具体实施方式
本发明中涉及如下定义:
本发明中所使用的碱基序列、核苷酸、氨基酸(单字母)的缩写,是基于国际理论和应用化学联合会及国际生物联合会对生物化学命名的规定,可参考European Journal ofBiochemistry 1984年底138卷第9期的"准备含碱基序列和氨基酸序列的说明书的指导"一文中的缩写,以及在生物技术领域中通用的缩写。
氨基酸位点的突变表示方法:
用原始氨基酸-氨基酸位置-突变后的氨基酸来表示突变的氨基酸位点。本发明中对赖氨酸脱羧酶氨基酸位置的编号参照大肠杆菌的CadA蛋白进行,其氨基酸序列可参考SEQ ID NO:1,因此,如N84Q表示赖氨酸脱羧酶蛋白序列中的第84位的天冬酰胺突变为谷氨酰胺,位置编号84对应于SEQ ID No:1中赖氨酸脱羧酶蛋白的氨基酸序列编号。
在一些实施方案中,编码赖氨酸脱羧酶(LDC)的基因可以来自微生物、动物或植物的细胞,包括但不局限于大肠杆菌(E.coli)、枯草芽孢杆菌(Bacillus subtilis)、嗜碱芽孢杆菌(Bacillus halodurans)、天蓝色链霉菌(Streptomyces coelicolor)、蜂房哈夫尼菌(H.alvei)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、奥克西托克雷白杆菌(Klebsiella oxytoca)等。赖氨酸脱羧酶也可以来自于上述菌株经过诱变或随机突变之后的菌株或基因工程菌。优选来自于大肠杆菌的诱导型赖氨酸脱羧酶基因cadA/ldcI(SEQ IDNo:2)。赖氨酸脱羧酶还可以是上述来源的赖氨酸脱羧酶的突变体(包括天然突变体和人工重组突变体)或者活性片段(保留了赖氨酸脱羧酶活性的截短形式的蛋白片段)。
本发明的赖氨酸脱羧酶突变体具有相对于野生型赖氨酸脱羧酶或其来源的赖氨酸脱羧酶母体而言降低的赖氨酸脱羧酶活性,优选地,具有相对于野生型赖氨酸脱羧酶或其来源的赖氨酸脱羧酶母体13%~72%的赖氨酸脱羧酶活性,例如,20~70%、30~68%、35~66%或40~65%,更优选地,46%~64%,例如,46%、47%、48%、49%、50%、51%、52%、53%、55%、60%、61%、62%、63%或64%。
本发明的实验数据表明N84、D470和/或D330是赖氨酸脱羧酶的酶活性中起关键作用的位点,这三个位点的氨基酸发生突变后都可能会导致其酶活性降低。在一些实施方案中,所述突变不是保守突变。所述保守突变是指其中氨基酸被其它具有类似性质的氨基酸所替换,使得肽化学领域中技术人员可预期多肽的二级结构和亲水(hydropathic)性质基本上不发生变化。氨基酸替换通常是基于氨基酸侧链取代基的相对相似性,如它们的疏水性、亲水性、电荷、大小等。考虑了各种前述特征的示例性替换是本领域技术人员公知的并包括:精氨酸和赖氨酸;谷氨酸和天冬氨酸;丝氨酸和苏氨酸;谷氨酰胺和天冬酰胺;以及缬氨酸、亮氨酸和异亮氨酸。在一些实施方案中,所述三个位点的氨基酸仅有一个被替换。在一些实施方案中,所述三个位点的氨基酸有两个被替换。在一些实施方案中,所述三个位点的氨基酸都被替换。在一些实施方案中,所述替换为D470C或N84Q。
赖氨酸脱羧酶突变体来源的赖氨酸脱羧酶母体是指赖氨酸脱羧酶突变体是在现有的赖氨酸脱羧酶的基础上进一步进行突变获得的,该现有的赖氨酸脱羧酶也许是野生型的,也许已经经历了某种突变或改造。
“序列同一性”是指在序列比对和引入缺口(如果必要的话,以获得最大百分比同源性)后,多核苷酸或多肽序列突变体的残基与非变体序列的相同的百分比。在一些实施方案中,多肽突变体与本文所述的多肽具有至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、至少99%、至少99.5%、至少99.58%、至少99.72%或至少99.86%的多肽同一性。在一些实施方案中,多核苷酸变体与本文所述的多核苷酸具有至少85%、至少90%、至少95%、至少96%、至少97%、至少98%、至少99%、至少99.5%、至少99.58%、至少99.63%、至少99.67%、至少99.72%、至少99.77%、至少99.81%、至少99.86%或至少99.95%的多核苷酸同一性。
本领域技术人员知晓,上述序列同一性分别基于赖氨酸脱羧酶的氨基酸序列和核苷酸序列计算而来,由于不能整除,因此,均为约数,这样的约数与其对应的精确计算值是对应的,具有相同的意义。例如,当其氨基酸序列发生一个取代替换时,序列同一性的准确值为99.86014%,对应于上述的至少99.86%。当其核苷酸序列发生一个取代替换时,序列同一性的准确值为99.9534451%,对应于上述的至少99.95%。
本领域技术人员公知,在不改变所编码的氨基酸的情况下,所述核苷酸序列中的一个或多个密码子可以进行等义替换,如一个或几个密码子,如1、2、3、4、5、6、8、9、10、15、20、30、40、50个密码子。密码子使用表是本领域公知的。
本发明的一些实施方案提供了能够在中度至高度严谨性条件下与本发明提供的多核苷酸序列杂交的多核苷酸。杂交技术是分子生物学领域公知的。为了说明的目的,用于测试本发明的多核苷酸与其他多核苷酸杂交的合适中等严谨性条件包括在5×SSC、0.5%SDS、1.0mM EDTA(pH8.0)的溶液中预洗;在50-60℃、5×SSC、过夜(16h)的条件下杂交;再于65℃下20分钟用含0.1%SDS的2×、0.5×和0.2×的SSC各洗涤两次。本领域技术人员理解,可容易地操纵杂交的严谨性,例如通过改变杂交溶液的盐含量和/或进行杂交的温度。例如,在另一个实施方式中,合适的高严谨性杂交条件包括上述的条件,所不同的是杂交温度升高了,例如达到60-65℃或65-70℃。
在一些实施方案中,构建的含有编码赖氨酸脱羧酶基因的重组表达质粒载体由在宿主细胞中可复制的骨架质粒衍生而成。
可复制的骨架质粒可以是能够在宿主菌中复制的任何一种质粒,在其中一个实施方案中,稳定的重组表达质粒可以是由一个可以在大肠杆菌和哈夫尼菌中复制的骨架质粒衍生而来,骨架质粒包括但不局限于pUC例如pUC18和pUC19、pBR322质粒和它们的衍生质粒。
本发明所用的术语“有效连接”是指这样的连接方式,其中所述多核苷酸置于载体的适当位置,使得所述多核苷酸正确地、顺利地复制、转录或表达。
在一些实施案例中,对编码赖氨酸脱羧酶基因进行随机突变采用的是易错PCR的方法;在对筛选到的突变株进行重复验证时,可以通过定点突变的方法对关键氨基酸位点进行突变。
其中随机突变属于非理性设计的方法,其不需要了解酶的空间结构和催化机制,通过将酶蛋白的基因克隆到一个合适的表达载体中,再利用基因突变的方法,在体外改造酶基因的序列,并通过合适的酶活性检测方法,来获得具有预期表型的突变蛋白。
易错PCR对基因进行扩增是比较简便、有效的对目的基因进行随机突变的方法。易错PCR的原理是采用Taq PCR扩增酶对目的基因进行扩增时,对反应体系进行调整(例如在反应体系中加入Mn2+,增加Mg2+,并改变加入到体系中的4种dNTPs的浓度,或者改变Taq酶及其浓度等),使得目的基因在扩增过程中引入一定量的突变,然后通过合适的高通量筛选方法获得具有目的表型的突变蛋白。
在一些实施方案中,易错PCR的反应条件为:
易错PCR体系(50μl):
5μl 10×Mutazyme II反应缓冲液
1μl 40mM dNTP混合物(dATP、dTTP、dCTP和dGTP各200μM终浓度)
1μl上游引物cadA-SacI-F(10μM)
1μl下游引物cadA-Xbal-R(10μM)
1μl Mutazyme II DNA聚合酶(购自安捷伦科技(中国)有限公司)
1μl模板DNA(400ng)
40μl ddH2O至总体积为50μl。
PCR程序:95摄氏度变性2min;95摄氏度变性30s,58摄氏度退火30s,72摄氏度延伸2min 10s,28个循环;72摄氏度终延伸,7min。
高通量筛选是指可以快速直观地从众多突变菌株中筛选出具有特定表型的突变菌株的方法,在本发明的一些实施方案中,采用抗生素和pH指示剂筛选的方法获得赖氨酸脱羧酶活性与对照菌株相比发生显著下降的菌株高通量筛选按如下步骤进行:
a.大量抽提突变体文库中的重组赖氨酸脱羧酶突变基因的质粒,并重新转化至E.coli BL21菌株中,同时转化pCIB71质粒作为对照菌株,涂布在含有100μg/ml氨苄青霉素的LB抗性平板中进行筛选,随机挑取若干个单菌落于含有液体LB(+100μg/ml氨苄青霉素)的96孔深孔板中,37摄氏度,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)培养过夜(16h)。利用排枪各取600μl的LB培养液至一个新的96孔深孔板中(同时设置反应开始不加入,反应结束后加入对照菌液的反应孔作为空白对照),加入400μl(浓度为400g/l)的赖氨酸盐酸盐溶液和辅酶PLP(终浓度为0.1mM),于37℃,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)反应2hrs,根据赖氨酸脱羧酶催化过程中1,5-戊二胺的生成会伴随pH的升高,反应结束后于每个反应孔中滴加酸碱指示剂(指示剂B:1mg溴百里香酚蓝溶于10ml去离子水中;指示剂C:10mg中性红溶于10ml乙醇中;指示剂C和B按1:1混合),根据指示剂颜色筛选明显没有对照pH升高的多(但相对空白对照仍有颜色变化)的突变株进行复筛。
b.复筛的程序与初筛相同。
在一些实施方案中,用于筛选赖氨酸脱羧酶突变体时所选的宿主细胞可以是微生物、植物或动物细胞,优选微生物细胞。
在一些实施方案中,用于筛选赖氨酸脱羧酶突变体时选择作为宿主的微生物可以是大肠杆菌(E.coli)、枯草芽孢杆菌(B.subtilis)、天蓝色链霉菌(S.coelicolor)、蜂房哈夫尼菌(H.alvei)或谷氨酸棒状杆菌(C.glutamicum),优选大肠杆菌和蜂房哈夫尼菌。
在一些实施例中,用于筛选赖氨酸脱羧酶突变体的宿主菌是一株不含有内源性质粒的菌株,不含有内源性质粒可以是天然没有内源性质粒的菌株,也可以是通过技术手段筛选到的消除内源性质粒的菌株。
在一些实施案例中,L-赖氨酸生产菌株为通过基因工程手段改造后可以生产L-赖氨酸的菌株,可供使用的赖氨酸生产菌株包括大肠杆菌(E.coli)和谷氨酸棒杆菌(C.glutamicum),在一些实施方案中,宿主菌优先选择为大肠杆菌(E.coli)。在一些实施方案中,L-赖氨酸生产菌株为适用于工业生产规模的工业菌株。工业菌株可以在放大实验中进行培养,包括摇瓶和发酵罐中进行培养,培养的规模可以足够大以进行工业化生产。
在一些实施方案中,培养宿主细胞或L-赖氨酸生产菌株的温度可以是任何能够让其生长的温度,较合适的温度是20~40摄氏度,优选30~40摄氏度,更优选35~40摄氏度。培养的时间可以是约1天,约2天,约3天,约4天,约5天,约6天,约7天,约8天,约9天或者约10天。
在一些实施方案中,培养宿主细胞的培养基含有肽、蛋白胨、维生素、微量元素和矿物质,这样的培养基包括但不局限于常用的LB培养基(由胰蛋白胨,酵母提取物和氯化钠溶解于水制成)。
在一些实施方案中,重组L-赖氨酸生产菌株可以在含有碳源和非碳营养源的培养基中培养。碳源包含但不限于糖(碳水化合物,如葡萄糖和果糖等)。非碳营养源包括但不限于氮源,无机盐和其他有机营养源。例如,培养基包含转化子可吸收的碳源,还可以包含一种或一种以上的其他营养源,如氮源/无机盐和其他有机营养源。氮源可以包括氨,铵盐(如氯化铵,硫酸铵和磷酸铵),蛋白胨,肉类提取物,酵母提取物等。无机盐包括但不限于磷酸二氢钾,硫酸镁,氯化钠等。其他有机营养源,包括但不限于氨基酸(如甘氨酸,丙氨酸,丝氨酸,苏氨酸和脯氨酸)等等。
在一些实施方案中,培养基中L-赖氨酸和1,5-戊二胺的量可以通过核磁共振方法检测,细胞L-赖氨酸转化率采用核磁共振方法检测赖氨酸脱羧酶催化L-赖氨酸转化生成的1,5-戊二胺的量来计算。
以下结合具体的实施案例对本发明做进一步说明,以下实施例仅用于说明本发明而不能限定本发明的范围。对于本领域的技术人员来说,在本发明范围内所做的任何变更,修改或直接采用实施例中的同等条件而实施的例子,都应理解为在本发明的涵盖范围内。
以下实施例、对比例中提到的PCR扩增、质粒提取、酶切、酶切产物连接的具体步骤、条件参数等均按所购相关酶和试剂的说明书建议的条件进行。其中PCR扩增所用的DNA聚合酶、酶切所用的限制性内切酶、酶切产物连接所用的连接酶均购自宝生物工程(大连)有限公司;质粒提取试剂盒、DNA胶回收试剂盒、PCR纯化试剂盒均购自康宁生命科学(吴江)有限公司,商标Axygen;引物均购自赛默飞世尔科技(中国)有限公司,商标INVITROGEN;有特别说明的除外。
如无特殊说明,以下实施例、对比例中提到的转化方法如下:将10μl的连接产物或2μl的质粒加入至100μl的大肠杆菌BL21(DE3)感受态细胞中,冰浴20min后在42℃中热激90s。冰上孵育5min后加入1ml的LB培养基。涂布至相对应的抗性平板上。
OD600(OD562nm)测量方法:将3ml的未培养细胞的培养基加入到1cm宽的比色皿中,在UV-8000紫外可见分光光度计(上海元析仪器有限公司)中600nm(562nm)下吸收光(ABS)校零。将比色皿洗净晾干后加入2.9ml未培养细菌的培养基,加入0.1ml的发酵液混匀后在UV-8000紫外可见分光光度计中测试600nm(562nm)下的吸收光值。仪器显示数值乘以30即为发酵液的OD600(OD562nm)。
实施例1
赖氨酸脱羧酶基因cadA的克隆
使用引物cadA-SacI-F和cadA-XbaI-R(表1所示的引物)将赖氨酸脱羧酶(SEQ IDNo:1)编码基因cadA(SEQ ID No:2)从大肠杆菌MG1655K12(E.coli MG1655K12,购自北京天恩泽生物技术有限公司)的基因组中扩增出来,通过SacI和XbaI双酶切后,连入同样双酶切的pUC18质粒(购自宝生物工程(大连)有限公司)中。利用CaCl2法制备感受态,并用热激法将连接产物转化至大肠杆菌E.coli BL21(购自宝生物工程(大连)有限公司)细胞中,利用LB培养基中添加氨苄抗生素进行筛选,克隆PCR和测序验证正确(验证CadA序列正确)后,抽提质粒即得到pCIB60质粒。
以质粒pCIB60为模板,进一步利用引物cadA-F2和cadA-R2对pCIB60质粒中cadA基因的5’序列进行优化,使CadA可以顺利表达,cadA基因的5’端上游序列由5’-ATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC-3’(SEQ ID No:56)替换为5’-ATTTCACACAGGAAACAGCT-3’(SEQ ID No:57)。PCR扩增后,利用DpnI限制性内切酶对PCR产物进行消化,同样利用热激法转化至E.coli BL21中,测序验证(验证5’端已优化)后得到质粒pCIB71。
表1.本发明中所用到的引物。
实施例2
赖氨酸脱羧酶基因CadA易错PCR突变库的构建及其筛选
利用实施例1中构建的质粒pCIB71作为模板,利用引物cadA-SacI-F和cadA-Xbal-R以及GeneMorph II Random Mutagenesis error-prone PCR试剂盒(购自安捷伦科技(中国)有限公司),按照该试剂盒的使用说明对cadA基因进行随机突变并在基因两端引入酶切位点,使用该试剂盒能够保证A/T/C/G这四个碱基有相等的频率发生突变,并且仅仅需要通过改变反应中初始的靶标DNA的量或者PCR的循环数来控制得到的PCR产物中平均每个基因发生点突变的个数。本实施例中通过使用较高的初始靶标DNA的量(450ng),较少的PCR循环次数(20次),控制每个基因中有1-2个核苷酸发生突变,从而控制氨基酸突变频率为平均每个基因有1个氨基酸发生突变,这种突变频率对于研究蛋白结构与功能之间的关系较为理想。
易错PCR体系(50μl):
5μl 10×Mutazyme II反应缓冲液
1μl 40mM dNTP混合物(dATP、dTTP、dCTP和dGTP各200μM终浓度)
1μl上游引物cadA-SacI-F(10μM)
1μl下游引物cadA-Xbal-R(10μM)
1μl Mutazyme II DNA聚合酶
1μl模板DNA(400ng)
40μl ddH2O至总体积为50μl。
PCR程序:95摄氏度变性2min;95摄氏度变性30s,58摄氏度退火30s,72摄氏度延伸2min 10s,28个循环;72摄氏度终延伸,7min。
1%(质量百分数)的琼脂糖凝胶检测PCR产物,并将目的大小(2.1~2.2kb)的条带切下来,利用DNA凝胶回收试剂盒(购自康宁生命科学(吴江)有限公司,商标Axygen)进行DNA片段的回收。
利用SacI和XbaI对回收的DNA片段进行酶切,与同样双酶切的实施例中提到的pCIB71进行连接。将连接产物转化至E.coli DH5α(购自宝生物工程(大连)有限公司)中,构建赖氨酸脱羧酶基因的随机突变体库。
大量抽提突变体文库中的重组赖氨酸脱羧酶突变基因的质粒,并重新转化至E.coli BL21菌株中,同时转化pCIB71质粒作为对照,涂布在含有100μg/ml氨苄青霉素的LB抗性平板中进行初筛,具体而言,随机挑取约2000个单菌落于含有液体LB(+100μg/ml氨苄青霉素)的96孔深孔板中,37摄氏度,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)培养过夜(16h)。利用排枪各取600μl的LB培养液至一个新的96孔深孔板中(同时设置反应开始不加入,反应结束后加入对照菌株的菌液的反应孔作为空白对照),加入400μl(浓度为400g/l)的赖氨酸盐酸溶液和辅酶磷酸吡哆醛(PLP,终浓度为0.1mM),于37℃,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)反应2hr,根据赖氨酸脱羧酶催化过程中1,5-戊二胺的生成会伴随pH的升高,反应结束后于每个反应孔中滴加酸碱指示剂(指示剂B:1mg溴百里香酚蓝溶于10ml去离子水中;指示剂C:10mg中性红溶于10ml乙醇中;指示剂C和B按1:1体积比混合),根据指示剂颜色筛选明显没有对照pH升高的多(但仍有颜色变化)的突变株进行复筛和测序鉴定突变位点。
实施例3
赖氨酸脱羧酶CadA易错PCR突变体文库筛选结果及突变位点测序鉴定
对构建的CadA易错PCR突变体文库进行初筛,2000个克隆中有31个在96孔深孔板中生长不起来的判定为假阳性克隆;其余1969个克隆中共筛选到19个克隆颜色变化明显弱于对照菌株的。对这19个克隆进行两次复筛(复筛方法与初筛同)。复筛后从中确定了4个克隆明显有低于对照pCIB71的赖氨酸脱羧酶活性。
进一步将这4个克隆接种至5ml LB液体培养基(含100μg/ml氨苄青霉素)进行培养,37摄氏度,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)过夜(16h)。抽提质粒后对赖氨酸脱羧酶基因进行测序鉴定突变位点,测序结果如下表2所示:
表2:CadA易错突变体文库中筛选到的4株突变体突变位点鉴定。
突变体编号 |
突变碱基及序列编号 |
突变氨基酸及序列编号 |
cadA‐1 |
A250G,T252A(SEQ ID No:3) |
N84E(SEQ ID No:4) |
cadA‐2 |
A250C,T252G(SEQ ID No:5) |
N84Q(SEQ ID No:6) |
cadA‐3 |
G1048T,A1049G(SEQ ID No:7) |
D470C(SEQ ID No:8) |
cadA‐4 |
C990A(SEQ ID No:9) |
D330E(SEQ ID No:10) |
由上述表2中所列出的4株突变体的氨基酸测序结果可见,cadA-1,cadA-2和cadA-3这三个突变体相比cadA均发生了两个核苷酸的突变,而cadA-4只发生了一个核苷酸的突变,符合在易错PCR设计时控制条件保证在PCR过程中只引入1-2个突变。由此共产生了三个突变CadA蛋白,突变分别发生在CadA的N84位点,D470位点和D330位点。利用PredUs软件(Zhang QC et al.,Nucleic Acids Res.39,283-287,2011)对CadA蛋白进行3D结构的预测,发现N84和D470位点都出现在CadA单体的表面上,因此推测当不带电荷的天冬酰胺突变为带负电荷的谷氨酸(N84E)或R基团变长的谷氨酰胺(N84Q);或当带负电荷的天冬氨酸突变为不带电荷的半胱氨酸(D470C)时,都可能会严重影响CadA蛋白和蛋白之间的相互作用,进一步影响CadA蛋白多聚体的形成,因此使CadA的活性出现了显著下降。另外,推测D330位点的突变(D330E)可能会影响到CadA蛋白与辅酶磷酸吡哆醛(PLP)的结合,其突变之后,CadA突变蛋白可能会由于结合辅酶PLP的能力减弱而导致其进行L-赖氨酸脱羧反应时活性发生显著下降。
实施例4
赖氨酸脱羧酶CadA突变蛋白赖氨酸转化率测定
将实施例3中提取的4个克隆的质粒和对照pCIB71质粒分别转化至蜂房哈夫尼(蜂房哈夫尼菌CGMCC1.1009,购自于中国微生物菌种保藏管理委员会普通微生物中心)的感受态细胞中,涂布于含有100μg/ml氨苄青霉素的LB抗性平板中进行筛选。进一步分别挑取5个单克隆,利用5ml LB液体培养基(含100μg/ml氨苄青霉素)进行培养,37摄氏度,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)过夜(16h)。分别取600μl的各克隆的LB培养液至EP管中,并加入400μl(浓度为400g/l)的赖氨酸盐酸溶液和辅酶PLP(终浓度为0.1mM),自然pH(约为6.7),于37摄氏度,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)反应2hrs,反应结束后对反应液进行12000rpm离心3min(常温离心机,型号Beckman coultermicrofuge 16centrifuge,下同),取500μl的反应液上清于核磁管中,并加入100μl D2O:DMSO=30:1(质量比)作为内标,利用核磁共振(BRUKER ULTRASHIEDTM400PLUS,Beckman NMR)检测反应体系中剩余的L-赖氨酸的量,以此计算生成的1,5-戊二胺的量和L-赖氨酸转化率(计算1,5-戊二胺的摩尔量除以剩余的赖氨酸和1,5-戊二胺的总摩尔量),进而计算相对CIB71的相对赖氨酸转化率,结果如表3所示。
表3.核磁检测3株CadA突变株与野生型菌株相比的L-赖氨酸转化率及OD。
如表3所示,相同的反应时间内,CadA-1-N84E,CadA-2-N84Q CadA-3-D470C和CadA-4-D330E突变株的L-赖氨酸转化率分别是对照CIB71菌株的72%,64%,46%和13%。而CadA-1-N84E,CadA-2-N84Q和CadA-4-D330E这三株突变株的生长OD均比对照要高;突变株CadA-3-D470C的OD比对照稍微有所降低,但差异并不显著。由此可以分析导致这四株突变菌株的L-赖氨酸转化率低于对照菌株的原因并非因为突变菌株的生长变化,而是蛋白本身的突变造成的,还需要进一步分析是否因为突变蛋白的表达出现问题导致细胞的L-赖氨酸转化率较对照出现了下降。
实施例5
突变赖氨酸脱羧酶CadA的提取及SDS-PAGE电泳分析
利用实施例4中CIB71、CadA-1-N84E、CadA-2-N84Q和CadA-4-D330E突变株的菌液,并根据实施例4中测定的菌株的OD值将这几株菌的OD调整为相同的水平,取同样体积(4ml)的同样OD的野生型和突变株的菌液,12000rpm离心10min,弃上清。按如下步骤提取各菌株的胞内蛋白:
1)用1ml的Buffer 1(Buffer 1(pH 8):50mM Tris HCl pH 8;25mM NaCl;2mMEDTA)重悬菌体(以下简称样品)。
2)-20℃冻存1hr。
3)37℃,15min融化样品。
4)加5μl溶菌酶(购自宝生物工程(大连)有限公司),并继续在37摄氏度孵育3hr,期间颠倒混匀几次。
5)-20℃冻存1hr。
6)37℃,15min融化样品。
7)加入1μl的DNase I(购自宝生物工程(大连)有限公司),继续在37摄氏度孵育1hr。
8)12000rpm离心10min,取上清至一个新的EP管中,确保上清不含有细胞碎片。
9)上清保存在4℃冰箱备用。沉淀用1ml的Buffer 1悬浮,备用。
分别取各菌株的胞内蛋白上清和沉淀各64μl,加入16μl的5×SDS-PAGE上样缓冲液(配方:250mM Tris-HCl(pH6.8),10%(w/v)SDS,0.5%(w/v)溴酚蓝,50%(v/v)甘油,5%(v/v)β-巯基乙醇)于沸水浴中煮沸变性10min,短暂冷却后,12000rpm离心2min,取10μl上清进行SDS-PAGE分析,结果如图1所示。可以看出CadA-1-N84E、CadA-2-N84Q和CadA-4-D330E突变株上清中的赖氨酸脱羧酶CadA表达量与对照CIB71相比无下降,反而有增多的趋势;而沉淀中的赖氨酸脱羧酶和对照菌株相比CadA-1-N84E无明显差异;CadA-2-N84Q和CadA-4-D330E突变株与对照相比也有明显增加。由此可以推断导致CadA-1-N84E、CadA-2-N84Q、CadA-3-D470C和CadA-4-D330E突变株表达的赖氨酸脱羧酶蛋白与对照相比L-赖氨酸转化率下降的原因是由于特定的氨基酸位点突变后对赖氨酸脱羧酶的结构产生了影响从而导致了赖氨酸脱羧酶的催化能力出现下降。
实施例6
不同浓度的1,5-戊二胺对微生物生长的抑制
将能够表达赖氨酸脱羧酶CadA的质粒pCIB71转化至大肠杆菌CIB103-2菌株中,得到CIB103-71菌株。其中CIB103-2菌株的具体构建方法如下:
1.构建四环素外排泵(tetracycline efflux pump)表达载体骨架:
利用引物psyn-1和psyn-2合成启动子序列(SEQ ID NO:11),其中引物psyn-1包启动子序列和一段与pUC18同源的序列,引物psyn-2包含一段与pUC18同源的序列,这两个PCR引物用于扩增合成的启动子序列和pUC18载体的一部分序列,其中包括多克隆位点,限制性内切酶EcoRI和ScaI用于消化扩增出来的DNA片段,进一步连接入pUC18中构建得到pCIB10。
2.构建四环素外排泵表达载体:
利用引物tetA-F和tetA-R四环素外排泵TetA(SEQ ID NO:12)编码基因(tetA,SEQID NO:13),从大肠杆菌克隆载体pBR322中扩增得到,利用SacI和XbaI双酶切后连接至pCIB10质粒中得到pCIB20。
3.L-赖氨酸合成途径上三个蛋白LysC,DapA和LysA阅读框的构建:
大肠杆菌基因组中编码L-赖氨酸生物合成途径上的三个蛋白的基因,lysC,dapA和lysA:天冬氨酸激酶(LysC或AKIII,由lysC编码),二氢吡啶二羧酸合酶(DapA或DHDPS,由dapA编码),二氨基庚酸脱羧酶(LysA,由lysA编码),这三个基因克隆至质粒载体中,由此三个蛋白LysC(SEQ ID NO:14),DapA(SEQ ID NO:16),LysA(SEQ ID NO:18)可以实现在大肠杆菌中的过表达。首先利用引物lysC-F和lysC-R将基因lysC(SEQ ID NO:15)从大肠杆菌E.coli MG1655K12的基因组中扩增出来,用SacI和BamHI双酶切消化之后连接至同样双酶切的质粒pUC18中得到质粒pCIB7。利用引物dapA-F和dapA-R将基因dapA(SEQ ID NO:17)从大肠杆菌E.coli MG1655K12的基因组中扩增出来,用BamHI和XbaI双酶切消化之后连接至同样双酶切的质粒pCIB7中得到质粒pCIB8。利用引物lysA-F和lysA-R将基因lysA(SEQ IDNO:19)从大肠杆菌E.coli MG1655K12的基因组中扩增出来,用XbaI和SalI双酶切消化之后连接至同样双酶切的质粒pCIB8中得到质粒pCIB9。再进一步利用lysC-F和lysA-R将这三个基因一并从pCIB9中扩增出来,并利用SacI和SalI双酶切消化,连接至同样双酶切的质粒pCIB10中得到pCIB32。
4.天冬氨酸激酶编码基因突变载体构建:
为了提高L-赖氨酸的产量,进一步对天冬氨酸激酶基因(LysC或AKIII,由lysC,SEQ ID NO:15编码)进行突变,具体而言,利用引物318-F,318-R,323-F,323-R扩增得到LysC-1(LysC-M318I,G323D,SEQ.ID NO:20)编码基因lysC-1(SEQ ID NO:21),并通过替换pCIB32中的野生型lysC,得到质粒pCIB43。突变后的LysC-1能够进一步减弱L-赖氨酸的反馈抑制。
5.构建L-赖氨酸合成通路中另外三种蛋白Asd,DapB和AspC的共表达载体:
进一步,对L-赖氨酸合成通路中的另外三个基因asd,dapB和aspC进行过表达。这些基因分别编码下列三个蛋白:天冬氨酸盐半醛脱氢酶(Asd(SEQ ID NO:22,由asd编码),二氢吡啶二羧酸还原酶(DapB或DHDPR(SEQ ID NO:24),由dapB编码),天冬氨酸转氨酶(AspC(SEQ ID NO:26),由aspC编码)。利用引物asd-F和asd-R将asd(SEQ ID NO:23)从大肠杆菌MG1655K12基因组中扩增出来,利用SacI和BamHI双酶切后,连接至pUC18中构建得到pCIB12;利用引物dapB-F和dapB-R将dapB(SEQ ID NO:25)从大肠杆菌MG1655K12基因组中扩增出来,利用BamHI和XbaI双酶切后,连接至pCIB12中构建得到pCIB13;利用引物aspC-F和aspC-R将aspC(SEQ ID NO:27)从大肠杆菌MG1655K12基因组中扩增出来,利用XbaI和SalI双酶切后,连接至pCIB13中构建得到pCIB31;利用引物tetA-F3和tetA-R3,以质粒pCIB20为模板,扩增得到tetA编码基因,利用XhoI和SphI双酶切后连入pCIB31构建得到pCIB59。
6.构建L-赖氨酸合成通路中六个基因共表达质粒:
利用引物LAL-F and LAL-R从pCIB43中将lysC-1,dapA,and lysA一起扩增出来,ApaI和KpnI双酶切后,与同样双酶切的质粒pCIB59连接,得到pCIB103-2质粒。
7.构建CIB103-2菌株:
将构建好的pCIB103-2质粒转化至大肠杆菌BL21中,得到大肠杆菌CIB103-2菌株。
分别挑取CIB103-71菌株的单克隆至含有不同浓度(0、5、10、15、20、25、30、35、40g/L)1,5-戊二胺的培养基(5mL液体培养基中含有4%<质量百分数,以下组分同>葡萄糖,0.1%KH2PO4,0.1%MgSO4,1.6%(NH4)2SO4,0.001%FeSO4,0.001%MnSO4,0.2%酵母提取物<购自英格兰OXOID LTD.>,0.05%L-甲硫氨酸,0.01%L-苏氨酸,0.005%L-异亮氨酸,10μg/mL四环素)中,于37℃,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)培养过夜(24h),利用分光光度计测定各1,5-戊二胺浓度条件下菌体的OD562nm。结果如图2所示,可以看出,当1,5-戊二胺浓度从0至10g/L逐渐增加时,菌体的OD急剧下降;当1,5-戊二胺浓度超过20g/L后,几乎检测不到菌体生长。
实施例7
表达突变赖氨酸脱羧酶CadA的赖氨酸生产菌株1,5-戊二胺产量的检测
将随机突变筛选到的能够表达野生型赖氨酸脱羧酶cadA和突变赖氨酸脱羧酶cadA-1、cadA-2、cadA-3和cadA-4的质粒分别转化至能够生产L-赖氨酸的大肠杆菌CIB103-2中,分别得到CIB103-71、CIB103-72、CIB103-73、CIB103-74和CIB103-75菌株。以CIB103-2菌株作为对照,分别挑取三个单克隆至5mL液体培养基中(含有4%<质量百分数,以下组分同>葡萄糖,0.1%KH2PO4,0.1%MgSO4,1.6%(NH4)2SO4,0.001%FeSO4,0.001%MnSO4,0.2%酵母提取物<购自英格兰OXOID LTD.>,0.05%L-甲硫氨酸,0.01%L-苏氨酸,0.005%L-异亮氨酸,10μg/mL四环素,100μg/mL氨苄西林),于37℃,250rpm(仪器购自上海世平实验设备有限公司,超凡型小容量全温度恒温培养振荡器,型号SPH-200B)培养过夜(24h)。第二天,每个菌株再分别转接至100ml新鲜的含有30g/L葡萄糖,0.7%(质量百分数)Ca(HCO3)2,10μg/mL四环素和100μg/mL氨苄西林的培养基中于37℃,170rpm(仪器购自上海世平实验设备有限公司,标准型大容量恒温培养摇床,型号SPH-211B)继续培养72小时,利用核磁检测并计算各培养基中的L-赖氨酸和1,5-戊二胺的含量(表4)。如表4所示,随着突变株CIB103-72、CIB103-73、CIB103-74表达的突变的赖氨酸脱羧酶CadA-N84E,CadA-N84Q和CadA-D470C的活性逐渐降低,1,5-戊二胺的产量逐渐升高,也就意味着更多的L-赖氨酸发生了转化,由此可能降低了L-赖氨酸对L-赖氨酸合成相关的酶的阻遏,L-赖氨酸的产量也会进一步增加并转化为戊二胺,体现在测定的L-赖氨酸和戊二胺的总量逐渐升高。但是当表达活性非常低的CadA-D330E时,虽然在降低戊二胺对重组菌株CIB103-75毒害作用方面有一些效果,表现在生成的戊二胺的量比CIB103-71菌株有所增加,但是剩余了比较多的L-赖氨酸,这将意味着后续需要额外添加较多的赖氨酸脱羧酶将这些L-赖氨酸转化完。据此综合分析,能够表达突变赖氨酸脱羧酶CadA-D470C的突变菌株CIB103-74表现最好。
表4.检测能同时表达赖氨酸合成蛋白及赖氨酸脱羧酶的菌株L-赖氨酸和1,5-戊二胺的水平。
序列表
<110> 上海凯赛生物技术有限公司
<120> 赖氨酸脱羧酶突变体及其应用
<130> LZ1700069CN01
<160> 55
<170> PatentIn version 3.3
<210> 1
<211> 715
<212> PRT
<213> 大肠杆菌
<400> 1
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 2
<211> 2148
<212> DNA
<213> 大肠杆菌
<400> 2
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 3
<211> 2148
<212> DNA
<213> 人工序列
<220>
<223> cadAN84E 突变基因序列
<400> 3
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgctg aaacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 4
<211> 715
<212> PRT
<213> 人工序列
<220>
<223> CadAN84E 突变氨基酸序列
<400> 4
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Glu Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 5
<211> 2148
<212> DNA
<213> 人工序列
<220>
<223> cadAN84Q 突变基因序列
<400> 5
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgctc agacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 6
<211> 715
<212> PRT
<213> 人工序列
<220>
<223> CadAN84Q 突变氨基酸序列
<400> 6
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Gln Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 7
<211> 2148
<212> DNA
<213> 人工序列
<220>
<223> cadAD470C 突变基因序列
<400> 7
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttcttgc agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 8
<211> 715
<212> PRT
<213> 人工序列
<220>
<223> CadAD470C 突变氨基酸序列
<400> 8
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Cys Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 9
<211> 2148
<212> DNA
<213> 人工序列
<220>
<223> cadAD330E突变基因序列
<400> 9
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgaa tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 10
<211> 715
<212> PRT
<213> 人工序列
<220>
<223> CadAD330E突变氨基酸序列
<400> 10
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Glu Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 11
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 合成的启动子核酸序列
<400> 11
agtttattct tgacatgtag tgagggggct ggtataat 38
<210> 12
<211> 396
<212> PRT
<213> 人工序列
<220>
<223> TetA多肽序列
<400> 12
Met Lys Ser Asn Asn Ala Leu Ile Val Ile Leu Gly Thr Val Thr Leu
1 5 10 15
Asp Ala Val Gly Ile Gly Leu Val Met Pro Val Leu Pro Gly Leu Leu
20 25 30
Arg Asp Ile Val His Ser Asp Ser Ile Ala Ser His Tyr Gly Val Leu
35 40 45
Leu Ala Leu Tyr Ala Leu Met Gln Phe Leu Cys Ala Pro Val Leu Gly
50 55 60
Ala Leu Ser Asp Arg Phe Gly Arg Arg Pro Val Leu Leu Ala Ser Leu
65 70 75 80
Leu Gly Ala Thr Ile Asp Tyr Ala Ile Met Ala Thr Thr Pro Val Leu
85 90 95
Trp Ile Leu Tyr Ala Gly Arg Ile Val Ala Gly Ile Thr Gly Ala Thr
100 105 110
Gly Ala Val Ala Gly Ala Tyr Ile Ala Asp Ile Thr Asp Gly Glu Asp
115 120 125
Arg Ala Arg His Phe Gly Leu Met Ser Ala Cys Phe Gly Val Gly Met
130 135 140
Val Ala Gly Pro Val Ala Gly Gly Leu Leu Gly Ala Ile Ser Leu His
145 150 155 160
Ala Pro Phe Leu Ala Ala Ala Val Leu Asn Gly Leu Asn Leu Leu Leu
165 170 175
Gly Cys Phe Leu Met Gln Glu Ser His Lys Gly Glu Arg Arg Pro Met
180 185 190
Pro Leu Arg Ala Phe Asn Pro Val Ser Ser Phe Arg Trp Ala Arg Gly
195 200 205
Met Thr Ile Val Ala Ala Leu Met Thr Val Phe Phe Ile Met Gln Leu
210 215 220
Val Gly Gln Val Pro Ala Ala Leu Trp Val Ile Phe Gly Glu Asp Arg
225 230 235 240
Phe Arg Trp Ser Ala Thr Met Ile Gly Leu Ser Leu Ala Val Phe Gly
245 250 255
Ile Leu His Ala Leu Ala Gln Ala Phe Val Thr Gly Pro Ala Thr Lys
260 265 270
Arg Phe Gly Glu Lys Gln Ala Ile Ile Ala Gly Met Ala Ala Asp Ala
275 280 285
Leu Gly Tyr Val Leu Leu Ala Phe Ala Thr Arg Gly Trp Met Ala Phe
290 295 300
Pro Ile Met Ile Leu Leu Ala Ser Gly Gly Ile Gly Met Pro Ala Leu
305 310 315 320
Gln Ala Met Leu Ser Arg Gln Val Asp Asp Asp His Gln Gly Gln Leu
325 330 335
Gln Gly Ser Leu Ala Ala Leu Thr Ser Leu Thr Ser Ile Ile Gly Pro
340 345 350
Leu Ile Val Thr Ala Ile Tyr Ala Ala Ser Ala Ser Thr Trp Asn Gly
355 360 365
Leu Ala Trp Ile Val Gly Ala Ala Leu Tyr Leu Val Cys Leu Pro Ala
370 375 380
Leu Arg Arg Gly Ala Trp Ser Arg Ala Thr Ser Thr
385 390 395
<210> 13
<211> 1191
<212> DNA
<213> 人工序列
<220>
<223> tetA核酸序列
<400> 13
atgaaatcta acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc 60
ataggcttgg ttatgccggt actgccgggc ctcttgcggg atatcgtcca ttccgacagc 120
atcgccagtc actatggcgt gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca 180
cccgttctcg gagcactgtc cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta 240
cttggagcca ctatcgacta cgcgatcatg gcgaccacac ccgtcctgtg gatcctctac 300
gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc 360
gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc 420
ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat 480
gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta 540
atgcaggagt cgcataaggg agagcgtcga ccgatgccct tgagagcctt caacccagtc 600
agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt 660
atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc 720
tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc 780
ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt 840
atcgccggca tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc 900
tggatggcct tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg 960
caggccatgc tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc 1020
gcggctctta ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc 1080
gcctcggcga gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc 1140
tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg a 1191
<210> 14
<211> 449
<212> PRT
<213> 人工序列
<220>
<223> LysC多肽序列
<400> 14
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 15
<211> 1350
<212> DNA
<213> 人工序列
<220>
<223> lysC核酸序列
<400> 15
atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60
aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120
tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180
ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240
ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300
gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca cggcgagctg 360
atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420
gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480
gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540
acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600
agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660
accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720
gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780
ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840
gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900
gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960
tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020
ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080
actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140
gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200
ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260
ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320
caaaaactgc atagtaattt gtttgagtaa 1350
<210> 16
<211> 292
<212> PRT
<213> 人工序列
<220>
<223> DapA多肽序列
<400> 16
Met Phe Thr Gly Ser Ile Val Ala Ile Val Thr Pro Met Asp Glu Lys
1 5 10 15
Gly Asn Val Cys Arg Ala Ser Leu Lys Lys Leu Ile Asp Tyr His Val
20 25 30
Ala Ser Gly Thr Ser Ala Ile Val Ser Val Gly Thr Thr Gly Glu Ser
35 40 45
Ala Thr Leu Asn His Asp Glu His Ala Asp Val Val Met Met Thr Leu
50 55 60
Asp Leu Ala Asp Gly Arg Ile Pro Val Ile Ala Gly Thr Gly Ala Asn
65 70 75 80
Ala Thr Ala Glu Ala Ile Ser Leu Thr Gln Arg Phe Asn Asp Ser Gly
85 90 95
Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Tyr Asn Arg Pro Ser Gln
100 105 110
Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu His Thr Asp Leu
115 120 125
Pro Gln Ile Leu Tyr Asn Val Pro Ser Arg Thr Gly Cys Asp Leu Leu
130 135 140
Pro Glu Thr Val Gly Arg Leu Ala Lys Val Lys Asn Ile Ile Gly Ile
145 150 155 160
Lys Glu Ala Thr Gly Asn Leu Thr Arg Val Asn Gln Ile Lys Glu Leu
165 170 175
Val Ser Asp Asp Phe Val Leu Leu Ser Gly Asp Asp Ala Ser Ala Leu
180 185 190
Asp Phe Met Gln Leu Gly Gly His Gly Val Ile Ser Val Thr Ala Asn
195 200 205
Val Ala Ala Arg Asp Met Ala Gln Met Cys Lys Leu Ala Ala Glu Gly
210 215 220
His Phe Ala Glu Ala Arg Val Ile Asn Gln Arg Leu Met Pro Leu His
225 230 235 240
Asn Lys Leu Phe Val Glu Pro Asn Pro Ile Pro Val Lys Trp Ala Cys
245 250 255
Lys Glu Leu Gly Leu Val Ala Thr Asp Thr Leu Arg Leu Pro Met Thr
260 265 270
Pro Ile Thr Asp Ser Gly Arg Glu Thr Val Arg Ala Ala Leu Lys His
275 280 285
Ala Gly Leu Leu
290
<210> 17
<211> 879
<212> DNA
<213> 人工序列
<220>
<223> dapA核酸序列
<400> 17
atgttcacgg gaagtattgt cgcgattgtt actccgatgg atgaaaaagg taatgtctgt 60
cgggctagct tgaaaaaact gattgattat catgtcgcca gcggtacttc ggcgatcgtt 120
tctgttggca ccactggcga gtccgctacc ttaaatcatg acgaacatgc tgatgtggtg 180
atgatgacgc tggatctggc tgatgggcgc attccggtaa ttgccgggac cggcgctaac 240
gctactgcgg aagccattag cctgacgcag cgcttcaatg acagtggtat cgtcggctgc 300
ctgacggtaa ccccttacta caatcgtccg tcgcaagaag gtttgtatca gcatttcaaa 360
gccatcgctg agcatactga cctgccgcaa attctgtata atgtgccgtc ccgtactggc 420
tgcgatctgc tcccggaaac ggtgggccgt ctggcgaaag taaaaaatat tatcggaatc 480
aaagaggcaa cagggaactt aacgcgtgta aaccagatca aagagctggt ttcagatgat 540
tttgttctgc tgagcggcga tgatgcgagc gcgctggact tcatgcaatt gggcggtcat 600
ggggttattt ccgttacggc taacgtcgca gcgcgtgata tggcccagat gtgcaaactg 660
gcagcagaag ggcattttgc cgaggcacgc gttattaatc agcgtctgat gccattacac 720
aacaaactat ttgtcgaacc caatccaatc ccggtgaaat gggcatgtaa ggaactgggt 780
cttgtggcga ccgatacgct gcgcctgcca atgacaccaa tcaccgacag tggtcgtgag 840
acggtcagag cggcgcttaa gcatgccggt ttgctgtaa 879
<210> 18
<211> 419
<212> PRT
<213> 人工序列
<220>
<223> LysA多肽序列
<400> 18
Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn
1 5 10 15
Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp
20 25 30
Ala Gln Ile Ile Arg Arg Gln Ile Ala Ala Leu Lys Gln Phe Asp Val
35 40 45
Val Arg Phe Ala Gln Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu
50 55 60
Met Arg Glu Gln Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu Ile
65 70 75 80
Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp
85 90 95
Ile Val Phe Thr Ala Asp Val Ile Asp Gln Ala Thr Leu Glu Arg Val
100 105 110
Ser Glu Leu Gln Ile Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp
115 120 125
Gln Leu Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn
130 135 140
Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu
145 150 155 160
Asn Ser Lys His Gly Ile Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp
165 170 175
Val Ile Gln Arg His His Leu Gln Leu Val Gly Ile His Met His Ile
180 185 190
Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met
195 200 205
Val Arg Gln Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala
210 215 220
Gly Gly Gly Leu Ser Val Pro Tyr Gln Gln Gly Glu Glu Ala Val Asp
225 230 235 240
Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gln Ile Ala
245 250 255
Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro Gly Arg Phe
260 265 270
Leu Val Ala Gln Ser Gly Val Leu Ile Thr Gln Val Arg Ser Val Lys
275 280 285
Gln Met Gly Ser Arg His Phe Val Leu Val Asp Ala Gly Phe Asn Asp
290 295 300
Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu
305 310 315 320
Ala Ala Asp Gly Arg Ser Leu Glu His Ala Pro Thr Val Glu Thr Val
325 330 335
Val Ala Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu
340 345 350
Gly Gly Asn Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala Gly Asp
355 360 365
Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser
370 375 380
Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly
385 390 395 400
Gln Ala Arg Leu Ile Arg Arg Arg Gln Thr Ile Glu Glu Leu Leu Ala
405 410 415
Leu Glu Leu
<210> 19
<211> 1263
<212> DNA
<213> 人工序列
<220>
<223> lysA核酸序列
<400> 19
atgccacatt cactgttcag caccgatacc gatctcaccg ccgaaaatct gctgcgtttg 60
cccgctgaat ttggctgccc ggtgtgggtc tacgatgcgc aaattattcg tcggcagatt 120
gcagcgctga aacagtttga tgtggtgcgc tttgcacaga aagcctgttc caatattcat 180
attttgcgct taatgcgtga gcagggcgtg aaagtggatt ccgtctcgtt aggcgaaata 240
gagcgtgcgt tggcggcggg ttacaatccg caaacgcacc ccgatgatat tgtttttacg 300
gcagatgtta tcgatcaggc gacgcttgaa cgcgtcagtg aattgcaaat tccggtgaat 360
gcgggttctg ttgatatgct cgaccaactg ggccaggttt cgccagggca tcgggtatgg 420
ctgcgcgtta atccggggtt tggtcacgga catagccaaa aaaccaatac cggtggcgaa 480
aacagcaagc acggtatctg gtacaccgat ctgcccgccg cactggacgt gatacaacgt 540
catcatctgc agctggtcgg cattcacatg cacattggtt ctggcgttga ttatgcccat 600
ctggaacagg tgtgtggtgc tatggtgcgt caggtcatcg aattcggtca ggatttacag 660
gctatttctg cgggcggtgg gctttctgtt ccttatcaac agggtgaaga ggcggttgat 720
accgaacatt attatggtct gtggaatgcc gcgcgtgagc aaatcgcccg ccatttgggc 780
caccctgtga aactggaaat tgaaccgggt cgcttcctgg tagcgcagtc tggcgtatta 840
attactcagg tgcggagcgt caaacaaatg gggagccgcc actttgtgct ggttgatgcc 900
gggttcaacg atctgatgcg cccggcaatg tacggtagtt accaccatat cagtgccctg 960
gcagctgatg gtcgttctct ggaacacgcg ccaacggtgg aaaccgtcgt cgccggaccg 1020
ttatgtgaat cgggcgatgt ctttacccag caggaagggg gaaatgttga aacccgcgcc 1080
ttgccggaag tgaaggcagg tgattatctg gtactgcatg atacaggggc atatggcgca 1140
tcaatgtcat ccaactacaa tagccgtccg ctgttaccag aagttctgtt tgataatggt 1200
caggcgcggt tgattcgccg tcgccagacc atcgaagaat tactggcgct ggaattgctt 1260
taa 1263
<210> 20
<211> 449
<212> PRT
<213> 人工序列
<220>
<223> LysC-1多肽序列
<400> 20
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Ile Leu His
305 310 315 320
Ser Arg Asp Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 21
<211> 1349
<212> DNA
<213> 人工序列
<220>
<223> lysC-1核酸序列
<400> 21
tgtctgaaat tgttgtctcc aaatttggcg gtaccagcgt agctgatttt gacgccatga 60
accgcagcgc tgatattgtg ctttctgatg ccaacgtgcg tttagttgtc ctctcggctt 120
ctgctggtat cactaatctg ctggtcgctt tagctgaagg actggaacct ggcgagcgat 180
tcgaaaaact cgacgctatc cgcaacatcc agtttgccat tctggaacgt ctgcgttacc 240
cgaacgttat ccgtgaagag attgaacgtc tgctggagaa cattactgtt ctggcagaag 300
cggcggcgct ggcaacgtct ccggcgctga cagatgagct ggtcagccac ggcgagctga 360
tgtcgaccct gctgtttgtt gagatcctgc gcgaacgcga tgttcaggca cagtggtttg 420
atgtacgtaa agtgatgcgt accaacgacc gatttggtcg tgcagagcca gatatagccg 480
cgctggcgga actggccgcg ctgcagctgc tcccacgtct caatgaaggc ttagtgatca 540
cccagggatt tatcggtagc gaaaataaag gtcgtacaac gacgcttggc cgtggaggca 600
gcgattatac ggcagccttg ctggcggagg ctttacacgc atctcgtgtt gatatctgga 660
ccgacgtccc gggcatctac accaccgatc cacgcgtagt ttccgcagca aaacgcattg 720
atgaaatcgc gtttgccgaa gcggcagaga tggcaacttt tggtgcaaaa gtactgcatc 780
cggcaacgtt gctacccgca gtacgcagcg atatcccggt ctttgtcggc tccagcaaag 840
acccacgcgc aggtggtacg ctgatgtgca ataaaactga aaatccgccg ctgttccgcg 900
ctctggcgct tcgtcgcaat cagactctgc tcactttgca cagcctgaat atactgcatt 960
ctcgcgattt cctcgcggaa gttttcggca tcctcgcgcg gcataatatt tcggtagact 1020
taatcaccac gtcagaagtg agcgtggcat taacccttga taccaccggt tcaacctcca 1080
ctggcgatac gttgctgacg caatctctgc tgatggagct ttccgcactg tgtcgggtgg 1140
aggtggaaga aggtctggcg ctggtcgcgt tgattggcaa tgacctgcca aaagcctgcg 1200
gcgttggcaa agaggtattc ggcgtactgg aaccgttcaa cattcgcatg atttgttatg 1260
gcgcatccag ccataacctg tgcttcctgg tgcccggcga agatgccgag caggtggtgc 1320
aaaaactgca tagtaatttg tttgagtaa 1349
<210> 22
<211> 350
<212> PRT
<213> 人工序列
<220>
<223> Asd多肽序列
<400> 22
Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val
1 5 10 15
Leu Met Gln Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro
20 25 30
Val Phe Phe Ser Thr Ser Gln Leu Gly Gln Ala Ala Pro Ser Phe Gly
35 40 45
Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys
50 55 60
Ala Leu Asp Ile Ile Val Thr Cys Gln Gly Gly Asp Tyr Thr Asn Glu
65 70 75 80
Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp
85 90 95
Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp
100 105 110
Pro Val Asn Gln Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg
115 120 125
Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu
130 135 140
Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr
145 150 155 160
Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu
165 170 175
Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr
180 185 190
Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr
195 200 205
Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly
210 215 220
Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg
225 230 235 240
Glu Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser
245 250 255
Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg
260 265 270
Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile
275 280 285
Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val
290 295 300
Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala
305 310 315 320
Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn
325 330 335
Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gln
340 345 350
<210> 23
<211> 1104
<212> DNA
<213> 人工序列
<220>
<223> asd核酸序列
<400> 23
atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60
atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120
ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180
gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240
atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300
ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360
gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420
ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480
taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540
catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600
gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660
ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagccgc 720
gaagagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780
gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840
aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900
tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960
gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020
ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080
cggatgcttc gtcaactggc gtaa 1104
<210> 24
<211> 273
<212> PRT
<213> 人工序列
<220>
<223> DapB多肽序列
<400> 24
Met His Asp Ala Asn Ile Arg Val Ala Ile Ala Gly Ala Gly Gly Arg
1 5 10 15
Met Gly Arg Gln Leu Ile Gln Ala Ala Leu Ala Leu Glu Gly Val Gln
20 25 30
Leu Gly Ala Ala Leu Glu Arg Glu Gly Ser Ser Leu Leu Gly Ser Asp
35 40 45
Ala Gly Glu Leu Ala Gly Ala Gly Lys Thr Gly Val Thr Val Gln Ser
50 55 60
Ser Leu Asp Ala Val Lys Asp Asp Phe Asp Val Phe Ile Asp Phe Thr
65 70 75 80
Arg Pro Glu Gly Thr Leu Asn His Leu Ala Phe Cys Arg Gln His Gly
85 90 95
Lys Gly Met Val Ile Gly Thr Thr Gly Phe Asp Glu Ala Gly Lys Gln
100 105 110
Ala Ile Arg Asp Ala Ala Ala Asp Ile Ala Ile Val Phe Ala Ala Asn
115 120 125
Phe Ser Val Gly Val Asn Val Met Leu Lys Leu Leu Glu Lys Ala Ala
130 135 140
Lys Val Met Gly Asp Tyr Thr Asp Ile Glu Ile Ile Glu Ala His His
145 150 155 160
Arg His Lys Val Asp Ala Pro Ser Gly Thr Ala Leu Ala Met Gly Glu
165 170 175
Ala Ile Ala His Ala Leu Asp Lys Asp Leu Lys Asp Cys Ala Val Tyr
180 185 190
Ser Arg Glu Gly His Thr Gly Glu Arg Val Pro Gly Thr Ile Gly Phe
195 200 205
Ala Thr Val Arg Ala Gly Asp Ile Val Gly Glu His Thr Ala Met Phe
210 215 220
Ala Asp Ile Gly Glu Arg Leu Glu Ile Thr His Lys Ala Ser Ser Arg
225 230 235 240
Met Thr Phe Ala Asn Gly Ala Val Arg Ser Ala Leu Trp Leu Ser Gly
245 250 255
Lys Glu Ser Gly Leu Phe Asp Met Arg Asp Val Leu Asp Leu Asn Asn
260 265 270
Leu
<210> 25
<211> 822
<212> DNA
<213> 人工序列
<220>
<223> dapB核酸序列
<400> 25
atgcatgatg caaacatccg cgttgccatc gcgggagccg gggggcgtat gggccgccag 60
ttgattcagg cggcgctggc attagagggc gtgcagttgg gcgctgcgct ggagcgtgaa 120
ggatcttctt tactgggcag cgacgccggt gagctggccg gagccgggaa aacaggcgtt 180
accgtgcaaa gcagcctcga tgcggtaaaa gatgattttg atgtgtttat cgattttacc 240
cgtccggaag gtacgctgaa ccatctcgct ttttgtcgcc agcatggcaa agggatggtg 300
atcggcacta cggggtttga cgaagccggt aaacaagcaa ttcgtgacgc cgctgccgat 360
attgcgattg tctttgctgc caattttagc gttggcgtta acgtcatgct taagctgctg 420
gagaaagcag ccaaagtgat gggtgactac accgatatcg aaattattga agcacatcat 480
agacataaag ttgatgcgcc gtcaggcacc gcactggcaa tgggagaggc gatcgcccac 540
gcccttgata aagatctgaa agattgcgcg gtctacagtc gtgaaggcca caccggtgaa 600
cgtgtgcctg gcaccattgg ttttgccacc gtgcgtgcag gtgacatcgt tggtgaacat 660
accgcgatgt ttgccgatat tggcgagcgt ctggagatca cccataaggc gtccagccgt 720
atgacatttg ctaacggcgc ggtaagatcg gctttgtggt tgagtggtaa ggaaagcggt 780
ctttttgata tgcgagatgt acttgatctc aataatttgt aa 822
<210> 26
<211> 396
<212> PRT
<213> 人工序列
<220>
<223> AspC多肽序列
<400> 26
Met Phe Glu Asn Ile Thr Ala Ala Pro Ala Asp Pro Ile Leu Gly Leu
1 5 10 15
Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Leu Gly
20 25 30
Ile Gly Val Tyr Lys Asp Glu Thr Gly Lys Thr Pro Val Leu Thr Ser
35 40 45
Val Lys Lys Ala Glu Gln Tyr Leu Leu Glu Asn Glu Thr Thr Lys Asn
50 55 60
Tyr Leu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Gln Glu
65 70 75 80
Leu Leu Phe Gly Lys Gly Ser Ala Leu Ile Asn Asp Lys Arg Ala Arg
85 90 95
Thr Ala Gln Thr Pro Gly Gly Thr Gly Ala Leu Arg Val Ala Ala Asp
100 105 110
Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro
115 120 125
Ser Trp Pro Asn His Lys Ser Val Phe Asn Ser Ala Gly Leu Glu Val
130 135 140
Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu Asn His Thr Leu Asp Phe Asp
145 150 155 160
Ala Leu Ile Asn Ser Leu Asn Glu Ala Gln Ala Gly Asp Val Val Leu
165 170 175
Phe His Gly Cys Cys His Asn Pro Thr Gly Ile Asp Pro Thr Leu Glu
180 185 190
Gln Trp Gln Thr Leu Ala Gln Leu Ser Val Glu Lys Gly Trp Leu Pro
195 200 205
Leu Phe Asp Phe Ala Tyr Gln Gly Phe Ala Arg Gly Leu Glu Glu Asp
210 215 220
Ala Glu Gly Leu Arg Ala Phe Ala Ala Met His Lys Glu Leu Ile Val
225 230 235 240
Ala Ser Ser Tyr Ser Lys Asn Phe Gly Leu Tyr Asn Glu Arg Val Gly
245 250 255
Ala Cys Thr Leu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe
260 265 270
Ser Gln Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Asn Pro Pro Ala
275 280 285
His Gly Ala Ser Val Val Ala Thr Ile Leu Ser Asn Asp Ala Leu Arg
290 295 300
Ala Ile Trp Glu Gln Glu Leu Thr Asp Met Arg Gln Arg Ile Gln Arg
305 310 315 320
Met Arg Gln Leu Phe Val Asn Thr Leu Gln Glu Lys Gly Ala Asn Arg
325 330 335
Asp Phe Ser Phe Ile Ile Lys Gln Asn Gly Met Phe Ser Phe Ser Gly
340 345 350
Leu Thr Lys Glu Gln Val Leu Arg Leu Arg Glu Glu Phe Gly Val Tyr
355 360 365
Ala Val Ala Ser Gly Arg Val Asn Val Ala Gly Met Thr Pro Asp Asn
370 375 380
Met Ala Pro Leu Cys Glu Ala Ile Val Ala Val Leu
385 390 395
<210> 27
<211> 1191
<212> DNA
<213> 人工序列
<220>
<223> aspC核酸序列
<400> 27
atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60
cgtgccgatg aacgtcccgg caaaattaac ctcgggattg gtgtctataa agatgagacg 120
ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180
accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240
ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300
ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360
aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420
ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480
gcactgatta acagcctgaa tgaagctcag gctggcgacg tagtgctgtt ccatggctgc 540
tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600
tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660
ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720
gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780
gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840
gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900
gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960
atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020
atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080
ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140
acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191
<210> 28
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> cadA-SacI-F
<400> 28
tccgagctca tgaacgttat tgcaatattg 30
<210> 29
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> cadA-XbaI-R
<400> 29
gcctctagac cacttccctt gtacgagc 28
<210> 30
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> cadA-F2
<400> 30
atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat 44
<210> 31
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> cadA-R2
<400> 31
agctgtttcc tgtgtgaaat 20
<210> 32
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> psyn-1
<400> 32
ggcgaattca gtttattctt gacatgtagt gagggggctg gtataatgag ctcggtaccc 60
ggggat 66
<210> 33
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> psyn-2
<400> 33
ggcagtactc aaccaagtca ttctgagaat agtg 34
<210> 34
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> tetA-F
<400> 34
ggcgagctca cacaggaaac agaccatgaa atctaacaat gcgctcatc 49
<210> 35
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> tetA-R
<400> 35
ggctctagat caacgacagg agcacgatc 29
<210> 36
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> lysC-F
<400> 36
ggcgagctca cacaggaaac agaccatgtc tgaaattgtt gtctcc 46
<210> 37
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> lysC-R
<400> 37
ggcggatcct tactcaaaca aattactatg cag 33
<210> 38
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> dapA-F
<400> 38
ggcggatcca cacaggaaac agaccatgtt cacgggaagt attgtc 46
<210> 39
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> dapA-R
<400> 39
ggctctagat tacagcaaac cggcatgc 28
<210> 40
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> lysA-F
<400> 40
ggctctagaa cacaggaaac agaccatgcc acattcactg ttcagc 46
<210> 41
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> lysA-R
<400> 41
ggcgtcgact taaagcaatt ccagcgccag 30
<210> 42
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> tetA-F3
<400> 42
ggcctcgaga gtttattctt gacatgtagt gagg 34
<210> 43
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> tetA-R3
<400> 43
ggcgcatgct caacgacagg agcacgatc 29
<210> 44
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 318-F
<400> 44
cagcctgaat atactgcatt ctc 23
<210> 45
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 318-R
<400> 45
gagaatgcag tatattcagg ctg 23
<210> 46
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 323-F
<400> 46
gcattctcgc gatttcctcg 20
<210> 47
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 323-R
<400> 47
cgaggaaatc gcgagaatgc 20
<210> 48
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> asd-F
<400> 48
ggcgagctca cacaggaaac agaccatgaa aaatgttggt tttatcgg 48
<210> 49
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> asd-R
<400> 49
ggcggatcct tacgccagtt gacgaagc 28
<210> 50
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> dapB-F
<400> 50
ggcggatcca cacaggaaac agaccatgca tgatgcaaac atccg 45
<210> 51
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> dapB-R
<400> 51
ggctctagat tacaaattat tgagatcaag tacatctc 38
<210> 52
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> aspC-F
<400> 52
ggctctagaa cacaggaaac agaccatgtt tgagaacatt accgcc 46
<210> 53
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> aspC-R
<400> 53
ggcgcatgcg acctcgaggt agtcgactta cagcactgcc acaatcg 47
<210> 54
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> LAL-F
<400> 54
ggcggtacca gtttattctt gacatgtagt gagg 34
<210> 55
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> LAL-R
<400> 55
ggcgggccct taaagcaatt ccagcgcca 29
<210> 56
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> cada基因原5'序列
<400> 56
atttcacaca ggaaacagct atgaccatga ttacgaattc gagctc 46
<210> 57
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> cada基因修饰后的5'序列
<400> 57
atttcacaca ggaaacagct 20