JPH02174699A - Hla type discrimination and reagent - Google Patents
Hla type discrimination and reagentInfo
- Publication number
- JPH02174699A JPH02174699A JP63311979A JP31197988A JPH02174699A JP H02174699 A JPH02174699 A JP H02174699A JP 63311979 A JP63311979 A JP 63311979A JP 31197988 A JP31197988 A JP 31197988A JP H02174699 A JPH02174699 A JP H02174699A
- Authority
- JP
- Japan
- Prior art keywords
- hla
- antigen
- restriction enzyme
- allospecificity
- type
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 238000001962 electrophoresis Methods 0.000 claims abstract description 9
- 102000006354 HLA-DR Antigens Human genes 0.000 claims description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 claims description 2
- 208000034454 F12-related hereditary angioedema with normal C1Inh Diseases 0.000 claims 3
- 208000016861 hereditary angioedema type 3 Diseases 0.000 claims 3
- 102000015789 HLA-DP Antigens Human genes 0.000 claims 1
- 108010010378 HLA-DP Antigens Proteins 0.000 claims 1
- 229940039227 diagnostic agent Drugs 0.000 claims 1
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- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000005549 deoxyribonucleoside Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 2
- 101100263837 Bovine ephemeral fever virus (strain BB7721) beta gene Proteins 0.000 description 1
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- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 1
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- 230000029087 digestion Effects 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
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- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
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Abstract
Description
【発明の詳細な説明】
〈産業上の利用分野〉
ヒトの主要組織適合性複合体である白血球膜抗原（ＨＬ
Ａ）は、臓器等の移植に重要なかかわりを持つ。本発明
は、この）ＩＬＡの型判別方法及びそれに用いられる試
薬にに関する。[Detailed Description of the Invention] <Industrial Application Field> Leukocyte membrane antigen (HL), which is a major histocompatibility complex in humans,
A) has an important role in organ transplantation. The present invention relates to a method for determining the type of ILA and reagents used therein.
〈従来の技術〉
ＨＬ Ａの型判別法は、従来、血清学的又は細胞学的方
法によって行われている。すなわち、ＨＬＡのクラス１
抗原であるＡ、Ｂ及びＣ並びにクラス■抗原のうちＤＲ
とＤＱの各抗原は血清学的方法、換言すれば特異抗体と
補体を用いたリンパ球障害テストにより型判別が行われ
ている。またクラ、Ｚ、 ［１抗原のうちＤ抗原は、Ｍ
Ｌ Ｃ（Ｍｉｘｅｄ ｌｙｍｐｈｏｃｙｔｅ ｃｕｌ
ｔｕｒｅ）法でＤＰ膜抗は、Ｐ Ｌ Ｔ （Ｐｒｉｍｅ
ｄｌｙｍｐｈｏｃｙｔｅ ｔｅｓｔｉｎｇ）法でそれぞ
れ判定されている。しかしながら、これらの方法は、判
定に要する時間及び手間が大きく、又、判定の精度も必
ずしも満足できるものではなかった。<Prior Art> HLA typing has conventionally been carried out by serological or cytological methods. That is, HLA class 1
Among the antigens A, B, and C and class II antigens, DR
and DQ antigens are typed by serological methods, in other words by lymphocyte damage tests using specific antibodies and complement. Also, Kura, Z. [Among 1 antigens, D antigen is M
L C (Mixed lymphocyte cul)
DP membrane anti-oxidant is prepared using PLT (Prime
They were determined using the dlymphocyte testing method. However, these methods require a large amount of time and effort to make the determination, and the accuracy of the determination is not necessarily satisfactory.
最近、ＨＬＡに関する研究が進展し、ＨＬＡの遺伝子の
同定、さらには遺伝子のクローニングが行われるように
なった。ＨＬＡは高度の遺伝的冬型性を持つ抗原である
ので、クローニングされた遺伝子をプローブとして用い
、遺伝子ＤＮＡの制限酵素切断パターンの違いからＨＬ
Ａ型の判別をする試みがなされた（例えば、ＨＬＡク
ラスＩＩＤＲ抗原に対して試みた特開昭５８−１３００
００号）。これはいわゆるＲ ＦＬ ｌ）法といイつれ
る方法である。Recently, research on HLA has progressed, and the HLA gene has been identified and furthermore, the gene has been cloned. Since HLA is an antigen with a high degree of genetic winter-type characteristic, we used the cloned gene as a probe to detect HL based on the difference in the restriction enzyme cleavage pattern of the genetic DNA.
Attempts were made to differentiate type A (e.g., Japanese Patent Application Laid-open No. 1300-1983, which attempted to identify HLA class II DR antigen).
No. 00). This is a method called the RFL l) method.
ＲＦＬ、 Ｐ法は比較的複雑な方法であって、制限酵素
切断、Ｉ） Ｎ Ａの固定化及び固定化されたＤＮＡ制
限断片の電気泳動的に分離された列への標識されたプロ
ーブのハイブリダイゼーションを必要とする。これらの
操作は繁雑であり、全工程で１〜２週間を要する。The RFL,P method is a relatively complex method that involves restriction enzyme cleavage, I) immobilization of NA and the mobilization of labeled probes onto electrophoretically separated columns of immobilized DNA restriction fragments. Requires hybridization. These operations are complicated and require 1 to 2 weeks for the entire process.
また、Ｉ−Ｉ Ｌ Ａクラス■抗原遺伝子内で多望性に
富む部分を２つのオリゴヌクレオチドプライマーとＴ
ａ ｑ ｌ） Ｎ Ａポリメラーゼを用いて、変性（９
５℃）、アニーリング（５５℃）、ＤＮＡ伸長（７０℃
）反応のサイクルを繰り返し、増幅後、アロ抗原特異的
オリゴヌクレオチドプローブとハイブリダイゼーシヨン
を行ない、ＨＬ Ａの型判別をする試みもなされている
（特開昭６２−２１４３５５）。しかし、この方法では
、プローブ毎にハイブリダイゼーションや、その後の洗
浄の条件を変える必要か生ずる（Ｓｃｈａｒｆ等、Ｐｒ
ｏｃ、 Ｎａ１１． Ａｃａｄ、 Ｓｃｉ、Ｌｌ、Ｓ、
Ａ、８５：３５０４−３５０８（＋９８８））。In addition, a highly versatile part of the I-I LA class ■ antigen gene was isolated using two oligonucleotide primers and T
aq l) Denaturation (9
5℃), annealing (55℃), DNA extension (70℃)
) There has also been an attempt to identify the HLA type by repeating the reaction cycle and, after amplification, performing hybridization with an alloantigen-specific oligonucleotide probe (Japanese Patent Application Laid-Open No. 214355/1983). However, this method requires changing hybridization and subsequent washing conditions for each probe (Scharf et al., Pr.
oc, Na11. Acad, Sci, Ll, S,
A, 85:3504-3508 (+988)).
〈発明か解決しようとしている問題点〉従って、本発明
の目的は、従来のＨＬＡ型判別法よりも簡便でかつ十分
に精度の高い、新規なＩ（ＬＡ型判別方法及びそれに用
いられる試薬を提供することである。<Problems to be Solved by the Invention> Therefore, an object of the present invention is to provide a novel I (LA type determination method and reagents used therein) that is simpler and sufficiently accurate than conventional HLA type determination methods. It is to be.
本願発明者等は鋭意研究の結果、ＨＬＡ抗原のアロタイ
プに特異的な制限酵素が存在することを見出し、かっ、
このような酵素でＰＣＲ法により増幅させたＤＮＡを切
断した後に、電気泳動を行なってＤＮＡの断片をその長
さに従って分離することによってＨＬＡ ＤＲ，ＤＱ
、Ｄ及びＤＰ膜抗の型判別を行なうことができることを
見出しこの発明を完成した。As a result of intensive research, the inventors of the present application discovered that there are restriction enzymes specific to allotypes of HLA antigens.
After cutting the DNA amplified by PCR with such an enzyme, electrophoresis is performed to separate the DNA fragments according to their length, thereby producing HLA DR, DQ.
, D and DP membrane resistors, and completed this invention.
すなわち、この発明は、ＰＣＲ法によって増幅したＤＮ
Ａを、ＨＬ Ａ抗原アロ特異性に特異的な制限酵素で切
断し、その断片の長さを電気泳動で分別、検出した後、
その長さの違いによってＨＬＡの型判別を行なうことか
ら成るＨＬＡ型判別方法を提供する。That is, the present invention provides DNA amplified by PCR method.
After cutting A with a restriction enzyme specific for HLA antigen allospecificity and fractionating and detecting the length of the fragment by electrophoresis,
An HLA type discrimination method is provided, which comprises performing HLA type discrimination based on the difference in length.
また、本発明は、ＨＬＡ抗原アロ特異性に特異的な制限
酵素から成るＨＬＡ型判別試薬を提供する。The present invention also provides an HLA typing reagent comprising a restriction enzyme specific for HLA antigen allospecificity.
〈発明の効果〉
１） ＣＲ法で増幅したＤＮＡをＤＮＡプローブを用い
て検出する従来の方法と比較して、本発明の方法は制限
酵素の反応を利用するため、塩基配列の変化をより高精
度で識別することか可能である。<Effects of the Invention> 1) Compared to the conventional method of detecting DNA amplified by the CR method using a DNA probe, the method of the present invention uses a restriction enzyme reaction, so changes in the base sequence can be detected at a higher rate. It is possible to identify it with precision.
また、放射性物質又は非放射性物質で標識された各種の
型特異的オリゴヌクレオチドプローブを用意する必要が
なく、より簡便なＨＬＡの型判別が可能になる。In addition, there is no need to prepare various type-specific oligonucleotide probes labeled with radioactive or non-radioactive substances, making HLA type discrimination easier.
〈発明の詳細な説明〉
本発明の方法の第１段階はｆＪ ＣＲ法によるＩ）ＮＡ
の増幅である。ＰＣＲ法はオリゴヌクレオチドプライマ
ーとＤＮＡポリメラーゼを用いて、変性、アニーリング
、ＤＮＡ伸長反応のサイクルを繰り返し、ＤＮＡを短時
間の間に急速に増幅させる方法であり、特開昭６２−２
１４３５５に記載されている。<Detailed Description of the Invention> The first step of the method of the present invention is I) NA by fJ CR method.
It is an amplification of The PCR method uses oligonucleotide primers and DNA polymerase to repeat cycles of denaturation, annealing, and DNA extension reactions to rapidly amplify DNA in a short period of time.
14355.
ＨＬ Ａ型判別にＰＣＲ法を適用する場合には、例えば
約２ Ｘ １０’個の血球を集め、リン酸緩衝溶液に分
散させたちのに、３催量の緩衝液（５０ｍλ１）・すス
ー塩酸、ｐＨ８，０，２０ｍＭ ＥＤＴＡ、 １００ｍ
Ｍ ＮａＣ１！ＳＩ％５ＤＳ）を加えて細胞を溶解させ
、プロテアーゼ処理、フェノール抽出によりタンパク質
を除いた後、２倍量の冷エタノールを添加して不溶化し
た高分子ＤＮＡを得、このＤＮＡのうち、ＨＬＡ抗原遺
伝子、特にＤＲ，ＤＱ及びＤＰ抗抗原遺伝山内多量性に
富む部分に上記ＰＣＲ法を適用して増幅させる。When applying the PCR method to HLA type A determination, for example, approximately 2 x 10' blood cells are collected, dispersed in a phosphate buffer solution, and then mixed with 3 volumes of a buffer solution (50 mλ1) and sulfuric acid. , pH 8, 0, 20mM EDTA, 100m
M NaC1! SI%5DS) was added to lyse the cells, proteins were removed by protease treatment and phenol extraction, and then twice the amount of cold ethanol was added to obtain insolubilized polymeric DNA. In particular, the above-mentioned PCR method is applied to amplify the DR, DQ, and DP anti-antigen gene portions with high abundance.
次に、ＰＣＲ法で増幅したＤＮＡをＨＬＡ抗原アロ特異
性に特異的な制限酵素で切断する。Next, the DNA amplified by the PCR method is cut with a restriction enzyme specific for HLA antigen allospecificity.
＋（Ｌ Ａ抗原アロ特異的な制限酵素の例及びその制限
酵素を適用した場合に生じるＤＮＡ断片の大きさを、酵
素処理されるＨＬＡ ＤＲ，ＤＱ、Ｄ。+ (Examples of LA antigen allospecific restriction enzymes and the size of DNA fragments generated when the restriction enzymes are applied.
ＤＰＰ原アロタイプと共に以下に示す。もっとも、トＩ
ＬＡ ＤＲ，ＤＱ、Ｄ及びＤＰ膜抗アロ持異的制限酵
素はこれらに限定されるものではない。It is shown below along with the original DPP allotype. However,
LA DR, DQ, D and DP membrane anti-allogenetic restriction enzymes are not limited to these.
１、ＤＲＩ鎖
（Ｄｉ（ｐｈｉ
■１２１ｂｐ、１１８ｂｐ、７１ｂｐ ＤＲ４■１８
９ｂｐ、１２１ｂｐ ＤＲＩ、ＤＲ２，ＤＲ３，
ＤＲ５ＤＲＷ６ ＤＲ７ＤＲｗ８ ＤＩ？ｗ９（２
）ｌｌｈａｌ
■ ＋４６ｂｐ、１ｏ１ｂｐ、６３ｂｐ ＤＲ２■
１６４ｂｐ、１４６ｂｐ ＤＩＩｌ Ｄ！
ン３ ＤＲ４ＤＲ５ＤＲｗ６．ＤＲ７，ＤＲｗ８
ＤＲｗ９（３）Ａｐａ １
■ ２４９ｂｐ、６１ｂｐ
■ ３＋１）ｂｐ
（４）Ｋｐｎ １
■ ２０２ｂｐ、１０８ｂｐ
■ ３１０ｂｐ
（５）Ｓａｃ ＩＴ
■ ２５１ｂｐ、５９ｂｐ
■ ３ｔＯｂｐ
（６）Ｆｏｋ Ｉ
Ｒｗ８
ＤＲＩ ＤＲ２ＤＲ７３Ｄｌｉ４
ＤＲ５，ＤＲｗ６．ＤＲ７，ＤＲｗ９
Ｒ３
Ｉ）ＲＩ ＤＲ２ＤＲ４ＤＲ５
ＤＲｗ６．ＤＲ７，ＤＲｗ８ ＤＲＷ９ＤＲＩ、ＤＲ
２，ＤＩ＋５゜
ＤＲ４Ｄｖ１３
ＤＲ３Ｄｆ？ｗ６ ＤＩ？７ Ｄｆ？ｗ９Ｄ Ｉｌ
４ Ｄ Ｗ ４ Ｄ １＋ ４ Ｄ Ｗ ｌ ０Ｄ
Ｒ４ＤＷ１４ ＤＲ４ＤＷＩ５
■ ２１４ｂｐ、！１８ｂｐ
■ ３１０ｂｐ
（７）Ｉ（ｉｎｆ ［
■ ｌ１８ｂｐ、１０５ｂｐ、８９ｂｐ■ ２０５ｂｐ
、 １０５ｂｐ
■ １７４ｂｐ、 １３６ｂｐ
■ ３１０ｂｐ
■ ＤＱ α鎖
（１）ｉｌａｅｌｌＴ
■ ］１４ｂｐ、７６ｂｐ、３５ｂｐ
■ １９０ｂｐＪ５ｂｐ
■ ２２５ｂｐ
（２）Ｄｄｅ １
ＤＢ２ＡＩＨ，ＤＲ７
ＤＲ５ＬＤＶ口、ＤＲ４ＤｗｌＯ
ＤＲｌ、ＤＲ３，ＤＩ？４ ＤＲ５
ＤＲｖ８．ＤＲｗ９
ＤＲ２Ｄｖ２ ＤＲ２Ｄｗ１２
ＤＲ？
Ｒｖ９
ＤＲＩ、ＤＲ２ＡＺＨ
ＤＲ３ＤＲ４，ＤＲ５，ＤＲＷ６
ＤＲ冑８
ＤＲ２Ｄｖ２ ＤＲ２Ｄｖｉ２
ＤＲＩＤＱｗｌ、１．ＤＲ２ＤＱｗ１．ＩＤＲ６ＤＱｖ
＋、１．ＤＲｗ８ＤＱｖ
ＤＲ３，ＤＱｖ２．ＤＲ７ＤＱｗ２
ＤＲ５ＤＱ豐３
ＤＲ４ＤＱｖ３．ＤＲｗ９ＤＱｗ３
■ ｌｌ３ｂｌ）
７１ｂｐ、４１ｂｐ
■ Ｉ［８ｂｐ、ｌ０７ｂｐ
■ １２７ｂＰ、９８ｂｐ
■ ２２５ｂｐ
（３）Ｍｂ○口
■ ＋４３ｂｐ、８２ｂｐ
■ ２２５ｂｐ
（４）ＳｃｒＦ Ｉ
■ １２３ｂｐ、８２ｂｐ、２０ｂｒｌ■ ２２５ｂｐ
（５）Ｆｏｋ （
■ １７５ｂｐ、５０ｂｐ
ＤＲＩＤＱｗｌ、ｌ ＤＲ２Ｄｆｈ１．ＩＤＲｗ６ＤＱ
ｗ１．！
ＤＲ３ＤＱｗ２．υＲ５ＤＱｗ３
ＤＲｖ８ＤＱｗ
ＤＲ７ＤＱｗ２
ＤＲ４ＤＱｗ３．ＤＲｗ９ＤＱｗ３
ＤＲ４ＤＱｗ３．Ｄ？９ＤＱｗ３
ＤＲＩＤＱｗｌ、ｔ、ＤＲ２ＤＱｗｌ
ＤＲ３ＤＱｖ２．Ｉ’ｌｌｉ５ＤＱｗ３ＤＲｗ６ＤＱｖ
１．、ｌ、ＤＲ７Ｄ（ｈ２ＤＲｗ８ＤＱｗ
ＤＲＩＤＱｗｌ、Ｉ 、ＯＲ２ＤＱｗ１．ＩＤＲｗ６Ｄ
Ｑｗ１．Ｉ
ＤＲ３ＤＱｗ２．ＤＲ４ＤＱｗ３
ＤＲ５ＤＱｗ：！、、ＤＲ７ＤＱｄ
ＤＲｗ８ＤＱｗ−、ＤＲｖ９ＤＱｗ３
ＤＲ４ＤＱｗ３．ＤＲ？ＤＱｗ２
■ ２２５ｂｐ
ＩＩｌ、 ＤＱβ鎖
（１）ｌｌａｅＩ［Ｉ
■ １２９ｂｐ、４６ｂｐ、３９ｂｐ
■ １２９ｂｐ、８５ｂｐ
■ １２６ｂｐ、８８ｂｐ
■ ２＋４ｂＰ
（２）Ｆｏｋ １
■ ＋４８ｂｐ、６６ｂｐ
■ ２１４ｂｐ
ＤＲｗ８ＤＱｖ−
ＤＲＩＤＱｗｌ、１．ＤＲ２ＤＱｗ１．ＩＤＲ３ＤＱｗ
２．ＤＲ５ＤＱｗ２
ＤＲ６ＤＱｗ１．ｌ、ＤＲ９ＤＱｖ３
（３）Ｓａｕ３Ａ １
■ １４３ｂｐ、７１ｂｐ
■ ２１４ｂｐ
ＤＲＩＤＱｗｌ、１．ＤＲ２ＤＱｗ１．ＩＡ２Ｈ，ＤＲ
ｗ６ＤＱｗ１．Ｉ
ＤＲ２Ｄｖ２．ＤＲ２Ｄｖ１２
ＤＲ４ＤＱｗ３．１．ＤＲ４ＤＱｗ３．２ＤＲ５ＤＱｗ
３．ｌ、ＤＲｖ９ＤＱｖ３．３ＤＲ４ＤＱｗ−、ＤＲｖ
８ＤＱｖ
ＤＲ３ＤＱｗ２．ＤＲ７ＤＱｗ２
（４）ＢｓｓｌｌＩＩ
■ １３８ｂｐ、７８ｂｐ
■ ２１４ｂｐ
ＤＲ２Ｄｗ２．ＤＲ２Ｄｖ！２
ＤＲ４ＤＱｗ−、ＤＲｖ８ＤＱｖ
ＤＲ３ＤＱｗ２．ＤＲ７ＤＱｖ２
ＤＲＩＤＴｏｌ、ｌ、ＤＲ２ＡＺＨ
ＤＲｖ８ＤＱｖ１．１．ＤＲ４ＤＱｖ３．ＩＤＲ４ＤＱ
ｖ（，２，ＤＲ５ＤＱｖ３．１（５）ｋｌａｅ Ｉ
■ １６６ｂｐ、４８ｂｐ
ＤＲｗ９ＤＱｗ３．３
ＤＲ３ＤＱｗ２．ＤＲ７ＤＱｗ２
ＤＲＩＤＱｖｌ、ｌ ＤＲ２Ｄｖ２
ＤＲ２Ｄｗ１２ ＤＲ２ＡＺＨ
ＤＲ４ＤＱｗ３．１．ＤＲ４ＤＱｗ３．２ＤＲ４ＤＱｗ
−、ＤＲ５ＤＱｗ３．Ｉ
ＤＲｖ６ＤＱｖ１．Ｉ
Ｄｋｖ８ＤＱｗ−、ＤＲｖ９ＤＱｗ３．３Ｒ２Ｄｖ２
ＤＲ４ＤＱｗ−、ＤＲｗ８ＤＱｖ
ＤＲＩＤＱｖｌ、１゜
ＤＲ２Ｄｖ１２．ＤＲ２ＡＺＨ
ＤＲ３ＤＱｗ２．ＤＲ４ＤＱｗ３．Ｉ
ＤＲ４ＤＱｗ３．２．ＤＲ５ＤＱｗ３．１ＤＲｖ６ＤＱ
ｗ１．ｌ、ＤＲ７ＤＱｖ２ＤＲｗ９ＤＱｗ３．３゜
ＤＲｖ６ＤＱｖ１．１
■ ２＋４ｂｐ
■ ＤＰβ鎖
（１）ＳｃｒＦ Ｉ
■７８ｈｐ、５８ｂｐ
■７１ｂｐ、５８ｂｐ
（２）Ｓａｕ １
■９７ｂｐ、３９ｂｐ
■１３６ｂｐ
（３）Ｍａｅｌｎ
■８１ｂｐ、６２ｂｐ、２７ｂｐ
■８９ｂｐ、８１ｂｐ
（４）ＢｓｔＵＵ
■１１４ｂｐ、５４ｂｐ
■ｔ７０ｂｐ
（５）＾ｖａＩＩ
ＤＲＩＤＱｗｌ、１゜
ＤＲ２Ｄｖ２．ＤＲ２Ｄｗ１２
ＤＲ２Ａ２■、ＤＲ３ＤＱｗ２
ＤＲ４ＤＱｗ３．１．ＤＲ４ＤＱｗ３．２ＤＲ５ＤＱｖ
３．１．ＤＲ７ＤＱｖ２
ＤＲｄＤＱｗ３Ｊ。1, DRI chain (Di(phi ■121bp, 118bp, 71bp DR4■18
9bp, 121bp DRI, DR2, DR3,
DR5DRW6 DR7DRw8 DI? w9(2
)llhal ■ +46bp, 1o1bp, 63bp DR2■
164bp, 146bp DIIl D!
3 DR4DR5DRw6. DR7, DRw8
DRw9 (3) Apa 1 ■ 249bp, 61bp ■ 3+1)bp (4) Kpn 1 ■ 202bp, 108bp ■ 310bp (5) Sac IT ■ 251bp, 59bp ■ 3tObp (6) Fok I Rw8 DRI DR2DR73Dli4 DR5, DRw6. DR7, DRw9 R3 I) RI DR2DR4DR5 DRw6. DR7, DRw8 DRW9DRI, DR
2, DI+5゜DR4Dv13 DR3Df? w6 DI? 7 Df? w9D Il
4 D W 4 D 1+ 4 D W l 0D
R4DW14 DR4DWI5 ■ 214bp,! 18bp ■ 310bp (7) I(inf [ ■ l18bp, 105bp, 89bp ■ 205bp
, 105bp ■ 174bp, 136bp ■ 310bp ■ DQ α chain (1) ilaellT ■ ]14bp, 76bp, 35bp ■ 190bpJ5bp ■ 225bp (2) Dde 1 DB2AIH, DR7 DR5LDV mouth, DR4DwlO DRl, DR 3.DI? 4 DR5 DRv8. DRw9 DR2Dv2 DR2Dw12 DR? Rv9 DRI, DR2AZH DR3DR4, DR5, DRW6 DR helmet 8 DR2Dv2 DR2Dvi2 DRIDQwl, 1. DR2DQw1. IDR6DQv
+, 1. DRw8DQv DR3,DQv2. DR7DQw2 DR5DQ豐3 DR4DQv3. DRw9DQw3 ■ ll3bl) 71bp, 41bp ■ I [8bp, l07bp ■ 127bP, 98bp ■ 225bp (3) Mb○mouth■ +43bp, 82bp ■ 225bp (4) ScrF I ■ 123bp, 82bp, 20brl ■ 225bp (5) Fok (■ 175bp, 50bp DRIDQwl, l DR2Dfh1.IDRw6DQ
w1. ! DR3DQw2. υR5DQw3 DRv8DQw DR7DQw2 DR4DQw3. DRw9DQw3 DR4DQw3. D? 9DQw3 DRIDQwl, t, DR2DQwl DR3DQv2. I'lli5DQw3DRw6DQv
1. ,l,DR7D(h2DRw8DQw DRIDQwl,I ,OR2DQw1.IDRw6D
Qw1. I DR3DQw2. DR4DQw3 DR5DQw:! ,,DR7DQd DRw8DQw-, DRv9DQw3 DR4DQw3. DR? DQw2 ■ 225bp IIl, DQβ chain (1) llaeI [I ■ 129bp, 46bp, 39bp ■ 129bp, 85bp ■ 126bp, 88bp ■ 2+4bP (2) Fok 1 ■ +48bp, 66bp ■ 214bp DRw8DQv- DRIDQ wl, 1. DR2DQw1. IDR3DQw
2. DR5DQw2 DR6DQw1. l, DR9DQv3 (3) Sau3A 1 ■ 143bp, 71bp ■ 214bp DRIDQwl, 1. DR2DQw1. IA2H,DR
w6DQw1. IDR2Dv2. DR2Dv12 DR4DQw3.1. DR4DQw3.2DR5DQw
3. l, DRv9DQv3.3DR4DQw-, DRv
8DQv DR3DQw2. DR7DQw2 (4) BssllII ■ 138bp, 78bp ■ 214bp DR2Dw2. DR2Dv! 2 DR4DQw-, DRv8DQv DR3DQw2. DR7DQv2 DRIDTol, l, DR2AZH DRv8DQv1.1. DR4DQv3. IDR4DQ
v(,2,DR5DQv3.1(5)klae I ■ 166bp, 48bp DRw9DQw3.3 DR3DQw2.DR7DQw2 DRIDQvl,l DR2Dv2 DR2Dw12 DR2AZH DR4DQw3.1.DR4DQw3.2DR4DQ lol
-, DR5DQw3. IDRv6DQv1. I Dkv8DQw-, DRv9DQw3.3R2Dv2 DR4DQw-, DRw8DQv DRIDQvl, 1°DR2Dv12. DR2AZH DR3DQw2. DR4DQw3. I DR4DQw3.2. DR5DQw3.1DRv6DQ
w1. l, DR7DQv2DRw9DQw3.3゜DRv6DQv1.1 ■ 2+4bp ■ DP β chain (1) ScrF I ■ 78hp, 58bp ■ 71bp, 58bp (2) Sau 1 ■ 97bp, 39bp ■ 136bp (3) Maeln ■ 81bp, 62bp, 27bp ■89bp , 81bp (4) BstUU ■114bp, 54bp ■t70bp (5)＾vaII DRIDQwl, 1°DR2Dv2. DR2Dw12 DR2A2■, DR3DQw2 DR4DQw3.1. DR4DQw3.2DR5DQv
3.1. DR7DQv2 DRdDQw3J.
ＤＰｗ２．ＤＰｗ４
ＤＰｗｌ、ＤＰｖ３．ＤＰｖ５
ＤＰｖ２．ＤＰｗ４
ＤＰｖｌ、ＤＰｖ３．ＤＰｖ５
ｐ６３
ＤＰｗ２．ＤＰｖ３．ＤＰｖ４
ＤＰ曹４
ＤＰｗ２．ＤＰｖ４．Ｃｐ６３
■１３７ｂｐ、３３ｂｐ ＤＰｖ３■１
７０ｂｐ ＤＰｗ２．ＤＰｗ４．
Ｃｐ６３（６）口ＩＩ
■１０６ｂｐ、６４ｂｐ Ｃｐ６３．Ｄ
Ｐｗ２．ＤＰｗ３■１７０ｂｐ
ＤＰｗ４これらの制限酵素自体は市販されており、Ｄ
ＮＡの消化もその指示書に基づいて行なうことができる
。DPw2. DPw4 DPwl, DPv3. DPv5 DPv2. DPw4 DPvl, DPv3. DPv5 p63 DPw2. DPv3. DPv4 DP Sergeant 4 DPw2. DPv4. Cp63 ■137bp, 33bp DPv3■1
70bp DPw2. DPw4.
Cp63 (6) Mouth II ■106bp, 64bp Cp63. D
Pw2. DPw3■170bp
DPw4 These restriction enzymes themselves are commercially available, and D
Digestion of NA can also be performed based on the instructions.
次に、このようなＨＬＡ抗原特異性にアロ特異的な制限
酵素で消化したＤＮＡを電気泳動法により、ＤＮＡ断片
の長さに基づいて分離する。ＤＮＡの分離のための電気
泳動法自体はこの分野において周知であり、種々の方法
が可能であるが、例えばポリアクリルアミドゲル電気泳
動法により行なうことができる。Next, the DNA digested with such a restriction enzyme allospecific to HLA antigen specificity is separated by electrophoresis based on the length of the DNA fragments. The electrophoretic method itself for separating DNA is well known in this field, and various methods are possible, for example, polyacrylamide gel electrophoresis can be used.
電気泳動法により分離したＤＮＡ断片の長さから、ＨＬ
Ａ抗原のアロタイプを判別する。すなわち、各制限酵素
を用いた場合に、上述のようにどの）′ロタイブであれ
ばどのような大きさの断片が生じるかわかっているので
、電気泳動法により検出されたＤＮＡ断片の大きさから
そのＨＬＡ型を判別することができる。なお、上述のよ
うに、ある制限酵素を用いた場合に、複数のアロタイプ
において同じ大きさのＤＮＡ断片を生じる場合が多いが
、複数の制限酵素を併用して適用することによりＨＬＡ
型判別を行なうことができる。From the length of DNA fragments separated by electrophoresis, HL
Determine the allotype of A antigen. In other words, when using each restriction enzyme, we know the size of the fragment produced by the rotaib as described above, so it can be determined from the size of the DNA fragment detected by electrophoresis. Its HLA type can be determined. As mentioned above, when a certain restriction enzyme is used, DNA fragments of the same size are often generated for multiple allotypes, but by applying multiple restriction enzymes in combination, HLA
Type determination can be performed.
以下、実施例に基づき本発明をより具体的に説明するが
、本発明は下記実施例に限定されるものではない。EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.
〈実施例１〉 下記の細胞株を増幅のために利用した。<Example 1> The following cell lines were utilized for amplification.
ＪＥＳＴＨＯＭ株 ＤＲＩＤＱｗｌ
ＭＧＡＲ株 ＤＲ２ＤＱｖｌ
ＡＫＩＢＡ 株 ＤＲ２ＤＱｖｌＬ ０
０８１７８５ 株 ＤＲ３ＤＱｗ２１３
Ｍ＋４ 株 ＤＲ４ＤＱｗ３ＭＯＵ
株 ＤＲ７ＤＱｗ２ＭＡＤＵＲ
Ａ 株 ＤＲ８ＤＱｗＤＸＢ
株 ＤＲ９ＤＱ豐３培養細胞を約２ Ｘ ｔｏ
’個集め、リン酸緩衝溶液に分散させたしのに、３倍量
の緩衝液（５０ｆｆ１Ｍトリスー塩酸、ｐＨ８，０１２
０ｍＭ ＥＤＴＡ、 １００ｍＭ ＮａＣ（１，１％５
ＤＳ）を加えて細胞を溶解させた。常法に従い、プロテ
アーゼ処理、フェノール抽出によりタンパク質を除いた
後、リボヌクレアーゼ処理してＲＮＡを除いた。これに
２倍量の冷エタノールを添加して不溶化した高分子ＤＮ
Ａを得た。このＤＮＡを用いて、ＰＣＲ法によりＨＬＡ
−ＤＱα遺伝子の第２エクソンを増幅した。下記のＤＮ
Ａ及び反応液を混合しミネラルオイルを数滴重層した反
応混合液中で増幅を行った。JESTHOM stock DRIDQwl MGAR stock DR2DQvl AKIBA stock DR2DQvlL 0
081785 stock DR3DQw213
M+4 shares DR4DQw3MOU
Stock DR7DQw2MADUR
A stock DR8DQwDXB
Approximately 2 X to 3 cultured cells of strain DR9DQ
Collect the pieces and disperse them in a phosphate buffer solution.
0mM EDTA, 100mM NaC (1,1%5
DS) was added to lyse the cells. According to a conventional method, proteins were removed by protease treatment and phenol extraction, and then RNA was removed by ribonuclease treatment. Polymer DN was insolubilized by adding twice the amount of cold ethanol to this.
I got an A. Using this DNA, HLA was detected by PCR method.
-The second exon of the DQα gene was amplified. DN below
Amplification was performed in a reaction mixture where A and the reaction solution were mixed and several drops of mineral oil were layered.
高分子 ＤＮＡ ＋ １μｌ（ｌμｌ
）オリゴヌクレオチドブライマー、 ＱＡ−１（５−
ＧＧＴＧＴＡ＾＾ＣＴＴＧＴＡＣＣＡＧＴＣ−３’ ）
： ｌμｌ（１００μＭ）オリゴヌクレオチドブラ
イマー、 Ｑ１２（５’ −ＧＧＴＡＧＣＡＧＣＧＧ
ＴＡＧＡＧＴＴＧＧ−３’ ） ＋ ｌμｌ（１００
μＭ）デオキシリボヌクレオシドトリホスフェート（５
００μＭずっｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰおよびｄＴ
ＴＰを含む）４０μｌ
ゼラチン ： ｌμｌ（２０ｍｇ
／ｍ１）Ｔａｑ ＤＮ＾ポリメラーゼ： ｌ
μｌ（２，５ｕ）水
４５μｌ増幅は、変性（９５℃）、アニーリング（
５５℃）、ＤＮＡ伸長（７０℃）をそれぞれ１分間ずっ
３０サイクル行なった。Polymer DNA + 1μl (lμl
) Oligonucleotide Brimer, QA-1 (5-
GGTGTA^^CTTGTACCAGTC-3')
: l μl (100 μM) oligonucleotide primer, Q12 (5′-GGTAGCAGCGG
TAGAGTTGG-3') + lμl (100
μM) deoxyribonucleoside triphosphate (5
00 μM each dATP, dCTP, dGTP and dT
(including TP) 40μl Gelatin: lμl (20mg
/m1) Taq DN^ Polymerase: l
μl (2,5u) water
45 μl amplification, denaturation (95°C), annealing (
55°C) and DNA extension (70°C) for 30 cycles of 1 minute each.
増幅したＤＮＡ約２００ｎｇ（８μｌ）を制限酵素Ｄｄ
ｅｌ。Approximately 200 ng (8 μl) of the amplified DNA was treated with restriction enzyme Dd.
el.
およびＨａｅｌ［ｌでそれぞれ切断した。常法に従い、
１２％アクリルアミドゲル電気泳動後、エチジウムブロ
マイド染色してバンドを検出した。and Hael[l, respectively. According to common law,
After 12% acrylamide gel electrophoresis, bands were detected by ethidium bromide staining.
その結果第１図に示すようにＤｄｅ Ｉで切断した場合
、ＤＱｖｌでは１１３ｂｐ、７１ｂｐ、４１ｂｐの３本
のバンドが、ＤＱｗ３では２２５ｂｐのバンドが検出さ
れた。As a result, as shown in FIG. 1, when cut with Dde I, three bands of 113 bp, 71 bp, and 41 bp were detected in DQvl, and a 225 bp band was detected in DQw3.
ＤＱｗ２・ＤＲ３では、１１８ｂｐと１０７ｂｐの２本
のバンドが、ＤＱｖ２・ＤＲ７では１２７ｂｐと９８ｂ
ｐの２本のバンドが検出され、ＤＱｖ抗原及びＤＲ抗原
アロ特異性によって区別ができた。DQw2/DR3 has two bands of 118bp and 107bp, DQv2/DR7 has two bands of 127bp and 98bp.
Two bands of p were detected and could be distinguished by DQv antigen and DR antigen allospecificity.
また第２図に示すようにＨａｅｍで切断した場合、ＤＱ
ｗｌでは１１４ｂｐ、７６ｂｐ、３５ｂｐの３本のバン
ドが、ＤＱｖ２では１９０ｂｐと３５ｂｐの２本のバン
ドが、ＤＱｖ３では２２５ｂｐのバンドが、ＤＱｖ抗原
アロ特異性に従ってそれぞれ検出された。In addition, when cutting with Haem as shown in Figure 2, DQ
Three bands of 114 bp, 76 bp, and 35 bp were detected for wl, two bands of 190 bp and 35 bp for DQv2, and a 225 bp band for DQv3 were detected according to the DQv antigen allospecificity.
〈実施例２〉
ＤＲアロ抗原特異性が既知の細胞を約２ Ｘ １０’個
集め、リン酸緩衝溶液に分散さけたものに、３倍１１（
７）緩衝液（５０ＩＩＩＭトリスー塩酸、ｐｔ＋８．０
．２０ｍＭ ＥＤＴＡ、 １００ｍＭ ＮａＣＱ、
１％５ＤＳ）を加えて細胞を溶解させた。常法に従い、
プロテアーゼ処理、フェノール抽出によりタンパク質を
除いた後、リボヌクレアーゼ処理してＲＮＡを除いた。<Example 2> Approximately 2 x 10' cells of known DR alloantigen specificity were collected and dispersed in a phosphate buffer solution with 3x 11 (
7) Buffer (50IIIM Tris-HCl, pt+8.0
．． 20mM EDTA, 100mM NaCQ,
1% 5DS) was added to lyse the cells. According to common law,
After removing proteins by protease treatment and phenol extraction, RNA was removed by ribonuclease treatment.
これに２倍量の冷エタノールを添加して不溶化１、た高
分子ＤＮＡを得た。このＤＮＡを用いて、ＰＣＲ法によ
り１１ＬＡ−ＤＩ’（β遺伝子の第２エクソンを増幅し
た。Twice the amount of cold ethanol was added to this to obtain insolubilized polymeric DNA. Using this DNA, the second exon of 11LA-DI' (β gene) was amplified by PCR.
下記のＤＮＡ及び反応液を混合しミネラルオイルを数滴
重層した反応混合液中で増幅を行った。The following DNA and reaction solution were mixed and amplification was performed in a reaction mixture overlaid with several drops of mineral oil.
高分子 ＤＮＡ： １μｌ（ｌμｌ）
オリゴヌクレオチドブライマー ＲＢ−８（５’−Ｃ
ＣＣＧＧＡＧＧＣＣＧＣＴＴＣＴＧＴ＾＾ＣＣＧＧ−３
°） ： ｌμｌ（１０ｈＭ）オリゴヌクレオチドブ
ライマー ＲＢ−９（５’−ＧＣＣＧＣＴＧＣＡＣＴ
ＧＴＧＡＡＧＣＴＣＴＣＣＣ−３°） ： ｌμｌ（１
０ｈｉりデオキシリボヌクレ才ソドトリホスフェート（
５００μＭずつｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰおよびｄ
ＴＴＰを含む）４０μｌ
ゼラチン ： ｌμｌ（２０ｍｇ
／ｍ１）Ｔａｑ ＤＮＡポリメラーゼ： ｌ
μｌ（２，５ｕ）水：４５μｍ
増幅は、変性（９５°Ｃ）、アニーリング（５５°Ｃ）
、ＤＮＡ伸長（７０℃）をそれぞれ１分間ずつ３０サイ
クル行なった。Polymer DNA: 1μl (lμl)
Oligonucleotide Brimer RB-8 (5'-C
CCGGAGGCCGCTTCTGT^^CCGG-3
°): lμl (10 hM) oligonucleotide primer RB-9 (5'-GCCGCTGCACT
GTGAAGCTCTCC-3°) : lμl(1
0hiredeoxyribonuclease sodotriphosphate (
500 μM each of dATP, dCTP, dGTP and d
(including TTP) 40μl Gelatin: lμl (20mg
/m1) Taq DNA polymerase: l
μl (2,5u) water: 45μm Amplification: denaturation (95°C), annealing (55°C)
, DNA extension (70°C) was performed for 30 cycles for 1 minute each.
増幅したＤＮＡ約２００ｎｇ（８μｌ）を制限酵素Ｈｈ
ａｌ。Approximately 200 ng (8 μl) of the amplified DNA was treated with restriction enzyme Hh.
al.
１１ｐｈ＋およびＡｐａ Ｉでそれぞれ切断した。常法
に従い、１２％アクリルアミドゲル電気泳動後、エチジ
ウムブロマイド染色してバンドを検出した。11ph+ and Apa I, respectively. Following 12% acrylamide gel electrophoresis, bands were detected by staining with ethidium bromide according to a conventional method.
その結果１！ｈａｌで切断するとＤＲ２については、１
４６ｂｐ、ｌ０１ｂｐ、６３ｂｐの３本のバンドが他の
アロ抗原タイプについては、１．６４ｂｐと１４６ｂｐ
の２本のバンドが検出された。また、Ｈｐｈ Ｉで切断
するとＤＲ４については、１２１ｂｐ、］１８ｂｐ、７
１ｂｐの３本のバンドが他のアロ抗原タイプについては
、１８９ｂｐと１２１ｂｐの２本のバンドが検出された
。＾ｐａ■で切断するとＤＲ８についてだけ２４８ｂ
ｐと６１ｂｐの２本のバンドが検出され、池の場合は切
断を生じなかった。The result is 1! For DR2 when cut with hal, 1
Three bands of 46bp, 101bp and 63bp are present for other alloantigen types, 1.64bp and 146bp.
Two bands were detected. Furthermore, when cut with Hph I, DR4 has 121bp, ]18bp, 7
Three bands of 1 bp were detected; for other alloantigen types, two bands of 189 bp and 121 bp were detected. If you cut with ^pa■, only DR8 will be 248b.
Two bands, p and 61 bp, were detected, and no cleavage occurred in the case of pond.
〈実施例３〉
ヒトの血液から約２ Ｘ １０’血球を個集め、リン酸
緩衝溶液に分散させたらのに、３倍量の緩衝液（５０ｍ
Ｍ トリス−塩酸、ｐｌ＋８．０．２０ｍＭ ＥＤＴＡ
、 １００ｍＭ ＮａＣｅ、１％５ＤＳ）を加えて細胞
を溶解させた。常法に従い、プロテアーゼ処理、フェノ
ール抽出によりタンパク質を除いた後、リボヌクレアー
ゼ処理してＲＮＡを除いた。これに２倍量の冷エタノー
ルを添加して不溶化した高分子ＤＮＡを得た。このＤＮ
Ａを用いて、ＰＣＲ法によりＨＬ Ａ −Ｄ Ｐβ遺伝
子の第２エクソンを増幅した。下記のＤＮＡ及び反応液
を混合しミネラルオイルを数滴重層した反応混合液中で
増幅を行った。<Example 3> Approximately 2 x 10' blood cells were collected from human blood and dispersed in a phosphate buffer solution.
M Tris-HCl, pl+8.0.20mM EDTA
, 100 mM NaCe, 1% 5DS) was added to lyse the cells. According to a conventional method, proteins were removed by protease treatment and phenol extraction, and then RNA was removed by ribonuclease treatment. Double the amount of cold ethanol was added to this to obtain insolubilized polymeric DNA. This DN
A was used to amplify the second exon of the HLA-D Pβ gene by PCR. The following DNA and reaction solution were mixed and amplification was performed in a reaction mixture overlaid with several drops of mineral oil.
高分子 ＤＮＡ ： １μｌ（ｌμｇ
）オリゴヌクレオチドブライマー １”１（５’−Ｃ
ＡＧＧＧＴＴＣＣＴＧＡＣＣＴＣＡＣＣＧＡＡ＾−３°
）；ｌμｌ（１００μＭ）オリゴヌクレオチドブライマ
ー Ｐ−３（５−ＣＴＧＡＣＴＴＣＡＧＡＧＣ＾＾Ｃ
ＴＴＣＴＴＧＧＣ−３’） ： ｌμｌ（１００μＭ
）デオキシリボヌクレオシドトリホスフェート（５００
μＭずつｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰおよびｄＴＴＰ
を含む）４０μｌ
ゼラチン ： ｌμｌ（２０ｍｇ
／ｍ１）Ｔａｑ ＤＮ人ポリメラーゼ＋ ｌ
μｌ（２，５ｕ）水・ ４５μｌ
増幅は、変性（９５℃）、アニーリング（５５℃）、Ｄ
ＮＡ伸＆（７０６Ｃ）をそれぞれ１分間ずつ３０サイク
ル行なった。Polymer DNA: 1μl (lμg
) Oligonucleotide Brimer 1”1 (5'-C
AGGGTTCCTGACCTCACCGAA＾−3°
); lμl (100μM) Oligonucleotide Brimer P-3 (5-CTGACTTCAGAGC^^C
TTCTTGGC-3'): lμl (100μM
) Deoxyribonucleoside triphosphate (500
dATP, dCTP, dGTP and dTTP in μM
) 40 μl gelatin: 1 μl (20 mg
/m1) Taq DN polymerase + l
μl (2,5u) water/45μl Amplification includes denaturation (95°C), annealing (55°C), D
NA extension & (706C) were performed for 30 cycles for 1 minute each.
増幅したＤＮＡ約２００ｎｇ（８μｌ）を制限酵素５ｅ
ｒｆ。Approximately 200 ng (8 μl) of the amplified DNA was treated with restriction enzyme 5e.
rf.
およびＳａｕ Ｉでそれぞれ切断した。常法に従い、１
２％アクリルアミドゲル電気泳動後、エチジウムブロマ
イド染色してバンドを検出した。and Sau I, respectively. According to common law, 1
After 2% acrylamide gel electrophoresis, bands were detected by ethidium bromide staining.
結果を第３図及び第４図に示す。第３図に示すように、
Ｓａｕ ｌで切断した場合、ＤＰｖ２及びＤＰｗ４は９
７ｂｐと３９ｂｐ、 ＤＰｗｌＳＤＰｗ３及びＤＰｖ５
については１３６ｂｐのバンドが、また第４図に示ずよ
うに５ｃｒＦ Ｉで切断した場合ＤＰｗ２及びＤＰｖ４
については７８ｂｐと５８ｂｐ。The results are shown in FIGS. 3 and 4. As shown in Figure 3,
When cutting with Saul, DPv2 and DPw4 are 9
7bp and 39bp, DPwlSDPw3 and DPv5
DPw2 and DPv4 when cut with 5crF I as shown in Figure 4.
For 78bp and 58bp.
ＤＰｗｌ、ＤＰＷ３及びＤＰｗ５については７１ｂｐと
５８ｂｐのバンドがＤＰ抗原アロ特異性に従って検出さ
れた。For DPwl, DPW3 and DPw5, bands of 71 bp and 58 bp were detected according to DP antigen allospecificity.
〈実施例４〉
オリゴヌクレオチドブライマー
Ｐ −１４＋ ５−ＴＴＡＡＴＧＧＧＡＣＡＣＡＧＣＧ
ＣＴＴＣＣＴＧＧＡＧＡ−３Ｐ −１５： ５−ＧＴＣ
ＣＧＧＣＡＣＴＧＣＣＣＧＣＴＴＣＴＣＣＴＣＣＡＧ−
３を用いて実施例１．２及び３と同様な条件でＨＬＡ−
ＤＰβ遺伝子のβＩドメイン領域を増幅する。<Example 4> Oligonucleotide Brimer P-14+ 5-TTAATGGGACACAGCG
CTTCCTGGAGA-3P-15: 5-GTC
CGGCACTGCCCGCTTCTCCTCCAG-
HLA-3 was used under the same conditions as in Examples 1.2 and 3.
Amplify the βI domain region of the DPβ gene.
増幅されたＤ Ｎ Ａ （１７０ｂｐ）に対して、下記
の制限酵素により切断を行なう。各制限酵素について次
のようなバンドが、おのおのアロ抗原タイプごとに検出
される。The amplified DNA (170 bp) is cleaved using the following restriction enzymes. The following bands for each restriction enzyme are detected for each alloantigen type.
１１ａｅ ＩＩＩ
Ｃｐ６３ ： ８１ｂｐ、６２ｂｐ
、２７ｂｐＤＰｖ２．ＤＰｗ４．Ｃｐ６３
： ８９ｂｐ、８１ｂｐｓｔＵＩＩ
ＤＰｗ４ ： １１４ｂｐ、５４
ｂｐＤＰｖ２．ＤＰｗ４．Ｃｐ６３ ：
１７０ｂｐ＾ｖａＩ［
Ｐｖ３
： １３７ｔ＋ｐ、３３ｂｐ
ＤＰｖ２．ＤＰｖ４ 、Ｃｐ６３
７０ｂｐ
ｎ１ｌ
Ｃｐ６３．ＤＰｖ２．ＤＰｖ３
： １０６ｂｐ、６４ｂｐ
ＤＰｗ４ ： １７０ｂｐ〈実施
例５〉
オリゴヌクレオチドブライマー
Ｑ Ｂ −１＋ ５” −ＧＣＣＡＴＧＴＧＣＴＡＣＴ
ＴＣＡＣＣＡＡＴＧ−３Ｑ ！３−２ 、５’ −ＣＧ
ＴＡＧＴＴＧＴＧＴＣＴＧＣＡＣＡＣＣＧＴ−３’及び
ＨＬ Ａ抗原既知の細胞を用いて実施例１．２３及び４
と同様な条件でＨＬＡ−ＤＱβ遺伝子の第２エクソン領
域を増幅する。増幅されたＤＮＡ（２１４ｂｐ）に対し
て、下記のような制限酵素により切断を行ない、各制限
酵素について次のようなバンドが、おのおのアロ抗原タ
イプごとに検出された。11ae III Cp63: 81bp, 62bp
, 27bpDPv2. DPw4. Cp63
: 89bp, 81bpstUII DPw4: 114bp, 54
bpDPv2. DPw4. Cp63:
170bp^vaI [Pv3: 137t+p, 33bp DPv2. DPv4, Cp63 70bp n1l Cp63. DPv2. DPv3: 106bp, 64bp DPw4: 170bp <Example 5> Oligonucleotide Brimer Q B -1+ 5''-GCCATGTGCTACT
TCACCAATG-3Q! 3-2, 5'-CG
Examples 1.23 and 4 using cells with known TAGTTGTGTCTGCACACCGT-3' and HLA antigens
The second exon region of the HLA-DQβ gene is amplified under the same conditions as above. The amplified DNA (214 bp) was digested with the following restriction enzymes, and the following bands were detected for each restriction enzyme for each alloantigen type.
１１ａｅｆＩＩ
ＤＱｗｌ、Ｉ
Ｑｗ３
ＤＩ？２Ｄ賓２
ＤＲ２Ｄｗ１２
ａｕ３ＡＩ
ＤＱ＆２
ＢｓｓＨ口
１２９ｂｐ、４６ｂｐ、３９ｂｐ
１２６ｂｐ、８８ｂｐ
： １２９ｂｐ、８５ｂｐ
１４３ｂｐ、７１ｂｐ
Ｒ２Ｄｗ２
＋ａｇｂｐ、７６ｂｐ
第１図、第２図、第３図及び第４図は、制限酵素処理後
のアクリルアミドゲル電気泳動の結果を示す。11aefII DQwl, I Qw3 DI? 2D guest 2 DR2Dw12 au3AI DQ&2 BssH mouth 129bp, 46bp, 39bp 126bp, 88bp: 129bp, 85bp 143bp, 71bp R2Dw2 +agbp, 76bp Figures 1, 2, 3 and 4 show acrylamide after restriction enzyme treatment. de The results of gel electrophoresis are shown.
Claims (9)
アロ特異性に特異的な制限酵素で切断し、その断片の長
さを電気泳動で分別、検出した後、その長さの違いによ
ってＨＬＡの型判別を行う方法。(1) DNA amplified by the PCR method is cut with a restriction enzyme specific to HLA antigen allospecificity, and the length of the fragments is separated and detected by electrophoresis.The difference in length determines the type of HLA. How to make the determination.
を行う第１項記載の方法。(2) The method according to item 1, which performs type discrimination of HLA DR antigen, DQ antigen, and D antigen.
ｏｋ I 、ＨａｅIII、Ｈｈａ I 、Ｈｉｎｆ I 、Ｈｐｈ
I 、Ｋｐｎ I 、Ｍａｅ I 、ＭｂｏII、ＳａｃII、Ｓ
ａｕ３Ａ I またはＳｃｒＦ I である請求項４記載の方
法。(3) Restriction enzymes Apa I, BssHII, Dde I, F
ok I, HaeIII, Hha I, Hinf I, Hph
I, Kpn I, Mae I, MboII, SacII, S
5. The method according to claim 4, wherein the method is au3A I or ScrF I .
法。(4) The method according to item 1, which performs HLA DP antigen type determination.
ＢｓｔＵII、ＡｖａII、またはＭｎｌ I である請求項
２記載の方法。(5) Restriction enzymes Sau I, ScrF I, Mae III,
3. The method of claim 2, wherein BstUII, AvaII, or MnlI.
るＨＬＡ型判別診断薬。(6) A diagnostic agent for HLA type discrimination comprising a restriction enzyme specific to HLA antigen allospecificity.
I 、ＢｓｓＨII、Ｄｄｅ I 、Ｆｏｋ I 、ＨａｅIII、
Ｈｈａ I 、Ｈｉｎｆ I 、Ｈｐｈ I 、Ｋｐｎ I 、Ｍａ
ｅ I 、ＭｂｏII、ＳａｃII、Ｓａｕ３Ａ I 、ＳｃｒＦ
I 、Ｓａｕ I 、ＭａｅIIIまたはＢｓｔＵIIである請
求項１記載の試薬。(7) The restriction enzymes include Ava II, Mnl I, Apa
I, BssHII, Dde I, Fok I, HaeIII,
Hha I , Hinf I , Hph I , Kpn I , Ma
e I , MboII, SacII, Sau3A I , ScrF
2. The reagent according to claim 1, which is I, SauI, MaeIII or BstUII.
量に応じてあらかじめ分注してある容器から成るＨＬＡ
型判別キット。(8) HLA consisting of a container in which restriction enzymes specific to HLA antigen allospecificity are dispensed in advance according to the amount to be used.
Type identification kit.
I 、ＢｓｓＨII、Ｄｄｅ I 、Ｆｏｋ I 、ＨａｅIII、
Ｈｈａ I 、Ｈｉｎｆ I 、Ｈｐｈ I 、Ｋｐｎ I 、Ｍａ
ｅ I 、ＭｂｏII、ＳａｃII、Ｓａｕ３Ａ I 、ＳｃｒＦ
I 、Ｓａｕ I 、ＭａｅIII、またはＢｓｔＵIIである
第１項記載のＨＬＡ型判別用のキット。(9) The restriction enzyme is Ava II, Mnl I, Apa
I, BssHII, Dde I, Fok I, HaeIII,
Hha I , Hinf I , Hph I , Kpn I , Ma
e I , MboII, SacII, Sau3A I , ScrF
2. The kit for HLA type determination according to item 1, which is HLA type I, Sau I, MaeIII, or BstUII.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63311979A JPH02174699A (en) | 1988-09-27 | 1988-12-12 | Hla type discrimination and reagent |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24200088 | 1988-09-27 | ||
| JP63-242000 | 1988-09-27 | ||
| JP63311979A JPH02174699A (en) | 1988-09-27 | 1988-12-12 | Hla type discrimination and reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02174699A true JPH02174699A (en) | 1990-07-06 |
Family
ID=26535554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63311979A Pending JPH02174699A (en) | 1988-09-27 | 1988-12-12 | Hla type discrimination and reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02174699A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58130000A (en) * | 1982-01-22 | 1983-08-03 | エフ、ホフマン−ラ ロシュ アーゲー | Hla type deciding method and cdna probe used therein |
| JPS62214355A (en) * | 1986-03-13 | 1987-09-21 | エフ．ホフマン―ラ ロシュ アクチェンゲゼルシャフト | Method of detecting specific nucleotide change and genetic polymorphism existing in nucleic acid |
-
1988
- 1988-12-12 JP JP63311979A patent/JPH02174699A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58130000A (en) * | 1982-01-22 | 1983-08-03 | エフ、ホフマン−ラ ロシュ アーゲー | Hla type deciding method and cdna probe used therein |
| JPS62214355A (en) * | 1986-03-13 | 1987-09-21 | エフ．ホフマン―ラ ロシュ アクチェンゲゼルシャフト | Method of detecting specific nucleotide change and genetic polymorphism existing in nucleic acid |
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