JPS6349084A - Gene - Google Patents
GeneInfo
- Publication number
- JPS6349084A JPS6349084A JP19305886A JP19305886A JPS6349084A JP S6349084 A JPS6349084 A JP S6349084A JP 19305886 A JP19305886 A JP 19305886A JP 19305886 A JP19305886 A JP 19305886A JP S6349084 A JPS6349084 A JP S6349084A
- Authority
- JP
- Japan
- Prior art keywords
- batroxobin
- amino acid
- dna
- acid sequence
- gene
- 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.)
- Granted
Links
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- 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 6
- 229920001817 Agar Polymers 0.000 description 6
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- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
l!上二上月±1
この発明は新規な遺伝子に関するものである.さらに詳
細には、この発明はバトロキソビンのアミノ酸配列を含
むポリペプチドをコードする遺伝子に関するものである
.
従来の技術およびこの 明の解決しようとする間鼠A
バ1・ロキソビン( Batroxobin )は、南
米産ガラガラ蛇(旺肋二肚拙−OX, mo(山呂)の
産生ずるトロンビン様酵素で、フィブリノーゲンA鎖を
特異的に切断して可溶性フィブリンを生成する.このた
め、血栓症の予助・治療剤として有用である[例えば、
Stocker, K. Handbook ofEx
arimental Pharmacolo vo
l. 46. pp+ 451(1978), Spr
inger−’/erlag. Berlin参照コ.
しかし、バトロキソビンは上記毒蛇の毒液より単離・精
製するために、高価でありかつ安定的な供給に不安があ
る.
明の構成および効果
そこで、この発明者等はバトロキンビンの安定供給を確
保するために、遺伝子組み換え技術、すなわち、バトロ
キソビンに対応する遺伝子を適当なベクター(例えばプ
ラスミド)等に挿入し、次いで、この組み換えベクター
で宿主細胞(例えば微生物)を形質転換し、この形質転
換体を培養することによりバトロキソビンを製造する方
法に着目した.
このためには、バトロキソビンの遺伝子を単離すること
が必要である.そこで、この発明者等はバトロキソビン
を精製して、バトロキソビンのアミノ酸配列のうち、N
末端から229のアミノ酸配列を決定し、これに基づき
29mcrのオリゴヌクレ才デドからなるブローブを作
成し、このプローブを用いて、バトロキソビンのcDN
Aのクローン化に成功し、遺伝子組み換え技術によるバ
トロキソビンの製造の道をひらいた.すなわち、この発
明はバトロキソビンのアミノ酸配列を含むポリペプチド
をコードする遺伝子を提供するものである,ここで、バ
トロキソビンのアミノ酸配列を含むポリペプチドをコー
ドする遺伝子としては、アミノ酸配列《I):
ValI1eG1yG1yAspG1uCysAspI
1eAsnGluHisProPheLcuA1aPh
cMetTyrTyrScrProArgTyrPhe
CysG1yMctThrLeuI1eAsnG1nG
1uTrpValLeuThrA1aA1aHisCy
sAsnArgArgPheMetArgI1eHis
LeuG1yLysHisA1aG1ySerValA
1aAsnTyrAspG1uValVa IArgτ
yrProLysG1uLysPheI1aCysPr
oAsnLysLysLysAsnValI 1eTh
rAspLysAspI 1eMetLeuI 1eA
rgLauAspArgProValLysAsnSe
rG1uHisI1eAlaProLeuSerLeu
ProSerAsnProProSerValG1yS
erValCysArgI1eMetG1yTrpG1
yA1aI1eThrThrSerG1uAspThr
TyrProAspValProHisCysA1aA
snI 1eAsnLeuPheAsnAsnThrV
alCysArgG1uA1aTyrAsnG14lL
euProA1aLysThrLeuCysA1aG1
yValLauG1nG1yG1yI1eAspThr
CysG1yG1yAspSerG1yG1yProL
euI 1eCysAsnG1yG1nPheG1nG
1yI 1eLeuSerTrpG1ySarAspP
roCysA1aG1uProArgLysProA1
aPhcTyrThrLysValPhaAspTyr
LeuProTrpI1eG1nSerI1elleA
1aG1yAsnLysThrA1aThrCysPr
oで示きれるポリペプチドをフードするDNAおよび
アミノ酸配列(■》:
MetValLeuI 1eArgValI1aA1a
AsnLeuLeuI1eLauG1nValSerT
yrA1aG1nLysSerSarG1uLeuVa
lI1eG1yG1yAspG 1uCysAspI
1eAsnG 1uHisProPheLeuA1aP
hcMetTyrTyrSerProArgTyrPh
eCysG1yMetThrLauI 1cAsnG1
nG1uTrpValLeuThrA1aA1a}Ii
sCysAsnArgArBPhaMetArgI1a
HisLeuG1yLys}IisA1aG1ySer
ValA1aAsnTyrAspG1uValValA
rgTyrProLysG1uLysPheI1eCy
sProAsnLysLysLysAsnValI1e
ThrAspLysAspI1eMetLeuI 1a
ArgLeuAspArgProValLysAsnS
erG1uHisl leA 1 aProLeuSe
rLeuProSerAsnProProServal
G1yserValCysArgI1eMetGlyT
rpG1yA1aI1eThrThrSerG1uAs
pThrTyrProAspValProHisCys
A1aAsnI leAsnLcuPheAsnAsn
ThrValCysArgG1uA1aTyrAsnG
1yLeuProA1aLysThrLeuCysA1
aG1yValLeuG1nG1yG1yI1eAsp
τhrCygG1yG1yAspSerG1yG1yP
roLauI1eCysAsnGLyG1nPhaG1
nG1yI1eLeuSarTrpG1ySerAsp
ProCysA1aG1uProArgLysProA
1aPheTyrThrLysValPhcAspTy
rLeuProTrplleG1nSerI1aI1@
A1aG1yAsnLysThrA1aThrCysP
ro
で示されるポリペプチドをコードするDNAまたは
塩基配列(I》:
GTCATTGGAGGTGATGAATGTGACA
IAAATGAACATCCTrTCCTTGCATT
CATGTACTACTCTCCCCGGTATTTC
TGTGGTATGACTTTGAICAACCAGG
AATGGGTGCTGACCGCTGCACACTG
TAACAGGAGATrTATGCGCATACAC
CTTG(;TAAACATGCCGGAAGTGTA
GCAAATIATGATGAGGIGGTAAGAr
ACCCAAAGGAGAAGTTCATTTGTCC
CAATAAGAAAAAAAATGTCATAACG
GACAAGGACATTATGTTGATCAGGC
TGGACAGACCTGTCAAAAACAGTGA
ACACATCGCGCCTCTCAGCTTGCCT
TCCAACCCTCCCAGTGTGGGCTCAG
TTIGCCGTATTATGGGAIGGGGCGC
AATCACAACTTC丁GAAGACACTTAT
CCCGATGTCCCTCATTGTGCrAACA
TTAACCTGITCAATAATACGGIGTG
TCGTGAAGC工TACAATGGGTIGCCG
GCGAAAACATTGrGTGCAGGTGTCC
TGCMGGAGGCATAGATACATGTGGG
GGTGACTCTGGGGGACCCCTCATCT
GTAATGGACAArTCCAGGGCATTTT
A’rCTTGGGGAAGIGATCCCTGTGC
CGAACCGCGTAAGCCTGCCTTCTAC
ACCAAGGTCTTTGATτATCTTCCCT
GGATCCAGAGCATTATTGCAGGAAA
TAAAACTGCGACTTGCCCGで示されるD
NAおよび
塩基配列(I[):
ATGGTGCTGATCAGAGTGATAGCAA
ACCrTCTGATAττACAGGTTTCTTA
CGCACAAAAGTCTTCTGAACTGGTC
ATTGGAGGrGATGAATGTGACArAA
ATGAACA工CCT’fTCCTTGCATTCA
TGTACTACTCTCCCCGGTATTTCTG
TGGTATGACTTTGATCAACCAGGAA
TGGGTGCTGACCGCTGCACACTGTA
ACAGGAGAITIATGCGCATACACCT
TGGIAAACATGCCGGAAGTGτAGCA
AA工丁ATGATGAGGTGGTAAGAIACC
CAAAGGAGAAGTTCA工TTGTCCCAA
TAAGAAAAAAAAIGTCATAACGGAC
AAGGACATTATGττGAτCAGGCTGG
ACAGACCTGICAAAAACAGTGAACA
CATCGCGCCTCTCAGCT’rGCCTTC
CAACCCTCCCAGTGTGGGCTCAGTT
TGCCGTATTAτGGGATGGGGCGCAA
TCACAACTTCTGAAGACACTTATCC
CGATGTCCCTCATTGTGCTAACATT
AACCTGrTCAATAATACGGTGTGTC
GTGAAGCTTACAATGG(JTGCCGGC
GAAAACA工τGTGTGCAGGTGTCCTG
CAAGGAGGCATAGATACATGTGGGG
GrGACTCTGGGGGACCCCTCATCTG
TAATGGACAATTC(:AGGGCAITTT
ATCTTGGGGAAGTGATCCCTGTGCC
GAACCGCG工AAGCCTGCCTTCTACA
CCAAGGrCTTTGATTATCTTCCCTG
GATCCAGAGCATTATTGCAGGAAAI
AAAACTGCGACTTGCCCG
で示されるDNAを含み、上記アミノ酸配列(I)のN
一末端にメテ才二ン(Met)が、また上記塩基配列(
1)の5′一末端にメチ才二ンに対応するコドン(AT
G)が付加していてもよく、上記アミノ酸配Jl(1)
および(I[)をコードするDNAおよび塩基配列(1
)および(I[)で示されるDNAの3′一末端に終止
フドン(例えば、τGA)を有していてもよく、要する
にバトロキソピン活性中心部分のベブテドをコードする
ものであればいずれでもよく、そのようなものとしては
、例えば上記遺伝子の一部が切断されたDNA,上記遺
伝子の一部が対立遺伝子変異、遺伝フードの縮重または
一部の修飾により他のヌクレ才チドに置換されたDNA
を含む.
この発明のバトロギソビンのアミノ酸配列を含むポリベ
プデドをコードする遺伝子は、例えば次のような方法に
より製造される.
まず、南米産ガラガラ蛇(む工敗」ソ牡μV.7)の毒
腺よりフェノール法[ Eur. J.Bioche
m.. 97. 1 (1979)]でRNAを単離し
、このRNAから才リコ゛(d丁)セルロース法[江匹
,麺口.紋り.≦1,並. 1408 (1972)コ
によりポリ(A)m−RNAを単離する.このポリ(A
>+n−RNAを鋳型、才リゴ(dT)をブライマー
として、逆転写酵素、大腸菌リボヌクレアーゼHと大腸
菌DNAポリメラーゼIを用いる方法[蝕凹,翻. 2
63 (1983)]により二重鎖cDNAを作成する
.この二重鎖cDNAよりcDNAライブラリーを作成
する.すなわち、例えばこの二重鎖cDNAの両端にE
coRIリンカーを付加し、これをT4DNAリガーゼ
を用いる方法[ Huynh,τ.■.ら. DNA
cloningvol.I. pp49 (1985)
IRLPress. Oxford ]により, E
coRIで消化したλgtllDNA ( Proa+
ega Biotec社製)と結合ξせ組み換えλgt
llDNAを作成し、これからパッケージング・ミック
スチJP − ( Packaging Mixtur
e ) ( vectorCloning Syst@
ms社製)を用いてcDNAライブラリーを作成する.
他方、バトロキソピンの市販品( Pentaphar
m社製)をヘバリンーセフ7ロース( Haparin
−Sepharose) CL−68, DEAE−
セファセル( DEAE−Saphacel )および
セファデックス( Saphadex ) G−100
(すべてPharmacia社製)を用いて精製する.
精製バトロキソビンを還元力ルポキシアミドメチル化法
[ Biochemistry Z. 1959 (1
968)コによりジスルフィド結合を切断し、下記実施
例に示すような常法によりN末端の22個のアミノ酸配
列を決定する(第1図参照).次いで、このようにして
決定されたバトロキソビンのN末端アミノ酸配列の2番
目から12番目のアミノ酸配列の遺伝暗号に対応する2
9mer合成オリゴヌタレオデド(第1図参WA)を全
自動DNA合成機を用いて合成する。[Detailed Description of the Invention] l! 1st month ± 1st month This invention relates to a new gene. More specifically, the present invention relates to a gene encoding a polypeptide containing the amino acid sequence of batroxobin. Conventional technology and the problem to be solved by this invention Batroxobin (Batroxobin) is a thrombin-like enzyme produced by the South American rattlesnake (OX, mo), and it is a fibrinogen-produced enzyme. It specifically cleaves the A chain to produce soluble fibrin.Therefore, it is useful as a prophylactic and therapeutic agent for thrombosis [e.g.
Stocker, K. Handbook ofEx
arimental Pharmacolo vo
l. 46. pp+ 451 (1978), Spr.
inger-'/erlag. Berlin reference co.
However, because batroxobin is isolated and purified from the venom of the above-mentioned viper, it is expensive and there are concerns about stable supply. Therefore, in order to ensure a stable supply of batroxobin, the inventors used genetic recombination technology to insert a gene corresponding to batroxobin into an appropriate vector (e.g., a plasmid), and then use this recombinant We focused on a method for producing batroxobin by transforming host cells (eg, microorganisms) with a vector and culturing the transformant. For this purpose, it is necessary to isolate the batroxobin gene. Therefore, the inventors purified batroxobin and found that among the amino acid sequences of batroxobin,
The terminal 229 amino acid sequence was determined, and based on this, a probe consisting of a 29mcr oligonucleotide was created. Using this probe, batroxobin cDNA
We succeeded in cloning A, paving the way for the production of batroxobin using genetic recombination technology. That is, the present invention provides a gene encoding a polypeptide containing the amino acid sequence of batroxobin. Here, the gene encoding the polypeptide containing the amino acid sequence of batroxobin includes the amino acid sequence <<I): ValI1eG1yG1yAspG1uCysAspI
1eAsnGluHisProPheLcuA1aPh
cMetTyrTyrScrProArgTyrPhe
CysG1yMctThrLeuI1eAsnG1nG
1uTrpValLeuThrA1aA1aHisCy
sAsnArgArgPheMetArgI1eHis
LeuG1yLysHisA1aG1ySerValA
1aAsnTyrAspG1uValVa IArgτ
yrProLysG1uLysPheI1aCysPr
oAsnLysLysLysAsnValI 1eTh
rAspLysAspI 1eMetLeuI 1eA
rgLauAspArgProValLysAsnSe
rG1uHisI1eAlaProLeuSerLeu
ProSerAsnProProSerValG1yS
erValCysArgI1eMetG1yTrpG1
yA1aI1eThrThrSerG1uAspThr
TyrProAspValProHisCysA1aA
snI 1eAsnLeuPheAsnAsnThrV
alCysArgG1uA1aTyrAsnG14lL
euProA1aLysThrLeuCysA1aG1
yValLauG1nG1yG1yI1eAspThr
CysG1yG1yAspSerG1yG1yProL
euI 1eCysAsnG1yG1nPheG1nG
1yI 1eLeuSerTrpG1ySarAspP
roCysA1aG1uProArgLysProA1
aPhcTyrThrLysValPhaAspTyr
LeuProTrpI1eG1nSerI1elleA
1aG1yAsnLysThrA1aThrCysPr
DNA and amino acid sequence that encodes the polypeptide represented by o (■): MetValLeuI 1eArgValI1aA1a
AsnLeuLeuI1eLauG1nValSerT
yrA1aG1nLysSerSarG1uLeuVa
lI1eG1yG1yAspG 1uCysAspI
1eAsnG 1uHisProPheLeuA1aP
hcMetTyrTyrSerProArgTyrPh
eCysG1yMetThrLauI 1cAsnG1
nG1uTrpValLeuThrA1aA1a}Ii
sCysAsnArgArBPhaMetArgI1a
HisLeuG1yLys}IisA1aG1ySer
ValA1aAsnTyrAspG1uValValA
rgTyrProLysG1uLysPheI1eCy
sProAsnLysLysLysAsnValI1e
ThrAspLysAspI1eMetLeuI 1a
ArgLeuAspArgProValLysAsnS
erG1uHisl leA 1 aProLeuSe
rLeuProSerAsnProProServal
G1yserValCysArgI1eMetGlyT
rpG1yA1aI1eThrThrSerG1uAs
pThrTyrProAspValProHisCys
A1aAsnI leAsnLcuPheAsnAsn
ThrValCysArgG1uA1aTyrAsnG
1yLeuProA1aLysThrLeuCysA1
aG1yValLeuG1nG1yG1yI1eAsp
τhrCygG1yG1yAspSerG1yG1yP
roLauI1eCysAsnGLyG1nPhaG1
nG1yI1eLeuSarTrpG1ySerAsp
ProCysA1aG1uProArgLysProA
1aPheTyrThrLysValPhcAspTy
rLeuProTrplleG1nSerI1aI1@
A1aG1yAsnLysThrA1aThrCysP
DNA or base sequence (I) encoding the polypeptide shown by ro: GTCATTGGAGGTGATGAATGTGACA
IAAATGAAACATCCTrTCCTTGCATT
CATGTACTACTCTCCCCCGGTATTTC
TGTGGTATGACTTTGAICAACCAGG
AATGGGTGCTGACCGCTGCAACTG
TAACAGGAGATrTATGCGCATACAC
CTTG(;TAAACATGCCGGAAGTGTA
GCAAATIATGATGAGGGIGGTAAGAr
ACCCAAAGGAGAAGTTCATTTGTCC
CAATAAGAAAAAAATGTCATAACG
GACAAGGACATTATGTTGATCAGGC
TGGACAGACCTGTCAAAAAACAGTGA
ACACATCGCGCCTCTCCAGCTTGCCT
TCCAAACCCTCCCCAGTGTGGGCTCAG
TTIGCCGTATTATGGGAIGGGGCGC
AATCACAACTTCDINGGAAGACACTTAT
CCCGATGTCCCCTCATTGTGCrAACA
TTAACCTGITCAAATAATACGGIGTG
TCGTGAAGCENG TACAATGGGTIGCCG
GCGAAACATTGrGTGCAGGTGTCC
TGCMGGAGGCATAGATACATGTGGGG
GGTGACTCTGGGGACCCCTCATCT
GTAATGGACAArTCCAGGGCATTTT
A'rCTTGGGGAAGIGATCCCTGTGC
CGAACCGCGTAAGCCTGCCTTCTAC
ACCAAGGTCTTTGATτATCTTCCCCT
GGATCCAGAGCATTATTGCAGGAAA
D indicated by TAAAACTGCGACTTGCCCG
NA and base sequence (I[): ATGGTGCTGATCAGAGTGATAGCAA
ACCrTCTGATAττACAGGTTTCTTTA
CGCACAAAAGTCTTCTGAACTGGTC
ATTGGAGGrGATGAATGTGACArAA
ATGAACA ENG CCT'fTCCTTGCATTCA
TGTACTACTCTCCCCCGGTATTTCTG
TGGTATGACTTTGATCAACCAGGAA
TGGGTGCTGACCGCTGCACACTGTA
ACAGGAGAITIATGCGCATACACCT
TGGIAAACATGCCGGAAGTGτAGCA
AA Kocho ATGATGAGGTGGTAAGAIACC
CAAAGGAGAAGTTCAENGTTGTCCCAA
TAAGAAAAAAAAAIGTCATAACGGAC
AAGGACATTATGττGAτCAGGCTGG
ACAGACCTGICAAAAAACAGTGAACA
CATCGCGCCTCTCAGCT'rGCCTTC
CAACCCTCCCCAGTGTGGGCTCAGTT
TGCCGTATTAτGGGATGGGGCGCAA
TCACAACTTTCTGAAGACACTTATCC
CGATGTCCCTCATTGTGCTAACATT
AACCTGrTCAATAATACGGTGTGTC
GTGAAGCTTACAATGG (JTGCCGGC
GAAAACA EngineeringτGTGTGCAGGTGTCCTG
CAAGGAGGCATAGATACATGTGGGG
GrGACTCTGGGGGACCCCTCCATCTG
TAATGGACAATTC(:AGGGCAITTT
ATCTTGGGGAAGTGATCCCTGTGCC
GAACCGCG EngineeringAAGCCTGCCTTCTACA
CCAAGGrCTTTGATTATCTTCCCTG
GATCCAGAGCATTATTGCAGGAAAAI
Contains the DNA shown by AAAACTGCGACTTGCCCG, and N of the above amino acid sequence (I).
At one end, there is Met, and the above base sequence (
At the 5' end of 1), there is a codon (AT
G) may be added, and the above amino acid sequence Jl(1)
DNA and base sequence (1
) and (I[) may have a terminating fudon (e.g., τGA) at one end of the 3' end. For example, DNA in which part of the above gene has been cut, DNA in which part of the above gene has been replaced with another nucleotide due to allelic mutation, degeneracy of the genetic food, or some modification.
including. The gene encoding polybepdide containing the amino acid sequence of batrogysobin of the present invention can be produced, for example, by the following method. First, the phenol method [Eur. J. Bioche
m. .. 97. 1 (1979)], and from this RNA using the cellulose method [Ebi, Menguchi. Crest. ≦1, average. 1408 (1972) to isolate poly(A) m-RNA. This poly(A
A method using reverse transcriptase, Escherichia coli ribonuclease H, and Escherichia coli DNA polymerase I using +n-RNA as a template and Saigo (dT) as a primer. 2
63 (1983)] to create double-stranded cDNA. A cDNA library is created from this double-stranded cDNA. That is, for example, E is added to both ends of this double-stranded cDNA.
A method of adding a coRI linker and using T4 DNA ligase [Huynh, τ. ■. and others. DNA
cloningvol. I. pp49 (1985)
IRLPress. Oxford], E.
λgtll DNA digested with coRI (Proa+
(manufactured by ega Biotec) and combined ξ recombinant λgt
Create llDNA, and from now on, Packaging Mixture JP - (Packaging Mixtur
e) (vector Cloning System@
ms) to create a cDNA library. On the other hand, a commercially available product of batroxopin (Pentahar
Hebarin-Cef 7 loin (manufactured by Haparin Co., Ltd.)
-Sepharose) CL-68, DEAE-
DEAE-Saphacel and Saphadex G-100
(all manufactured by Pharmacia).
Purified batroxobin was purified by reducing lupoxiamide methylation method [Biochemistry Z. 1959 (1
968), and the N-terminal 22 amino acid sequence is determined by a conventional method as shown in the example below (see Figure 1). Next, 2 genes corresponding to the genetic code of the 2nd to 12th amino acid sequences of the N-terminal amino acid sequence of batroxobin determined in this way were
A 9mer synthetic oligonucleotide (WA in Figure 1) is synthesized using a fully automatic DNA synthesizer.
この29marの合成オリゴヌクし・オチドをプローブ
として上記で調製したcDNAライブラリーをプラーク
・ハイブリダイゼーション法[Maniatis。Using this 29-mar synthetic oligonucleotide as a probe, the cDNA library prepared above was subjected to plaque hybridization [Maniatis.
工らMo1ecular 仙イ」pp、 320 (1
982)、 ColdSpring Habor La
boratory、 New York]でスクリーニ
ングし、陽性のファージ・クローンを単離する。"Kura Mo1ecular Sen'i" pp, 320 (1
982), ColdSpring Harbor La
laboratory, New York] and positive phage clones are isolated.
このクローンより、プレート・リゼート法(上記Mo1
ecular 肋イ5pp、 371 )を用いて・組
み換えλgtllDNAを単離する。この組み換え入g
t 11DNAよりEcoRI消化法(上記Mo1ec
ular q圧山」pp、 104)により、DNA
インサートを単離して、ベクター(例えばプラスミドp
LIc13)に挿入して組換えベクターを作成し、この
組換えベクターで宿主細胞(例えばエシェリヒア・コリ
DHI )を形質転換して形質転換体を得、常法(上記
Mo1ecularqy状邦pp、 390)により、
再クローニングする。From this clone, plate lysate method (Mo1
Recombinant λgtll DNA is isolated using ecular ribs 5pp, 371). This recombinant g
EcoRI digestion method (Mo1ec
DNA
The insert is isolated and inserted into a vector (e.g. plasmid p
LIc13) to create a recombinant vector, transform a host cell (for example, Escherichia coli DHI) with this recombinant vector to obtain a transformant, and transform it by a conventional method (the above-mentioned Molecular Journal, pp. 390). ,
Re-cloning.
[なお、このようにしてクローン化きれたもののうち、
最長のDNAインサート(すなわち、第2図に示す塩基
配列を膚するバトロキソビンのcDNA )が挿入され
たプラスミドpUc13(この組み換えDNAをpBa
t−2と命名する)を含有するエシェリヒア・フリ(E
scherichia coli ) DHI (pB
at−2)と命名し、工業技術院微生物工業技術研究所
にFERMP−8913として寄8モした。コ
上記で再クローニングした形質転換体より、組み換えベ
クターをアルカリ溶菌法[Nucleic Ac1ds
独 Z、 1513 (1979)]により単離し、こ
の組み換えベクターよりEcoRI消化法とアガロース
ゲル電気泳動法(上記Mo1ecular 摩庄状邦p
p、 104およびpp、 173)により、cDNA
インサートを単離する。[Incidentally, among those that were cloned in this way,
Plasmid pUc13 (this recombinant DNA was transformed into pBa
Escherichia furi (named t-2) containing E.
scherichia coli ) DHI (pB
at-2) and submitted it to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMP-8913. From the transformant recloned above, the recombinant vector was extracted using the alkaline lysis method [Nucleic Ac1ds
German Z, 1513 (1979)], and this recombinant vector was isolated by EcoRI digestion and agarose gel electrophoresis (Molecular, 1513 (1979)).
p, 104 and pp, 173), cDNA
Isolate the insert.
実施例に示す方法により単離芒れたcDNAインサート
の塩基配列を常法に従い制限酵素で切断した後、得られ
たDNA断片をM13mp18とM13mpL9ファー
ジ・ベクター(宝酒造社製)のマルチプル・クローニン
グサイトにサブクローンする。この組み換えM13mp
18とM13mp19ファージを用い、 サンガー(S
anger )法[Sanger、 Fら、 Proc
、 Natl。After cleaving the base sequence of the cDNA insert isolated by the method shown in the example with restriction enzymes according to a conventional method, the obtained DNA fragment was inserted into the multiple cloning site of M13mp18 and M13mpL9 phage vectors (manufactured by Takara Shuzo Co., Ltd.). Subclone. This recombinant M13mp
18 and M13mp19 phage, Sanger (S
Anger) method [Sanger, F et al., Proc.
, Natl.
インサートの全塩基配列を決定する。Determine the entire nucleotide sequence of the insert.
pBat −2より単離されたcDNAインサートの全
塩基配列は、第2図に示きれている通り、全長1.52
3のヌクレオチドからなる。そして、ヌクレオチド25
2〜317によってフードきれるアミノ酸配列は天然バ
トロキソビンのN末端の22個のアミノ酸配列と一致す
る。ヌクレオチド945〜947の終止コドンTGAの
前のCCGでコードきれるプロリン(Pro)は天然バ
トロキソビンのC末端(下記実施例参照)と一致する。As shown in Figure 2, the complete base sequence of the cDNA insert isolated from pBat-2 has a total length of 1.52
Consists of 3 nucleotides. and nucleotide 25
The amino acid sequence covered by 2 to 317 matches the N-terminal 22 amino acid sequence of natural batroxobin. The proline (Pro) encoded by the CCG before the stop codon TGA at nucleotides 945-947 corresponds to the C-terminus of native batroxobin (see Examples below).
啓らにヌクレオチド252〜944に対応するアミノ酸
配列はは乳動物のセリン・プロテアーゼであるトリプシ
ン(trypsin)、バンクレアチック・カリクレイ
ン(pancreatickallikrein )お
よびトロンビン(thrombin )ならびにクロタ
ラーゼ(crotalase )の公知の酵素のアミノ
酸配列とかなりの相同性が見られる(第3図参照)。以
上の事実より、ヌクレオチド252〜944はバトロキ
ンピンのアミノ酸配列をコードする遺伝子であることは
確実である。きらに、ヌクレオチド180〜182のA
TGフドン(メチオニンの遺伝暗号でありかつ翻訳開始
コドンでもある)から始まり231〜233のGCAコ
ドン(アラニンの遺伝暗号)で終るヌクレオチド180
〜233に対応するアミン末端疎水性ペプチドはプレペ
プチド(シグナル・ペプチド)であり、ヌクレオチド2
34〜236のCAAロドン(グルタミンの遺伝暗号)
から初まりヌクレオチド249〜251のCTGコドン
(ロイシンの遺伝暗号)で終るヌクレオチド234〜2
51に対応する親水性ペプチドはプロペプチド(zym
ogen −ペプチド)と推定される。従って、ヌクレ
オチド180〜944は255個のアミノ酸残基からな
るプレプロバトロキソビンをコードし、ヌクレオチド2
34〜944は237個のアミノ酸残基からなるプロバ
トロキソビンをコードする遺伝子である。The amino acid sequence corresponding to nucleotides 252 to 944 was found in the mammalian serine proteases trypsin, pancreatic kallikrein and thrombin, and the known enzymes crotalase. Considerable homology with the amino acid sequence is observed (see Figure 3). From the above facts, it is certain that nucleotides 252 to 944 are the gene encoding the amino acid sequence of batloquinpin. Kirani, A of nucleotides 180-182
180 nucleotides starting from TG fudon (genetic code for methionine and translation start codon) and ending at GCA codons 231-233 (genetic code for alanine)
The amine-terminated hydrophobic peptide corresponding to ~233 is a prepeptide (signal peptide), and the nucleotide 2
34-236 CAA lodon (genetic code for glutamine)
Starting from nucleotides 249-251, ending with the CTG codon (genetic code for leucine), nucleotides 234-2
The hydrophilic peptide corresponding to 51 is the propeptide (zym
gen-peptide). Thus, nucleotides 180-944 encode preprobatroxobin, which consists of 255 amino acid residues, and nucleotides 2
34-944 is a gene encoding probatroxobin consisting of 237 amino acid residues.
このようにして、バトロキソピンの遺伝子が明らかにさ
れたことにより、バトロキソビンのアミノ酸配列を含む
ポリペプチドをコードするDNAは本明細書で具体的に
開示した方法によらなくとも、制限酵素による切断やD
NA化学合成を用いる常法により、当業者ならばきわめ
て容易にV造することができる。In this way, the gene for batroxopin has been clarified, and DNA encoding a polypeptide containing the amino acid sequence of batroxobin can be obtained by cleavage with restriction enzymes or DNA encoding without using the method specifically disclosed herein.
V can be produced very easily by those skilled in the art by a conventional method using NA chemical synthesis.
次に実施例によりこの発明の詳細な説明する。Next, the present invention will be explained in detail with reference to Examples.
実施例1 (mRNA(7) m製)
南米産ガラガラ蛇(取置こ鵠史!、吃山典)の毒腺をハ
サミで細切し、0.0IMEDIA、 2%ドデンル硫
酸ナトリウム(5DS)、 0.1Mジテオスレイトー
ル(DTT)を含有する0、1Mトリス塩酸緩衝液(T
risHCl ) (pH9,0)に懸濁する。この懸
濁液に同量のフェノール、クロロホルムおよびイソアミ
ルアルコール(50:50:2)の混液を加えてホモジ
ナイズし、10分間激しく攪拌後、10.00Orpm
、10分間遠心分離して、水層を分離した。この水層を
同量のフェノール、クロロホルムおよびイソアミルアル
コール(50:50:2)で3回抽出後、水層に0.1
倍量の2M酢酸ナトリウム水溶液(pH55)と2.5
倍量のエタノールを加え、全核酸成分を沈殿させた。こ
の沈殿を水に溶解し、同量の4Mリチウムクロライド水
溶液をカロえ、 RNAを沈殿させ、回収した。0.4
M塩化ナトリウム、1mMEDIA、 0.1%SDS
を含む10mM Tris−HCI (pH7,4)に
平fl化させたオリゴ(dτ)セルロースカラムに上記
RNAを加え、上記と同じTris−HCIでカラムを
fc浄後、1 mMEDTA、0.1%SDSを含む1
0mM Tris−HCl(pH7,4)でmRNAを
溶出した。この溶出液に0.1倍量の2M#酸ナトナト
リウム水溶液pH5,5)と2.5倍量のエタノールを
加え、ポリ(A )mRNAを沈殿させ、回収した。Example 1 (manufactured by mRNA (7) m) The venom gland of a rattlesnake from South America (Torikikogeshi!, Noriyuki Uyama) was cut into small pieces with scissors, and mixed with 0.0 IMEDIA, 2% sodium dodenle sulfate (5DS), 0 .0, 1M Tris-HCl buffer (T) containing 1M ditheothreitol (DTT)
risHCl) (pH 9,0). A mixture of equal amounts of phenol, chloroform and isoamyl alcohol (50:50:2) was added to this suspension, homogenized, stirred vigorously for 10 minutes, and then heated to 10.00 rpm.
The aqueous layer was separated by centrifugation for 10 minutes. After extracting this aqueous layer three times with equal amounts of phenol, chloroform and isoamyl alcohol (50:50:2), 0.1
Double the amount of 2M sodium acetate aqueous solution (pH 55) and 2.5
Double the amount of ethanol was added to precipitate all the nucleic acid components. This precipitate was dissolved in water and added with the same amount of 4M lithium chloride aqueous solution to precipitate and collect RNA. 0.4
M Sodium Chloride, 1mMEDIA, 0.1%SDS
Add the above RNA to an oligo(dτ) cellulose column flattened with 10mM Tris-HCI (pH 7,4) containing 1mM EDTA, 0.1% SDS after fc cleaning the column with the same Tris-HCI as above. including 1
mRNA was eluted with 0mM Tris-HCl (pH 7,4). To this eluate were added 0.1 times the amount of 2M sodium chloride aqueous solution (pH 5.5) and 2.5 times the amount of ethanol to precipitate and collect poly(A) mRNA.
実施例2 (cDNAライブラリーの調製)実施例1で
調製されたポリ(A)mRNAを鋳型とし、グブラー(
Gubler )らの方法[Gubler at al
。Example 2 (Preparation of cDNA library) Poly(A) mRNA prepared in Example 1 was used as a template, and Gubler (
[Gubler at al.
.
Gene 25.263 (1983)コに準じて、二
重%cDHAを調製した。すなわち、上記で得られたm
RNA (5K )を50mM塩化ナトリウム、8 m
M塩化マグネシウム、1mMの各dATP、 dCTP
、 dGTP、 dTTP、 10mMDTTを含むT
ris−MCI (pH8,3)に溶解させ、オリゴ(
dr)12−18(2< )(Pharmacia社製
)、 RNasin(60ユニツト)(和光純薬社製)
、逆転写酵素(生化学工業製)(100x二wト)を加
え、42℃に60分間保ち、mRNA−cDNA混成体
を調製した。反応終了後、反応液をフェノール処理1回
を行ない、同量の4M酢酸アンモニウム、2.5倍量の
エタノールを加え、mRNA・cDNA混成体を沈殿さ
せ、回収した。きらに、このncRNA−cDNA混成
体から、Huynhらの方法[Huynh atat、
DNA qy状邦Vo1.1. PP49(198
5)、 IRLPress、 0xford ]に準じ
て二重鎖cDNAを調製した。Double% cDHA was prepared according to Gene 25.263 (1983). That is, m obtained above
RNA (5K) in 50mM sodium chloride, 8 m
M magnesium chloride, 1mM each dATP, dCTP
, dGTP, dTTP, T containing 10mM DTT
Dissolve in ris-MCI (pH 8,3) and oligo(
dr) 12-18 (2< ) (manufactured by Pharmacia), RNasin (60 units) (manufactured by Wako Pure Chemical Industries)
, reverse transcriptase (manufactured by Seikagaku Corporation) (100 x 2 wt) was added and kept at 42°C for 60 minutes to prepare an mRNA-cDNA hybrid. After the reaction was completed, the reaction solution was treated with phenol once, and the same amount of 4M ammonium acetate and 2.5 times the amount of ethanol were added to precipitate and collect the mRNA/cDNA complex. From this ncRNA-cDNA hybrid, the method of Huynh et al.
DNA qy state Vo1.1. PP49 (198
Double-stranded cDNA was prepared according to [5), IRLPress, Oxford].
1なわち、mRNA−cDNA混成体を5mM塩化マグ
ネシウム、10mM硫酸アンモニウム、 100mM塩
化カリウム、1 mMDTT、0.15mMβ−NAD
、50 Hg / mQ牛血清アルブミン(BSA)、
75μHの各dATP、 dcTP、 dGTP。1, the mRNA-cDNA complex was mixed with 5mM magnesium chloride, 10mM ammonium sulfate, 100mM potassium chloride, 1mM DTT, 0.15mM β-NAD.
, 50 Hg/mQ bovine serum albumin (BSA),
75 μH each of dATP, dcTP, dGTP.
dTTPを含む20mM Tris−HCl (pH7
,4)に溶かし、E、coli RNase H(
2,5x二yト)(Pharmaciaネ土製)、E、
coli DNAポリメラーゼ(75ユニツト)(
Pharmacia7土製)B、 coli DN
Aリ ガーゼ(2,5ユニツト)(Pharmacia
社製)を加え、13℃に60分間さらに22℃に60分
間保ち、二重鎖cDNAを調製した。反応終了後、反応
液をフェノール処理1回を行ない、同量の4M酢酸アン
モニウム、2.5倍量のエタノールを加え、二重鎖cD
NAを沈殿させ、回収した。さらに、この二重鎖cDN
Aを60mM酢酸カリウム、10mM酢i9−/グネン
ウム、5mMDTI、 1005g/mlウシ血清アル
ブミン(BSA)、0.15mM(7)各dArP、
dcTP、dGTP%dTTPを含む30mM Ir1
s−HCI(pH7,9)に溶かし、T4DNAポリメ
ラーゼ(37x二yト) (Pharn+acia社製
)を加え、37°Cに30分間保ち、二重鎖cDNAの
両端を平滑末端(Flush ends )にした。反
応終了後、反応液を上記と同様な操作で、フェノール処
理し、エタノールにより、二重鎖CDNAを沈殿きせ、
回収した。さらに、この二重鎖DNAを80μMS−ア
デノシルメチオニン、1005g/ muBsA、
1 mM EDTAを含むTrisHcI(pH8,0
>に溶かし、200に、トのEcoRIメチラーゼを加
え、37℃に60分間保ち、二重鎖cDNAをメチル化
した。反応終了後、反応液を上記と同様な操作で、フェ
ノール処理し、エタノールにより、二重鎖cDNAを沈
殿させ、回収した。さらに、この二重鎖cDNAを5
xEcoR1リンカ−(Boehringer社製)
、50mM塩化マグネシウム、5mMスペルミジン、5
0mMDTI、1mMアゾンシン3リン酸(ATP )
を含む60mM TrisHCl(pH7,4)に溶か
し、30ユニツトのT4DNAリガーゼ(Pharma
cla社製)を加え、37℃に60分間保ち、二重鎖c
DNAにEcoRIリンカ−を付加した1反応終了後、
反応液
を70℃に10分間保ち、次いで、0℃に冷却した。20mM Tris-HCl (pH 7) containing dTTP
, 4) and E. coli RNase H (
2.5x2yt) (Pharmacia clay), E,
coli DNA polymerase (75 units) (
Pharmacia7 clay) B, coli DN
A ligase (2,5 units) (Pharmacia
Co., Ltd.) and kept at 13°C for 60 minutes and then at 22°C for 60 minutes to prepare double-stranded cDNA. After the reaction was completed, the reaction solution was treated with phenol once, and the same amount of 4M ammonium acetate and 2.5 times the amount of ethanol were added, and the double-chain cD
NA was precipitated and collected. Furthermore, this double-stranded cDN
A was mixed with 60mM potassium acetate, 10mM vinegar i9-/gnenium, 5mM DTI, 1005g/ml bovine serum albumin (BSA), 0.15mM (7) each dArP,
30mM Ir1 containing dcTP, dGTP% dTTP
Dissolved in s-HCI (pH 7,9), added T4 DNA polymerase (37x2t) (manufactured by Pharn+acia), and kept at 37°C for 30 minutes to make both ends of the double-stranded cDNA blunt (Flush ends). . After the reaction was completed, the reaction solution was treated with phenol in the same manner as above, and the double-stranded CDNA was precipitated with ethanol.
Recovered. Furthermore, this double-stranded DNA was treated with 80 μM S-adenosylmethionine, 1005 g/muBsA,
TrisHcI (pH 8,0
The double-stranded cDNA was methylated by adding 200% EcoRI methylase and keeping at 37°C for 60 minutes. After the reaction was completed, the reaction solution was treated with phenol in the same manner as above, and the double-stranded cDNA was precipitated with ethanol and recovered. Furthermore, this double-stranded cDNA was
xEcoR1 linker (manufactured by Boehringer)
, 50mM magnesium chloride, 5mM spermidine, 5
0mM DTI, 1mM azonsine triphosphate (ATP)
60mM TrisHCl (pH 7.4) containing 30 units of T4 DNA ligase (Pharma
(manufactured by CLA) and kept at 37°C for 60 minutes.
After completing one reaction in which an EcoRI linker was added to DNA,
The reaction was kept at 70°C for 10 minutes and then cooled to 0°C.
きらに、反応液に750ユニフ)のEcoRI (東洋
紡社製)を力Uえ、37℃に4時間保ち、過剰のEeo
RIリンカ−を分解した0分解された過剰のEcoRI
リンカ−をセフ y O−ス(Sepharosa)
4 B (Pharmacia社製)のクロマトグラフ
ィにより反応液より除き、上記と同様な操作でエタノー
ルにより、二重鎖cDNAを沈殿させ、回収した。この
二重鎖cDNAを10mM0mM塩化マグネラムmMA
YP、 10mMDTTを含む50mM TrisHc
l(pH7,4)に溶かし、EcoRIで消化したλg
tlIDNA (1,54)CPromega Bi
otecネ土製)と14x二v)のT4DNAリガーゼ
を加え、13℃に7時間保ち、組み換えλgt 11
DNAを調製した。この組み換えλgtlIDNAヲバ
ッケージング・ミックスチャ−(Packaging
m1xture、Vector Cloning Sy
stems社製)と混合し、室温で1時間保ち、組み換
えDNAをファージ粒子内に挿入した。Then, add 750 units of EcoRI (manufactured by Toyobo Co., Ltd.) to the reaction solution and keep at 37°C for 4 hours to remove excess EeoRI.
0 degraded excess EcoRI that degraded the RI linker
Sepharosa linker
The double-stranded cDNA was removed from the reaction solution using 4B (Pharmacia) chromatography, and the double-stranded cDNA was precipitated and recovered using ethanol in the same manner as above. This double-stranded cDNA was dissolved in 10mM 0mM Magnelum Chloride mM.
YP, 50mM TrisHc with 10mM DTT
λg dissolved in l (pH 7,4) and digested with EcoRI
tlIDNA (1,54)CPromega Bi
Add T4 DNA ligase (14 x 2 v) and keep at 13°C for 7 hours to transform the recombinant λgt 11
DNA was prepared. Packaging mixture of this recombinant λgtlI DNA
m1xture, Vector Cloning Sy
stems) and kept at room temperature for 1 hour to insert the recombinant DNA into the phage particles.
このファージを大腸菌Y1088に感染きせ、寒天プレ
ート上で、42°Cに15時間保ち、増幅芒せ [r;
!NA 懇圧状IVo1. L pp、 49 (
1985)、 IRLPress、 0xfordコ、
cDNAライブラリーを調製した。このcDNAライ
ブラリーは6×107個の独立したファージを含んでい
た。This phage was infected with E. coli Y1088, kept at 42°C for 15 hours on an agar plate, and amplified.
! NA Pressure IVo1. L pp, 49 (
1985), IRLPress, Oxford Co., Ltd.
A cDNA library was prepared. This cDNA library contained 6 x 107 independent phages.
実施例3(バトロキソビンの精製)
バトロキソビン標品(Pentapharm社製)を0
.1Mグリシン・水酸化ナトリウム緩衝液(pH8,2
)で平衡化したのち、ヘパリン−セファ0−ス(I(e
parin−5epharose ) CL−6B (
Pharmacia社製)のカラム・クロマトグラフィ
ーで分画した。各画分を、合成基質Pro−Phe−A
rg−MCA (ペプチド研究所1)を用いてバトロキ
ソビン活性を測定し、0.25M塩化ナトリウム水溶液
で溶出きれるバトロキソビン画分を得た。さらに、この
画分をダイアフロー・メンプランPM−10(Amlc
on社製)で限外a、過して、濃縮した後、20mM
Tris−HCI (pH8,2)に対して透析後、D
EAE−セファセル(DEAE−5aphacel)(
Pharmacia社製)のカラム・クロマトグラフィ
に付し、上記と同様な方法でバトロキソビン活性を測定
し、004M塩化ナトリウムで溶出きれるバトロキソビ
ンを含む画分を得た。この画分をグイアフロ−・メンブ
ランPト10で濃縮後、100+r+M塩化ナトリウム
および10mM塩化カルシウムを含む50n+M Tr
is−Hcl(pH8,0)で平衡化したセファデック
ス(Sephadax) G−100(pharmac
ia社製)で分画し、上記と同様な方法でバトロキソビ
ン活性を測定し、精製バトロキンビン画分を得た。Example 3 (Purification of Batroxobin) Batroxobin preparation (manufactured by Pentapharm) was
.. 1M glycine/sodium hydroxide buffer (pH 8,2
) and then equilibrated with heparin-Sepha0-se (I(e
parin-5epharose) CL-6B (
It was fractionated by column chromatography (manufactured by Pharmacia). Each fraction was divided into synthetic substrate Pro-Phe-A
Batroxobin activity was measured using rg-MCA (Peptide Institute 1), and a batroxobin fraction that could be completely eluted with a 0.25 M sodium chloride aqueous solution was obtained. Furthermore, this fraction was added to Diaflow Menpuran PM-10 (Amlc
on) and concentrated to 20mM.
After dialysis against Tris-HCI (pH 8,2), D
EAE-5aphacel (DEAE-5aphacel) (
The product was subjected to column chromatography (manufactured by Pharmacia), and batroxobin activity was measured in the same manner as above to obtain a fraction containing batroxobin that could be completely eluted with 004M sodium chloride. After concentrating this fraction on a Guiaflo membrane P10, 50n+M Tr containing 100+r+M sodium chloride and 10mM calcium chloride was added.
Sephadex G-100 (pharmac) equilibrated with is-Hcl (pH 8,0)
(manufactured by IA) and the batroxobin activity was measured in the same manner as above to obtain a purified batroxobin fraction.
実施例4(バトロキソビンのN末端アミノ酸配列および
C末端アミノ酸の決定)
上記で得たバトロキソピンをワクスダル(Waxdal
)らの方法[Biochemistry 7. 19
59<1968) ]に準じて還元カルボキシアミノエ
チル化した。すなわち、バトロキソビンを8M尿素、0
.2%EDTAを含む4M Tris−HCl (pH
8,5)に溶かし、バトロキソビンに含まれるハーフ・
シスチン(half−cystine )の140倍量
のβ−メルカプトエタ7/−ルを加え、室温で6時間保
ち、還元した。β−メルカプトエタノールの5倍量の2
−ブロモエチルアミンを室温で10分ごとに3回に分け
て加えた。その後、室温で30分間77置したのち、p
Hを3゜0に調整した。反応終了後、反応液を0.01
M酢酸で透析し、その透析物を凍結乾燥して、還元カル
ボキシアミノエチル
した.この還元カルボキシアミノエチル化バトロキ・ソ
ビンのN末端アミノ酸を全自動気相アミノ酸配列決定機
( Model 470A, Applied Bio
systems社製)を用いて、順次、PTH−アミノ
酸として遊離させた.遊離したPTH−アミノ酸をPT
H−アミノ酸分析機( Model 120A. Ap
plied Biosystem社製)を用いて同定し
た.その結果、第1図のごとく、22番目までのN末端
アミノ酸配列を決定した。また、バトロキソビンのC末
端アミノ酸についてのヒドラジツリシス法[ Bull
. Chem Sac. 3g2. 29。Example 4 (Determination of N-terminal amino acid sequence and C-terminal amino acid of batroxobin) The batroxopin obtained above was
) et al. [Biochemistry 7. 19
59<1968)]. That is, batroxobin was mixed with 8M urea, 0
.. 4M Tris-HCl (pH
8,5) and half-contained in batroxobin.
β-Mercaptoethanol 7/-ol was added in an amount 140 times that of half-cystine, and the mixture was kept at room temperature for 6 hours for reduction. 2 of 5 times the amount of β-mercaptoethanol
- Bromoethylamine was added in three portions every 10 minutes at room temperature. After that, after leaving it at room temperature for 30 minutes, p
H was adjusted to 3°0. After the reaction is completed, the reaction solution is diluted to 0.01
Dialysis was performed against M acetic acid, and the dialysate was lyophilized to reduce carboxyaminoethyl. The N-terminal amino acid of this reduced carboxyaminoethylated Batroki sobin was analyzed using a fully automatic gas-phase amino acid sequencer (Model 470A, Applied Bio
Systems) to sequentially release PTH-amino acids. The released PTH-amino acid is converted into PT
H-amino acid analyzer (Model 120A. Ap
(manufactured by plied Biosystem). As a result, as shown in FIG. 1, the N-terminal amino acid sequence up to the 22nd position was determined. In addition, the hydrazitolysis method for the C-terminal amino acid of batroxobin [Bull
.. Chem Sac. 3g2. 29.
507 (1956)コによりプロリン(Pro)と決
定した。507 (1956), it was determined to be proline (Pro).
実施例5(cDNAライブラリーのスクリーニング)ハ
トロキソビンのN末端アミノ酸配列の2番目から12番
目のアミノ酸配列に対応する遺伝暗号を有する29me
rの合成オリボタクレオチド(第1図参照)を全自動D
NA合成機(Model 391A、 Applied
Biosy=tem社製)を用いて合成した。なお、ス
クリーニング効率をよくするために合成オリボタクレオ
チドの塩基の一部をデオキシイノシン(I>に置き換え
た[江匹、匣且、とり、辷、婬、 1931(1985
) コ 。Example 5 (cDNA library screening) 29me having a genetic code corresponding to the 2nd to 12th amino acid sequence of the N-terminal amino acid sequence of hatroxobin
Synthetic olibotacreotide (see Figure 1) of r
NA synthesizer (Model 391A, Applied
Synthesis was performed using Biosy (manufactured by TEM). In order to improve the screening efficiency, some of the bases of the synthetic oligotacreotide were replaced with deoxyinosine (I>) [Edu, Sakashi, Tori, Tatsu, and Tsun, 1931 (1985
) Ko .
cDNAライブラリーのスクリーニングはプラーク・ハ
イブリダイゼーション法[Maniatis T、 a
t 旦L Mo1ecular (A工状邦pp、 3
20 (1982)、 Co1d SpringHar
bor Laboratory (Naw York)
コに準じて行な−)た、実施例1で得たcDNAライブ
ラリーを大腸菌Y1090 [Huynh at al
、 DNA 四庄吐邦Vo1. pp49 (1985
ン、IRL Press、 0xfordコに感染させ
、寒天平板上にまき、42℃に5時間保ち、プラークを
形成さセた。その後、寒天平板上にニトロセルロース・
フィルターをのせ、各プラークに含まれルDNAヲニト
ロセルロース・フィルター上に転写シタ。ニトロセルロ
ース・フィルター上0.5M 水酸化ナトリウム、1.
5M塩化ナトリウム中、室温で10分間、許らに1,5
M塩化ナトリウムを含む0.5M TrisHCl (
pH7,4)中、室温で10分間処理し、風乾後、80
℃に2時間保ら、DNAを固定した。Screening of the cDNA library was performed using the plaque hybridization method [Maniatis T, a
t DanL Mo1ecular (A Engineering Department pp, 3
20 (1982), Co1d SpringHar
bor Laboratory (Naw York)
The cDNA library obtained in Example 1 was transformed into Escherichia coli Y1090 [Huynh at al.
, DNA Shisho Toho Vol.1. pp49 (1985
(IRL Press, Oxford), plated on an agar plate, and kept at 42°C for 5 hours to allow plaque formation. Then, place nitrocellulose on an agar plate.
Place the filter on the filter and transfer the DNA contained in each plaque onto the nitrocellulose filter. 0.5M sodium hydroxide on nitrocellulose filter, 1.
1,5 min in 5M sodium chloride at room temperature for 10 min.
0.5M TrisHCl containing M sodium chloride (
pH 7.4) at room temperature for 10 minutes, and after air drying,
The DNA was fixed by keeping at ℃ for 2 hours.
上記の29mar合成オリゴヌクレオチドの5′末端を
[rj2 PコATP (ICN社製)とT4ポリヌク
レオチド・キナーゼ(pl+1rmacia社製)を用
イテ、32Fで標識後、フィルター上のDNAとハイブ
リダイズさせ、この合成オリボタクレオチドとハイブリ
ダイズするプラークを検出し、上記スクリーニングを3
回繰り返して、10個の陽性ファージを単離・精製した
。The 5' end of the above 29mar synthetic oligonucleotide was labeled with 32F using rj2PcoATP (ICN) and T4 polynucleotide kinase (PL+1Rmacia), and then hybridized with the DNA on the filter. Plaques that hybridize with this synthetic oligotacreotide were detected, and the above screening was carried out in 3 steps.
Ten positive phages were isolated and purified in duplicate.
この10個の陽性ファージより、プレート・リゼート法
CManLat−1s、 T、 et al、
MolecularCloning pp、 371
(1982) Co1d Spring Harbor
Laboratory (New York)コより組
み換えλgtllDNAを調製した。すなわち、陽性フ
ァージを大腸菌Y1090に感染させ、寒天平板上にま
き、42℃に15時間保ち、ファージを増幅させた。増
幅したファージを0.1M塩化ナトリウム、10mM
MgSO4,01%ゼラチンを含む50mM Ir1s
−HCI (pH7,5)(SM、I液液)で寒天平板
より抽出した。この抽出液に4P@のRNaseA (
Sigma社製)と4 pgDNase I (Sig
ma社製)を加え、37℃に30分間保ち、大腸菌由来
のRNA及びDNAを分解した。きらに、この抽出液に
同量の20%ポリエチレングリコール、2M塩化ナトリ
ウムを含むSM、ill液液 Maniatis、 T
、 et。From these 10 positive phages, plate lysate method CManLat-1s, T. et al.
Molecular Cloning pp, 371
(1982) Co1d Spring Harbor
Recombinant λgtll DNA was prepared from Laboratory (New York). That is, E. coli Y1090 was infected with the positive phage, spread on an agar plate, and kept at 42°C for 15 hours to amplify the phage. The amplified phage was added to 0.1M sodium chloride, 10mM
50mM Ir1s with MgSO4,01% gelatin
Extracted from an agar plate with -HCI (pH 7.5) (SM, I liquid). Add 4P@RNaseA (
Sigma) and 4 pgDNase I (Sig
(manufactured by MA) and kept at 37°C for 30 minutes to degrade E. coli-derived RNA and DNA. Maniatis, T
, etc.
al、 Mo1ecular ユニ状」、 pp、 7
0 (1982)、 ColdSpring Harb
or Laboratory (New York)コ
を加え、0℃に1時間保ち、ファージを沈殿させた。こ
のファージを5M緩衝液に懸濁させ、SDS (最終濃
度0.1%)とEDTA (最終濃度5mM)を加え、
68℃に15分間保った。その後、この溶液をフェノー
ル処理を行ない、同量のイソプロパツールを加え、組み
換え入gtllDNAを沈殿し、回収した。al, Mo1ecular, pp, 7
0 (1982), ColdSpring Harb
or Laboratory (New York) and kept at 0°C for 1 hour to precipitate the phages. The phages were suspended in 5M buffer, SDS (final concentration 0.1%) and EDTA (final concentration 5mM) were added,
It was kept at 68°C for 15 minutes. Thereafter, this solution was treated with phenol, and the same amount of isopropanol was added to precipitate and collect the recombinant gtll DNA.
この組み換えDNAを100+nM塩化ナトリウム、1
0mM塩化マグネンウム、1 mMDTTを含む50m
M Tris・)ICI (pH7,s )に溶かし、
10ユニyトのEcoRI (東洋紡社製)を加え、3
7℃に60分間保ち、反応終了後、反応液を1%アガロ
ース・ゲル1気泳動法[Maniatis、 T、 e
t al、 Mo1ecular 駐イシ、 pp、
157 (1982)、 Co1d Spring
Harbor Laboratory(law Yor
k)コで分画し、分画後、ゲルを0.5eg /1エチ
ジウム・ブロマイドで染色し、cDNAインサートのサ
イズを決定した。そのうち、最長のcDNAインサート
(約1.50(lbp)を常法[ManiatisT、
et al、 Mo1ecular 4
pp、 390 (1982)。This recombinant DNA was mixed with 100+nM sodium chloride, 1
50m containing 0mM Magnenium Chloride, 1mM DTT
Dissolve in M Tris・)ICI (pH 7, s),
Add 10 units of EcoRI (manufactured by Toyobo Co., Ltd.) and
The reaction solution was kept at 7°C for 60 minutes, and after the reaction was completed, the reaction solution was subjected to 1% agarose gel 1 pneumophoresis [Maniatis, T.
tal, Molecular Resident, pp.
157 (1982), Co1d Spring
Harbor Laboratory (law
k) After fractionation, the gel was stained with 0.5eg/1 ethidium bromide to determine the size of the cDNA insert. Among them, the longest cDNA insert (approximately 1.50 (lbp)) was inserted using the conventional method [ManiatisT,
et al, Mo1ecular 4
pp. 390 (1982).
Co1d Spring Harbor Labo
ratory (New York) コ に従
い、プラスミドpUc13 (Boehringer
Mannheim社製)のEcoRIサイトに再クロー
ニングした。すなわち、最長のインサートをもつ組み換
えλgtllDNAを上記の同様な方法でEcoRIで
消化した。同様に、プラスミドDNApUC13もEC
0RIで消化した。これらの消化きれた組み換えλgt
llDNAとプラスミドDNApUC13を混ぜ、実施
例2と同様の条件で、T4DNAリガーゼを月いて、ラ
イゲーションした。このライゲーションされた組み換え
プラスミドDNAを用いて、大腸菌DHI [Mani
atis、 ’r at al。Co1d Spring Harbor Labo
Plasmid pUc13 (Boehringer
Mannheim) EcoRI site. That is, the recombinant λgtll DNA with the longest insert was digested with EcoRI in the same manner as described above. Similarly, plasmid DNA pUC13 also
Digested with 0RI. These digested recombinant λgt
llDNA and plasmid DNA pUC13 were mixed, and ligation was carried out under the same conditions as in Example 2 by adding T4 DNA ligase. Using this ligated recombinant plasmid DNA, E. coli DHI [Mani
atis, 'r at al.
Mo1ecular q工佳邦pp、505 (198
2)、 Co1d SpringHarbor La
boratory (New York)コを形質転換
し、アンピシリンを含む寒天平板上にまき、形質転換し
た大腸菌DHIを単離した。この形質転換きれた大腸菌
DHIよりアルカリ・抽出法[Maniatis、 T
et al、 Mo1ecular 4 pp、36B
(1982)、 ColdSpring Harbo
r Laboratory (New York) ]
に従い組み換えプラスミドDNAを調製した。すなわち
、大腸菌DHIを4 mg/ mQリゾチーム(Sig
n+a社製〕、50mMグルコース、10mMEDIA
を含む25mM Tris−HQ(pH8,0)に懸濁
させ、室温で、5分間保った。Mo1ecular q engineering pp, 505 (198
2), Colorado Spring Harbor La
laboratory (New York) was transformed and plated on agar plates containing ampicillin, and the transformed E. coli DHI was isolated. From this transformed E. coli DHI, the alkaline extraction method [Maniatis, T.
et al, Molecular 4 pp, 36B
(1982), ColdSpring Harbo
r Laboratory (New York) ]
Recombinant plasmid DNA was prepared according to the procedure. That is, E. coli DHI was mixed with 4 mg/mQ lysozyme (Sig
manufactured by n+a], 50mM glucose, 10mMEDIA
The suspension was suspended in 25mM Tris-HQ (pH 8,0) containing 100% chloride and kept at room temperature for 5 minutes.
その後、2倍量0.2tJ7に酸化ナトリウム水溶液、
1%5DSを加え、0℃に5分間保ち、さらに、1.5
倍量の5M酢酸カリウム(pH4,8)を加え、O″C
に5分間保ち、生じた沈殿を遠心分離で除去した、得ら
れた上清をフェノール処理1回し、組み換えプラスミド
DNAをエタノールで沈殿させ、回収した。Then, double the amount of 0.2tJ7 with sodium oxide aqueous solution,
Add 1% 5DS, keep at 0°C for 5 minutes, and add 1.5
Add twice the amount of 5M potassium acetate (pH 4,8), and
The resulting precipitate was removed by centrifugation. The resulting supernatant was treated with phenol once, and the recombinant plasmid DNA was precipitated with ethanol and collected.
得られた組み換えプラスミドDNAを、上記と同様な方
法で、EcoRI消化法、アガロース・ゲル電気泳動法
により分析し、このプラスミドDNAにcDNAインサ
ートが含まれていることを確認した。The obtained recombinant plasmid DNA was analyzed by EcoRI digestion and agarose gel electrophoresis in the same manner as above, and it was confirmed that this plasmid DNA contained a cDNA insert.
実施例6 (cDNAインサートの塩基配列の決定)実
施例5で形質転換したDHIより、実施例5と同様な方
法でcDNAインサートを含むプラスミドDNApUC
13を調製した。この組み換えプラスミドDNAを実施
例5と同様な方法で、EcoRI消化アガロース・ゲル
電気泳動法で分画した。 cDNAインサートを含むゲ
ル断片を透析チューブに加え、1 mMEDIAを含む
Tris・ホウ酸緩衝液存在下で電気泳動的に溶出し、
回収した。Example 6 (Determination of base sequence of cDNA insert) From DHI transformed in Example 5, plasmid DNA pUC containing cDNA insert was obtained in the same manner as in Example 5.
13 was prepared. This recombinant plasmid DNA was fractionated by EcoRI-digested agarose gel electrophoresis in the same manner as in Example 5. The gel fragment containing the cDNA insert was added to a dialysis tube and eluted electrophoretically in the presence of Tris-borate buffer containing 1 mM MEDIA.
Recovered.
得られたcDNAインサートは、実施例5と同様な方法
で)find m、BamHI、5au 3 AI、H
pal[(すべて東洋紡社製)で切断した。The obtained cDNA insert was purified using the same method as in Example 5) find m, BamHI, 5au 3 AI, H
It was cut with pal [all manufactured by Toyobo Co., Ltd.].
一方、ファージ・ベクターM13mp18とM13+n
p19(すべて宝酒造社製)を同時に、HindlI[
、BamHI、シoR1,Accl (すべて東洋紡社
製)で消化し、上記のcDNAインサート断片をT4D
NAリガーゼを用いて、上記のファージベクターのマル
チプル・クローニングサイトに、上記と同様な方法でサ
ブクローニングした。On the other hand, phage vectors M13mp18 and M13+n
p19 (all manufactured by Takara Shuzo Co., Ltd.) was simultaneously mixed with HindlI[
, BamHI, ShioR1, Accl (all manufactured by Toyobo), and the above cDNA insert fragment was digested with T4D.
Using NA ligase, it was subcloned into the multiple cloning site of the above phage vector in the same manner as above.
この組み換えMl 3mp18とM13mp19ファー
ジを用い、サンガー(Sanger )法[Sange
r、 F、 et al、 Proc、 Natl、
Acad、 Sci、 74.5463 <1977)
コに準じ、塩基配列決定キット(日本ジーン社製)を使
用し、cDNAインサートの全塩基配列(第2図参WA
)を決定した。このcDNAノンサートとプラスミドp
uct 3の組み換え体を含有する大腸菌DHIをエシ
ェリヒア・コリ(Escherichia coli
) DHI<pBat−2)と命名し、工業技術院微生
物工業技術研究所にFERMP−8913として寄託し
た。Using the recombinant Ml 3mp18 and M13mp19 phages, the Sanger method [Sange
r, F, et al, Proc, Natl,
Acad, Sci, 74.5463 <1977)
The entire base sequence of the cDNA insert (see Figure 2 WA
)It was determined. This cDNA nonsert and plasmid p
Escherichia coli DHI containing the recombinant uct 3 was transformed into Escherichia coli.
) DHI<pBat-2) and deposited as FERMP-8913 at the Institute of Microbial Technology, Agency of Industrial Science and Technology.
第1図はバトロキソビンのアミン末端アミノ酸配列とそ
れから想定されるm−RNAの配列と29marのプロ
ーブの塩基配列を示す。
第2図は本実施例で単離されたバトロ率ソビンのcDN
Aの塩基配列と翻訳領域の塩基配列に対応するアミノ酸
配列を示す、ヌクレオチド塩基は5′から3′方向に番
号を付し、アミノ酸配列はバトロキソビンのアミン末端
のアミノ酸Valを1として、これを基に番号を付した
。
第3図はバトロキソピンとは乳類のセリン・プロテアー
ゼおよびクロタラーゼ゛のアミノ酸配列の比較を示す0
番号はバトロキソビンのアミン末端アミノ酸から順次付
した。アミノ酸配列のうち、かっこで囲む領域はバトロ
キソピンのそれと一致していることを示す。
7 ミ/i配列Val lie GlyPro Ph
e Leu
Δ Δ
CCI CTI CTT ACA CTA 、
TへI TTI CTI GTCG G
第1図FIG. 1 shows the amine-terminal amino acid sequence of batroxobin, the m-RNA sequence assumed from it, and the base sequence of the 29-mar probe. Figure 2 shows the cDNA of Batolo Sobin isolated in this example.
The amino acid sequence corresponds to the base sequence of A and the base sequence of the translated region. Nucleotide bases are numbered from 5' to 3' direction, and the amino acid sequence is based on the amine terminal amino acid Val of batroxobin as 1. numbered. Figure 3 shows a comparison of the amino acid sequences of batroxopin, mammalian serine protease and crotalase.
Numbers were assigned sequentially starting from the amine-terminal amino acid of batroxobin. In the amino acid sequence, the region enclosed in parentheses indicates that it matches that of batroxopin. 7 Mi/i array Val lie GlyPro Ph
e Leu Δ Δ CCI CTI CTT ACA CTA,
To T I TTI CTI GTCG G Figure 1
Claims (8)
ドをコードする遺伝子(1) Gene encoding a polypeptide containing the amino acid sequence of batroxobin
第18番目のAはGとそれぞれ置換されていてもよい) で示されるオリゴヌクレオチドとハイブリ ダイズする特許請求の範囲第1項記載の遺 伝子。(2) Base sequence: [There is a gene sequence] (In the formula, the 12th T is C, the 15th A is G,
The gene according to claim 1, which hybridizes with the oligonucleotide represented by (the 18th A may be replaced with G).
1項記載の遺伝子。(3) Amino acid sequence (I): [There is a gene sequence] The gene according to claim 1, which encodes the polypeptide shown in the following.
1項記載の遺伝子。(5) Amino acid sequence (II): [There is a gene sequence] The gene according to claim 1, which encodes the polypeptide shown in the following.
ドをコードする遺伝子を含有する組み換えベクター。(7) A recombinant vector containing a gene encoding a polypeptide containing the amino acid sequence of batroxobin.
ドをコードする遺伝子を含有する形質転換体。(8) A transformant containing a gene encoding a polypeptide containing the amino acid sequence of batroxobin.
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JP61193058A JP2517241B2 (en) | 1986-08-19 | 1986-08-19 | gene |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991002814A1 (en) * | 1988-02-24 | 1991-03-07 | Akzo N.V. | Nucleic acid amplification process |
JPH08116980A (en) * | 1994-10-11 | 1996-05-14 | Mogam Biotechnol Res Inst | Gene carrying code for proteinases analogous to novel thrombin |
JP2005281145A (en) * | 2004-03-26 | 2005-10-13 | Yamada Bee Farm | New angiotensin i converting enzyme inhibiting peptide derived rrom royal jelly |
WO2008119203A1 (en) * | 2007-03-30 | 2008-10-09 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A purified recombinant batroxobin with high specific activity |
WO2009084841A3 (en) * | 2007-12-28 | 2009-09-17 | Biobud Co., Ltd. | Mutated nucleotide sequences of batroxobin, mutated alpha factor secretion signal sequence and processes for preparing batroxobin using the same |
-
1986
- 1986-08-19 JP JP61193058A patent/JP2517241B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
THE JOURNAL OF BIOLOGICAL=1976US * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991002814A1 (en) * | 1988-02-24 | 1991-03-07 | Akzo N.V. | Nucleic acid amplification process |
JPH08116980A (en) * | 1994-10-11 | 1996-05-14 | Mogam Biotechnol Res Inst | Gene carrying code for proteinases analogous to novel thrombin |
JP2005281145A (en) * | 2004-03-26 | 2005-10-13 | Yamada Bee Farm | New angiotensin i converting enzyme inhibiting peptide derived rrom royal jelly |
WO2008119203A1 (en) * | 2007-03-30 | 2008-10-09 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A purified recombinant batroxobin with high specific activity |
US7939067B2 (en) | 2007-03-30 | 2011-05-10 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | Purified recombinant batroxobin with high specific activity |
WO2009084841A3 (en) * | 2007-12-28 | 2009-09-17 | Biobud Co., Ltd. | Mutated nucleotide sequences of batroxobin, mutated alpha factor secretion signal sequence and processes for preparing batroxobin using the same |
KR101005821B1 (en) * | 2007-12-28 | 2011-01-05 | (주)바이오버드 | Mutated Nucleotide Sequences of Batroxobin, Mutated ??Factor Secretion Signal Sequence and Processes for Preparing Batroxobin Using the Same |
US8377676B2 (en) | 2007-12-28 | 2013-02-19 | Biobud Co., Ltd | Mutated nucleotide sequences of batroxobin, mutated α factor secretion signal sequence and processes for preparing batroxobin using the same |
Also Published As
Publication number | Publication date |
---|---|
JP2517241B2 (en) | 1996-07-24 |
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