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

专利名称：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
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。
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。
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。
编码[1]所述的任一种多肽的核酸。
编码[2]所述的任一种多肽的核酸。
保有[3]或[4]所述核酸的载体。
用[5]所述载体转化的转化体。
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。
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。
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。
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。
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。
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。
一种与催吐素合成酶特异结合的抗体。
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。
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。
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。
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。
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。
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。
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。
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。
[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。
[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/mlPerkin 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，MichioAGATA，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>1Phe Ile Lys Ser Met Asn Gln Leu Gly Lys Ser Lys Asn Leu His Asn1 5 10 15Gly Gly Met Met Glu Met Lys Arg Val Glu Glu His Asp His Ile His20 25 30Val Leu Asn Glu Ile Glu Asn Glu Cys Glu Arg Arg Tyr Gly Arg Ser35 40 45Asn Ile Ala Ile Met Leu Glu Lys His Gly Val His Glu Gln Pro Leu50 55 60His Ile Glu Asp Leu Phe His Glu Val Glu Met Gln Glu His Ser Arg65 70 75 80
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Phe Phe Val Pro Val Ile Asn His Val Ser Val Met Phe Ala Thr Met610 615 620Gln Gln Ile Lys Gln Ile Val Ala Arg Lys Met Gly Ile Thr Pro Lys625 630 635 640Val Ile Ile Pro Ile Gln Lys Glu Ala Phe Phe Lys Thr Asp Ser Gly645 650 655Lys Ile Thr Arg Asn Ala Phe Gln Lys Gln Phe Glu Asn Gly Ala Tyr660 665 670Arg Glu Ile Thr Gln Lys Ile Asp Cys His Leu Gln Asn Glu Lys Thr675 680 685Leu Ser Gln Trp Phe Tyr Arg Glu Lys Leu Val Glu Ser Lys Leu Gly690 695 700Lys Ser Val Ser Ser Gln Lys Glu Thr Tyr Val Phe Phe Arg Gln Gly705 710 715 720Lys Ser Phe His His Val Leu Lys Glu Lys Leu Thr Gln His Ser Val725 730 735Val Ile Val Asp Val Gly Glu Thr Phe Gly Glu Ile His Pro Asn His740 745 750Tyr Gln Ile Asn Pro Lys Asn Lys Met Asp Tyr Val Arg Leu Phe Glu755 760 765Glu Leu Ala Lys Arg Asn Val Glu Asp Gln Val Phe His Leu Leu His770 775 780
Ala Trp Asn Tyr Cys Asp Thr Val Pro Thr Phe Arg Ser Val Glu Asp785 790 795 800Leu Ala Asn Ala Gln Tyr Leu Gly Val Phe Ser Val Met Phe Ala Leu805 810 815Gln Ala Ile Met His Ala Lys Leu Pro Leu Arg Arg Val Thr Val Ile820 825 830Ala Thr Asn Ser Val Gly Leu Glu Ala Lys Glu Met Asn Tyr Ser Cys835 840 845Ser Thr Leu G1u G1y Tyr Val Lys Thr Leu Pro Ala Glu Phe Glu Asn850 855 860Leu Gln Val Lys Tyr Ile Asp I1e G1u G1y Lys Asp I1e G1n Phe Asp865 870 875 880Thr Glu Thr Val Trp Lys Glu Leu Gln G1n Gln G1u Thr I1e Pro Val885 890 895Val Leu Tyr Arg Asp Glu Lys Arg Tyr Lys Ile Gly Leu G1u Lys Val900 905 910Pro Met Leu Glu Gln Lys Glu Lys Asn Ile Pro Phe Gln Gln Gln Gly915 920 925Phe Tyr Ile Ile Thr Gly Gly Leu Gly Gly Leu Gly Thr Leu Val Ala930 935 940Lys Leu Leu Leu Glu Arg Tyr Ser Ala Asn Val Leu Leu Leu Gly Arg945 950 955 960
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Ala Thr Tyr Asn Gly Asn Gln Cys Ile His Glu Leu Phe Glu Ala179017951800Ala Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn180518101815Lys Glu Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu182018251830Ala His Lys Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val183518401845Gly Val Leu Cys Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu185018551860Ala Val Leu Lys Ala Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala186518701875Tyr Pro Met Gln Arg Thr Glu Tyr Val Leu Gln Asn Ser Glu Ala188018851890Thr Ile Leu Leu Thr Lys Glu Cys Tyr Leu Lys Glu Ser Leu Asp189519001905Phe Glu Gly Glu Val Phe Tyr Leu Asp Asp Ala Arg Leu Phe Glu191019151920Gly Asp Arg Arg Asp Leu Gln Asn Ile Asn Asn Pro Thr Asn Leu192519301935Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr Gly Asn Pro Lys Gly194019451950
Val Met Val Ala His Gln Ser Val Val Asn Leu Leu Leu Asp Leu195519601965Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His Leu Leu Lys197019751980Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe Gly Trp198519901995Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp Glu200020052010Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val201520202025Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln203020352040Ala Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr204520502055Ile Ile Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg206020652070Leu Tyr Ala His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly207520802085Pro Thr Glu Gly Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys209020952100Glu Met Asn Val Ala Asn Val Pro Ile Gly Lys Pro Leu Ser His210521102115
Val Glu Thr Tyr Ile Leu Asp Gln Asn Asn Gln Ile Val Pro Ile212021252130Gly Val Pro Gly Glu Leu Cys Leu Gly Gly Ile Cys Val Ala Lys213521402145Gly Tyr Met Lys Glu Pro Val Leu Thr Glu Glu Lys Phe Val Val215021552160Asn Pro Met Lys Gln Ser Glu Arg Met Tyr Arg Thr Gly Asp Leu216521702175Val Arg Trp Leu Ala Asp Gly Asn Ile Glu Tyr Leu Gly Arg Ile218021852190Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Gly Glu219522002205Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val Gln Thr Ile221022152220Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile Val Ala222522302235Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu His224022452250Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val225522602265Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg227022752280
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Lys Thr Tyr His Gly Asp Thr Leu Ser Tyr Lys Val Asn Ser Gln261526202625Leu His Gln Lys Leu Lys Asp Phe Ser His Ala Asn Gly Val Thr263026352640Met Phe Met Thr Leu Leu Thr Ala Tyr Asn Ile Met Leu Asn Lys264526502655Leu Thr Asn Glu Thr Asp Ile Val Val Gly Ser Pro Val Ala Gly266026652670Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly Met Phe Val Asn267526802685Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro Thr Val Asp269026952700Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala Tyr Asn270527102715His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp Pro272027252730His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly273527402745Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val275027552760Arg Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr276527702775
Leu His Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu278027852790Tyr Asn Thr Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln279528002805Asn His Phe Glu Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr281028152820Lys Arg Ile Ser Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr282528302835Glu Ile Leu Glu Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His284028452850Glu Ser Val Ala Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln285528602865Ala Lys Leu Ala Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala287028752880Glu Leu Ser Glu Lys Ala Asn Ala Leu Ala His Thr Leu Lys Arg288528902895Arg Gly Val Ala Gln His Asp Val Val Gly Ile Val Ala Glu Arg290029052910Ser Pro Glu Thr Ile Ile Gly Ile Leu Ala Ile Leu Lys Val Gly291529202925Ala Ile Tyr Leu Pro Ile Asp Pro Lys Leu Pro Gln Leu Thr Leu293029352940
Gln His Ile Trp Arg Asp Ser Gly Ala Lys Val Leu Leu Gly Lys294529502955Asn Glu Thr Thr Val Glu Val Gly Lys Glu Val Pro Phe Val Asp296029652970Ile Glu Gly Asp Lys Gly Lys Gln Glu Glu Leu Val Cys Pro Ile297529802985Ser Pro Glu Asp Thr Ala Tyr Ile Met Tyr Thr Ser Gly Ser Thr299029953000Gly Lys Pro Lys Gly Val Met Val Thr His Arg Asn Ile Val Arg300530103015Leu Val Lys Asn Thr Asn Phe Val Ser Leu Gln Glu Gln Asp Val302030253030Leu Leu Gln Thr Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu303530403045Ile Trp Gly Ala Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu305030553060Asp Tyr Val Ile Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln306530703075Gln Asn Lys Ala Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn308030853090Gln Leu Ala Asp Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln309531003105
Leu Leu Ile Gly Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys311031153120Val Met Asp His Cys Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly312531303135Pro Thr Glu Asn Thr Thr Phe Ser Thr Ser Phe Val Ile Asp Gln314031453150Met Tyr Gln Asp Ser Ile Pro Ile Gly Thr Pro Ile Ala Asn Ser315531603165Ser Ala Tyr Ile Leu Asp Val His Gln Asn Ile Gln Pro Ile Gly317031753180Val Val Gly Glu Leu Cys Val Gly Gly Asp Gly Val Ala Lys Gly318531903195Tyr Val Asn Leu Glu Gln Leu Thr Glu Glu Arg Phe Ile Ala Asp320032053210Pro Phe Leu Lys Gly Ser Thr Met Tyr Arg Thr Gly Asp Tyr Val321532203225Lys Leu Leu Pro Asn Gly Asn Ile Gln Tyr Ile Gly Arg Val Asp323032353240Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Glu Ala Ile324532503255Met Asn Thr Leu Lys Gln Cys Glu Ser Ile Lys Asp Val Ile Val326032653270
Val Val Gln Glu Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val327532803285Val Gly Glu Glu Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala329032953300Lys Lys His Leu Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe330533103315Ile Glu Glu Ile Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser332033253330Lys Leu Pro Lys Val Gln Glu Val Leu His Lys Lys Val Glu Thr333533403345Leu Leu Pro Glu Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr335033553360Arg Asp Val Leu Glu Lys Glu Asp Phe Gly Val Thr Asp Ser Phe336533703375Phe Ala Tyr Gly Gly Asp Ser Leu Leu Ser Ile Gln Val Val Ser338033853390Met Leu Lys Lys Glu Glu Ile Ala Val Asp Pro Lys Met Ile Phe339534003405Met His Thr Thr Val Arg Glu Leu Ala Lys Ala Cys Glu Asn Arg341034153420Pro Val Met Glu Glu Thr Lys Arg Thr Glu Lys Asp Tyr Leu Ile342534303435
Gln Met Arg Glu Gly Ser Glu Glu Asp Ser Cys Ile Ile Phe Ala344034453450Pro Pro Ala Gly Gly Thr Val Leu Gly Tyr Ile Glu Leu Ala Arg345534603465Tyr Phe Glu Gly Ile Gly Asn Val Tyr Gly Leu Gln Ala Pro Gly347034753480Leu Tyr Asp Asp Glu Glu Pro Thr Phe Leu Asp Tyr Asp Glu Leu348534903495Val Gln Val Phe Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro Gly350035053510Gln Asp Tyr Leu Gly Gly His Ser Leu Gly Gly His Ile Ala Phe351535203525Gly Met Cys Cys Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly353035353540Leu Leu Ile Leu Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly354535503555Ala Lys Asp Glu Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val356035653570Leu Ala Ala Gly Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu357535803585Leu Lys Gln Leu Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala359035953600
Val Ala Gln Lys Asp Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr360536103615Tyr Leu Asp Lys Tyr Leu Lys Leu Gln Ile His Ser Leu Leu Met362036253630Ser Arg Thr Leu Glu Leu Glu Lys Thr Gln Leu Asp Ile Pro Ile363536403645Lys Val Phe Lys Thr Gln Phe His Thr Glu Glu Leu Val Glu Arg365036553660Phe Asp Ala Trp His Asn Tyr Thr Asn Gln Ala Cys Thr Phe Ile366536703675Asp Ile Pro Gly Thr His Thr Thr Met Met Arg Leu Pro His Val368036853690Lys Glu Val Ala Lys Lys Ile Glu Glu Gln Leu36953700<210>2<211>1804<212>PRT<213>蜡状芽孢杆菌<400>2Phe Ile Lys Ser Met Asn Gln Leu Gly Lys Ser Lys Asn Leu His Asn1 5 10 15Gly Gly Met Met Glu Met Lys Arg Val Glu Glu His Asp His Ile His20 25 30Val Leu Asn Glu Ile Glu Asn Glu Cys Glu Arg Arg Tyr Gly Arg Ser
35 40 45Asn Ile Ala Ile Met Leu Glu Lys His Gly Val His Glu Gln Pro Leu50 55 60His Ile Glu Asp Leu Phe His Glu Val Glu Met Gln Glu His Ser Arg65 70 75 80Val Ser Arg His Glu Thr Val Leu Met Thr Asp Lys Gln Cys Ile Asp85 90 95Glu Ser Gly Lys Pro Leu Ala Leu Arg Phe Gly Glu Pro Leu His Leu100 105 110Asp Asp Cys Thr Pro Lys Thr Leu Gln Glu Ile Leu Lys Arg Ala Ala115 120 125Lys Gln Ala Lys Asp Lys Gly Met Thr Phe Val Tyr Glu Asp Gly His130 135 140Glu Glu Tyr Leu Ser Tyr Gln Glu Met Leu Ala Asp Ala Glu Arg Leu145 150 155 160Leu Lys Gly Leu Arg Asn Leu Gly Ile Gln Pro Gly Glu Ser Ile Leu165 170 175Phe Gln Phe Lys Asp Asn Lys His Phe Val Thr Ala Phe Trp Ala Cys180 185 190Ile Leu Gly Gly Phe Leu Pro Thr Pro Leu Gly Thr Ala Pro Ile Tyr195 200 205Ser Glu Gln Asn Ala Gln Val Leu Lys Leu Tyr Asn Thr Trp Gln Leu
210 215 220Leu Glu Gln Pro Ile Ile Leu Thr Glu Phe Glu Leu Lys Glu Glu Ile225 230 235 240Ala Ala Ile Arg Thr Thr Leu Gln Arg Gln Glu Ile Val Ile His Ser245 250 255Ile Glu Asn Val Met Asp Thr Ala Arg Asp Thr Asn Trp Phe Pro Cys260 265 270Thr Glu Asp Thr Ile Val Leu Asn Leu Leu Thr Ser Gly Ser Thr Gly275 280 285Val Pro Lys Cys Val Gln His Lys Ser Lys Ser Ile Ile Ala Arg Thr290 295 300Val Ser Asn Cys Ile Asp Arg Gln Leu Asp Glu Lys Glu Val Ser Leu305 310 315 320Asn Trp Met Pro Leu Asp His Val Gly Gly Ile Val Met Cys His Ile325 330 335Arg Asp Thr Tyr Leu Met Cys Gln Gln Val Asn Cys Leu Ile Ser Ala340 345 350Phe Ile Glu Asn Pro Leu Asn Trp Leu His Trp Ile Asp Ala Tyr Ser355 360 365Ala Thr Phe Thr Trp Ala Pro Asn Phe Ala Phe Ser Leu Ile Asn Gln370 375 380Tyr Glu Glu Glu Ile Lys Ser Ser Ser Trp Asn Leu Ser Ser Met Arg
385 390 395 400Tyr Ile Val Asn Gly Gly Glu Ala Val Ile Ser Ser Val Gly Met Lys405 410 415Phe Leu Gln Leu Leu Gln Gln His Gln Leu Pro Ser Asn Cys Leu Ile420 425 430Pro Thr Phe Gly Met Ser Glu Val Ser Ser Gly Ile Ile Glu Cys His435 440 445Ser Phe Tyr Thr Gln Thr Thr Asn Thr Gly Met Leu Tyr Val Asp Lys450 455 460Asn Ser Leu Asp Gly Asn Leu Gln Phe Thr Tyr Glu Gly His Gln Asn465 470 475 480Ala Ile Val Phe Thr Glu Val Gly Arg Pro Met Pro Gly Ile Gly Ile485 490 495Arg Ile Val Asp Glu Asp Asn Gln Cys Leu Ser Glu Asp Arg Ile Gly500 505 510Arg Phe Gln Ile His Gly Pro Thr Val Met Asn Gly Tyr Phe Lys Asn515 520 525Asp Glu Ala Asn Ala Glu Ser Phe Thr Glu Asp Gly Trp Phe Asp Ser530 535 540Gly Asp Leu Gly Phe Ile His Asn Gly Asn Leu Val Ile Thr Gly Arg545 550 555 560Lys Lys Asp Met Ile Val Val His Gly Ala Asn Tyr Tyr Asn Tyr Glu
565 570 575Ile Glu Ala Leu Val Glu Gln Val Pro Gly Val Glu Thr Thr Phe Val580 585 590Cys Ala Thr Ser Val Lys Ser Ala Glu Gly Ala Glu Glu Leu Ala Ile595 600 605Phe Phe Val Pro Val Ile Asn His Val Ser Val Met Phe Ala Thr Met610 615 620Gln Gln Ile Lys Gln Ile Val Ala Arg Lys Met Gly Ile Thr Pro Lys625 630 635 640Val Ile Ile Pro Ile Gln Lys Glu Ala Phe Phe Lys Thr Asp Ser Gly645 650 655Lys Ile Thr Arg Asn Ala Phe Gln Lys Gln Phe Glu Asn Gly Ala Tyr660 665 670Arg Glu Ile Thr Gln Lys Ile Asp Cys His Leu Gln Asn Glu Lys Thr675 680 685Leu Ser Gln Trp Phe Tyr Arg Glu Lys Leu Val Glu Ser Lys Leu Gly690 695 700Lys Ser Val Ser Ser Gln Lys Glu Thr Tyr Val Phe Phe Arg Gln Gly705 710 715 720Lys Ser Phe His His Val Leu Lys Glu Lys Leu Thr Gln His Ser Val725 730 735Val Ile Val Asp Val Gly Glu Thr Phe Gly Glu Ile His Pro Asn His
740 745 750Tyr Gln Ile Asn Pro Lys Asn Lys Met Asp Tyr Val Arg Leu Phe Glu755 760 765Glu Leu Ala Lys Arg Asn Val Glu Asp Gln Val Phe His Leu Leu His770 775 780Ala Trp Asn Tyr Cys Asp Thr Val Pro Thr Phe Arg Ser Val Glu Asp785 790 795 800Leu Ala Asn Ala Gln Tyr Leu Gly Val Phe Ser Val Met Phe Ala Leu805 810 815Gln Ala Ile Met His Ala Lys Leu Pro Leu Arg Arg Val Thr Val Ile820 825 830Ala Thr Asn Ser Val Gly Leu Glu Ala Lys Glu Met Asn Tyr Ser Cys835 840 845Ser Thr Leu Glu Gly Tyr Val Lys Thr Leu Pro Ala Glu Phe Glu Asn850 855 860Leu Gln Val Lys Tyr Ile Asp Ile Glu Gly Lys Asp Ile Gln Phe Asp865 870 875 880Thr Glu Thr Val Trp Lys Glu Leu Gln Gln Gln Glu Thr Ile Pro Val885 890 895Val Leu Tyr Arg Asp Glu Lys Arg Tyr Lys Ile Gly Leu Glu Lys Val900 905 910Pro Met Leu Glu Gln Lys Glu Lys Asn Ile Pro Phe Gln Gln Gln Gly
915 920 925Phe Tyr Ile Ile Thr Gly Gly Leu Gly Gly Leu Gly Thr Leu Val Ala930 935 940Lys Leu Leu Leu Glu Arg Tyr Ser Ala Asn Val Leu Leu Leu Gly Arg945 950 955 960Thr Glu Ile Glu Thr Asn Ala Glu Lys Met Arg Leu Leu Asp Ser Leu965 970 975Lys Glu Tyr Glu Gln Tyr Gly Gly Thr Val Gln Tyr Lys Met Cys Asn980 985 990Val Met Asp Leu Asp Ala Met Arg Lys Val Val His Ser Gln Glu Glu995 10001005Arg Leu Gln Gln Lys Val Asn Gly Ile Ile His Leu Ala Gly Ile101010151020Ile Gln Glu Ile Leu Ile Glu Lys Gln Thr Glu Lys Glu Leu His102510301035Ala Met Phe Glu Ala Lys Val Tyr Ala Ser Trp Val Leu His Glu104010451050Ile Val Lys Glu Arg Gln Asp Cys Leu Tyr Ile Thr Thr Ser Ser105510601065Ala Arg Thr Leu Leu Pro Gly Met Thr Ile Ser Ala Tyr Cys Ser107010751080Ala Asn Arg Phe Val Glu Asn Phe Ala Tyr Tyr Gln Arg Ser Gln
108510901095Asn Val Asn Ser Tyr Cys Phe Ser Trp Ser Phe Trp Asn Glu Ile110011051110Gly Met Gly Thr Asn Leu Leu Ile Lys Asn Ala Leu Ile Ala Lys111511201125Gly Phe Gln Leu Ile Asp Asp Gln Lys Gly Ile Tyr Ser Leu Leu113011351140Ala Gly Leu Lys Gly Asn Glu Pro Asn Val Phe Val Gly Ile Asn114511501155His Glu Lys Glu Glu Met Ala His Leu Ile Gly Thr Glu Glu Gln116011651170Glu Thr Gln Gln Leu Thr Ile Tyr Ile Thr Pro Glu Tyr Leu His117511801185Ile Leu Glu Glu Val Phe Ser Ile Leu Asn Arg Glu Glu Phe Gly119011951200Gly Leu Glu Lys Glu Ile Val Ile Leu Pro Lys Leu Pro Leu Asp120512101215Glu Tyr Gly Lys Val Asp Gln Thr Arg Leu Ala His Ala Ser Asp122012251230Ser Arg Phe Gly Lys Lys Gln His Ile Val Pro Arg Asn Asp Ile123512401245Glu Glu Lys Ile Ala Phe Ile Trp Glu Gly Leu Leu Asn Lys Lys
125012551260Asp Ile Ser Val Leu Asp His Phe Phe Glu Leu Gly Gly Asp Ser126512701275Leu Lys Ala Thr Gln Met Ile Ser Ala Leu Lys Lys Asn Phe Ala128012851290Val Thr Ile Thr Gln Gln Glu Phe Phe Gln Ser Ser Thr Val Glu129513001305Glu Leu Ala Ser Leu Val Glu Lys Lys Leu Ser Arg Thr Arg Thr131013151320His Glu Met Asp Ile Val Thr Phe Ser Asp Arg Gly Asn Val Val132513301335Glu Met Ser Ser Ala Gln Lys Arg Gln Trp Phe Leu Tyr Glu Met134013451350Asp Arg Glu Asn Pro Tyr Tyr Asn Asn Thr Leu Val Ile Arg Leu135513601365Thr Gly Glu Ile His Leu Pro Ile Leu Arg Ser Ser Ile Ile Glu137013751380Leu Val Asn Lys His Glu Thr Leu Arg Thr Thr Phe Val Met Val138513901395Asp Gly Ile Pro Ser Gln Ile Ile Ala Asp Glu Glu Leu Val Glu140014051410Ile Glu Glu Ile Asp Leu Lys His Leu Ser Ala Glu Glu Thr Leu
141514201425Gln Lys Leu Glu Gly Leu Arg Gln Arg Glu Ala Asn Thr Ala Phe143014351440Lys Ile Glu Asn Ser Ala Phe Arg Ala Lys Val Ile Leu Ile Asp144514501455Glu Lys Arg Val Glu Ile Leu Leu Ser Val His His Ile Val Ser146014651470Asp Gly Trp Ser Met Gly Ile Leu Val Lys Asp Ile Ala Glu Ile147514801485Tyr Glu Asp Ile Arg Gln Trp Gly Glu Ser Lys Gln Glu Pro Leu149014951500Pro Ile Gln Tyr Ala Asp Tyr Thr Leu Trp Gln Asn Glu Phe Met150515101515Lys Gly Glu Glu Phe Ser Lys Gln Leu Ser Tyr Trp Lys Glu Lys152015251530Leu Ala Glu Asp Ile Pro Val Leu Asp Leu Pro Leu Asp Lys Pro153515401545Arg Pro Pro Ile Gln Thr Tyr Arg Gly Lys Val Lys Thr Phe Thr155015551560Leu His Glu Asn Met Thr Arg Met Leu Lys Glu Ile Cys Gln Glu156515701575Glu Glu Cys Thr Leu Phe Met Leu Leu Leu Ser Ala Phe Ser Ser
158015851590Leu Leu His Arg Tyr Thr Gly Gln Glu Asp Leu Val Val Gly Ser159516001605Leu Val Ala Asn Arg Asn Arg Glu Gln Ile Glu Lys Leu Ile Gly161016151620Phe Phe Val Asn Thr Leu Pro Leu Arg Ile Asn Leu His Arg Glu162516301635Met Gln Phe Thr Glu Leu Leu Ser Gln Val Lys Lys Thr Thr Ile164016451650Asp Ala Tyr Asp His Gln Asp Val Pro Phe Glu Leu Leu Val Asp165516601665Glu Leu Gln Ile Glu Arg Asp Ser Ser Arg Asn Ala Leu Phe Gln167016751680Val Leu Phe Val Leu Gln Asn Ala Gln Leu Gln Ala Val Asp Leu168516901695Glu Lys Ala Thr Met Glu Leu Glu Ile Leu Asp Ser Asp Thr Ala170017051710Lys Phe Asp Met Ser Val Gln Ile Phe Glu Leu Glu Asp Thr Leu171517201725Ser Ile Lys Leu Glu Tyr Asn Thr Asp Leu Phe Phe Asp Asp Thr173017351740Ile Glu Arg Phe Leu Ala His Tyr Glu Thr Ile Leu Ala Ser Val
174517501755Ile His Asn Gln Lys Ala Lys Ile Gly Glu Leu Ser Ile Leu Pro176017651770Gln Ser Glu Tyr Thr Lys Leu Val Ser Glu Trp Asn Glu Lys Ser177517801785Ala Thr Tyr Asn Gly Asn Gln Cys Ile His Glu Leu Phe Glu Ala179017951800Ala<210>3<211>1900<212>PRT<213>蜡状芽孢杆菌<400>3Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn Lys Glu1 5 10 15Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu Ala His Lys20 25 30Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val Gly Val Leu Cys35 40 45Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu Ala Val Leu Lys Ala50 55 60Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala Tyr Pro Met Gln Arg Thr65 70 75 80
Glu Tyr Val Leu Gln Asn Ser Glu Ala Thr Ile Leu Leu Thr Lys Glu85 90 95Cys Tyr Leu Lys Glu Ser Leu Asp Phe Glu Gly Glu Val Phe Tyr Leu100 105 110Asp Asp Ala Arg Leu Phe Glu Gly Asp Arg Arg Asp Leu Gln Asn Ile115 120 125Asn Asn Pro Thr Asn Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr130 135 140Gly Asn Pro Lys Gly Val Met Val Ala His Gln Ser Val Val Asn Leu145 150 155 160Leu Leu Asp Leu Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His165 170 175Leu Leu Lys Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe180 185 190Gly Trp Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp195 200 205Glu Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val210 215 220Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln Ala225 230 235 240Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr Ile Ile245 250 255
Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg Leu Tyr Ala260 265 270His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly Pro Thr Glu Gly275 280 285Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys Glu Met Asn Val Ala290 295 300Asn Val Pro Ile Gly Lys Pro Leu Ser His Val Glu Thr Tyr Ile Leu305 310 315 320Asp Gln Asn Asn Gln Ile Val Pro Ile Gly Val Pro Gly Glu Leu Cys325 330 335Leu Gly Gly Ile Cys Val Ala Lys Gly Tyr Met Lys Glu Pro Val Leu340 345 350Thr Glu Glu Lys Phe Val Val Asn Pro Met Lys Gln Ser Glu Arg Met355 360 365Tyr Arg Thr Gly Asp Leu Val Arg Trp Leu Ala Asp Gly Asn Ile Glu370 375 380Tyr Leu Gly Arg Ile Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile385 390 395 400Glu Leu Gly Glu Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val405 410 415Gln Thr Ile Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile420 425 430
Val Ala Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu435 440 445His Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val450 455 460Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg Lys465 470 475 480Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr Val Ile485 490 495Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val Ile Trp Gln500 505 510Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp Asn Phe Phe Lys515 520 525Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu Val Ser Lys Ile Tyr530 535 540Ser Val Cys Arg Val Arg Met Pro Leu Lys Asn Val Phe Gln Tyr Thr545 550 555 560Thr Leu Ala Thr Met Ala Arg Val Leu Glu Glu Leu Leu Val Ser Ala565 570 575Val Asp Glu Val Ala Val Thr Thr Glu Arg Ile Pro Lys Ile Leu Pro580 585 590Arg Thr Tyr Tyr Asp Leu Ser Tyr Ser Gln Gln Arg Ile Tyr Phe Leu595 600 605
Ser Thr Met Glu Lys Glu Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp610 615 620Asp Ile Tyr Gly Lys Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln625 630 635 640Leu Leu Met Lys Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val645 650 655Asp Gly Lys Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val660 665 670Gln Phe Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu675 680 685Val Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala690 695 700Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly Glu705 710 715 720His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly Trp Ser725 730 735Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp Ala Phe Val740 745 750Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile Ser Ile Met Asp755 760 765Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu Asp Gln Asp Asp Arg770 775 780
Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu Leu Pro Thr Leu Glu Leu785 790 795 800Pro Thr Asp Arg Gln Arg Pro Leu Leu Lys Thr Tyr His Gly Asp Thr805 810 815Leu Ser Tyr Lys Val Asn Ser Gln Leu His Gln Lys Leu Lys Asp Phe820 825 830Ser His Ala Asn Gly Val Thr Met Phe Met Thr Leu Leu Thr Ala Tyr835 840 845Asn Ile Met Leu Asn Lys Leu Thr Asn Glu Thr Asp Ile Val Val Gly850 855 860Ser Pro Val Ala Gly Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly865 870 875 880Met Phe Val Asn Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro885 890 895Thr Val Asp Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala900 905 910Tyr Asn His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp915 920 925Pro His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly930 935 940Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val Arg945 950 955 960
Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr Leu His965 970 975Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu Tyr Asn Thr980 985 990Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln Asn His Phe Glu995 10001005Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr Lys Arg Ile Ser101010151020Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr Glu Ile Leu Glu102510301035Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His Glu Ser Val Ala104010451050Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln Ala Lys Leu Ala105510601065Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala Glu Leu Ser Glu107010751080Lys Ala Asn Ala Leu Ala His Thr Leu Lys Arg Arg Gly Val Ala108510901095Gln His Asp Val Val Gly Ile Val Ala Glu Arg Ser Pro Glu Thr110011051110Ile Ile Gly Ile Leu Ala Ile Leu Lys Val Gly Ala Ile Tyr Leu111511201125
Pro Ile Asp Pro Lys Leu Pro Gln Leu Thr Leu Gln His Ile Trp113011351140Arg Asp Ser Gly Ala Lys Val Leu Leu Gly Lys Asn Glu Thr Thr114511501155Val Glu Val Gly Lys Glu Val Pro Phe Val Asp Ile Glu Gly Asp116011651170Lys Gly Lys Gln Glu Glu Leu Val Cys Pro Ile Ser Pro Glu Asp117511801185Thr Ala Tyr Ile Met Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys119011951200Gly Val Met Val Thr His Arg Asn Ile Val Arg Leu Val Lys Asn120512101215Thr Asn Phe Val Ser Leu Gln Glu Gln Asp Val Leu Leu Gln Thr122012251230Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu Ile Trp Gly Ala123512401245Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu Asp Tyr Val Ile125012551260Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln Gln Asn Lys Ala126512701275Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn Gln Leu Ala Asp128012851290
Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln Leu Leu Ile Gly129513001305Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys Val Met Asp His131013151320Cys Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly Pro Thr Glu Asn132513301335Thr Thr Phe Ser Thr Ser Phe Val Ile Asp Gln Met Tyr Gln Asp134013451350Ser Ile Pro Ile Gly Thr Pro Ile Ala Asn Ser Ser Ala Tyr Ile135513601365Leu Asp Val His Gln Asn Ile Gln Pro Ile Gly Val Val Gly Glu137013751380Leu Cys Val Gly Gly Asp Gly Val Ala Lys Gly Tyr Val Asn Leu138513901395Glu Gln Leu Thr Glu Glu Arg Phe Ile Ala Asp Pro Phe Leu Lys140014051410Gly Ser Thr Met Tyr Arg Thr Gly Asp Tyr Val Lys Leu Leu Pro141514201425Asn Gly Asn Ile Gln Tyr Ile Gly Arg Val Asp Asn Gln Val Lys143014351440Ile Arg Gly Phe Arg Ile Glu Leu Glu Ala Ile Met Asn Thr Leu144514501455
Lys Gln Cys Glu Ser Ile Lys Asp Val Ile Val Val Val Gln Glu146014651470Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val Val Gly Glu Glu147514801485Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala Lys Lys His Leu149014951500Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe Ile Glu Glu Ile150515101515Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser Lys Leu Pro Lys152015251530Val Gln Glu Val Leu His Lys Lys Val Glu Thr Leu Leu Pro Glu153515401545Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr Arg Asp Val Leu155015551560Glu Lys Glu Asp Phe Gly Val Thr Asp Ser Phe Phe Ala Tyr Gly156515701575Gly Asp Ser Leu Leu Ser Ile Gln Val Val Ser Met Leu Lys Lys158015851590Glu Glu Ile Ala Val Asp Pro Lys Met Ile Phe Met His Thr Thr159516001605Val Arg Glu Leu Ala Lys Ala Cys Glu Asn Arg Pro Val Met Glu161016151620
Glu Thr Lys Arg Thr Glu Lys Asp Tyr Leu Ile Gln Met Arg Glu162516301635Gly Ser Glu Glu Asp Ser Cys Ile Ile Phe Ala Pro Pro Ala Gly164016451650Gly Thr Val Leu Gly Tyr Ile Glu Leu Ala Arg Tyr Phe Glu Gly165516601665Ile Gly Asn Val Tyr Gly Leu Gln Ala Pro Gly Leu Tyr Asp Asp167016751680Glu Glu Pro Thr Phe Leu Asp Tyr Asp Glu Leu Val Gln Val Phe168516901695Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro Gly Gln Asp Tyr Leu170017051710Gly Gly His Ser Leu Gly Gly His Ile Ala Phe Gly Met Cys Cys171517201725Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly Leu Leu Ile Leu173017351740Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly Ala Lys Asp Glu174517501755Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val Leu Ala Ala Gly176017651770Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu Leu Lys Gln Leu177517801785
Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala Val Ala Gln Lys179017951800Asp Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr Tyr Leu Asp Lys180518101815Tyr Leu Lys Leu Gln Ile His Ser Leu Leu Met Ser Arg Thr Leu182018251830Glu Leu Glu Lys Thr Gln Leu Asp Ile Pro Ile Lys Val Phe Lys183518401845Thr Gln Phe His Thr Glu Glu Leu Val Glu Arg Phe Asp Ala Trp185018551860His Asn Tyr Thr Asn Gln Ala Cys Thr Phe Ile Asp Ile Pro Gly186518701875Thr His Thr Thr Met Met Arg Leu Pro His Val Lys Glu Val Ala188018851890Lys Lys Ile Glu Glu Gln Leu18951900<210>4<211>1020<212>PRT<213>蜡状芽孢杆菌<400>4Val His Lys Thr Pro Ser Ala Thr Ala Leu Ile Tyr Arg Asn Lys Glu1 5 10 15
Met Thr Tyr Glu Asp Val Asn Ala Gln Ala Asn Ala Leu Ala His Lys20 25 30Leu Arg Asp Ala Gly Val Gly Pro Asn Gln Val Val Gly Val Leu Cys35 40 45Asp Arg Ser Phe Glu Met Val Val Gly Ile Leu Ala Val Leu Lys Ala50 55 60Gly Gly Ala Tyr Leu Pro Ile Asp Thr Ala Tyr Pro Met Gln Arg Thr65 70 75 80Glu Tyr Val Leu Gln Asn Ser Glu Ala Thr Ile Leu Leu Thr Lys Glu85 90 95Cys Tyr Leu Lys Glu Ser Leu Asp Phe Glu Gly Glu Val Phe Tyr Leu100 105 110Asp Asp Ala Arg Leu Phe Glu Gly Asp Arg Arg Asp Leu Gln Asn Ile115 120 125Asn Asn Pro Thr Asn Leu Ala Tyr Ile Ile Tyr Thr Ser Gly Ser Thr130 135 140Gly Asn Pro Lys Gly Val Met Val Ala His Gln Ser Val Val Asn Leu145 150 155 160Leu Leu Asp Leu Gln Glu Lys Tyr Pro Val Leu Ala Glu Asp Lys His165 170 175Leu Leu Lys Thr Thr Tyr Thr Phe Asp Val Ser Val Ala Glu Ile Phe180 185 190
Gly Trp Phe His Ala Gly Gly Thr Leu Val Ile Ala Gly His Gly Asp195 200 205Glu Lys Asp Pro Glu Lys Leu Ile Gln Leu Ile Gln Cys His Lys Val210 215 220Thr His Ile Asn Phe Val Pro Ser Met Leu His Ala Met Leu Gln Ala225 230 235 240Leu Asp Glu Lys Asp Phe Ala Ile Met Asn Arg Leu Lys Tyr Ile Ile245 250 255Val Ala Gly Glu Ala Val Ser Pro Glu Leu Cys Asn Arg Leu Tyr Ala260 265 270His Cys Pro Asn Val Lys Leu Glu Asn Leu Tyr Gly Pro Thr Glu Gly275 280 285Thr Ile Tyr Ala Thr Gly Phe Ser Ile His Lys Glu Met Asn Val Ala290 295 300Asn Val Pro Ile Gly Lys Pro Leu Ser His Val Glu Thr Tyr Ile Leu305 310 315 320Asp Gln Asn Asn Gln Ile Val Pro Ile Gly Val Pro Gly Glu Leu Cys325 330 335Leu Gly Gly Ile Cys Val Ala Lys Gly Tyr Met Lys Glu Pro Val Leu340 345 350Thr Glu Glu Lys Phe Val Val Asn Pro Met Lys Gln Ser Glu Arg Met355 360 365
Tyr Arg Thr Gly Asp Leu Val Arg Trp Leu Ala Asp Gly Asn Ile Glu370 375 380Tyr Leu Gly Arg Ile Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile385390 395 400Glu Leu Gly Glu Ile Glu Ala Ala Ile Ala Ala Leu Glu Asp Val Val405 410 415Gln Thr Ile Val Thr Thr Met Thr Asp His Lys Gly Ala Asn Lys Ile420 425 430Val Ala Tyr Val Val Ser Glu Lys Tyr Asp Glu Glu Arg Ile Arg Glu435 440 445His Val Lys Lys Thr Leu Pro Gln Tyr Met Val Pro Ser Tyr Phe Val450 455 460Ser Met Lys Ala Leu Pro Leu Asn Lys Asn Gly Lys Val Asp Arg Lys465 470 475 480Gln Leu His Ser Val Asp Leu Tyr Glu Thr Ser Met Asp Thr Val Ile485 490 495Val Gly Pro Arg Asn Glu Lys Glu Ala Met Leu Ser Val Ile Trp Gln500 505 510Glu Leu Leu Gly Leu Glu Asn Ile Ser Val His Asp Asn Phe Phe Lys515 520 525Leu Gly Gly His Ser Ile Asn Ala Thr Gln Leu Val Ser Lys Ile Tyr530 535 540
Ser Val Cys Arg Val Arg Met Pro Leu Lys Asn Val Phe Gln Tyr Thr545 550 555 560Thr Leu Ala Thr Met Ala Arg Val Leu Glu Glu Leu Leu Val Ser Ala565 570 575Val Asp Glu Val Ala Val Thr Thr Glu Arg Ile Pro Lys Ile Leu Pro580 585 590Arg Thr Tyr Tyr Asp Leu Ser Tyr Ser Gln Gln Arg Ile Tyr Phe Leu595 600 605Ser Thr Met Glu Lys Glu Thr Asn Tyr Tyr Asn Ile Leu Gly Ala Trp610 615 620Asp Ile Tyr Gly Lys Leu Asp Val Thr Leu Phe Glu Lys Ala Ile Gln625 630 635 640Leu Leu Met Lys Lys His His Ser Leu Arg Ala Thr Phe Glu Ile Val645 650 655Asp Gly Lys Pro Val Gln Ile Ile His Asp Asp Met Glu Ile Pro Val660 665 670Gln Phe Ile Asp Leu Thr Val Met Pro Glu Gly Leu Arg Ile Glu Glu675 680 685Val Asp Glu Leu Met Leu Lys Glu Ser Lys Arg Val Tyr Asn Leu Ala690 695 700Asn Gly Pro Leu Met His Cys Thr Ile Val Lys Ile Lys Glu Gly Glu705 710 715 720
His Val Leu Leu Ile Gly Gln His His Ile Ile Ser Asp Gly Trp Ser725 730 735Leu Gly Ile Phe Val Lys Glu Leu Asn Glu Met Tyr Asp Ala Phe Val740 745 750Gln His Lys Pro Val Ala Glu Thr Pro Ser Thr Ile Ser Ile Met Asp755 760 765Phe Thr Ala Trp His Asn Ser Lys Val Asp Glu Asp Gln Asp Asp Arg770 775 780Gln Tyr Trp Leu Gln Arg Phe Glu Gly Glu Leu Pro Thr Leu Glu Leu785 790 795 800Pro Thr Asp Arg Gln Arg Pro Leu Leu Lys Thr Tyr His Gly Asp Thr805 810 815Leu Ser Tyr Lys Val Asn Ser Gln Leu His Gln Lys Leu Lys Asp Phe820 825 830Ser His Ala Asn Gly Val Thr Met Phe Met Thr Leu Leu Thr Ala Tyr835 840 845Asn Ile Met Leu Asn Lys Leu Thr Asn Glu Thr Asp Ile Val Val Gly850 855 860Ser Pro Val Ala Gly Arg Asn Glu Pro Glu Ser Lys Asp Leu Ile Gly865 870 875 880Met Phe Val Asn Thr Leu Ala Leu Arg Ser His Leu Gly Asp Asn Pro885 890 895
Thr Val Asp Val Leu Leu Lys Gln Ile Lys Gln Asn Thr Leu Glu Ala900 905 910Tyr Asn His Gln Asp Tyr Pro Phe Asp Lys Leu Val Asp Asp Leu Asp915 920 925Pro His Arg Asp Leu Ser Arg Thr Pro Ile Phe Gln Val Met Met Gly930 935 940Tyr Met Asn Met Pro Leu Met Val Ala Phe Arg Glu Ala Glu Val Arg945 950 955 960Glu Arg Phe Val Arg His Lys Val Ala Arg Phe Asp Leu Thr Leu His965 970 975Val Phe Glu Asp Glu Asp Gln Met Lys Ile Phe Phe Glu Tyr Asn Thr980 985 990Asp Leu Phe Asp Glu Ser Thr Ile Met Arg Trp Gln Asn His Phe Glu995 10001005Thr Leu Leu Gln Glu Ile Val Ser Asn Pro Thr Lys101010151020<210>5<211>880<212>PRT<213>蜡状芽孢杆菌<400>5Arg Ile Ser Glu Leu Asn Ile Leu Thr Asn Glu Glu Lys Tyr Glu Ile1 5 10 15
Leu Glu Met Asn Asn Asn Ser Thr Glu Tyr Pro Gln His Glu Ser Val20 25 30Ala Glu Ile Phe Arg Glu Thr Lys Ile Lys His Gln Ala Lys Leu Ala35 40 45Ile Thr Tyr Lys Asp Arg Lys Leu Thr Tyr Ala Glu Leu Ser Glu Lys50 55 60Ala Asn Ala Leu Ala His Thr Leu Lys Arg Arg Gly Val Ala Gln His65 70 75 80Asp Val Val Gly Ile Val Ala Glu Arg Ser Pro Glu Thr Ile Ile Gly85 90 95Ile Leu Ala Ile Leu Lys Val Gly Ala Ile Tyr Leu Pro Ile Asp Pro100 105 110Lys Leu Pro Gln Leu Thr Leu Gln His Ile Trp Arg Asp Ser Gly Ala115 120 125Lys Val Leu Leu Gly Lys Asn Glu Thr Thr Val Glu Val Gly Lys Glu130 135 140Val Pro Phe Val Asp Ile Glu Gly Asp Lys Gly Lys Gln Glu Glu Leu145 150 155 160Val Cys Pro Ile Ser Pro Glu Asp Thr Ala Tyr Ile Met Tyr Thr Ser165 170 175Gly Ser Thr Gly Lys Pro Lys Gly Val Met Val Thr His Arg Asn Ile180 185 190
Val Arg Leu Val Lys Asn Thr Asn Phe Val Ser Leu Gln Glu Gln Asp195 200 205Val Leu Leu Gln Thr Gly Ser Leu Thr Phe Asp Ala Ala Thr Phe Glu210 215 220Ile Trp Gly Ala Leu Leu Asn Gly Leu Thr Leu His Leu Val Glu Asp225 230 235 240Tyr Val Ile Leu Asp Gly Glu Ala Leu Gln Glu Glu Ile Gln Gln Asn245 250 255Lys Ala Thr Ile Met Trp Val Ser Ala Pro Leu Phe Asn Gln Leu Ala260 265 270Asp Gln Asn Pro Ala Met Phe Thr Gly Ile Lys Gln Leu Leu Ile Gly275 280 285Gly Asp Val Leu Ser Pro Lys His Ile Asn Lys Val Met Asp His Cys290 295 300Ala Pro Ile Asn Ile Ile Asn Gly Tyr Gly Pro Thr Glu Asn Thr Thr305 310 315 320Phe Ser Thr Ser Phe Val Ile Asp Gln Met Tyr Gln Asp Ser Ile Pro325 330 335Ile Gly Thr Pro Ile Ala Asn Ser Ser Ala Tyr Ile Leu Asp Val His340 345 350Gln Asn Ile Gln Pro Ile Gly Val Val Gly Glu Leu Cys Val Gly Gly355 360 365
Asp Gly Val Ala Lys Gly Tyr Val Asn Leu Glu Gln Leu Thr Glu Glu370 375 380Arg Phe Ile Ala Asp Pro Phe Leu Lys Gly Ser Thr Met Tyr Arg Thr385 390 395 400Gly Asp Tyr Val Lys Leu Leu Pro Asn Gly Asn Ile Gln Tyr Ile Gly405 410 415Arg Val Asp Asn Gln Val Lys Ile Arg Gly Phe Arg Ile Glu Leu Glu420 425 430Ala Ile Met Asn Thr Leu Lys Gln Cys Glu Ser Ile Lys Asp Val Ile435 440 445Val Val Val Gln Glu Gln Asn Gly Tyr Lys Thr Leu Val Ala Tyr Val450 455 460Val Gly Glu Glu Ser Leu Ser Ile Glu Thr Val Arg Ala Tyr Ala Lys465 470 475 480Lys His Leu Ala Glu Tyr Met Val Pro Ser Gln Phe Ile Phe Ile Glu485 490 495Glu Ile Pro Leu Ser Ile Asn Gly Lys Val Gln Tyr Ser Lys Leu Pro500 505 510Lys Val Gln Glu Val Leu His Lys Lys Val Glu Thr Leu Leu Pro Glu515 520 525Asn Arg Leu Glu Glu Ile Ile Leu Arg Val Tyr Arg Asp Val Leu Glu530 535 540
Lys Glu Asp Phe Gly Val Thr Asp Ser Phe Phe Ala Tyr Gly Gly Asp545 550 555 560Ser Leu Leu Ser Ile Gln Val Val Ser Met Leu Lys Lys Glu Glu Ile565 570 575Ala Val Asp Pro Lys Met Ile Phe Met His Thr Thr Val Arg Glu Leu580 585 590Ala Lys Ala Cys Glu Asn Arg Pro Val Met Glu Glu Thr Lys Arg Thr595 600 605Glu Lys Asp Tyr Leu Ile Gln Met Arg Glu Gly Ser Glu Glu Asp Ser610 615 620Cys Ile Ile Phe Ala Pro Pro Ala Gly Gly Thr Val Leu Gly Tyr Ile625 630 635 640Glu Leu Ala Arg Tyr Phe Glu Gly Ile Gly Asn Val Tyr Gly Leu Gln645 650 655Ala Pro Gly Leu Tyr Asp Asp Glu Glu Pro Thr Phe Leu Asp Tyr Asp660 665 670Glu Leu Val Gln Val Phe Leu Arg Ser Ile Glu Gly Thr Tyr Arg Pro675 680 685Gly Gln Asp Tyr Leu Gly Gly His Ser Leu Gly Gly His Ile Ala Phe690 695 700Gly Met Cys Cys Glu Leu Ile Lys Gln Gly Lys Ala Pro Lys Gly Leu705 710 715 720
Leu Ile Leu Asp Thr Thr Pro Ser Leu Gln Val Val Lys Gly Ala Lys725 730 735Asp Glu Lys Ile Ala Glu Glu Asp Phe Lys Met Met Val Leu Ala Ala740 745 750Gly Ile Gly Asn Met Met Gly Val Asp Pro Glu Glu Leu Lys Gln Leu755 760 765Ser Tyr Glu Glu Ala Lys Thr Arg Val Val Ala Val Ala Gln Lys Asp770 775 780Glu Lys Leu Lys Thr Phe Ile Asn Glu Thr Tyr Leu Asp Lys Tyr Leu785 790 795 800Lys Leu Gln Ile His Ser Leu Leu Met Ser Arg Thr Leu Glu Leu Glu805 810 815Lys Thr Gln Leu Asp Ile Pro Ile Lys Val Phe Lys Thr Gln Phe His820 825 830Thr Glu Glu Leu Val Glu Arg Phe Asp Ala Trp His Asn Tyr Thr Asn835 840 845Gln Ala Cys Thr Phe Ile Asp Ile Pro Gly Thr His Thr Thr Met Met850 855 860Arg Leu Pro His Val Lys Glu Val Ala Lys Lys Ile Glu Glu Gln Leu865 870 875 880<210>6<211>11115<212>DNA<213>蜡状芽孢杆菌
<400>6tttataaaat caatgaatca gttgggaaaa tcaaaaaatt tacataatgg ggggatgatg60gagatgaaac gagtggaaga acatgatcac attcatgtgt taaatgaaat agaaaacgaa120tgcgaaagaa gatatgggag aagtaatatt gcaattatgc ttgaaaagca tggtgttcat180gaacagccgc ttcatataga agacttattt catgaggtag agatgcaaga acattcacga240gtatcgcgcc acgaaacagt tttgatgaca gataaacaat gtatagatga gagtggaaaa300ccgttagctc ttcgttttgg tgagccactt catcttgatg actgtacccc aaaaacacta360caagaaattt taaagcgtgc cgctaagcaa gcaaaagata aagggatgac atttgtatat420gaagatggac atgaagagta cctctcctac caagagatgt tggcagatgc ggagcggtta480ctaaaagggt tgcgaaatct tggtatacaa ccaggagaaa gtattttgtt tcaatttaag540gacaataagc attttgttac tgcgttttgg gcatgtatac ttgggggatt tttaccaacg600ccgttaggaa cggcccctat ctatagtgag caaaatgcac aagtattaaa actttataac660acatggcagc tattagaaca accgattatt ttaacggaat ttgaattgaa agaagagatt720gctgcaattc gaacaacatt gcaacgtcaa gagattgtta tacatagtat tgagaatgtt780atggatacag cgcgcgatac aaactggttt ccttgtaccg aagatactat tgttttgaat840ttattaacgt ctggtagcac aggagtaccc aaatgtgtgc agcacaaaag taaatccatt900attgcgcgca cagtttccaa ttgtattgac cgccagctag atgaaaaaga agtatcgtta960aattggatgc cgcttgatca tgttggaggc atcgtaatgt gtcacattcg tgatacctat1020ttaatgtgcc aacaggtaaa ctgtcttatt tcggcattta ttgaaaatcc gctaaattgg1080ttgcactgga ttgatgctta ctcagcgaca tttacatggg cgccaaactt cgctttttca1140ttaattaacc agtatgaaga agagattaaa tcatcttcat ggaatctttc ttcgatgaga1200tacatcgtaa atggtgggga agctgttatt tcaagtgttg ggatgaaatt tttacaattg1260
ttacagcaac atcaattgcc ttcgaactgt cttattccta cgtttgggat gtctgaagtt1320tcttcgggta ttattgaatg tcattcgttt tatacgcaaa cgacaaatac aggaatgttg1380tatgttgata agaattcttt agatggtaat ttacaattca catatgaggg gcaccaaaat1440gccattgttt ttacggaagt agggagaccg atgcctggta ttggcattcg tattgttgat1500gaggacaatc agtgcctgtc agaagatcgt attggacgat tccaaattca tggtccaacg1560gttatgaatg gttatttcaa aaatgatgaa gcaaatgcgg aaagttttac tgaagatggc1620tggttcgata gtggagatct ggggtttata cataacggta atcttgtcat tacaggaaga1680aaaaaggata tgattgttgt tcatggtgca aattactaca actatgaaat tgaagccttg1740gtagaacaag tacctggggt ggaaaccacg tttgtatgtg caacgagtgt gaagtcggct1800gaaggagcag aggaattagc tattttcttt gtcccagtaa ttaatcatgt ttctgtgatg1860tttgcgacga tgcaacaaat caaacaaatt gttgcgcgca aaatgggtat cacgccgaaa1920gtgattatac caattcagaa ggaagcattc tttaaaacgg atagtgggaa aataacgaga1980aatgcatttc aaaaacagtt tgagaacggg gcatatagag agattacaca aaagattgat2040tgccatttac aaaatgaaaa aacactatct cagtggtttt atcgtgaaaa attagtcgaa2100agtaagttgg gcaaaagcgt atcctcccaa aaagaaacgt atgttttctt tcgacaaggt2160aaatcatttc atcatgtcct aaaagaaaag ttaacgcaac attctgttgt tattgtggat2220gtaggagaaa cattcggtga gatccatcca aatcattatc aaattaatcc taaaaacaaa2280atggattacg ttcgattatt tgaagaactc gcaaaaagaa atgtagaaga tcaagttttt2340catctcttgc atgcttggaa ttattgcgat acagttccaa cttttagatc ggtagaagat2400ttagctaatg cgcaatatct tggtgtgttt agtgtgatgt tcgcacttca agctattatg2460catgcgaaat tgccactacg tcgtgttacg gtgattgcga caaatagtgt tggattagaa2520gcgaaggaaa tgaactattc atgttcaaca ttagaaggtt atgtgaaaac tttgccagct2580
gagtttgaaa atttacaagt gaagtatatt gatatagaag gaaaagatat acagtttgat2640actgagaccg tatggaaaga acttcagcaa caagaaacca ttcctgtcgt attgtatcgt2700gatgagaaaa gatacaaaat aggtttagaa aaagtaccaa tgttagaaca gaaagaaaaa2760aatattccgt ttcaacagca agggttttac atcattacag gtggtcttgg tggtttgggg2820acgcttgtag ccaaattact tttagaacga tacagcgcaa atgttctttt acttggtcga2880acagaaattg aaacaaatgc agaaaaaatg cgccttcttg attcattaaa agagtatgaa2940caatatggtg gtacagtcca atataaaatg tgcaatgtaa tggatttaga tgcgatgcga3000aaagttgttc attcacagga agaacgtctg caacaaaagg taaatgggat tatccacctt3060gcagggatta ttcaagaaat actgatagaa aagcaaaccg aaaaagaact gcatgctatg3120tttgaagcta aggtatatgc atcttgggtg ctacatgaaa tcgtaaaaga aaggcaagat3180tgtctctaca ttacaacttc ttcagcaaga acgttgttac cggggatgac catctcagct3240tattgtagtg cgaatcgatt tgttgaaaat tttgcatatt atcaacgaag tcaaaatgta3300aatagctact gtttttcatg gagtttctgg aatgagattg gaatgggtac aaatttactt3360attaaaaatg cgttgatagc aaaaggattt caattgatcg atgatcaaaa aggtatatat3420tcccttttgg cgggattaaa agggaacgaa cctaatgttt ttgttggaat caatcatgaa3480aaagaagaaa tggctcatct gattggaacc gaggaacaag aaacacaaca attaacaatc3540tatattacac cagaatactt acatattctt gaagaagtgt tctctatact aaatagagaa3600gaatttggtg gattggagaa agagattgtc attctaccaa aattaccgct tgatgaatat3660ggtaaagtag atcaaactcg tttggctcat gcgtcggata gccgttttgg aaagaaacaa3720catatcgtac caagaaatga tatagaagag aaaattgcat tcatttggga aggtcttttg3780aataaaaagg atattagtgt acttgaccat ttcttcgaat taggtggtga ttctttaaaa3840gcgacacaaa tgatttctgc gttgaaaaag aattttgctg ttacgattac gcaacaggaa3900
ttttttcaat cgagtacagt agaagagctt gctagtttag tagaaaagaa actttctcgt3960actcgtacgc atgaaatgga catagttact tttagtgacc gaggtaacgt agtagagatg4020tcttctgcac aaaagcggca atggttttta tatgaaatgg atcgagaaaa tccttattac4080aataatacac ttgtaattcg tttgacggga gaaattcatc ttcctatttt aagaagttct4140attattgagt tagtaaataa gcatgaaaca ttgcgaacaa catttgtgat ggtggatggt4200ataccatcac aaattattgc agatgaagag ttagttgaaa tagaggaaat tgatttgaaa4260cacctatctg ctgaggagac gttgcaaaaa ctagagggtt tacgacaacg ggaagcaaat4320acggcgttta aaatcgaaaa tagcgctttt cgtgcaaaag tgattttaat tgatgagaag4380agagtggaga ttttactttc cgtgcatcac attgtttcgg atggttggtc gatggggatt4440ttagtgaagg acattgcgga aatctatgaa gatattcggc agtggggaga aagtaagcaa4500gagccattac cgattcaata cgcagattat actttgtggc aaaatgagtt tatgaaaggt4560gaggaattta gcaagcaact gtcttattgg aaggagaaat tagctgaaga tatacctgta4620cttgatcttc cgttagataa accacggcca ccaattcaaa catatcgtgg gaaggttaag4680actttcacgt tacatgaaaa catgacaagg atgctaaaag aaatatgtca agaagaagaa4740tgcacgctct ttatgttgtt actttcggct ttctcatcat tattacatcg ttatacaggt4800caggaggatc ttgttgttgg ttcgctagtt gcaaatcgaa accgtgagca aatcgagaaa4860ttgattggtt tctttgttaa tacgttaccg ctacgtatta atcttcatcg ggaaatgcaa4920tttactgaat tgctttcgca agtaaagaaa acgaccattg atgcatatga tcatcaagat4980gtgccttttg agctactagt cgatgaatta cagattgaga gagattcgag tcgtaatgcg5040ctattccaag tgttgtttgt cttacaaaac gcacaattac aagcagtaga cttagagaaa5100gcgacaatgg aactcgaaat tttagatagt gacacggcca agtttgatat gtcagtgcaa5160attttcgaat tggaggacac tttatctatc aaattagagt acaatacgga tttatttttt5220
gatgatacaa tagaacgctt tcttgctcat tatgaaacca tattagcaag cgttattcat5280aatcaaaagg caaaaatagg ggaattgtca attttaccac aatctgaata tacgaaactt5340gtatctgagt ggaatgaaaa gagtgccact tataatggaa atcagtgtat tcatgaattg5400ttcgaagcag ctgttcacaa aacgccatct gcaacagcgc ttatttatcg caacaaagag5460atgacatacg aggatgttaa tgcgcaggca aatgcacttg cacataaatt aagagatgca5520ggtgttggac caaaccaggt agttggcgtg ttatgtgatc gctctttcga gatggttgtt5580ggtatattag ctgttttaaa agcaggtggt gcgtatttgc caattgatac agcgtacccg5640atgcaacgaa cagaatacgt cctgcaaaat agtgaggcaa ctattctctt aacaaaggaa5700tgttacctta aggagtcttt agattttgag ggggaagttt tttacttaga tgatgcaaga5760ctgtttgaag gggatagaag agatttacaa aatatcaata atcctactaa ccttgcttat5820atcatttata catcaggatc cacgggaaat ccaaaaggtg ttatggtagc gcatcaaagt5880gttgtgaatt tgctactcga tttacaagag aaatatccgg tgctagcaga agataagcac5940ttgttaaaaa caacatatac gtttgatgtt tctgtagccg aaatttttgg atggtttcat6000gcaggtggca cacttgttat tgctggacat ggtgatgaaa aagacccaga gaaactgatt6060caattgattc aatgccacaa ggttacacat attaacttcg taccatcgat gctacatgca6120atgttacagg ccttggatga aaaagatttt gcaattatga atcggttgaa atatattatc6180gtcgcaggag aagctgtttc accagaactt tgtaatcgac tgtacgctca ttgtccaaat6240gtaaaactag aaaatctata tgggccaacg gaaggaacga tttatgcgac agggttttct6300attcataaag aaatgaatgt agctaatgta ccgattggaa aaccactttc tcatgtggaa6360acgtatattc ttgatcaaaa caatcaaatt gtaccaattg gtgtaccagg tgaattgtgt6420ctgggaggaa tatgtgtagc aaaaggttat atgaaagagc cggtgttaac agaagaaaaa6480ttcgtcgtca atcctatgaa acaaagtgaa agaatgtacc gaacgggtga tttggtacgc6540
tggttagcag atgggaatat tgaatattta ggaagaatag ataaccaagt caagataaga6600ggcttccgaa ttgagcttgg tgaaattgaa gcggcaattg ctgcattaga agatgtagta6660caaacaattg ttacaacaat gacggatcat aaaggtgcga acaagattgt cgcatatgtt6720gtgagcgaaa agtatgatga agaacgaatt cgtgaacatg tgaaaaagac gttgccgcaa6780tatatggtac caagttattt cgtttcgatg aaggcattgc ctcttaataa aaatggaaaa6840gttgatcgca aacagttgca ttcggttgat ctttatgaaa cgagtatgga tacagtcatt6900gtgggaccaa gaaacgaaaa agaagcaatg ctttcggtta tttggcaaga gcttttggga6960ttagagaata tcagtgttca cgataatttc tttaagcttg gtggtcattc cattaatgcg7020acacaattgg tatcaaaaat ttatagtgtt tgccgagtga gaatgcctct taaaaatgtg7080tttcagtata caacgttagc tacaatggca cgggtgttag aagagttgtt ggtaagcgct7140gttgacgaag tagctgtaac aacggagcgc attccaaaga tactaccgag aacatattac7200gatttgtcgt attcacaaca aagaatttat ttcttatcta caatggagaa agaaaccaat7260tactataata ttcttggtgc ttgggatatt tatgggaaac tagatgttac gctatttgaa7320aaagcaatcc aactattaat gaagaaacac cattcattac gtgcaacatt tgaaatcgtg7380gacggcaaac ctgtgcaaat catccacgat gatatggaaa ttcctgtgca atttattgac7440cttactgtga tgccagaagg attacggata gaagaagtag atgaacttat gttaaaagag7500tctaaaagag tatacaatct cgcaaatggt ccgttaatgc attgtacaat tgttaagata7560aaagaaggtg agcatgtatt attgattgga caacatcata tcattagtga tggttggtca7620cttggtattt ttgtaaaaga gttaaatgaa atgtatgatg cctttgtgca acacaaacca7680gttgctgaaa caccatcaac aatctccatt atggacttta ctgcttggca caatagtaaa7740gtagatgaag atcaagatga tcgacaatat tggttacagc gatttgaggg agagttaccg7800acgttagagt tgccgacaga cagacaacgt ccacttttga aaacatatca tggtgacaca7860
ttatcatata aggtgaattc tcaattgcat caaaaattaa aggactttag tcatgcaaat7920ggtgtaacga tgtttatgac gctattaacg gcgtataata ttatgttgaa taagttaaca7980aatgaaacag acattgttgt tggctcccct gtagcaggta gaaatgaacc agaatcaaaa8040gatttaatcg ggatgtttgt gaatacgtta gcgttacgtt cgcatttagg agataatccg8100acagttgatg tcttattaaa acaaataaaa cagaatactt tagaagcata caatcatcaa8160gattatccat ttgataagtt ggttgatgac ttggatccac atcgagattt aagtaggaca8220ccaattttcc aagtgatgat gggatatatg aatatgccat tgatggttgc atttcgtgaa8280gcagaggttc gcgaacgatt tgttcgacat aaagtcgcaa ggtttgattt aacacttcat8340gtgtttgaag atgaagatca gatgaaaata ttctttgagt ataatacaga tttatttgat8400gaatcaacga ttatgcgttg gcagaatcat ttcgaaacgc tattacagga aattgtatcg8460aatccgacaa aacgtatttc ggaattgaat atacttacaa atgaggagaa atatgaaatt8520ctagagatga acaataattc aacggagtat cctcagcatg aatctgttgc ggagattttt8580agagaaacga agataaagca tcaagcaaaa ctagcaatta cgtacaaaga tagaaagtta8640acgtatgcag agttgagtga aaaagcaaat gcgttggcac atacattgaa acgtcgaggt8700gttgcgcagc atgatgttgt tggaattgtc gcagagcgtt cgcctgaaac aattattgga8760atactcgcaa tcttaaaagt aggagcaatt tatttgccaa ttgatccaaa actaccgcaa8820ttaacactgc aacacatttg gcgagatagc ggtgcaaaag tcctcctagg gaaaaatgaa8880acaactgtag aagttggcaa ggaagttccg tttgtggaca tcgaagggga taaagggaag8940caagaggagt tagtgtgtcc aattagtcca gaagatacgg catatattat gtatacgtca9000ggcagtactg gaaaaccaaa aggggttatg gtgacacata gaaatattgt tcgtttagta9060aaaaatacga atttcgtttc tttgcaagag caagatgtgt tgttacagac aggttcgctt9120acttttgacg ctgcaacatt tgaaatttgg ggcgcattgc taaatggact tacgcttcat9180
ttagtagaag attacgtaat tttagatggg gaggcgcttc aggaagagat tcagcagaac9240aaagcaacca ttatgtgggt gagtgcaccg ctgtttaatc aattggcgga tcaaaaccca9300gcgatgttta caggcattaa gcaattgctc attggtggtg atgttttatc gccaaaacat9360attaacaaag tgatggacca ttgtgcacca atcaatatca ttaatggata cggtccaaca9420gaaaatacga cgttctcgac gtcatttgta attgatcaaa tgtatcaaga cagcattccg9480attggaacac cgattgctaa ttctagtgct tacattttag atgtacatca aaatatacaa9540cctattggtg tagttggcga actatgtgtt ggtggtgatg gagttgcaaa aggttatgtg9600aaccttgaac aattaacaga agaacggttt atagcagatc cgttcctaaa gggttctaca9660atgtacagaa ccggcgatta tgtgaaatta ttgcctaatg gaaatattca atacattgga9720cgtgtggaca atcaagtgaa aattcgtgga ttccgcatcg aattagaagc cattatgaac9780acattaaaac aatgtgaatc aatcaaagat gtaattgttg ttgtacaaga acagaatggg9840tataaaacac tggttgcata tgttgtggga gaagaatcgc tttcaataga aacagtgagg9900gcctatgcaa aaaaacattt ggctgaatat atggtacctt ctcaatttat atttatagaa9960gaaattccgc tctcaataaa tgggaaagta cagtatagta agttaccgaa agtacaagaa10020gtattgcata aaaaagtaga aacgctgtta ccagaaaaca gattagaaga aattattcta10080cgtgtgtatc gtgatgtatt agagaaagaa gattttggcg taacagattc attcttcgct10140tatggtggtg actctttact aagtattcaa gtcgtttcga tgttgaaaaa agaggagatt10200gcagtagatc cgaaaatgat ttttatgcat acaacggtta gagagttagc aaaggcatgt10260gaaaatcgtc cggttatgga agaaacaaaa aggactgaga aggattattt aattcaaatg10320cgtgaaggta gtgaagaaga tagttgtatc atttttgctc ctccggcagg tggaacggta10380cttggatata tagaattagc aaggtatttc gagggaattg gcaatgttta cggcctacaa10440gcaccgggac tgtatgacga tgaagagcct acgttcttag attacgatga acttgtacaa10500
gtgtttcttc gctcgattga agggacatat cgtccaggtc aagattattt aggtggccac10560tccttagggg gacatatcgc atttggaatg tgctgtgaac tgattaagca aggaaaggca10620ccaaagggat tgctaattct agatacaaca ccatcacttc aagttgtaaa gggggccaag10680gatgaaaaaa tagccgagga ggactttaaa atgatggtac tggctgccgg tatcggaaat10740atgatgggtg ttgatccaga agaattaaag caactgtcgt atgaagaagc aaaaacaaga10800gttgtcgcag tggcacaaaa ggatgaaaag ttaaaaactt ttataaatga aacatatttg10860gataagtatt tgaagttaca aattcatagt ttactaatgt cacgaacatt agaattggag10920aaaacacaat tagatattcc gattaaggta tttaaaacac agtttcatac agaagagcta10980gtagaaagat ttgatgcttg gcataactat acaaatcaag cctgcacatt cattgatata11040ccaggcacac atacgacgat gatgcgttta ccacatgtga aagaggtagc gaaaaaaata11100gaagaacagc tataa 11115<210>7<211>5700<212>DNA<213>蜡状芽孢杆菌<400>7cacaaaacgc catctgcaac agcgcttatt tatcgcaaca aagagatgac atacgaggat60gttaatgcgc aggcaaatgc acttgcacat aaattaagag atgcaggtgt tggaccaaac120caggtagttg gcgtgttatg tgatcgctct ttcgagatgg ttgttggtat attagctgtt180ttaaaagcag gtggtgcgta tttgccaatt gatacagcgt acccgatgca acgaacagaa240tacgtcctgc aaaatagtga ggcaactatt ctcttaacaa aggaatgtta ccttaaggag300tctttagatt ttgaggggga agttttttac ttagatgatg caagactgtt tgaaggggat360agaagagatt tacaaaatat caataatcct actaaccttg cttatatcat ttatacatca420ggatccacgg gaaatccaaa aggtgttatg gtagcgcatc aaagtgttgt gaatttgcta480
ctcgatttac aagagaaata tccggtgcta gcagaagata agcacttgtt aaaaacaaca540tatacgtttg atgtttctgt agccgaaatt tttggatggt ttcatgcagg tggcacactt600gttattgctg gacatggtga tgaaaaagac ccagagaaac tgattcaatt gattcaatgc660cacaaggtta cacatattaa cttcgtacca tcgatgctac atgcaatgtt acaggccttg720gatgaaaaag attttgcaat tatgaatcgg ttgaaatata ttatcgtcgc aggagaagct780gtttcaccag aactttgtaa tcgactgtac gctcattgtc caaatgtaaa actagaaaat840ctatatgggc caacggaagg aacgatttat gcgacagggt tttctattca taaagaaatg900aatgtagcta atgtaccgat tggaaaacca ctttctcatg tggaaacgta tattcttgat960caaaacaatc aaattgtacc aattggtgta ccaggtgaat tgtgtctggg aggaatatgt1020gtagcaaaag gttatatgaa agagccggtg ttaacagaag aaaaattcgt cgtcaatcct1080atgaaacaaa gtgaaagaat gtaccgaacg ggtgatttgg tacgctggtt agcagatggg1140aatattgaat atttaggaag aatagataac caagtcaaga taagaggctt ccgaattgag1200cttggtgaaa ttgaagcggc aattgctgca ttagaagatg tagtacaaac aattgttaca1260acaatgacgg atcataaagg tgcgaacaag attgtcgcat atgttgtgag cgaaaagtat1320gatgaagaac gaattcgtga acatgtgaaa aagacgttgc cgcaatatat ggtaccaagt1380tatttcgttt cgatgaaggc attgcctctt aataaaaatg gaaaagttga tcgcaaacag1440ttgcattcgg ttgatcttta tgaaacgagt atggatacag tcattgtggg accaagaaac1500gaaaaagaag caatgctttc ggttatttgg caagagcttt tgggattaga gaatatcagt1560gttcacgata atttctttaa gcttggtggt cattccatta atgcgacaca attggtatca1620aaaatttata gtgtttgccg agtgagaatg cctcttaaaa atgtgtttca gtatacaacg1680ttagctacaa tggcacgggt gttagaagag ttgttggtaa gcgctgttga cgaagtagct1740gtaacaacgg agcgcattcc aaagatacta ccgagaacat attacgattt gtcgtattca1800
caacaaagaa tttatttctt atctacaatg gagaaagaaa ccaattacta taatattctt1860ggtgcttggg atatttatgg gaaactagat gttacgctat ttgaaaaagc aatccaacta1920ttaatgaaga aacaccattc attacgtgca acatttgaaa tcgtggacgg caaacctgtg1980caaatcatcc acgatgatat ggaaattcct gtgcaattta ttgaccttac tgtgatgcca2040gaaggattac ggatagaaga agtagatgaa cttatgttaa aagagtctaa aagagtatac2100aatctcgcaa atggtccgtt aatgcattgt acaattgtta agataaaaga aggtgagcat2160gtattattga ttggacaaca tcatatcatt agtgatggtt ggtcacttgg tatttttgta2220aaagagttaa atgaaatgta tgatgccttt gtgcaacaca aaccagttgc tgaaacacca2280tcaacaatct ccattatgga ctttactgct tggcacaata gtaaagtaga tgaagatcaa2340gatgatcgac aatattggtt acagcgattt gagggagagt taccgacgtt agagttgccg2400acagacagac aacgtccact tttgaaaaca tatcatggtg acacattatc atataaggtg2460aattctcaat tgcatcaaaa attaaaggac tttagtcatg caaatggtgt aacgatgttt2520atgacgctat taacggcgta taatattatg ttgaataagt taacaaatga aacagacatt2580gttgttggct cccctgtagc aggtagaaat gaaccagaat caaaagattt aatcgggatg2640tttgtgaata cgttagcgtt acgttcgcat ttaggagata atccgacagt tgatgtctta2700ttaaaacaaa taaaacagaa tactttagaa gcatacaatc atcaagatta tccatttgat2760aagttggttg atgacttgga tccacatcga gatttaagta ggacaccaat tttccaagtg2820atgatgggat atatgaatat gccattgatg gttgcatttc gtgaagcaga ggttcgcgaa2880cgatttgttc gacataaagt cgcaaggttt gatttaacac ttcatgtgtt tgaagatgaa2940gatcagatga aaatattctt tgagtataat acagatttat ttgatgaatc aacgattatg3000cgttggcaga atcatttcga aacgctatta caggaaattg tatcgaatcc gacaaaacgt3060atttcggaat tgaatatact tacaaatgag gagaaatatg aaattctaga gatgaacaat3120
aattcaacgg agtatcctca gcatgaatct gttgcggaga tttttagaga aacgaagata3180aagcatcaag caaaactagc aattacgtac aaagatagaa agttaacgta tgcagagttg3240agtgaaaaag caaatgcgtt ggcacataca ttgaaacgtc gaggtgttgc gcagcatgat3300gttgttggaa ttgtcgcaga gcgttcgcct gaaacaatta ttggaatact cgcaatctta3360aaagtaggag caatttattt gccaattgat ccaaaactac cgcaattaac actgcaacac3420atttggcgag atagcggtgc aaaagtcctc ctagggaaaa atgaaacaac tgtagaagtt3480ggcaaggaag ttccgtttgt ggacatcgaa ggggataaag ggaagcaaga ggagttagtg3540tgtccaatta gtccagaaga tacggcatat attatgtata cgtcaggcag tactggaaaa3600ccaaaagggg ttatggtgac acatagaaat attgttcgtt tagtaaaaaa tacgaatttc3660gtttctttgc aagagcaaga tgtgttgtta cagacaggtt cgcttacttt tgacgctgca3720acatttgaaa tttggggcgc attgctaaat ggacttacgc ttcatttagt agaagattac3780gtaattttag atggggaggc gcttcaggaa gagattcagc agaacaaagc aaccattatg3840tgggtgagtg caccgctgtt taatcaattg gcggatcaaa acccagcgat gtttacaggc3900attaagcaat tgctcattgg tggtgatgtt ttatcgccaa aacatattaa caaagtgatg3960gaccattgtg caccaatcaa tatcattaat ggatacggtc caacagaaaa tacgacgttc4020tcgacgtcat ttgtaattga tcaaatgtat caagacagca ttccgattgg aacaccgatt4080gctaattcta gtgcttacat tttagatgta catcaaaata tacaacctat tggtgtagtt4140ggcgaactat gtgttggtgg tgatggagtt gcaaaaggtt atgtgaacct tgaacaatta4200acagaagaac ggtttatagc agatccgttc ctaaagggtt ctacaatgta cagaaccggc4260gattatgtga aattattgcc taatggaaat attcaataca ttggacgtgt ggacaatcaa4320gtgaaaattc gtggattccg catcgaatta gaagccatta tgaacacatt aaaacaatgt4380gaatcaatca aagatgtaat tgttgttgta caagaacaga atgggtataa aacactggtt4440
gcatatgttg tgggagaaga atcgctttca atagaaacag tgagggccta tgcaaaaaaa4500catttggctg aatatatggt accttctcaa tttatattta tagaagaaat tccgctctca4560ataaatggga aagtacagta tagtaagtta ccgaaagtac aagaagtatt gcataaaaaa4620gtagaaacgc tgttaccaga aaacagatta gaagaaatta ttctacgtgt gtatcgtgat4680gtattagaga aagaagattt tggcgtaaca gattcattct tcgcttatgg tggtgactct4740ttactaagta ttcaagtcgt ttcgatgttg aaaaaagagg agattgcagt agatccgaaa4800atgattttta tgcatacaac ggttagagag ttagcaaagg catgtgaaaa tcgtccggtt4860atggaagaaa caaaaaggac tgagaaggat tatttaattc aaatgcgtga aggtagtgaa4920gaagatagtt gtatcatttt tgctcctccg gcaggtggaa cggtacttgg atatatagaa4980ttagcaaggt atttcgaggg aattggcaat gtttacggcc tacaagcacc gggactgtat5040gacgatgaag agcctacgtt cttagattac gatgaacttg tacaagtgtt tcttcgctcg5100attgaaggga catatcgtcc aggtcaagat tatttaggtg gccactcctt agggggacat5160atcgcatttg gaatgtgctg tgaactgatt aagcaaggaa aggcaccaaa gggattgcta5220attctagata caacaccatc acttcaagtt gtaaaggggg ccaaggatga aaaaatagcc5280gaggaggact ttaaaatgat ggtactggct gccggtatcg gaaatatgat gggtgttgat5340ccagaagaat taaagcaact gtcgtatgaa gaagcaaaaa caagagttgt cgcagtggca5400caaaaggatg aaaagttaaa aacttttata aatgaaacat atttggataa gtatttgaag5460ttacaaattc atagtttact aatgtcacga acattagaat tggagaaaac acaattagat5520attccgatta aggtatttaa aacacagttt catacagaag agctagtaga aagatttgat5580gcttggcata actatacaaa tcaagcctgc acattcattg atataccagg cacacatacg5640acgatgatgc gtttaccaca tgtgaaagag gtagcgaaaa aaatagaaga acagctataa5700
<210>8<211>11115<212>RNA<213>蜡状芽孢杆菌<400>8uuuauaaaau caaugaauca guugggaaaa ucaaaaaauu uacauaaugg ggggaugaug60gagaugaaac gaguggaaga acaugaucac auucaugugu uaaaugaaau agaaaacgaa120ugcgaaagaa gauaugggag aaguaauauu gcaauuaugc uugaaaagca ugguguucau180gaacagccgc uucauauaga agacuuauuu caugagguag agaugcaaga acauucacga240guaucgcgcc acgaaacagu uuugaugaca gauaaacaau guauagauga gaguggaaaa300ccguuagcuc uucguuuugg ugagccacuu caucuugaug acuguacccc aaaaacacua360caagaaauuu uaaagcgugc cgcuaagcaa gcaaaagaua aagggaugac auuuguauau420gaagauggac augaagagua ccucuccuac caagagaugu uggcagaugc ggagcgguua480cuaaaagggu ugcgaaaucu ugguauacaa ccaggagaaa guauuuuguu ucaauuuaag540gacaauaagc auuuuguuac ugcguuuugg gcauguauac uugggggauu uuuaccaacg600ccguuaggaa cggccccuau cuauagugag caaaaugcac aaguauuaaa acuuuauaac660acauggcagc uauuagaaca accgauuauu uuaacggaau uugaauugaa agaagagauu720gcugcaauuc gaacaacauu gcaacgucaa gagauuguua uacauaguau ugagaauguu780auggauacag cgcgcgauac aaacugguuu ccuuguaccg aagauacuau uguuuugaau840uuauuaacgu cugguagcac aggaguaccc aaaugugugc agcacaaaag uaaauccauu900auugcgcgca caguuuccaa uuguauugac cgccagcuag augaaaaaga aguaucguua960aauuggaugc cgcuugauca uguuggaggc aucguaaugu gucacauucg ugauaccuau1020uuaaugugcc aacagguaaa cugucuuauu ucggcauuua uugaaaaucc gcuaaauugg1080uugcacugga uugaugcuua cucagcgaca uuuacauggg cgccaaacuu cgcuuuuuca1140
uuaauuaacc aguaugaaga agagauuaaa ucaucuucau ggaaucuuuc uucgaugaga1200uacaucguaa auggugggga agcuguuauu ucaaguguug ggaugaaauu uuuacaauug1260uuacagcaac aucaauugcc uucgaacugu cuuauuccua cguuugggau gucugaaguu1320ucuucgggua uuauugaaug ucauucguuu uauacgcaaa cgacaaauac aggaauguug1380uauguugaua agaauucuuu agaugguaau uuacaauuca cauaugaggg gcaccaaaau1440gccauuguuu uuacggaagu agggagaccg augccuggua uuggcauucg uauuguugau1500gaggacaauc agugccuguc agaagaucgu auuggacgau uccaaauuca ugguccaacg1560guuaugaaug guuauuucaa aaaugaugaa gcaaaugcgg aaaguuuuac ugaagauggc1620ugguucgaua guggagaucu gggguuuaua cauaacggua aucuugucau uacaggaaga1680aaaaaggaua ugauuguugu ucauggugca aauuacuaca acuaugaaau ugaagccuug1740guagaacaag uaccuggggu ggaaaccacg uuuguaugug caacgagugu gaagucggcu1800gaaggagcag aggaauuagc uauuuucuuu gucccaguaa uuaaucaugu uucugugaug1860uuugcgacga ugcaacaaau caaacaaauu guugcgcgca aaauggguau cacgccgaaa1920gugauuauac caauucagaa ggaagcauuc uuuaaaacgg auagugggaa aauaacgaga1980aaugcauuuc aaaaacaguu ugagaacggg gcauauagag agauuacaca aaagauugau2040ugccauuuac aaaaugaaaa aacacuaucu cagugguuuu aucgugaaaa auuagucgaa2100aguaaguugg gcaaaagcgu auccucccaa aaagaaacgu auguuuucuu ucgacaaggu2160aaaucauuuc aucauguccu aaaagaaaag uuaacgcaac auucuguugu uauuguggau2220guaggagaaa cauucgguga gauccaucca aaucauuauc aaauuaaucc uaaaaacaaa2280auggauuacg uucgauuauu ugaagaacuc gcaaaaagaa auguagaaga ucaaguuuuu2340caucucuugc augcuuggaa uuauugcgau acaguuccaa cuuuuagauc gguagaagau2400uuagcuaaug cgcaauaucu ugguguguuu agugugaugu ucgcacuuca agcuauuaug2460
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<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>10ggtgaattgt 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>11atttttatta agaggcaatg20<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>12gtcaagataa gaggcttccg aatt 24<210>13<211>20<212>DNA<213>人工序列
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<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>15cacataacct tttgcaactc 20<210>16<211>20<212>DNA<213>人工序列<220>
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<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
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<221>修饰的碱基<222>(14)..(14)<223>I<220>
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<221>修饰的碱基
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<221>修饰的碱基<222>(33)..(33)<223>I<400>21ggaattcctt taggattacc agttgtacca gaagtgtaaat 418权利要求
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。
全文摘要
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。
文档编号C07K14/32GK1653173SQ0381126
公开日2005年8月10日 申请日期2003年5月16日 优先权日2002年5月17日
发明者太田美智男, 安形则雄 申请人:股份有限公司生物控制研究所