CN114250204A - 一种羧酸还原酶突变体及酶法合成脱羧肌肽的方法 - Google Patents
一种羧酸还原酶突变体及酶法合成脱羧肌肽的方法 Download PDFInfo
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Abstract
本发明提供了一种酶法合成脱羧肌肽的方法，包括：将组胺、氨基保护的β‑丙氨酸、还原酶、活化剂与辅酶在碱性溶液中反应，脱保护后，得到脱羧肌肽或其盐；所述还原酶包括野生型NiCAR和/或NiCAR突变体；野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。与现有技术相比，本发明利用酶催化生产脱羧肌肽，具有效率高、步骤短、成本低、操作简便、收率高、副产物少等优点，将其用于工业生产，可以简化工艺流程、降低耗能、节省资源、减少污染等。
Description
技术领域
本发明属于有机合成技术领域，尤其涉及一种羧酸还原酶突变体及酶法合成脱羧肌肽的方法。
背景技术
脱羧肌肽因具有抗氧化、抗糖基化、抗衰、美白祛斑、晒后修复等功效，被广泛应用于美容护肤品中。脱羧肌肽是由组织胺与β-丙氨酸缩合而成的二肽，市售脱羧肌肽主要由化学合成，其生产过程主要经历氨基酸的保护、活化、分子间缩合、脱保护等步骤，存在合成路线长、操作复杂、副产物多、不易纯化、生产成本高、污染环境等问题。因此开发一种绿色、高效、成本低的合成方法具有重要的意义。
生物酶在有机合成中的应用是20世纪80年代发展的生化技术，由于它具有许多优点，如反应条件温和(常温、近中温)，具有高度的区域选择性、立体选择性和对映体选择性，可避免敏感官能团发生变化，可产生许多光学活性物质，尚可完成一些用传统的化学反应很难完成的化学反应，另外还有产品纯、无三废、无环境污染等雨点，因此越来越收到有机化学研究者的青睐。
羧酸还原酶主要由3个模块化的结构域组成，分别为N-端的腺苷酰化结构域(N-terminal adenylation domain，A domain)、硫醇化结构域(thiolation domain，Tdomain)和还原酶结构域(reductase domain，R domain)。相较于已经广泛研究的非核糖体多肽合成酶(nonribosomal peptide synthase，NRPS)，羧酸还原酶仅仅缺少了N端的缩合结构域。NRPS的缩合结构域与AAR的腺苷酸激活结构域有一定的序列相似性，但后者缺少一些保守序列，表明其在维持重要结构域完整性的同时已经丧失了部分缩合催化功能。Finnigan等将羧酸还原酶的腺苷酰化结构域与腺苷形成酶超家族的酰基CoA连接酶(acyl-CoA synthetase)、萤火虫荧光素酶(luciferase)及非核糖体多肽合成酶进行序列比对，发现其序列相似性达20％，且拥有保守的氨基酸序列。
2017年，Sabine L.Flitsch课题组发表文章，用NiCAR(from Nocardia iowensis)酶催化酰胺键的合成。反应式如下，但其催化效率较差。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种催化活性较高的羧酸还原酶突变体及酶法合成脱羧肌肽的方法。
本发明提供了一种酶法合成脱羧肌肽的方法，包括：
将组胺、氨基保护的β-丙氨酸、还原酶、活化剂与辅酶在碱性溶液中反应，脱保护后，得到脱羧肌肽或其盐；
所述还原酶包括野生型NiCAR和/或NiCAR突变体；
野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；
NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。
优选的，所述NiCAR突变体的氨基酸序列如SEQ ID NO：2～11任一项所示。
优选的，以室温一分钟转化1μmol的底物所需酶量为U，所述还原酶的用量为1000～10000U。
优选的，所述活化剂选自镁盐；所述活化剂的摩尔数为组胺摩尔数的5％～20％；所述辅酶选自腺苷三磷酸；所述辅酶的摩尔数为组胺摩尔数的1％～10％。
优选的，所述碱性溶液的pH值为8～10；所述碱性溶液中的碱性物质选自碳酸氢钠、碳酸氢钾、碳酸钠、碳酸钾、碳酸锂、氢氧化钠、氢氧化钾与氢氧化锂中的一种或多种。
优选的，所述反应的温度为30℃～40℃；所述反应的时间为20～30h。
本发明还提供了一种羧酸还原酶突变体，所述羧酸还原酶突变体为NiCAR突变体，其野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；
所述NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。
本发明还提供了上述羧酸还原酶突变体在催化合成酰胺键中的应用。
本发明还提供了编码上述羧酸还原酶突变体的核酸。
优选的，其序列如SEQ ID NO：13～22任一项所示。
本发明提供了一种酶法合成脱羧肌肽的方法，包括：将组胺、氨基保护的β-丙氨酸、还原酶、活化剂与辅酶在碱性溶液中反应，脱保护后，得到脱羧肌肽或其盐；所述还原酶包括野生型NiCAR和/或NiCAR突变体；野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。与现有技术相比，本发明利用酶催化生产脱羧肌肽，具有效率高、步骤短、成本低、操作简便、收率高、副产物少等优点，将其用于工业生产，可以简化工艺流程、降低耗能、节省资源、减少污染等。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了羧酸还原酶突变体，所述羧酸还原酶突变体为NiCAR突变体，其野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；
所述NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。
优选地，所述NiCAR突变体的氨基酸序列如SEQ ID NO：2～11任一项所示。
本发明还提供了一种编码上述羧酸还原酶突变体的核酸。
优选地，其野生型NiCAR的核酸序列如SEQ ID NO：12所示。
优选地，所述编码上述羧酸还原酶突变体的核酸序列如13～22任一项所示。
在本发明中所述酶合成的方法为本领域技术人员熟知的方法即可，并无特殊的限制，本发明中所用的酶均是合成相应基因后构建在特定的表达质粒上，再通过大肠杆菌发酵生产制得；其具体包含以下步骤：在pET21质粒中对Nocardia iowensis CAR(CARni)进行了针对大肠杆菌的密码子优化，优化方式为在N末端增加了多个组氨酸标签，然后转移至E.Coil BL21(DE3)细胞(NEB)的PCDF1质粒中。转化的BL21 DE3细胞随后接种到600mL自动诱导培养基(Formedium)，100μg/mL氨苄青霉素，100μg/mL大霉素，置于2L烧瓶中，20℃，250rpm孵育48h。随后，细胞离心(4000rpm,30分钟，4℃)成球并在-20℃下冷冻，直到需要裂解时才解冻。用100mM Tris-HCl pH 7.5、10mM咪唑、1mM PMSF、0.2mg/mL溶菌酶、2mMMgCl2、2.5U/mL(NEB)裂解细胞。溶菌酶在37℃下裂解1小时，然后在冰上超声。然后通过离心(18000rpm，25分钟，4℃)澄清裂解液，用0.45μm注射器过滤器过滤，并应用于5mlHisTrap FF Crude nickel亲和力柱(GE Healthcare)。采用AKTA自动净化系统进行净化。使用两种缓冲液，洗涤缓冲液(缓冲液A)为100mM Tris-HCl pH 7.5,10mM咪唑和洗脱缓冲液，洗脱缓冲液(缓冲液B)为100mM Tris-HCl(pH 7.5)1M咪唑。用99％缓冲液A和1％缓冲液B对色谱柱进行20柱体积(CVs)的洗涤，10mL馏分中收集洗涤馏分。随后，用超过10CVs的洗脱相，缓冲液B的梯度从1％增加到100％，收集了2mL洗脱馏分。2mL洗脱组分通过SDS-PAGE和快速考马斯蓝染色(Expedeon)进行分析。CAR酶可见条带最大的部分被合并并透析。将纯化的蛋白质等量用液氮快速冷冻，并储存在-80℃。
本发明还提供了一种上述羧酸还原酶突变体在催化合成酰胺键中的应用。
本发明还提供了一种酶法合成脱羧肌肽的方法，包括：将组胺、氨基保护的β-丙氨酸、还原酶、活化剂与辅酶在碱性溶液中反应，脱保护后，得到脱羧肌肽或其盐；所述还原酶包括野生型NiCAR和/或NiCAR突变体；野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。
其中，本发明对所有原料的来源并没有特殊的限制，为市售即可；所述还原酶同上所述，在此不再赘述。
在本发明中，优选先将组胺、氨基保护的β-丙氨酸与活化剂在碱性溶液中混合，然后加入还原酶与辅酶，进行反应；所述氨基保护的β-丙氨酸中的氨基保护基团为本领域技术人员熟知的氨基保护基团即可，并无特殊的限制，本发明中优选为烷氧羰基类保护基团，更优选为Cbz、Boc或Fmoc；所述组胺与氨基保护的β-丙氨酸的摩尔比优选为1：(0.8～1.2)，更优选为1：(0.9～1.1)，再优选为1：1；所述活化剂优选为镁盐，更优选为氯化镁；所述活化剂的摩尔数优选为组胺摩尔数的5％～20％，更优选为8％～15％，再优选为10％～12％；所述碱性溶液的pH值优选为8～10，更优选为8～9；所述碱性溶液的碱性物质优选为碳酸氢钠、碳酸氢钾、碳酸钠、碳酸钾、碳酸锂、氢氧化钠、氢氧化钾与氢氧化锂中的一种或多种；所述辅酶优选为腺苷三磷酸；所述辅酶的摩尔数优选为组胺摩尔数的1％～10％，更优选为2％～8％，再优选为4％～6％，最优选为5％；以室温一分钟转化1μmol的底物所需酶量为U，所述还原酶的用量优选为1000～10000U，更优选为2000～8000U，再优选为3000～6000U，最优选为4000～5000U；所述反应的温度优选为30℃～40℃，更优选为30℃～36℃，再优选为32℃～35℃，最优选为33℃；所述反应的时间优选为20～40h，更优选为24～40h；所述反应优选在搅拌的条件下进行。
反应结束后，优选离心，取上清液纳滤除盐后，进行脱保护；所述脱保护的方法为本领域技术人员熟知的方法即可，并无特殊的限制，可根据氨基保护基团的种类选择相应的脱保护反应，在本发明中所述氨基保护基团优选为Boc，因此优选在酸性溶液中进行脱保护；所述酸性溶液优选为盐酸；所述盐酸的浓度优选为1～3mol/L，更优选为1.5～2.5mol/L，再优选为2mol/L；所述酸性溶液的pH值优选为1～2。
脱保护后，除去溶剂，进一步纯化，得到脱羧肌肽或其盐；所述进一步纯化的方法优选为将除去溶剂后的固体用甲醇与二氯甲烷的混合溶液重结晶，更优选为将除去溶剂后的固体在甲醇与二氯甲烷的混合溶液搅拌，然后静置；所述甲醇与二氯甲烷的体积比优选为1：(3～5)，更优选为1：4；所述搅拌的时间优选为20～60min，更优选为30～40min；然后静置10分钟，过滤得纯品。所述洗涤优选在搅拌的条件下进行。
本发明利用酶催化生产脱羧肌肽，具有效率高、步骤短、成本低、操作简便、收率高、副产物少等优点，将其用于工业生产，可以简化工艺流程、降低耗能、节省资源、减少污染等。
为了进一步说明本发明，以下结合实施例对本发明提供的一种羧酸还原酶突变体及酶法合成脱羧肌肽的方法进行详细描述。
以下实施例中所用的试剂均为市售。
NiCAR from Nocardia iowensis(源于艾阿华诺卡氏菌的羧酸还原酶)。
基因表达及纯化
在pET21质粒中对Nocardia iowensis CAR(CARni)进行了针对大肠杆菌的密码子优化，优化方式为在N末端增加了多个组氨酸标签，然后转移至E.Coil BL21(DE3)细胞(NEB)的PCDF1质粒中。转化的BL21 DE3细胞随后接种到600mL自动诱导培养基(Formedium)，100μg/mL氨苄青霉素，100μg/mL大霉素，置于2L烧瓶中，20℃，250rpm孵育48h。随后，细胞离心(4000rpm,30分钟，4℃)成球并在-20℃下冷冻，直到需要裂解时才解冻。用100mM Tris-HCl pH7.5、10mM咪唑、1mM PMSF、0.2mg/mL溶菌酶、2mM MgCl2、2.5U/mL(NEB)裂解细胞。溶菌酶在37℃下裂解1小时，然后在冰上超声。然后通过离心(18000rpm，25分钟，4℃)澄清裂解液，用0.45μm注射器过滤器过滤，并应用于5ml HisTrap FF Crudenickel亲和力柱(GE Healthcare)。采用AKTA自动净化系统进行净化。使用两种缓冲液，洗涤缓冲液(缓冲液A)为100mM Tris-HCl pH7.5,10mM咪唑和洗脱缓冲液，洗脱缓冲液(缓冲液B)为100mM Tris-HCl(pH 7.5)1M咪唑。用99％缓冲液A和1％缓冲液B对色谱柱进行20柱体积(CVs)的洗涤，10mL馏分中收集洗涤馏分。随后，用超过10CVs的洗脱相，缓冲液B的梯度从1％增加到100％，收集了2mL洗脱馏分。2mL洗脱组分通过SDS-PAGE和快速考马斯蓝染色(Expedeon)进行分析。CAR酶可见条带最大的部分被合并并透析。将纯化的蛋白质等量用液氮快速冷冻，并储存在-80℃。
实施例1
在1L 100mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g (100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCAR(4000U),0.92gATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节pH为1。真空蒸除溶剂得白色固体。白色固体在40毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐3.7克，产率14.5％，纯度98.6％。
利用质谱对实施例1中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.11[M+H]+。
利用核磁共振对实施例1中得到的脱羧肌肽二盐酸盐进行检测，得到1H NMR(400MHz,D2O)δ＝8.66(s,1H),7.34(s,1H),3.56(t,J＝6.5,2H),3.28(t,J＝6.5,2H),3.00(t,J＝6.5,2H),2.70(t,J＝6.5,2H)。
实施例2
在1L 100mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARA762V(SEQ ID NO：2，4000U),0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在300毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐23.6克，产率92.5％，纯度98.7％。
利用质谱对实施例2中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.11[M+H]+。
实施例3
在1L 100mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARQ365N(SEQ ID NO：3，4000U),0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在70毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐6.3克，产率24.7％，纯度98.7％。
利用质谱对实施例3中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.20[M+H]+。
实施例4
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARR371H(SEQ ID NO：4，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在120毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐11.2克，产率43.9％，纯度98.6％。
利用质谱对实施例4中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.07[M+H]+。
实施例5
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARE489D(SEQ ID NO：5，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在210毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐20.6克，产率80.8％，纯度98.7％。
利用质谱对实施例5中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.01[M+H]+。
实施例6
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30-40℃加入粗酶NiCARA675T(SEQ ID NO：6，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在210毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐20.3克，产率79.6％，纯度98.2％。
利用质谱对实施例6中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.16[M+H]+。
实施例7
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARD256R(SEQ ID NO：7，4000U),0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在200毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐19.4克，产率76.1％，纯度99.2％。
利用质谱对实施例7中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.20[M+H]+。
实施例8
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(SEQ ID NO：8，100mmol)，0.95g MgCl2(10mM)。恒温30-40℃加入粗酶NiCARY485F(SEQ ID NO：8，4000U),0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在240毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐23.7克，产率92.9％，纯度98.7％。
利用质谱对实施例8中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.03[M+H]+。
实施例9
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30-40℃加入粗酶NiCART580C(SEQ ID NO：9，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体进在230毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐22.4克，产率87.8％，纯度98.3％。
利用质谱对实施例9中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.13[M+H]+。
实施例10
在1L100 mM pH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARG395P(SEQ ID NO：10，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在190毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐18.6克，产率72.9％，纯度98.9％。
利用质谱对实施例10中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.11[M+H]+。
实施例11
在1L100 mMpH＝9的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30～40℃加入粗酶NiCARG866D(SEQ ID NO：11，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在200毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐19.7克，产率77.2％，纯度99.1％。
利用质谱对实施例11中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.13[M+H]+。
实施例12
在1L100 mM pH＝8的碳酸钠缓冲溶液中依次加入组胺11.1g(100mmol)，Boc-β-丙氨酸18.9g(100mmol)，0.95g MgCl2(10mM)。恒温30-40℃加入粗酶NiCAR-7(4000U),0.92gATP(5mM)，33℃缓慢搅拌反应24小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在230毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐22.6克，产率88.6％，纯度99.1％。
利用质谱对实施例12中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.01[M+H]+。
实施例13
在1L100 mM pH＝8的碳酸钠缓冲溶液中依次加入化合物1组胺11.1g(100mmol)，Boc-β-丙氨酸20.8g(110mmol)，0.95gMgCl2(10mM))。恒温30～40℃加入粗酶NiCARD256R(SEQID NO：7，4000U)，0.92g ATP(5mM)，33℃缓慢搅拌反应40小时。反应结束后，离心、上清液纳滤除盐。除盐后的母液中加入2N盐酸，调节PH为1。真空蒸除溶剂得白色固体。白色固体在250毫升甲醇：二氯甲烷＝1:4的混合溶液中搅拌30分钟，静置10分钟，过滤得纯产品脱羧肌肽二盐酸盐24.2克，产率94.9％，纯度99.1％。
利用质谱对实施例13中得到的脱羧肌肽二盐酸盐进行检测，得到MS(ESI):m/z:183.25[M+H]+。
序列表
<110> 深圳瑞德林生物技术有限公司
<120> 一种羧酸还原酶突变体及酶法合成脱羧肌肽的方法
<130> S21P003413
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
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Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
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Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
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Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
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Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
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Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
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Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
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Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
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Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
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Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
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Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
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Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
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Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
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Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
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Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
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Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
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Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
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Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
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Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
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Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
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Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
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Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
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Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
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Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
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Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
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Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
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Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
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Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
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Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
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His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
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Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
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Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
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Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
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Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
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Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
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His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
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Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
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Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
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Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
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Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
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Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
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<210> 2
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
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Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
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Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
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Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
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Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
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Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
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Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
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Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
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Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
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Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
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Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
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Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
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Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
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Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
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Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
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Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
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Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
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Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Val Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
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His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 3
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Asn Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 4
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg His Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 5
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Asp Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 6
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Thr Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 7
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Arg
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 8
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Phe Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 9
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Cys Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 10
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Pro Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 11
<211> 1005
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ser His His His His His His Gly Thr Met Ala Val Asp Ser Pro
1 5 10 15
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln Leu Phe Ala Glu Asp Glu
20 25 30
Gln Val Lys Ala Ala Arg Pro Leu Glu Ala Val Ser Ala Ala Val Ser
35 40 45
Ala Pro Gly Met Arg Leu Ala Gln Ile Ala Ala Thr Val Met Ala Gly
50 55 60
Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg Ala Phe Glu Leu Asn Thr
65 70 75 80
Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg Leu Leu Pro Arg Phe Glu
85 90 95
Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg Val Gly Glu Val Ala Ala
100 105 110
Ala Trp His His Asp Pro Glu Asn Pro Leu Arg Ala Gly Asp Phe Val
115 120 125
Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr Ala Thr Leu Asp Leu Ala
130 135 140
Asp Ile His Leu Gly Ala Val Thr Val Pro Leu Gln Ala Ser Ala Ala
145 150 155 160
Val Ser Gln Leu Ile Ala Ile Leu Thr Glu Thr Ser Pro Arg Leu Leu
165 170 175
Ala Ser Thr Pro Glu His Leu Asp Ala Ala Val Glu Cys Leu Leu Ala
180 185 190
Gly Thr Thr Pro Glu Arg Leu Val Val Phe Asp Tyr His Pro Glu Asp
195 200 205
Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala Arg Arg Arg Leu Ala Asp
210 215 220
Ala Gly Ser Leu Val Ile Val Glu Thr Leu Asp Ala Val Arg Ala Arg
225 230 235 240
Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe Val Pro Asp Thr Asp Asp
245 250 255
Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro
260 265 270
Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala Ala Thr Met Trp Gln Gly
275 280 285
Asn Ser Met Leu Gln Gly Asn Ser Gln Arg Val Gly Ile Asn Leu Asn
290 295 300
Tyr Met Pro Met Ser His Ile Ala Gly Arg Ile Ser Leu Phe Gly Val
305 310 315 320
Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala Ala Lys Ser Asp Met Ser
325 330 335
Thr Leu Phe Glu Asp Ile Gly Leu Val Arg Pro Thr Glu Ile Phe Phe
340 345 350
Val Pro Arg Val Cys Asp Met Val Phe Gln Arg Tyr Gln Ser Glu Leu
355 360 365
Asp Arg Arg Ser Val Ala Gly Ala Asp Leu Asp Thr Leu Asp Arg Glu
370 375 380
Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu Gly Gly Arg Phe Leu Val
385 390 395 400
Ala Val Val Gly Ser Ala Pro Leu Ala Ala Glu Met Lys Thr Phe Met
405 410 415
Glu Ser Val Leu Asp Leu Pro Leu His Asp Gly Tyr Gly Ser Thr Glu
420 425 430
Ala Gly Ala Ser Val Leu Leu Asp Asn Gln Ile Gln Arg Pro Pro Val
435 440 445
Leu Asp Tyr Lys Leu Val Asp Val Pro Glu Leu Gly Tyr Phe Arg Thr
450 455 460
Asp Arg Pro His Pro Arg Gly Glu Leu Leu Leu Lys Ala Glu Thr Thr
465 470 475 480
Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Glu Ile Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile Val Ala Glu Leu Glu His
500 505 510
Asp Arg Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Val Ala His Leu Glu Ala Val Phe Ala Ser
530 535 540
Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Asp Asp Ala Leu Arg Gly Arg
565 570 575
Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala Glu Ser Ile Gln Arg Ile
580 585 590
Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu Ile Pro Arg Asp Phe Leu
595 600 605
Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn Gly Leu Leu Ser Gly Ile
610 615 620
Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu Arg Tyr Gly Ala Gln Leu
625 630 635 640
Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly Gln Ala Asp Glu Leu Leu
645 650 655
Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro Val Leu Glu Thr Val Ser
660 665 670
Arg Ala Ala Lys Ala Met Leu Gly Val Ala Ser Ala Asp Met Arg Pro
675 680 685
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
690 695 700
Phe Ser Asn Leu Leu His Glu Ile Phe Gly Val Glu Val Pro Val Gly
705 710 715 720
Val Val Val Ser Pro Ala Asn Glu Leu Arg Asp Leu Ala Asn Tyr Ile
725 730 735
Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg Pro Thr Phe Thr Ser Val
740 745 750
His Gly Gly Gly Ser Glu Ile Arg Ala Ala Asp Leu Thr Leu Asp Lys
755 760 765
Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala Asp Ser Ile Pro His Ala
770 775 780
Pro Val Pro Ala Gln Thr Val Leu Leu Thr Gly Ala Asn Gly Tyr Leu
785 790 795 800
Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu Arg Leu Asp Lys Thr Gly
805 810 815
Gly Thr Leu Ile Cys Val Val Arg Gly Ser Asp Ala Ala Ala Ala Arg
820 825 830
Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly Asp Pro Gly Leu Leu Glu
835 840 845
His Tyr Gln Gln Leu Ala Ala Arg Thr Leu Glu Val Leu Ala Gly Asp
850 855 860
Ile Asp Asp Pro Asn Leu Gly Leu Asp Asp Ala Thr Trp Gln Arg Leu
865 870 875 880
Ala Glu Thr Val Asp Leu Ile Val His Pro Ala Ala Leu Val Asn His
885 890 895
Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala
900 905 910
Glu Ile Val Arg Leu Ala Ile Thr Ala Arg Arg Lys Pro Val Thr Tyr
915 920 925
Leu Ser Thr Val Gly Val Ala Asp Gln Val Asp Pro Ala Glu Tyr Gln
930 935 940
Glu Asp Ser Asp Val Arg Glu Met Ser Ala Val Arg Val Val Arg Glu
945 950 955 960
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
965 970 975
Leu Arg Glu Ala His Asp Leu Cys Gly Leu Pro Val Ala Val Phe Arg
980 985 990
Ser Asp Met Ile Leu Ala His Ser Arg Met Arg Ala Ser
995 1000 1005
<210> 12
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 13
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggtcgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 14
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt ataacagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 15
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt catagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 16
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggatgtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 17
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cgaccaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 18
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatcgtga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 19
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attttaaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 20
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgtgt 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 21
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgccgggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattggcga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
<210> 22
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atgagccatc atcatcatca tcatggcacc atggcggtgg atagcccgga tgaacgtctg 60
cagcgtcgta ttgcgcagct gtttgcggaa gatgaacagg tgaaagcagc acgcccgctg 120
gaagcggtta gcgcagcggt gagcgcaccg ggtatgcgtc tggcccagat tgcggcgacc 180
gtgatggcgg gctatgcgga tcgtccggca gcgggtcagc gtgcgtttga actgaacacc 240
gatgatgcga ccggccgtac cagcctgcgt ctgctgccgc gttttgaaac cattacctat 300
cgtgaactgt ggcagcgtgt gggtgaagtt gcggcagcgt ggcatcacga tccggaaaat 360
ccgctgcgtg cgggcgattt tgtggcgctg ctgggcttta ccagcattga ttatgcgacc 420
ctggatctgg ccgatattca tctgggcgcg gtgaccgttc cgctgcaggc gagcgcagca 480
gtcagccaac tgattgcgat tctgaccgaa acgagtccgc gcctgctggc atctaccccg 540
gaacatctgg atgcggcggt ggaatgtctg ctggcaggta cgacgccgga acgcctggtg 600
gtgtttgatt atcatccgga agatgatgat cagcgtgcgg cgtttgaaag cgcgcgtcgt 660
cgtctggccg atgcgggcag cctggtgatt gtggaaaccc tggatgcggt gcgtgcgcgt 720
ggtcgtgatc tgccggctgc tccgctgttt gtgccggata ccgatgatga tccgctggcc 780
ctgctgattt atacctctgg tagcacgggt acgccgaaag gcgccatgta taccaaccgc 840
ctggcagcaa cgatgtggca aggtaacagc atgctgcagg gcaatagcca gcgtgtgggc 900
attaacctga actatatgcc gatgagccat attgcgggcc gtattagcct gtttggcgtg 960
ctggcccgtg gtggcaccgc gtattttgcg gcgaaaagcg atatgagcac cctgtttgaa 1020
gatattggcc tggtgcgtcc gaccgaaatt ttttttgtgc cgcgtgtgtg cgatatggtg 1080
tttcagcgtt atcagagcga actggatcgt cgtagcgtgg cgggtgcgga tctggatacc 1140
ctggatcgtg aagtgaaagc ggatctgcgt cagaactatc tgggcggtcg ttttctggtg 1200
gcggtggtgg gtagcgcacc gctggccgcg gaaatgaaaa cctttatgga aagcgtgctg 1260
gatctgccgc tgcatgatgg ctatggcagc accgaagcgg gtgcgagcgt gctgctggat 1320
aaccagattc agcgtccgcc ggtgctggat tataaactgg tggacgtccc ggaactgggc 1380
tattttcgta ccgatcgtcc gcatccgcgt ggcgaactgc tgctgaaagc ggaaaccacc 1440
attccgggct attataaacg tccggaagtg accgcggaaa tttttgatga agatggcttc 1500
tataaaaccg gcgatattgt ggcggaactg gaacatgatc gtctggtgta tgtggatcgt 1560
cgcaacaacg tgctgaaact gagccagggc gaatttgtga ccgtggcgca tctggaagcg 1620
gtgtttgcga gcagcccgct gattcgtcag atttttatct acggctctag tgaacgctct 1680
tatctgctgg cagtgattgt gccgaccgat gatgccctgc gtggccgtga taccgcgacc 1740
ctgaaaagcg cgctggccga aagcattcag cgtattgcga aagatgcgaa cctgcagccg 1800
tatgaaattc cgcgtgattt tctgattgaa accgaaccgt tcaccattgc gaacggcctg 1860
ctgtctggca ttgcgaaact gctgcgtccg aacctgaaag aacgttatgg cgcgcagctg 1920
gaacaaatgt ataccgatct ggccaccggc caggcggatg aactgctggc cctgcgtcgt 1980
gaagcggcgg atctgccggt tctggaaacc gttagccgtg cggcgaaagc catgctgggt 2040
gtggcgagcg cggatatgcg tccggatgcg cattttaccg atctgggcgg cgatagcctg 2100
agcgccctga gctttagcaa cctgctgcat gaaatttttg gcgtggaagt gccggtgggt 2160
gtggttgtga gcccggcaaa cgaactgcgt gacctggcca actatattga agcggaacgt 2220
aacagcggcg cgaaacgtcc gacctttacc agcgtgcatg gcggcggtag cgaaattcgt 2280
gcggccgatc tgaccctgga taaatttatt gatgcgcgta ccctggccgc agcggatagc 2340
attccgcatg caccggttcc ggcacagacc gtcctgctga cgggcgcaaa tggctatctg 2400
ggccgttttc tgtgcctgga atggctggaa cgtctggata aaaccggtgg caccctgatt 2460
tgcgtggtgc gtggcagcga tgcggcggca gcccgtaaac gcctggatag cgcgtttgat 2520
agcggcgatc cgggcctgct ggaacattat cagcagctgg ccgcacgcac cctggaagtt 2580
ctggccggtg atattgatga tccgaacctg ggcctggatg atgccacctg gcagcgtctg 2640
gccgaaaccg tggatctgat tgtgcacccg gctgctctgg tgaatcatgt gctgccgtat 2700
acccagctgt ttggcccgaa cgttgtgggc accgcggaaa tcgttcgtct ggctattacc 2760
gcgcgtcgta aaccggtgac ctatctgagc accgtgggcg tggcggatca ggttgatccg 2820
gcggaatatc aggaagatag cgacgtccgc gaaatgagcg cagtccgcgt cgttcgcgaa 2880
agttatgcaa acggttatgg taacagcaaa tgggcgggtg aagtgctgct gcgtgaagcg 2940
catgatctgt gcggtctgcc ggtggcggtg tttcgtagcg atatgattct ggcccatagc 3000
cgtatgcggg ccagctga 3018
Claims (10)
1.一种酶法合成脱羧肌肽的方法，其特征在于，包括：
将组胺、氨基保护的β-丙氨酸、还原酶、活化剂与辅酶在碱性溶液中反应，脱保护后，得到脱羧肌肽或其盐；
所述还原酶包括野生型NiCAR和/或NiCAR突变体；
野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；
NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。
2.根据权利要求1所述的方法，其特征在于，所述NiCAR突变体的氨基酸序列如SEQ IDNO：2～11任一项所示。
3.根据权利要求1所述的方法，其特征在于，以室温一分钟转化1μmol的底物所需酶量为U，所述还原酶的用量为1000～10000U。
4.根据权利要求1所述的方法，其特征在于，所述活化剂选自镁盐；所述活化剂的摩尔数为组胺摩尔数的5％～20％；所述辅酶选自腺苷三磷酸；所述辅酶的摩尔数为组胺摩尔数的1％～10％。
5.根据权利要求1所述的方法，其特征在于，所述碱性溶液的pH值为8～10；所述碱性溶液中的碱性物质选自碳酸氢钠、碳酸氢钾、碳酸钠、碳酸钾、碳酸锂、氢氧化钠、氢氧化钾与氢氧化锂中的一种或多种。
6.根据权利要求1所述的方法，其特征在于，所述反应的温度为30℃～40℃；所述反应的时间为20～30h。
7.一种羧酸还原酶突变体，所述羧酸还原酶突变体为NiCAR突变体，其野生型NiCAR的氨基酸序列如SEQ ID NO：1所示；
所述NiCAR突变体包括如下突变位点中的至少一个：A762V、Q365N、R371H、E489D、A675T、D256R、Y485F、T580C、G395P与G866D。
8.权利要求7所述的羧酸还原酶突变体在催化合成酰胺键中的应用。
9.编码权利要求7所述羧酸还原酶突变体的核酸。
10.根据权利要求9所述的核酸，其特征在于，其序列如SEQ ID NO：13～22任一项所示。
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Address after: 518000 floor 3, building 2, Shenzhen biological incubation base, No. 10, Gaoxin Zhongyi Road, Maling community, Yuehai street, Nanshan District, Shenzhen, Guangdong Applicant after: SHENZHEN READLINE BIOTECHNOLOGY Co.,Ltd. Address before: 518000 Room 101, 1st floor, building 2, Shenzhen biological incubation base, No.10, Gaoxin middle 1st Road, Maling community, Yuehai street, Nanshan District, Shenzhen City, Guangdong Province Applicant before: SHENZHEN READLINE BIOTECHNOLOGY Co.,Ltd. |
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