CN1080308C - 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 PDF

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CN1080308C
CN1080308C CN95115824A CN95115824A CN1080308C CN 1080308 C CN1080308 C CN 1080308C CN 95115824 A CN95115824 A CN 95115824A CN 95115824 A CN95115824 A CN 95115824A CN 1080308 C CN1080308 C CN 1080308C
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汤不二夫
加藤真美
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Mitsubishi Rayon Co Ltd
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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DNA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

一种源于红球菌属微生物 的卡那霉素抗性基因
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DNA具有编码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ATCC 12674。
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IFO3338，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 MY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pK 4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PRC 004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ATCC 12674，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 kpnI片段，然后自身连接，并通过电穿孔导入玫瑰色红球菌ATCC 12674，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 GAC AAC TCC   384GTC GCC ATC GCG TGC TAC CGA TCG GTG GGG 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  TTTGCCGTGG      120AGGATCGTTG  CAGTCACGGC  GACCATTGTT  CCAGGTTAGG  GTCGATGAGT  GACAACGGCT      180CCGGAACTAC  GCGGCCCGAG  GGTGCTCCTC  TCCCCCGTCG  CGCCCGATCA  TCACGCCCGT      240CTGCGGGCAA  TTCACCTGCA  CCCGGACGTG  GTGCAGTGGT  GGCAAAATCC  CGACGACGAC      300TGGCTGCGGC  GCCAGAAGCC  GGAACCACGC  ACTACAGCAT  CTTCCACGGC  GACCAACTGA      360TCGGCTTCAT  CCAGTGGTAC  GAAGCGGAAG  ACAACCCCGA  CTTCCGCCAC  GCCGGGCTCG      420ACTTGTTCCT  CGATCCCGAC  TTCGACGGCC  GAGGGTTCGG  TCGCGAATCG  ATTCGCGTGC      480TGTGTGCCCA  CCTGATCGAC  GACGTCGCAT  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，所述DNA具有编码SEQ NO.1的氨基酸序列的DNA序列。
2.根据权利要求1的DNA，其中所述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的DNA，其中所述宿主微生物是红球菌属微生物或大肠杆菌。
4.一种质粒载体，该质粒载体含有权利要求1-3中任一项的DNA和能在红球菌属微生物中复制的DNA区域。
5.根据权利要求4的质粒载体，其中能在红球菌属微生物中复制的DNA区域源于一个选自pRC001、pRC002、pRC003和pRC004的质粒。
CN95115824A 1994-08-04 1995-08-04 一种源于红球菌属微生物的卡那霉素抗性基因 Expired - Lifetime CN1080308C (zh)

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JPH0838184A (ja) 1996-02-13
CN1133342A (zh) 1996-10-16
DE69532024T2 (de) 2004-06-24
KR100343424B1 (ko) 2002-11-14
EP0704530B1 (en) 2003-10-29
TW338069B (en) 1998-08-11
KR960007778A (ko) 1996-03-22
EP0704530A2 (en) 1996-04-03
US5776771A (en) 1998-07-07
JP3235934B2 (ja) 2001-12-04
EP0704530A3 (en) 1997-12-29
DE69532024D1 (de) 2003-12-04

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