CN1653173A - 蜡状芽孢杆菌产生的呕吐毒素的合成酶、编码该酶的基因以及呕吐毒素的检测方法 - Google Patents
蜡状芽孢杆菌产生的呕吐毒素的合成酶、编码该酶的基因以及呕吐毒素的检测方法 Download PDFInfo
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Abstract
提供蜡状芽孢杆菌(Bacillus cereus)产生的呕吐毒素(催吐素)的简便而且迅速的检测法。以检体中的催吐素合成酶的存在作为指标，对催吐素进行检测。通过检测编码催吐素的核酸、或通过使用对该酶特异的抗体的免疫学方法进行催吐素合成酶的存在的检测。
Description
技术领域
本发明涉及蜡状芽孢杆菌(Bacillus cereus)产生的呕吐毒素(催吐素)及其检测方法。在临床检查或食品检查等中的催吐素的检测中可以利用本发明。
背景技术
人们都知道混入食品成为食物中毒原因的细菌毒素中黄色葡萄球菌泻肚毒素和蜡状芽孢杆菌呕吐毒素作为耐热性毒素，进行加热处理是无效的。关于在食品卫生上极其重要的黄色葡萄球菌毒素已经确立了检测方法，但关于蜡状芽孢杆菌呕吐毒素的检测到目前为止还没有开发出适当的方法。蜡状芽孢杆菌由于形成能够耐受在100℃下加热30分钟的耐热性芽孢，通过煮沸完全杀死他是困难的，非加热食品本来就存在，即使在加热食品中也存在由蜡状芽孢杆菌呕吐毒素造成的污染的问题。蜡状芽孢杆菌在世界范围内以食物中毒菌闻名，即使在我国由于该菌引起的食物中毒也报告了多起。在1994年，从蜡状芽孢杆菌分离、纯化了该呕吐毒素(命名为催吐素，セレウリド)，确定了其化学构造(Agat，N.，等FEMS Microbiol.Lett.121，31-34(1994))。随之而来开发了利用HEp-2细胞检测催吐素的方法(Agat，N.，等FEMS Microbiol.Lett.121，31-34(1994))。
弄清楚在食品以及其他检体中有无呕吐毒素(催吐素)在通过HACCP进行的食品制造管理上是非常重要的，在世界范围内一直探求其检测法的开发。然而，直到现在简便而迅速地进行蜡状芽孢杆菌的检测以及催吐素检测的方法也没有开发出来。由于利用上述的HEp-2细胞的方法也需要熟练的技术，所以简便而且正确的检测，以及多个检体同时处理是困难的。另外，检体为患者的呕吐物、粪便、食品或擦拭样品时，连蜡状芽孢杆菌的鉴定都必须进行经增菌培养、分离培养直至纯培养、确认培养的操作。在各个培养阶段需要的时间都各为18～24小时，总共需要的时间约4天。
本发明是在以上背景下完成的发明，目的在于提供在催吐素的检测中可以利用的多肽、核酸等，以及利用他们的简便、而迅速的催吐素的检测法。
发明内容
本发明人鉴于上述目的，进行了不断研究。结果首先发现参与催吐素生物合成的酶，并且在其鉴定中获得了成功。当对编码该酶的基因的碱基序列和不产生催吐素的蜡状芽孢杆菌保有的对应基因进行比较时，发现了在两者之间存在不同序列，直至获得了利用这一不同部分可以进行催吐素的检测的想法。本发明是根据以上想法完成的发明，提供以下构成。
[1]一种含有序列编号1的氨基酸序列的多肽，或含有序列编号1的氨基酸序列的一部分被改变后构成的序列、而且具有催吐素合成活性的多肽。
[2]一种含有序列编号3的氨基酸序列的多肽，或含有序列编号3的氨基酸序列的一部分被改变后构成的序列、而且保持上述多肽所具有的有关催吐素合成的功能构造的多肽。
[3]编码[1]所述的任一种多肽的核酸。
[4]编码[2]所述的任一种多肽的核酸。
[5]保有[3]或[4]所述核酸的载体。
[6]用[5]所述载体转化的转化体。
[7]含有序列6的碱基序列中与催吐素合成活性直接有关的区域的至少部分的序列、或含有与上述区域的碱基序列相互补的序列的至少部分的核酸。
[8]含有序列7碱基序列的至少部分、或含有与上述碱基序列互补的序列的至少部分的核酸。
[9]含有序列8的碱基序列中与催吐素合成活性直接有关的区域的碱基序列互补的序列的至少部分的核酸。
[10]含有与序列编号9的碱基序列互补的序列的至少部分的核酸。
[11]被设计成可以对含有在编码具有催吐素合成活性的多肽的DNA中直接与催吐素合成活性有关的区域的至少部分的DNA区域进行特异扩增的一组核酸。
[12]一种将[7]～[10]任一项所述的核酸固定于不溶性支持体后构成的固相化核酸。
[13]一种与催吐素合成酶特异结合的抗体。
[14]一种抗体，其特征是对含有序列编号1的氨基酸序列的多肽具有结合性、而对由序列编号2的氨基酸序列构成的多肽没有结合性。
[15]一种含有[7]～[10]任一项所述的核酸、[11]所述的一组核酸或[12]所述的固相化核酸的催吐素检测用试剂盒。
[16]一种含有[11]所述的一组核酸、DNA扩增用酶以及DNA合成试剂的催吐素检测用试剂盒。
[17]一种含有[13]或[14]所述抗体和抗原抗体反应用试剂的催吐素检测用试剂盒。
[18]一种包括研究检体中的(a)或(b)存在的步骤的催吐素检测方法：
(a)含有序列编号1的氨基酸序列的多肽，或含有序列编号1的氨基酸序列的一部分被改变后构成的序列、而且具有催吐素合成活性的多肽，
(b)编码(a)中任一种多肽的核酸。
[19]一种包括以下步骤的催吐素检测方法：
(i)以检体中的DNA为模板，使用[11]所述的一组核酸进行DNA扩增反应的步骤，
(ii)对被扩增的DNA进行检测的步骤。
[20]一种包括以下步骤的催吐素检测方法：
(iii)以检体中的DNA为模板制备cDNA的步骤，
(iv)使用[11]所述的一组核酸进行DNA扩增反应的步骤，
(v)对被扩增的DNA进行检测的步骤。
[21]一种包括以下步骤的催吐素检测方法：
(I)使检体与[13]或[14]所述抗体接触的步骤，
(II)I的步骤后，对抗原抗体反应物进行检测的步骤。
[22][18]～[21]中任一项所述的催吐素检测方法，其中作为前处理进行以下步骤：
(A)将检体接种到蜡状芽孢杆菌的增菌培养基中进行培养的步骤。
[23][18]～[21]中任一项所述的催吐素检测方法，其中作为前处理进行以下步骤：
(A)将检体接种到蜡状芽孢杆菌的增菌培养基中进行培养的步骤，
(B)对增殖的蜡状芽孢杆菌进行溶菌或破碎的步骤。
另外，本发明中的DNA不限于双链DNA，也可使用包括构成双链的单链DNA(有义链和反义链)。另外在本发明的DNA中包含含有考虑了密码子的简并性的任意的碱基序列的DNA。另外其形式也没有限定，可以含有cDNA、基因组DNA、合成DNA。
另外，在本发明中所谓多肽指的是广义上的多肽，即包括使用表现为多个氨基酸以肽键连接形成的肽在内的寡肽、狭义多肽以及蛋白质。
在本发明中，将蜡状芽孢杆菌产生的呕吐毒素称为催吐素。
附图的简单说明
图1是表示在本发明的实施例中对PCR扩增产物进行电泳后的凝胶染色后的状态图。从左边的带开始依次为对NC7401株、NC-T株、NC-G15株、NC327株、NC-1-55株(以上为催吐素产生株)、ATCC14579株、B-4ac株、PHLS2668株、PHLS4433株、NC1225株(以上为催吐素非产生株)、苏云金芽孢杆菌(HD73)、以及枯草芽孢杆菌(ATCC21332)的PCR反应后的溶液进行电泳后的结果。
图2是表示对来自DNA浓度不同的样品的PCR扩增产物进行电泳后的凝胶染色后的状态图。带编号1、2、3和4分别为对来自300ng、30ng、3ng、以及0.3ng的样品的PCR扩增产物进行电泳后的带。
实施发明的最佳方式
本发明的第1个方面涉及到具有催吐素合成活性的多肽(以下也称为「催吐素合成酶」)。本发明中提供的催吐素合成酶含有序列编号1的氨基酸序列。就象下面实施例所示那样，该酶是用蜡状芽孢杆菌的cDNA文库鉴定的。催吐素合成酶参与催吐素的合成，只出现在产生催吐素的蜡状芽孢杆菌中。因此，检体中该酶的存在可以反映有无催吐素产生。这样一来，催吐素合成酶在作为催吐素检测的指标方面是有用的。
另外，如果获得与催吐素合成酶特异结合的抗体，利用该抗体提供免疫学方法也可以进行该酶的检测、即催吐素的检测。因此，在可以作为用于制备这样的抗体的免疫源(抗原)利用方面，本发明提供的多肽(催吐素合成酶)也是有用的。
其中作为含有序列编号1的氨基酸序列的一部分被改变后构成的氨基酸序列的多肽(以下称为「改变多肽」)只要是具有催吐素合成活性的，可以与上述多肽同样在催吐素的检测等中利用。作为这样的多肽的例子，如关于含有序列编号1的氨基酸序列的多肽、保存与催吐素合成活性有关的立体构造的多肽。
这里所谓的「氨基酸序列的一部分被改变」指的是在氨基酸序列中缺失、置换、附加、和/或插入1个或数个氨基酸。只要是保持催吐素合成活性，氨基酸序列的改变(变异)位置没有特别限定，也可以在多个位置进行改变。改变的氨基酸数例如可以是相当于总氨基酸的10％以内的数，优选是相当于总氨基酸的5％以内的数。更优选的是相当于总氨基酸的1％以内的数。以上那样改变的多肽可以用众所周知的基因工程的手段进行制作。
象下面叙述的实施例所示的那样，由序列编号1的氨基酸序列构成的多肽可以认为是由4个结构域构成的。在本发明中为了方便，从N末端一侧开始依次将这些结构域称为CRS1、CRS2、CRS3和CRS4。根据本发明人的研究结果，催吐素产生菌中特征性的结构域是CRS3(1805位～2824位的氨基酸序列)和CRS4(2825位～3704位的氨基酸序列)。因此，可以认为这些结构域直接参与催吐素生物合成。由此可以认为为了制备对催吐素合成酶特异的抗体，含有上述特异结构域中的任一个或两者的多肽可以成为特别有效的抗原。本发明也提供含有序列编号3、序列编号4、或序列编号5记载的氨基酸序列的多肽。另外在这些多肽中，只要是保持他们具有的与催吐素生物合成有关的功能构造，即使一部分氨基酸被改变也可以。
另外，只要是没有特别限定，以下说明中，称之为「催吐素生物合成直接相关的区域」指的都是序列编号1的氨基酸序列中1805位～3704位的区域，即CRS3和CRS4(对于DNA是编码CRS3和CRS4的DNA区域)。
本发明的多肽中，若是存在于自然界中的，可以通过提取纯化等操作作为天然的多肽制备。例如，可以从产生催吐素的蜡状芽孢杆菌的菌体内制备。
另外，本发明的多肽(包括改变多肽)也可以用基因工程手法作为重组多肽制备。即，用编码本发明的多肽的DNA转化适当的宿主细胞，通过回收在转化体内表达的多肽进行制备。回收的多肽可以根据目的进行适当纯化。作为重组多肽制备时，可以进行各种修饰。例如，将编码本发明的多肽与其他的适当DNA同时插入到载体中，可以得到本发明的多肽和上述另一个DNA编码的肽或多肽连接的重组多肽。通过这样的修饰，可以使重组多肽的提取、纯化简便，或附加生物学上的功能。
本发明的多肽也可以通过化学合成制备。例如，可以通过众所周知的肽合成法-固相合成法进行合成。
本发明的第2个方面提供编码上述本发明的多肽的核酸。作为这样的核酸的具体例子，如具有序列编号6或序列编号7的碱基序列的DNA、或序列编号8或序列编号9的RNA。或者是这些DNA中一部分被改变的DNA等。这里的所谓「一部分被改变」指的是构成DNA或RNA的碱基的一部分被缺失、置换、插入或附加。改变的碱基数可以是例如1～100个、优选1～20个、更优选1～10个。
本发明的核酸可以用作对催吐素进行检测时的样品，即在给予有关有无催吐素存在的指标方面是有用的。另外，为了制备与上述本发明的多肽结合的抗体、即在催吐素的检测中可以利用的抗体的抗原制备过程方面也是有用的。
以上的核酸可以适当利用可与编码催吐素合成酶的基因(具有序列编号6的碱基序列的DNA)特异杂交的探针、引物等从适当的基因组DNA文库或cDNA文库、或催吐素产生菌的菌体内提取液制备。另外也可以通过以至少编码催吐素合成酶的基因的一部分作为模板，以dNTP(dATP、dGTP、dCTP、dTTP)作为原料的PCR法等进行合成。
为了制备本发明的DNA使用的基因组DNA文库或cDNA文库可以根据常规方法从蜡状芽孢杆菌NC7401株制作。
本发明还提供保持上述本发明的DNA(包括改变DNA)的载体。只要是可以保持本发明的DNA的载体，可以使用任一种载体，但最好是根据目的(克隆、多肽的表达)，或考虑宿主细胞的种类之后选择适当的载体。本发明的DNA向载体插入可以通过使用限制酶和DNA连接酶的众所周知的方法(Molecular Cloning，ThirdEdition，1.84，Cold Spring Harbor Laboratory Press，New York)进行。
本发明还提供保持上述本发明的DNA(包括改变DNA)的转化体。即涉及到用本发明的DNA转化宿主细胞得到的转化体。例如利用磷酸钙法、电穿孔(Potter，H.等，Proc.Natl.Acad.Sci.U.S.A.81，7161-7165(1984))、脂转染法(Felgner，P.L.等，Proc.Natl.Acad.Sci.U.S.A.84，7413-7417(1984))、微注射(Graessmann，M.&Graessmann，A.，Proc.Natl.Acad.Sci.
U.S.A.73，366-370(1976))等众所周知的基因导入方法将本发明的DNA导入到宿主细胞后进行转化。用上述本发明的载体转化宿主细胞后可以得到本发明的转化体。根据目的可以使用各种各样的宿主细胞，例如可以使用大肠杆菌等原核细胞、酵母等真核细胞。在利用大肠杆菌系时，作为表达载体可以使用pET-3c或pET-8c等pET载体(Novagen公司)、pBAD质粒(Invitrogen公司)、pGEX质粒(AmershamPharmacia biotech公司)等。
通过在适当的条件下对本发明的转化体进行培养，可以大量生产本发明的DNA表达产物(多肽)，该表达产物可在催吐素合成酶的检测用抗体的制备中利用。另外通过作为与由数个组氨酸构成的His-Tag、β-D-半乳糖苷酶、GST(谷胱甘肽S-转移酶)、硫氧还蛋白、麦芽糖结合蛋白质、Myc、Xpress、FLAG等标记分子的融合蛋白质(肽)表达，可容易进行表达产物的纯化。
本发明的第3个方面涉及到催吐素的检测方法，其特征是包括研究检体中是否存在以下(a)或(b)的步骤。(a)：含有序列编号1的氨基酸序列的多肽，或含有序列编号1的氨基酸序列的一部分被改变后构成的序列、而且具有催吐素合成活性的多肽，或(b)：编码(a)中任一种多肽的核酸。另外，由于检体中催吐素的存在即意味着产生催吐素的蜡状芽孢杆菌的存在，所以本发明中的「催吐素的检测方法」可以作为与「产生催吐素的蜡状芽孢杆菌的检测方法」同义的概念使用。
研究(a)存在的方法没有特别限定，可以利用使用对检测对象的多肽特异的抗体的免疫学方法。
同样，研究(b)存在的方法没有特别限定，例如可以利用使用对编码检测对象多肽的核酸特异的核酸引物和/或核酸探针的方法，使用设计成对催吐素特异的核酸区域进行特异扩增的一组引物(核酸)的PCR法(聚合酶链式反应)以及其改变法或应用方法(PCR-RFLP限制片段长度多态性)法、RT-PCR(逆转录酶PCR)法等)，Soutern blot杂交法、点杂交法(Southern，E.，J.Mol.Biol.98，503-517(1975))，Northern blot法等。
以下给出了本发明提供的催吐素检测方法的更具体的例子。首先，作为利用PCR法等的核酸扩增反应的例子，如包括(i)以检体中的DNA为模板，使用一组核酸进行DNA扩增反应的步骤，该组核酸被设计成可以对含有编码催吐素合成活性的多肽的DNA(例如由序列编号6的碱基序列构成的DNA)中，与催吐素合成活性直接相关的至少一部分的DNA区域(例如由序列编号7的碱基序列构成的DNA区域)进行特异扩增，以及(ii)对被扩增的DNA进行检测的步骤的方法。另外作为利用RT-PCR法的方法，如包括(iii)以检体中的mRNA为模板制备cDNA的步骤，(iv)使用一组核酸进行DNA扩增反应的步骤，该组核酸被设计成可以对含有编码催吐素合成活性的多肽的DNA中与催吐素合成活性直接有关的至少一部分DNA区域进行特异扩增，以及(v)对被扩增的DNA进行检测的步骤的方法。
由于必须是可通过PCR法等扩增得到的大小，所以由一组引物规定的扩增区域最好是约4,000bp以下。另外，当扩增区域过小时，由于对扩增产物和引物的二聚体难于进行区别，所以扩增区域最好是50bp以上。另外，为了有效进行扩增，扩增区域优选100bp～1,000bp大小。
作为在PCR法等中使双链核酸热变性时的温度，例如可以是约90℃～约95℃，作为使引物杂交的退火温度，例如可以为约37℃～约65℃，而作为聚合反应时的温度例如可以为50℃～约80℃。在PCR法以及其变更方法等中可以将这些热变性、退火、聚合作为一个循环反复进行直至扩增到可以检测到扩增产物为止。扩增产物的检测可以利用琼脂糖凝胶电泳进行。即，通过对酶反应液进行琼脂糖凝胶电泳，可以确认扩增的核酸片段的存在及其长度。由该电泳结果可以判定在检体中是否存在含有引物识别的序列的核酸，由此可以判定有无催吐素存在，即有无产生催吐素的蜡状芽孢杆菌的存在。在扩增产物的检测中不限定于琼脂糖凝胶电泳，也可以利用其他电泳或各种层析。
作为在本发明的催吐素检测方法中能够利用的核酸(引物或探针用)，只要是在对编码催吐素合成酶的基因进行特异检测中可以利用的，没有特别限定，例如至少含有序列编号6的碱基序列中与催吐素合成活性直接有关的区域的一部分序列的核酸、或与上述区域的碱基序列互补的序列的一部分的核酸。这里所谓「与催吐素合成活性直接有关的区域」如上所述，具体指的是编码CRS3和CRS4的区域，即具有序列编号7的碱基序列的区域。在用检体中的mRNA作为检测对象时，在催吐素检测方法中也同样可以利用至少含有与序列编号8的碱基序列中与催吐素合成活性直接有关区域的碳基序列互补的序列的一部分的核酸。更具体讲，如至少含有与序列编号9的碱基序列互补的序列的一部分的核酸。
至于探针、引物，可以根据解析方法使用合适的DNA片段或RNA片段。探针、引物的碱基长度只要是能够发挥各自功能的长度就可以，如果考虑到选择性或检测灵敏度和再现性，作为引物的碱基长度为10bp以上、优选在15bp以上，具体来说如10～30bp左右，优选15～25bp程度。
另外，对于引物，只要是可以与扩增对象特异杂交、能够扩增目的DNA片段，即使与作为模板的序列多少有些错配也可以。错配的程度可以是1～n个，优选1～5个，更优选1～3个。对于探针也同样，在对检测没有影响的范围内，即使对检测对象的序列多少有点错配也可以。
以下给出了在运用PCR法等伴随特定DNA区域的扩增的方法的催吐素检测方法中能够利用的核酸(引物组)的具体例子。
引物组1
有义链用引物：5’-GGTGAATTGTGTCTGGGAGG-3’(序列编号10)
反义链用引物：5’-ATTTTTATTAAGAGGCAATG-3’(序列编号11)
引物组2
有义链用引物：5’-GTCAAGATAAGAGGCTTCCGAATT-3’(序列编号12)
反义链用引物：5’-AATGGAATGACCACCAAGCT-3’(序列编号13)
引物组3
有义链用引物：5’-AGGAAGTTCCGTTTGTGGAC-3’(序列编号14)
反义链用引物：5’-CACATAACCTTTTGCAACTC-3’(序列编号15)
引物组4
有义链用引物：5’-GGCGAACTATGTGTTGGTGG-3’(序列编号16)
反义链用引物：5’-TAAAGAGTCACCACCATAAG-3’(序列编号17)
引物组5
有义链用引物：5’-ACGTCAGGCAGTACTGGAAA-3’(序列编号18)
反义链用引物：5’-TTCGATGCGGAATCCACGAA-3’(序列编号19)
本发明中的核酸(引物、探针)可以通过磷酸二酯法等众所周知的方法合成。另外，作为探针使用时的标记物质、标记方式可以采用众所周知的物质。作为标记物质如32P等放射性同位素、异硫氰酸荧光素、异硫氰酸四甲基罗丹明等荧光物质，作为标记方法如使用T4DNA聚合酶或Klenow片段的3’末端标记法、切口移位法、随机引物法(Molecular Cloning，Third Edition，Chapter 9，Cold SpringHarbor Laboratory Press，New York)等。
以下就利用免疫学方法的催吐素的检测方法进行说明。作为利用免疫学方法的催吐素的检测方法，如包括I)使检体与对催吐素合成酶特异的抗体接触的步骤，(II)I的步骤后，抗原抗体反应物的检出步骤在内的方法。这里的所谓「对催吐素合成酶特异的抗体」指的是具有与催吐素合成酶特异结合性的抗体，作为具体例子，如对含有序列编号1的氨基酸序列的多肽具有结合性、而对由序列编号2的氨基酸序列构成的多肽没有结合性的抗体。可以使用的抗体的类别没有特别限定，例如可以使用属于IgG类、IgM类等的抗体。另外也可以使用Fab、Fab’、F(ab’)2，scFv、dsFv等抗体片段。
作为测定方法，如ELISA(酶联免疫吸附定量法)法、放射免疫分析、FACS、免疫沉淀法、免疫印迹等定性的或定量的方法。另外作为抗原抗体反应的种类可以采用使检体中的催吐素合成酶和另外添加的催吐素合成酶与催吐素合成酶特异的抗体竞争反应的方法(竞争法)，以及不使他们进行竞争反应的方法(非竞争法)。
作为对催吐素合成酶特异的抗体优选使用单克隆抗体。由于单克隆抗体的特异性高，所以可以进行高灵敏度测定。另外，利用使用对催吐素合成酶特异的而且识别互不相同的抗原表位的2种抗体的夹层法在灵敏度、特异性方面更好。
抗体可以进行固相化后使用。作为固相化使用的不溶性支持体，可以使用聚苯乙烯树脂、聚碳酸酯树脂、硅树脂、尼龙树脂等树脂或玻璃等对水不溶性的物质，其材料没有特别限定。抗体向这些不溶性支持体的负载可以通过物理吸附或化学吸附进行。
作为在免疫学测定法中可使用的标记物质，过氧化物酶、碱性磷酸酶、β-D-半乳糖苷酶、葡萄糖氧化酶、葡萄糖-6-磷酸脱氢酶、以及微过氧化物酶等酶、异硫氰酸荧光素(FITC)、异硫氰酸四甲基罗丹明(TRITC)、以及铕等荧光物质、氨基苯二酰肼、异氨基苯二酰肼、以及吖啶鎓衍生物等化学发光物质、NAD等辅酶、生物素以及131I和125I等放射性物质等可以作为标记物质使用。特别是作为标记物质使用生物素，使用荧光色素或酶标记的抗生物素蛋白(例如抗生物素蛋白过氧化物酶)反应的方法，可以进行更高灵敏度的测定。
与催吐素合成酶特异结合的单克隆抗体可以通过常规方法获得。以下给出了一个单克隆抗体的制作方法的例子。首先取得催吐素合成酶，作为抗原免疫小鼠等动物。然后从被免疫的动物摘出抗体产生细胞，使该细胞与骨髓瘤细胞融合得到杂交瘤细胞。接下来，使该杂交瘤单克隆化后，选择产生与催吐素合成酶特异结合的克隆。
作为抗原可以使用从产生催吐素的蜡状芽孢杆菌的菌体内分离、纯化的催吐素合成酶。另外也可以使用利用用编码催吐素合成酶的碱基序列，通过来自大肠杆菌的表达体系得到的重组多肽。
作为免疫方法，例如可以采用将上述抗原与弗氏完全或不完全佐剂混合，进行乳化，注射到小鼠等的腹腔内、皮下或肌肉，隔一定时间注射一次，进行数次注射的方法。作为免疫的动物可以使用小鼠以及大鼠、仓鼠、兔、豚鼠、鸡、绵羊、山羊等。免疫完成后，取出免疫的动物的脾脏，取得抗体产生细胞。也可以从淋巴节、末梢血液等采取抗体产生细胞。
使用的骨髓瘤细胞的种类没有特别限定，选择与免疫使用的动物的相关的合适的种类。最好是使用来自与抗体产生细胞同种动物的骨髓瘤细胞，例如当使用小鼠时可以使用骨髓瘤细胞株PAI。细胞融合，例如可以通过按照一定比例将抗体产生细胞与骨髓瘤细胞混合，然后加入聚乙二醇后搅拌处理进行细胞融合。另外也可以使用用电脉冲进行细胞融合。
在只选择进行了细胞融合的杂交瘤时，可以运用使用一般的HAT培养基(按给定比例含有次黄嘌呤、氨基蝶呤、胸腺嘧啶的选择培养基)的方法。含有杂交瘤的培养液为了以后的选择，可以在96孔板中培育。
然后取各个容器内的培养上清，通过使用催吐素合成酶的ELISA法等选择产生抗催吐素合成酶的抗体的杂交瘤。抗体阳性的容器内的杂交瘤通过有限稀释法进行克隆，可以得到单克隆化的杂交瘤细胞株。
通过对杂交瘤的培养液进行纯化可以得到所期望的抗体。另外使杂交瘤增殖到所期望的数以上后，将他们移植到动物(例如小鼠)的腹腔内，使其在腹水内增殖，通过纯化腹水，也可以获得所期望的抗体。在上述培养液的纯化或腹水的纯化中适合采用使用蛋白G、蛋白A等的亲和层析。另外，也可以使用将抗原固相化的亲和层析。另外也可以使用离子交换层析、凝胶过滤层析、硫铵分级、以及离心分离等方法。这些方法可以单独或任意组合使用。
通过以上方法得到的抗体是否特异识别催吐素合成酶，例如可以通过使用将催吐素合成酶固相化的板的ELISA法进行确认。
在本发明的催吐素检测方法中可以将利用的核酸(引物或探针)固定于不溶性支持体后使用。同样，在本发明的催吐素检测方法中也可以将可利用的抗体固定于不溶性支持体后使用。可以将固定化中使用的不溶性支持体事先加工成片状、颗粒状等，使用这些固定化核酸或固定化抗体可以简便地进行检体中的催吐素的检测。
本发明的另一个方面提供在催吐素检测中可以利用的试剂盒。即，使用在催吐素的检测中可以利用的核酸(含有一组的核酸、固相化核酸)构建催吐素检测用试剂盒。在催吐素检测用试剂盒中除了这些核酸之外，还可以含有核酸扩增用酶(例如PCR法中使用的DNA合成酶)以及作为底物的核酸(dATP、dCTP、dGTP、dTTP)、反应用的试剂等。另外，作为标准物质可以含有催吐素合成酶(可以是部分纯化品)、催吐素产生菌株的菌体内提取物(可以是进行了部分纯化提取物)。
利用以上的催吐素检测用试剂盒，以检体中的核酸作为靶可以检测催吐素，也可以构建以检体中的多肽、即催吐素合成酶作为靶，用于对催吐素进行检测的试剂盒。在这样的试剂盒中含有对上述的催吐素合成酶特异的抗体(包括固相化抗体)。此外，也可以构建包含与该抗体结合的第二抗体、抗原抗体反应用试剂(缓冲液、发色底物、发色试剂、发色反应终止液等)等的试剂盒。另外作为标准物质可以含有催吐素合成酶(可以是部分纯化品)、催吐素产生菌株的菌体内提取物(可以是进行了部分纯化的提取物)。
提供给本发明的催吐素检测方法的检体没有特别限定，作为检体例如可以使用各种食品、人或动物的呕吐物或粪便、或擦拭样品等。这些检体可以预先进行用溶菌酶等酶处理、加压处理、加热处理或超声处理等。通过这些处理，检体中的菌体被溶菌或破碎。但是当预料在采取检体时，检体中的蜡状芽孢杆菌的细胞膜破碎时，就不必进行溶菌处理等。
对于从检查对象采取的样品最好是用蜡状芽孢杆菌的增殖培养基(选择培养基)预先进行培养。通过采用该培养步骤，可以进行可靠性更高的检测。
另外，检查对象为液状时，可以将采取的样品用于直接溶菌等处理或培养步骤，当检查对象为固体状时，优选是用适当的溶剂对菌体进行提取后，用于这些处理。
以下用实施例对本发明进行更详细说明。
<实施例1>从蜡状芽孢杆菌基因组DNA文库克隆呕吐毒素(催吐素)合成酶基因
使用来自蜡状芽孢杆菌NC7401株(保管在名古屋市卫生研究所、国立医药品食品卫生研究所)的EMBL3(Promega公司生产)制作噬菌体文库。对得到的约400个白色蚀斑进行筛选，作为在不借助于核糖体的氨基酸合成酶中特异存在的区域，用BSCI(GGAATTCCTTAAAIGCIGGAGGAGCITATGTGCCGCTT GATCC：序列编号20)以及II(GGAATTCCTTTIGGITTICCIGTTGTICCI GAIGTGTAAAT：序列编号21)作为引物(Kathrin，M.，等，FEMSMicrobiol.Lett.135，295-303(1996))特异扩增的DNA片段通过Southern杂交法进行分析。另外对通过作为模板使用NC7401株的染色体DNA，作为引物使用BSC I和II的PCR法扩增的DNA片段用标记物标记试剂盒(Roch Diagnostics公司生产)进行标记的探针进行分析。分析结果，选择多个表达量大的插入DNA，分别用限制性内切酶SaIl切出，亚克隆到克隆载体pHSG299(宝酒造株式会社生产)的多克隆位点。
<实施例2>呕吐毒素(催吐素)合成酶cDNA的序列分析
亚克隆的各个DNA片段的序列通过使用自动测序仪(AppliedBiosystems公司生产)循环测序反应进行分析。考虑到重复的序列后，通过对各个DNA片段的序列信息进行分析，决定催吐素合成酶的全长DNA序列以及氨基酸序列(序列编号1)。当对该序列进行详细研究时，发现催吐素合成酶由合成各个氨基酸的的4个结构域构成，N末端一侧的2个结构域虽然也广泛地存在于不产生催吐素的蜡状芽孢杆菌株中，但C末端一侧的2个结构域是产生催吐素的菌株所特异的。
<实施例3>利用PCR法进行催吐素的检测
(3-1)检体的制备
从图1的表表示的芽孢杆菌(催吐素产生株和非产生株各5株)按照以下顺序制备检体。作为对照组使用苏云金芽孢杆菌(Bacillusthuringienesis)、以及枯草芽孢杆菌(Bacillus subtilis)。
将各个菌体分别接种到适当的增菌培养基(LB培养基)，于37℃下在好氧条件下培养过夜，通过离心操作从培养后的1.5ml培养基中回收菌体。将回收的菌体用10mMTris-HCl缓冲液(pH7.5)清洗一次后，悬浮于0.5ml溶解了终浓度为1mg/ml的溶菌酶的同一缓冲液中。在该状态下于37℃下放置10分钟，使其溶菌。然后，向溶菌液中添加等量被上述缓冲液饱和的酚，进行充分搅拌。离心处理后，回收上层液，进行醇沉淀处理，使核酸成分沉淀。将得到的沉淀物溶解于1ml上述缓冲液，作为以下检测方法中的检体。
(3-2)PCR用引物的合成
以序列编号6给出的催吐素合成酶的碱基序列信息为基础，选择对催吐素产生株特异的序列，化学合成以下所示引物(寡核苷酸)。
有义链用引物：5’-GGTGAATTGTGTCTGGGAGG-3’(序列编号10)
反义链用引物：5’-ATTTTTATTAAGAGGCAATG-3’(序列编号11)
(3-3)PCR法
向上述各个检体3μl加入灭菌蒸馏水16.05μl、10×反应用缓冲液3μl、dNTP溶液4.8μl、有义链用引物1.5μl、反义链用引物1.5μl、以及耐热性DNA聚合酶0.150μl，将总量调整到约30μl的反应液。而10×反应用缓冲液的组成为500mM KCl、100mM Tris-HCl(pH8.3)、15mM MgCl2、0.1％(w/v)明胶，dNTP溶液是使终浓度各为1.25mM的dATP、dCTP、dGTP和dTTP混合的溶液。另外各个引物是(3-2)中得到的化学合成纯化品的水溶液(50DU/ml)。至于耐热性DNA聚合酶使用TaqDNA聚合酶(5unit/ml：Perkin Elmer Cetus公司生产)。
PCR反应条件如下。即热变性：94℃1分钟，退火：55℃1分钟，聚合反应：72℃1分钟。由热变性开始经退火直至聚合反应的过程作为1个循环，共进行35次循环。另外，使用DNA Thermal Cycler(Perkin Elmer Cetus公司生产)进行PCR反应。
(3-4)PCR扩增产物的检测
为了检测由PCR反应液扩增的DNA片段，在以下条件下进行琼脂糖电泳。作为琼脂糖凝胶使用凝胶浓度为2％(w/v)的凝胶。使用溴乙锭(0.5μg/ml)进行电泳后的凝胶染色。电泳条件为加100V电压，电泳30分钟。其他的电泳条件以及操作方法根据MolecularCloning，Third Edition，Cold Spring Harbor LaboratoryPress，New York记载的方法。
图1表示染色后的凝胶。在图1中，从左边的带开始依次为对NC7401株、NC-T株、NC-G15株、NC327株、NC-1-55株(以上为产生催吐素株)、ATCC14579株、B-4ac株、PHLS2668株、PHLS4433株、NC1225株(以上为不产生催吐素的菌株)、苏云金芽孢杆菌(HD73)、以及枯草芽孢杆菌(ATCC21332)的PCR反应后的溶液进行电泳后的结果。就象图1所表示的那样，可知在催吐素产生菌株(带1～5)可以得到约450bp的PCR扩增产物。而在催吐素非产生菌株和苏云金芽孢杆菌HD73、以及枯草芽孢杆菌ATCC21332中没有检测到相当于该PCR扩增产物的带。由以上结果可以确认通过本实施例的方法可特异进行催吐素产生菌株的检测、即催吐素的检测。
<实施例4>利用PCR法进行催吐素产生菌的微量检测
(4-1)检体的DNA量的计算
使用图1表示的蜡状芽孢杆菌NC7401株，用实施例的(3-1)给出的方法制备检体，得到纯化DNA标准品。然后通过测定波长260nm的吸光度计算出该标准品中的DNA量。
(4-2)PCR扩增产物的检测
以(4-1)求出的DNA量作为参考，对检体进行稀释，制备分别含有300ng(分子数：约1×102)、30ng(分子数：约1×101)、3ng(分子数：约1)、0.3ng(分子数：约1×10-1)的样品。利用这些样品通过实施例3的(3-2)和(3-3)给出的方法进行PCR，然后用(3-4)给出的方法进行PCR扩增产物的检测。图2给出了对PCR扩增产物电泳后的凝胶进行染色后的状态。道编号1、2、3和4数分别对应来自300ng、3ng、3ng以及0.3的样品的PCR扩增产物进行电泳后的道。道3，即使在使用含有3ng的DNA的样品时也可以确认目的带。这意味着可以对相当于蜡状芽孢杆菌染色体的1～数个分子的DNA量进行检测，由此可认为理论上如果在检体中存在大约几个催吐素产生蜡状芽孢杆菌，也可以其存在、即催吐素进行检测。
本发明并不受上述发明的实施方式以及实施例的说明的任何限定。只要是没有脱离开专利申请范围的记载，本领域技术人员容易想到的范围中种种变化方式都包括在本发明中。
产业上利用的可能性
本发明提供蜡状芽孢杆菌产生的呕吐毒素(催吐素)的氨基酸序列以及碱基序列。利用这些序列，使用核酸探针或抗体有可能进行催吐素的检测。通过使用对催吐素特异的抗体或核酸探针，对催吐素可以简便而且迅速地进行检测。在实施例中给出的本发明的检测方法的一个例子中，对检体中的核酸进行扩增的反应所需要的时间约3小时，直至获得后来的检测结果大约为30分钟，可以在极短的时间内对到目前为止不能检测的催吐素进行检测。
另外利用本发明的方法可以对催吐素进行高灵敏度检测。这意味着可以对少量检体进行检测，以及可以使检体的前处理简便化。另外通过本发明的催吐素检测方法不仅可以对催吐素直接进行检测，而且可以对催吐素合成酶的有无进行研究，由研究结果判别催吐素是否存在于检体中。作为在蜡状芽孢杆菌中产生催吐素的菌株的共通的特征，认为是具有在本发明中鉴定的催吐素合成酶活性，即催吐素产生株必然有催吐素合成酶基因。然而，由于不知道在其他生物种中是否产生与催吐素同源的毒素，所以通过将催吐素合成酶或编码该酶的基因作为检测对象，可以有选择地或特异地对检体中的催吐素的存在(催吐素产生菌的存在)进行检测。因此，可以得到可靠性高的结果，适用于食品检查、临床检查。
序列表
<110>股份有限公司生物控制研究所
OHTA，Michio
AGATA，Norio
<120>催吐素合成酶，其基因，和催吐素的检测方法
<130>P0202401
<150>JP P2002-142398
<151>2002-05-17
<160>21
<170>PatentIn version 3.1
<210>1
<211>3704
<212>PRT
<213>蜡状芽孢杆菌
<400>1
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Glu Arg Ile Pro Lys Ile Leu Pro Arg Thr Tyr Tyr Asp Leu Ser
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Tyr Ser Gln Gln Arg Ile Tyr Phe Leu Ser Thr Met Glu Lys Glu
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Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp Asp Ile Tyr Gly Lys
2420 2425 2430
Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln Leu Leu Met Lys
2435 2440 2445
Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val Asp Gly Lys
2450 2455 2460
Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val Gln Phe
2465 2470 2475
Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu Val
2480 2485 2490
Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala
2495 2500 2505
Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly
2510 2515 2520
Glu His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly
2525 2530 2535
Trp Ser Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp
2540 2545 2550
Ala Phe Val Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile
2555 2560 2565
Ser Ile Met Asp Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu
2570 2575 2580
Asp Gln Asp Asp Arg Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu
2585 2590 2595
Leu Pro Thr Leu Glu Leu Pro Thr Asp Arg Gln Arg Pro Leu Leu
2600 2605 2610
Lys Thr Tyr His Gly Asp Thr Leu Ser Tyr Lys Val Asn Ser Gln
2615 2620 2625
Leu His Gln Lys Leu Lys Asp Phe Ser His Ala Asn Gly Val Thr
2630 2635 2640
Met Phe Met Thr Leu Leu Thr Ala Tyr Asn Ile Met Leu Asn Lys
2645 2650 2655
Leu Thr Asn Glu Thr Asp Ile Val Val Gly Ser Pro Val Ala Gly
2660 2665 2670
Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly Met Phe Val Asn
2675 2680 2685
Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro Thr Val Asp
2690 2695 2700
Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala Tyr Asn
2705 2710 2715
His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp Pro
2720 2725 2730
His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly
2735 2740 2745
Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val
2750 2755 2760
Arg Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr
2765 2770 2775
Leu His Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu
2780 2785 2790
Tyr Asn Thr Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln
2795 2800 2805
Asn His Phe Glu Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr
2810 2815 2820
Lys Arg Ile Ser Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr
2825 2830 2835
Glu Ile Leu Glu Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His
2840 2845 2850
Glu Ser Val Ala Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln
2855 2860 2865
Ala Lys Leu Ala Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala
2870 2875 2880
Glu Leu Ser Glu Lys Ala Asn Ala Leu Ala His Thr Leu Lys Arg
2885 2890 2895
Arg Gly Val Ala Gln His Asp Val Val Gly Ile Val Ala Glu Arg
2900 2905 2910
Ser Pro Glu Thr Ile Ile Gly Ile Leu Ala Ile Leu Lys Val Gly
2915 2920 2925
Ala Ile Tyr Leu Pro Ile Asp Pro Lys Leu Pro Gln Leu Thr Leu
2930 2935 2940
Gln His Ile Trp Arg Asp Ser Gly Ala Lys Val Leu Leu Gly Lys
2945 2950 2955
Asn Glu Thr Thr Val Glu Val Gly Lys Glu Val Pro Phe Val Asp
2960 2965 2970
Ile Glu Gly Asp Lys Gly Lys Gln Glu Glu Leu Val Cys Pro Ile
2975 2980 2985
Ser Pro Glu Asp Thr Ala Tyr Ile Met Tyr Thr Ser Gly Ser Thr
2990 2995 3000
Gly Lys Pro Lys Gly Val Met Val Thr His Arg Asn Ile Val Arg
3005 3010 3015
Leu Val Lys Asn Thr Asn Phe Val Ser Leu Gln Glu Gln Asp Val
3020 3025 3030
Leu Leu Gln Thr Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu
3035 3040 3045
Ile Trp Gly Ala Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu
3050 3055 3060
Asp Tyr Val Ile Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln
3065 3070 3075
Gln Asn Lys Ala Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn
3080 3085 3090
Gln Leu Ala Asp Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln
3095 3100 3105
Leu Leu Ile Gly Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys
3110 3115 3120
Val Met Asp His Cys Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly
3125 3130 3135
Pro Thr Glu Asn Thr Thr Phe Ser Thr Ser Phe Val Ile Asp Gln
3140 3145 3150
Met Tyr Gln Asp Ser Ile Pro Ile Gly Thr Pro Ile Ala Asn Ser
3155 3160 3165
Ser Ala Tyr Ile Leu Asp Val His Gln Asn Ile Gln Pro Ile Gly
3170 3175 3180
Val Val Gly Glu Leu Cys Val Gly Gly Asp Gly Val Ala Lys Gly
3185 3190 3195
Tyr Val Asn Leu Glu Gln Leu Thr Glu Glu Arg Phe Ile Ala Asp
3200 3205 3210
Pro Phe Leu Lys Gly Ser Thr Met Tyr Arg Thr Gly Asp Tyr Val
3215 3220 3225
Lys Leu Leu Pro Asn Gly Asn Ile Gln Tyr Ile Gly Arg Val Asp
3230 3235 3240
Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Glu Ala Ile
3245 3250 3255
Met Asn Thr Leu Lys Gln Cys Glu Ser Ile Lys Asp Val Ile Val
3260 3265 3270
Val Val Gln Glu Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val
3275 3280 3285
Val Gly Glu Glu Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala
3290 3295 3300
Lys Lys His Leu Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe
3305 3310 3315
Ile Glu Glu Ile Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser
3320 3325 3330
Lys Leu Pro Lys Val Gln Glu Val Leu His Lys Lys Val Glu Thr
3335 3340 3345
Leu Leu Pro Glu Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr
3350 3355 3360
Arg Asp Val Leu Glu Lys Glu Asp Phe Gly Val Thr Asp Ser Phe
3365 3370 3375
Phe Ala Tyr Gly Gly Asp Ser Leu Leu Ser Ile Gln Val Val Ser
3380 3385 3390
Met Leu Lys Lys Glu Glu Ile Ala Val Asp Pro Lys Met Ile Phe
3395 3400 3405
Met His Thr Thr Val Arg Glu Leu Ala Lys Ala Cys Glu Asn Arg
3410 3415 3420
Pro Val Met Glu Glu Thr Lys Arg Thr Glu Lys Asp Tyr Leu Ile
3425 3430 3435
Gln Met Arg Glu Gly Ser Glu Glu Asp Ser Cys Ile Ile Phe Ala
3440 3445 3450
Pro Pro Ala Gly Gly Thr Val Leu Gly Tyr Ile Glu Leu Ala Arg
3455 3460 3465
Tyr Phe Glu Gly Ile Gly Asn Val Tyr Gly Leu Gln Ala Pro Gly
3470 3475 3480
Leu Tyr Asp Asp Glu Glu Pro Thr Phe Leu Asp Tyr Asp Glu Leu
3485 3490 3495
Val Gln Val Phe Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro Gly
3500 3505 3510
Gln Asp Tyr Leu Gly Gly His Ser Leu Gly Gly His Ile Ala Phe
3515 3520 3525
Gly Met Cys Cys Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly
3530 3535 3540
Leu Leu Ile Leu Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly
3545 3550 3555
Ala Lys Asp Glu Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val
3560 3565 3570
Leu Ala Ala Gly Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu
3575 3580 3585
Leu Lys Gln Leu Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala
3590 3595 3600
Val Ala Gln Lys Asp Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr
3605 3610 3615
Tyr Leu Asp Lys Tyr Leu Lys Leu Gln Ile His Ser Leu Leu Met
3620 3625 3630
Ser Arg Thr Leu Glu Leu Glu Lys Thr Gln Leu Asp Ile Pro Ile
3635 3640 3645
Lys Val Phe Lys Thr Gln Phe His Thr Glu Glu Leu Val Glu Arg
3650 3655 3660
Phe Asp Ala Trp His Asn Tyr Thr Asn Gln Ala Cys Thr Phe Ile
3665 3670 3675
Asp Ile Pro Gly Thr His Thr Thr Met Met Arg Leu Pro His Val
3680 3685 3690
Lys Glu Val Ala Lys Lys Ile Glu Glu Gln Leu
3695 3700
<210>2
<211>1804
<212>PRT
<213>蜡状芽孢杆菌
<400>2
Phe Ile Lys Ser Met Asn Gln Leu Gly Lys Ser Lys Asn Leu His Asn
1 5 10 15
Gly Gly Met Met Glu Met Lys Arg Val Glu Glu His Asp His Ile His
20 25 30
Val Leu Asn Glu Ile Glu Asn Glu Cys Glu Arg Arg Tyr Gly Arg Ser
35 40 45
Asn Ile Ala Ile Met Leu Glu Lys His Gly Val His Glu Gln Pro Leu
50 55 60
His Ile Glu Asp Leu Phe His Glu Val Glu Met Gln Glu His Ser Arg
65 70 75 80
Val Ser Arg His Glu Thr Val Leu Met Thr Asp Lys Gln Cys Ile Asp
85 90 95
Glu Ser Gly Lys Pro Leu Ala Leu Arg Phe Gly Glu Pro Leu His Leu
100 105 110
Asp Asp Cys Thr Pro Lys Thr Leu Gln Glu Ile Leu Lys Arg Ala Ala
115 120 125
Lys Gln Ala Lys Asp Lys Gly Met Thr Phe Val Tyr Glu Asp Gly His
130 135 140
Glu Glu Tyr Leu Ser Tyr Gln Glu Met Leu Ala Asp Ala Glu Arg Leu
145 150 155 160
Leu Lys Gly Leu Arg Asn Leu Gly Ile Gln Pro Gly Glu Ser Ile Leu
165 170 175
Phe Gln Phe Lys Asp Asn Lys His Phe Val Thr Ala Phe Trp Ala Cys
180 185 190
Ile Leu Gly Gly Phe Leu Pro Thr Pro Leu Gly Thr Ala Pro Ile Tyr
195 200 205
Ser Glu Gln Asn Ala Gln Val Leu Lys Leu Tyr Asn Thr Trp Gln Leu
210 215 220
Leu Glu Gln Pro Ile Ile Leu Thr Glu Phe Glu Leu Lys Glu Glu Ile
225 230 235 240
Ala Ala Ile Arg Thr Thr Leu Gln Arg Gln Glu Ile Val Ile His Ser
245 250 255
Ile Glu Asn Val Met Asp Thr Ala Arg Asp Thr Asn Trp Phe Pro Cys
260 265 270
Thr Glu Asp Thr Ile Val Leu Asn Leu Leu Thr Ser Gly Ser Thr Gly
275 280 285
Val Pro Lys Cys Val Gln His Lys Ser Lys Ser Ile Ile Ala Arg Thr
290 295 300
Val Ser Asn Cys Ile Asp Arg Gln Leu Asp Glu Lys Glu Val Ser Leu
305 310 315 320
Asn Trp Met Pro Leu Asp His Val Gly Gly Ile Val Met Cys His Ile
325 330 335
Arg Asp Thr Tyr Leu Met Cys Gln Gln Val Asn Cys Leu Ile Ser Ala
340 345 350
Phe Ile Glu Asn Pro Leu Asn Trp Leu His Trp Ile Asp Ala Tyr Ser
355 360 365
Ala Thr Phe Thr Trp Ala Pro Asn Phe Ala Phe Ser Leu Ile Asn Gln
370 375 380
Tyr Glu Glu Glu Ile Lys Ser Ser Ser Trp Asn Leu Ser Ser Met Arg
385 390 395 400
Tyr Ile Val Asn Gly Gly Glu Ala Val Ile Ser Ser Val Gly Met Lys
405 410 415
Phe Leu Gln Leu Leu Gln Gln His Gln Leu Pro Ser Asn Cys Leu Ile
420 425 430
Pro Thr Phe Gly Met Ser Glu Val Ser Ser Gly Ile Ile Glu Cys His
435 440 445
Ser Phe Tyr Thr Gln Thr Thr Asn Thr Gly Met Leu Tyr Val Asp Lys
450 455 460
Asn Ser Leu Asp Gly Asn Leu Gln Phe Thr Tyr Glu Gly His Gln Asn
465 470 475 480
Ala Ile Val Phe Thr Glu Val Gly Arg Pro Met Pro Gly Ile Gly Ile
485 490 495
Arg Ile Val Asp Glu Asp Asn Gln Cys Leu Ser Glu Asp Arg Ile Gly
500 505 510
Arg Phe Gln Ile His Gly Pro Thr Val Met Asn Gly Tyr Phe Lys Asn
515 520 525
Asp Glu Ala Asn Ala Glu Ser Phe Thr Glu Asp Gly Trp Phe Asp Ser
530 535 540
Gly Asp Leu Gly Phe Ile His Asn Gly Asn Leu Val Ile Thr Gly Arg
545 550 555 560
Lys Lys Asp Met Ile Val Val His Gly Ala Asn Tyr Tyr Asn Tyr Glu
565 570 575
Ile Glu Ala Leu Val Glu Gln Val Pro Gly Val Glu Thr Thr Phe Val
580 585 590
Cys Ala Thr Ser Val Lys Ser Ala Glu Gly Ala Glu Glu Leu Ala Ile
595 600 605
Phe Phe Val Pro Val Ile Asn His Val Ser Val Met Phe Ala Thr Met
610 615 620
Gln Gln Ile Lys Gln Ile Val Ala Arg Lys Met Gly Ile Thr Pro Lys
625 630 635 640
Val Ile Ile Pro Ile Gln Lys Glu Ala Phe Phe Lys Thr Asp Ser Gly
645 650 655
Lys Ile Thr Arg Asn Ala Phe Gln Lys Gln Phe Glu Asn Gly Ala Tyr
660 665 670
Arg Glu Ile Thr Gln Lys Ile Asp Cys His Leu Gln Asn Glu Lys Thr
675 680 685
Leu Ser Gln Trp Phe Tyr Arg Glu Lys Leu Val Glu Ser Lys Leu Gly
690 695 700
Lys Ser Val Ser Ser Gln Lys Glu Thr Tyr Val Phe Phe Arg Gln Gly
705 710 715 720
Lys Ser Phe His His Val Leu Lys Glu Lys Leu Thr Gln His Ser Val
725 730 735
Val Ile Val Asp Val Gly Glu Thr Phe Gly Glu Ile His Pro Asn His
740 745 750
Tyr Gln Ile Asn Pro Lys Asn Lys Met Asp Tyr Val Arg Leu Phe Glu
755 760 765
Glu Leu Ala Lys Arg Asn Val Glu Asp Gln Val Phe His Leu Leu His
770 775 780
Ala Trp Asn Tyr Cys Asp Thr Val Pro Thr Phe Arg Ser Val Glu Asp
785 790 795 800
Leu Ala Asn Ala Gln Tyr Leu Gly Val Phe Ser Val Met Phe Ala Leu
805 810 815
Gln Ala Ile Met His Ala Lys Leu Pro Leu Arg Arg Val Thr Val Ile
820 825 830
Ala Thr Asn Ser Val Gly Leu Glu Ala Lys Glu Met Asn Tyr Ser Cys
835 840 845
Ser Thr Leu Glu Gly Tyr Val Lys Thr Leu Pro Ala Glu Phe Glu Asn
850 855 860
Leu Gln Val Lys Tyr Ile Asp Ile Glu Gly Lys Asp Ile Gln Phe Asp
865 870 875 880
Thr Glu Thr Val Trp Lys Glu Leu Gln Gln Gln Glu Thr Ile Pro Val
885 890 895
Val Leu Tyr Arg Asp Glu Lys Arg Tyr Lys Ile Gly Leu Glu Lys Val
900 905 910
Pro Met Leu Glu Gln Lys Glu Lys Asn Ile Pro Phe Gln Gln Gln Gly
915 920 925
Phe Tyr Ile Ile Thr Gly Gly Leu Gly Gly Leu Gly Thr Leu Val Ala
930 935 940
Lys Leu Leu Leu Glu Arg Tyr Ser Ala Asn Val Leu Leu Leu Gly Arg
945 950 955 960
Thr Glu Ile Glu Thr Asn Ala Glu Lys Met Arg Leu Leu Asp Ser Leu
965 970 975
Lys Glu Tyr Glu Gln Tyr Gly Gly Thr Val Gln Tyr Lys Met Cys Asn
980 985 990
Val Met Asp Leu Asp Ala Met Arg Lys Val Val His Ser Gln Glu Glu
995 1000 1005
Arg Leu Gln Gln Lys Val Asn Gly Ile Ile His Leu Ala Gly Ile
1010 1015 1020
Ile Gln Glu Ile Leu Ile Glu Lys Gln Thr Glu Lys Glu Leu His
1025 1030 1035
Ala Met Phe Glu Ala Lys Val Tyr Ala Ser Trp Val Leu His Glu
1040 1045 1050
Ile Val Lys Glu Arg Gln Asp Cys Leu Tyr Ile Thr Thr Ser Ser
1055 1060 1065
Ala Arg Thr Leu Leu Pro Gly Met Thr Ile Ser Ala Tyr Cys Ser
1070 1075 1080
Ala Asn Arg Phe Val Glu Asn Phe Ala Tyr Tyr Gln Arg Ser Gln
1085 1090 1095
Asn Val Asn Ser Tyr Cys Phe Ser Trp Ser Phe Trp Asn Glu Ile
1100 1105 1110
Gly Met Gly Thr Asn Leu Leu Ile Lys Asn Ala Leu Ile Ala Lys
1115 1120 1125
Gly Phe Gln Leu Ile Asp Asp Gln Lys Gly Ile Tyr Ser Leu Leu
1130 1135 1140
Ala Gly Leu Lys Gly Asn Glu Pro Asn Val Phe Val Gly Ile Asn
1145 1150 1155
His Glu Lys Glu Glu Met Ala His Leu Ile Gly Thr Glu Glu Gln
1160 1165 1170
Glu Thr Gln Gln Leu Thr Ile Tyr Ile Thr Pro Glu Tyr Leu His
1175 1180 1185
Ile Leu Glu Glu Val Phe Ser Ile Leu Asn Arg Glu Glu Phe Gly
1190 1195 1200
Gly Leu Glu Lys Glu Ile Val Ile Leu Pro Lys Leu Pro Leu Asp
1205 1210 1215
Glu Tyr Gly Lys Val Asp Gln Thr Arg Leu Ala His Ala Ser Asp
1220 1225 1230
Ser Arg Phe Gly Lys Lys Gln His Ile Val Pro Arg Asn Asp Ile
1235 1240 1245
Glu Glu Lys Ile Ala Phe Ile Trp Glu Gly Leu Leu Asn Lys Lys
1250 1255 1260
Asp Ile Ser Val Leu Asp His Phe Phe Glu Leu Gly Gly Asp Ser
1265 1270 1275
Leu Lys Ala Thr Gln Met Ile Ser Ala Leu Lys Lys Asn Phe Ala
1280 1285 1290
Val Thr Ile Thr Gln Gln Glu Phe Phe Gln Ser Ser Thr Val Glu
1295 1300 1305
Glu Leu Ala Ser Leu Val Glu Lys Lys Leu Ser Arg Thr Arg Thr
1310 1315 1320
His Glu Met Asp Ile Val Thr Phe Ser Asp Arg Gly Asn Val Val
1325 1330 1335
Glu Met Ser Ser Ala Gln Lys Arg Gln Trp Phe Leu Tyr Glu Met
1340 1345 1350
Asp Arg Glu Asn Pro Tyr Tyr Asn Asn Thr Leu Val Ile Arg Leu
1355 1360 1365
Thr Gly Glu Ile His Leu Pro Ile Leu Arg Ser Ser Ile Ile Glu
1370 1375 1380
Leu Val Asn Lys His Glu Thr Leu Arg Thr Thr Phe Val Met Val
1385 1390 1395
Asp Gly Ile Pro Ser Gln Ile Ile Ala Asp Glu Glu Leu Val Glu
1400 1405 1410
Ile Glu Glu Ile Asp Leu Lys His Leu Ser Ala Glu Glu Thr Leu
1415 1420 1425
Gln Lys Leu Glu Gly Leu Arg Gln Arg Glu Ala Asn Thr Ala Phe
1430 1435 1440
Lys Ile Glu Asn Ser Ala Phe Arg Ala Lys Val Ile Leu Ile Asp
1445 1450 1455
Glu Lys Arg Val Glu Ile Leu Leu Ser Val His His Ile Val Ser
1460 1465 1470
Asp Gly Trp Ser Met Gly Ile Leu Val Lys Asp Ile Ala Glu Ile
1475 1480 1485
Tyr Glu Asp Ile Arg Gln Trp Gly Glu Ser Lys Gln Glu Pro Leu
1490 1495 1500
Pro Ile Gln Tyr Ala Asp Tyr Thr Leu Trp Gln Asn Glu Phe Met
1505 1510 1515
Lys Gly Glu Glu Phe Ser Lys Gln Leu Ser Tyr Trp Lys Glu Lys
1520 1525 1530
Leu Ala Glu Asp Ile Pro Val Leu Asp Leu Pro Leu Asp Lys Pro
1535 1540 1545
Arg Pro Pro Ile Gln Thr Tyr Arg Gly Lys Val Lys Thr Phe Thr
1550 1555 1560
Leu His Glu Asn Met Thr Arg Met Leu Lys Glu Ile Cys Gln Glu
1565 1570 1575
Glu Glu Cys Thr Leu Phe Met Leu Leu Leu Ser Ala Phe Ser Ser
1580 1585 1590
Leu Leu His Arg Tyr Thr Gly Gln Glu Asp Leu Val Val Gly Ser
1595 1600 1605
Leu Val Ala Asn Arg Asn Arg Glu Gln Ile Glu Lys Leu Ile Gly
1610 1615 1620
Phe Phe Val Asn Thr Leu Pro Leu Arg Ile Asn Leu His Arg Glu
1625 1630 1635
Met Gln Phe Thr Glu Leu Leu Ser Gln Val Lys Lys Thr Thr Ile
1640 1645 1650
Asp Ala Tyr Asp His Gln Asp Val Pro Phe Glu Leu Leu Val Asp
1655 1660 1665
Glu Leu Gln Ile Glu Arg Asp Ser Ser Arg Asn Ala Leu Phe Gln
1670 1675 1680
Val Leu Phe Val Leu Gln Asn Ala Gln Leu Gln Ala Val Asp Leu
1685 1690 1695
Glu Lys Ala Thr Met Glu Leu Glu Ile Leu Asp Ser Asp Thr Ala
1700 1705 1710
Lys Phe Asp Met Ser Val Gln Ile Phe Glu Leu Glu Asp Thr Leu
1715 1720 1725
Ser Ile Lys Leu Glu Tyr Asn Thr Asp Leu Phe Phe Asp Asp Thr
1730 1735 1740
Ile Glu Arg Phe Leu Ala His Tyr Glu Thr Ile Leu Ala Ser Val
1745 1750 1755
Ile His Asn Gln Lys Ala Lys Ile Gly Glu Leu Ser Ile Leu Pro
1760 1765 1770
Gln Ser Glu Tyr Thr Lys Leu Val Ser Glu Trp Asn Glu Lys Ser
1775 1780 1785
Ala Thr Tyr Asn Gly Asn Gln Cys Ile His Glu Leu Phe Glu Ala
1790 1795 1800
Ala
<210>3
<211>1900
<212>PRT
<213>蜡状芽孢杆菌
<400>3
Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn Lys Glu
1 5 10 15
Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu Ala His Lys
20 25 30
Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val Gly Val Leu Cys
35 40 45
Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu Ala Val Leu Lys Ala
50 55 60
Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala Tyr Pro Met Gln Arg Thr
65 70 75 80
Glu Tyr Val Leu Gln Asn Ser Glu Ala Thr Ile Leu Leu Thr Lys Glu
85 90 95
Cys Tyr Leu Lys Glu Ser Leu Asp Phe Glu Gly Glu Val Phe Tyr Leu
100 105 110
Asp Asp Ala Arg Leu Phe Glu Gly Asp Arg Arg Asp Leu Gln Asn Ile
115 120 125
Asn Asn Pro Thr Asn Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr
130 135 140
Gly Asn Pro Lys Gly Val Met Val Ala His Gln Ser Val Val Asn Leu
145 150 155 160
Leu Leu Asp Leu Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His
165 170 175
Leu Leu Lys Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe
180 185 190
Gly Trp Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp
195 200 205
Glu Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val
210 215 220
Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln Ala
225 230 235 240
Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr Ile Ile
245 250 255
Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg Leu Tyr Ala
260 265 270
His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly Pro Thr Glu Gly
275 280 285
Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys Glu Met Asn Val Ala
290 295 300
Asn Val Pro Ile Gly Lys Pro Leu Ser His Val Glu Thr Tyr Ile Leu
305 310 315 320
Asp Gln Asn Asn Gln Ile Val Pro Ile Gly Val Pro Gly Glu Leu Cys
325 330 335
Leu Gly Gly Ile Cys Val Ala Lys Gly Tyr Met Lys Glu Pro Val Leu
340 345 350
Thr Glu Glu Lys Phe Val Val Asn Pro Met Lys Gln Ser Glu Arg Met
355 360 365
Tyr Arg Thr Gly Asp Leu Val Arg Trp Leu Ala Asp Gly Asn Ile Glu
370 375 380
Tyr Leu Gly Arg Ile Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile
385 390 395 400
Glu Leu Gly Glu Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val
405 410 415
Gln Thr Ile Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile
420 425 430
Val Ala Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu
435 440 445
His Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val
450 455 460
Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg Lys
465 470 475 480
Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr Val Ile
485 490 495
Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val Ile Trp Gln
500 505 510
Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp Asn Phe Phe Lys
515 520 525
Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu Val Ser Lys Ile Tyr
530 535 540
Ser Val Cys Arg Val Arg Met Pro Leu Lys Asn Val Phe Gln Tyr Thr
545 550 555 560
Thr Leu Ala Thr Met Ala Arg Val Leu Glu Glu Leu Leu Val Ser Ala
565 570 575
Val Asp Glu Val Ala Val Thr Thr Glu Arg Ile Pro Lys Ile Leu Pro
580 585 590
Arg Thr Tyr Tyr Asp Leu Ser Tyr Ser Gln Gln Arg Ile Tyr Phe Leu
595 600 605
Ser Thr Met Glu Lys Glu Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp
610 615 620
Asp Ile Tyr Gly Lys Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln
625 630 635 640
Leu Leu Met Lys Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val
645 650 655
Asp Gly Lys Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val
660 665 670
Gln Phe Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu
675 680 685
Val Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala
690 695 700
Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly Glu
705 710 715 720
His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly Trp Ser
725 730 735
Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp Ala Phe Val
740 745 750
Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile Ser Ile Met Asp
755 760 765
Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu Asp Gln Asp Asp Arg
770 775 780
Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu Leu Pro Thr Leu Glu Leu
785 790 795 800
Pro Thr Asp Arg Gln Arg Pro Leu Leu Lys Thr Tyr His Gly Asp Thr
805 810 815
Leu Ser Tyr Lys Val Asn Ser Gln Leu His Gln Lys Leu Lys Asp Phe
820 825 830
Ser His Ala Asn Gly Val Thr Met Phe Met Thr Leu Leu Thr Ala Tyr
835 840 845
Asn Ile Met Leu Asn Lys Leu Thr Asn Glu Thr Asp Ile Val Val Gly
850 855 860
Ser Pro Val Ala Gly Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly
865 870 875 880
Met Phe Val Asn Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro
885 890 895
Thr Val Asp Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala
900 905 910
Tyr Asn His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp
915 920 925
Pro His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly
930 935 940
Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val Arg
945 950 955 960
Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr Leu His
965 970 975
Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu Tyr Asn Thr
980 985 990
Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln Asn His Phe Glu
995 1000 1005
Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr Lys Arg Ile Ser
1010 1015 1020
Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr Glu Ile Leu Glu
1025 1030 1035
Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His Glu Ser Val Ala
1040 1045 1050
Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln Ala Lys Leu Ala
1055 1060 1065
Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala Glu Leu Ser Glu
1070 1075 1080
Lys Ala Asn Ala Leu Ala His Thr Leu Lys Arg Arg Gly Val Ala
1085 1090 1095
Gln His Asp Val Val Gly Ile Val Ala Glu Arg Ser Pro Glu Thr
1100 1105 1110
Ile Ile Gly Ile Leu Ala Ile Leu Lys Val Gly Ala Ile Tyr Leu
1115 1120 1125
Pro Ile Asp Pro Lys Leu Pro Gln Leu Thr Leu Gln His Ile Trp
1130 1135 1140
Arg Asp Ser Gly Ala Lys Val Leu Leu Gly Lys Asn Glu Thr Thr
1145 1150 1155
Val Glu Val Gly Lys Glu Val Pro Phe Val Asp Ile Glu Gly Asp
1160 1165 1170
Lys Gly Lys Gln Glu Glu Leu Val Cys Pro Ile Ser Pro Glu Asp
1175 1180 1185
Thr Ala Tyr Ile Met Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys
1190 1195 1200
Gly Val Met Val Thr His Arg Asn Ile Val Arg Leu Val Lys Asn
1205 1210 1215
Thr Asn Phe Val Ser Leu Gln Glu Gln Asp Val Leu Leu Gln Thr
1220 1225 1230
Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu Ile Trp Gly Ala
1235 1240 1245
Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu Asp Tyr Val Ile
1250 1255 1260
Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln Gln Asn Lys Ala
1265 1270 1275
Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn Gln Leu Ala Asp
1280 1285 1290
Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln Leu Leu Ile Gly
1295 1300 1305
Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys Val Met Asp His
1310 1315 1320
Cys Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly Pro Thr Glu Asn
1325 1330 1335
Thr Thr Phe Ser Thr Ser Phe Val Ile Asp Gln Met Tyr Gln Asp
1340 1345 1350
Ser Ile Pro Ile Gly Thr Pro Ile Ala Asn Ser Ser Ala Tyr Ile
1355 1360 1365
Leu Asp Val His Gln Asn Ile Gln Pro Ile Gly Val Val Gly Glu
1370 1375 1380
Leu Cys Val Gly Gly Asp Gly Val Ala Lys Gly Tyr Val Asn Leu
1385 1390 1395
Glu Gln Leu Thr Glu Glu Arg Phe Ile Ala Asp Pro Phe Leu Lys
1400 1405 1410
Gly Ser Thr Met Tyr Arg Thr Gly Asp Tyr Val Lys Leu Leu Pro
1415 1420 1425
Asn Gly Asn Ile Gln Tyr Ile Gly Arg Val Asp Asn Gln Val Lys
1430 1435 1440
Ile Arg Gly Phe Arg Ile Glu Leu Glu Ala Ile Met Asn Thr Leu
1445 1450 1455
Lys Gln Cys Glu Ser Ile Lys Asp Val Ile Val Val Val Gln Glu
1460 1465 1470
Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val Val Gly Glu Glu
1475 1480 1485
Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala Lys Lys His Leu
1490 1495 1500
Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe Ile Glu Glu Ile
1505 1510 1515
Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser Lys Leu Pro Lys
1520 1525 1530
Val Gln Glu Val Leu His Lys Lys Val Glu Thr Leu Leu Pro Glu
1535 1540 1545
Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr Arg Asp Val Leu
1550 1555 1560
Glu Lys Glu Asp Phe Gly Val Thr Asp Ser Phe Phe Ala Tyr Gly
1565 1570 1575
Gly Asp Ser Leu Leu Ser Ile Gln Val Val Ser Met Leu Lys Lys
1580 1585 1590
Glu Glu Ile Ala Val Asp Pro Lys Met Ile Phe Met His Thr Thr
1595 1600 1605
Val Arg Glu Leu Ala Lys Ala Cys Glu Asn Arg Pro Val Met Glu
1610 1615 1620
Glu Thr Lys Arg Thr Glu Lys Asp Tyr Leu Ile Gln Met Arg Glu
1625 1630 1635
Gly Ser Glu Glu Asp Ser Cys Ile Ile Phe Ala Pro Pro Ala Gly
1640 1645 1650
Gly Thr Val Leu Gly Tyr Ile Glu Leu Ala Arg Tyr Phe Glu Gly
1655 1660 1665
Ile Gly Asn Val Tyr Gly Leu Gln Ala Pro Gly Leu Tyr Asp Asp
1670 1675 1680
Glu Glu Pro Thr Phe Leu Asp Tyr Asp Glu Leu Val Gln Val Phe
1685 1690 1695
Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro Gly Gln Asp Tyr Leu
1700 1705 1710
Gly Gly His Ser Leu Gly Gly His Ile Ala Phe Gly Met Cys Cys
1715 1720 1725
Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly Leu Leu Ile Leu
1730 1735 1740
Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly Ala Lys Asp Glu
1745 1750 1755
Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val Leu Ala Ala Gly
1760 1765 1770
Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu Leu Lys Gln Leu
1775 1780 1785
Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala Val Ala Gln Lys
1790 1795 1800
Asp Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr Tyr Leu Asp Lys
1805 1810 1815
Tyr Leu Lys Leu Gln Ile His Ser Leu Leu Met Ser Arg Thr Leu
1820 1825 1830
Glu Leu Glu Lys Thr Gln Leu Asp Ile Pro Ile Lys Val Phe Lys
1835 1840 1845
Thr Gln Phe His Thr Glu Glu Leu Val Glu Arg Phe Asp Ala Trp
1850 1855 1860
His Asn Tyr Thr Asn Gln Ala Cys Thr Phe Ile Asp Ile Pro Gly
1865 1870 1875
Thr His Thr Thr Met Met Arg Leu Pro His Val Lys Glu Val Ala
1880 1885 1890
Lys Lys Ile Glu Glu Gln Leu
1895 1900
<210>4
<211>1020
<212>PRT
<213>蜡状芽孢杆菌
<400>4
Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn Lys Glu
1 5 10 15
Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu Ala His Lys
20 25 30
Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val Gly Val Leu Cys
35 40 45
Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu Ala Val Leu Lys Ala
50 55 60
Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala Tyr Pro Met Gln Arg Thr
65 70 75 80
Glu Tyr Val Leu Gln Asn Ser Glu Ala Thr Ile Leu Leu Thr Lys Glu
85 90 95
Cys Tyr Leu Lys Glu Ser Leu Asp Phe Glu Gly Glu Val Phe Tyr Leu
100 105 110
Asp Asp Ala Arg Leu Phe Glu Gly Asp Arg Arg Asp Leu Gln Asn Ile
115 120 125
Asn Asn Pro Thr Asn Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr
130 135 140
Gly Asn Pro Lys Gly Val Met Val Ala His Gln Ser Val Val Asn Leu
145 150 155 160
Leu Leu Asp Leu Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His
165 170 175
Leu Leu Lys Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe
180 185 190
Gly Trp Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp
195 200 205
Glu Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val
210 215 220
Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln Ala
225 230 235 240
Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr Ile Ile
245 250 255
Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg Leu Tyr Ala
260 265 270
His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly Pro Thr Glu Gly
275 280 285
Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys Glu Met Asn Val Ala
290 295 300
Asn Val Pro Ile Gly Lys Pro Leu Ser His Val Glu Thr Tyr Ile Leu
305 310 315 320
Asp Gln Asn Asn Gln Ile Val Pro Ile Gly Val Pro Gly Glu Leu Cys
325 330 335
Leu Gly Gly Ile Cys Val Ala Lys Gly Tyr Met Lys Glu Pro Val Leu
340 345 350
Thr Glu Glu Lys Phe Val Val Asn Pro Met Lys Gln Ser Glu Arg Met
355 360 365
Tyr Arg Thr Gly Asp Leu Val Arg Trp Leu Ala Asp Gly Asn Ile Glu
370 375 380
Tyr Leu Gly Arg Ile Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile
385 390 395 400
Glu Leu Gly Glu Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val
405 410 415
Gln Thr Ile Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile
420 425 430
Val Ala Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu
435 440 445
His Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val
450 455 460
Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg Lys
465 470 475 480
Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr Val Ile
485 490 495
Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val Ile Trp Gln
500 505 510
Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp Asn Phe Phe Lys
515 520 525
Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu Val Ser Lys Ile Tyr
530 535 540
Ser Val Cys Arg Val Arg Met Pro Leu Lys Asn Val Phe Gln Tyr Thr
545 550 555 560
Thr Leu Ala Thr Met Ala Arg Val Leu Glu Glu Leu Leu Val Ser Ala
565 570 575
Val Asp Glu Val Ala Val Thr Thr Glu Arg Ile Pro Lys Ile Leu Pro
580 585 590
Arg Thr Tyr Tyr Asp Leu Ser Tyr Ser Gln Gln Arg Ile Tyr Phe Leu
595 600 605
Ser Thr Met Glu Lys Glu Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp
610 615 620
Asp Ile Tyr Gly Lys Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln
625 630 635 640
Leu Leu Met Lys Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val
645 650 655
Asp Gly Lys Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val
660 665 670
Gln Phe Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu
675 680 685
Val Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala
690 695 700
Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly Glu
705 710 715 720
His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly Trp Ser
725 730 735
Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp Ala Phe Val
740 745 750
Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile Ser Ile Met Asp
755 760 765
Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu Asp Gln Asp Asp Arg
770 775 780
Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu Leu Pro Thr Leu Glu Leu
785 790 795 800
Pro Thr Asp Arg Gln Arg Pro Leu Leu Lys Thr Tyr His Gly Asp Thr
805 810 815
Leu Ser Tyr Lys Val Asn Ser Gln Leu His Gln Lys Leu Lys Asp Phe
820 825 830
Ser His Ala Asn Gly Val Thr Met Phe Met Thr Leu Leu Thr Ala Tyr
835 840 845
Asn Ile Met Leu Asn Lys Leu Thr Asn Glu Thr Asp Ile Val Val Gly
850 855 860
Ser Pro Val Ala Gly Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly
865 870 875 880
Met Phe Val Asn Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro
885 890 895
Thr Val Asp Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala
900 905 910
Tyr Asn His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp
915 920 925
Pro His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly
930 935 940
Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val Arg
945 950 955 960
Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr Leu His
965 970 975
Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu Tyr Asn Thr
980 985 990
Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln Asn His Phe Glu
995 1000 1005
Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr Lys
1010 1015 1020
<210>5
<211>880
<212>PRT
<213>蜡状芽孢杆菌
<400>5
Arg Ile Ser Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr Glu Ile
1 5 10 15
Leu Glu Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His Glu Ser Val
20 25 30
Ala Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln Ala Lys Leu Ala
35 40 45
Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala Glu Leu Ser Glu Lys
50 55 60
Ala Asn Ala Leu Ala His Thr Leu Lys Arg Arg Gly Val Ala Gln His
65 70 75 80
Asp Val Val Gly Ile Val Ala Glu Arg Ser Pro Glu Thr Ile Ile Gly
85 90 95
Ile Leu Ala Ile Leu Lys Val Gly Ala Ile Tyr Leu Pro Ile Asp Pro
100 105 110
Lys Leu Pro Gln Leu Thr Leu Gln His Ile Trp Arg Asp Ser Gly Ala
115 120 125
Lys Val Leu Leu Gly Lys Asn Glu Thr Thr Val Glu Val Gly Lys Glu
130 135 140
Val Pro Phe Val Asp Ile Glu Gly Asp Lys Gly Lys Gln Glu Glu Leu
145 150 155 160
Val Cys Pro Ile Ser Pro Glu Asp Thr Ala Tyr Ile Met Tyr Thr Ser
165 170 175
Gly Ser Thr Gly Lys Pro Lys Gly Val Met Val Thr His Arg Asn Ile
180 185 190
Val Arg Leu Val Lys Asn Thr Asn Phe Val Ser Leu Gln Glu Gln Asp
195 200 205
Val Leu Leu Gln Thr Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu
210 215 220
Ile Trp Gly Ala Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu Asp
225 230 235 240
Tyr Val Ile Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln Gln Asn
245 250 255
Lys Ala Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn Gln Leu Ala
260 265 270
Asp Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln Leu Leu Ile Gly
275 280 285
Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys Val Met Asp His Cys
290 295 300
Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly Pro Thr Glu Asn Thr Thr
305 310 315 320
Phe Ser Thr Ser Phe Val Ile Asp Gln Met Tyr Gln Asp Ser Ile Pro
325 330 335
Ile Gly Thr Pro Ile Ala Asn Ser Ser Ala Tyr Ile Leu Asp Val His
340 345 350
Gln Asn Ile Gln Pro Ile Gly Val Val Gly Glu Leu Cys Val Gly Gly
355 360 365
Asp Gly Val Ala Lys Gly Tyr Val Asn Leu Glu Gln Leu Thr Glu Glu
370 375 380
Arg Phe Ile Ala Asp Pro Phe Leu Lys Gly Ser Thr Met Tyr Arg Thr
385 390 395 400
Gly Asp Tyr Val Lys Leu Leu Pro Asn Gly Asn Ile Gln Tyr Ile Gly
405 410 415
Arg Val Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Glu
420 425 430
Ala Ile Met Asn Thr Leu Lys Gln Cys Glu Ser Ile Lys Asp Val Ile
435 440 445
Val Val Val Gln Glu Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val
450 455 460
Val Gly Glu Glu Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala Lys
465 470 475 480
Lys His Leu Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe Ile Glu
485 490 495
Glu Ile Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser Lys Leu Pro
500 505 510
Lys Val Gln Glu Val Leu His Lys Lys Val Glu Thr Leu Leu Pro Glu
515 520 525
Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr Arg Asp Val Leu Glu
530 535 540
Lys Glu Asp Phe Gly Val Thr Asp Ser Phe Phe Ala Tyr Gly Gly Asp
545 550 555 560
Ser Leu Leu Ser Ile Gln Val Val Ser Met Leu Lys Lys Glu Glu Ile
565 570 575
Ala Val Asp Pro Lys Met Ile Phe Met His Thr Thr Val Arg Glu Leu
580 585 590
Ala Lys Ala Cys Glu Asn Arg Pro Val Met Glu Glu Thr Lys Arg Thr
595 600 605
Glu Lys Asp Tyr Leu Ile Gln Met Arg Glu Gly Ser Glu Glu Asp Ser
610 615 620
Cys Ile Ile Phe Ala Pro Pro Ala Gly Gly Thr Val Leu Gly Tyr Ile
625 630 635 640
Glu Leu Ala Arg Tyr Phe Glu Gly Ile Gly Asn Val Tyr Gly Leu Gln
645 650 655
Ala Pro Gly Leu Tyr Asp Asp Glu Glu Pro Thr Phe Leu Asp Tyr Asp
660 665 670
Glu Leu Val Gln Val Phe Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro
675 680 685
Gly Gln Asp Tyr Leu Gly Gly His Ser Leu Gly Gly His Ile Ala Phe
690 695 700
Gly Met Cys Cys Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly Leu
705 710 715 720
Leu Ile Leu Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly Ala Lys
725 730 735
Asp Glu Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val Leu Ala Ala
740 745 750
Gly Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu Leu Lys Gln Leu
755 760 765
Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala Val Ala Gln Lys Asp
770 775 780
Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr Tyr Leu Asp Lys Tyr Leu
785 790 795 800
Lys Leu Gln Ile His Ser Leu Leu Met Ser Arg Thr Leu Glu Leu Glu
805 810 815
Lys Thr Gln Leu Asp Ile Pro Ile Lys Val Phe Lys Thr Gln Phe His
820 825 830
Thr Glu Glu Leu Val Glu Arg Phe Asp Ala Trp His Asn Tyr Thr Asn
835 840 845
Gln Ala Cys Thr Phe Ile Asp Ile Pro Gly Thr His Thr Thr Met Met
850 855 860
Arg Leu Pro His Val Lys Glu Val Ala Lys Lys Ile Glu Glu Gln Leu
865 870 875 880
<210>6
<211>11115
<212>DNA
<213>蜡状芽孢杆菌
<400>6
tttataaaat caatgaatca gttgggaaaa tcaaaaaatt tacataatgg ggggatgatg 60
gagatgaaac gagtggaaga acatgatcac attcatgtgt taaatgaaat agaaaacgaa 120
tgcgaaagaa gatatgggag aagtaatatt gcaattatgc ttgaaaagca tggtgttcat 180
gaacagccgc ttcatataga agacttattt catgaggtag agatgcaaga acattcacga 240
gtatcgcgcc acgaaacagt tttgatgaca gataaacaat gtatagatga gagtggaaaa 300
ccgttagctc ttcgttttgg tgagccactt catcttgatg actgtacccc aaaaacacta 360
caagaaattt taaagcgtgc cgctaagcaa gcaaaagata aagggatgac atttgtatat 420
gaagatggac atgaagagta cctctcctac caagagatgt tggcagatgc ggagcggtta 480
ctaaaagggt tgcgaaatct tggtatacaa ccaggagaaa gtattttgtt tcaatttaag 540
gacaataagc attttgttac tgcgttttgg gcatgtatac ttgggggatt tttaccaacg 600
ccgttaggaa cggcccctat ctatagtgag caaaatgcac aagtattaaa actttataac 660
acatggcagc tattagaaca accgattatt ttaacggaat ttgaattgaa agaagagatt 720
gctgcaattc gaacaacatt gcaacgtcaa gagattgtta tacatagtat tgagaatgtt 780
atggatacag cgcgcgatac aaactggttt ccttgtaccg aagatactat tgttttgaat 840
ttattaacgt ctggtagcac aggagtaccc aaatgtgtgc agcacaaaag taaatccatt 900
attgcgcgca cagtttccaa ttgtattgac cgccagctag atgaaaaaga agtatcgtta 960
aattggatgc cgcttgatca tgttggaggc atcgtaatgt gtcacattcg tgatacctat 1020
ttaatgtgcc aacaggtaaa ctgtcttatt tcggcattta ttgaaaatcc gctaaattgg 1080
ttgcactgga ttgatgctta ctcagcgaca tttacatggg cgccaaactt cgctttttca 1140
ttaattaacc agtatgaaga agagattaaa tcatcttcat ggaatctttc ttcgatgaga 1200
tacatcgtaa atggtgggga agctgttatt tcaagtgttg ggatgaaatt tttacaattg 1260
ttacagcaac atcaattgcc ttcgaactgt cttattccta cgtttgggat gtctgaagtt 1320
tcttcgggta ttattgaatg tcattcgttt tatacgcaaa cgacaaatac aggaatgttg 1380
tatgttgata agaattcttt agatggtaat ttacaattca catatgaggg gcaccaaaat 1440
gccattgttt ttacggaagt agggagaccg atgcctggta ttggcattcg tattgttgat 1500
gaggacaatc agtgcctgtc agaagatcgt attggacgat tccaaattca tggtccaacg 1560
gttatgaatg gttatttcaa aaatgatgaa gcaaatgcgg aaagttttac tgaagatggc 1620
tggttcgata gtggagatct ggggtttata cataacggta atcttgtcat tacaggaaga 1680
aaaaaggata tgattgttgt tcatggtgca aattactaca actatgaaat tgaagccttg 1740
gtagaacaag tacctggggt ggaaaccacg tttgtatgtg caacgagtgt gaagtcggct 1800
gaaggagcag aggaattagc tattttcttt gtcccagtaa ttaatcatgt ttctgtgatg 1860
tttgcgacga tgcaacaaat caaacaaatt gttgcgcgca aaatgggtat cacgccgaaa 1920
gtgattatac caattcagaa ggaagcattc tttaaaacgg atagtgggaa aataacgaga 1980
aatgcatttc aaaaacagtt tgagaacggg gcatatagag agattacaca aaagattgat 2040
tgccatttac aaaatgaaaa aacactatct cagtggtttt atcgtgaaaa attagtcgaa 2100
agtaagttgg gcaaaagcgt atcctcccaa aaagaaacgt atgttttctt tcgacaaggt 2160
aaatcatttc atcatgtcct aaaagaaaag ttaacgcaac attctgttgt tattgtggat 2220
gtaggagaaa cattcggtga gatccatcca aatcattatc aaattaatcc taaaaacaaa 2280
atggattacg ttcgattatt tgaagaactc gcaaaaagaa atgtagaaga tcaagttttt 2340
catctcttgc atgcttggaa ttattgcgat acagttccaa cttttagatc ggtagaagat 2400
ttagctaatg cgcaatatct tggtgtgttt agtgtgatgt tcgcacttca agctattatg 2460
catgcgaaat tgccactacg tcgtgttacg gtgattgcga caaatagtgt tggattagaa 2520
gcgaaggaaa tgaactattc atgttcaaca ttagaaggtt atgtgaaaac tttgccagct 2580
gagtttgaaa atttacaagt gaagtatatt gatatagaag gaaaagatat acagtttgat 2640
actgagaccg tatggaaaga acttcagcaa caagaaacca ttcctgtcgt attgtatcgt 2700
gatgagaaaa gatacaaaat aggtttagaa aaagtaccaa tgttagaaca gaaagaaaaa 2760
aatattccgt ttcaacagca agggttttac atcattacag gtggtcttgg tggtttgggg 2820
acgcttgtag ccaaattact tttagaacga tacagcgcaa atgttctttt acttggtcga 2880
acagaaattg aaacaaatgc agaaaaaatg cgccttcttg attcattaaa agagtatgaa 2940
caatatggtg gtacagtcca atataaaatg tgcaatgtaa tggatttaga tgcgatgcga 3000
aaagttgttc attcacagga agaacgtctg caacaaaagg taaatgggat tatccacctt 3060
gcagggatta ttcaagaaat actgatagaa aagcaaaccg aaaaagaact gcatgctatg 3120
tttgaagcta aggtatatgc atcttgggtg ctacatgaaa tcgtaaaaga aaggcaagat 3180
tgtctctaca ttacaacttc ttcagcaaga acgttgttac cggggatgac catctcagct 3240
tattgtagtg cgaatcgatt tgttgaaaat tttgcatatt atcaacgaag tcaaaatgta 3300
aatagctact gtttttcatg gagtttctgg aatgagattg gaatgggtac aaatttactt 3360
attaaaaatg cgttgatagc aaaaggattt caattgatcg atgatcaaaa aggtatatat 3420
tcccttttgg cgggattaaa agggaacgaa cctaatgttt ttgttggaat caatcatgaa 3480
aaagaagaaa tggctcatct gattggaacc gaggaacaag aaacacaaca attaacaatc 3540
tatattacac cagaatactt acatattctt gaagaagtgt tctctatact aaatagagaa 3600
gaatttggtg gattggagaa agagattgtc attctaccaa aattaccgct tgatgaatat 3660
ggtaaagtag atcaaactcg tttggctcat gcgtcggata gccgttttgg aaagaaacaa 3720
catatcgtac caagaaatga tatagaagag aaaattgcat tcatttggga aggtcttttg 3780
aataaaaagg atattagtgt acttgaccat ttcttcgaat taggtggtga ttctttaaaa 3840
gcgacacaaa tgatttctgc gttgaaaaag aattttgctg ttacgattac gcaacaggaa 3900
ttttttcaat cgagtacagt agaagagctt gctagtttag tagaaaagaa actttctcgt 3960
actcgtacgc atgaaatgga catagttact tttagtgacc gaggtaacgt agtagagatg 4020
tcttctgcac aaaagcggca atggttttta tatgaaatgg atcgagaaaa tccttattac 4080
aataatacac ttgtaattcg tttgacggga gaaattcatc ttcctatttt aagaagttct 4140
attattgagt tagtaaataa gcatgaaaca ttgcgaacaa catttgtgat ggtggatggt 4200
ataccatcac aaattattgc agatgaagag ttagttgaaa tagaggaaat tgatttgaaa 4260
cacctatctg ctgaggagac gttgcaaaaa ctagagggtt tacgacaacg ggaagcaaat 4320
acggcgttta aaatcgaaaa tagcgctttt cgtgcaaaag tgattttaat tgatgagaag 4380
agagtggaga ttttactttc cgtgcatcac attgtttcgg atggttggtc gatggggatt 4440
ttagtgaagg acattgcgga aatctatgaa gatattcggc agtggggaga aagtaagcaa 4500
gagccattac cgattcaata cgcagattat actttgtggc aaaatgagtt tatgaaaggt 4560
gaggaattta gcaagcaact gtcttattgg aaggagaaat tagctgaaga tatacctgta 4620
cttgatcttc cgttagataa accacggcca ccaattcaaa catatcgtgg gaaggttaag 4680
actttcacgt tacatgaaaa catgacaagg atgctaaaag aaatatgtca agaagaagaa 4740
tgcacgctct ttatgttgtt actttcggct ttctcatcat tattacatcg ttatacaggt 4800
caggaggatc ttgttgttgg ttcgctagtt gcaaatcgaa accgtgagca aatcgagaaa 4860
ttgattggtt tctttgttaa tacgttaccg ctacgtatta atcttcatcg ggaaatgcaa 4920
tttactgaat tgctttcgca agtaaagaaa acgaccattg atgcatatga tcatcaagat 4980
gtgccttttg agctactagt cgatgaatta cagattgaga gagattcgag tcgtaatgcg 5040
ctattccaag tgttgtttgt cttacaaaac gcacaattac aagcagtaga cttagagaaa 5100
gcgacaatgg aactcgaaat tttagatagt gacacggcca agtttgatat gtcagtgcaa 5160
attttcgaat tggaggacac tttatctatc aaattagagt acaatacgga tttatttttt 5220
gatgatacaa tagaacgctt tcttgctcat tatgaaacca tattagcaag cgttattcat 5280
aatcaaaagg caaaaatagg ggaattgtca attttaccac aatctgaata tacgaaactt 5340
gtatctgagt ggaatgaaaa gagtgccact tataatggaa atcagtgtat tcatgaattg 5400
ttcgaagcag ctgttcacaa aacgccatct gcaacagcgc ttatttatcg caacaaagag 5460
atgacatacg aggatgttaa tgcgcaggca aatgcacttg cacataaatt aagagatgca 5520
ggtgttggac caaaccaggt agttggcgtg ttatgtgatc gctctttcga gatggttgtt 5580
ggtatattag ctgttttaaa agcaggtggt gcgtatttgc caattgatac agcgtacccg 5640
atgcaacgaa cagaatacgt cctgcaaaat agtgaggcaa ctattctctt aacaaaggaa 5700
tgttacctta aggagtcttt agattttgag ggggaagttt tttacttaga tgatgcaaga 5760
ctgtttgaag gggatagaag agatttacaa aatatcaata atcctactaa ccttgcttat 5820
atcatttata catcaggatc cacgggaaat ccaaaaggtg ttatggtagc gcatcaaagt 5880
gttgtgaatt tgctactcga tttacaagag aaatatccgg tgctagcaga agataagcac 5940
ttgttaaaaa caacatatac gtttgatgtt tctgtagccg aaatttttgg atggtttcat 6000
gcaggtggca cacttgttat tgctggacat ggtgatgaaa aagacccaga gaaactgatt 6060
caattgattc aatgccacaa ggttacacat attaacttcg taccatcgat gctacatgca 6120
atgttacagg ccttggatga aaaagatttt gcaattatga atcggttgaa atatattatc 6180
gtcgcaggag aagctgtttc accagaactt tgtaatcgac tgtacgctca ttgtccaaat 6240
gtaaaactag aaaatctata tgggccaacg gaaggaacga tttatgcgac agggttttct 6300
attcataaag aaatgaatgt agctaatgta ccgattggaa aaccactttc tcatgtggaa 6360
acgtatattc ttgatcaaaa caatcaaatt gtaccaattg gtgtaccagg tgaattgtgt 6420
ctgggaggaa tatgtgtagc aaaaggttat atgaaagagc cggtgttaac agaagaaaaa 6480
ttcgtcgtca atcctatgaa acaaagtgaa agaatgtacc gaacgggtga tttggtacgc 6540
tggttagcag atgggaatat tgaatattta ggaagaatag ataaccaagt caagataaga 6600
ggcttccgaa ttgagcttgg tgaaattgaa gcggcaattg ctgcattaga agatgtagta 6660
caaacaattg ttacaacaat gacggatcat aaaggtgcga acaagattgt cgcatatgtt 6720
gtgagcgaaa agtatgatga agaacgaatt cgtgaacatg tgaaaaagac gttgccgcaa 6780
tatatggtac caagttattt cgtttcgatg aaggcattgc ctcttaataa aaatggaaaa 6840
gttgatcgca aacagttgca ttcggttgat ctttatgaaa cgagtatgga tacagtcatt 6900
gtgggaccaa gaaacgaaaa agaagcaatg ctttcggtta tttggcaaga gcttttggga 6960
ttagagaata tcagtgttca cgataatttc tttaagcttg gtggtcattc cattaatgcg 7020
acacaattgg tatcaaaaat ttatagtgtt tgccgagtga gaatgcctct taaaaatgtg 7080
tttcagtata caacgttagc tacaatggca cgggtgttag aagagttgtt ggtaagcgct 7140
gttgacgaag tagctgtaac aacggagcgc attccaaaga tactaccgag aacatattac 7200
gatttgtcgt attcacaaca aagaatttat ttcttatcta caatggagaa agaaaccaat 7260
tactataata ttcttggtgc ttgggatatt tatgggaaac tagatgttac gctatttgaa 7320
aaagcaatcc aactattaat gaagaaacac cattcattac gtgcaacatt tgaaatcgtg 7380
gacggcaaac ctgtgcaaat catccacgat gatatggaaa ttcctgtgca atttattgac 7440
cttactgtga tgccagaagg attacggata gaagaagtag atgaacttat gttaaaagag 7500
tctaaaagag tatacaatct cgcaaatggt ccgttaatgc attgtacaat tgttaagata 7560
aaagaaggtg agcatgtatt attgattgga caacatcata tcattagtga tggttggtca 7620
cttggtattt ttgtaaaaga gttaaatgaa atgtatgatg cctttgtgca acacaaacca 7680
gttgctgaaa caccatcaac aatctccatt atggacttta ctgcttggca caatagtaaa 7740
gtagatgaag atcaagatga tcgacaatat tggttacagc gatttgaggg agagttaccg 7800
acgttagagt tgccgacaga cagacaacgt ccacttttga aaacatatca tggtgacaca 7860
ttatcatata aggtgaattc tcaattgcat caaaaattaa aggactttag tcatgcaaat 7920
ggtgtaacga tgtttatgac gctattaacg gcgtataata ttatgttgaa taagttaaca 7980
aatgaaacag acattgttgt tggctcccct gtagcaggta gaaatgaacc agaatcaaaa 8040
gatttaatcg ggatgtttgt gaatacgtta gcgttacgtt cgcatttagg agataatccg 8100
acagttgatg tcttattaaa acaaataaaa cagaatactt tagaagcata caatcatcaa 8160
gattatccat ttgataagtt ggttgatgac ttggatccac atcgagattt aagtaggaca 8220
ccaattttcc aagtgatgat gggatatatg aatatgccat tgatggttgc atttcgtgaa 8280
gcagaggttc gcgaacgatt tgttcgacat aaagtcgcaa ggtttgattt aacacttcat 8340
gtgtttgaag atgaagatca gatgaaaata ttctttgagt ataatacaga tttatttgat 8400
gaatcaacga ttatgcgttg gcagaatcat ttcgaaacgc tattacagga aattgtatcg 8460
aatccgacaa aacgtatttc ggaattgaat atacttacaa atgaggagaa atatgaaatt 8520
ctagagatga acaataattc aacggagtat cctcagcatg aatctgttgc ggagattttt 8580
agagaaacga agataaagca tcaagcaaaa ctagcaatta cgtacaaaga tagaaagtta 8640
acgtatgcag agttgagtga aaaagcaaat gcgttggcac atacattgaa acgtcgaggt 8700
gttgcgcagc atgatgttgt tggaattgtc gcagagcgtt cgcctgaaac aattattgga 8760
atactcgcaa tcttaaaagt aggagcaatt tatttgccaa ttgatccaaa actaccgcaa 8820
ttaacactgc aacacatttg gcgagatagc ggtgcaaaag tcctcctagg gaaaaatgaa 8880
acaactgtag aagttggcaa ggaagttccg tttgtggaca tcgaagggga taaagggaag 8940
caagaggagt tagtgtgtcc aattagtcca gaagatacgg catatattat gtatacgtca 9000
ggcagtactg gaaaaccaaa aggggttatg gtgacacata gaaatattgt tcgtttagta 9060
aaaaatacga atttcgtttc tttgcaagag caagatgtgt tgttacagac aggttcgctt 9120
acttttgacg ctgcaacatt tgaaatttgg ggcgcattgc taaatggact tacgcttcat 9180
ttagtagaag attacgtaat tttagatggg gaggcgcttc aggaagagat tcagcagaac 9240
aaagcaacca ttatgtgggt gagtgcaccg ctgtttaatc aattggcgga tcaaaaccca 9300
gcgatgttta caggcattaa gcaattgctc attggtggtg atgttttatc gccaaaacat 9360
attaacaaag tgatggacca ttgtgcacca atcaatatca ttaatggata cggtccaaca 9420
gaaaatacga cgttctcgac gtcatttgta attgatcaaa tgtatcaaga cagcattccg 9480
attggaacac cgattgctaa ttctagtgct tacattttag atgtacatca aaatatacaa 9540
cctattggtg tagttggcga actatgtgtt ggtggtgatg gagttgcaaa aggttatgtg 9600
aaccttgaac aattaacaga agaacggttt atagcagatc cgttcctaaa gggttctaca 9660
atgtacagaa ccggcgatta tgtgaaatta ttgcctaatg gaaatattca atacattgga 9720
cgtgtggaca atcaagtgaa aattcgtgga ttccgcatcg aattagaagc cattatgaac 9780
acattaaaac aatgtgaatc aatcaaagat gtaattgttg ttgtacaaga acagaatggg 9840
tataaaacac tggttgcata tgttgtggga gaagaatcgc tttcaataga aacagtgagg 9900
gcctatgcaa aaaaacattt ggctgaatat atggtacctt ctcaatttat atttatagaa 9960
gaaattccgc tctcaataaa tgggaaagta cagtatagta agttaccgaa agtacaagaa 10020
gtattgcata aaaaagtaga aacgctgtta ccagaaaaca gattagaaga aattattcta 10080
cgtgtgtatc gtgatgtatt agagaaagaa gattttggcg taacagattc attcttcgct 10140
tatggtggtg actctttact aagtattcaa gtcgtttcga tgttgaaaaa agaggagatt 10200
gcagtagatc cgaaaatgat ttttatgcat acaacggtta gagagttagc aaaggcatgt 10260
gaaaatcgtc cggttatgga agaaacaaaa aggactgaga aggattattt aattcaaatg 10320
cgtgaaggta gtgaagaaga tagttgtatc atttttgctc ctccggcagg tggaacggta 10380
cttggatata tagaattagc aaggtatttc gagggaattg gcaatgttta cggcctacaa 10440
gcaccgggac tgtatgacga tgaagagcct acgttcttag attacgatga acttgtacaa 10500
gtgtttcttc gctcgattga agggacatat cgtccaggtc aagattattt aggtggccac 10560
tccttagggg gacatatcgc atttggaatg tgctgtgaac tgattaagca aggaaaggca 10620
ccaaagggat tgctaattct agatacaaca ccatcacttc aagttgtaaa gggggccaag 10680
gatgaaaaaa tagccgagga ggactttaaa atgatggtac tggctgccgg tatcggaaat 10740
atgatgggtg ttgatccaga agaattaaag caactgtcgt atgaagaagc aaaaacaaga 10800
gttgtcgcag tggcacaaaa ggatgaaaag ttaaaaactt ttataaatga aacatatttg 10860
gataagtatt tgaagttaca aattcatagt ttactaatgt cacgaacatt agaattggag 10920
aaaacacaat tagatattcc gattaaggta tttaaaacac agtttcatac agaagagcta 10980
gtagaaagat ttgatgcttg gcataactat acaaatcaag cctgcacatt cattgatata 11040
ccaggcacac atacgacgat gatgcgttta ccacatgtga aagaggtagc gaaaaaaata 11100
gaagaacagc tataa 11115
<210>7
<211>5700
<212>DNA
<213>蜡状芽孢杆菌
<400>7
cacaaaacgc catctgcaac agcgcttatt tatcgcaaca aagagatgac atacgaggat 60
gttaatgcgc aggcaaatgc acttgcacat aaattaagag atgcaggtgt tggaccaaac 120
caggtagttg gcgtgttatg tgatcgctct ttcgagatgg ttgttggtat attagctgtt 180
ttaaaagcag gtggtgcgta tttgccaatt gatacagcgt acccgatgca acgaacagaa 240
tacgtcctgc aaaatagtga ggcaactatt ctcttaacaa aggaatgtta ccttaaggag 300
tctttagatt ttgaggggga agttttttac ttagatgatg caagactgtt tgaaggggat 360
agaagagatt tacaaaatat caataatcct actaaccttg cttatatcat ttatacatca 420
ggatccacgg gaaatccaaa aggtgttatg gtagcgcatc aaagtgttgt gaatttgcta 480
ctcgatttac aagagaaata tccggtgcta gcagaagata agcacttgtt aaaaacaaca 540
tatacgtttg atgtttctgt agccgaaatt tttggatggt ttcatgcagg tggcacactt 600
gttattgctg gacatggtga tgaaaaagac ccagagaaac tgattcaatt gattcaatgc 660
cacaaggtta cacatattaa cttcgtacca tcgatgctac atgcaatgtt acaggccttg 720
gatgaaaaag attttgcaat tatgaatcgg ttgaaatata ttatcgtcgc aggagaagct 780
gtttcaccag aactttgtaa tcgactgtac gctcattgtc caaatgtaaa actagaaaat 840
ctatatgggc caacggaagg aacgatttat gcgacagggt tttctattca taaagaaatg 900
aatgtagcta atgtaccgat tggaaaacca ctttctcatg tggaaacgta tattcttgat 960
caaaacaatc aaattgtacc aattggtgta ccaggtgaat tgtgtctggg aggaatatgt 1020
gtagcaaaag gttatatgaa agagccggtg ttaacagaag aaaaattcgt cgtcaatcct 1080
atgaaacaaa gtgaaagaat gtaccgaacg ggtgatttgg tacgctggtt agcagatggg 1140
aatattgaat atttaggaag aatagataac caagtcaaga taagaggctt ccgaattgag 1200
cttggtgaaa ttgaagcggc aattgctgca ttagaagatg tagtacaaac aattgttaca 1260
acaatgacgg atcataaagg tgcgaacaag attgtcgcat atgttgtgag cgaaaagtat 1320
gatgaagaac gaattcgtga acatgtgaaa aagacgttgc cgcaatatat ggtaccaagt 1380
tatttcgttt cgatgaaggc attgcctctt aataaaaatg gaaaagttga tcgcaaacag 1440
ttgcattcgg ttgatcttta tgaaacgagt atggatacag tcattgtggg accaagaaac 1500
gaaaaagaag caatgctttc ggttatttgg caagagcttt tgggattaga gaatatcagt 1560
gttcacgata atttctttaa gcttggtggt cattccatta atgcgacaca attggtatca 1620
aaaatttata gtgtttgccg agtgagaatg cctcttaaaa atgtgtttca gtatacaacg 1680
ttagctacaa tggcacgggt gttagaagag ttgttggtaa gcgctgttga cgaagtagct 1740
gtaacaacgg agcgcattcc aaagatacta ccgagaacat attacgattt gtcgtattca 1800
caacaaagaa tttatttctt atctacaatg gagaaagaaa ccaattacta taatattctt 1860
ggtgcttggg atatttatgg gaaactagat gttacgctat ttgaaaaagc aatccaacta 1920
ttaatgaaga aacaccattc attacgtgca acatttgaaa tcgtggacgg caaacctgtg 1980
caaatcatcc acgatgatat ggaaattcct gtgcaattta ttgaccttac tgtgatgcca 2040
gaaggattac ggatagaaga agtagatgaa cttatgttaa aagagtctaa aagagtatac 2100
aatctcgcaa atggtccgtt aatgcattgt acaattgtta agataaaaga aggtgagcat 2160
gtattattga ttggacaaca tcatatcatt agtgatggtt ggtcacttgg tatttttgta 2220
aaagagttaa atgaaatgta tgatgccttt gtgcaacaca aaccagttgc tgaaacacca 2280
tcaacaatct ccattatgga ctttactgct tggcacaata gtaaagtaga tgaagatcaa 2340
gatgatcgac aatattggtt acagcgattt gagggagagt taccgacgtt agagttgccg 2400
acagacagac aacgtccact tttgaaaaca tatcatggtg acacattatc atataaggtg 2460
aattctcaat tgcatcaaaa attaaaggac tttagtcatg caaatggtgt aacgatgttt 2520
atgacgctat taacggcgta taatattatg ttgaataagt taacaaatga aacagacatt 2580
gttgttggct cccctgtagc aggtagaaat gaaccagaat caaaagattt aatcgggatg 2640
tttgtgaata cgttagcgtt acgttcgcat ttaggagata atccgacagt tgatgtctta 2700
ttaaaacaaa taaaacagaa tactttagaa gcatacaatc atcaagatta tccatttgat 2760
aagttggttg atgacttgga tccacatcga gatttaagta ggacaccaat tttccaagtg 2820
atgatgggat atatgaatat gccattgatg gttgcatttc gtgaagcaga ggttcgcgaa 2880
cgatttgttc gacataaagt cgcaaggttt gatttaacac ttcatgtgtt tgaagatgaa 2940
gatcagatga aaatattctt tgagtataat acagatttat ttgatgaatc aacgattatg 3000
cgttggcaga atcatttcga aacgctatta caggaaattg tatcgaatcc gacaaaacgt 3060
atttcggaat tgaatatact tacaaatgag gagaaatatg aaattctaga gatgaacaat 3120
aattcaacgg agtatcctca gcatgaatct gttgcggaga tttttagaga aacgaagata 3180
aagcatcaag caaaactagc aattacgtac aaagatagaa agttaacgta tgcagagttg 3240
agtgaaaaag caaatgcgtt ggcacataca ttgaaacgtc gaggtgttgc gcagcatgat 3300
gttgttggaa ttgtcgcaga gcgttcgcct gaaacaatta ttggaatact cgcaatctta 3360
aaagtaggag caatttattt gccaattgat ccaaaactac cgcaattaac actgcaacac 3420
atttggcgag atagcggtgc aaaagtcctc ctagggaaaa atgaaacaac tgtagaagtt 3480
ggcaaggaag ttccgtttgt ggacatcgaa ggggataaag ggaagcaaga ggagttagtg 3540
tgtccaatta gtccagaaga tacggcatat attatgtata cgtcaggcag tactggaaaa 3600
ccaaaagggg ttatggtgac acatagaaat attgttcgtt tagtaaaaaa tacgaatttc 3660
gtttctttgc aagagcaaga tgtgttgtta cagacaggtt cgcttacttt tgacgctgca 3720
acatttgaaa tttggggcgc attgctaaat ggacttacgc ttcatttagt agaagattac 3780
gtaattttag atggggaggc gcttcaggaa gagattcagc agaacaaagc aaccattatg 3840
tgggtgagtg caccgctgtt taatcaattg gcggatcaaa acccagcgat gtttacaggc 3900
attaagcaat tgctcattgg tggtgatgtt ttatcgccaa aacatattaa caaagtgatg 3960
gaccattgtg caccaatcaa tatcattaat ggatacggtc caacagaaaa tacgacgttc 4020
tcgacgtcat ttgtaattga tcaaatgtat caagacagca ttccgattgg aacaccgatt 4080
gctaattcta gtgcttacat tttagatgta catcaaaata tacaacctat tggtgtagtt 4140
ggcgaactat gtgttggtgg tgatggagtt gcaaaaggtt atgtgaacct tgaacaatta 4200
acagaagaac ggtttatagc agatccgttc ctaaagggtt ctacaatgta cagaaccggc 4260
gattatgtga aattattgcc taatggaaat attcaataca ttggacgtgt ggacaatcaa 4320
gtgaaaattc gtggattccg catcgaatta gaagccatta tgaacacatt aaaacaatgt 4380
gaatcaatca aagatgtaat tgttgttgta caagaacaga atgggtataa aacactggtt 4440
gcatatgttg tgggagaaga atcgctttca atagaaacag tgagggccta tgcaaaaaaa 4500
catttggctg aatatatggt accttctcaa tttatattta tagaagaaat tccgctctca 4560
ataaatggga aagtacagta tagtaagtta ccgaaagtac aagaagtatt gcataaaaaa 4620
gtagaaacgc tgttaccaga aaacagatta gaagaaatta ttctacgtgt gtatcgtgat 4680
gtattagaga aagaagattt tggcgtaaca gattcattct tcgcttatgg tggtgactct 4740
ttactaagta ttcaagtcgt ttcgatgttg aaaaaagagg agattgcagt agatccgaaa 4800
atgattttta tgcatacaac ggttagagag ttagcaaagg catgtgaaaa tcgtccggtt 4860
atggaagaaa caaaaaggac tgagaaggat tatttaattc aaatgcgtga aggtagtgaa 4920
gaagatagtt gtatcatttt tgctcctccg gcaggtggaa cggtacttgg atatatagaa 4980
ttagcaaggt atttcgaggg aattggcaat gtttacggcc tacaagcacc gggactgtat 5040
gacgatgaag agcctacgtt cttagattac gatgaacttg tacaagtgtt tcttcgctcg 5100
attgaaggga catatcgtcc aggtcaagat tatttaggtg gccactcctt agggggacat 5160
atcgcatttg gaatgtgctg tgaactgatt aagcaaggaa aggcaccaaa gggattgcta 5220
attctagata caacaccatc acttcaagtt gtaaaggggg ccaaggatga aaaaatagcc 5280
gaggaggact ttaaaatgat ggtactggct gccggtatcg gaaatatgat gggtgttgat 5340
ccagaagaat taaagcaact gtcgtatgaa gaagcaaaaa caagagttgt cgcagtggca 5400
caaaaggatg aaaagttaaa aacttttata aatgaaacat atttggataa gtatttgaag 5460
ttacaaattc atagtttact aatgtcacga acattagaat tggagaaaac acaattagat 5520
attccgatta aggtatttaa aacacagttt catacagaag agctagtaga aagatttgat 5580
gcttggcata actatacaaa tcaagcctgc acattcattg atataccagg cacacatacg 5640
acgatgatgc gtttaccaca tgtgaaagag gtagcgaaaa aaatagaaga acagctataa 5700
<210>8
<211>11115
<212>RNA
<213>蜡状芽孢杆菌
<400>8
uuuauaaaau caaugaauca guugggaaaa ucaaaaaauu uacauaaugg ggggaugaug 60
gagaugaaac gaguggaaga acaugaucac auucaugugu uaaaugaaau agaaaacgaa 120
ugcgaaagaa gauaugggag aaguaauauu gcaauuaugc uugaaaagca ugguguucau 180
gaacagccgc uucauauaga agacuuauuu caugagguag agaugcaaga acauucacga 240
guaucgcgcc acgaaacagu uuugaugaca gauaaacaau guauagauga gaguggaaaa 300
ccguuagcuc uucguuuugg ugagccacuu caucuugaug acuguacccc aaaaacacua 360
caagaaauuu uaaagcgugc cgcuaagcaa gcaaaagaua aagggaugac auuuguauau 420
gaagauggac augaagagua ccucuccuac caagagaugu uggcagaugc ggagcgguua 480
cuaaaagggu ugcgaaaucu ugguauacaa ccaggagaaa guauuuuguu ucaauuuaag 540
gacaauaagc auuuuguuac ugcguuuugg gcauguauac uugggggauu uuuaccaacg 600
ccguuaggaa cggccccuau cuauagugag caaaaugcac aaguauuaaa acuuuauaac 660
acauggcagc uauuagaaca accgauuauu uuaacggaau uugaauugaa agaagagauu 720
gcugcaauuc gaacaacauu gcaacgucaa gagauuguua uacauaguau ugagaauguu 780
auggauacag cgcgcgauac aaacugguuu ccuuguaccg aagauacuau uguuuugaau 840
uuauuaacgu cugguagcac aggaguaccc aaaugugugc agcacaaaag uaaauccauu 900
auugcgcgca caguuuccaa uuguauugac cgccagcuag augaaaaaga aguaucguua 960
aauuggaugc cgcuugauca uguuggaggc aucguaaugu gucacauucg ugauaccuau 1020
uuaaugugcc aacagguaaa cugucuuauu ucggcauuua uugaaaaucc gcuaaauugg 1080
uugcacugga uugaugcuua cucagcgaca uuuacauggg cgccaaacuu cgcuuuuuca 1140
uuaauuaacc aguaugaaga agagauuaaa ucaucuucau ggaaucuuuc uucgaugaga 1200
uacaucguaa auggugggga agcuguuauu ucaaguguug ggaugaaauu uuuacaauug 1260
uuacagcaac aucaauugcc uucgaacugu cuuauuccua cguuugggau gucugaaguu 1320
ucuucgggua uuauugaaug ucauucguuu uauacgcaaa cgacaaauac aggaauguug 1380
uauguugaua agaauucuuu agaugguaau uuacaauuca cauaugaggg gcaccaaaau 1440
gccauuguuu uuacggaagu agggagaccg augccuggua uuggcauucg uauuguugau 1500
gaggacaauc agugccuguc agaagaucgu auuggacgau uccaaauuca ugguccaacg 1560
guuaugaaug guuauuucaa aaaugaugaa gcaaaugcgg aaaguuuuac ugaagauggc 1620
ugguucgaua guggagaucu gggguuuaua cauaacggua aucuugucau uacaggaaga 1680
aaaaaggaua ugauuguugu ucauggugca aauuacuaca acuaugaaau ugaagccuug 1740
guagaacaag uaccuggggu ggaaaccacg uuuguaugug caacgagugu gaagucggcu 1800
gaaggagcag aggaauuagc uauuuucuuu gucccaguaa uuaaucaugu uucugugaug 1860
uuugcgacga ugcaacaaau caaacaaauu guugcgcgca aaauggguau cacgccgaaa 1920
gugauuauac caauucagaa ggaagcauuc uuuaaaacgg auagugggaa aauaacgaga 1980
aaugcauuuc aaaaacaguu ugagaacggg gcauauagag agauuacaca aaagauugau 2040
ugccauuuac aaaaugaaaa aacacuaucu cagugguuuu aucgugaaaa auuagucgaa 2100
aguaaguugg gcaaaagcgu auccucccaa aaagaaacgu auguuuucuu ucgacaaggu 2160
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cauaucguac caagaaauga uauagaagag aaaauugcau ucauuuggga aggucuuuug 3780
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caaacaauug uuacaacaau gacggaucau aaaggugcga acaagauugu cgcauauguu 6720
gugagcgaaa aguaugauga agaacgaauu cgugaacaug ugaaaaagac guugccgcaa 6780
uauaugguac caaguuauuu cguuucgaug aaggcauugc cucuuaauaa aaauggaaaa 6840
guugaucgca aacaguugca uucgguugau cuuuaugaaa cgaguaugga uacagucauu 6900
gugggaccaa gaaacgaaaa agaagcaaug cuuucgguua uuuggcaaga gcuuuuggga 6960
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acacaauugg uaucaaaaau uuauaguguu ugccgaguga gaaugccucu uaaaaaugug 7080
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aaagcaaucc aacuauuaau gaagaaacac cauucauuac gugcaacauu ugaaaucgug 7380
gacggcaaac cugugcaaau cauccacgau gauauggaaa uuccugugca auuuauugac 7440
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aaagaaggug agcauguauu auugauugga caacaucaua ucauuaguga ugguugguca 7620
cuugguauuu uuguaaaaga guuaaaugaa auguaugaug ccuuugugca acacaaacca 7680
guugcugaaa caccaucaac aaucuccauu auggacuuua cugcuuggca caauaguaaa 7740
guagaugaag aucaagauga ucgacaauau ugguuacagc gauuugaggg agaguuaccg 7800
acguuagagu ugccgacaga cagacaacgu ccacuuuuga aaacauauca uggugacaca 7860
uuaucauaua aggugaauuc ucaauugcau caaaaauuaa aggacuuuag ucaugcaaau 7920
gguguaacga uguuuaugac gcuauuaacg gcguauaaua uuauguugaa uaaguuaaca 7980
aaugaaacag acauuguugu uggcuccccu guagcaggua gaaaugaacc agaaucaaaa 8040
gauuuaaucg ggauguuugu gaauacguua gcguuacguu cgcauuuagg agauaauccg 8100
acaguugaug ucuuauuaaa acaaauaaaa cagaauacuu uagaagcaua caaucaucaa 8160
gauuauccau uugauaaguu gguugaugac uuggauccac aucgagauuu aaguaggaca 8220
ccaauuuucc aagugaugau gggauauaug aauaugccau ugaugguugc auuucgugaa 8280
gcagagguuc gcgaacgauu uguucgacau aaagucgcaa gguuugauuu aacacuucau 8340
guguuugaag augaagauca gaugaaaaua uucuuugagu auaauacaga uuuauuugau 8400
gaaucaacga uuaugcguug gcagaaucau uucgaaacgc uauuacagga aauuguaucg 8460
aauccgacaa aacguauuuc ggaauugaau auacuuacaa augaggagaa auaugaaauu 8520
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agagaaacga agauaaagca ucaagcaaaa cuagcaauua cguacaaaga uagaaaguua 8640
acguaugcag aguugaguga aaaagcaaau gcguuggcac auacauugaa acgucgaggu 8700
guugcgcagc augauguugu uggaauuguc gcagagcguu cgccugaaac aauuauugga 8760
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caagaggagu uagugugucc aauuagucca gaagauacgg cauauauuau guauacguca 9000
ggcaguacug gaaaaccaaa agggguuaug gugacacaua gaaauauugu ucguuuagua 9060
aaaaauacga auuucguuuc uuugcaagag caagaugugu uguuacagac agguucgcuu 9120
acuuuugacg cugcaacauu ugaaauuugg ggcgcauugc uaaauggacu uacgcuucau 9180
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gaaaauacga cguucucgac gucauuugua auugaucaaa uguaucaaga cagcauuccg 9480
auuggaacac cgauugcuaa uucuagugcu uacauuuuag auguacauca aaauauacaa 9540
ccuauuggug uaguuggcga acuauguguu gguggugaug gaguugcaaa agguuaugug 9600
aaccuugaac aauuaacaga agaacgguuu auagcagauc cguuccuaaa ggguucuaca 9660
auguacagaa ccggcgauua ugugaaauua uugccuaaug gaaauauuca auacauugga 9720
cguguggaca aucaagugaa aauucgugga uuccgcaucg aauuagaagc cauuaugaac 9780
acauuaaaac aaugugaauc aaucaaagau guaauuguug uuguacaaga acagaauggg 9840
uauaaaacac ugguugcaua uguuguggga gaagaaucgc uuucaauaga aacagugagg 9900
gccuaugcaa aaaaacauuu ggcugaauau augguaccuu cucaauuuau auuuauagaa 9960
gaaauuccgc ucucaauaaa ugggaaagua caguauagua aguuaccgaa aguacaagaa 10020
guauugcaua aaaaaguaga aacgcuguua ccagaaaaca gauuagaaga aauuauucua 10080
cguguguauc gugauguauu agagaaagaa gauuuuggcg uaacagauuc auucuucgcu 10140
uaugguggug acucuuuacu aaguauucaa gucguuucga uguugaaaaa agaggagauu 10200
gcaguagauc cgaaaaugau uuuuaugcau acaacgguua gagaguuagc aaaggcaugu 10260
gaaaaucguc cgguuaugga agaaacaaaa aggacugaga aggauuauuu aauucaaaug 10320
cgugaaggua gugaagaaga uaguuguauc auuuuugcuc cuccggcagg uggaacggua 10380
cuuggauaua uagaauuagc aagguauuuc gagggaauug gcaauguuua cggccuacaa 10440
gcaccgggac uguaugacga ugaagagccu acguucuuag auuacgauga acuuguacaa 10500
guguuucuuc gcucgauuga agggacauau cguccagguc aagauuauuu agguggccac 10560
uccuuagggg gacauaucgc auuuggaaug ugcugugaac ugauuaagca aggaaaggca 10620
ccaaagggau ugcuaauucu agauacaaca ccaucacuuc aaguuguaaa gggggccaag 10680
gaugaaaaaa uagccgagga ggacuuuaaa augaugguac uggcugccgg uaucggaaau 10740
augaugggug uugauccaga agaauuaaag caacugucgu augaagaagc aaaaacaaga 10800
guugucgcag uggcacaaaa ggaugaaaag uuaaaaacuu uuauaaauga aacauauuug 10860
gauaaguauu ugaaguuaca aauucauagu uuacuaaugu cacgaacauu agaauuggag 10920
aaaacacaau uagauauucc gauuaaggua uuuaaaacac aguuucauac agaagagcua 10980
guagaaagau uugaugcuug gcauaacuau acaaaucaag ccugcacauu cauugauaua 11040
ccaggcacac auacgacgau gaugcguuua ccacauguga aagagguagc gaaaaaaaua 11100
gaagaacagc uauaa 11115
<210>9
<211>5700
<212>RNA
<213>蜡状芽孢杆菌
<400>9
cacaaaacgc caucugcaac agcgcuuauu uaucgcaaca aagagaugac auacgaggau 60
guuaaugcgc aggcaaaugc acuugcacau aaauuaagag augcaggugu uggaccaaac 120
cagguaguug gcguguuaug ugaucgcucu uucgagaugg uuguugguau auuagcuguu 180
uuaaaagcag guggugcgua uuugccaauu gauacagcgu acccgaugca acgaacagaa 240
uacguccugc aaaauaguga ggcaacuauu cucuuaacaa aggaauguua ccuuaaggag 300
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ggauccacgg gaaauccaaa agguguuaug guagcgcauc aaaguguugu gaauuugcua 480
cucgauuuac aagagaaaua uccggugcua gcagaagaua agcacuuguu aaaaacaaca 540
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guuauugcug gacaugguga ugaaaaagac ccagagaaac ugauucaauu gauucaaugc 660
cacaagguua cacauauuaa cuucguacca ucgaugcuac augcaauguu acaggccuug 720
gaugaaaaag auuuugcaau uaugaaucgg uugaaauaua uuaucgucgc aggagaagcu 780
guuucaccag aacuuuguaa ucgacuguac gcucauuguc caaauguaaa acuagaaaau 840
cuauaugggc caacggaagg aacgauuuau gcgacagggu uuucuauuca uaaagaaaug 900
aauguagcua auguaccgau uggaaaacca cuuucucaug uggaaacgua uauucuugau 960
caaaacaauc aaauuguacc aauuggugua ccaggugaau ugugucuggg aggaauaugu 1020
guagcaaaag guuauaugaa agagccggug uuaacagaag aaaaauucgu cgucaauccu 1080
augaaacaaa gugaaagaau guaccgaacg ggugauuugg uacgcugguu agcagauggg 1140
aauauugaau auuuaggaag aauagauaac caagucaaga uaagaggcuu ccgaauugag 1200
cuuggugaaa uugaagcggc aauugcugca uuagaagaug uaguacaaac aauuguuaca 1260
acaaugacgg aucauaaagg ugcgaacaag auugucgcau auguugugag cgaaaaguau 1320
gaugaagaac gaauucguga acaugugaaa aagacguugc cgcaauauau gguaccaagu 1380
uauuucguuu cgaugaaggc auugccucuu aauaaaaaug gaaaaguuga ucgcaaacag 1440
uugcauucgg uugaucuuua ugaaacgagu auggauacag ucauuguggg accaagaaac 1500
gaaaaagaag caaugcuuuc gguuauuugg caagagcuuu ugggauuaga gaauaucagu 1560
guucacgaua auuucuuuaa gcuugguggu cauuccauua augcgacaca auugguauca 1620
aaaauuuaua guguuugccg agugagaaug ccucuuaaaa auguguuuca guauacaacg 1680
uuagcuacaa uggcacgggu guuagaagag uuguugguaa gcgcuguuga cgaaguagcu 1740
guaacaacgg agcgcauucc aaagauacua ccgagaacau auuacgauuu gucguauuca 1800
caacaaagaa uuuauuucuu aucuacaaug gagaaagaaa ccaauuacua uaauauucuu 1860
ggugcuuggg auauuuaugg gaaacuagau guuacgcuau uugaaaaagc aauccaacua 1920
uuaaugaaga aacaccauuc auuacgugca acauuugaaa ucguggacgg caaaccugug 1980
caaaucaucc acgaugauau ggaaauuccu gugcaauuua uugaccuuac ugugaugcca 2040
gaaggauuac ggauagaaga aguagaugaa cuuauguuaa aagagucuaa aagaguauac 2100
aaucucgcaa augguccguu aaugcauugu acaauuguua agauaaaaga aggugagcau 2160
guauuauuga uuggacaaca ucauaucauu agugaugguu ggucacuugg uauuuuugua 2220
aaagaguuaa augaaaugua ugaugccuuu gugcaacaca aaccaguugc ugaaacacca 2280
ucaacaaucu ccauuaugga cuuuacugcu uggcacaaua guaaaguaga ugaagaucaa 2340
gaugaucgac aauauugguu acagcgauuu gagggagagu uaccgacguu agaguugccg 2400
acagacagac aacguccacu uuugaaaaca uaucauggug acacauuauc auauaaggug 2460
aauucucaau ugcaucaaaa auuaaaggac uuuagucaug caaauggugu aacgauguuu 2520
augacgcuau uaacggcgua uaauauuaug uugaauaagu uaacaaauga aacagacauu 2580
guuguuggcu ccccuguagc agguagaaau gaaccagaau caaaagauuu aaucgggaug 2640
uuugugaaua cguuagcguu acguucgcau uuaggagaua auccgacagu ugaugucuua 2700
uuaaaacaaa uaaaacagaa uacuuuagaa gcauacaauc aucaagauua uccauuugau 2760
aaguugguug augacuugga uccacaucga gauuuaagua ggacaccaau uuuccaagug 2820
augaugggau auaugaauau gccauugaug guugcauuuc gugaagcaga gguucgcgaa 2880
cgauuuguuc gacauaaagu cgcaagguuu gauuuaacac uucauguguu ugaagaugaa 2940
gaucagauga aaauauucuu ugaguauaau acagauuuau uugaugaauc aacgauuaug 3000
cguuggcaga aucauuucga aacgcuauua caggaaauug uaucgaaucc gacaaaacgu 3060
auuucggaau ugaauauacu uacaaaugag gagaaauaug aaauucuaga gaugaacaau 3120
aauucaacgg aguauccuca gcaugaaucu guugcggaga uuuuuagaga aacgaagaua 3180
aagcaucaag caaaacuagc aauuacguac aaagauagaa aguuaacgua ugcagaguug 3240
agugaaaaag caaaugcguu ggcacauaca uugaaacguc gagguguugc gcagcaugau 3300
guuguuggaa uugucgcaga gcguucgccu gaaacaauua uuggaauacu cgcaaucuua 3360
aaaguaggag caauuuauuu gccaauugau ccaaaacuac cgcaauuaac acugcaacac 3420
auuuggcgag auagcggugc aaaaguccuc cuagggaaaa augaaacaac uguagaaguu 3480
ggcaaggaag uuccguuugu ggacaucgaa ggggauaaag ggaagcaaga ggaguuagug 3540
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ccaaaagggg uuauggugac acauagaaau auuguucguu uaguaaaaaa uacgaauuuc 3660
guuucuuugc aagagcaaga uguguuguua cagacagguu cgcuuacuuu ugacgcugca 3720
acauuugaaa uuuggggcgc auugcuaaau ggacuuacgc uucauuuagu agaagauuac 3780
guaauuuuag auggggaggc gcuucaggaa gagauucagc agaacaaagc aaccauuaug 3840
ugggugagug caccgcuguu uaaucaauug gcggaucaaa acccagcgau guuuacaggc 3900
auuaagcaau ugcucauugg uggugauguu uuaucgccaa aacauauuaa caaagugaug 3960
gaccauugug caccaaucaa uaucauuaau ggauacgguc caacagaaaa uacgacguuc 4020
ucgacgucau uuguaauuga ucaaauguau caagacagca uuccgauugg aacaccgauu 4080
gcuaauucua gugcuuacau uuuagaugua caucaaaaua uacaaccuau ugguguaguu 4140
ggcgaacuau guguuggugg ugauggaguu gcaaaagguu augugaaccu ugaacaauua 4200
acagaagaac gguuuauagc agauccguuc cuaaaggguu cuacaaugua cagaaccggc 4260
gauuauguga aauuauugcc uaauggaaau auucaauaca uuggacgugu ggacaaucaa 4320
gugaaaauuc guggauuccg caucgaauua gaagccauua ugaacacauu aaaacaaugu 4380
gaaucaauca aagauguaau uguuguugua caagaacaga auggguauaa aacacugguu 4440
gcauauguug ugggagaaga aucgcuuuca auagaaacag ugagggccua ugcaaaaaaa 4500
cauuuggcug aauauauggu accuucucaa uuuauauuua uagaagaaau uccgcucuca 4560
auaaauggga aaguacagua uaguaaguua ccgaaaguac aagaaguauu gcauaaaaaa 4620
guagaaacgc uguuaccaga aaacagauua gaagaaauua uucuacgugu guaucgugau 4680
guauuagaga aagaagauuu uggcguaaca gauucauucu ucgcuuaugg uggugacucu 4740
uuacuaagua uucaagucgu uucgauguug aaaaaagagg agauugcagu agauccgaaa 4800
augauuuuua ugcauacaac gguuagagag uuagcaaagg caugugaaaa ucguccgguu 4860
auggaagaaa caaaaaggac ugagaaggau uauuuaauuc aaaugcguga agguagugaa 4920
gaagauaguu guaucauuuu ugcuccuccg gcagguggaa cgguacuugg auauauagaa 4980
uuagcaaggu auuucgaggg aauuggcaau guuuacggcc uacaagcacc gggacuguau 5040
gacgaugaag agccuacguu cuuagauuac gaugaacuug uacaaguguu ucuucgcucg 5100
auugaaggga cauaucgucc aggucaagau uauuuaggug gccacuccuu agggggacau 5160
aucgcauuug gaaugugcug ugaacugauu aagcaaggaa aggcaccaaa gggauugcua 5220
auucuagaua caacaccauc acuucaaguu guaaaggggg ccaaggauga aaaaauagcc 5280
gaggaggacu uuaaaaugau gguacuggcu gccgguaucg gaaauaugau ggguguugau 5340
ccagaagaau uaaagcaacu gucguaugaa gaagcaaaaa caagaguugu cgcaguggca 5400
caaaaggaug aaaaguuaaa aacuuuuaua aaugaaacau auuuggauaa guauuugaag 5460
uuacaaauuc auaguuuacu aaugucacga acauuagaau uggagaaaac acaauuagau 5520
auuccgauua agguauuuaa aacacaguuu cauacagaag agcuaguaga aagauuugau 5580
gcuuggcaua acuauacaaa ucaagccugc acauucauug auauaccagg cacacauacg 5640
acgaugaugc guuuaccaca ugugaaagag guagcgaaaa aaauagaaga acagcuauaa 5700
<210>10
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>10
ggtgaattgt gtctgggagg 20
<210>11
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>11
atttttatta agaggcaatg 20
<210>12
<211>24
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>12
gtcaagataa gaggcttccg aatt 24
<210>13
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>13
aatggaatga ccaccaagct 20
<210>14
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>14
aggaagttcc gtttgtggac 20
<210>15
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>15
cacataacct tttgcaactc 20
<210>16
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>16
ggcgaactat gtgttggtgg 20
<210>17
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>17
taaagagtca ccaccataag 20
<210>18
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>18
acgtcaggca gtactggaaa 20
<210>19
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<400>19
ttcgatgcgg aatccacgaa 20
<210>20
<211>43
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<220>
<221>修饰的碱基
<222>(14)..(14)
<223>I
<220>
<221>修饰的碱基
<222>(17)..(17)
<223>I
<220>
<221>修饰的碱基
<222>(26)..(26)
<223>I
<400>20
ggaattcctt aaaagcagga ggagcatatg tgccgcttga tcc 43
<210>21
<211>41
<212>DNA
<213>人工序列
<220>
<223>人工序列描述：用于扩增催吐素合成酶基因特异的DNA片段的PCR引物
<220>
<221>修饰的碱基
<222>(12)..(12)
<223>I
<220>
<221>修饰的碱基
<222>(15)..(15)
<223>I
<220>
<221>修饰的碱基
<222>(18)..(18)
<223>I
<220>
<221>修饰的碱基
<222>(21)..(21)
<223>I
<220>
<221>修饰的碱基
<222>(27)..(27)
<223>I
<220>
<221>修饰的碱基
<222>(30)..(30)
<223>I
<220>
<221>修饰的碱基
<222>(33)..(33)
<223>I
<400>21
ggaattcctt taggattacc agttgtacca gaagtgtaaat 41
87
Claims (23)
1.一种含有序列编号1的氨基酸序列的多肽，或含有序列编号1的氨基酸序列的一部分被改变后构成的序列、而且具有催吐素合成活性的多肽。
2.一种含有序列编号3的氨基酸序列的多肽，或含有序列编号3的氨基酸序列的一部分被改变后构成的序列、而且保持上述多肽所具有的有关催吐素合成的功能构造的多肽。
3.编码权利要求1所述的任一种多肽的核酸。
4.编码权利要求2所述的任一种多肽的核酸。
5.保有权利要求3所述的核酸的载体。
6.用权利要求5所述的载体转化的转化体。
7.含有序列6的碱基序列中与催吐素合成活性直接有关的区域的至少部分的序列、或含有与上述区域的碱基序列相互补的序列的至少部分的核酸。
8.含有序列7碱基序列的至少部分、或含有与上述碱基序列互补的序列的至少部分的核酸。
9.含有序列9的碱基序列中与催吐素合成活性直接有关的区域的碱基序列互补的序列的至少部分的核酸。
10.含有与序列编号9的碱基序列互补的序列的至少部分的核酸。
11.被设计成可以对含有在编码具有催吐素合成活性的多肽的DNA中直接与催吐素合成活性有关的区域的至少部分的DNA区域进行特异扩增的一组核酸。
12.一种将权利要求7所述的核酸固定于不溶性支持体后构成的固相化核酸。
13.一种与催吐素合成酶特异结合的抗体。
14.一种抗体，其特征是对含有序列编号1的氨基酸序列的多肽具有结合性、而对由序列编号2的氨基酸序列构成的多肽没有结合性。
15.一种含有权利要求7所述核酸的催吐素检测用试剂盒。
16.一种含有权利要求11所述的一组核酸、DNA扩增用酶以及DNA合成试剂的催吐素检测用试剂盒。
17.一种含有权利要求13所述的抗体和抗原抗体反应用试剂的催吐素检测用试剂盒。
18.一种包括研究检体中的(a)或(b)存在的步骤的催吐素检测方法：
(a)含有序列编号1的氨基酸序列的多肽，或含有序列编号1的氨基酸序列的一部分被改变后构成的序列、而且具有催吐素合成活性的多肽，
(b)编码(a)中任一种多肽的核酸。
19.一种包括以下步骤的催吐素检测方法：
(i)以检体中的DNA为模板，使用权利要求11所述的一组核酸进行DNA扩增反应的步骤，
(ii)对被扩增的DNA进行检测的步骤。
20.一种包括以下步骤的催吐素检测方法：
(iii)以检体中的DNA为模板制备cDNA的步骤，
(iv)使用权利要求11所述的一组核酸进行DNA扩增反应的步骤，
(v)对被扩增的DNA进行检测的步骤。
21.一种包括以下步骤的催吐素检测方法：
(I)使检体与权利要求13所述的抗体接触的步骤，
(II)I的步骤后，对抗原抗体反应物进行检测的步骤。
22.权利要求18所述的催吐素检测方法，其中作为前处理进行以下步骤：
(A)将检体接种到蜡状芽孢杆菌的增菌培养基中进行培养的步骤。
23.权利要求18所述的催吐素检测方法，其中作为前处理进行以下步骤：
(A)将检体接种到蜡状芽孢杆菌的增菌培养基中进行培养的步骤，
(B)对增殖的蜡状芽孢杆菌进行溶菌或破碎的步骤。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002142398 | 2002-05-17 | ||
| JP142398/2002 | 2002-05-17 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1653173A true CN1653173A (zh) | 2005-08-10 |
| CN100359005C CN100359005C (zh) | 2008-01-02 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB038112663A Expired - Fee Related CN100359005C (zh) | 2002-05-17 | 2003-05-16 | 蜡状芽孢杆菌产生的呕吐毒素的合成酶、编码该酶的基因以及呕吐毒素的检测方法 |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US7264959B2 (zh) |
| EP (1) | EP1512740B1 (zh) |
| JP (1) | JP4340227B2 (zh) |
| KR (1) | KR20050008722A (zh) |
| CN (1) | CN100359005C (zh) |
| AT (1) | ATE409742T1 (zh) |
| AU (1) | AU2003234928A1 (zh) |
| DE (1) | DE60323834D1 (zh) |
| WO (1) | WO2003097821A1 (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0324675D0 (en) * | 2003-10-23 | 2003-11-26 | Univ Glasgow | Identification of emetic toxin producing bacterial strains |
| JP2006006256A (ja) * | 2004-06-29 | 2006-01-12 | Meiji Milk Prod Co Ltd | 嘔吐型Ｂａｃｉｌｌｕｓｃｅｒｅｕｓの迅速検出法 |
| JP5146978B2 (ja) * | 2006-05-24 | 2013-02-20 | 国立大学法人九州大学 | 嘔吐毒産生セレウス菌の検出方法及び当該方法のためのプライマーセット |
| EP2813513B1 (en) | 2012-02-08 | 2019-07-03 | Stella Pharma Corporation | Method for producing cereulide and derivative thereof, intermediate for production of cereulide, and cereulide derivative |
| EP3207145A4 (en) * | 2014-10-13 | 2018-04-25 | Dow AgroSciences LLC | Copi coatomer gamma subunit nucleic acid molecules that confer resistance to coleopteran and hemipteran pests |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6582908B2 (en) * | 1990-12-06 | 2003-06-24 | Affymetrix, Inc. | Oligonucleotides |
| FI104737B (fi) | 1998-04-06 | 2000-03-31 | Locus Genex Oy | Menetelmä toksiinien osoittamiseksi ja pakkaus sitä varten |
| DE19937241A1 (de) | 1999-08-06 | 2001-02-15 | Siegfried Scherer | Nachweis des Toxin-Operons nhe aus Bacillus cereus durch PCR |
| AU4463101A (en) | 2000-03-29 | 2001-10-08 | Amano Enzyme Inc | Process for producing aglycon by using diglycosidase and flavor-improved food containing the aglycon and converting agent to be used in the process |
| EP1508622A1 (en) * | 2003-08-22 | 2005-02-23 | Siegfried Prof. Dr. Scherer | Detection of the peptide synthase responsible for emetic toxin production in bacillus cereus |
-
2003
- 2003-05-16 US US10/513,786 patent/US7264959B2/en not_active Expired - Fee Related
- 2003-05-16 KR KR10-2004-7018592A patent/KR20050008722A/ko active IP Right Grant
- 2003-05-16 AU AU2003234928A patent/AU2003234928A1/en not_active Abandoned
- 2003-05-16 WO PCT/JP2003/006132 patent/WO2003097821A1/ja active IP Right Grant
- 2003-05-16 AT AT03752910T patent/ATE409742T1/de not_active IP Right Cessation
- 2003-05-16 DE DE60323834T patent/DE60323834D1/de not_active Expired - Lifetime
- 2003-05-16 JP JP2004506479A patent/JP4340227B2/ja not_active Expired - Lifetime
- 2003-05-16 EP EP03752910A patent/EP1512740B1/en not_active Expired - Lifetime
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2003097821A1 (fr) | 2003-11-27 |
| CN100359005C (zh) | 2008-01-02 |
| AU2003234928A1 (en) | 2003-12-02 |
| US7264959B2 (en) | 2007-09-04 |
| ATE409742T1 (de) | 2008-10-15 |
| DE60323834D1 (de) | 2008-11-13 |
| KR20050008722A (ko) | 2005-01-21 |
| EP1512740B1 (en) | 2008-10-01 |
| EP1512740A4 (en) | 2005-09-21 |
| JPWO2003097821A1 (ja) | 2005-09-15 |
| US20050260589A1 (en) | 2005-11-24 |
| JP4340227B2 (ja) | 2009-10-07 |
| EP1512740A1 (en) | 2005-03-09 |
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