CN101245343A - 新的纤维素酶及其应用 - Google Patents
新的纤维素酶及其应用 Download PDFInfo
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- CN101245343A CN101245343A CNA2007100374599A CN200710037459A CN101245343A CN 101245343 A CN101245343 A CN 101245343A CN A2007100374599 A CNA2007100374599 A CN A2007100374599A CN 200710037459 A CN200710037459 A CN 200710037459A CN 101245343 A CN101245343 A CN 101245343A
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Abstract
本发明涉及一种新的纤维素酶、编码该酶的多核苷酸和经DNA重组技术产生这种酶的方法。本发明还涉及含该纤维素酶的载体和宿主细胞及其在生产简单糖和葡萄糖方面的用途。
Description
技术领域
本发明涉及生物领域。更具体地,本发明涉及一种新的纤维素酶、编码该酶的多核苷酸和经DNA重组技术产生这种酶的方法。本发明还涉及含该纤维素酶的载体和宿主细胞及其在生产简单糖和葡萄糖方面的用途。
背景技术
纤维素是自然界最丰富的可再生的能源。将未经任何化学处理的纤维素生物转化成简单糖或葡萄糖,作为发酵碳源生产酒精,是最理想和有效利用天然纤维素资源的方法之一。
通常认为,生物转化纤维素生成葡萄糖至少需要三种不同的酶的协同作用:(1)葡聚糖内切酶(Endo-1,4-β-D-glucanase,E.C.3.2.1.4,也称之为纤维素内切酶)。它作用于纤维素的非结晶区,随机水解β-1,4-糖苷键生成带非还原端的较短的寡聚糖,(2)葡聚糖外切酶(Exo-1,4-β-D-glucanase,E.C.3.2.1.91),它作用于纤维素分子的非还原末端水解β-1,4-糖苷键产生纤维二糖,(3)β-葡萄糖苷酶(β-D-glucosidase,E.C.3.2.1.21),它将纤维二糖水解为葡萄糖。
目前,研究较为集中的是真菌和细菌的纤维素酶系。丝状真菌中的李氏木霉(T.reesei)和绿色木霉(T.viride)以及热纤梭菌(C.thermocellum)和cellulomonas fimi等物种的纤维素酶系较复杂,有多种亚类的内切酶,外切酶及葡萄糖苷酶[ThomasM.Wood,Biochemical.Society Transactions.1992,20,46-53],例如绿色木霉有6种亚型的内切酶[Beldman,G.et al Eur.J.Biochem.1985,146:301-308]。热纤梭菌的纤维素酶需要同多个蛋白质形成分子量巨大的纤维体(Cellulosome)才能起催化反应[Felix C.R and L.G.Ljiungdahl,Annu.Rev.Microbiol.1993,47:791-819],显然这样复杂的酶系必然会给研究和应用带来极大的困难。另一方面,纯化的单一组分的内切酶和外切酶要么不能水解未经化学处理的天然纤维素生成简单糖,要么水解活力极低,如李氏木霉(T.reesei)的纤维素酶系统中至少需14类纤维素酶的协同作用才能水解将未经化学处理的植物纤维。
传统的观点认为动物没有自己的纤维素酶系,需依赖共生菌的纤维素酶将纤维素水解成单糖,供生命活动需要。但是90年代末发现了白蚁和小龙虾内在的纤维素内切酶[Hirofumi,W.Gaku,T,nathan,L Nature,1998,394:330-331;Keren A.B.et al.,Gene,1999,239,317-324]。此外,在拟南芥中也发现了12种内切酶基因[del Compillo,Curr.Top.Dev.Boil.1999,46:39-61]。动物纤维素酶可能成为新的应用研究的热点。
在地球上每年由固定CO2的光合作用就能形成100亿吨以是上的干植物物质,其中这些物质的组成一半以上是由纤维素,其次是半纤维素(主要由木聚糖所构成)。如果再加上人类活动所造成的废弃的纤维素,如稻草,麦秸等,其存在量则要以天文数字来计算。如何采用生物技术综合利用植物干物质或废弃的纤维素的问题上,纤维素酶起着关键作用。有效地将这些天然的纤维素转化为简单糖是纤维素作为再生能源的关键。目前的纤维素酶还远不能适应将植物纤维素转化为简单糖作为再生能源的需求。因此,本领域迫切需要开发不同来源的具有能高效率水解纤维素的新的纤维素酶,以及新的生产葡萄糖的工艺。
发明内容
本发明的目的就是提供一种新的纤维素酶以及其片段、类似物和衍生物,及其编码序列。
本发明的另一目的是提供生产纤维素酶的方法以及其用途。
本发明的另一目的是提供新的生产简单糖和葡萄糖的工艺。
本发明一方面提供一种分离的纤维素酶,该纤维素酶选自(a)具有SEQ ID NO:2第1-713位氨基酸序列的多肽;(b)具有SEQ ID NO:4第1-723位氨基酸序列的多肽;(c)具有SEQID NO:6第1-722位氨基酸序列的多肽;(d)具有SEQ ID NO:8、9、10、11或12所述多核苷酸序列编码的氨基酸序列的多肽;(e)将SEQ ID NO:2、4、6的氨基酸序列或(d)的氨基酸序列经过1-10个氨基酸残基的取代、缺失或添加而形成的,且具有纤维素酶功能的由(a)、(b)、(c)或(d)衍生的多肽。
在一个优选实施例中,所述纤维素酶的氨基酸序列为SEQ ID NO:2、4或6所示的氨基酸序列。
本发明另一方面涉及一种分离的多核苷酸,该多核苷酸选自:(a)编码本发明分离的纤维素酶的多核苷酸;(b)在严格条件下与(a)所述多核苷酸序列杂交且编码具有纤维素酶活性的蛋白质的多核苷酸。
在一个优选的实施例中,所述纤维素酶选自((a)具有SEQ ID NO:2第1-713位氨基酸序列的多肽;
(b)具有SEQ ID NO:4第1-723位氨基酸序列的多肽;
(c)具有SEQ ID NO:6第1-722位氨基酸序列的多肽;
(d)具有SEQ ID NO:8、9、10、11或12所述多核苷酸序列编码的氨基酸序列的多肽;
(e)将SEQ ID NO:2、4、6的氨基酸序列或(d)的氨基酸序列经过1-10个氨基酸残基的取代、缺失或添加而形成的,且具有纤维素酶功能的由(a)、(b)、(c)或(d)衍生的多肽。
在另一优选实施例中,所述的多核苷酸选自下组:(i)具有SEQ ID NO:1中1-2142位的核苷酸序列;(ii)具有SEQ ID NO:3中1-2172位的核苷酸序列;(iii)具有SEQ ID NO:5中1-2169位的核苷酸序列;(iv)具有SEQ ID NO:8中1-2172位的核苷酸序列;(v)具有SEQ ID NO:9中1-2172位的核苷酸序列;(vi)具有SEQ ID NO:10中1-2169位的核苷酸序列;(vii)具有SEQ ID NO:11中1-2169位的核苷酸序列;或(viii)具有SEQ ID NO:12中1-2169位的核苷酸序列;
在另一方面,本发明涉及一种含有本发明多核苷酸序列的重组表达载体。
在另一方面,本发明涉及一种含有本发明重组载体的转化的宿主细胞。
在又一方面,本发明涉及一种制备本发明纤维素酶的方法,该方法包含:
(a)在适合表达所述纤维素酶的条件下,培养本发明所述的宿主细胞;和
(b)从培养物中分离出所述的纤维素酶。
又一方面,本发明涉及所述的纤维素酶用于生产简单糖的工艺,所述的简单糖是纤维二糖、葡萄糖及其混合物中的用途。
本发明还涉及一种生产简单糖的方法,该方法包括步骤:
(a)用本发明所述的纤维素酶或宿主细胞处理纤维素,从而产生简单糖;
(b)分离出所述的简单糖,所述的简单糖包括纤维二糖、葡萄糖及其混合物。
所述的纤维素材料是未经任何化学预处理的纤维素材料。
本发明还涉及一种提高本发明纤维素酶的酶活力的方法,该方法包括用含NO3 -、HSO3 -、SO4 2-、S2O3 2-、Mn2+、C6H5O7 3-或EDTA的溶液处理所述纤维素酶,从而提高其酶活力。
本发明的其它方面由于本文的技术的公开,对本领域的技术人员而言是显而易见的。
附图说明
图1显示从软体动物福寿螺(Ampullaria crossean)的胃液中分离纯化到的纤维素酶的组份的SDS-PAGE结果。图中,泳道“M”表示分子质量标准,包括兔磷酸化酶B(97.4kD)、牛血清白蛋白(66.2kD)、兔肌动蛋白(43.0kD)、牛碳酸酐酶(31.0kD)和胰蛋白酶抑制剂(20.1kD)。泳道1为纯化的纤维素酶EG65。
图2A显示EG65在15℃在不同pH中培养不同时间所得的相对活力(%)。图2B显示RG65在50℃在不同pH中培养15分钟的相对活力(%)。
图3显示在50℃测量的纯化的EG65在不同时间的相对酶活性。
图4显示甲醇酵母表达载体pPIC9K,共9276个核苷酸,其中,5′AOX1启动子片段:第1-948核苷酸碱基;5′AOX1引物位点:第855-875位碱基;a-因子分泌信号:第949-1218位碱基;a-因子引物位点:第1152-1172位碱基;多克隆位点:第1216-1241位碱基:3′AOX1引物位点:第1327-1347位碱基;3′AOX1转录终止(TT):第1253-1286位碱基;HIS4 ORF:第4514-1980位碱基;卡那霉素抗性基因(Kanamycin):第5743-4928位碱基;3′AOX1片段:第6122-6879位碱基;pBR322起点:第7961-7288位碱基;氨苄青霉素抗性基因(Ampicillin):第8966-8106位碱基。
图5显示用引物F1和R1扩增得到的cDNA片段及其预测的氨基酸序列。
图6显示eg65-a、eg65-b、eg65-c的图谱及测序策略。
图7显示EG65-a、EG65-b、EG65-c蛋白的同源性比较。
图8显示EG-65a的氨基酸序列与其它纤维素酶的氨基酸序列的比较结果。其中,Termite.1:家白蚁(Coptotermes formosanus,BAB40697);Termite.2:达尔文澳白蚁(Mastotermes darwiniensis,CAD54729);Crayfish:鳌虾(Cherax quadricarinatus,AAO61672);A.crossean:EG65-a;Haliotis:自鲍鱼(Haliotis discus,BAC67186)。
图9显示EG65-a、EG65-b和EG65-c的结构域。途中,“Glyco_hydro_9”表示糖苷水解酶第9家族。
图10A-10F显示采用NetNGlyc 1.0进行的EG65-a、EG65-b和EG65-c的O-糖基化和N-糖基化预测,其中图10A-10C分别显示EG65-a、EG65-b和EG65-c的O-糖基化预测位点,而图10D-10F分别显示EG65-a、EG65-b和EG65-c的N-糖基化预测位点。
图11A-11C分别显示EG65-a、EG65-b和EG65-c的SignalP-NN预测结果。图中,较浅曲线代表S分值(S score),较深曲线代表C分值(C score),竖线(丨)代表Y分值(Y score)。图11A显示,在EG65-a中在位置16和17之间存在最有可能的解离位点:VLS-SV;图11B显示,在EG65-b中在位置16和17之间存在最有可能的解离位点:GFG-SI;图11C显示在EG65-c中在位置16和17之间存在最有可能的解离位点:SFA-IT。
图12显示纯化的重组酶的SDS-PAGE分析。图中泳道1为EG65-b-16;泳道2为EG65-b-18;泳道3为EG65-a-137;泳道4为EG65-c-147;M为分子质量标准,包括兔磷酸化酶B(97.4kD)、牛血清白蛋白(66.2kD)、兔肌动蛋白(43.0kD)、牛碳酸酐酶(31.0kD)。左面的凝胶(10%SDS-PAGE)用考马斯亮蓝G-250染色,而右面的凝胶(10%SDS-PAGE)用硝酸银染色。
图13A和图13B显示重组酶EG65-b-16(◆)和EG65-b-148(○)的最适pH(图13A)和最适温度(图13B)。
图14A-14D显示EG65-a-137和EG65-c-147的最适pH和最适温度。其中,图14A和14C分别显示EG65-a-137的最适温度和最适pH,图14B和14D分别显示EG65-c-147的最适温度和最适pH。
图15A和15B分别显示重组酶EG65-b-16(◆)和EG65-b-148(○)的温度稳定性。
图16A和16B分别显示重组酶EG65-b-16(◆)和EG65-b-148(○)的pH稳定性。
图17A显示纯化的EG65的双向凝胶电泳。其中,横向显示IPG条带,pH3-10,L=24cm;纵向为12.5%SDS-PAGE,260×200×1mm。图17B显示用MALDI-TOF多肽质量指纹图谱进行分析所得结果。
图18A和18B分别显示EG65-b1、b2、b3的氨基酸序列比较,和EG65-c1、c2、c3、c4的氨基酸序列比较。
图19显示纯化的EG65的双向凝胶电泳。其中,横向显示IPG条带,pH3-10,L=24cm;纵向为12.5%SDS-PAGE,260×200×1mm。
具体实施方式
如本文所用,“分离的”是指物质从其原始环境中分离出来(如果是天然的物质,原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的,但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开,则为分离纯化的。
如本文所用,“分离的纤维素酶蛋白或多肽”是指纤维素酶基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化该纤维素酶。基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的带。
本发明的纤维素酶可以是重组的、天然的、合成的,优选是重组的。本发明的纤维素酶可以是天然纯化的产物,或是化学合成的产物,或使用重组技术从原核或真核宿主(例如,细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主,本发明的多肽可以是糖基化的,或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括该纤维素酶的片段、衍生物和类似物。如本文所用,术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然纤维素酶相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的多肽,或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成的多肽,或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列)。根据本文的教导,这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中,术语“福寿螺纤维素酶”指具有纤维素酶活性的SEQ ID NO:2第1-713位氨基酸序列、SEQ ID NO:4第1-723位氨基酸序列或SEQ ID NO:6第1-722位氨基酸序列的多肽,以及具有SEQ ID NO:8、9、10、11或12编码的氨基酸序列的多肽;该术语还包括SEQ ID NO:2、4、6序列以及SEQ ID NO:8、9、10、11或12编码的氨基酸序列的变异形式。这些变异形式包括(但并不限于):若干个(通常为1-50个,较佳地1-30个,更佳地1-20个,最佳地1-10个)氨基酸的缺失、插入和/或取代,以及在C末端和/或N末端添加一个或数个(通常为20个以内,较佳地为10个以内,更佳地为5个以内)氨基酸。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能。又比如,在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括福寿螺纤维素酶的活性片段和活性衍生物。
该多肽的变异形式包括:同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与福寿螺纤维素酶DNA杂交的DNA所编码的蛋白。本发明还提供了其他多肽,如包含福寿螺纤维素酶或其片段的融合蛋白。除了几乎全长的多肽外,本发明还包括了福寿螺纤维素酶的可溶性片段。通常,该片段具有福寿螺纤维素酶序列的至少约30个连续氨基酸,较佳地至少约50个连续氨基酸,更佳地至少约80个连续氨基酸,最佳地至少约100个连续氨基酸。
发明还提供福寿螺纤维素酶或多肽的类似物。这些类似物与天然福寿螺纤维素酶的差别可以是氨基酸序列上的差异,也可以是不影响序列的修饰形式上的差异,或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到,如通过辐射或暴露于诱变剂而产生随机诱变,还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物,以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解,本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括:体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化,如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中,“福寿螺纤维素酶的保守性变异多肽”指与SEQ ID NO:2、4、6的氨基酸序列以及SEQ ID NO:8、9、10、11或12编码的氨基酸序列相比,有至多10个,较佳地至多8个,更佳地至多5个,最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 | 代表性的取代 | 优选的取代 |
Ala(A) | Val;Leu;Ile | Val |
Arg(R) | Lys;Gln;Asn | Lys |
Asn(N) | Gln;His;Lys;Arg | Gln |
Asp(D) | Glu | Glu |
Cys(C) | Ser | Ser |
Gln(Q) | Asn | Asn |
Glu(E) | Asp | Asp |
Gly(G) | Pro;Ala | Ala |
His(H) | Asn;Gln;Lys;Arg | Arg |
Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
Lys(K) | Arg;Gln;Ash | Arg |
Met(M) | Leu;Phe;Ile | Leu |
Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
Pro(P) | Ala | Ala |
Ser(S) | Thr | Thr |
Thr(T) | Ser | Ser |
Trp(W) | Tyr;Phe | Tyr |
Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。如本文所用,“简并的变异体”在本发明中是指编码具有SEQ ID NO:2、4、6或SEQ IDNO:8-12所编码的氨基酸序列的蛋白质,但与SEQ ID NO:1、3、5、8-12所示的编码区序列有差别的核酸序列。
编码福寿螺纤维素酶成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列;成熟多肽的编码序列+各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)+非编码序列。
术语“编码福寿螺纤维素酶的多核苷酸”可以是包括仅编码福寿螺纤维素酶的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50%,较佳地至少70%,更佳地至少80%相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中,“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,60℃;或(2)杂交时加有变性剂,如50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的相同性至少在90%以上,更好是95%以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽具有纤维素酶活性。
本发明还涉及与上述的序列杂交的核酸片段。如本文所用,“核酸片段”的长度至少含15个核苷酸,较好是至少30个核苷酸,更好是至少50个核苷酸,最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码福寿螺纤维素酶的多聚核苷酸。
本发明的福寿螺纤维素酶核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据本发明所公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用按常规方法所制备的福寿螺cDNA库作为模板,扩增而得有关序列。
一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。
目前,已经可以完全通过化学合成来得到编码本发明蛋白(或其片段,或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外,还可通过化学合成将突变引入本发明蛋白序列中。
本发明也涉及包含本发明的多核苷酸的载体,以及用本发明的载体或纤维素酶编码序列转化的宿主细胞,以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术(Science,1984;224:1431),可利用本发明的多聚核苷酸序列可用来表达或生产重组的纤维素酶。一般来说有以下步骤:
(1).用本发明的编码纤维素酶的多核苷酸(或变异体),或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞;
(2).在合适的培养基中培养的宿主细胞;
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中,纤维素酶多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、或其他载体。在本发明中适用的载体包括但不限于:在细菌中表达的基于T7的表达载体(Rosenberg,et al.Gene,1987,56:125)。总之,只要能在宿主体内复制和稳定,任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含纤维素酶编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等(Sambroook,et al.Molecular Cloning,a Laboratory Manual,cold Spring Harbor Laboratory.New York,1989)。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。这些启动子的代表性例子有:大肠杆菌的lac或trp启动子;λ噬菌体PL启动子;真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外,表达载体优选地包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如真核细胞培养用的二氢叶酸还原酶、以及新霉素抗性,或用于大肠杆菌的四环素、卡那霉素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体,可以用于转化适当的宿主细胞,以使其能够表达蛋白质。
宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞。较佳地是大肠杆菌。代表性例子有:大肠杆菌,链霉菌属;鼠伤寒沙门氏菌的细菌细胞;真菌细胞如酵母等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的酶还包括固定在固相载体上的固定化酶。本领域熟知的各种固定化酶技术都可用于制备本发明的固定化的纤维素酶。
此外,本发明提供了纤维素酶、其编码序列、载体或宿主细胞的用途,它们被用于生产简单糖,尤其是葡萄糖的工艺。所述的简单糖包括纤维二糖、简单戊糖、葡萄糖及其混合物。
本发明的生产简单糖的工艺包括步骤:(a)用本发明上述的纤维素酶或转化或转导的宿主细胞处理纤维素材料,从而产生简单糖;(b)分离出所述的简单糖。本发明酶可直接产生纤维二糖、简单戊糖等简单糖。优选的工艺还包括加入其他酶,例如β-葡萄糖苷酶(可将纤维二糖水解为葡萄糖),这样就可直接产生葡萄糖。
一种代表性的工艺包括步骤:(a)将用机械铰碎稻草与含0.1M氯化钠的0.05M pH5.2醋酸缓冲液按20-30%混合(按重量/体积)在45℃预热10-15分钟。(b)然后加入0.05-0.2%纤维素酶和β-葡萄糖苷酶,缓慢搅动,在45℃水浴反应12-24小时,收集自然沉降的90%上清部分。蒸干得到葡萄糖粗品。(c)沉淀部分补加含0.1M氯化钠的0.05M pH5.2醋酸缓冲液至原体积,缓慢搅动在45℃水浴反应24小时,收集自然沉降的90%上清部分。蒸干得到葡萄糖粗品。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1:福寿螺EG65的分离纯化
1.材料和试剂
福寿螺广泛分布在中国福建、广东,广西,浙江,江苏等省。将从福建厦门购得的福寿螺作为实验材料。实验中,层析介质DEAE-Sepharose CL-6B,DEAE-Sepharose fastflow DEAE-Sephadex A-50,Phenyl-Sepharose CL-4B购自Pharmacia AB(Sweden)公司,Bio-gel P-100购自Bio-Rad(USA)公司。电泳试剂丙稀酰胺、甲叉双丙稀酰胺、十二烷基磺酸钠(SDS)购自Pharmacia AB(Sweden)公司,蛋白分子量标准购自上海西巴斯公司。测活试剂pNPC、Sigmacell 101、木聚糖(xylan from birchwood or oat spelt)、CMC-Na(中等粘度),淀粉(from potato)购自Sigma公司;β-水杨素、Avicel pH101购自Fluka公司。糖蛋白荧光染料荧光酰肼(dansyl glycyl hydtazide,DNS-GLY-NHNH2)由上海生化细胞所刘望夷教授馈赠。其余试剂为国产试剂分析纯。
2.分离纯化
1)硫铵分级沉淀
取18g福寿螺的胃,用剪刀将螺胃剪粹,在4℃用24ml(1∶1.5,w/v)缓冲液A(10mMNa2HPO4-NaH2PO4 pH6.8-100mM NaCl,1mM EDTA)抽提30分钟,抽提液12000rpm离心15分钟后上清加入(NH4)2SO4至55%饱和度,4℃静置过夜。
2)离子交换柱层析
将静置过夜的上清离心,沉淀对缓冲液A(自配,10mM Na2HPO4-NaH2PO4 pH6.8-100mM NaCl,1mM EDTA)进行透析,透析后的酶液离心,上清为用A预先平衡好的DEAE-Sephadex A-50柱(2.6×16cm)的上样液,收集用A洗涤下来的有CMC-Na活性的组份。
CMC-Na水解活力测定:取适量的酶加入到50℃预热10min的200μl 1%(w/v)CMC-Na(100mM NaAC-HAC,pH5.2-100mM NaCl)中,混匀,50℃反应10分钟后,加入0.5mlDNS试剂终止反应,沸水浴5min显色,冷水浴冷却,再补入0.5ml二次蒸馏水,混匀后测定540nm处光吸收值(此测活方法被定义为标准方法)。水解CMC-Na的活力单位定义为:在上述条件下,每分钟水解生成1μmol葡萄糖还原当量所需要的酶量为一个单位(Unit)。
3)凝胶过滤柱层析
Ultrafilter PM10超滤管(pall)浓缩后上Bio-gel P-100柱(2.7×92cm,预先用A平衡好),收集下来的具有CMC-Na活性的组份。
4)疏水相互作用层析
在收集得到的活性组份中加入(NH4)2SO4至0.5M,上疏水柱Phenyl-sepharose CL-4B柱(1.0×6.0cm),经柱平衡缓冲液B(自配,10mM Na2HPO4-NaH2PO4 pH6.8-0.5M(NH4)2SO4,100mM NaCl,1mM EDTA)洗去未结合的蛋白质后,用0.5-0M(NH4)2SO4的B缓冲液梯度洗脱,收集活性具有CMC-Na活性的组份。
5)离子交换柱层析
用Ultrafilter PM10超滤管更换缓冲液为C(自配,50mM Tris-HCl pH8.0),上柱DEAE-sepharose fast flow柱(1.0×6.0cm,预先用C平衡好),平衡洗脱除去未结合的蛋白质后,用含0-0.2M NaCl的溶液洗脱,收集纯酶,并用Ultrafilter PM10超滤管浓缩以备下面实验用。
3.结果
通过硫铵分级沉淀、离子交换(2次)、凝胶过滤以及疏水相互作用等一系列的柱层析,从软体动物福寿螺的胃液中分离纯化到了纤维素酶的组份,该组份在SDS-PAGE上显示的分子量为65kDa(图1),命名为EG65。EG65对CMC-Na的相对活力为13.3IU/mg。下表显示了对福寿螺胃液进行分离纯化所得结果。
纯化步骤 | 总蛋白(mg) | 总活性(U) | 相对活力(U/mg) | 纯化系数(倍) | 产率(%) |
粗抽提物 | 663.7 | 2414.4 | 3.64 | 1 | 100 |
硫铵分级分离 | 410.5 | 1722.6 | 4.20 | 1.15 | 71.3 |
DEAE Sephadex A-50 | 100.4 | 557.2 | 5.55 | 1.52 | 23.1 |
Bio-gel P-100 | 14.9 | 80.11 | 5.38 | 1.48 | 3.32 |
Phenyl Sepharose CL-4B | 5.98 | 51.24 | 8.57 | 2.35 | 2.12 |
DEAE Sepharose fast flow | 1.0 | 13.30 | 13.3 | 3.65 | 0.55 |
实施例2:EG65的活性测定
1.活性测定方法
(1)糖蛋白鉴定
选用荧光酰肼(DNS-Gly-NHNH2)特异标记糖蛋白上的糖链,根据W.Y.Liu,R.Q.Jiang在“The fluorescent labeling of glycoproteins on sodium dodecyl sulfate-polyacrylamide gel”(Bioorg.Chem.22(1994)29-35)中公开的方法鉴定糖蛋白。
(2)最适pH、最适温度以及pH稳定性和温度稳定性测定
1)最适pH:取适量的EG65加入到的不同pH值的200μl、溶解在pH3.6-7.2 100mMC6H8O7(柠檬酸)/Na2HPO4·2H2O的浓度为1%(w/v)的CMC-Na中按标准方法测活。
2)最适温度:在25℃到70℃范围内按标准方法测活。
3)pH稳定性:将EG65酶于pH2.7-9.7的广泛pH缓冲液中50℃保温15min或者50℃保温不同时间后按标准方法测活。
4)温度稳定性:将EG65在pH4.6的广泛pH缓冲液中50℃保温不同的时间按标准方法测活。
(3)金属离子、阴离子以及螯合剂对酶活性的影响
金属离子、阴离子以及螯合剂加入到酶溶液中,然后按标准方法测活。
(4)结合实验
取一定量的酶加入到1.0ml不溶性的微晶纤维素Avicel pH101(50mM NaAC-HAC,pH5.2)中,于4℃放置并不时摇动,上清中活性按标准方法测定。离心后的沉淀用NaAC-HAC溶液洗涤三次,每次1ml,尽量将上清去干净,洗涤后的沉淀用1′SDS上样缓冲液重悬,沸水浴2-3min后,用15%的SDS-PAGE分析。
2.结果
(1)糖蛋白分析实验表明,EG65为糖蛋白。
(2)pH和温度对酶活性的影响
(2.1)最适pH和最适温度
EG65水解CMC-Na的最适温度和最适pH分别为:50-55℃,5.5-6.5。
(2.2)pH稳定性实验表明,EG65在不同pH条件下15℃保温100h之后仍没有明显的活力下降(图2A),然而,50℃保温15min之后,活力有很明显的变化:处于pH4.6的酶活力达到最高;pH5.6时达到相对最高活力的93%;在pH4.6-5.6范围之外,酶水解CMC-Na的活力就明显下降,到pH2.7和pH7.5时,酶基本失活(图2B)。
(2.3)EG65的温度稳定性测定是将pH4.6的酶溶液在50℃保温不同的时间后测残留的酶活,结果发现EG65具有很高的温度稳定性,50℃保温3h,保留有>70%的活力(图3)。
(2.4)不同结构的多糖,如CMC-Na、pNPC、桦树木聚糖(birch wood xylan)、燕麦木聚糖(oat spelt xylan)、β-水杨素、Sigmacell 101以及淀粉等分别作为EG65的底物来检测其底物特异性,结果发现EG65对CMC-Na有很特异的催化活性,对pNPC、β-水杨素和淀粉均无水解能力(见下表)。这些实验表明,这三种酶属于典型的纤维素内切酶。
底物 | CMC | pNPC | 木聚糖a | Sigmacell 101 | 淀粉 | β-水杨素 |
EG65活性(IU/mg) | 13.3 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
上标“a”表示分别使用桦树木聚糖和燕麦木聚糖。
(2.5)将金属离子、阴离子以及螯合剂加入到酶溶液中使其终浓度为10mM,然后再在标准条件下测其相对活力。从实验结果可以看出,Na+、Li+、NH4+、Mg2+、Ca2+、Ni2+、F-、Cl-、Br-、I-、Ac-和CO3 2-等离子的存在对酶活力的变化没有明显的影响,NO3 -、HSO3 -、SO4 2-、S2O3 2-、Mn2+、C6H5O7 3-和EDTA的存在可以提高酶的活力。下表列出了实验结果。
阴离子或螯合剂 | 相对活力 | 金属离子 | 相对活力 |
NaFNaClNaBrNaINaACNaNO3NaHSO3Na2CO3Na2SO4Na2S2O3柠檬酸三钠 | 102.1101.9106.9101.5106.3118.7122.5106.6119.0135.2114.8 | LiClKClNH4ClMgCl2CaCl2NiCl2MnCl2NiSO4MgSO4EDTA | 108.4119.295.6103.489.8110.1121.9114.8100.1124.4 |
上表中,在标准条件下没有加入所述试剂所测得的相对活力为100。
实施例3:EG65的基因及其相关基因的克隆和表达
1.材料、试剂和方法
(1)材料和试剂
3-环己氨基-1-丙磺酸(CAPS)转膜缓冲液购自Amresco公司;V8蛋白水解酶购自Pierce公司;二硫苏糖醇(DTT)购自Sigma公司;PVDF膜购自Millipore公司;Trizol购自上海生工(加拿大BioBasic Inc.进口分装);反转录试剂购自Promega公司;福寿螺胃组织cDNA文库由本实验室构建;限制性内切酶、PCR所用的酶(Taq E、Pyrobest E、La-Taq E等)以及载体pMD18-T Vector购自Takara公司;胶回收试剂盒、丽春红购自上海华舜生物工程有限公司;质粒抽提试剂盒购自博大泰克;IPTG购自BBI公司;X-gal购自Takara公司;E.coli培养所用蛋白胨(Tryptone)、酵母抽提物(Yeast Extract)购自Oxoid公司;甲醇酵母P.pastoris培养所用蛋白胨(BactoTM Peptone)、酵母基本氮源(Yeast Nitrogen Base W/O amino acid)购自B&D公司(原Difco公司产品);D-山梨醇、生物素、琼脂糖等都是国产分析纯;PCR仪为PTC-150 Mini轮rTM;蛋白测序仪为491型蛋白测序仪;酵母电转化仪为Bio-Rad公司MicroPulser电转化仪。
试剂配方包括:YPD:1%酵母抽提物,2%蛋白胨(peptone),2%葡萄糖;灭菌。RBD平板:1M山梨醇,2%葡萄糖,1.34%YNB,0.005%氨基酸,4×10-5%生物素,2%琼脂糖;灭菌。BMGY(或BMMY):1%酵母抽提物,2%蛋白胨(peptone),100mM磷酸钾,pH6.0,1.34%YNB,4×10-5%生物素,1%甘油或者0.5%甲醇;灭菌。需要注意的是2%葡萄糖,100mM磷酸钾,pH6.0要单独灭菌;1.34%YNB,0.005%氨基酸,4×10-5%生物素过滤除菌;0.5%甲醇要在使用前加。其它试剂同实施例1。
(2)方法
(2.1)N-端及中间部分氨基酸序列测定
EG65纯酶和用V8蛋白酶水解产生的14kD片段,分别经10%SDS-PAGE电泳之后,通过半干法将蛋白条带转移到PVDF膜上,丽春红显色之后用自动测序仪通过Edman降解法进行N-端序列测定。
(2.2)福寿螺胃组织总RNA分离及反转录
取新鲜福寿螺胃组织100mg,用Trizol试剂按照GIBCOBRL的总RNA分离Protocol抽提胃组织的总RNA,利用oligodT-15作为引物反转录,得到福寿螺胃组织cDNA第一链,-20℃保存,作为PCR反应的模板。
(2.3)福寿螺EG65基因(eg65-a、eg65-b、eg65-c)cDNA的克隆
1)核心序列的确定
根据EG65 N-端氨基酸序列以及对糖苷水解酶第九家族的保守序列并对鲍鱼[K.Suzuki,T.Ojima,K.Nishita,Purification and cDNA cloning of a cellulase from abaloneHaliotis discus hannai,Eur.J.Biochem.270(2003)771-778]、鳌虾葡聚糖内切酶的序列[K.A.Byrne,S.A.Lehnert,S.E.Johnson,S.S.Moore,Isolation of a cDNA encoding a putativecellulase in the red claw crayfish Cherax quadricarinatus,Gene.239(1999)317-324]作比较选取一段保守序列(DAGDHVKFG),设计兼并引物F1(041016-F1),R1(041105r),以cDNA为模板,94℃,5min;94℃,1min;48℃,45s;72℃,1min10s;35轮;72℃,10min PCR扩增,测序。
2)3′-RACE,即尾部序列的确定
a.根据糖苷水解酶第九家族的保守序列并对鲍鱼、鳌虾葡聚糖内切酶的序列作比较选取一段保守序列(HNEVACDYN),设计兼并引物F2(041105F4);另选取福寿螺胃组织cDNA文库载体上的一段序列,设计特异性引物λC(040428-2),以福寿螺胃组织cDNA文库为模板,94℃,5min;94℃,1min;58℃,45s;72℃,1min30s;30轮;72℃,10min PCR扩增,测序。
b.根据核心序列,设计引物F3(041213-f4)和cDNA文库的特异性引物λC,以福寿螺胃组织cDNA文库为模板,94℃,4min;94℃,30s;52℃,30s;72℃,1min10s,5轮;94℃,30s;57℃,30s;72℃,1min10s,25轮;72℃,10min PCR扩增,测序。
3)5′-RACE,即前半部分序列的确定
根据核心序列,设计引物R2(041127-r),另选取福寿螺胃组织cDNA文库载体臂上的特异的一段序列,设计特异性引物λN,以福寿螺胃组织cDNA文库为模板,94℃,5min;94℃,1min;62℃,45s;72℃,2min10s,30轮;72℃,10min PCR扩增,测序。
4)通拉,即全基因序列的确定
a.根据前半部分序列以及尾部序列(a),分别设计引物F4(0412133041-f2),F5(0412133042-f1)以及R3(1822r1),以福寿螺胃组织cDNA文库为模板,94℃,5min;94℃,30s;60℃,30s;72℃,2min30s,30轮;72℃,10min PCR扩增,测序,得到两段DNA序列,分别为eg65-a和eg65-b的cDNA全长序列。
b.根据前半部分序列以及由尾部序列(b)设计的引物设计的引物F6(0412133042-f3)以及R4(50113166),以福寿螺胃组织cDNA文库为模板,94℃,5min;94℃,30s;60℃,30s;72℃,2min30s,30轮;72℃,10min PCR扩增,测序,得到eg65-c的cDNA全长。
上述PCR扩增得到的片段经胶回收试剂盒纯化之后与pMD18-T载体16℃连接4h,然后转化大肠杆菌DH12S。挑克隆,菌体PCR以及酶切鉴定之后,测序。经测序验证序列正确的含质粒的甘油菌于-70℃待用。所使用到的引物序列如下:
“*”表示包括丰余部分(R=AG;Y=CT;N=ATCG),I=次黄嘌呤
(2.4)eg65-a,eg65-b以及eg65-c在甲醇酵母(Pichia pastoris)中的表达
1)表达质粒的构建
以上述含eg65-a、eg65-b、eg65-c cDNA全长的质粒为模板,分别以Fex(-16a)即Fex(-160923),Rex;Fex(-137a)即Fex(-1370923),Rex;Fex(-16b),Rex;Fex(-148b),Rex;Fex(-16c)即Fex(-16F2A),Rex1即Rex(-1470121F2B)和Fex(-147c)即Fex(-1470121F2A),Rex1为引物,扩增eg65-a,eg65-b,eg65-c的开放阅读框。所得产物通过EcoR I和Not I酶切位点接入P.pastoris表达载体pPIC9K(图4),转化E.coli DH12S感受态细胞,构建表达质粒。挑选阳性克隆,测定插入片段DNA序列,筛选包含正确序列的克隆。
2)P.pastoris表达质粒的转化
表达质粒线性化
用质粒抽提试剂盒抽提相当于3-4个小抽量的P.pastoris表达质粒,限制性内切酶SacI酶切线性化后,无水乙醇沉淀,70%乙醇洗涤两次,晾干后以10μl TE溶解,以备转化。
甲醇酵母GS115感受态的制备
将GS115菌株接种于2ml YPD培养基,30℃培养12~20小时,以1∶100比例接种于100ml YPD培养基中,30℃培养至OD600=1.3~1.5。4℃,1500g离心10min,沉淀用预冷的无菌水悬浮洗涤2次,4℃,1500g离心10min,再用预冷的1M D-山梨醇悬浮洗涤沉淀2次,4℃,1500g离心10min后,所得GS115重悬于0.3ml预冷的1M D-山梨醇中,冰上放置待用。
电转
每管取GS115感受态80μl,分别加入5-20μg线性化的DNA(于5-10μl TE中),小心混匀,加入电转化杯(电极间距0.2cm),冰浴5分钟后,使用MicroPulserTM电转化仪(Bio-Rad)转化。电极化后的细胞加200μl冰冷的1M D-山梨醇,混匀后铺于RDB平板,30℃倒置培养3~5天。
3)eg65-a、eg65-b以及eg65-c在P.pastoris中的表达
挑取单克隆于2ml YPD培养基中,30℃过夜培养,一部分保存甘油菌,一部分以1∶500比例接种于25ml BMGY培养基中,30℃培养至OD600=2-6。1500g离心10min,收集下来的菌体重悬于100ml BMMY培养基中,30℃诱导表达4天,每天补加甲醇至终浓度0.5%。然后4℃,15000g离心15分钟,收集培养基上清待分离纯化用。
表达所用引物如下表所示:
(2.5)重组蛋白EG65-a,EG65-b以及EG65-c的纯化(Ni柱纯化)
离心后的培养基上清,用1M Tris调pH至7.5-8.O后,上Ni柱,先用20mM Tris-HCl,0.5M NaCl,pH7.5的结合缓冲液洗脱,然后再20mM Tris-HCl,0.5M NaCl,20mM咪唑,pH7.5的洗涤缓冲液洗脱,最后用含20mM Tris-HCl,0.5M NaCl,200mM咪唑,pH7.5的洗脱缓冲液洗脱。洗脱下来的组份实施例1的“CMC-Na水解活力测定”方法,50℃,2h测活并且经SDS-PAGE分析之后,收集对应于SDS-PAGE上显示均一的活性组份,对50mM NaAC-HAC,pH5.2缓冲液透析之后,用Ultrafilter PM1O超滤管浓缩以备下面性质分析用。
(2.6)性质分析
1)最适温度、最适pH
最适pH:在pH2.5-7.5,100mM C6H8O7(柠檬酸)/Na2HPO4·2H2O的1%CMC-Na中进行;
最适温度:在20℃到70℃范围内测定;
方法与实施例1的“CMC-Na水解活力测定”相同,只是酶反应的时间长短不同。
2)底物特异性
不同的底物:CMC-Na(1%,中等粘度)、0.9mg/ml pNPC、Sigmacell 101(1%)、AvicelpH101(1%)、桦树木聚糖(1%)、燕麦木聚糖(1%)、淀粉(0.5%)、β-水杨素(0.5%)分别按照实施例1所述方法测活,不同的是酶反应的时间。
3)温度以及pH对酶稳定性的影响
温度稳定性:酶在50℃保温不同的时间或者在不同温度保温1h后,50℃,30min按标准方法测活。
pH稳定性:酶于不同pH值的广泛pH缓冲液中50℃保温15min或者30℃保温20h之后,50℃,30min按标准方法测活。
(2.7)福寿螺卵巢组织基因组DNA抽提
取新鲜福寿螺卵巢组织100mg,用Trizol试剂按照GIBCOBRL的总DNA分离Protocol抽提得到卵巢组织的总DNA。
(2.8)福寿螺eg65-a、eg65-b以及eg65-c的基因组片段扩增
选取引物F5和R2,以福寿螺卵巢基因组DNA为模板,选用La-Taq扩增系统,94℃,5min;94℃,1min;55℃,45s;72℃,7min,30轮;72℃,10min,PCR扩增得到的片段胶回收后与pMD18-T载体16℃连接4h,然后转化大肠杆菌DH12S。挑克隆,菌体PCR以及酶切鉴定之后,测序。
2.结果
(1)EG65 N-端及其中间部分氨基酸序列测定
EG65经Edman降解法测定得到N-末端序列为VSVPDGNGFPATTRVAN。用V8蛋白水解酶对EG65进行酶解,选取酶解产生的14kD肽段测其N-末端序列,结果显示的顺序为:AQRSGALPAN。这段序列与来自鲍鱼(BAC67186,Haliotisdiscus)164~173区域的序列具有90%的同源性(见K.Suzuki等,同上);与鼻白蚁(BAA33708,Nasutitermes takasagoensis)的33~42区域(G.Tokuda,N.Lo,H.Watanabe,M.Slaytor,T.Matsumoto,H.Noda,Metazoan cellulase genes from termites:intron/exonstructures and sites of expression,Biochim Biophys Acta.1447(1999)146-159)以及鳌虾(AAD38027,Cherax quadricarinatus)的53~62区域(K.A.Byrne等,同上)分别具有80%和70%的同源性,并且对比的这三个葡聚糖内切酶均属于糖苷水解酶第九家族。
(2)福寿螺eg65-a、eg65-b以及eg65-c cDNA的克隆
1)核心序列的克隆
根据N-端氨基酸序列VPDGNGFP以及对第九家族保守序列,尤其是对鲍鱼,鳌虾葡聚糖内切酶等序列分析得到的序列DAGDHVKFG,分别设计兼并引物F1和R1,PCR扩增,经测序得到一段552bp的序列(图5),即核心序列,其中包括EG65经V8蛋白水解酶水解产生的14kD肽段的N-末端序列。
2)5′-RACE和3′-RACE
在PCR扩增核心序列的同时,根据第九家族保守序列,尤其是对鲍鱼,鳌虾葡聚糖内切酶的序列分析得到的另一段保守序列HNEVACDYN,设计引物F2与根据cDNA文库载体序列设计的引物λC进行PCR扩增,测序后得到一段尾部序列(a);然后如图6所示,设计特异性的引物R2,F3,分别与根据cDNA文库载体序列设计的引物λN和λC进行PCR扩增,经测序分别得到3段(记为a1,b1,c1)不同的且具有较高同源性的前半部分序列和1段尾部序列(b)。
3)全长cDNA的克隆
根据5′-RACE得到的3段前半部分序列分别设计引物:F4,F5和F6;另外根据3′-RACE得到的2段序列设计引物R3和R4。F4与R3,F5与R3,F6与R4分别进行PCR扩增,得到的PCR片段测序后,分别得到3个不同的基因——eg65-a、eg65-b和eg65-c(SEQ ID NO:1、3和5)。
(3)EG65-a、EG65-b和EG65-c的蛋白序列分析
1)序列相似性分析
eg65-a、eg65-b和eg65-c的开放阅读框分别编码713、723和722个氨基酸,利用ClustalW序列分析软件(http://www.ebi.ac.uk/clustalw/)对这三个序列进行分析,结果显示它们相互之间的同源性分别达到89%,88%和87%(见下表和图7)。
将得到的EG65-a、EG65-b和EG65-c用保守结构域搜索软件(Conserved DomainSearch,http://www.ncbi.nlm.nih.gov/BLAST/)分析,发现它们均与糖苷水解酶第9家族催化结构域有相当高的序列相似性,因此归属于糖苷水解酶第9家族(GHF9)。将EG65-a的氨基酸序列与糖苷水解酶第10家族的其它成员比较发现,来自福寿螺的葡聚糖内切酶与另一种软体动物鲍鱼的同源性可以达到49%(见K.Suzuki等,同上),与两种不同来源的白蚁(Nakashima K,Watanabe H,Saitoh H,Tokuda G,Azuma JI.Dual cellulose-digesting system ofthe wood-feeding termite,Coptotermes formosanus Shiraki.Insect Biochem Mol Biol.2002Jul;32(7):777-84;Li L,Frohlich J,Pfeiffer P,Konig H.Termite gut symbiotic archaezoa arebecoming living metabolic fossils.Eukaryot Cell.2003 Oct;2(5):1091-8)以及鳌虾(K.A.Byrne等,同上)的同源性分别达52%,51%以及46%(见下表和图8)。
另外,EG65-c除含有一个明显第9家族催化结构域外,还含有一个属纤维素酶第∏家族的纤维素结合结构域(CBD);而EG65-a,EG65-b则不含明显的纤维素结合结构域(图9)。
2)糖基化分析
分别用O-糖基化预测软件NetOGlyc 3.1Server(http://www.cbs.dtu.dk/services/NetOGlyc/)以及N-糖基化预测软件NetNGlyc 1.0 Server对EG65-a、EG65-b和EG65-c的糖基化情况作了分析图10A-10F),结果表明:EG65-a 125位的Thr;EG65-b 125,130,131,139,140,146,150位的Thr以及EG65-c 130,139,145位的Thr均为可能的O-糖基化位点。另外,EG65-a序列中N-糖基化的可能性:183,232位Asn大于97,154,188,190,211,246,251,266,305,373,469,472,667位Asn,其中251和472位Asn位于Asn-XAA-Ser/Thr序列中。同样,EG65-b:37,194,243,277位Asn>79,83,165,199,211,222,257,262,479,482,677位Asn,其中262和482位于Asn-XAA-Ser/Thr序列中。EG65-c:200,210位Asn>82,164,198,220,276,382,478,481,676位Asn;其中481位于Asn-XAA-Ser/Thr序列中。
3)信号肽分析
从全长cDNA对应的蛋白序列和分泌到福寿螺胃液中的蛋白,即成熟蛋白N-末端测序得到的结果分析发现,EG65-a较其相应的成熟蛋白EG65多了137个氨基酸残基。而EG65-b和EG65-c蛋白相对应的成熟蛋白N-末端序列可能分别为:LTVPDGNGFPAT和LSVPDGNGFPAT(分别与蛋白测序得到的N-末端序列VSVPDGNGFPAT相比较之后推测),这样,EG65-b和EG65-c分别较成熟蛋白多了148和147个氨基酸残基。但蛋白质信号肽的长度一般为20-30aa,为了确定这3个蛋白的信号肽,用SignalP 3.0 Server信号肽序列预测软件对EG65-a,EG65-b和EG65-c可能的信号肽剪切位点作了预测(图11A-11C),结果发现,它们最可能的剪切位点均在第16和17位氨基酸残基之间。这也说明了这3蛋白很有可能在分泌到胞外之后又进一步被剪切掉了100多个氨基酸残基。
(4)EG65-a、EG65-b和EG65-c在甲醇酵母中的表达及纯化
由上面的信号肽预测软件分析我们可以知道,这3个蛋白N-端都含有16个氨基酸的信号肽,因此我们对每个蛋白质设计两条正向引物,一条反向引物,一对引物PCR得到的是去掉N-端16个信号肽序列的开放阅读框,另一对PCR得到的是成熟蛋白对应的序列;另外,为了纯化重组蛋白的方便,我们在设计正向引物的同时,引入了6个His的密码子。分别构建表达质粒,经测序正确之后,电转到甲醇酵母GS115中,然后甲醇诱导表达。将诱导表达所得到的蛋白分别对应的命名为:EG65-a-16,EG65-a-137;EG65-b-16,EG65-b-148;EG65-c-16,EG65-c-147。
用不含插入基因的质粒pPIC9K为对照组,发现甲醇诱导4天时,上清中葡聚糖内切酶的活力达到最高,因此取诱导4天的培养基4℃,15000g,15min离心后的上清用1M Tris调pH后过Ni柱来纯化蛋白,纯化结果见图12。其中EG65-b-16和EG65-b-148重组蛋白的活力相对最高且水解底物CMC-Na的相对活力相当,分别为:6.67IU/mg和6.45IU/mg。对于EG65-a-16,EG65-a-137和EG65-c-16,EG65-c-147这两组重组蛋白,分别是EG65-a-137和EG65-c-147的活力要高于EG65-a-16和EG65-c-16。而EG65-a-16和EG65-c-16只表现出极其微弱的水解CMC-Na的活力。另外,虽然是在甲醇酵母中诱导表达的,但是从SDS-PAGE电泳图上我们可以看出,重组蛋白并没有发生很严重的糖基化现象。
(5)性质分析
1)最适温度和最适pH
a.EG65-b-16和EG65-b-148
以CMC-Na为底物,从图13A和13B可以看出,EG65-b-16和EG65-b-148的最适反应温度和最适pH曲线走势相似。其最适温度均为50℃,与从福寿螺胃液中得到的组织酶EG65的最适温度相似。当温度高于50℃或者低于50℃时,酶的活力很快下降(图13A)。其最适pH均在pH5.5-6.5之间,在此范围之外,酶的活力明显下降,并且在pH2.5-4.5之间,酶的活力很弱,其相对活力均<20%(图13B)。
b.EG65-a-137和EG65-c-147
EG65-a-137的最适温度为50℃,当温度升高5℃到55℃时,EG65-a-137的活力迅速下降至<20%。而EG65-c-147的最适温度则为40℃,当温度升高时,酶的活力迅速下降,到50℃时,酶的活力下降至18%左右,当温度升高至70℃,EG65-c-147几乎完全丧失活力(图14A,B)。EG65-a-137和EG65-c-147的最适pH见图14C、D,它们的最适pH分别为5.5-6.5和5.5。在此范围之外,活力很快下降。
2)底物特异性
不同结构的多糖,如CMC-Na、pNPC、桦树木聚糖、燕麦木聚糖、β-水杨素、Sigmacell 101、Avicel pH101以及淀粉等作为底物来检测6种不同的重组酶的底物特异性,结果发现它们与从福寿螺胃液中得到的组织酶EG65相似,除CMC-Na之外,对其它底物均无水解活性。而对葡聚糖内切酶的底物CMC-Na而言,EG65-b-16和EG65-b-148的水解活力最高且相对活力相当,EG65-a-137和EG65-c-147的水解活力次之,而EG65-a-137和EG65-c-147对应的只去掉16个氨基酸残基,即信号肽的重组蛋白EG65-a-16和EG65-c-16,仅仅能检测到极其微弱的水解CMC-Na的能力。因此它们属于典型的葡聚糖内切酶。
3)温度和pH对EG65-b-16和EG65-b-148稳定性的影响
将酶溶液在50℃保温不同的时间后测残留的酶活,结果发现两种酶随保温时间的变化,相对酶活力的变化趋势相同。但相对而言,EG65-b-16较EG65-b-148稍稍稳定一些。50℃保温180min之后,EG65-b-16还残留约60%的活性,而EG65-b-148则只剩余约35%的活性(图15A)。但是,将酶在不同温度保温1h之后,发现这两种均在20-40℃的范围内比较稳定,当温度高于40℃时,酶的活力迅速下降,当温度升到60℃时,酶的活力基本为0;同样,我们从图15B中可以看出,EG65-b-16较EG65-b-148的热稳定性要稍稍好一些。
pH稳定性实验表明,50℃保温15min之后,活力有很明显的变化:在pH5.5-6.5范围之间,EG65-b-16和EG65-b-148的酶活力达到最高;在此范围之外,酶水解CMC-Na的活力就明显下降,到pH3.5和pH7.5时,这两个酶基本失活(图16A)。但EG65-b-16和EG65-b-148在不同pH条件下30℃保温20h之后,从图16B可以看出,两者的稳定pH范围均很广,最稳定pH均为pH9.5,但是EG65-b-148在此条件下的pH稳定性较EG65-b-16好。EG65-b-148在pH4.5-10.5仍保留有>70%的活力,而EG65-b-16达到>70%相对活力的pH范围为pH7.5-10.5。
(6)eg65-c对应的福寿螺基因组片段的扩增
为了进一步验证从福寿螺胃组织cDNA文库中得到的eg65-a、eg65-b和eg65-c三个基因来源于福寿螺自身,我们以F5和R2为引物,用福寿螺卵巢抽提出的基因组DNA为模板,PCR扩增,测序,得到一段长约1.6kb的片段(SEQ ID NO:7),Blast之后发现,这段序列是包含4个内含子的eg65-c的部分基因组序列,内含子的剪切位置分别在176bp,296bp,370bp和442bp之后。上述实验进一步验证了这三个基因是来源于福寿螺本身,而非其体内的肠道微生物所产生。
实施例4:EG65蛋白的多态性分析
本实施例讨论福寿螺纤维素酶的多态性,分析引起其多态性的可能的原因。
1.材料、试剂和方法
(1)材料试剂
双向凝胶电泳系统购自Amersham Biosciences;凝胶扫描成像系统(D2000 Uniscanscanner)购自Tsinghua Uniscab;脱色摇床(TY-20型)购自西巴斯;Voyager-DE STR MALDI-TOF质谱仪购自PerSeptive Biosystems,Framinham,MA;胰蛋白酶(sequencing-grademodified trypsin)购自Promega;葡聚糖内切酶为实施例1所得;EG65-b-148为实施例3所得;其它试剂和材料见实施例1和3。
(2)方法
(2.1)双向凝胶电泳
双向电泳的方法主要根据Amersham Biosciences的仪器使用手册(T.Berkelman,T.Stenstedt,2-D electrophoresis:using immobilized pH gradients;principles and methods,Uppsala,Sweden:Amersham Biosciences.(1998))和等改进的方法(A.C.Obermaier,G.Boguth,A.Harder,B.Scheibe,R.Wildgruber,W.Weiss,The current state oftwo-dimensional electrophoresis with immobilized pH gradients,Electro-phoresis.21(2000)1037-1053)。IPG-IEF在IPGphor等电聚焦系统上进行。等电聚焦后,IPG胶条在15ml的平衡液中平衡两次,每次15min(等,同上)。SDS-PAGE在Ettan DALT twelve系统上进行。双向电泳后,分析型的电泳凝胶利用Yan等改进的硝酸银染色方法进行染色(J.X.Yan,R.Wait,T.Berkelman,R.A.Harry,J.A.Westbrook,C.H.Wheeler,M.J.Dunn,A modifiedsilver staining protocol for visualization of proteins compatible with matrix-assisted laserdesorption/ionization and electrospray ionizationmass spectrometry,Electophoresis.21(2000)3666-3672),制备型的电泳凝胶利用Neuhoff等的考马斯亮蓝染色方法进行染色(V.Neuhoff,N.Arold,D.Taube,W.Ehrhardt,Improved staining of proteins in polyacrylamide gelsincluding isoelectric focusing gels with clear background at nanogram sensitivity usingCoomassie Brilliant Blue G-250 and R-250,Electrophoresis.9(1988)255-262)。
(2.2)MALDI-TOF多肽质量指纹图谱分析
用来进行MALDI多肽质量指纹图谱分析的蛋白质取自考马斯亮蓝G250染色24小时的制备型凝胶。用切点仪(The Gel Company,USA)切取目标蛋白质点,
每个胶片被切成1mm3得胶块然后放到0.65ml硅烷化的EP管中,胶内酶解的方法是根据Shevchenko等(A.Shevchenko,M.Wilm,O.Vorm,M.Mann,Mass spectrometricsequencing of proteins silver-stained polyacrylamide gels,Anal Chem.68(1996)850-858)的方法进行。胰蛋白酶酶解的样品用Voyager-DE STR MALDI-TOF质谱仪在延滞抽提线性正向模式下进行分析。
(2.3)eg65-b1、b2、b3以及eg65-c1、c2、c3、c4基因的克隆(eg65-b1即eg65-b,eg65-c1即eg65-c)
方法与实施例3所述的方法相同。
通拉时F4、F5、F6分别与R3和R4组成引物对,以福寿螺胃组织cDNA文库为模板,94℃,5min; 94℃,30s;60℃,30s;72℃,2min30s,30轮;72℃,10min PCR扩增,测序,得到eg65-b1、b2、b3以及eg65-c1、c2、c3、c4基因。
2.结果
(1)EG65蛋白的多态性
为了证明福寿螺胃液中的葡聚糖内切酶是否也具有蛋白多态性,取SDS-PAGE上显示一条带的EG65进行双向凝胶电泳,结果呈现的是分子量相似而等电点不同的一串点(图17A),这表明EG65至少在等电点不同方面具有蛋白多态性。为了进一步验证这些点是否属于不同翻译后修饰的同一个核心蛋白,随机取双向电泳凝胶上的6个点用MALDI-TOF多肽质量指纹图谱进行分析,结果发现这六个点的多肽质量指纹图谱很相似,这暗示了这些点很可能是属于同一个核心蛋白或者是同源性很高,即氨基酸序列几乎相同的多个蛋白(图17B),如整联蛋白β-3(Integrin beta 3)的多态性只表现在第33位氨基酸是Leu还是Pro(Vijayan KV,Liu Y,Sun W,Ito M,Bray PF.The Pro33 isoform of integrin beta 3enhancesoutside-in signaling in human platelets by regulating the activation of serine/threoninephosphatases.J Biol Chem.2005 Apr 11),因此不会对这两种异构体的多肽质量指纹图谱造成太大的差别一样。
(2)eg65-b1、b2、b3以及eg65-c1、c2、c3、c4基因序列以及蛋白序列分析
以F4,F5,F6与R3或R4为引物,分别从福寿螺胃组织cDNA文库中得到了同源性很高的eg65-a(SEQ ID NO:1)、eg65-b1(SEQ ID NO:3)、b2(SEQ ID NO:8)、b3(SEQ ID NO:9)以及eg65-c1(SEQ ID NO:5)、c2(SEQ ID NO:10)、c3(SEQ ID NO:11)、c4(SEQ ID NO:12)三组基因。蛋白序列分析发现,除这三组蛋白之间具有很高的同源性之外,eg65-b和eg65-c这两组蛋白编码的氨基酸序列相互之间的同源性可高达98%以上(图18A、B)。与EG65葡聚糖内切酶的N-末端序列相比较发现,这三组蛋白可能具有相同或极其相似的成熟蛋白N-末端。另外,用蛋白质初级结构分析软件ProtParam(http://www.expasy.ch/tools/protparam.html)预测了eg65-a,eg65-b1、b2、b3以及eg65-c1、c2、c3、c4编码的成熟蛋白的分子量以及对应的理论等电点的大小(见下表),结果发现这些成熟蛋白的分子量极其相似,而等电点却不同,这与组织酶EG65进行双向凝胶电泳得到的结果很相似,另外,这也与能够与组织酶EG65的多肽质量指纹图谱相对应。从而进一步证明了这种蛋白多态性的原因可能是由于蛋白序列的微观不同造成的。
采用ProtParam获得的分子量和理论pI预测结果
(3)EG65-b-148蛋白的多态性
为了证明真核生物中翻译后修饰很容易引起蛋白的多态性,将含有eg65-b-148基因的质粒转化到甲醇酵母P.pastoris之后表达出的重组蛋白EG65-b-148经Ni柱纯化,SDS-PAGE上显示的是像“双眼皮”样的挨得很近的两条带(见实施例3),双向凝胶电泳图上呈现的是分子量很相近的而等电点不同的一串点(图19),这充分证明了EG65-b-148存在蛋白多态性,并且造成这种多态性的原因是翻译后修饰的不同。
软体动物福寿螺体内含有大量的翻译后修饰系统,并且组织酶EG65经荧光酰肼染色之后发现是一个糖蛋白(见实施例2),并且双向电泳图谱上呈现的也是一串点(图17A),因此造成福寿螺纤维素酶多态性的另一个原因可能是翻译后修饰。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110>中国科学院上海生命科学研究院
浙江理工大学
<120>新的纤维素酶及其应用
<130>065777
<160>32
<170>PatentIn version 3.3
<210>1
<211>2142
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>1
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cccgtcacca accattggcc gggtggattc caggcaaaag tctgtttcac tatcgacaaa 120
gagatgactt cttgggtcgt cgatcttgtc ttcgatcacc ctgtggacac actgagcctg 180
tggacggccg atgcgaagag caccagcgca gacaagacga aatggacgct gaccagcaag 240
acgtggaact cccaggagca tgtgggagat gagctgtgca tcgatatcaa tggccaaggt 300
agcggtgatg actggcgtgt tgtaaaagcc accctggaag gagcagcaga tggcgggtca 360
taccaagtcg ttacaagcgc ccctctccca ccaggtgtct ctgcagctcc agtgtctgtc 420
cccgacggaa acggtttccc cgccaccacc agagtcgcca acgtcaggga cgggctgtcg 480
gagcagtggc tgaccttcac catcaccggc cccaccgtca tgggctgggt ggtcaagttc 540
cgctgcaaca agccagtcac caacctcaac gtggcagatg ccgacgccct cagccacaac 600
gccgacatga cggagtggct gctggtgaac aacgacaaca agatcgccct caaggcggga 660
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tcggcgatcc tcatcaacat gggggtagat aactacacct gcggcgagct acccaacaag 780
gccaactcca agtacaacta cgacgacctt ctgtacaaat ccatcttgtt ctacgaggcg 840
cagcgctcgg gcaaacttcc ggccaacaac cgcatcccct ggcgaggcga ctccgccctc 900
aacgaccacg gcaatgccgg cgaggacttg actggcggct ggtacgacgc gggagacttc 960
gtcaagttca acttccccat ggcctggtca acggccgtct tgacctgggg cttgctgcag 1020
ttcaaggacg cctaccaggc cgcaggtcag ctggagtgga tgtacgagag catcaagtgg 1080
ccgctggact acctgctcaa gtgtcacgtg tctgacaacg tgctgtacgt gcaggtgggt 1140
gatggaggtg tggaccacgg atcatggggg agacccgagg acatgaagat ggccagaccc 1200
gccttcaaga tcgacgccag caaacccgga tctgaagttg ccatggaaac agcagctgcc 1260
ttcgcggctg gacatttggc ttttaaagaa aaagatccgt catactcagc caaactgctg 1320
caacatgcca agtcgctgtg gcagttcgct gtcacacaca agggcaagta cagtgacagt 1380
gtgtcagctg ctgccggcta ctacaattcc gccaacgtca cggacgagtt gtgctggggg 1440
tcgctgtggt tgtacaaggc caccaaggaa cccaagtacc tggaggaggc cctcaagcac 1500
tatgatgctt ctcccgactg gggcatgtcc tgggacgatg tcttcatcgg caatcaggtg 1560
tcgctgtacg aactcacgaa ggaggccaag tacaaagcag ctgtagaggg caccttcaag 1620
gagtggttcc ctggtgggac tgtcccctac acccctaagg gtctggcgta cagactgcag 1680
tggggcgccc tacggtacgc atccaacatg gctatggccg cgctgatggc tgcagaagcg 1740
ggtatccacc cggacgagta tcgccactgg gccatgtgtc agatccacta cgccctgggg 1800
gacactggcc gcagctttgt cgtgggtttt ggcaaaaatc cacccgtcag tcctcaccac 1860
cgctctagct cctgccccaa cctacctgtg cggtgtaaca tgaactacct ccacctggac 1920
acccccaaca ctcacatgct gtgcggggcg ctggtgggtg gccccgatag ctcggatggt 1980
tacaaggaca gccgcgagaa ctacgtcaac aacgaggtgg cctgcgacta caacgccggc 2040
ttccagacag ccgtggccgg tcttcgctcg ctgctgatca gacacctgca tcccgagcag 2100
aagggcggcg ccacgtgtcc ctaccatggg gcagcccctt ga 2142
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Asn Gly Gln Gly Ser Gly Asp Asp Trp Arg Val Val Lys Ala Thr Leu
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Leu Pro Pro Gly Val Ser Ala Ala Pro Val Ser Val Pro Asp Gly Asn
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Gly Phe Pro Ala Thr Thr Arg Val Ala Asn Val Arg Asp Gly Leu Ser
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Glu Gln Trp Leu Thr Phe Thr Ile Thr Gly Pro Thr Val Met Gly Trp
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Val Val Lys Phe Arg Cys Asn Lys Pro Val Thr Asn Leu Asn Val Ala
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Asp Ala Asp Ala Leu Ser His Asn Ala Asp Met Thr Glu Trp Leu Leu
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Val Asn Asn Asp Asn Lys Ile Ala Leu Lys Ala Gly Thr Leu Glu Met
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Lys Ile Glu Val Lys Leu Val Asn Val His Asp Ser Ala Pro Gln Cys
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Ser Ala Ile Leu Ile Asn Met Gly Val Asp Asn Tyr Thr Cys Gly Glu
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Leu Pro Asn Lys Ala Asn Ser Lys Tyr Asn Tyr Asp Asp Leu Leu Tyr
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Lys Ser Ile Leu Phe Tyr Glu Ala Gln Arg Ser Gly Lys Leu Pro Ala
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Asn Asn Arg Ile Pro Trp Arg Gly Asp Ser Ala Leu Asn Asp His Gly
290 295 300
Asn Ala Gly Glu Asp Leu Thr Gly Gly Trp Tyr Asp Ala Gly Asp Phe
305 310 315 320
Val Lys Phe Asn Phe Pro Met Ala Trp Ser Thr Ala Val Leu Thr Trp
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Gly Leu Leu Gln Phe Lys Asp Ala Tyr Gln Ala Ala Gly Gln Leu Glu
340 345 350
Trp Met Tyr Glu Ser Ile Lys Trp Pro Leu Asp Tyr Leu Leu Lys Cys
355 360 365
His Val Ser Asp Asn Val Leu Tyr Val Gln Val Gly Asp Gly Gly Val
370 375 380
Asp His Gly Ser Trp Gly Arg Pro Glu Asp Met Lys Met Ala Arg Pro
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Ala Phe Lys Ile Asp Ala Ser Lys Pro Gly Ser Glu Val Ala Met Glu
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Thr Ala Ala Ala Phe Ala Ala Gly His Leu Ala Phe Lys Glu Lys Asp
420 425 430
Pro Ser Tyr Ser Ala Lys Leu Leu Gln His Ala Lys Ser Leu Trp Gln
435 440 445
Phe Ala Val Thr His Lys Gly Lys Tyr Ser Asp Ser Val Ser Ala Ala
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Ala Gly Tyr Tyr Asn Ser Ala Asn Val Thr Asp Glu Leu Cys Trp Gly
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Ser Leu Trp Leu Tyr Lys Ala Thr Lys Glu Pro Lys Tyr Leu Glu Glu
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Ala Leu Lys His Tyr Asp Ala Ser Pro Asp Trp Gly Met Ser Trp Asp
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Asp Val Phe Ile Gly Asn Gln Val Ser Leu Tyr Glu Leu Thr Lys Glu
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Trp Gly Ala Leu Arg Tyr Ala Ser Asn Met Ala Met Ala Ala Leu Met
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Ala Ala Glu Ala Gly Ile His Pro Asp Glu Tyr Arg His Trp Ala Met
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Cys Gln Ile His Tyr Ala Leu Gly Asp Thr Gly Arg Ser Phe Val Val
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Gly Phe Gly Lys Asn Pro Pro Val Ser Pro His His Arg Ser Ser Ser
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Thr Pro Asn Thr His Met Leu Cys Gly Ala Leu Val Gly Gly Pro Asp
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Ser Ser Asp Gly Tyr Lys Asp Ser Arg Glu Asn Tyr Val Asn Asn Glu
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Val Ala Cys Asp Tyr Asn Ala Gly Phe Gln Thr Ala Val Ala Gly Leu
675 680 685
Arg Ser Leu Leu Ile Arg His Leu His Pro Glu Gln Lys Gly Gly Ala
690 695 700
Thr Cys Pro Tyr His Gly Ala Ala Pro
705 710
<210>3
<211>2172
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>3
atgttctcgc tggtcctgtg ggcggtgcta ccgctcttgg gctttggcag catcactgta 60
cccgtcaaca accactgggc gggtggattc caggctagag tctgctttaa catcgacaag 120
gacatgagtt cctgggtcat ccatctggct ttcgatcaac ctgtacagac ccttgatgtg 180
tggacggcca atgcgcagag caccagcgcg gacaagaagg aatggtcgct gactaacaag 240
gagtggaacg cagtggagca tgtcggggac gagctgtgca ttgacctcat gggtcacggc 300
gacggagata tcgcgcctgt gatcacagcc accctggagg gagcggaagg tggcgggtcg 360
caccaagtta ttacaagtgc accccgcacc acagtcaacc tccctccagg tgtcaccacc 420
gctgaacctc agcctacagc cccattgact gtccccgacg gaaacggttt ccccgccacc 480
accagagtcg ccaacgtcag ggacgggctg tcggagcagt ggctgacctt caccatcacc 540
ggccccaccg tcatgggctg ggtggtcaag ttccgctgca acaagccagt caccaacctc 600
cacgtggcag atgccgacgc cctcagccac aacgaggaca tgacggagtg gctgctggtg 660
aacaacgaca acaagatcgc cctcaaggcg ggaaccttgg agatgaagat tgaagtcaag 720
ctggtgaatg tccaagactc agcccctcag tgctcggcga tcctcatcaa catgggcgta 780
gataactaca cctgcggcga gctacccaac aaggccaact ccaagtacaa ctatgacgat 840
gtcctgtaca agtccatctt gttctacgag gcgcagcgct cgggcaaact tccggccaac 900
aaccgcatcc cctggcgggg ggactccgcc ctcaatgatg gagatggcgg ggtggacctg 960
acaggcgggt ggtatgacgc gggagacttc gtcaagttca acttccccat ggcctggtcc 1020
acggccatct tgaactgggg cttgctgcag ttcaaggacg cctacgaggc cgcaggtcag 1080
ctggagtgga tgtacgagag tgtcaagtgg ccgctggact acctgctcaa gtgtcactcg 1140
tctgatgacg tgctgtacgt gcaggtgggt gatggaggtg cggaccacgg atcatggggg 1200
agacccgagg acatgaagat ggccagaccc gccttcaagg ttgacgccag cagacctggc 1260
tccgaagttg ccatggaaac agctgttgcc tttgtgactg gccacttggc tttcaaagaa 1320
aaagatccgc catactcagc caaactgctg caacatgcca agtccctgtg ggagttcgct 1380
gtcacacaca agggcaagta cagtgaaagt gtctccgccg ccgcaagtta ctacaattcc 1440
gtcaacgtca cagacgagtt gtgctggggg tcgctgtggt tgtacaaggc caccaaggaa 1500
cccaagtacc tggaggaggc cctcaagcac tatgatgctt ctcccgactg gggcatgtcc 1560
tgggacgatg tcttcatcgg caatcaggtg ttgctgtacg aactcacgaa ggaggagaag 1620
tacaaagcag ctgtggaggg caccttcaag gagtggttcc ctggcgggac tgtcccctac 1680
acccctaagg gtctggcgta cagactgcag tggggcgccc tacggtactc gtccaacatg 1740
gctatggccg cgctgatggc tgcagaagcg ggtatccacc cggacgagta tcgccactgg 1800
gccatgtgtc agatccacta cgccctgggg gacactggcc gcagctttgt cgtgggtttt 1860
ggcaaaaatc cacccgtcag tcctcaccac cgctctagct cctgccccaa cctccctgtg 1920
cggtgtaaca tgaactacct ccacctggac acccccaaca ctcacatgct gtgcggggcg 1980
ctggtgggtg gccccgatag ctcggatggt tacaaggaca gccgcgagaa ctacgtcaac 2040
aacgaggtgg cctgcgacta caacgccggc ttccagacag ccgtggccgg tcttcgctcg 2100
ctgttgctaa ggaacctgca tcccgagcag aagggcagcg ccacgtgtcc ctaccatggg 2160
gcagcccctt ga 2172
<210>4
<211>723
<212>PRT
<213>福寿螺(Ampullaria crossean)
<400>4
Met Phe Ser Leu Val Leu Trp Ala Val Leu Pro Leu Leu Gly Phe Gly
1 5 10 15
Ser Ile Thr Val Pro Val Asn Asn His Trp Ala Gly Gly Phe Gln Ala
20 25 30
Arg Val Cys Phe Asn Ile Asp Lys Asp Met Ser Ser Trp Val Ile His
35 40 45
Leu Ala Phe Asp Gln Pro Val Gln Thr Leu Asp Val Trp Thr Ala Asn
50 55 60
Ala Gln Ser Thr Ser Ala Asp Lys Lys Glu Trp Ser Leu Thr Asn Lys
65 70 75 80
Glu Trp Asn Ala Val Glu His Val Gly Asp Glu Leu Cys Ile Asp Leu
85 90 95
Met Gly His Gly Asp Gly Asp Ile Ala Pro Val Ile Thr Ala Thr Leu
100 105 110
Glu Gly Ala Glu Gly Gly Gly Ser His Gln Val Ile Thr Ser Ala Pro
115 120 125
Arg Thr Thr Val Asn Leu Pro Pro Gly Val Thr Thr Ala Glu Pro Gln
130 135 140
Pro Thr Ala Pro Leu Thr Val Pro Asp Gly Asn Gly Phe Pro Ala Thr
145 150 155 160
Thr Arg Val Ala Asn Val Arg Asp Gly Leu Ser Glu Gln Trp Leu Thr
165 170 175
Phe Thr Ile Thr Gly Pro Thr Val Met Gly Trp Val Val Lys Phe Arg
180 185 190
Cys Asn Lys Pro Val Thr Asn Leu His Val Ala Asp Ala Asp Ala Leu
195 200 205
Ser His Asn Glu Asp Met Thr Glu Trp Leu Leu Val Asn Asn Asp Asn
210 215 220
Lys Ile Ala Leu Lys Ala Gly Thr Leu Glu Met Lys Ile Glu Val Lys
225 230 235 240
Leu Val Asn Val Gln Asp Ser Ala Pro Gln Cys Ser Ala Ile Leu Ile
245 250 255
Asn Met Gly Val Asp Asn Tyr Thr Cys Gly Glu Leu Pro Asn Lys Ala
260 265 270
Asn Ser Lys Tyr Asn Tyr Asp Asp Val Leu Tyr Lys Ser Ile Leu Phe
275 280 285
Tyr Glu Ala Gln Arg Ser Gly Lys Leu Pro Ala Asn Asn Arg Ile Pro
290 295 300
Trp Arg Gly Asp Ser Ala Leu Asn Asp Gly Asp Gly Gly Val Asp Leu
305 310 315 320
Thr Gly Gly Trp Tyr Asp Ala Gly Asp Phe Val Lys Phe Asn Phe Pro
325 330 335
Met Ala Trp Ser Thr Ala Ile Leu Asn Trp Gly Leu Leu Gln Phe Lys
340 345 350
Asp Ala Tyr Glu Ala Ala Gly Gln Leu Glu Trp Met Tyr Glu Ser Val
355 360 365
Lys Trp Pro Leu Asp Tyr Leu Leu Lys Cys His Ser Ser Asp Asp Val
370 375 380
Leu Tyr Val Gln Val Gly Asp Gly Gly Ala Asp His Gly Ser Trp Gly
385 390 395 400
Arg Pro Glu Asp Met Lys Met Ala Arg Pro Ala Phe Lys Val Asp Ala
405 410 415
Ser Arg Pro Gly Ser Glu Val Ala Met Glu Thr Ala Val Ala Phe Val
420 425 430
Thr Gly His Leu Ala Phe Lys Glu Lys Asp Pro Pro Tyr Ser Ala Lys
435 440 445
Leu Leu Gln His Ala Lys Ser Leu Trp Glu Phe Ala Val Thr His Lys
450 455 460
Gly Lys Tyr Ser Glu Ser Val Ser Ala Ala Ala Ser Tyr Tyr Asn Ser
465 470 475 480
Val Asn Val Thr Asp Glu Leu Cys Trp Gly Ser Leu Trp Leu Tyr Lys
485 490 495
Ala Thr Lys Glu Pro Lys Tyr Leu Glu Glu Ala Leu Lys His Tyr Asp
500 505 510
Ala Ser Pro Asp Trp Gly Met Ser Trp Asp Asp Val Phe Ile Gly Asn
515 520 525
Gln Val Leu Leu Tyr Glu Leu Thr Lys Glu Glu Lys Tyr Lys Ala Ala
530 535 540
Val Glu Gly Thr Phe Lys Glu Trp Phe Pro Gly Gly Thr Val Pro Tyr
545 550 555 560
Thr Pro Lys Gly Leu Ala Tyr Arg Leu Gln Trp Gly Ala Leu Arg Tyr
565 570 575
Ser Ser Asn Met Ala Met Ala Ala Leu Met Ala Ala Glu Ala Gly Ile
580 585 590
His Pro Asp Glu Tyr Arg His Trp Ala Met Cys Gln Ile His Tyr Ala
595 600 605
Leu Gly Asp Thr Gly Arg Ser Phe Val Val Gly Phe Gly Lys Asn Pro
610 615 620
Pro Val Ser Pro His His Arg Ser Ser Ser Cys Pro Asn Leu Pro Val
625 630 635 640
Arg Cys Asn Met Asn Tyr Leu His Leu Asp Thr Pro Asn Thr His Met
645 650 655
Leu Cys Gly Ala Leu Val Gly Gly Pro Asp Ser Ser Asp Gly Tyr Lys
660 665 670
Asp Ser Arg Glu Asn Tyr Val Asn Asn Glu Val Ala Cys Asp Tyr Asn
675 680 685
Ala Gly Phe Gln Thr Ala Val Ala Gly Leu Arg Ser Leu Leu Leu Arg
690 695 700
Asn Leu His Pro Glu Gln Lys Gly Ser Ala Thr Cys Pro Tyr His Gly
705 710 715 720
Ala Ala Pro
<210>5
<211>2169
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>5
atgttctcgc tagtcttgtg ggcggggcta ccgctgctga gctttgccat cactgtaccc 60
gtcaccaacc actgggcggg tggattccag gctagagtct gcttcaaaat cgacaaagag 120
atgagttcct gggtcatcca tctgaccttt gaccaccctg tagagacaat tgatgtgtgg 180
acggcggatg cgcagagcac cagcgcggac aagaagcaat ggacgctgac cagcaagaac 240
tggaacgcag tggagcatgt cggggacgag ctgtgcatcg acctcatggg tcacggaact 300
ggtgatttcg ctcctgtcat cacagcaacc ctggaaggag caagtggcgg cggttcacat 360
caagttgcta caagtgcccc agctaccaca caagctcacc atcaaggtgt cgccactgct 420
gctcctgtgc atacaacccc attgtctgtc cccgacggaa atggattccc cgccactacc 480
cgagtctcca acgtgagaga cggacgctcg gaccattacc tgaccttcac catcagcggc 540
ccaaccgtca tgggctgggt ggtcaagttc cggtgcagca agccagtgca caacctcaac 600
gttcgcgagg ccgatccact cagccacaac gccgatcaga cggagtggct gctggtgaac 660
aacgacaacc agatcaccat caagccagga acattcgagt tgaaaattga actggagctg 720
gtgaccggca gagactcggc ccctcagtgc tccgccacac tcaccaacat gggcgtcgac 780
aaacacacct gcggacagct gcccaacaaa gccaactcca agtacaacta tgacgatgtc 840
ctgtacaagt ccatcttgtt ctacgaggcg cagcgctcgg gcaaacttcc ggccaacaac 900
cgcatcccct ggcgagggga ctccgccctc aatgatggag atggcggggt ggacctgact 960
ggcgggtggt atgacgcggg agacttcgtc aagttcaact tccccatggc ctggtccacg 1020
gccgtcttga cctggggctt gctgcagttc aaggacgcct accaggccgc aggtcagctg 1080
gagtggatgt acgagagtgt caagtggccg ctggactacc tgctcaagtg tcacgtgtct 1140
gacaacgtgc tgtacgtgca ggtgggtgat ggaggtgtgg accacggatc atgggggaga 1200
cccgaggaca tgaagatggc cagacccgca ttcaagatcg acgccaccaa gccaggatct 1260
gaagttgcca tggaaacagc ggctgccttt gcggctggac atttggcatt caaagaaaaa 1320
gatccgtcat actcagccaa actgctgcaa catgccaagt cgctgtggca gttcgctgtc 1380
acacacaagg gcaagtacag tgacagtgtg tcagctgctg ccggctacta caattccgcc 1440
aacgtcacgg acgagttgtg ctgggggtcg ctgtggttgt acgaggccac caaggaaccc 1500
aagtacctgg aggaggccct caagcactat gatgcttctc ccgactgggg catgtcctgg 1560
gacgatgtct tcatcggcaa tcaggtgttg ctgtacgaac tcacgaagga ggccaagtac 1620
aaaacagctg tggaaggcac cttcagggag tggttccctg gcgggacggt cccctacacc 1680
cctaagggtc tggcgtacag actgcagtgg gggtccctgc ggtacgcatc caacatggct 1740
atggccgcgc tgatggctgc agaagcgggt atccacccgg acgagtatcg ccactgggcc 1800
atgtgtcaga tccactacgc cctgggggac actggccgca gctttgtcgt gggttttggc 1860
aaaaatccac ccgtcagtcc tcaccaccgc tctagctcct gccccaacct acctgtgcgg 1920
tgtaacatga actacctcca cctggacacc cccaacactc acatgctgtg cggggcgctg 1980
gtgggtggcc ccgatagctc ggatggttac aaggacagcc gcgagaacta cgtcaacaac 2040
gaggtggcct gcgactacaa cgccggcttc cagacagccg tggccggcct tcgctccttg 2100
ctggtccgac acttgcatcc cgagcagaaa ggcggcgcca cgtgtcccta ccacgggaga 2160
gcaccgtga 2169
<210>6
<211>722
<212>PRT
<213>福寿螺(Ampullaria crossean)
<400>6
Met Phe Ser Leu Val Leu Trp Ala Gly Leu Pro Leu Leu Ser Phe Ala
1 5 10 15
Ile Thr Val Pro Val Thr Asn His Trp Ala Gly Gly Phe Gln Ala Arg
20 25 30
Val Cys Phe Lys Ile Asp Lys Glu Met Ser Ser Trp Val Ile His Leu
35 40 45
Thr Phe Asp His Pro Val Glu Thr Ile Asp Val Trp Thr Ala Asp Ala
50 55 60
Gln Ser Thr Ser Ala Asp Lys Lys Gln Trp Thr Leu Thr Ser Lys Asn
65 70 75 80
Trp Asn Ala Val Glu His Val Gly Asp Glu Leu Cys Ile Asp Leu Met
85 90 95
Gly His Gly Thr Gly Asp Phe Ala Pro Val Ile Thr Ala Thr Leu Glu
100 105 110
Gly Ala Ser Gly Gly Gly Ser His Gln Val Ala Thr Ser Ala Pro Ala
115 120 125
Thr Thr Gln Ala His His Gln Gly Val Ala Thr Ala Ala Pro Val His
130 135 140
Thr Thr Pro Leu Ser Val Pro Asp Gly Asn Gly Phe Pro Ala Thr Thr
145 150 155 160
Arg Val Ser Asn Val Arg Asp Gly Arg Ser Asp His Tyr Leu Thr Phe
165 170 175
Thr Ile Ser Gly Pro Thr Val Met Gly Trp Val Val Lys Phe Arg Cys
180 185 190
Ser Lys Pro Val His Asn Leu Asn Val Arg Glu Ala Asp Pro Leu Ser
195 200 205
His Asn Ala Asp Gln Thr Glu Trp Leu Leu Val Asn Asn Asp Asn Gln
210 215 220
Ile Thr Ile Lys Pro Gly Thr Phe Glu Leu Lys Ile Glu Leu Glu Leu
225 230 235 240
Val Thr Gly Arg Asp Ser Ala Pro Gln Cys Ser Ala Thr Leu Thr Asn
245 250 255
Met Gly Val Asp Lys His Thr Cys Gly Gln Leu Pro Asn Lys Ala Asn
260 265 270
Ser Lys Tyr Asn Tyr Asp Asp Val Leu Tyr Lys Ser Ile Leu Phe Tyr
275 280 285
Glu Ala Gln Arg Ser Gly Lys Leu Pro Ala Asn Asn Arg Ile Pro Trp
290 295 300
Arg Gly Asp Ser Ala Leu Asn Asp Gly Asp Gly Gly Val Asp Leu Thr
305 310 315 320
Gly Gly Trp Tyr Asp Ala Gly Asp Phe Val Lys Phe Asn Phe Pro Met
325 330 335
Ala Trp Ser Thr Ala Val Leu Thr Trp Gly Leu Leu Gln Phe Lys Asp
340 345 350
Ala Tyr Gln Ala Ala Gly Gln Leu Glu Trp Met Tyr Glu Ser Val Lys
355 360 365
Trp Pro Leu Asp Tyr Leu Leu Lys Cys His Val Ser Asp Asn Val Leu
370 375 380
Tyr Val Gln Val Gly Asp Gly Gly Val Asp His Gly Ser Trp Gly Arg
385 390 395 400
Pro Glu Asp Met Lys Met Ala Arg Pro Ala Phe Lys Ile Asp Ala Thr
405 410 415
Lys Pro Gly Ser Glu Val Ala Met Glu Thr Ala Ala Ala Phe Ala A1a
420 425 430
Gly His Leu Ala Phe Lys Glu Lys Asp Pro Ser Tyr Ser Ala Lys Leu
435 440 445
Leu Gln His Ala Lys Ser Leu Trp Gln Phe Ala Val Thr His Lys Gly
450 455 460
Lys Tyr Ser Asp Ser Val Ser Ala Ala Ala Gly Tyr Tyr Asn Ser Ala
465 470 475 480
Asn Val Thr Asp Glu Leu Cys Trp Gly Ser Leu Trp Leu Tyr Glu Ala
485 490 495
Thr Lys Glu Pro Lys Tyr Leu Glu Glu Ala Leu Lys His Tyr Asp Ala
500 505 510
Ser Pro Asp Trp Gly Met Ser Trp Asp Asp Val Phe Ile Gly Asn Gln
515 520 525
Val Leu Leu Tyr Glu Leu Thr Lys Glu Ala Lys Tyr Lys Thr Ala Val
530 535 540
Glu Gly Thr Phe Arg Glu Trp Phe Pro Gly Gly Thr Val Pro Tyr Thr
545 550 555 560
Pro Lys Gly Leu Ala Tyr Arg Leu Gln Trp Gly Ser Leu Arg Tyr Ala
565 570 575
Ser Asn Met Ala Met Ala Ala Leu Met Ala Ala Glu Ala Gly Ile His
580 585 590
Pro Asp Glu Tyr Arg His Trp Ala Met Cys Gln Ile His Tyr Ala Leu
595 600 605
Gly Asp Thr Gly Arg Ser Phe Val Val Gly Phe Gly Lys Asn Pro Pro
610 615 620
Val Ser Pro His His Arg Ser Ser Ser Cys Pro Asn Leu Pro Val Arg
625 630 635 640
Cys Asn Met Asn Tyr Leu His Leu Asp Thr Pro Asn Thr His Met Leu
645 650 655
Cys Gly Ala Leu Val Gly Gly Pro Asp Ser Ser Asp Gly Tyr Lys Asp
660 665 670
Ser Arg Glu Asn Tyr Val Asn Asn Glu Val Ala Cys Asp Tyr Asn Ala
675 680 685
Gly Phe Gln Thr Ala Val Ala Gly Leu Arg Ser Leu Leu Val Arg His
690 695 700
Leu His Pro Glu Gln Lys Gly Gly Ala Thr Cys Pro Tyr His Gly Arg
705 710 715 720
Ala Pro
<210>7
<211>1597
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>7
atgttctcgc tagtcttgtg ggcggggcta ctgctgctga gctttgccat cactgtaccc 60
gtcaccaacc actgggcggg tagattccag gctagagtct gcttcaaaat cgacaaagag 120
atgagttcct gggtcatcca tctgaccttt gaccaccctg tagagacaat tgatgtaagg 180
ctcacctgtc ataggtagat gtatcctacc atagtctaca cctggaagag actgtgtcgg 240
tgagttcagt agttgataac cgtgctgccc tctgtcggtg caggtgtgga cggcggatgc 300
gcagagcacc agcgcggaca agaagcaatg gacgctgacc agcaagaact ggaacgcagt 360
ggagcatgtc ggggacgagc tgtgcatcga cctcatgggt cacggtacgt taggtctact 420
gtcctcagtg tcaccatacg acaatgatga tgatgatgat gatgttgatg atgatgatga 480
tgatgatgat gatgatgatg atgatgatga tttcaggaac tggtgatttc gctcctgtca 540
tcacagcaac cctggaagga gcaagtggcg gcggttcaca tcaagttgct agtgagtact 600
tcacactcgc actctctctc acacacacac acacacgcac atgcgcgcgc acacaaacac 660
acaatgacag aaggacatac agctatttct actctatccc aatacacaaa taaacatata 720
ttttataaat aaagtagtgt ggtgagtgtg tttccgcata tcagtacttc accaacgtgt 780
acctctgtat acgtacataa aggtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 840
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt ttgtttgtgt gtgtgtgtgt 900
gttctctgta tacaattgcc ttcaaactac aaccactaca cttctcttga tcagcaagtg 960
ccccagctac cacacaagct caccaccaag gtgtcgccac tgctgctcct gtgcatacaa 1020
ccccatgtaa gtattcattc accctcgtac ccccatacct gtcgcagtca tacacattgc 1080
cagtgtcatc agaacacacc tcgcagtcac acccacccac tgctacctca gacgtgtgca 1140
actgtggcca gtacaacaga caggaggaga cagtcgagtg tctaataatg attacagtga 1200
agtccagcgt ctgctagcgg ccttctgtcc atctcagtag tttcaaactc actttctgac 1260
aggtgtgggc gtgtcgtaca cacagtcaaa cacgtttcat aagtttcttg cagtcaggtg 1320
tccagacacc aattcaagta cgggtaaggg tagtgatcca cgctatcgca ccccgggtac 1380
atgtcgtgga cctcaagcac agcgtctcta cccacaatgc actcgtacca ttgtatgcat 1440
cacatgtgtt gtcatctctc tttctctccc agtgtctgtc cccgacggaa atggattccc 1500
cgccactacc cgagtctcca acgtgagaga cggacgctca aggccattac ctgaccttca 1560
ccatcagcgg cccaaccgtc atgggctggg tggtcaa 1597
<210>8
<211>2172
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>8
atgttctcgc tagtcttgtg ggcggggcta ccgctcttgg gctttggcag catcactgta 60
cccgtcaaca accactgggc gggtggattc caggctagag tctgctttaa catcgacaag 120
gacatgagtt cctgggtcat ccatctggct ttcgatcaac ctgtacagac ccttgatgtg 180
tggacggcca atgcgcagag caccagcgcg gacaagaagg aatggtcgct gactaacaag 240
gagtggaacg cagtggagca tgtcggggac gagctgtgca ttgacctcat gggtcacggc 300
gacggagata tcgcgcctgt gatcacagcc accctggagg gagcggaagg tggcgggtcg 360
caccaagtta ttacaagtgc accccgcacc acagtcaacc tccctccagg tgtcaccacc 420
gctgaacctc agcctacagc cccattgact gtccccgacg gaaacggttt ccccgccacc 480
accagagtcg ccaacgtcag ggacgggctg tcggagcagt ggctgacctt caccatcacc 540
ggccccaccg tcatgggctg ggtggtcaag ttccgctgca acaagccagt caccaacctc 600
cacgtggcag atgccgacgc cctcagccac aacgaggaca tgacggagtg gctgctggtg 660
aacaacgaca acaagatcgc cctcaaggcg ggaaccttgg agatgaagat tgaagtcaag 720
ctggtgaatg tccaagactc agcccctcag tgctcggcga tcctcatcaa catgggcgta 780
gataactaca cctgcggcga gctacccaac aaggccaact ccaagtacaa ctatgacgat 840
gtcctgtaca agtccatctt gttctacgag gcgcagcgct cgggcaaact tccggccaac 900
aaccgcatcc cctggcgggg ggactccgcc ctcaatgatg gagatggcgg ggtggacctg 960
acaggcgggt ggtatgacgc gggagacttc gtcaagttca acttccccat ggcctggtcc 1020
acggccatct tgaactgggg cttgctgcag ttcaaggacg cctacgaggc tgcaggtcag 1080
ctggagtgga tgtacgagag tgtcaagtgg ccgctggact acctgctcaa gtgtcactcg 1140
tctgatgacg tgctgtacgt gcaggtgggt gatggaggtg cggaccacgg atcatggggg 1200
agacccgagg acatgaagat ggccagaccc gccttcaagg ttgacgccag cagacctggc 1260
tccgaagttg ccatggaaac agctgttgcc tttgtgactg gccacttggc tttcaaagaa 1320
aaagatccgc catactcagc caaactgctg caacatgcca agtccctgtg ggagttcgct 1380
gtcacacaca agggcaagta cagtgaaagt gtctccgccg ccgcaagtta ctacaattcc 1440
gtcaacgtca cagacgagtt gtgctggggg tcgctgtggt tgtacaaggc caccaaggaa 1500
cccaagtacc tggaggaggc cctcaagcac tatgatgctt ctcccgactg gggcatgtcc 1560
tgggacgatg tcttcatcgg caatcaggtg ttgctgtacg aactcacgaa ggaggagaag 1620
tacaaagcag ctgtggaggg caccttcaag gagtggttcc ctggcgggac tgtcccctac 1680
acccctaagg gtctggcgta cagactgcag tggggcgccc tacggtactc gtccaacatg 1740
gctatggccg cgctgatggc tgcagaagcg ggtatccacc cggacgagta tcgccactgg 1800
gccatgtgtc agatccacta cgccctgggg gacactggcc gcagctttgt cgtgggtttt 1860
ggcaaaaatc cacccgtcag tcctcaccac cgctctagct cctgccccaa cctccctgtg 1920
cggtgtaaca tgaactacct ccacctggac acccccaaca ctcacatgct gtgcggggcg 1980
ctggtgggtg gccccgatag ctcggatggt tacaaggaca gccgcgagaa ctacgtcaac 2040
aacgaggtgg cctgcgacta caacgccggc ttccagacag ccgtggccgg tcttcgctcg 2100
ctgttgctaa ggaacctgca tcccgagcag aagggcagcg ccacgtgtcc ctaccatggg 2160
gcagcccctt ga 2172
<210>9
<211>2172
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>9
atgttctcgc tagtcttgtg ggcggggctg ccgctcttgg gctttggcag catcactgta 60
cccgtcaaca accactgggc gggtggattc caggctagag tctgttttaa catcgacaag 120
gacatgagtt cctgggtcat ccatctggct ttcgatcaac ctgtacagac ccttgatgtg 180
tggacggcca atgcgcagag caccagcgcg gacaagaagg aatggtcgct gaccaacaag 240
gagtggaacg cagtggagca tgtcggggac gagctgtgca ttgacctcat gggtcacggc 300
gacggagata tcgcgcctgt gatcacagcc accctggagg gagcggaagg tggcgggtcg 360
caccaagtta ttacaagtgc accccgcacc acagtcaacc tccctccagg tgtcaccacc 420
gctgaacctc agcctacagc cccattgtct gtccccgacg gaaacggttt ccccgccacc 480
accagagtcg ccaacgtcag ggacgggctg tcggagcagt ggctgacctt caccatcacc 540
ggccccaccg tcacgggctg ggtggtcaag ttccgctgca acaagccagt caccaacctc 600
cacgtggcag atgccgacgc cctcagccac aacgaggaca tgacagagtg gctgctggtg 660
aacaacgaca acaagatcgc cctcaaggcg ggaacattgg agatgaagat tgaagtcaag 720
ctggtgaatg tccaagactc agcccctcag tgctcggcga tcctcatcaa catgggcgta 780
gataactaca cgtgcggcga gctacccaac aaggccaact ccaagtacaa ctatgacgat 840
gtcctgtaca agtccatctt gttctacgag gcgcagcgct cgggcaaact tccggccaac 900
aaccgcatcc cctggcgagg cgactccgcc ctcaatgatg gagatggcgg ggtggacctg 960
actggcgggt ggtatgacgc gggagacttc gtcaagttca acttccccat ggcctggtcc 1020
acggccatct tgaactgggg cttgctgcag ttcaaggacg cgtacgaggc cgcaggtcag 1080
ctggagtgga tgtacgagag tgtcaagtgg ccgctggact acctgctcaa gtgtcacgcg 1140
tctgatgacg tgctgtacgt gcaggtgggt gatggaggtg cggaccacgg atcatggggg 1200
agacccgagg acatgaagat ggccagaccc gccttcaaga tcgacgccag caaacctggc 1260
tccgaagttg ccatggaaac agctgttgcc tttgtgactg gccacttggc tttcaaagaa 1320
aaagatccgc catactcagc caaactgctg cagcatgcca agtccctgtg ggagttcgct 1380
gtcacccaca agggcaagta cagtgaaagt gtctccgctg ccgcaagtta ctacaattcc 1440
gtcaacgtca cggacgagtt gtgctggggg tcgctgtggt tgtacaaggc caccaaggaa 1500
cccaagtacc tggaggaggc cctcaagcac tatgatgctt ctcccgactg gggcatgtcc 1560
tgggacgatg tcttcatcgg caatcaggtg ttgctgtacg aactcacgaa ggaggagaag 1620
tacaaagcag ctgtggaggg caccttcaag gagtggttcc ctggcgggac tgtcccctac 1680
acccctaagg gtctggcgta cagactgcag tggggcgccc tacggtactc gtccaacatg 1740
gctatggccg cgctgatggc tgcagaagcg ggtatccacc cggacgagta tcgccactgg 1800
gccatgtgtc agatccacta cgccctgggg gacactggcc gcagctttgt cgtgggtttt 1860
ggcaaaaatc cacccgtcag tcctcaccac cgctctagct cccgccccaa cctacctgtg 1920
cggtgtaaca tgaactacct ccacctggac acccccaaca ctcacatgct gtgcggggcg 1980
ctggtgggtg gccccgatag ctcggatggt tacaaggaca gccgcgagaa ctacgtcaac 2040
aacgaggtgg cctgcgacta caacgccggc ttccagacag ccgtggccgg tcttcgctcg 2100
ctgttgctaa ggaacctgca tcccgagcag aagggcagcg ccacgtgtcc ctaccatggg 2160
gcagcccctt ga 2172
<210>10
<211>2169
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>10
atgttctcgc tggtcctgtg ggcggtgcta ccgctgctga gctttgccat cactgtaccc 60
gtcaccaacc actgggcggg tggattccag gctagagtct gcttcaaaat cgacaaagag 120
atgagttcct gggtcatcca tctgaccttt gaccaccctg tagagacaat tgatgtgtgg 180
acggcggatg cgcagagcac cagcgcggac aagaagcaat ggacgctgac cagcaagaac 240
tggaacgcag tggagcatgt cggggacgag ctgtgcatcg acctcatggg tcacggaact 300
ggtgatttcg ctcctgtcat cacagcaacc ctggaaggag caagtggcgg cggttcacat 360
caagttgcta caagtgcccc agctaccaca caagctcacc atcaaggtgt cgccactgct 420
gctcctgtgc atacaacccc attgtctgtc cccgacggaa atggattccc cgccactacc 480
cgagtctcca acgtgagaga cggacgctcg gaccattacc tgaccttcac catcagcggc 540
ccaaccgtca tgggctgggt ggtcaagttc cggtgcagca agccagtgca caacctcaac 600
gttcgcgagg ccgatccact cagccacaac gccgatcaga cggagtggct gctggtgaac 660
aacaacaacc agatcaccat caagccagga acattcgagt tgaaaattga actggagctg 720
gtgaccggca gagactcggc ccctcagtgc tccgccacac tcaccaacat gggcgtcgac 780
aaacacacct gcggacagct gcccaacaaa gccaactcca agtacaacta tgacgatgtc 840
ctgtacaagt ccatcttgtt ctacgaggcg cagcgctcgg gcaaacttcc ggccaacaac 900
cgcatcccct ggcgagggga ctccgccctc aatgatggag atggcggggt ggacctgact 960
ggcgggtggt atgacgcggg agacttcgtc aagttcaact tccccatggc ctggtccacg 1020
gccgtcttga cctggggctt gctgcagttc aaggacgcct accaggccgc aggtcagctg 1080
gagtggatgt acgagagtgt caagtggccg ctggactacc tgctcaagtg tcacgtgtct 1140
gacaacgtgc tgtacgtgca ggtgggtgat ggaggtgtgg accacggatc atgggggaga 1200
cccgaggaca tgaagatggc cagacccgca ttcaagatcg acgccaccaa gccaggatct 1260
gaagttgcca tggaaacagc ggctgccttt gcggctggac atttggcatt caaagaaaaa 1320
gatccgtcat actcagccaa actgctgcaa catgccaagt cgctgtggca gttcgctgtc 1380
acacacaagg gcaagtacag tgacagtgtg tcagctgctg ccggctacta caattccgcc 1440
aacgtcacgg acgagttgtg ctgggggtcg ctgtggttgt acaaggccac caaggaaccc 1500
aagtacctgg aggaggccct caagcactat gatgcttctc ccgactgggg catgtcctgg 1560
gacgatgtct tcatcggcaa tcaggtgttg ctgtacgaac tcacgaagga ggccaagtac 1620
aaaacagctg tggaaggcac cttcagggag tggttccctg gcgggacggt cccctacacc 1680
cctaagggtc tggcgtacag actgcagtgg gggtccctgc ggtacgcatc caacatggct 1740
atggccgcgc tgatggctgc agaagcgggt atccacccgg acgagtatcg ccactgggcc 1800
atgtgtcaga tccactacgc cctgggggac actggccgca gctttgtcgt gggttttggc 1860
aaaaatccac ccgtcagtcc tcaccaccgc tctagctcct gccccaacct acctgtgcgg 1920
tgtaacatga actacctcca cctggacacc cccaacactc acatgctgtg cggggcgctg 1980
gtgggcggcc ccgatagctc ggatggttac aaggacagcc gcgagaacta cgtcaacaac 2040
gaggtggcct gcgactacaa cgccggcttc cagacagccg tggccggcct tcgctccttg 2100
ctggtccgac acttgcatcc cgagcagaaa ggcggcgcca cgtgtcccta ccacgggaga 2160
gcaccgtga 2169
<210>11
<211>2169
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>11
atgttctcgc tagtcttgtg ggcggggcta ctgctgctga gctttgccat cgctgtaccc 60
gtcaccaacc actgggcggg tggattccag gctagagtct gcttcaaaat cgacaaagag 120
atgagttcct gggtcatcca tctgaccttt gaccaccctg tagagacaat tgatgtgtgg 180
acggcggatg cgcagagcac cagcgcggac aagaagcaat ggacgctgac cagcaagaac 240
tggaacgcag tggagcatgt cggggacgag ctgtgcatcg acctcatggg tcacggaact 300
ggtgatttcg ctcctgtcat cacagcaacc ctggaaggag caagtggcgg cggttcacat 360
caagttgcta caagtgcccc agctaccaca caagctcacc accaaggtgt cgccactgct 420
gctcctgtgc atacaacccc attgtctgtc cccgacggaa atggattccc cgccactacc 480
cgagtctcca acgtgagaga cggacgctcg gaccattacc tgaccttcac catcagcggc 540
ccaaccgtca tgggctgggc ggtcaagttc cggtgcagca agccagtgca caacctcaac 600
gttcgcgagg ccgatccact cagccacaac gccgatcaga cggagtggct gctggtgaac 660
aacgacaacc agatcaccat caagccagga acattcgagt taaaaattga actggagctg 720
gtgaccggca gagactcggc ccctcagtgc tccgccacac tcaccaacat gggggtcgac 780
aaacacacct gcggacagcc gcccaacaag gccaactcca agtacaacta tgacgatgtc 840
ctgtacaagt ccatcttgtt ctacgaggcg cagcgctcgg gcaaacttcc ggccaacaac 900
cgcatcccct ggcgaggcga ctccgccctc aatgatggag atggcggggt ggacctgact 960
ggcgggtggt atgacgcggg agacttcgtc aagttcaact tccccatggc ctggtccacg 1020
gccgtcttga cctggggctt gctgcagttc aaggacgcct accaggccgc aggtcagctg 1080
gagtggatgt acgagagtgt caagtggccg ctggactacc tgctcaagtg tcacgcgtcc 1140
gacaacgtgc tgtacgtgca ggtgggtgac gggggtgcag accacggatc atgggggaga 1200
cccgaggaca tgaagatggc cagacccgcc ttcaagatcg acgccaccaa gccaggatct 1260
gaagttgcca tggaaacagc agctgccttt gcggctgggt atttggcatt caaagaaaaa 1320
gatccgtcat actcagccaa actgctgcaa catgccaagt ccctgtggca gttcgctgtc 1380
acccacaagg gcaagtacag tgacagtgtg tcagctgctg ccggctacta caattccgcc 1440
aacgtcacgg acgagttgtg ctgggggtcg ctgtggttgt acaaggccac caaggaaccc 1500
aagtacctgg aggaggccct caagcactat gatgcttctc cggactgggg catgtcctgg 1560
gacgatgtct tcatcggcaa tcaggtgttg ctgtacgaac tcacgaagga ggccaagtac 1620
aaagcagctg tggagggcac cttcagggag tggttccctg gcgggactgt cccctacacc 1680
cctaagggtc tggcgtacag actgcagtgg gggtccctgc ggtacgcatc caacatggct 1740
atggccgcgc tgatggctgc agaagtgggt atccacccgg acgagtaccg ccactgggcc 1800
atgtgtcaga tccactacgc cctgggcgac actggccgca gctttgtcgt gggttttggc 1860
aaaaatccac ccgtcagtcc tcaccaccgc tctagctcct gtcccaacct acctgtgcgg 1920
tgtaacatga actacctcca cctggacacc cccaacactc acatgctgtg cggggcgctg 1980
gtgggtggcc ccgatagctc ggatggttac aaggacagcc gcgagaacta cgtcaacaac 2040
gaggtggcct gcgactacaa cgccggcttc cagacagccg tggccggcct tcgctccttg 2100
ctggtccgac acttgcatcc cgagcagaaa ggcggcgcca cgtgtcccta ccacgggaga 2160
gcaccgtga 2169
<210>12
<211>2169
<212>DNA
<213>福寿螺(Ampullaria crossean)
<400>12
atgttctcgt tagtcgtgtg ggcggcgcta ccgctgctga gctttgccat cactgtaccc 60
gtcaccaacc actgggcggg tggattccag gctagagtct gcttcaaaat cgacaaagag 120
atgagttcct gggtcatcca tctgaccttt gaccaccctg tagagacaat tgatgtgtgg 180
acggcggatg cgcagagcac cagcgcggac aagaagcaat ggacgctgac cagcaagaac 240
tggaacgcag tggagcatgt cggggacgag ctgtgcatcg acctcatggg tcacggaact 300
ggtgatttcg ctcctgtcat cacagcaacc ctggaaggag caagtggcgg cggttcacat 360
caagttgcta caagtgcccc ggctaccaca caagctcacc atcaaggtgt cgccactgct 420
gctcctgtgc atacaacccc attgtctgtc cccgacggaa atggattccc cgccactacc 480
cgagtctcca acgtgagaga cggacgctcg gaccattacc tgaccttcac catcagcggc 540
ccaaccgtca tgggctgggt ggtcaagttc cggtgcagca agccagtgca caacctcaac 600
gttcgcgagg ccgatccact cagccacaac gccgatcaga cggagtggct actggtgaac 660
aacgacaacc agatcaccat caagccagga acattcgagt tgaaaattga actggagctg 720
gtgaccggca gagactcggc ccctcagtgc tccgccacac tcaccaacat gggcgtcgac 780
aaacacacct gcggacagct gcccaacaaa gccaactcca agtacaacta tgacgatgtc 840
ctgtacaagt ccatcttgtt ctacgaggcg cagcgctcgg gcaaacttcc ggccaacaac 900
cgcatcccct ggcgagggga ctccgccctc aatgatggag atggcggggt ggacctgact 960
ggcgggtggt atgacgcggg agacttcgtc aagttcaact tccccatggc ctggtccacg 1020
gccgtcttga cctggggctt gctgcagttc aaggacgcct accaggccgc aggtcagctg 1080
gagtggatgt acgagagtgt caagtggccg ctggactacc tgctcaagtg tcacgtgtct 1140
gacaacgtgc tgtacgtgca ggtgggtgat ggaggtgtgg accacggatc atgggggaga 1200
cccgaggaca tggagatggc cagacccgca ttcaagatcg acgccaccaa gccaggatct 1260
gaagttgcca tggaaacagc ggctgccttt gcggctggac atttggcatt caaagaaaaa 1320
gatccgtcat actcagccaa actgctgcaa catgccaagt cgctgtggca gttcgctgtc 1380
acacacaagg gcaagtacag tgacagtgtg tcagctgctg ccggctacta caattccgcc 1440
aacgtcacgg acgagttgtg ctgggggtcg ctgtggttgt acaaggccac caaggaaccc 1500
aagtacctgg aggaggccct caagcactat gatgcttctc ccgactgggg catgtcctgg 1560
gacgatgtct tcatcggcaa tcaggtgttg ctgtacgaac tcacgaagga ggccaagtac 1620
aaaacagctg tggaaggcac cttcagggag tggttccctg gcgggacggt cccctacacc 1680
cctaagggtc tggcgtacag actgcagtgg gggtccctgc ggtacgcatc caacatggct 1740
atggccgcgc tgatggctgc agaagcgggt atccacccgg acgagtatcg ccactgggcc 1800
atgtgtcaga tccactacgc cctgggggac actggccgca gctttgtcgt gggttttggc 1860
aaaaatccac ccgtcagtcc tcaccaccgc tctagctcct gccccaacct acctgtgcgg 1920
tgtaacatga actacctcca cctggacacc cccaacactc acatgctgtg cggggcgctg 1980
gtgggtggcc ccgatagctc ggatggttac aaggacagcc gcgagaacta cgtcaacaac 2040
gaggtggcct gcgactacaa cgccggcttc cagacagccg tggccggcct tcgctccttg 2100
ctggtccgac acttgcatcc cgagcagaaa ggcggcgcca cgtgtcccta ccacgggaga 2160
gcaccgtga 2169
<210>13
<211>23
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<220>
<221>misc_feature
<222>(3)..(3)
<223>n=次黄嘌呤
<220>
<221>misc_feature
<222>(6)..(6)
<223>n=次黄嘌呤
<220>
<221>misc_feature
<222>(12)..(12)
<223>n=次黄嘌呤
<220>
<221>misc_feature
<222>(18)..(18)
<223>n=a,t,c或g
<400>13
gtnccngayg gnaayggntt ycc 23
<210>14
<211>26
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>14
cacaacgagg trgcctgcga ctacaa 26
<210>15
<211>27
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>15
gcgctcgggc aaacttccgg ccaacaa 27
<210>16
<211>28
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>16
atgttctcgc tagtcttgtg ggcggggc 28
<210>17
<211>28
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>17
atgttctcgc tggtcctgtg ggcggtgc 28
<210>18
<211>28
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>18
atgttctcgt tagtcgtgtg ggcggcgc 28
<210>19
<211>23
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>19
ctcgggaagc gcgccattgt gtt 23
<210>20
<211>26
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<220>
<221>misc_feature
<222>(1)..(26)
<223>n=a,t,c或g
<400>20
cyraayttna crtgrtcncc ngcrtc 26
<210>21
<211>23
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>21
ttgaccaccc agcccatgac ggt 23
<210>22
<211>23
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>22
gctcaagggg ctgccccatg gta 23
<210>23
<211>28
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>23
cattcagaat gactgcaaac tatgtagc 28
<210>24
<211>28
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>24
atacgactca ctatagggcg aattggcc 28
<210>25
<211>49
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>25
tttgaattcc accaccacca ccaccacagc gtcactgtac ccgtcacca 49
<210>26
<211>48
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>26
tttgaattcc accaccacca ccaccacgtg tctgtccccg acggaaac 48
<210>27
<211>48
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>27
tttgaattcc accaccacca ccaccacagc atcactgtac ccgtcaac 48
<210>28
<211>48
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>28
tttgaattcc accaccacca ccaccacttg actgtccccg acggaaac 48
<210>29
<211>49
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>29
tttgaattcc accaccacca ccaccacatc actgtacccg tcaccaacc 49
<210>30
<211>49
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>30
tttgaattcc accaccacca ccaccacttg tctgtccccg acggaaatg 49
<210>31
<211>31
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>31
aaagcggccg ctcaaggggc tgccccatgg t 31
<210>32
<211>32
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>32
aaagcggccg ctcacggtgc tctcccgtgg ta 32
Claims (10)
1. 一种分离的纤维素酶,其特征在于,它选自:
(a)具有SEQ ID NO:2第1-713位氨基酸序列的多肽;
(b)具有SEQ ID NO:4第1-723位氨基酸序列的多肽;
(c)具有SEQ ID NO:6第1-722位氨基酸序列的多肽;
(d)具有SEQ ID NO:8、9、10、11或12所述多核苷酸序列编码的氨基酸序列的多肽;
(e)将SEQ ID NO:2、4、6的氨基酸序列或(d)的氨基酸序列经过1-10个氨基酸残基的取代、缺失或添加而形成的,且具有纤维素酶功能的由(a)、(b)、(c)或(d)衍生的多肽。
2. 如权利要求1所述的纤维素酶,其特征在于,该酶具有SEQ ID NO:2、4或6的氨基酸序列。
3. 一种分离的多核苷酸,其特征在于,它选自:
(a)编码如权利要求1所述酶的多核苷酸;
(b)在严格条件下与(a)所述多核苷酸序列杂交且编码具有纤维素酶活性的蛋白质的多核苷酸。
4. 如权利要求3所述的多核苷酸,其特征在于,该多核苷酸是编码具有SEQ IDNO:2、4或6所示氨基酸序列的多核苷酸。
5. 如权利要求4所述的多核苷酸,其特征在于,所述的多核苷酸选自下组:
(i)具有SEQ ID NO:1中1-2142位的核苷酸序列;
(ii)具有SEQ ID NO:3中1-2172位的核苷酸序列;
(iii)具有SEQ ID NO:5中1-2169位的核苷酸序列;
(iv)具有SEQ ID NO:8中1-2172位的核苷酸序列;
(v)具有SEQ ID NO:9中1-2172位的核苷酸序列;
(vi)具有SEQ ID NO:10中1-2169位的核苷酸序列;
(vii)具有SEQ ID NO:11中1-2169位的核苷酸序列;
(viii)具有SEQ ID NO:12中1-2169位的核苷酸序列。
6. 一种重组的表达载体,其特征在于,它含有权利要求3所述的多核苷酸。
7. 一种转化的宿主细胞,其特征在于,它含有权利要求6所述的重组的表达载体。
8. 一种权利要求1所述的纤维素酶的制备方法,其特征在于,该方法包含:
(a)在适合表达权利要求1所述的酶的条件下,培养权利要求7所述的宿主细胞;
(b)从培养物中分离出权利要求1所述的纤维素酶。
9. 权利要求1所述的纤维素酶的用途,其特征在于,用于生产简单糖的工艺,所述的简单糖是纤维二糖、葡萄糖及其混合物。
10. 一种生产简单糖的方法,其特征在于,包括步骤:
(a)用权利要求1所述的纤维素酶或权利要求7所述的宿主细胞处理纤维素,从而产生简单糖;
(b)分离出所述的简单糖,所述的简单糖包括纤维二糖、葡萄糖及其混合物。
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CNA2007100374599A CN101245343A (zh) | 2007-02-13 | 2007-02-13 | 新的纤维素酶及其应用 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010005551A2 (en) * | 2008-07-07 | 2010-01-14 | Mascoma Corporation | Heterologous expression of termite cellulases in yeast |
WO2013024021A1 (en) * | 2011-08-15 | 2013-02-21 | Novozymes A/S | Polypeptides having cellulase activity and polynucleotides encoding same |
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WO2010005551A2 (en) * | 2008-07-07 | 2010-01-14 | Mascoma Corporation | Heterologous expression of termite cellulases in yeast |
WO2010005551A3 (en) * | 2008-07-07 | 2010-03-18 | Mascoma Corporation | Heterologous expression of termite cellulases in yeast |
US8658398B2 (en) | 2008-07-07 | 2014-02-25 | Mascoma Corporation | Heterologous expression of termite cellulases yeast |
US9856465B2 (en) | 2008-07-07 | 2018-01-02 | Lallemand Hungary Liquidity Management Llc | Heterologous expression of termite cellulases in yeast |
US10214733B2 (en) | 2008-07-07 | 2019-02-26 | Lallemand Hungary Liquidity Management Llc | Heterologous expression of termite cellulases in yeast |
US10428322B2 (en) | 2008-07-07 | 2019-10-01 | Lallemand Hungary Liquidity Management Llc | Heterologous expression of termite cellulases in yeast |
WO2013024021A1 (en) * | 2011-08-15 | 2013-02-21 | Novozymes A/S | Polypeptides having cellulase activity and polynucleotides encoding same |
CN103748219A (zh) * | 2011-08-15 | 2014-04-23 | 诺维信公司 | 具有纤维素酶活性的多肽以及编码它的多核苷酸 |
US9000138B2 (en) | 2011-08-15 | 2015-04-07 | Novozymes A/S | Expression constructs comprising a Terebella lapidaria nucleic acid encoding a cellulase, host cells, and methods of making the cellulase |
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