CN111718915A - 一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用 - Google Patents
一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用 Download PDFInfo
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Abstract
本发明涉及生物科技技术领域,提供了一种提高酶活性的烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及在β‑烟酰胺单核苷酸合成中的应用。所述烟酰胺磷酸核糖转移酶突变体中,野生型烟酰胺磷酸核糖转移酶的氨基酸序列中选自156、174、203、231、242、258、272和405位的氨基酸相当位置的氨基酸中的1个或2个的氨基酸残基被其他氨基酸残基取代。本发明公开的烟酰胺磷酸核糖转移酶突变体提高了烟酰胺磷酸核糖转移酶的酶活,能够促进β‑烟酰胺单核苷酸的合成,有利于β‑烟酰胺单核苷酸的生产。
Description
技术领域
本发明涉及生物科技技术领域,具体涉及一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用。
背景技术
NMN是人体内固有的物质,也富含在一些水果和蔬菜中。在人体中NMN是NAD+的前体,其功能是通过NAD+体现。NAD+又叫辅酶,全称烟酰胺腺嘌呤二核苷酸,存在每一个细胞中参与上千项反应,NMN对人体细胞有重要的生理功能,能在细胞中天然合成,也可以来源于多种食物,包括西兰花、卷心菜、黄瓜、毛豆、鳄梨等。在人体中NMN是合成NAD+的前体,其生理功能主要通过提高NAD+水平来体现。NAD+又叫辅酶Ⅰ,全称烟酰胺腺嘌呤二核苷酸。NAD+在细胞中,不仅作为辅酶也作为多种信号反应的底物,参与几百项反应。华盛顿大学医学院的科学家在2016年发表的一篇论文中指出,小鼠摄取溶解NMN的饮用水后,10分钟内NMN在血液中的浓度逐渐上升,并且在30分钟内,NMN随血液循环进入多个组织中,并在组织中合成NAD+,提升NAD+水平。
β-烟酰胺单核苷酸(β-Nicotinamide mononuclotide,NMN),是哺乳动物体内烟酰;胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide,NAD+)合成途径的重要中间体;近年研究证明,NMN具有显著的抗衰老功能。
传统的NMN的生产是采用化学合成,以烟酰胺核糖为原料,用三氯氧磷进行磷酸化得到,然而化学合成磷酸化特异性不高,导致产品中杂质过多,分离纯化极其困难,总体收率很低;同时有机溶剂使用量大,环境污染严重,比如,2002年,Tanimori等人用乙酰基保护的核糖与烟酰胺在TMSOTf的催化下发生缩合反应;又比如2004年Palmarisa等人使用硅烷化试剂对烟酰胺进行硅烷化,因而,目前NMN需要采用生物酶法来制备,其中烟酰胺磷酸核糖转移酶是整个反应的限速酶,但是现有的烟酰胺磷酸核糖转移酶活性太低,使得NMN的生产效率不理想。
发明内容
有鉴于此,本发明的目的在于提供一种提高酶活性的烟酰胺磷酸核糖转移酶突变体及其应用,所述烟酰胺磷酸核糖转移酶突变体,提高了烟酰胺磷酸核糖转移酶的酶活,能够促进β-烟酰胺单核苷酸的合成。
为解决上述技术问题,本发明提供了以下技术方案:
一种提高酶活性的烟酰胺磷酸核糖转移酶的单点突变体,所述烟酰胺磷酸核糖转移酶的单点突变体选自突变体(1)~(8)中的任意一种:
(1)突变体T156G,氨基酸序列如SEQ ID NO:1所示;
(2)突变体D174T,氨基酸序列如SEQ ID NO:2所示;
(3)突变体A203L,氨基酸序列如SEQ ID NO:3所示;
(4)突变体F231Q,氨基酸序列如SEQ ID NO:4所示;
(5)突变体T242M,氨基酸序列如SEQ ID NO:5所示;
(6)突变体R258K,氨基酸序列如SEQ ID NO:6所示;
(7)突变体D272L,氨基酸序列如SEQ ID NO:7所示;
(8)突变体K405P,氨基酸序列如SEQ ID NO:8所示。
本发明还提供了所述的烟酰胺磷酸核糖转移酶的单点突变体的编码序列为:
突变体T156G的编码序列如SEQ ID NO:37所示;
突变体D174T的编码序列如SEQ ID NO:38所示;
突变体A203L的编码序列如SEQ ID NO:39所示;
突变体F231Q的编码序列如SEQ ID NO:40所示;
突变体T242M的编码序列如SEQ ID NO:41所示;
突变体R258K的编码序列如SEQ ID NO:42所示;
突变体D272L的编码序列如SEQ ID NO:43所示;
突变体K405P的编码序列如SEQ ID NO:44所示。
本发明还提供了一种提高酶活性的烟酰胺磷酸核糖转移酶的双点突变体,所述突变体选自突变体(M1)~(M4)中的任意一种:
(M1)突变体D174T-F231Q,氨基酸序列如SEQ ID NO:9所示;
(M2)突变体R258K-D272L,氨基酸序列如SEQ ID NO:11所示;
(M3)突变体D174T-K405P,氨基酸序列如SEQ ID NO:13所示;
(M4)突变体F231Q-R258K,氨基酸序列如SEQ ID NO:15所示。
本发明提供了所述的烟酰胺磷酸核糖转移酶的双点突变体的编码序列为:
突变体D174T-F231Q的编码序列如SEQ ID NO:10所示;
突变体R258K-D272L的编码序列如SEQ ID NO:12所示;
突变体D174T-K405P的编码序列如SEQ ID NO:14所示;
突变体F231Q-R258K的编码序列如SEQ ID NO:16所示;
本发明还提供了包括上述单点突变体编码序列或双点突变体编码序列的重组载体。
本发明还提供了包括所述的重组载体的重组菌株。
本发明还提供了一种上述烟酰胺磷酸核糖转移酶单点突变体、双点突变体、重组载体或重组菌株在合成β-烟酰胺单核苷酸中的应用。
优选的,所述应用包括以下步骤:
以PRPP和烟酰胺为底物,利用制备得到的所述烟酰胺磷酸核糖转移酶突变体作为催化用酶,催化合成β-烟酰胺单核苷酸。
优选的,所述催化合成β-烟酰胺单核苷酸反应的温度为35-40℃,pH值为7.6-8.2。
优选的,所述催化合成β-烟酰胺单核苷酸反应中添加镁离子作为辅基。
本发明提供了一种提高酶活性的烟酰胺磷酸核糖转移酶突变体,所述突变体提高了烟酰胺磷酸核糖转移酶的酶活,能够促进β-烟酰胺单核苷酸的合成,利用本发明的突变体合成烟酰胺磷酸核糖转移酶浓度达到25.7g/L,提高到5.98倍,有利于β-烟酰胺单核苷酸的生产。
附图说明
图1不同时间下NMN浓度,其中,横坐标代表时间,纵坐标代表NMN浓度。
具体实施方式
本发明旨在提供一种烟酰胺磷酸核糖转移酶(Nampt)的突变体及其在β-烟酰胺单核苷酸(NMN)合成中的应用。
本发明提供了一种提高酶活性的烟酰胺磷酸核糖转移酶的单点突变体,所述烟酰胺磷酸核糖转移酶的单点突变体选自突变体(1)~(8)中的任意一种:
(1)突变体T156G,氨基酸序列如SEQ ID NO:1所示;
(2)突变体D174T,氨基酸序列如SEQ ID NO:2所示;
(3)突变体A203L,氨基酸序列如SEQ ID NO:3所示;
(4)突变体F231Q,氨基酸序列如SEQ ID NO:4所示;
(5)突变体T242M,氨基酸序列如SEQ ID NO:5所示;
(6)突变体R258K,氨基酸序列如SEQ ID NO:6所示;
(7)突变体D272L,氨基酸序列如SEQ ID NO:7所示;
(8)突变体K405P,氨基酸序列如SEQ ID NO:8所示。
本发明还提供了一种提高酶活性的烟酰胺磷酸核糖转移酶的双点突变体,所述突变体选自突变体(M1)~(M4)中的任意一种:
(M1)突变体D174T-F231Q,氨基酸序列如SEQ ID NO:9所示;
(M2)突变体R258K-D272L,氨基酸序列如SEQ ID NO:11所示;
(M3)突变体D174T-K405P,氨基酸序列如SEQ ID NO:13所示;
(M4)突变体F231Q-R258K,氨基酸序列如SEQ ID NO:15所示。
在本发明中,所述烟酰胺磷酸核糖转移酶单点突变体和双点突变体是在野生型烟酰胺磷酸核糖转移酶的基础上进行突变而得到。在本发明中,该突变是指:在野生型烟酰胺磷酸核糖转移酶的氨基酸序列中选自156、174、203、231、242、258、272和405位的氨基酸相当位置的氨基酸中的1个或2个的氨基酸残基被其他氨基酸残基取代。本发明通过将野生型烟酰胺磷酸核糖转移酶进行突变,改变了野生型烟酰胺磷酸核糖转移酶活性,提高了酶活,加强了该酶合成β-烟酰胺单核苷酸的能力。
在本发明中,对野生型烟酰胺磷酸核糖转移酶的来源没有限制,可用于合成β-烟酰胺单核苷酸即可。本发明野生型烟酰胺磷酸核糖转移酶优选由SEQ ID NO:35所示氨基酸序列构成,由SEQ ID NO:36所示核苷酸序列编码得到。
本发明还提供了上述烟酰胺磷酸核糖转移酶单点突变体的编码序列,所述突变体T156G的编码序列如SEQ ID NO:37所示;所述突变体D174T的编码序列如SEQ ID NO:38所示;所述突变体A203L的编码序列如SEQ ID NO:39所示;所述突变体F231Q的编码序列如SEQID NO:40所示;所述突变体T242M的编码序列如SEQ ID NO:41所示;所述突变体R258K的编码序列如SEQ ID NO:42所示;所述突变体D272L的编码序列如SEQ ID NO:43所示;所述突变体K405P的编码序列如SEQ ID NO:44所示。
本发明还提供了上述烟酰胺磷酸核糖转移酶双点突变体的编码序列,所述突变体D174T-F231Q的编码序列如SEQ ID NO:10所示;所述突变体R258K-D272L的编码序列如SEQID NO:12所示;所述突变体D174T-K405P的编码序列如SEQ ID NO:14所示;所述突变体F231Q-R258K的编码序列如SEQ ID NO:16所示。
本发明还提供了包括上述单点突变体编码序列或双点突变体编码序列的重组载体。本发明所述重组载体优选的以pET30a作为原始表达载体;所述单点突变体或双点突变体的编码序列优选的***在质粒pET30a上的NcoI和BamHI酶切位点之间。本发明对构建重组载体的方法没有特殊限制,采用本领域常规方法即可。
本发明还提供了一种包括上述方案所述重组载体的重组菌株。所述重组菌优选的以大肠杆菌作为宿主菌;所述大肠杆菌优选的为E.coliBL21(DE3)。本发明对所述重组菌株的构建方法没有特殊限制,采用本领域常规方法即可。
本发明还提供了上述方案所述烟酰胺磷酸核糖转移酶单点突变体、烟酰胺磷酸核糖转移酶双点突变体、重组载体或重组菌株在合成β-烟酰胺单核苷酸中的应用。
本发明的具体实施过程中,以PRPP和烟酰胺为底物,所述的烟酰胺磷酸核糖转移酶突变体或其重组菌作为催化用酶,催化合成β-烟酰胺单核苷酸。本发明中,所述催化合成β-烟酰胺单核苷酸反应中底物初始浓度优选为90mM-110mM,更优选为100mM;所述调节pH值的试剂优选为PBS缓冲液。本发明所述催化合成β-烟酰胺单核苷酸反应的温度优选为35-40℃,更优选为37℃;所述催化合成β-烟酰胺单核苷酸反应的pH值优选为7.6-8.2,更优选为8.0。
本发明优选的在催化合成β-烟酰胺单核苷酸反应中添加金属离子作为辅基;所述的金属离子优选为镁离子。在本发明的具体实施过程中,本发明对所述底物或辅基的来源没有特殊限定,采用本领域常规市售产品即可。
为进一步详细说明本发明,下面结合具体实施例对本发明的技术方案进行详细描述,但实施例的具体描述并不限定本发明,通过采用了其他方式而达到本发明的突变效果,都属于本发明的保护范围。
本发明实施例所用的主要试剂中,所述的限制性内切酶和DNA连接酶均购自thermofisher公司,高保真DNA聚合酶KOD购自TOYOBO公司,质粒抽提试剂盒、DNA凝胶回收试剂盒购自上海捷瑞生物工程公司。大肠杆菌表达宿主BL21(DE3)和表达载体pET30a购自Merck公司。
本发明实施例中,未特别说明的各种分子生物学操作,包括PCR条件,DNA酶切条件,DNA连接条件,感受态细胞制备和转化方法,DNA回收纯化等操作,按照购买的酶或试剂盒的说明书进行操作或者按照《分子克隆实验指南》操作即可。
实施例1野生型Nampt表达载体的构建
根据GenBank数据库中Luteibactersp.UNCMF366Tsu5.1烟酰胺磷酸核糖转移酶的编码序列(GenBank:SFW68293,SEQ ID NO:36),委托苏州金唯智生物科技有限公司合成基因片段,命名为Nampt基因。为了便于后续克隆,基因片段5'端增加CC两个碱基,形成NcoI酶切位点,3'端增加GGATCC六个碱基,形成BamHI位点。
将合成的Nampt基因和pET30a载体分别用NcoI和BamHI进行双酶切,分别用DNA凝胶试剂盒回收片段,用T4DNA连接酶连接后,转化大肠杆菌DH5a,用卡那霉素抗性LB平板筛选,得到克隆子。
挑取8个克隆子,用质粒提取试剂盒抽提质粒后,由苏州金唯智生物科技有限公司测序,选择正确***载体且无突变的克隆子,得到Nampt表达载体,命名pET30a-Nampt。
实施例2 Nampt的表达和酶的制备
将实施例1中得到的表达载体pET30a-Nampt转化到大肠杆菌BL21(DE3)中,得到能高表达烟酰胺磷酸核糖转移酶的重组菌株。
将高表达烟酰胺磷酸核糖转移酶的重组菌株按常规方法在摇瓶中发酵培养,当发酵液OD600达到0.7时,加入终浓度为1mM的异丙基硫代半乳糖苷IPTG诱导,降温到25℃继续培养16小时,4000转离心20分钟,收集菌体,用50mM pH为7.4的PBS缓冲液洗两次,得到含有烟酰胺磷酸核糖转移酶的菌体。
实施例3烟酰胺磷酸核糖转移酶活性的测定
将实施例2收集到的含有烟酰胺磷酸核糖转移酶的菌体称重10g,另加入终浓度为0.5%的十六烷基三甲基溴化铵CTAB通透细胞,加100mM pH为8.0的PBS缓冲液至终体积50mL,得到烟酰胺磷酸核糖转移酶粗酶液。
1mL反应体系中,加入5-磷酸核糖-1-焦磷酸PRPP至终浓度5mM,加入ATP到终浓度1mM,加入镁离子至终浓度16mM,加入粗酶液50μL,加100mM pH为8.0的PBS缓冲液至终体积1mL,得到反应液。
将反应液于37℃反应30分钟,取出250μL反应液,加入100mM pH为8.0的PBS缓冲液250μL和500μL甲醇终止反应。12000转离心5分钟,收集上清液用于HPLC检测。
HPLC检测条件:Alltech LiChrosorb RP-18色谱柱(10mmparticle size,250*4mm),检测波长254nm,检测温度30℃,检测流速1mL/min。流动相A为含有8mM四丁基溴化铵的0.1M KH2PO4(pH6.0),流动相B为体积比A:甲醇=70:30。
根据测定得到的NMN浓度计算Nampt的酶活,并将酶活力单位(U)定义为:37℃,pH8.0条件下,1分钟内催化生成lμmol产物所需的酶量为1U。
实施例4 Nampt基因的定点突变
根据序列比对和结构模拟,对上述氨基酸序列为SEQ ID NO:35的野生型Nampt进行定点突变,得到8个烟酰胺磷酸核糖转移酶的单点突变体:(1)突变体T156G,氨基酸序列如SEQ ID NO:1所示;(2)突变体D174T,氨基酸序列如SEQ ID NO:2所示;(3)突变体A203L,氨基酸序列如SEQ ID NO:3所示;(4)突变体F231Q,氨基酸序列如SEQ ID NO:4所示;(5)突变体T242M,氨基酸序列如SEQ ID NO:5所示;(6)突变体R258K,氨基酸序列如SEQ ID NO:6所示;(7)突变体D272L,氨基酸序列如SEQ ID NO:7所示;(8)突变体K405P,氨基酸序列如SEQ ID NO:8所示;并检测所述单点突变体酶催化活性的变化。
全部突变采用全质粒扩增的方法获得,分别设计突变引物如下表1:
表1扩增8个烟酰胺磷酸核糖转移酶的单点突变体的引物
PCR扩增条件为:95℃30s,95℃15s,55℃30s,68℃5min,30个循环,68℃10min。
PCR结束后,加限制性内切酶DpnI除掉模板质粒,DNA凝胶回收试剂盒回收后,转化大肠杆菌DH5α感受态细胞,利用卡那霉素抗性平板筛选正确转化子。每种转化子挑取5个单克隆,送至苏州金唯智生物科技有限公司测序,确定为正确的突变。
实施例5Nampt单点突变体的活性测定
将实施例4中的正确单点突变质粒转化到大肠杆菌BL21(DE3)中,得到8个包括单点突变的Nampt高表达宿主菌株。
将8个单点突变的Nampt高表达宿主菌株连同野生型Nampt的宿主菌株分别按照实施例2和实施例3的方法,制备得到烟酰胺磷酸核糖转移酶并进行酶活性测定,按每克湿菌体计算酶活单位,得到结果如下表2:
表2烟酰胺磷酸核糖转移酶单点突变体的相对酶活
突变体 | 相对酶活 |
Wt | 100 |
T156G | 121 |
D174T | 124 |
A203V | 178 |
F231Q | 310 |
T242M | 231 |
R258K | 380 |
D272L | 455 |
K405P | 263 |
从表2相对酶活检测结果中可知,本发明提供的8个单点突变都能够提高烟酰胺磷酸核糖转移酶的酶活性。
实施例6Nampt的位点组合突变
从实施例5中得知,有8个位点的单点突变使Nampt活性提高,采用DNA Shuffling方法将这些位点相互组合,具体操作为:
根据Nampt序列设计引物,用于扩增整个基因,具体引物序列为:
F-Nampt-NcoI:GAGCCATGGGAATGCTGTGGGTTATGACCACCCACTC(SEQ ID NO:33)
R-Nampt-BamHI:CACGGATCCTTAACCACCAGCAGCAGCACGTTC(SEQ ID NO:34)
用上述引物对分别扩增T156G,D174T,A203V,F231Q,T242M,R258K,Y273L,K405P共8个突变基因,将得到的DNA片段分别用DNaseI处理2小时后,进行无模板PCR扩增,得到组合突变的Nampt片段,然后通过常规基因克隆方式,构建表达载体,转化到大肠杆菌BL21(DE3)菌株中。
分别挑取转化子进行小量酶制备后,测定其中烟酰胺磷酸核糖转移酶的酶活性,得到四个活性大幅提高的双点突变体:(M1)突变体D174T-F231Q,氨基酸序列如SEQ ID NO:9所示;(M2)突变体R258K-D272L,氨基酸序列如SEQ ID NO:11所示;(M3)突变体D174T-K405P,氨基酸序列如SEQ ID NO:13所示;(M4)突变体F231Q-R258K,氨基酸序列如SEQ IDNO:15所示;对四株双点突变体的重组菌株按照实施例2和3的方法进行酶制备和活性测定,与野生型Nampt酶活性对比可得下表3:
表3烟酰胺磷酸核糖转移酶双点突变体的相对酶活
突变体 | 相对酶活 |
wt | 100 |
M1(D174T,F231Q) | 525 |
M2(R258K,D272L) | 634 |
M3(D174T,K405P) | 567 |
M4(F231Q,R258K) | 357 |
由表3可以看出,本发明提供的烟酰胺磷酸核糖转移酶的双点突变体能够大幅度提高烟酰胺磷酸核糖转移酶的酶活性,单点突变的相互组合,使得活性提高得到叠加。
实施例7烟酰胺磷酸核糖转移酶突变体用于酶催化制备NMN
分别将野生型WT和活性最高的突变体M3按照实施例2的方法培养,制备得到烟酰胺磷酸核糖转移酶。
1L反应体系中,分别加入PRPP和ATP到终浓度100mM,加入镁离子至终浓度20mM,分别加入含有野生型WT或M3突变体的湿菌体40g,加pH为8.0的PBS缓冲液至终体积为1L,得到反应液。
将反应液于37℃反应,维持pH在8.0,每1小时取样500μL,并加入等体积的甲醇,12000转进行离心5分钟,得到上清液。测定上清液中NMN浓度得到附图1。
附图1中不同时间下得到的NMN浓度对应得到表4:
表4不同时间下上清液中NMN浓度
转化时间(h) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
WT(g/L) | 1.4 | 2 | 2.8 | 3.5 | 3.9 | 4 | 4.1 | 4.3 |
M3(g/L) | 5.1 | 10.7 | 15.3 | 16.1 | 19.8 | 23.5 | 24.6 | 25.7 |
可以看出,经过8个小时反应,野生型烟酰胺磷酸核糖转移酶合成NMN浓度为4.3g/L,本发明M3突变体合成的烟酰胺磷酸核糖转移酶浓度达到25.7g/L,提高了5.98倍,取得了很好的效果。
由以上实施例可以看出,本发明提供的烟酰胺磷酸核糖转移酶突变体的酶活显著提高,能够促进β-烟酰胺单核苷酸的合成,利用本发明的突变体合成烟酰胺磷酸核糖转移酶浓度达到25.7g/L,提高了5.98倍,有利于β-烟酰胺单核苷酸的生产。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
序列表
<110> 仙居两山生物科技有限公司
<120> 一种烟酰胺磷酸核糖转移酶突变体、包含该突变体的重组表达载体和重组菌及应用
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Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Asp Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Phe Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Lys His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Asp
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Lys Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 7
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Asp Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Phe Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Arg His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Leu
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Lys Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 8
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Asp Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Phe Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Arg His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Asp
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Pro Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 9
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Thr Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Gln Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Arg His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Asp
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Lys Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 10
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgaca ccccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt caatctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 11
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Asp Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Phe Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Lys His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Leu
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Lys Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 12
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gaaacacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctttgtctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 13
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Thr Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Phe Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Arg His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Asp
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Pro Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 14
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgaca ccccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggcctgaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 15
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Asp Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Gln Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Lys His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Asp
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Lys Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 16
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt caatctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gaaacacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 17
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cccggttacc gttgctaccg gcacctcttg gcacgctcgt c 41
<210> 18
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gacgagcgtg ccaagaggtg ccggtagcaa cggtaaccgg g 41
<210> 19
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ctacctggac accacctctg acaccccggc tgctcagatc ccgttca 47
<210> 20
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tgaacgggat ctgagcagcc ggggtgtcag aggtggtgtc caggtag 47
<210> 21
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ctgctggtct gggtggtatg gttcacctgg ttaacttcct ggg 43
<210> 22
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
cccaggaagt taaccaggtg aaccatacca cccagaccag cag 43
<210> 23
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
acggtgaacc gatggctggt caatctatcc cggctgctga aca 43
<210> 24
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tgttcagcag ccgggataga ttgaccagcc atcggttcac cgt 43
<210> 25
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ctgctgaaca ctctaccatc atgtcttggg gtcgtgacca cga 43
<210> 26
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tcgtggtcac gaccccaaga catgatggta gagtgttcag cag 43
<210> 27
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
acgcttaccg taacatgctg aaacacttcg ctaaaccggg ttc 43
<210> 28
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gaacccggtt tagcgaagtg attcagcatg ttacggtaag cgt 43
<210> 29
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ctctggttgc tgttgtttct ttgtcttacg acatctacca cgc 43
<210> 30
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gcgtggtaga tgtcgtaaga caaagaaaca acagcaacca gag 43
<210> 31
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gtgcttggcg tgacgtttgg cctgacccgg ttaccgacca ggg 43
<210> 32
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ccctggtcgg taaccgggtc aggccaaacg tcacgccaag cac 43
<210> 33
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gagccatggg aatgctgtgg gttatgacca cccactc 37
<210> 34
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
cacggatcct taaccaccag cagcagcacg ttc 33
<210> 35
<211> 482
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Met Leu Trp Val Met Thr Thr His Ser Val Ser Tyr Leu Asp Asn Pro
1 5 10 15
Ile Leu Asp Thr Asp Ser Tyr Lys Ala Ser His Trp Leu Gln Tyr Pro
20 25 30
Pro Asn Thr Asp Ala Thr Phe Phe Tyr Val Glu Ser Arg Gly Gly Thr
35 40 45
Tyr Asp Arg Thr Leu Phe Phe Gly Leu Gln Ala Val Leu Lys Ala Arg
50 55 60
Leu Glu Arg Pro Val Thr His Ala Asp Val Asp Glu Ala Arg Asp Phe
65 70 75 80
Phe Ala Ala His Gly Glu Pro Phe Asn Asp Glu Gly Trp Arg Tyr Ile
85 90 95
Val Asp Thr His Gly Gly Arg Leu Pro Val Arg Val Arg Ala Val Pro
100 105 110
Glu Gly Ser Val Val Pro Thr His Gln Ala Leu Val Thr Ile Glu Ser
115 120 125
Thr Asp Pro Arg Thr Tyr Trp Leu Pro Ser Tyr Leu Glu Thr Arg Leu
130 135 140
Leu Arg Leu Trp Tyr Pro Val Thr Val Ala Thr Thr Ser Trp His Ala
145 150 155 160
Arg Gln Thr Ile Ala His Tyr Leu Asp Thr Thr Ser Asp Asp Pro Ala
165 170 175
Ala Gln Ile Pro Phe Lys Leu His Asp Phe Gly Ala Arg Gly Val Ser
180 185 190
Ser Ala Glu Ser Ala Gly Leu Gly Gly Met Ala His Leu Val Asn Phe
195 200 205
Leu Gly Thr Asp Thr Val Ser Gly Val Leu Ala Ala Arg Ala Tyr Tyr
210 215 220
Gly Glu Pro Met Ala Gly Phe Ser Ile Pro Ala Ala Glu His Ser Thr
225 230 235 240
Ile Thr Ser Trp Gly Arg Asp His Glu Val Asp Ala Tyr Arg Asn Met
245 250 255
Leu Arg His Phe Ala Lys Pro Gly Ser Leu Val Ala Val Val Ser Asp
260 265 270
Ser Tyr Asp Ile Tyr His Ala Ile Lys Glu His Trp Gly Lys Thr Leu
275 280 285
Arg Asp Glu Val Ile Ala Ser Gly Ala Thr Val Val Val Arg Pro Asp
290 295 300
Ser Gly Asp Pro Val Glu Val Val His Arg Cys Val Ser Leu Leu Asp
305 310 315 320
Glu Ala Phe Gly Ser Thr Val Asn Gly Lys Gly Tyr Arg Val Leu Asn
325 330 335
His Val Arg Val Ile Gln Gly Asp Gly Val Asn Pro Asp Ser Ile Arg
340 345 350
Ala Ile Leu Glu Arg Ile Thr Thr Ala Gly Tyr Ser Ala Asp Asn Leu
355 360 365
Ala Phe Gly Met Gly Gly Ala Leu Leu Gln Lys Leu Thr Arg Asp Thr
370 375 380
Gln Lys Phe Ala Leu Lys Cys Ser Ala Ala Arg Val Asp Gly Ala Trp
385 390 395 400
Arg Asp Val Trp Lys Asp Pro Val Thr Asp Gln Gly Lys Leu Ser Lys
405 410 415
Arg Gly Arg Met Thr Leu Leu His His Arg Glu Ser Gly Thr Tyr Arg
420 425 430
Thr Val Pro Leu Pro Gly Asp Ala Ile Ala Met Pro Pro Glu Ala Ile
435 440 445
Glu Pro Gly Trp Glu Glu Ala Met Val Thr Val Trp Glu Asn Gly Glu
450 455 460
Pro Val Arg Glu Trp Ser Phe Ala Asp Val Arg Glu Arg Ala Ala Ala
465 470 475 480
Gly Gly
<210> 36
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 37
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccggctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 38
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgaca ccccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 39
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggttc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 40
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt caatctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 41
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcatgtctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 42
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gaaacacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 43
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctttgtctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggaaagaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
<210> 44
<211> 1449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
atgctgtggg ttatgaccac ccactctgtt tcttacctgg acaacccgat cctggacacc 60
gactcttaca aagcttctca ctggctgcag tacccgccga acaccgacgc taccttcttc 120
tacgttgaat ctcgtggtgg tacctacgac cgtaccctgt tcttcggtct gcaggctgtt 180
ctgaaagctc gtctggaacg tccggttacc cacgctgacg ttgacgaagc tcgtgacttc 240
ttcgctgctc acggtgaacc gttcaacgac gaaggttggc gttacatcgt tgacacccac 300
ggtggtcgtc tgccggttcg tgttcgtgct gttccggaag gttctgttgt tccgacccac 360
caggctctgg ttaccatcga atctaccgac ccgcgtacct actggctgcc gtcttacctg 420
gaaacccgtc tgctgcgtct gtggtacccg gttaccgttg ctaccacctc ttggcacgct 480
cgtcagacca tcgctcacta cctggacacc acctctgacg acccggctgc tcagatcccg 540
ttcaaactgc acgacttcgg tgctcgtggt gtttcttctg ctgaatctgc tggtctgggt 600
ggtatggctc acctggttaa cttcctgggt accgacaccg tttctggtgt tctggctgct 660
cgtgcttact acggtgaacc gatggctggt ttctctatcc cggctgctga acactctacc 720
atcacctctt ggggtcgtga ccacgaagtt gacgcttacc gtaacatgct gcgtcacttc 780
gctaaaccgg gttctctggt tgctgttgtt tctgactctt acgacatcta ccacgctatc 840
aaagaacact ggggtaaaac cctgcgtgac gaagttatcg cttctggtgc taccgttgtt 900
gttcgtccgg actctggtga cccggttgaa gttgttcacc gttgcgtttc tctgctggac 960
gaagctttcg gttctaccgt taacggtaaa ggttaccgtg ttctgaacca cgttcgtgtt 1020
atccagggtg acggtgttaa cccggactct atccgtgcta tcctggaacg tatcaccacc 1080
gctggttact ctgctgacaa cctggctttc ggtatgggtg gtgctctgct gcagaaactg 1140
acccgtgaca cccagaaatt cgctctgaaa tgctctgctg ctcgtgttga cggtgcttgg 1200
cgtgacgttt ggcctgaccc ggttaccgac cagggtaaac tgtctaaacg tggtcgtatg 1260
accctgctgc accaccgtga atctggtacc taccgtaccg ttccgctgcc gggtgacgct 1320
atcgctatgc cgccggaagc tatcgaaccg ggttgggaag aagctatggt taccgtttgg 1380
gaaaacggtg aaccggttcg tgaatggtct ttcgctgacg ttcgtgaacg tgctgctgct 1440
ggtggttaa 1449
Claims (10)
1.一种提高酶活性的烟酰胺磷酸核糖转移酶的单点突变体,所述烟酰胺磷酸核糖转移酶的单点突变体选自突变体(1)~(8)中的任意一种:
(1)突变体T156G,氨基酸序列如SEQ ID NO:1所示;
(2)突变体D174T,氨基酸序列如SEQ ID NO:2所示;
(3)突变体A203L,氨基酸序列如SEQ ID NO:3所示;
(4)突变体F231Q,氨基酸序列如SEQ ID NO:4所示;
(5)突变体T242M,氨基酸序列如SEQ ID NO:5所示;
(6)突变体R258K,氨基酸序列如SEQ ID NO:6所示;
(7)突变体D272L,氨基酸序列如SEQ ID NO:7所示;
(8)突变体K405P,氨基酸序列如SEQ ID NO:8所示。
2.权利要求1所述的烟酰胺磷酸核糖转移酶的单点突变体的编码序列,其特征在于,
所述突变体T156G的编码序列如SEQ ID NO:37所示;
所述突变体D174T的编码序列如SEQ ID NO:38所示;
所述突变体A203L的编码序列如SEQ ID NO:39所示;
所述突变体F231Q的编码序列如SEQ ID NO:40所示;
所述突变体T242M的编码序列如SEQ ID NO:41所示;
所述突变体R258K的编码序列如SEQ ID NO:42所示;
所述突变体D272L的编码序列如SEQ ID NO:43所示;
所述突变体K405P的编码序列如SEQ ID NO:44所示。
3.一种提高酶活性的烟酰胺磷酸核糖转移酶的双点突变体,其特征在于,所述突变体选自突变体(M1)~(M4)中的任意一种:
(M1)突变体D174T-F231Q,氨基酸序列如SEQ ID NO:9所示;
(M2)突变体R258K-D272L,氨基酸序列如SEQ ID NO:11所示;
(M3)突变体D174T-K405P,氨基酸序列如SEQ ID NO:13所示;
(M4)突变体F231Q-R258K,氨基酸序列如SEQ ID NO:15所示。
4.权利要求3所述的烟酰胺磷酸核糖转移酶的双点突变体的编码序列,其特征在于,
所述突变体D174T-F231Q的编码序列如SEQ ID NO:10所示;
所述突变体R258K-D272L的编码序列如SEQ ID NO:12所示;
所述突变体D174T-K405P的编码序列如SEQ ID NO:14所示;
所述突变体F231Q-R258K的编码序列如SEQ ID NO:16所示。
5.包括权利要求2所述的单点突变体编码序列或权利要求4所述的双点突变体编码序列的重组载体。
6.包括权利要求5所述重组载体的重组菌株。
7.权利要求1所述的烟酰胺磷酸核糖转移酶单点突变体、权利要求3所述的烟酰胺磷酸核糖转移酶双点突变体、权利要求5所述的重组载体或权利要求6所述的重组菌株在合成β-烟酰胺单核苷酸中的应用。
8.根据权利要求7所述的应用,其特征在于,所述应用包括以下步骤:
以PRPP和烟酰胺为底物,利用所述烟酰胺磷酸核糖转移酶突变体作为催化用酶,催化合成β-烟酰胺单核苷酸。
9.根据权利要求8所述的应用,其特征在于,所述催化合成β-烟酰胺单核苷酸反应的温度为35-40℃,pH值为7.6-8.2。
10.根据权利要求8所述的应用,其特征在于,所述催化合成β-烟酰胺单核苷酸反应中添加镁离子作为辅基。
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