JPH0479885A - New virulence plasmid and base sequence related to the same plasmid - Google Patents
New virulence plasmid and base sequence related to the same plasmidInfo
- Publication number
- JPH0479885A JPH0479885A JP2194069A JP19406990A JPH0479885A JP H0479885 A JPH0479885 A JP H0479885A JP 2194069 A JP2194069 A JP 2194069A JP 19406990 A JP19406990 A JP 19406990A JP H0479885 A JPH0479885 A JP H0479885A
- Authority
- JP
- Japan
- Prior art keywords
- pkdsc50
- salmonella
- plasmid
- virulence plasmid
- probe
- 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
- 239000013612 plasmid Substances 0.000 title claims abstract description 17
- 230000001018 virulence Effects 0.000 title claims abstract description 14
- 241001138501 Salmonella enterica Species 0.000 claims abstract description 12
- 241000607142 Salmonella Species 0.000 claims abstract description 9
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 9
- 239000000523 sample Substances 0.000 claims abstract description 8
- 239000012634 fragment Substances 0.000 claims abstract description 4
- 230000007918 pathogenicity Effects 0.000 claims abstract description 3
- 208000031729 Bacteremia Diseases 0.000 claims description 9
- 241000699670 Mus sp. Species 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 244000052616 bacterial pathogen Species 0.000 abstract description 3
- 102000016943 Muramidase Human genes 0.000 abstract description 2
- 108010014251 Muramidase Proteins 0.000 abstract description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 229960000274 lysozyme Drugs 0.000 abstract description 2
- 235000010335 lysozyme Nutrition 0.000 abstract description 2
- 239000004325 lysozyme Substances 0.000 abstract description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 abstract description 2
- 230000001420 bacteriolytic effect Effects 0.000 abstract 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 235000013372 meat Nutrition 0.000 abstract 1
- 239000000203 mixture Substances 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 206010040047 Sepsis Diseases 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 206010008631 Cholera Diseases 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000607132 Salmonella enterica subsp. enterica serovar Gallinarum Species 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241000392514 Salmonella enterica subsp. enterica serovar Dublin Species 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、サルモネラ・コレラスイス
（Ｓａｌｍｏｎｅｌｌａ ｃｈｏｌｅｒａｅｓｕｉｓ）
から単離された新規ビルレンスプラスミドＰＫＤＳＣ５
０及びこれに関連する塩基配列に関するものである。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is directed to Salmonella choleraesuis.
A novel virulence plasmid PKDSC5 isolated from
0 and related base sequences.
更に詳細には、ヒトやブタに敗血症を起すサルモネラ・
コレラスイスから分離されたビルレンスプラスミドｐＫ
ＤＳＣ５０とこれに含まれるマウス菌血症惹起能を有し
ているａ＋ｂａ領域塩基配列及びこれを適宜切断してサ
ルモネラ菌検出用に使用できるプローブなどに関するも
のである。More specifically, Salmonella, which causes sepsis in humans and pigs,
Virulence plasmid pK isolated from Cholera suis
This article relates to DSC50, the base sequence of the a+ba region contained therein that has the ability to induce bacteremia in mice, and probes that can be appropriately cleaved from this and used for detecting Salmonella.
本発明において、サルモネラ・コレラスイスから新規ビ
ルレンスプラスミドが見出されたことにより１敗血症に
対応する抗体の作成が考えられ、この抗体をもとに敗血
症の検査及び治癒も可能となるものと考えられる。また
、ビルレンスプラスミドρＫＤＳＣ５０の塩基配列の解
析によって、各種タンパク質が明らかとなり、サルモネ
ラ・コレラスイスの病原性の解明に貢献することとなり
、更には、ｐＫＤＳＣ５０を適宜切断してサルモネラ菌
検出用の各種プローブを提供でき、医療界及び食品界に
大きく寄与するものである。In the present invention, the discovery of a new virulence plasmid from Salmonella cholerae suis makes it possible to create an antibody that responds to sepsis, and it is thought that it will be possible to test and cure sepsis based on this antibody. . In addition, analysis of the base sequence of the virulence plasmid ρKDSC50 revealed various proteins, contributing to the elucidation of the pathogenicity of Salmonella cholerae suis.Furthermore, by cutting pKDSC50 appropriately, various probes for detecting Salmonella were provided. This will greatly contribute to the medical and food worlds.
（従来の技術）
一般に、サルモネラ・コレラスイスはヒトやブタに対し
て高頻度に敗血症を引き起こす病原菌として知られてい
る。(Prior Art) Salmonella cholerae suis is generally known as a pathogenic bacterium that frequently causes sepsis in humans and pigs.
また、その他サルモネラ属菌は広く腸チフスや腸炎症を
起す病原菌として知られている。実際に、サルモネラ・
チフイムリウム（Ｓ、 ｔｙｐｈｉｍｕｒｉｕｍ）、サ
ルモネラ・エンテリテイデイス
（Ｓ、ｅｎｔｅｒｉｔｉｄｉｓ）、サルモネラ・ダブリ
ン（Ｓ、 ｄｕｂｌｉｎ）、サルモネラ・ガリナラム（
Ｓ、 ｇａｌｌｉｎａｒｕｍ）などからはビルレンスプ
ラスミド（病原性プラスミド又は毒カブラスミド）が見
出されている。In addition, other Salmonella genus bacteria are widely known as pathogenic bacteria that cause typhoid fever and intestinal inflammation. In fact, Salmonella
Typhimurium (S, typhimurium), Salmonella enteritidis (S, enteritidis), Salmonella dublin (S, dublin), Salmonella gallinarum (
Virulence plasmids (virulence plasmids or poisonous kabrasmids) have been found in S. gallinarum and the like.
しかしながら、従来敗血症の原因菌であるサルモネラ・
コレラスイスからビルレンスプラスミドが見出されたこ
とはなかった。However, Salmonella, which is the causative agent of sepsis,
No virulence plasmid was found in Cholera suis.
（発明が解決しようとする課題）
本発明においては、サルモネラ・コレラスイスからビル
レンスプラスミドを見出し、塩基配列を解明し、生産さ
れる蛋白質のアミノ酸配列等を探索するものである。(Problems to be Solved by the Invention) In the present invention, a virulence plasmid is found from Salmonella cholerae suis, the base sequence is elucidated, and the amino acid sequence of the protein produced is searched.
（問題点紮解決するための手段）
本発明者らは、サルモネラ・コレラスイスからビルレン
スプラスミドを単離するために鋭意研究したところ、臨
床分離のサルモネラ・コレラスイスのすべての菌株から
５０Ｋｂのプラスミドを単離することに成功した。そし
て、この５０にｂのプラスミドをｐＫＤＳＣ５０と命名
した。(Means for resolving the problem) The present inventors conducted extensive research to isolate virulence plasmids from Salmonella cholerae suis, and found that a 50Kb plasmid was found from all clinically isolated strains of Salmonella cholerae suis. successfully isolated. This 50-b plasmid was named pKDSC50.
即ち、サルモネラ・コレラスイスＲＦ−１（北里研究所
保存菌株）を肉汁液体培地で３０℃で３日間培養し、
得られた培養物を集菌し、これにリゾチームを添加して
、溶菌し、溶菌物を除蛋白処理した後ＳＯＳポリアクリ
ルアミドゲル電気泳動にかけ、５０Ｋｂ部分に多量のｐ
ＫＤＳＣ５０を得ることができる。That is, Salmonella cholerae suis RF-1 (Kitasato Institute preserved strain) was cultured in a broth liquid medium at 30°C for 3 days,
The resulting culture was collected, lysozyme was added thereto, the bacterium was lysed, and the lysate was subjected to protein removal treatment, and then subjected to SOS polyacrylamide gel electrophoresis.
KDSC50 can be obtained.
ｐＫＤＳＣ５０を各種制限酵素で切断することによって
制限酵素地図を作成した。第１図はｐＫＤＳＣ５０（５
０Ｋｂ）の制限酵素地図である。A restriction enzyme map was created by cutting pKDSC50 with various restriction enzymes. Figure 1 shows pKDSC50 (5
0Kb) restriction enzyme map.
第１図において、外側は制限酵素ＥｃｏＲＩの切断部位
を示し、中間は制限酵素ＳａＱ Ｉの切断部位を示し、
内側は制限酵素Ｈｉｎｄ ＩＩＩの切断部位を示してい
る。また、 ｍｂａはＥｃｏＲＩ切断部位から５ａｆｉ
Ｉ切断部位までの約６．４Ｋｂのマウス菌血症惹起能
領域（＋ｗｏｕｓｅ ｂａｃｔｅｒｍｉａ領域： ｍｂ
ａ領域）を示している。In FIG. 1, the outside shows the cleavage site of the restriction enzyme EcoRI, the middle shows the cleavage site of the restriction enzyme SaQ I,
The inside shows the cleavage site of the restriction enzyme Hind III. In addition, mba is 5afi from the EcoRI cleavage site.
Approximately 6.4 Kb mouse bacteremia-inducing region up to I cleavage site (+mouse bacteremia region: mb
area a) is shown.
ｍｂａ領域の確定に際しては、ｐＴＨｌｏにあるトラン
スポゾンＴｎｌをＰＫＤＳＣ５０に挿入し、得られたＴ
ｎｌ挿入変異株を亜致死量（１０’ｃｅｌｌ） ＩＣＲ
マウスの腹腔内に投与し、３日目に心臓から採血し、投
与菌が血液中に存在するかどうかによって、マウス菌血
症惹起能をみることができる。血液中のサルモネラ・コ
レラスイスの変異株の培養にはＴｎｌのアンピシリン耐
性を利用して変異株のみ増殖させることができ、また、
Ｔｎｌの挿入個所の追跡によって、マウス菌血症惹起能
がどの領域に起因するかを確定することができる。To determine the mba region, transposon Tnl in pTHlo was inserted into PKDSC50, and the resulting Tnl
Sublethal dose (10'cell) ICR of nl insertion mutant strain
The drug is administered intraperitoneally to mice, blood is collected from the heart on the third day, and the ability to induce bacteremia in mice can be determined by whether or not the administered bacteria are present in the blood. For culturing mutant strains of Salmonella cholerae suis in the blood, only the mutant strains can be grown by utilizing the ampicillin resistance of Tnl, and
By tracing the insertion site of Tnl, it is possible to determine which region is responsible for the ability to induce bacteremia in mice.
本発明においては、このトランスポラン挿入変異法によ
ってマウス菌血症惹起能が第１図の■ｂａ領域（約６．
４Ｋｂ）に関与することを確定することができた。In the present invention, the ability to induce bacteremia in mice was determined by this transporan insertion mutation method in the ■ba region (approximately 6.5 mm) in Figure 1.
4Kb).
約６．４Ｋｂのｍｂａ領域の蛋白質については、大腸菌
ミニセル法によって数種類の蛋白質が同領域にコードさ
れているのがわかった。Regarding the protein in the mba region of about 6.4 Kb, several types of proteins were found to be encoded in the same region by E. coli minicell method.
即ち、ｐＫＤＳＣ５０のｍｂａ領域を切り出して、 ｐ
ＢＲ３２２又はｐＡｃＹｃ１８４に挿入し、クローニン
グした後、Ｅ。That is, the mba region of pKDSC50 was cut out and p
After insertion and cloning into BR322 or pAcYc184, E.
ｃｏｌｉ Ｍ２１４１株に形質転換し、この形質転換体
からミニセル（染色体のない細胞）を分離し、このミニ
セルに（３５ｓ）メチオニンを取り込ませ、菌体内住成
物をＳＤＳポリアクリルアミドゲル電気泳動にかけ、２
９Ｋ、３２Ｋ及び７０にの３種類の蛋白質が確認された
。E. coli M2141 strain was isolated, minicells (cells without chromosomes) were isolated from this transformant, methionine was incorporated into the minicells (35s), and the intracellular organisms were subjected to SDS polyacrylamide gel electrophoresis.
Three types of proteins, 9K, 32K and 70, were confirmed.
次いで、ｐＫＤＳＣ５０からｍｂａ領域を切り出し、ｐ
Ｕｃ１１８に挿入し、Ｅ、 ｃｏｌｉ ＪＭ１０９でク
ローニングし、ジデオキシ法により塩基配列を決定した
。Next, the mba region was excised from pKDSC50 and pKDSC50 was cut out.
It was inserted into Uc118, cloned in E. coli JM109, and the nucleotide sequence was determined by the dideoxy method.
第２図はｐＫＤＳＣ５０のｍｂａ領域のＤＮＡ配列及び
アミノ酸配列を示す図である。FIG. 2 is a diagram showing the DNA sequence and amino acid sequence of the mba region of pKDSC50.
第２図において、３２にの５ｔａｒｔ−＋５ｔｏｐの下
線部は３２にの蛋白質のＤＮＡ配列を示し、次に３２に
の５ｔａｒｔ→５ｔｏｐの下線部は別の３２にの蛋白質
のＤＮＡ配列を示し。In FIG. 2, the underlined portion of 5tart-+5top at 32 indicates the DNA sequence of the protein at 32, and the underlined section of 5tart→5top at 32 indicates the DNA sequence of another protein at 32.
次に７０にの５ｔａｒｔ４Ｓｔｏｐの下線部は７０にの
蛋白質のＤＮＡ配列を示し、更に２９にの５ｔａｒｔ→
５ｔｏｐの下線部は２９にの蛋白質のＤＮＡ配列を示し
ている。Next, the underlined part of 5tart4Stop at 70 shows the DNA sequence of the protein at 70, and 5tart4Stop at 29→
The underlined part of 5top shows the DNA sequence of protein 29.
第２図から明らかなように、ｍｂａ領域には、４種類の
蛋白質がコードされているのが分る。この４種類の蛋白
質のうち、１種類もしくはそれ以上がマウス菌血症に関
与するものと考えられるが、これら蛋白質の機能の解明
はヒトやブタの敗血症の機構解明にもつながり、更には
敗血症の診断、治療に用いられる抗体の作製へとつなが
ることが期待される。As is clear from FIG. 2, four types of proteins are encoded in the mba region. It is thought that one or more of these four types of proteins are involved in bacteremia in mice, and elucidation of the functions of these proteins will lead to elucidation of the mechanism of sepsis in humans and pigs. It is expected that this will lead to the production of antibodies used in diagnosis and treatment.
また、ｐＫＤＳＣ５０を適宜切断したＤＮＡ断片はサル
モネラ菌検出用プローブとして有用であり、また、本発
明によって塩基配列が明らかとなったｍｂａ領域のうち
、適宜の部分をＤＮＡ合成機にかけて合成してプローブ
とすることもできる。In addition, a DNA fragment obtained by appropriately cleaving pKDSC50 is useful as a probe for detecting Salmonella, and a probe can be obtained by synthesizing an appropriate portion of the mba region whose base sequence has been revealed by the present invention using a DNA synthesizer. You can also do that.
第１図は、ｐＫＤＳＣ５０の制限酵素地図を示し、第２
図はｐＫＤＳＣ５０のｍｂａ領域のＤＮＡ配列及びアミ
ノ酸配列を示す図である。
代理人 弁理士 戸 １）親 男
第
図
Ｑく
Ｑく
く−Figure 1 shows the restriction enzyme map of pKDSC50;
The figure shows the DNA sequence and amino acid sequence of the mba region of pKDSC50. Agent Patent attorney 1) Parents
Claims (6)
与する新規ビルレンスプラスミドｐＫＤＳＣ５０。(1) A novel virulence plasmid pKDSC50 possessed by Salmonella cholerae suis and involved in pathogenicity.
ｐＫＤＳＣ５０。(2) pKDSC50, which is about 50 Kb and is shown on the restriction enzyme map in FIG.
有し、第２図のアミノ酸配列で表わされる塩基配列。(3) A nucleotide sequence that is contained in pKDSC50, has the ability to induce bacteremia in mice, and is represented by the amino acid sequence shown in FIG.
有し、第２図の１行目に示される約６．４Ｋｂのｍｂａ
領域塩基配列。(4) mba of approximately 6.4 Kb, which is included in pKDSC50 and has the ability to induce bacteremia in mice, and is shown in the first line of Figure 2.
Region base sequence.
るプローブ。(5) A probe consisting of a DNA fragment obtained by appropriately cleaving pKDSC50.
るサルモネラ菌検出用プローブ。(6) A probe for detecting salmonella consisting of a DNA fragment obtained by appropriately cleaving pKDSC50.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2194069A JPH0479885A (en) | 1990-07-24 | 1990-07-24 | New virulence plasmid and base sequence related to the same plasmid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2194069A JPH0479885A (en) | 1990-07-24 | 1990-07-24 | New virulence plasmid and base sequence related to the same plasmid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0479885A true JPH0479885A (en) | 1992-03-13 |
Family
ID=16318446
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2194069A Pending JPH0479885A (en) | 1990-07-24 | 1990-07-24 | New virulence plasmid and base sequence related to the same plasmid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0479885A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011254829A (en) * | 2008-07-08 | 2011-12-22 | National Agriculture & Food Research Organization | Method for quickly identifying salmonella typhimurium, choleraesuis, enteritidis, dublin, and gallinarum by multiplex detection of specific gene |
-
1990
- 1990-07-24 JP JP2194069A patent/JPH0479885A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011254829A (en) * | 2008-07-08 | 2011-12-22 | National Agriculture & Food Research Organization | Method for quickly identifying salmonella typhimurium, choleraesuis, enteritidis, dublin, and gallinarum by multiplex detection of specific gene |
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