CN1133342A - 一种源于红球菌属微生物的卡那霉素抗性基因 - Google Patents
一种源于红球菌属微生物的卡那霉素抗性基因 Download PDFInfo
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Abstract
本发明涉及一种源于红球菌属微生物并对宿主赋予卡那霉素抗性的DNA,所说宿主携带可编码SEQ NO1的氨基酸序列或含其部分氨基酸序列的多肽的DNA序列。本发明的卡那霉素抗性基因用于构建红球菌属微生物载体,特别是玫瑰色红球菌的自主克隆载体。
Description
本发明涉及一种源于红球菌属微生物并赋予细菌以卡那霉素抗性的基因,以及含此基因的质粒载体。
称为细菌催化剂的红球菌属微生物,能水化或水解腈类生成相应的酰胺类或酸类(日本专利公告号4873/92和日本专利公开号91189/87、470/90和84198/90),特别是玫瑰色红球菌种微生物具有相当高效的水化腈的活性(日本专利公开号470/90)。
鉴于此,本发明的发明人之一从玫瑰色红球菌种的一个菌株中发现了隐匿的质粒,构建了杂种质粒载体以便建立红球菌属宿主载体体系(日本专利公开号148685/92、64589/93和68566/93)。
为建立具有较高可靠性的自主克隆体系,也有必要开发源于红球菌属微生物的标记基因。然而,源于玫瑰色红球菌种微生物的只有亚砷酸和镉抗性基因才称为所说的抗药性基因(质粒23,242-247(1990))。
为建立一种红球菌属的自主克隆体系,本发明人广泛研究了源于红球菌属微生物的抗药性基团,特别是玫瑰色红球菌种的抗药性基因,结果他们发现了本发明的卡那霉素抗性基因。
本发明涉及源于红球菌属微生物并对宿主菌赋予卡那霉素抗性的DNA,所说的宿主携带编码SEQ No.1的氨基酸序列或含部分此序列的多肽的DNA序列。
图1表示质粒pKM001的限制性酶切图谱。
图2表示质粒pKM002、pKM003和pKM004的构建过程。
图3表示质粒pKM011的限制性酶切图谱。
作为本发明的DNA供体,可提到的是,通过玫瑰色红球菌ATCC12674自发突变获得的卡那霉素突变菌株KM-02(保藏号为FERMBP-5137,国立生物科学和人类技术研究院,工业科技会社,日本)。
作为本发明用于克隆的载体,可提及的是,包括但不限于pTrc99A、pUC18等大肠杆菌的质粒载体,和如λgt11的噬菌体载体。宿主微生物包括但不局限于大肠杆菌JM109、大肠杆菌JM105和玫瑰色红球菌ATCC12674。
由携带能在红球菌属微生物中复制的区域的本发明卡那霉素抗性基因构建的、用于提供质粒载体的质粒包括但不局限于pRC001、pRC002、pRC003、和pRC004。质粒pRC001、pRC002、pRC003和pRC004分别源于玫瑰色红球菌ATCC4276、ATCC14349、ATCC14348和IF03338,而且这些质粒在上述日本特许专利公开号148685/92、64589/93和68566/93中分别作了描述。
源于红球菌属微生物的本发明的卡那霉素抗性基因用于构建红球菌属微生物载体,特别是玫瑰色红球菌自主克隆载体。
实施例
下面的实施例详述了本发明,但不是限制本发明范围。
实施例1源于突变菌株KM-02的卡那霉素抗性基因克隆进大肠杆菌JM109中(1)制备源于KM-02的基因组DNA和DNA文库
在100ml附培养基中(0.5%多蛋白胨、0.3%细菌酵母提取物、0.3%细菌麦芽提取物)于30℃摇动培养KM-02菌株,按照SaitoMiura法(Biochem.Biophys.Acta 72,619(1963))从细菌中制备基因组DNA。用限制性酶Sau 3AI部分消化提取的一部分DNA,然后插入到大肠杆菌载体PTrc 99A的BamHI位点,建立重组DNA文库。(2)转化子的制备和重组DNA的筛选
将步骤(1)制备的重组DNA文库通过CaCl2法转化大肠杆菌JM109,以下列方式筛选带有卡那霉素抗性的转化子。
上述转化子被涂到含40μg/ml盐酸卡那霉素和1mM IPTG(异丙基-β-硫代半乳糖苷)的LB培养基上(1%胰蛋白胨、0.5%细菌酵母提取物、0.5%NaCl、1.5%琼脂),37℃孵育过夜,移出培养基上长出的菌落并转接到同样的琼脂培养基上,使其稳定生长。
按Birnboim和Doly法(NuCleic Acid Res.7,1513-1523(1979)),从得到的转化子制备质粒DNA,命名为pKM001。将这种质粒再次引进大肠杆菌,结果产生带有卡那霉素抗性的转化子,命名为JM109/pKM001,保藏号为BP-5138,国立生物科学和人类技术研究院,工业科技会社。大肠杆菌JM109/pKM001的卡那霉素抗性的表达需要IPTG。(3)pKM001的限制性酶谱和卡那霉素抗性基因的定位。
绘制了经步骤(2)获得的质粒pKM001的限制性酶谱(图1)。此后,用质粒pKM001,制备小片段DNA质粒。以与步骤(2)同样的方法制备的转化子的卡那霉素抗性的有或无识别含靶基因的区域。此过程构建了质粒pKM002(图2)。(4)核苷酸测序
用Pharmacia公司生产的荧光测序仪ALFII测定质粒pKM002的卡那霉素抗性基因的核苷酸序列(SEQ NO 3)。
实施例2制备源于玫瑰色红球菌的带有卡那霉素抗性基因的杂种(大肠杆菌-红球菌)质粒载体。
杂种质粒载体pK4先由本发明人之一通过连接源于红球菌属的质粒pRC004和大肠杆菌载体pHSG299构建的,保藏号为FERMBP-3731,国立生物科学和人类技术研究院,工业科技会社(日本特许专利公开号64589/93和68566/93)。此载体用于制备一个含整个PRC004和部分pHSG299的3.1kb Hind III片段,将结果产生的片段和质粒pKM002连接起来。
结果,获得了携带插入子方向不同的两个质粒,分别命名为pKM003和pKM004(图2)。这些质粒不仅在红球菌属中,而且能在大肠杆菌中复制。经电穿孔法用这些质粒转化玫瑰色红球菌ATCC12674,获得了能在含75μg/ml卡那霉素的MY培养基上生长的转化子,从转化子获得的质粒和引进的质粒相同。当红球菌属微生物作为宿主时,表达卡那霉素抗性不需要IPTG。
实施例3红球菌属微生物载体的构建
用限制性酶kpnI切割杂种质粒载体pKM004,产生一4.3kb kpn I片段,然后自身连接,并通过电穿孔导入玫瑰色红球菌ATCC12674,所产生的转化子和实施例2产生的转化子的卡那霉素抗性程度相同。从此转化子获得了一个质粒,命名为pKM011(图3)。序列表序列号:1长度:171序列类型:氨基酸拓扑结构:线性性质:蛋白质起源
微生物:玫瑰红球菌
菌株:KM-02序列Met Ser Asp Asn Gly Ser Gly Thr Thr Arg Pro Glu Gly Ala Pro Leu1 5 10 15Pro Arg Arg Ala Arg Ser Ser Arg Pro Ser Ala Gly Asn Ser Pro Ala
20 25 30Pro Gly Arg Arg Ala Val Val Ala Lys Ser Arg Arg Arg Leu Ala Ala
35 40 45Ala Pro Glu Ala Gly Thr Thr His Tyr Ser Ile Phe His Gly Asp Gln
50 55 60Leu Ile Gly Phe Ile Gln Trp Tyr Glu Ala Glu Asp Asn Pro Asp Phe65 70 75 80Arg His Ala Gly Leu Asp Leu Phe Leu Asp Pro Asp Phe His Gly Arg
85 90 95Gly Phe Gly Arg Glu Ser Ile Arg Val Leu Cys Ala His Leu Ile Asp
100 105 110Asp Leu Ala Phe His Arg Leu Val Ile Asp Pro Glu Val Asp Asn Ser
115 120 125Val Ala Ile Ala Cys Tyr Arg Ser Val Gly Phe Lys Asp Val Gly Val
130 135 140序列号:2长度:516性质:核酸链形:双链拓扑结构:线性起源
微生物:玫瑰色红球菌
菌株:KM-02序列:ATG AGT GAC AAC GGC TCC GGA ACT ACG CGG CCC GAG GGT GCT CCT CTC 48CCC CGT CGC GCC CGA TCA TCA CGC CCG TCT GCG GGC AAT TCA CCT GCA 96CCC GGA CGT CGT GCA GTG GTG GCA AAA TCC CGA CGA CGA CTG GCT GCG 144GCG CCA GAA GCC GGA ACC ACG CAC TAC AGC ATC TTC CAC GGC GAC CAA 192CTG ATC GGC TTC ATC CAG TGG TAC GAA GCG GAA GAC AAC CCC GAC TTC 240CGC CAC GCC GGG CTC GAC TTG TTC CTC GAT CCC GAC TTC CAC GGC CGA 288GGG TTC GGT CGC GAA TCG ATT CGC GTG CTG TGT GCC CAC CTG ATC GAC 336GAC CTC GCA TTC CAC CGT CTG GTC ATC GAC CCG GAG GTC CAC AAC TCC 384GTC GCC ATC GCG TGC TAC CGA TCG GTG CGG TTC AAA GAC GTC GGG GTG 432ATG CGC GAG TAT TCG CGA GAT CGC CAT GGT GTG TGG AAG GAC GGA CTG 480CTG ATG GAT CTG CTC GCA CGG GAA TTC ATC CGC TGA 516序列号:3长度:748序列类型:核酸链形:双链拓扑结构:线性起源
微生物:玫瑰色红球菌
菌株:KM-2序列:GGATCCGGGG TCGTCGCCCA CCAGGATGGT ACCCAAGCCG GGTGTGATGC CCTCTGCCTT 60CGAGCGCTCA CCCGCACCTT CAGGTCTTCG AAGATTTCGT CGCGGGTAGC TTTGCCGTCG 120AGGATCGTTG CAGTCACGGC GACCATTGTT CCAGGTTAGG GTCGATGAGT GACAACGGCT 180CCGGAACTAC GCGGCCCGAG GGTGCTCCTC TCCCCCGTCG CGCCCGATCA TCACGCCCGT 240CTGCGGGCAA TTCACCTGCA CCCGGACGTC GTGCAGTGGT GGCAAAATCC CGACGACGAC 300TGGCTGCGGC GCCAGAAGCC GGAACCACGC ACTACAGCAT CTTCCACGGC GACCAACTGA 360TCGGCTTCAT CCAGTGGTAC GAAGCGGAAG ACAACCCCGA CTTCCGCCAC GCCGGGCTCG 420ACTTGTTCCT CGATCCCGAC TTCCACGGCC GAGGGTTCGG TCGCGAATCG ATTCGCGTGC 480TGTGTGCCCA CCTGATCGAC GACCTCGCAT TCCACCGTCT GGTCATCGAC CCGGAGGTCG 540ACAACTCCGT CGCCATCGCG TGCTACCGAT CGGTGGGGTT CAAAGACGTC GGGGTGATGC 600GCGAGTATTC GCGAGATCGC CATGGTGTGT GGAAGGACGG ACTGCTGATG GATCTGCTCG 660CACGGGAATT CATCCGCTGA TCGACTGGGA CGAGTTCGAA AGGACCGACA TCATGTTGCT 720GGACAAGGAA TTCACGGCCA CCCTGCAG 748
Claims (5)
1.一种源于红球菌属微生物并对宿主赋予卡那霉素抗性的DNA,所说宿主携带编码SEQ NO 1的氨基酸序列或包含其部分氨基酸序列的多肽的DNA序列。
2.根据权利要求1,其中的DNA序列包含Seq.NO.2的序列或其部分序列。
3.根据权利要求1或2,其中的宿主微生物是红球菌属或大肠杆菌微生物。
4.含有权利要求1-3的任一权利要求的DNA的质粒载体和能在红球菌属微生物中复制DNA区。
5.根据权利要求4,其中能在红球菌属微生物中复制的DNA区源于pRC001、pRC002、pRC003和pRC004质粒中筛选的一个质粒。
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JP201582/94 | 1994-08-04 | ||
JP20158294A JP3235934B2 (ja) | 1994-08-04 | 1994-08-04 | ロドコッカス属細菌由来カナマイシン耐性遺伝子 |
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EP (1) | EP0704530B1 (zh) |
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KR (1) | KR100343424B1 (zh) |
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US6586661B1 (en) | 1997-06-12 | 2003-07-01 | North Carolina State University | Regulation of quinolate phosphoribosyl transferase expression by transformation with a tobacco quinolate phosphoribosyl transferase nucleic acid |
DE19803219A1 (de) * | 1998-01-28 | 1999-08-12 | Fraunhofer Ges Forschung | Verfahren und Vorrichtung zur Bestimmung des Oberflächen-Reibungskoeffizienten von Körpern |
DE19926216A1 (de) * | 1999-06-09 | 2001-02-22 | Metallgesellschaft Ag | Verfahren zur Herstellung von Bariumsulfat, Bariumsulfat und Verwendung des Bariumsulfats |
CA2420724A1 (en) * | 2000-08-30 | 2002-03-07 | North Carolina State University | Transgenic plants containing molecular decoys that alter protein content therein |
JP2004533807A (ja) * | 2000-11-07 | 2004-11-11 | ノース・キャロライナ・ステイト・ユニヴァーシティ | プトレッシン−n−メチルトランスフェラーゼプロモーター |
US6949362B2 (en) | 2000-12-12 | 2005-09-27 | E. I. Du Pont De Nemours And Company | Rhodococcus cloning and expression vectors |
KR20040019301A (ko) * | 2001-06-08 | 2004-03-05 | 벡터 토바코 리미티드 | 담배에서 니코틴 및 니트로사민 수준의 조절 |
US20060157072A1 (en) * | 2001-06-08 | 2006-07-20 | Anthony Albino | Method of reducing the harmful effects of orally or transdermally delivered nicotine |
US20040115661A1 (en) * | 2001-12-12 | 2004-06-17 | Bramucci Michael G. | Rhodococcus cloning and expression vectors |
JP2005522201A (ja) * | 2002-04-09 | 2005-07-28 | ベクター、タバコ、リミテッド | ニコチンおよびニトロソアミンを減少させたタバコ |
JP4495429B2 (ja) * | 2003-09-24 | 2010-07-07 | 三菱レイヨン株式会社 | ロドコッカス属細菌の形質転換方法 |
JP4493011B2 (ja) * | 2004-06-11 | 2010-06-30 | 三菱レイヨン株式会社 | ロドコッカス属細菌由来トリメトプリム耐性ジヒドロ葉酸還元酵素及びその遺伝子 |
GB0606190D0 (en) | 2006-03-28 | 2006-05-10 | Isis Innovation | Construct |
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JPH0740948B2 (ja) | 1985-06-04 | 1995-05-10 | 旭化成工業株式会社 | アミドの微生物学的製造法 |
US4920054A (en) * | 1986-07-25 | 1990-04-24 | Allelix, Inc. | Shuttle vectors from rhodococcus equi |
DD274631A5 (de) | 1987-09-18 | 1989-12-27 | Kk | Verfahren zur biologischen herstellung von amiden |
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US5246857A (en) * | 1990-10-11 | 1993-09-21 | Nitto Chemical Industry Co., Ltd. | Circular plasmids derived from the genus rhodococcus |
JP3142349B2 (ja) | 1991-03-04 | 2001-03-07 | 輝彦 別府 | 複合プラスミドベクターおよび形質転換微生物 |
DE69231939T2 (de) * | 1991-03-04 | 2002-04-04 | Mitsubishi Rayon Co | Hybrid-Plasmid-Vektoren, die für Nitril-abbauende Enzyme kodierende Gene enthalten und Verfahren zur Herstellung von Amiden und Säuren |
JP3142348B2 (ja) | 1991-03-04 | 2001-03-07 | 輝彦 別府 | ニトリル分解酵素系の遺伝子を有する組換え体プラスミド、形質転換微生物、ならびに該形質転換微生物によるアミドおよび酸の製造法 |
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Publication number | Publication date |
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JP3235934B2 (ja) | 2001-12-04 |
EP0704530A2 (en) | 1996-04-03 |
EP0704530A3 (en) | 1997-12-29 |
DE69532024D1 (de) | 2003-12-04 |
TW338069B (en) | 1998-08-11 |
DE69532024T2 (de) | 2004-06-24 |
KR100343424B1 (ko) | 2002-11-14 |
KR960007778A (ko) | 1996-03-22 |
US5776771A (en) | 1998-07-07 |
EP0704530B1 (en) | 2003-10-29 |
JPH0838184A (ja) | 1996-02-13 |
CN1080308C (zh) | 2002-03-06 |
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