expression levels such as albumin, immunoglobulin G (IgG), transferrin, immunoglobulin A (IgA), haptoglobin, α1 antitrypsin, A pretreatment for removing fibrinogen, α2 macroglobulin, immunoglobulin M (IgM), α1-acid glycoprotein, complement C3, apolipoprotein AI, apolipoprotein AII, transthyretin and the like may be performed.
生体から分離した生体検体におけるＬＲＧの検出または定量は、該生体検体からＲＮＡ（例：全ＲＮＡ、ｍＲＮＡ）画分を調製し、該画分中に含まれるＬＲＧ遺伝子の転写産物を検出または定量することにより調べることができる。
従って、一実施態様において、本発明の検査方法は、ＬＲＧ遺伝子の転写産物を特異的に検出し得る核酸プローブまたは核酸プライマーを用いて測定することを特徴とする。
For detection or quantification of LRG in a biological specimen separated from a living body, an RNA (eg, total RNA, mRNA) fraction is prepared from the biological specimen, and a transcript of the LRG gene contained in the fraction is detected or quantified. Can be examined.
Therefore, in one embodiment, the test method of the present invention is characterized by measurement using a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of the LRG gene.
ＲＮＡ画分の調製は、グアニジン−ＣｓＣｌ超遠心法、ＡＧＰＣ法など公知の手法を用いて行うことができるが、市販のＲＮＡ抽出用キット（例：ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ；ＱＩＡＧＥＮ製等）を用いて、微量生体検体から迅速且つ簡便に高純度の全ＲＮＡを調製することができる。ＲＮＡ画分中のＬＲＧ遺伝子の転写産物を検出する手段としては、例えば、ハイブリダイゼーション（ノーザンブロット、ドットブロット、ＤＮＡチップ解析等）を用いる方法、あるいはＰＣＲ（ＲＴ−ＰＣＲ、競合ＰＣＲ、リアルタイムＰＣＲ等）を用いる方法などが挙げられる。微量生体検体から迅速且つ簡便に定量性よくＬＲＧ遺伝子の発現変動を検出できる点で競合ＰＣＲやリアルタイムＰＣＲなどの定量的ＰＣＲ法が好ましい。 The RNA fraction can be prepared using a known method such as guanidine-CsCl ultracentrifugation, AGPC, etc., but using a commercially available RNA extraction kit (eg, RNeasy Mini Kit; manufactured by QIAGEN, etc.) High-purity total RNA can be prepared quickly and easily from a small amount of biological specimen. As a means for detecting the transcription product of the LRG gene in the RNA fraction, for example, a method using hybridization (Northern blot, dot blot, DNA chip analysis, etc.) or PCR (RT-PCR, competitive PCR, real-time PCR, etc.) ) And the like. Quantitative PCR methods such as competitive PCR and real-time PCR are preferred because they can detect changes in the expression of the LRG gene quickly and easily from a small amount of biological specimen with high quantitativeness.
ノーザンブロットまたはドットブロットハイブリダイゼーションによる場合、ＬＲＧ遺伝子の検出は、例えば、ＬＲＧの転写産物を特異的に検出し得る核酸プローブを用いて行うことができる。そのような核酸プローブは、前述の公知のＬＲＧヌクレオチド配列に含まれる、約１５塩基以上、好ましくは約１８〜約５００塩基、より好ましくは約１８〜約２００塩基、いっそう好ましくは約１８〜約５０塩基の連続したヌクレオチド配列またはその相補配列を含むポリヌクレオチドである。該核酸はＤＮＡであってもＲＮＡであってもよく、あるいはＤＮＡ／ＲＮＡキメラであってもよい。好ましくはＤＮＡが挙げられる。また、プローブとして用いられる核酸は、二本鎖であっても一本鎖であってもよい。二本鎖の場合は、二本鎖ＤＮＡ、二本鎖ＲＮＡまたはＤＮＡ：ＲＮＡのハイブリッドでもよい。一本鎖の場合は、アンチセンス鎖を用いることができる。 In the case of Northern blot or dot blot hybridization, the detection of the LRG gene can be performed using, for example, a nucleic acid probe that can specifically detect the LRG transcript. Such a nucleic acid probe has about 15 bases or more, preferably about 18 to about 500 bases, more preferably about 18 to about 200 bases, more preferably about 18 to about 50, contained in the aforementioned known LRG nucleotide sequence. A polynucleotide comprising a contiguous nucleotide sequence of bases or a complementary sequence thereof. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The nucleic acid used as the probe may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, an antisense strand can be used.
また、本発明の検査方法に用いられる核酸プローブは、前述の公知のＬＲＧについて示されるヌクレオチド配列にストリンジェントな条件下でハイブリダイズするヌクレオチド配列を含むポリヌクレオチドである。ハイブリダイゼーションは、自体公知の方法あるいはそれに準じる方法、例えば、モレキュラー・クローニング（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ）第２版（Ｊ．Ｓａｍｂｒｏｏｋ ｅｔ ａｌ．，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂ．Ｐｒｅｓｓ，１９８９）に記載の方法などに従って行なうことができる。ストリンジェントな条件としては、例えば、６×ＳＳＣ（ｓｏｄｉｕｍ ｃｈｌｏｒｉｄｅ／ｓｏｄｉｕｍ ｃｉｔｒａｔｅ）中４５℃でのハイブリダイゼーション反応の後、０．２×ＳＳＣ／０．１％ＳＤＳ中６５℃での一回以上の洗浄などが挙げられる。当業者は、ハイブリダイゼーション溶液の塩濃度、ハイブリダゼーション反応の温度、プローブ濃度、プローブの長さ、ミスマッチの数、ハイブリダイゼーション反応の時間、洗浄液の塩濃度、洗浄の温度等を適宜変更することにより、所望のストリンジェンシーに容易に調節することができる。 Further, the nucleic acid probe used in the test method of the present invention is a polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence shown with the known LRG above. Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. The stringent conditions include, for example, a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by one or more times at 65 ° C. in 0.2 × SSC / 0.1% SDS. For example, washing. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.
ＬＲＧ遺伝子の発現を検出し得るプローブとして機能する核酸は、該遺伝子の転写産物の一部もしくは全部を増幅し得る後述するプライマーセットを用い、生体（例：ヒト、サル、マウス、ラット、イヌ、ウシ、ウマ、ブタ、ヒツジ、ヤギ、ネコ、ウサギ、ハムスター、モルモット等）のあらゆる細胞［例えば、肝細胞、脾細胞、神経細胞、グリア細胞、膵臓β細胞、骨髄細胞、メサンギウム細胞、ランゲルハンス細胞、表皮細胞、上皮細胞、杯細胞、内皮細胞、平滑筋細胞、線維芽細胞、線維細胞、筋細胞、脂肪細胞、免疫細胞（例、マクロファージ、Ｔ細胞、Ｂ細胞、ナチュラルキラー細胞、肥満細胞、好中球、好塩基球、好酸球、単球）、巨核球、滑膜細胞、軟骨細胞、骨細胞、骨芽細胞、破骨細胞、乳腺細胞もしくは間質細胞、またはこれら細胞の前駆細胞、幹細胞もしくは癌細胞など］もしくはそれらの細胞が存在するあらゆる組織［例えば、脳、脳の各部位（例、嗅球、扁桃核、大脳基底球、海馬、視床、視床下部、大脳皮質、延髄、小脳）、脊髄、下垂体、胃、膵臓、腎臓、肝臓、生殖腺、甲状腺、胆嚢、骨髄、副腎、皮膚、肺、消化管（例、大腸、小腸）、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子宮、骨、関節、脂肪組織、骨格筋など］由来のｃＤＮＡもしくはゲノムＤＮＡを鋳型としてＰＣＲ法によって所望の長さの核酸を増幅するか、前記した細胞・組織由来のｃＤＮＡもしくはゲノムＤＮＡライブラリーから、コロニーもしくはプラークハイブリダイゼーション等により上記ＬＲＧ遺伝子もしくはｃＤＮＡをクローニングし、必要に応じて制限酵素等を用いて適当な長さの断片とすることにより取得することができる。ハイブリダイゼーションは、例えば、モレキュラー・クローニング（Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ）第２版（前述）に記載の方法などに従って行なうことができる。あるいは、該核酸は、公知のＬＲＧについて示される塩基配列情報に基づいて、該塩基配列および／またはその相補鎖配列の一部もしくは全部を市販のＤＮＡ／ＲＮＡ自動合成機等を用いて化学的に合成することによっても得ることができる。 A nucleic acid that functions as a probe that can detect the expression of the LRG gene uses a primer set that can amplify a part or all of the transcription product of the gene, and a living body (eg, human, monkey, mouse, rat, dog, Any cell of bovine, horse, pig, sheep, goat, cat, rabbit, hamster, guinea pig, etc. [eg, hepatocyte, spleen cell, nerve cell, glial cell, pancreatic β cell, bone marrow cell, mesangial cell, Langerhans cell, Epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, favorable cells Neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells, or Progenitor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells exist [eg, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebrum) Cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, Spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue, skeletal muscle, etc.] Alternatively, the above LRG gene or cDNA is cloned from the above-mentioned cDNA or genomic DNA library derived from cells or tissues by colony or plaque hybridization. If necessary, it can be obtained by making a fragment of an appropriate length using a restriction enzyme or the like. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (described above). Alternatively, the nucleic acid based on the nucleotide sequence information shown with the known LRG, chemical part or all of the nucleotide sequence and / or its complementary strand sequence using a commercially available automated DNA / RNA synthesizer, etc. It can also be obtained by synthesis.
該核酸は、標的核酸の検出・定量を可能とするために、標識剤により標識されていることが好ましい。標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、〔３２Ｐ〕、〔３Ｈ〕、〔１４Ｃ〕などが用いられる。酵素としては、安定で比活性の大きなものが好ましく、例えば、β−ガラクトシダーゼ、β−グルコシダーゼ、アルカリホスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。さらに、プローブと標識剤との結合にビオチン−（ストレプト）アビジンを用いることもできる。 The nucleic acid is preferably labeled with a labeling agent in order to enable detection and quantification of the target nucleic acid. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ 32 P], [ 3 H], [ 14 C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, biotin- (strept) avidin can also be used for the binding between the probe and the labeling agent.
ノーザンハイブリダイゼーションによる場合は、上記のようにして調製したＲＮＡ画分をゲル電気泳動にて分離した後、ニトロセルロース、ナイロン、ポリビニリデンジフロリド等のメンブレンに転写し、上記のようにして調製された標識プローブを含むハイブリダイゼーション緩衝液中、特異的にハイブリダイゼーションさせた後、適当な方法でメンブレンに結合した標識量をバンド毎に測定することにより、ＬＲＧ遺伝子の発現量を測定することができる。ドットブロットの場合も、ＲＮＡ画分をスポットしたメンブレンを同様にハイブリダイゼーション反応に付し、スポットの標識量を測定することにより、ＬＲＧ遺伝子の発現量を測定することができる。 In the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis, then transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, and prepared as described above. After specifically hybridizing in a hybridization buffer containing the labeled probe, the amount of LRG gene expressed can be measured by measuring the amount of label bound to the membrane for each band by an appropriate method. it can. Also in the case of dot blotting, the expression level of the LRG gene can be measured by subjecting a membrane spotted with the RNA fraction to a hybridization reaction in the same manner and measuring the amount of the label on the spot.
別の好ましい実施態様によれば、ＬＲＧ遺伝子の発現を測定する方法として定量的ＰＣＲ法が用いられる。定量的ＰＣＲとしては、例えば、競合ＰＣＲやリアルタイムＰＣＲなどがある。
ＰＣＲにおいてプライマーとして用いられるオリゴヌクレオチドのセットとしては、例えば、ＬＲＧ遺伝子の転写産物を特異的に検出し得る核酸プライマーを挙げることができる。１つの好ましい態様においては、本発明の検査方法に用いられる核酸プライマーとしては、例えば、公知のＬＲＧについて示されるヌクレオチド配列に含まれる、約１５塩基以上、好ましくは約１５〜約５０塩基、より好ましくは約１５〜約３０塩基、いっそう好ましくは約１５〜約２５塩基の連続したヌクレオチド配列の長さを有し、約１００ｂｐ〜数ｋｂｐのＤＮＡ断片を増幅するようにデザインされたポリヌクレオチド（センス鎖）配列に相補的なポリヌクレオチド、及び前記のポリヌクレオチド配列に相補的なヌクレオチド配列を有するポリヌクレオチド（アンチセンス鎖）にハイブリダイズし得るポリヌクレオチドのオリゴヌクレオチドのセットが挙げられる。
According to another preferred embodiment, a quantitative PCR method is used as a method for measuring the expression of the LRG gene. Examples of quantitative PCR include competitive PCR and real-time PCR.
Examples of the set of oligonucleotides used as primers in PCR include nucleic acid primers that can specifically detect a transcription product of the LRG gene . In one preferred embodiment, the nucleic acid primer for use in the inspection method of the present invention, for example, included in the nucleotide sequence shown with the known LRG, about 15 bases or more, preferably about 15 to about 50 bases, more A polynucleotide designed to amplify a DNA fragment of about 100 bp to several kbp, preferably having a length of a continuous nucleotide sequence of about 15 to about 30 bases, more preferably about 15 to about 25 bases (sense And a set of polynucleotide oligonucleotides that can hybridize to a polynucleotide (antisense strand) having a nucleotide sequence complementary to the polynucleotide sequence.
あるいは、生体から分離した生体検体におけるＬＲＧの検出または定量は、該生体検体からタンパク質画分を調製し、該画分中に含まれる該遺伝子の翻訳産物（即ち、ＬＲＧ）を検出または定量することにより調べることができる。ＬＲＧの検出または定量は、ＬＲＧを特異的に認識する抗体を用いて、免疫学的測定法（例：ＥＬＩＳＡ、ＦＩＡ、ＲＩＡ、ウェスタンブロット等）によって行うこともできる。
従って、一実施態様において、本発明の検査方法は、ＬＲＧ遺伝子の翻訳産物を特異的に検出し得る抗体を用いて測定することを特徴とする。
Alternatively, the detection or quantification of LRG in a biological specimen separated from a living body involves preparing a protein fraction from the biological specimen and detecting or quantifying the translation product (ie, LRG) of the gene contained in the fraction. Can be examined. Detection or quantification of LRG can also be performed by an immunological assay (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody that specifically recognizes LRG.
Therefore, in one embodiment, the test method of the present invention is characterized by measuring using an antibody capable of specifically detecting the translation product of the LRG gene .
ＬＲＧを特異的に認識する抗体は、ＬＲＧポリペプチドやその抗原性を有する部分ペプチド、具体的には、前述の公知のＬＲＧに示されるペプチド配列の全部またはエピトープに当たる部分を有する部分ペプチドを免疫原として用い、既存の一般的な製造方法によって製造することができる。本明細書において、抗体には、ポリクローナル抗体、モノクローナル抗体（ｍＡｂ）等の天然型抗体、遺伝子組換技術を用いて製造され得るキメラ抗体、ヒト化抗体や一本鎖抗体、およびこれらの結合性断片が含まれるが、これらに限定されない。好ましくは、抗体はポリクローナル抗体、モノクローナル抗体又はこれらの結合性断片である。結合性断片とは、特異的結合活性を有する前述の抗体の一部分の領域を意味し、具体的には例えばＦ（ａｂ’）２、Ｆａｂ’、Ｆａｂ、Ｆｖ、ｓＦｖ、ｄｓＦｖ、ｓｄＡｂ等が挙げられる（Ｅｘｐ．Ｏｐｉｎ．Ｔｈｅｒ．Ｐａｔｅｎｔｓ，Ｖｏｌ．６，Ｎｏ．５，ｐ．４４１−４５６，１９９６）。抗体のクラスは、特に限定されず、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤあるいはＩｇＥ等のいずれのアイソタイプを有する抗体をも包含する。好ましくは、ＩｇＧ又はＩｇＭであり、精製の容易性等を考慮するとより好ましくはＩｇＧである。 An antibody specifically recognizing LRG is an immunogen derived from an LRG polypeptide or a partial peptide having antigenicity thereof, specifically, a partial peptide having a portion corresponding to all of the peptide sequence shown in the aforementioned known LRG or an epitope. And can be manufactured by an existing general manufacturing method. In the present specification, the antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody or a single chain antibody, and binding properties thereof. Fragments are included, but are not limited to these. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) 2 , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.
個々の免疫学的測定法を本発明の検査方法に適用するにあたっては、特別の条件、操作等の設定は必要とされない。それぞれの方法における通常の条件、操作法に当業者の通常の技術的配慮を加えてＬＲＧの測定系を構築すればよい。これらの一般的な技術手段の詳細については、総説、成書などを参照することができる。例えば、入江寛編「ラジオイムノアッセイ」（講談社、昭和４９年発行）、入江寛編「続ラジオイムノアッセイ」（講談社、昭和５４年発行）、石川栄治ら編「酵素免疫測定法」（医学書院、昭和５３年発行）、石川栄治ら編「酵素免疫測定法」（第２版）（医学書院、昭和５７年発行）、石川栄治ら編「酵素免疫測定法」（第３版）（医学書院、昭和６２年発行）、「Ｍｅｔｈｏｄｓ ｉｎ ＥＮＺＹＭＯＬＯＧＹ」Ｖｏｌ．７０（Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｔｅｃｈｎｉｑｕｅｓ（ＰａｒｔＡ））、同書Ｖｏｌ．７３（Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｔｅｃｈｎｉｑｕｅｓ（ＰａｒｔＢ））、同書Ｖｏｌ．７４（Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｔｅｃｈｎｉｑｕｅｓ（ＰａｒｔＣ））、同書Ｖｏｌ．８４（Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｔｅｃｈｎｉｑｕｅｓ（ＰａｒｔＤ：Ｓｅｌｅｃｔｅｄ Ｉｍｍｕｎｏａｓｓａｙｓ））、同書Ｖｏｌ．９２（Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｔｅｃｈｎｉｑｕｅｓ（ＰａｒｔＥ：Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｉｅｓ ａｎｄ Ｇｅｎｅｒａｌ Ｉｍｍｕｎｏａｓｓａｙ Ｍｅｔｈｏｄｓ））、同書Ｖｏｌ．１２１（Ｉｍｍｕｎｏｃｈｅｍｉｃａｌ Ｔｅｃｈｎｉｑｕｅｓ（ＰａｒｔＩ：Ｈｙｂｒｉｄｏｍａ Ｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｉｅｓ））（以上、アカデミックプレス社発行）などを参照することができる。 In applying each immunological measurement method to the test method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of LRG, adding the normal technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like. For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62)), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Technologies (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Technologies (Part E: Monoclonal Antibodies and General Immunoassay Methods)), Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technologies and Monoclonal Antibodies)) (published by Academic Press).
また、別の実施態様では、ＬＲＧの検出は、ハイスループットなタンパク質の発現・定量解析が可能なｉＴＲＡＱＴＭ試薬（ＡＢＩ社）及び質量分析計の組み合わせによりプロテオーム解析を用いて測定することを特徴とする。 In another embodiment, the detection of LRG is characterized by measurement using proteome analysis with a combination of iTRAQ TM reagent (ABI) capable of high-throughput protein expression / quantitative analysis and a mass spectrometer. To do.
本発明の結核検査方法において、ＬＲＧの検出又は定量により、結核の検査を行うことができる。結核の検査には、治療後の疾患活動性の評価や、生物学的製剤による治療をうける自己免疫疾患において結核発症の検査を行うことも含まれる。関節リウマチなどの自己免疫疾患においてＩＬ−６受容体阻害抗体など生物学的製剤による治療を受ける患者においては、ＩＬ−６シグナルが阻害されるため、副作用として感染症併発のリスクが高くなる。そして、感染症を併発した患者においてはＩＬ−６シグナルが遮断されているため、ＩＬ−６により発現誘導が起きるＣＲＰなどの感染症の血清マーカーは常に正常値を示すため、感染症検出マーカーとなり得ない。ＩＬ−６受容体阻害抗体など生物学的製剤治療を受ける自己免疫疾患患者において、疾患活動性に変化がないが、血中ＬＲＧ濃度が上昇した際には結核をはじめとする感染症併発が強く疑われるため、このような状況において血中ＬＲＧ濃度が上昇した際は結核をはじめとする感染症検出マーカーとなり得る。ここで、ＩＬ−６阻害療法とはＩＬ−６やその受容体に対する阻害抗体を含む治療法を意味する。 In the tuberculosis test method of the present invention, tuberculosis can be tested by detecting or quantifying LRG. Tuberculosis testing includes assessment of disease activity after treatment and testing for the development of tuberculosis in autoimmune diseases that are treated with biologics. In autoimmune diseases such as rheumatoid arthritis, in patients treated with biological preparations such as IL-6 receptor-inhibiting antibodies, IL-6 signaling is inhibited, which increases the risk of concurrent infection as a side effect. Since the IL-6 signal is blocked in patients with concurrent infections, serum markers for infections such as CRP, whose expression is induced by IL-6, always show normal values, and thus serve as detection markers for infections. I don't get it. There is no change in disease activity in patients with autoimmune diseases such as IL-6 receptor-inhibiting antibodies, but there is a strong complication of infections such as tuberculosis when the blood LRG concentration increases. Since it is suspected, when the blood LRG concentration increases in such a situation, it can be a marker for detection of infectious diseases including tuberculosis. Here, IL-6 inhibitory therapy means a therapeutic method including an inhibitory antibody against IL-6 or its receptor.
後述の実施例に示されるように、健常人と比較して結核の患者において血清中のＬＲＧ濃度が高い。結核の検査は、ＬＲＧの濃度と結核への罹患率との間のこのような正の相関に基づき行われる。 As shown in Examples described later, serum LRG concentrations are higher in tuberculosis patients than in healthy individuals. Tuberculosis testing is based on such a positive correlation between the concentration of LRG and the prevalence of tuberculosis.
例えば、健常人及び対象患者からの生体検体におけるＬＲＧの濃度を定量し、対象患者からの生体検体におけるＬＲＧの濃度を、健常人からの生体検体におけるＬＲＧの濃度と比較する。あるいは、ＬＲＧの濃度と結核の罹患の有無との相関図をあらかじめ作成しておき、対象患者におけるＬＲＧ濃度をその相関図と比較してもよい。濃度の比較は、好ましくは、有意差の有無に基づいて行われる。 For example, the LRG concentration in the biological sample from the healthy subject and the target patient is quantified, and the LRG concentration in the biological sample from the target patient is compared with the LRG concentration in the biological sample from the healthy subject. Alternatively, a correlation diagram between the LRG concentration and the presence or absence of tuberculosis may be created in advance, and the LRG concentration in the target patient may be compared with the correlation diagram. The concentration comparison is preferably performed based on the presence or absence of a significant difference.
そして、対象患者においてＬＲＧが、健常人に比べて高値で検出若しくは定量された場合には、上記のような結核に罹患している可能性が高いと判断することができる。 And when LRG is detected or quantified in a target patient at a high value compared with a healthy person, it can be judged that there is a high possibility of having tuberculosis as described above.
また、後述の実施例に示されるように、疾患活動性と、ＬＲＧの濃度との関係について解析した結果、結核患者の血清において、疾患活動性の高い患者は、ＬＲＧが有意に高値を示したことが確認された。結核の疾患活動性の評価は、ＬＲＧ濃度と疾患活動性との間のこのような正の相関に基づき行われる。 In addition, as shown in the examples described later, as a result of analyzing the relationship between disease activity and LRG concentration, patients with high disease activity showed significantly higher LRG values in the serum of tuberculosis patients. It was confirmed. Assessment of tuberculosis disease activity is based on such a positive correlation between LRG concentration and disease activity.
例えば、活動性の高い結核の患者及び、活動性の低い結核の患者から、生体検体を採取し、対象患者から採取された生体検体におけるＬＲＧ濃度が活動性の高い結核の患者及び活動性の低い結核の患者のそれと比較される。あるいは、生体検体におけるＬＲＧ濃度と結核の疾患活動性との間の相関図をあらかじめ作成しておき、対象患者から採取された生体検体におけるＬＲＧ濃度をその相関図と比較してもよい。濃度の比較は、好ましくは、有意差の有無に基づいて行われる。 For example, a biological sample is collected from a patient with high activity tuberculosis or a patient with low activity tuberculosis, and a patient with tuberculosis with high activity or low activity in an LRG concentration in the biological sample collected from the target patient. Compared with that of tuberculosis patients. Alternatively, a correlation diagram between the LRG concentration in the biological sample and the disease activity of tuberculosis may be prepared in advance, and the LRG concentration in the biological sample collected from the target patient may be compared with the correlation diagram. The concentration comparison is preferably performed based on the presence or absence of a significant difference.
そして、ＬＲＧ濃度の比較結果より、対象患者のＬＲＧ濃度が相対的に高い場合には、結核の疾患活動性が高いと判断することができる。 From the comparison result of the LRG concentration, when the target patient has a relatively high LRG concentration, it can be determined that the disease activity of tuberculosis is high.
したがって、イソニアジド（ＩＮＨ）、リファンピシン（ＲＩＦ）、ピラジナミド（ＰＺＡ）、エタンブトール（ＥＭＢ）またはストレプトマイシン（ＳＭ）などの抗結核治療薬投与に伴い、患者の血清ＬＲＧを定量し、解析することで、結核の疾患活動性を評価することができ、投与した結核治療薬の有効性や、薬剤抵抗性などを客観的に判断することができる。 Therefore, by quantifying and analyzing the patient's serum LRG with the anti-tuberculosis therapeutic agent such as isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (EMB) or streptomycin (SM), Therefore, the effectiveness of the administered tuberculosis drug and drug resistance can be objectively determined.
ここで、投与した結核治療薬の有効性や、薬剤抵抗性などを客観的に判断するための検査は、結核治療薬の投与前後に採取して得た血清ＬＲＧを定量し、各ＬＲＧ量を比較することにより、行うことができる。 Here, the test for objectively judging the effectiveness and drug resistance of the administered tuberculosis drug is performed by quantifying the serum LRG collected before and after the administration of the tuberculosis drug, and calculating the amount of each LRG. This can be done by comparison.
具体的には、以下の工程を含む方法による。
１）結核治療剤の投与前及び投与後に対象患者から生体検体（例、血清）を分離取得し、各検査用検体とする工程；
２）上記１）の検査用検体について、本発明の結核検査方法により検査を行う工程；
３）上記２）で得られた結果をもとに、結核治療剤の投与前及び投与後の各検査用検体中のＬＲＧ量比を算出する工程。
Specifically, the method includes the following steps.
1) A step of separating and obtaining a biological sample (eg, serum) from a target patient before and after administration of a tuberculosis therapeutic agent, and using it as a test sample;
2) A step of examining the test specimen of 1) by the tuberculosis test method of the present invention;
3) A step of calculating an LRG amount ratio in each test specimen before and after administration of the tuberculosis therapeutic agent based on the result obtained in 2) above.
更に、後述の実施例に示されるように、結核の患者において、生体検体中のＬＲＧ濃度が高いほど、結核の疾患活動性が高い。このような、ＬＲＧ濃度と結核の疾患活動性との間の正の相関に基づき、結核に対する抗結核薬による治療効果を評価することが出来る。 Furthermore, as shown in the Examples described later, in tuberculosis patients, the higher the LRG concentration in a biological sample, the higher the disease activity of tuberculosis. Based on such a positive correlation between the LRG concentration and the disease activity of tuberculosis, the therapeutic effect of antituberculous drugs against tuberculosis can be evaluated.
たとえば、結核治療薬による治療前、および治療後の患者から生体検体を採取し、対象患者から採取された生体検体におけるＬＲＧ発現量について治療前の発現量と比較される。濃度の比較は、好ましくは、有意差の有無に基づいて行われる。 For example, a biological sample is collected from a patient before and after treatment with a tuberculosis therapeutic agent, and the LRG expression level in the biological sample collected from the target patient is compared with the expression level before treatment. The concentration comparison is preferably performed based on the presence or absence of a significant difference.
そして、ＬＲＧ発現量の比較結果より治療後の患者のＬＲＧ発現量が、治療前の患者より相対的に低い場合には、結核治療薬による治療効果があったと判断することができる。 When the LRG expression level of the patient after treatment is relatively lower than that of the patient before treatment from the comparison result of the LRG expression level, it can be determined that there is a therapeutic effect by the tuberculosis therapeutic agent.
さらに、本発明は、結核検査用のキット（診断剤）にも及ぶ。本発明の結核検査用キットは、上述の本発明の検査方法（生物学的製剤による治療を受ける自己免疫疾患患者の結核発症の検出マーカーを含む）を簡便に実施するためのキットであればよく、特に限定されない。該検査するためのキットは、
（ａ）ＬＲＧ遺伝子の転写産物を特異的に検出し得る核酸プローブまたは核酸プライマー、または
（ｂ）ＬＲＧを特異的に認識する抗体
を含有してなる。該判定するためのキットが２以上の上記核酸および／または抗体を含む場合、各核酸または抗体は互いにＬＲＧ遺伝子の塩基配列上の異なる部分を特異的に認識、またはＬＲＧの異なるエピトープを特異的に認識し得るものである。
Furthermore, the present invention extends to a kit (diagnostic agent) for tuberculosis examination. The tuberculosis test kit of the present invention may be any kit for simply carrying out the above-described test method of the present invention (including a detection marker for the onset of tuberculosis in an autoimmune disease patient treated with a biological preparation). There is no particular limitation. The test kit is
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of the LRG gene, or (b) an antibody specifically recognizing LRG. When the determination kit includes two or more nucleic acids and / or antibodies, each nucleic acid or antibody specifically recognizes a different part of the base sequence of the LRG gene from each other or specifically recognizes a different epitope of LRG. It can be recognized.
本発明のキットが前記（ａ）の核酸を含有する試薬を構成として含む場合、該核酸としては、本発明の検査方法において前記したプローブ用核酸もしくはプライマー用オリゴヌクレオチドが挙げられる。 When the kit of the present invention includes the reagent containing the nucleic acid (a) as a component, examples of the nucleic acid include the probe nucleic acid or the primer oligonucleotide described above in the test method of the present invention.
ＬＲＧ遺伝子の発現を検出し得る核酸は、乾燥した状態もしくはアルコール沈澱の状態で、固体として提供することもできるし、水もしくは適当な緩衝液（例：ＴＥ緩衝液等）中に溶解した状態で提供することもできる。標識プローブとして用いられる場合、該核酸は予め上記のいずれかの標識物質で標識した状態で提供することもできるし、標識物質とそれぞれ別個に提供され、用時標識して用いることもできる。
あるいは、該核酸は、適当な固相に固定化された状態で提供することもできる。固相としては、例えば、ガラス、シリコン、プラスチック、ニトロセルロース、ナイロン、ポリビニリデンジフロリド等が挙げられるが、これらに限定されない。また、固定化手段としては、予め核酸にアミノ基、アルデヒド基、ＳＨ基、ビオチンなどの官能基を導入しておき、一方、固相上にも該核酸と反応し得る官能基（例：アルデヒド基、アミノ基、ＳＨ基、ストレプトアビジンなど）を導入し、両官能基間の共有結合で固相と核酸を架橋したり、ポリアニオン性の核酸に対して、固相をポリカチオンコーティングして静電結合を利用して核酸を固定化するなどの方法が挙げられるが、これらに限定されない。
The nucleic acid capable of detecting the expression of the LRG gene can be provided as a solid in a dry state or in an alcohol precipitated state, or dissolved in water or a suitable buffer (eg, TE buffer). It can also be provided. When used as a labeled probe, the nucleic acid can be provided in a state of being previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substance and can be used after labeling.
Alternatively, the nucleic acid can be provided in a state immobilized on an appropriate solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like. As immobilization means, a functional group such as an amino group, an aldehyde group, an SH group, or biotin is introduced into a nucleic acid in advance, and a functional group capable of reacting with the nucleic acid on a solid phase (eg, aldehyde) Group, amino group, SH group, streptavidin, etc.), and the solid phase and the nucleic acid are cross-linked by covalent bond between the two functional groups, or the polyanionic nucleic acid is coated with the polycation and the static phase is coated. Examples of the method include immobilization of nucleic acid using electric coupling, but are not limited thereto.
該検査するためのキットに含有される核酸は、同一の方法（例：ノーザンブロット、ドットブロット、ＤＮＡアレイ技術、定量ＲＴ−ＰＣＲ等）によりＬＲＧ遺伝子の発現を検出し得るように構築されていることが特に好ましい。 The nucleic acid contained in the test kit is constructed so that the expression of the LRG gene can be detected by the same method (eg, Northern blot, dot blot, DNA array technology, quantitative RT-PCR, etc.). It is particularly preferred.
本発明のキットが前記（ｂ）の抗体を含有する試薬を構成として含む場合、該抗体としては、本発明の検査方法において前記した抗体が挙げられる。 When the kit of the present invention contains the reagent containing the antibody (b) as a component, examples of the antibody include the antibodies described above in the test method of the present invention.
本発明のキットを構成する試薬は、ＬＲＧ遺伝子の発現を検出し得る核酸や抗体に加えて、該遺伝子の発現を検出するための反応において必要な他の物質であって、共存状態で保存することにより反応に悪影響を及ぼさない物質をさらに含有することができる。あるいは、該試薬は、ＬＲＧ遺伝子の発現を検出するための反応において必要な他の物質を含有する別個の試薬とともに提供されてもよい。例えば、ＬＲＧ遺伝子の発現を検出するための反応がＰＣＲの場合、当該他の物質としては、例えば、反応緩衝液、ｄＮＴＰｓ、耐熱性ＤＮＡポリメラーゼ等が挙げられる。競合ＰＣＲやリアルタイムＰＣＲを用いる場合は、ｃｏｍｐｅｔｉｔｏｒ核酸や蛍光試薬（上記インターカレーターや蛍光プローブ等）などをさらに含むことができる。また、ＬＲＧ遺伝子の発現を検出するための反応が抗原抗体反応の場合、当該他の物質としては、例えば、反応緩衝液、ｃｏｍｐｅｐｔｉｔｏｒ抗体、標識された二次抗体（例えば、一次抗体がウサギ抗ヒトＬＲＧ抗体の場合、ペルオキシダーゼやアルカリホスファターゼ等で標識されたマウス抗ウサギＩｇＧなど）、ブロッキング液等が挙げられる。プロテオーム解析による場合は、市販の試薬、例えばｉＴＲＡＱＴＭ試薬やＩＣＡＴＴＭ試薬（いずれもＡＢＩ社）などを用いることができる。 The reagent constituting the kit of the present invention is a substance other than a nucleic acid and an antibody that can detect the expression of the LRG gene and other substances necessary for the reaction for detecting the expression of the gene, and is stored in a coexisting state. Thus, a substance that does not adversely influence the reaction can be further contained. Alternatively, the reagent may be provided with a separate reagent containing other substances necessary in the reaction for detecting the expression of the LRG gene. For example, when the reaction for detecting the expression of the LRG gene is PCR, examples of the other substance include a reaction buffer, dNTPs, and a heat-resistant DNA polymerase. When competing PCR or real-time PCR is used, it can further include a competitor nucleic acid, a fluorescent reagent (such as the above intercalator and fluorescent probe), and the like. In addition, when the reaction for detecting the expression of the LRG gene is an antigen-antibody reaction, examples of the other substance include a reaction buffer, a competent antibody, and a labeled secondary antibody (for example, the primary antibody is a rabbit anti-human). In the case of an LRG antibody, mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), blocking solution, etc. may be mentioned. In the case of proteome analysis, commercially available reagents such as iTRAQ TM reagent and ICAT TM reagent (both ABI) can be used.
以下に、実施例を示して本発明をさらに具体的に説明するが、本発明はこれらにより限定されるものではないことは明らかである。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, it is obvious that the present invention is not limited to these examples.
（実施例１）結核患者のＥＬＩＳＡ法による血清ＬＲＧの評価
結核患者（ＴＢ患者）４０名、健常者（ＨＣ）５０名、合計９０名より採血し、血清を調製した。血清は１０００倍に希釈し、血清中ＬＲＧ濃度をＥＬＩＳＡ法により測定した。ＥＬＩＳＡにはＨｕｍａｎ ＬＲＧ Ａｓｓａｙ Ｋｉｔ（ＩＢＬ社）を使用した。
(Example 1) Evaluation of serum LRG by the ELISA method for tuberculosis patients Blood was collected from 40 tuberculosis patients (TB patients) and 50 healthy persons (HC) in total, and serum was prepared. The serum was diluted 1000 times, and the serum LRG concentration was measured by ELISA. For the ELISA, Human LRG Assay Kit (IBL) was used.
その結果、血清中ＬＲＧ濃度が、結核患者において有意に高いことが確認できた（表１・図１）。また、治療に伴って有意に血中ＬＲＧ濃度が低下することが確認された（表１・図１）。これにより、ＬＲＧ濃度の検査は、結核を診断するバイオマーカーとなり得ることが確認された。 As a result, it was confirmed that the serum LRG concentration was significantly higher in tuberculosis patients (Table 1 and FIG. 1). It was also confirmed that the blood LRG concentration significantly decreased with treatment (Table 1 and FIG. 1). Thereby, it was confirmed that the test | inspection of a LRG density | concentration can become a biomarker which diagnoses tuberculosis.
（実施例２）結核患者の治療前・治療後におけるＥＬＩＳＡ法による血清ＬＲＧの評価 ４０名の結核患者（ＴＢ患者）より結核治療薬による治療前と治療後（１ヶ月後、３ヶ月後）に採血し、血清を調製し、それぞれ血清は１０００倍に希釈し、血清ＬＲＧ濃度を測定した。 (Example 2) Evaluation of serum LRG by ELISA method before and after treatment of tuberculosis patients Before and after treatment with tuberculosis drugs (1 month later, 3 months later) from 40 tuberculosis patients (TB patients) Blood was collected and serum was prepared. Each serum was diluted 1000 times and the serum LRG concentration was measured.
その結果、血清中ＬＲＧ濃度が、結核治療薬による治療後の結核患者において、治療前に比べて血清中ＬＲＧ濃度が有意に低いことが確認された（表２・図２）。これにより、ＬＲＧ濃度の検査は、結核の疾患活動性・治療効果を評価するバイオマーカーとなり得ることが確認された。 As a result, it was confirmed that the serum LRG concentration was significantly lower in tuberculosis patients after treatment with a tuberculosis therapeutic agent than before treatment (Table 2 and FIG. 2). Thereby, it was confirmed that the test | inspection of a LRG density | concentration can become a biomarker which evaluates the disease activity and therapeutic effect of tuberculosis.
（実施例３）結核患者の疾患活動性とＥＬＩＳＡ法による血清ＬＲＧ
実施例２の結核患者の治療前・後の血清中ＬＲＧ濃度測定結果と、それぞれのＣＲＰ値及び塗抹陽性度を比較した。その結果、血清中ＬＲＧ濃度は、塗抹陽性度と相関がみられ、ＣＲＰ値は塗抹陽性度と相関がみられなかった（表３・図３）。血清中ＬＲＧ濃度は、ＣＲＰ値と比較し、より疾患活動性を反映することが示された。
(Example 3) Disease activity of tuberculosis patients and serum LRG by ELISA
The measurement results of serum LRG before and after treatment of the tuberculosis patient of Example 2 were compared with the respective CRP values and smear positivity. As a result, the serum LRG concentration was correlated with the smear positive degree, and the CRP value was not correlated with the smear positive degree (Table 3 and FIG. 3). Serum LRG concentrations were shown to reflect more disease activity compared to CRP values.
以上詳述したように、本発明の結核検査用バイオマーカーであるＬＲＧは、結核の検査用バイオマーカーとなりうる。ＬＲＧをバイオマーカーとして、検出又は定量することにより、上記結核の検査を行うことができ、疾患活動性を評価することができる。結核治療薬を投与したときの治療効果を評価しうるバイオマーカーともなる。具体的には、結核患者に結核治療薬を投与した場合の疾患活動性や、生物学的低製剤投与時の自己免疫疾患において結核発症の診断を本発明のバイオマーカーにより評価することができる。これにより、結核治療薬の有効性を確認したり、自己免疫疾患患者において生物学的製剤治療時の感染症合併を有する患者を早期に発見することができる。その結果、例えば薬剤投与終了時の判断や投与薬剤の変更などに対して、最も効果的な方法を選択することができる。 As described above in detail, the LRG that is a biomarker for tuberculosis testing of the present invention can be a biomarker for tuberculosis testing. By detecting or quantifying LRG as a biomarker, the above tuberculosis can be examined, and disease activity can be evaluated. It is also a biomarker that can evaluate the therapeutic effect when a tuberculosis therapeutic agent is administered. Specifically, the biomarker of the present invention can evaluate the disease activity when a tuberculosis therapeutic agent is administered to a tuberculosis patient and the diagnosis of the onset of tuberculosis in an autoimmune disease at the time of administration of a low biological preparation. Thereby, the effectiveness of a tuberculosis therapeutic agent can be confirmed, or a patient having an infectious disease complication at the time of treatment with a biologic agent can be found early in a patient with an autoimmune disease. As a result, for example, it is possible to select the most effective method for determination at the end of drug administration, change of administered drug, and the like.
Claims (18)
(i)疾患活動性の高い結核の患者および疾患活動性の低い結核の患者から分離した生体検体のＬＲＧ遺伝子産物の濃度、
(ii)予め作成された結核の疾患活動性とＬＲＧ遺伝子産物の濃度との間の相関図
と前記検出または定量されたＬＲＧ遺伝子産物の濃度を比較することをさらに含む、請求項９〜１４のいずれか１項に記載の方法。 (I) or (ii) below:
(i) the concentration of LRG gene product in biological specimens isolated from patients with tuberculosis with high disease activity and those with low disease activity;
(ii) pre further comprising correlation diagram between the concentration of disease activity and LRG gene product created tuberculosis and comparing the concentration of the detected or quantified LRG gene product of claim 9 to 14 The method according to any one of the above.
（ａ）ＬＲＧ遺伝子の転写産物を特異的に検出し得る核酸プローブまたは核酸プライマー
（ｂ）ＬＲＧを特異的に認識する抗体
を含む、結核の疾患活動性を判断するためのキット。 Below (a) or (b):
(A) a nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of an LRG gene; and (b) a kit for determining tuberculosis disease activity , comprising an antibody that specifically recognizes LRG.
（ａ）ＬＲＧ遺伝子の転写産物を特異的に検出し得る核酸プローブまたは核酸プライマー(A) a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of the LRG gene
（ｂ）ＬＲＧを特異的に認識する抗体(B) an antibody specifically recognizing LRG
を含む、結核治療薬を投与したときの治療効果を評価するためのキット。A kit for evaluating a therapeutic effect when a tuberculosis therapeutic agent is administered.
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