腈水解酶突变体及其编码基因和应用的制作方法

文档序号:11145155阅读:701来源:国知局
本发明涉及一种腈水解酶突变体,特别涉及一种腈水解酶突变体及其编码基因,以及其在制备光学活性2-芳基丙酸中的应用。
背景技术
:在有机合成中,氰基易作为“水稳定负碳离子”引入有机化合物,而腈类化合物则是一种多用途的中间体,能转化为胺、酰胺、羧酸和羧基化合物等。用微生物酶水解腈为酰胺和羧酸时,由于反应条件温和以及高纯度的产物使之成为一种备受关注的方法,迄今在工业生产上已有许多成功的例子。近年来,颇有意义的是研究用微生物酶立体选择性水解腈类制备光学活性2-芳基丙酸(2-APA),光学活性2-APA可作为药物活性基质,特别是作为非甾体抗炎药(NSAID)应用。由于药理临床的研究表明2-APA类的NSAID的对映体中只有右旋的构型显示高的药理活性,左旋构型活性低或无作用。鉴于一般化学反应所产生的手性中心总是得到等量的一对对映体混合物,称之为外消旋体。欲得到光学纯右旋2-APA,拆分外消旋体是主要方法之一。一般来说采用化学拆分需用手性衍生试剂,价格昂贵,且过程冗长,较难得到满意的结果。而用微生物酶法拆分则可提供一个经济的选择。酮洛芬(Ketoprofen,KP)又名酮基布洛芬、苯酮苯丙酸、优布芬、优洛芬或Profenid,化学名为α-甲基-3-苯甲酰基苯乙酸,是由法国化学家Farge、Messer和Moutounier于1967年开发的优良的2-芳基丙酸类非甾体抗炎镇痛药物。其作用机制主要是通过抑制体内环氧合酶(COXs)、脂氧化酶(LOXs)的生物活性,从而抑制致炎物质前列腺素(PGs)、白三烯(LTs)的合成,具有对抗缓激肽释放、清除羟自由基以及稳定溶酶体膜活性,从而产生良好的解热、镇痛和抗炎作用,并增强其外周止痛效果。临床研究表明,酮洛芬作为重要的非甾体抗炎药物,与同类药物相比,具有剂量小、疗效高、耐受性好和毒副作用轻微等显著优点,成为治疗类风湿性关节炎、风湿性关节炎、骨关节炎、强硬性脊椎炎以及痛风的理想药物,广泛适用于治疗痛经、牙痛、术后疼痛、癌性疼痛和神经炎、红斑狼疮、咽喉及支气管炎等疾病,对软组织受伤的治疗效果更好。酮洛芬是2-芳基丙酸类非甾体抗炎药(NSAIDs),只有右旋体才具有抗炎抗风湿和镇痛作用,左旋体几乎没有药理活性。酮洛芬的主要生产企业,国外除法国Rhone-Poulenc公司外,还有德国Sanofi-Aventis、意大利SIMS、Cosma和BidaChem、印度BECChemicals等公司;国内西南合成制药股份有限公司(原西南合成制药厂)、中国科学院上海药物研究所实验药厂较早开展了酮洛芬的产品研制和工艺开发工作。目前化学合成酮洛芬的工艺路线已非常成熟,但是合成的酮洛芬缺乏手型选择性。由于左旋酮洛芬没有药理活性,并且有很大副作用。因此,直接合成右旋酮洛芬或者将左旋酮洛芬转变成右旋酮洛芬都具有巨大的经济效益。定点突变是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组,也可以是质粒)中引入所需变化(通常是表征有利方向的变化),包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征,是基因研究工作中一种非常有用的手段。体外定点突变技术是当前生物、医学各领域研究中的一种重要实验手段,是改造、优化基因的便捷方案,是探索启动子调节位点的有效手段,也是研究蛋白质结构和功能之间的复杂关系的有力工具。定点突变改造粪产碱菌腈水解酶的研究具有极佳的理论和应用价值。技术实现要素:本发明的目的是通过定点突变的方法对粪产碱菌腈水解酶基因进行改造,使改造后的基因工程腈水解酶在酶活和手性选择性方面有所提高,使其符合在催化生产右旋2-芳基丙酸工业化应用的需求。本发明采用的技术方案是:本发明提供一种腈水解酶突变体,所述突变体包含SEQIDNO:1所示氨基酸序列中下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点为135、41、192、120;其中,第135位的缬氨酸被突变为丙氨酸或酪氨酸;第41位的天冬氨酸被突变为丙氨酸;第192位亮氨酸被突变为缬氨酸;第120位的赖氨酸被突变为异亮氨酸。优选地,在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、134、120、82、192、319。优选的第41位的天冬氨酸被突变为丙氨酸,第134位的组氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸,第82位的丙氨酸被突变为色氨酸,第192位亮氨酸被突变为缬氨酸,第319位的丝氨酸被突变为异亮氨酸。优选地,在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、192、120。优选的第41位的天冬氨酸被突变为丙氨酸,第192位亮氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸。优选地,在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第134位的组氨酸被突变为缬氨酸,第135位的缬氨酸被突变为丙氨酸或酪氨酸。优选地,在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第134位的组氨酸被突变为缬氨酸,第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、82、192、120、319,优选的第41位的天冬氨酸被突变为丙氨酸,第82位的丙氨酸被突变为色氨酸,第192位亮氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸,第319位的丝氨酸被突变为异亮氨酸。优选地,在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第134位的组氨酸被突变为缬氨酸,第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、192、120,优选的第41位的天冬氨酸被突变为丙氨酸,第192位亮氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸。更优选地,在一些实施例中,所述腈水解酶突变体,包含SEQIDNO:1所示氨基酸序列中选自以下氨基酸突变组成的组:1)V135A+D41A;2)V135A+K120I;3)V135A+L192V;4)H134V+V135A;5)H134V+V135A+L192V;6)H134V+V135A+L192V+D41A;7)H134V+V135A+L192V+K120I。本发明还涉及一种多核苷酸,其编码以上所述的腈水解酶突变体。本发明还涉及一种包含上述多核苷酸的重组载体。本发明还提供一种由所述重组载体转化制备的重组基因工程菌。此外,本发明还提供一种所述腈水解酶突变体在制备光学活性2-APA中的应用。本发明通过定点突变的方式提供了一种腈水解酶突变体,该突变体与野生型腈水解酶相比具有更高的酶活和/或立体选择性,该腈水解酶突变体在制备光学活性2-APA中的应用,较化学合成法,能够降低生产成本,提高生产效率,适应工业化生产的需求。附图说明附图1实施例1野生型腈水解酶琼脂糖凝胶电泳图;附图2实施例5腈水解酶突变体立体选择性催化水解氰基酮洛芬;附图3实施例5腈水解酶突变体琼脂糖凝胶电泳图;附图4野生型腈水解酶三维结构模拟图。具体实施方式本发明的其他特点和优越性可通过以下详细的描述体现。但是,应该理解的是,仅以示例性的方式给出详细描述和具体实施例以表明本发明的实施方案,这是因为对于本领域熟练技术人员从所述的详细描述中,在本发明的实质和范围内多种改变和改良是显而易见的。术语定义本发明所述“2-芳基丙酸(2-APA)”是指具有式Ⅰ结构的化合物,也被称为芳基-2-丙酸。术语“芳基”可以是单环,双环,和三环的碳环体系,其中,至少一个环体系是芳香族的,其中每一个环体系包含3-7个原子。具体来说,本文所述具有消炎作用的2-芳基丙酸类药物包括但不限于如下举例,例如酮洛芬(Ketoprofen),甲氧基甲基萘乙酸(naproxene)、异丁苯丙酸(Ibrufen)、舒洛芬(Sutoprofen)、非诺洛芬(Fenoprofen)、苯恶洛芬(Bennoxaprofen)、卡布洛芬(Carprofen)、环洛芬(Cicloprofen)、氟比洛芬(Flurbiprofen)和氟洛芬(Fluprofen)等。本文所述“腈水解酶的活性”是指,腈水解酶催化水解腈为酰胺和羧酸,本文中优选水解为羧酸。本文所述“酶活”或“转化率”是指腈水解酶催化水解腈为羧酸的能力,或腈水解酶催化水解腈转化为羧酸的比例。腈被催化水解为羧酸的比例越高,酶活越高,即转化率越高,酶活越高。本文所述“光学活性2-芳基丙酸”,是指具有药理活性的2-芳基丙酸,更进一步的,为S(+)-2-APA。本文所述“ee%”意为对映体超量,即在手性合成中,生成目标产物(某一种特定的立体异构体)的百分含量减去副产物(另一种异构体)的百分含量。例如,ee%为98%,即表示生成的目标产物的含量为99%。本文所述“右旋ee%”=(右旋%-左旋%)/(右旋%+左旋%)本文中所述的腈水解酶突变体,除文中提到的氨基酸取代,还包含普通意义上的保守氨基酸取代,所述“保守氨基酸取代”可包括天然氨基酸残基被非天然残基取代,使得对该位置上的氨基酸残基的极性或电荷几乎没有或没有影响。保守氨基酸取代还包括通常通过化学上的肽合成而不是通过在生物系统中的合成而掺入的非天然存在的氨基酸残基。这些包括肽模拟物(peptidomimetics)和氨基酸部分的其它反转形式或反向形式。常见的保守氨基酸取代见表A。表A氨基酸氨基酸取代AlanincD-Ala,Gly,Aib,β-Ala,L-Cys,D-CysArginincD-Arg,Lys,D-Lys,OrnD-OmAsparagineD-Asn.Asp,D-Asp,Glu,D-GluGln,D-GlnAsparticAcidD-Asp,D-Asn.Asn.Glu.D-Glu,Gln,D-GlnCysteineD-Cys,S-Me-Cys,Met,D-Met,Thr,D-Thr,L-Ser,D-SerGlutamincD-Gln,Asn,D-Asn,Glu,D-Glu,Asp,D-AspGlutamicAcidD-Glu,D-Asp,Asp,Asn,D-Asn,Gln,D-GlnGlycincAla,D-Ala,Pro,D-Pro,Aib,β-AlaIsoleucineD-Ile,Val,D-Val,leu,D-Leu,Met,D-MetLeucineVal,D-Val,Met,D-Met,D-Ile,D-Leu,lleLysincD-Lys,Arg,D-Arg,Orn,D-OmMethionineD-Met,S-Me-Cys,lle,D-Ile,Leu,D-Leu,Val,D-ValPhcnylalanincD-Phc,Tyr,D-Tyr,His,D-His,Trp,D-Trp腈水解酶突变体、制备方法及应用NagasawaT等发现玫瑰色红球菌(Rhodococcusrhodochrous)J1产生的腈水解酶既可以用作脂肪族腈,也可以作用于芳香族水解为相应羧酸,此后,MaugerJ等分离出粪产碱杆菌(Alcaligenesfaecalis)JM3能够将芳基乙腈水解为相应羧酸。发明人在土壤中分离出一株粪产碱杆菌,并提取出野生型腈水解酶,发现其在制备光学活性2-APA中具有较低的转化率且立体选择性不佳。发明人通过对该腈水解酶的三维结构进行模拟(见图4),筛选出可能对催化水解起关键作用的25个位点:R127、V135、L167、V57、W164、S189、L192、K203、M206、S106、K131、P169、M314、L322、P330、I344、V12、D41、A82、K120、H134、V139、S319、W58、H134,这些位点所在的区域被称为催化口袋,在与底物结合或立体选择性方面可能存在较大影响,发明人对它们分别进行定点突变,意外获得7个有益的突变位点V135、L192、D41、A82、K120、S319、H134,其中包含这些位点突变的腈水解酶突变体,在催化转化率和/或立体选择性方面有所增强。在一些实施例中,本发明提供一种腈水解酶突变体,所述突变体包含SEQIDNO:1所示氨基酸序列中下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点为135、41、192、120。其中第135位的缬氨酸被突变为丙氨酸或酪氨酸;第41位的天冬氨酸被突变为丙氨酸;第192位亮氨酸被突变为缬氨酸;第120位的赖氨酸被突变为异亮氨酸。在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、134、120、82、192、319。优选的第41位的天冬氨酸被突变为丙氨酸,第134位的组氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸,第82位的丙氨酸被突变为色氨酸,第192位亮氨酸被突变为缬氨酸,第319位的丝氨酸被突变为异亮氨酸。在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、192、120。优选的第41位的天冬氨酸被突变为丙氨酸,第192位亮氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸。在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第134位的组氨酸被突变为缬氨酸,第135位的缬氨酸被突变为丙氨酸或酪氨酸。在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第134位的组氨酸被突变为缬氨酸,第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、82、192、120、319,优选的第41位的天冬氨酸被突变为丙氨酸,第82位的丙氨酸被突变为色氨酸,第192位亮氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸,第319位的丝氨酸被突变为异亮氨酸。在一些实施例中,所述腈水解酶突变体包含SEQIDNO:1所示氨基酸序列中第134位的组氨酸被突变为缬氨酸,第135位的缬氨酸被突变为丙氨酸或酪氨酸。还包含下述任何一个或多个氨基酸位点上的氨基酸取代,所述位点选自41、192、120,优选的第41位的天冬氨酸被突变为丙氨酸,第192位亮氨酸被突变为缬氨酸,第120位的赖氨酸被突变为异亮氨酸。在一些优选实施例中,所述腈水解酶突变体,包含SEQIDNO:1所示氨基酸序列中选自以下氨基酸突变组成的组:1)V135A+D41A;2)V135A+K120I;3)V135A+L192V;4)H134V+V135A;5)H134V+V135A+L192V;6)H134V+V135A+L192V+D41A;7)H134V+V135A+L192V+K120I;本发明还涉及一种多核苷酸,其编码以上所述的腈水解酶突变体。本发明还涉及一种包含上述多核苷酸的重组载体。本发明还提供一种由所述重组载体转化制备的重组基因工程菌。此外,本发明还提供一种所述腈水解酶突变体制备光学活性2-APA中的应用。本文中所用到的试剂均可通过市售得到。实施例实施例1野生型腈水解酶基因的克隆(1)野生型腈水解酶基因的获得从工业废水附近土壤中筛选出一株粪产碱杆菌HEC-AF001使用OMEGA细菌基因组试剂盒进行总基因组DNA的提取,使用下述引物扩增长约1.1kb大小的基因片段。HEC-AF001-F:CCCATATGCAGACAAGAAAAATC(SEQIDNO:17)HEC-AF001-R:CCAAGCTTTCAGGACGGTTCTTG(SEQIDNO:18)PCR反应体系见表1表1成分体系(μl)5×PrimeSTARPCRHSBuffer10引物11引物21模板(<0.2μg)1dNTPs(2.5mM)4PrimeSTARPCRHSPolymerase0.5ddH2O50PCR扩增程序:98℃,预变性20s;98℃,变性10s;60℃退火10s;72℃延伸60s;重复30个循环;72℃继续延伸10min。PCR产物经琼脂糖凝胶电泳纯化,利用琼脂糖凝胶DNA回收试剂盒回收1100bp左右的目标条带(见图1)。送广州艾基生物公司测序,确认是目的条带,如此获得一条完整的腈水解酶全长基因序列,命名为NIT1,全长1071bp,碱基序列如序列表中SEQIDNo:2所示,利用软件对该基因序列进行分析,得到如SEQIDNo.2所示的野生型腈水解酶氨基酸序列SEQIDNo:1。实施例2重组表达质粒和重组表达转化体的制备将实施例1所得的PCR产物在37℃用限制性内切酶NdeI和HindⅢ双酶切4h,经琼脂糖凝胶电泳纯化,利用琼脂糖凝胶DNA回收试剂盒回收目标片段,其中含有正确的插入片段。将目标片段与同样经NdeI和HindⅢ酶切后的质粒pET28a混合,在T4DNA连接酶的作用下,16℃连接4h得到重组表达质粒pET28a-NIT1。将上述重组表达质粒转化到E.coliTop10感受态细胞中。在含有卡那霉素的抗性平板(培养基成分LB培养基,蛋白胨10g/L,酵母膏5g/L,氯化钠10g/L和琼脂2%,抗生素含量50mg/L)上对阳性重组体进行筛选,挑取单克隆,培养重组菌株,待质粒扩增后提取质粒,经测序验证正确后,重新转化至E.coliBL21(DE3)感受态细胞中。转化液涂布到含有卡那霉素(50mg/L)的LB平板上,37℃倒置培养过夜,获得阳性重组大肠杆菌E.coliBL21(DE3)/pET28a-NIT1,命名为HEC-NIT1。实施例3重组腈水解酶的表达将实施例2所得的重组大肠杆菌,接种至含卡那霉素(50mg/L)的LB培养基中,37℃振荡培养过夜。按2‰(v/v)的接种量接入装有50mlLB培养基的250ml三角烧瓶中,置37℃、180rpm摇床震荡培养。当培养液的OD600达到0.8时,加入终浓度为0.5mmol/L的IPTG进行诱导。30℃诱导8h后,将培养液离心,收集细胞,得0.3g湿细胞。实施例4利用NIT1重组腈水解酶催化水解氰基酮洛芬酶催化反应:分别将实施例4得到的腈水解酶湿菌体加入至含25ml底物溶液(工作浓度为1mM,称取0.235g氰基酮洛芬,使用甲醇90ml、1M磷酸钾缓冲液(pH7.8)90ml、H2O720ml进行溶解)的三角瓶中,于30℃、250rpm条件下反应48h,取出三角瓶,分别加入10ml甲醇溶液使氰基酮洛芬底物完全溶解,8000rpm、10min离心,弃除菌渣,收集上清。反应体系后处理:将得到的上清液分别用旋转蒸发仪于40℃条件下挥除甲醇,用1MHCl调溶液pH至2~3左右;加入等体积的乙酸乙酯、混合均匀,收集乙酸乙酯相;取乙酸乙酯层吹干,取200μl90%正己烷(异丙醇)复溶,过0.45um滤膜,待用。HPLC检测转化率:色谱柱,WatersXBridgeC18;柱温,30℃;流动相,0.02MNaClO4-乙腈,结果见表5。HPLC检测手性::色谱柱,ChiralND柱;柱温,30℃;流动相,正己烷(0.15%TFA):异丙醇=93:7,检测结果见表5。实施例5:野生型腈水解酶定向改造参考野生型腈水解酶基因序列(SEQIDNO:2),设计并合成两条寡核苷酸引物,并采用未突变的重组质粒,通过PCR扩增的方法获得表2中腈水解酶突变体:表2涉及到的引物见表3表3PCR反应体系见表4表4成分体系(μl)PrimeSTARMaxPremix25引物12引物22模板(约300ng)3灭菌水Upto50PCR程序条件设定为:98℃预变性5min;98℃变性20s,60℃退火20s,72℃延伸2min,30个循环;72℃终延伸5min。1%核酸电泳检测结果见图3。对扩增获得的PCR产物进行醇沉处理,再使用DpnⅠ对模板进行消化处理,最后使用DNA胶回收试剂盒进行割胶回收。将回收处理得到的产物电转化至电感受态细胞E.coliBL21(DE3)中,转移细胞至含kana抗性(终浓度为50μg/ml)平板在37℃条件下培养过夜获得大量转化子。扩培后进行测序验证,验证正确的转化子即为基因工程腈水解酶产生菌。将基因工程腈水解酶产生菌接入50ml/250ml的液体LB培养基中(含kana抗性,终浓度为50μg/ml),37℃培养OD600至0.8左右时,使用IPTG在30℃条件下进行诱导培养6h,收菌。菌体在离心机最大转速中4℃离心10min收集蛋白。测定蛋白的酶转化率及手性选择性(方法与实施例4)相同,测定结果见表5。表5突变情况转化率(酶活)右旋ee%值野生型18%8.9%D41A90%86%A82W84%77%K120I89%85%V135A90%90%L192V96%67%S319I82%70%V135A、D41A93%91%V135A、K120I92%92%V135A、L192V98%80%V135Y65%70%H134V、V135A93%98%H134V、V135A、L192V96%99%H134V、V135A、L192V、D41A99%99%H134V、V135A、L192V、K120I98%99%结果分析:在单一位点突变中,D41A、A82W、K120I、V135A、V135Y、L192V、S319I显示出优于野生型腈水解酶的酶活或立体选择性,L192V在提高酶活方面最优,V135A在立体选择性方面最优,在多位点突变中,135、192、41、120也表现出有益的效果。在三位点突变组合中加入第四个突变位点H134,可以使效果进一步提高,根据三维立体结构推测H134V、V135A位点的突变,破坏有利于R型底物结合的因素,降低对R型催化活力。His134可能在R型底物优势构象的定位中具有重要作用,His134的咪唑基与底物苯环间可能存在π-π相互作用或阳离子-π相互作用,不利于左旋产物的形成。SEQUENCELISTING<110>东莞东阳光药物研发有限公司;黄石世星药业有限责任公司<120>腈水解酶突变体及其编码基因和应用<130>2017<160>34<170>PatentInversion3.5<210>1<211>356<212>PRT<213>Alcaligenesfaecalis<400>1MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisValGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205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rPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>6<211>356<212>PRT<213>人工序列<400>6MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>7<211>356<212>PRT<213>人工序列<400>7MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisValGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>8<211>356<212>PRT<213>人工序列<400>8MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisValGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisIleGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>9<211>356<212>PRT<213>人工序列<400>9MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAlaLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>10<211>356<212>PRT<213>人工序列<400>10MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspIleGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>11<211>356<212>PRT<213>人工序列<400>11MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>12<211>356<212>PRT<213>人工序列<400>12MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrHisTyrGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>13<211>356<212>PRT<213>人工序列<400>13MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerLeu180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>14<211>356<212>PRT<213>人工序列<400>14MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>15<211>356<212>PRT<213>人工序列<400>15MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAlaLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspLysGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>16<211>356<212>PRT<213>人工序列<400>16MetGlnThrArgLysIleValArgAlaAlaAlaValGlnAlaAlaSer151015ProAsnTyrAspLeuAlaAlaGlyValAspLysThrIleGluLeuAla202530ArgGlnAlaArgAspGluGlyCysAspLeuIleValPheGlyGluThr354045TrpLeuProGlyTyrProPheHisValTrpLeuGlyAlaProAlaTrp505560SerLeuLysTyrSerAlaArgTyrTyrAlaAsnSerLeuSerLeuAsp65707580SerAlaGluPheGlnArgIleAlaGlnAlaAlaArgThrLeuGlyIle859095PheIleAlaLeuGlyTyrSerGluArgSerGlyGlySerLeuTyrLeu100105110GlyGlnCysLeuIleAspAspIleGlyGluMetLeuTrpSerArgArg115120125LysLeuLysProThrValAlaGluArgThrValPheGlyGluGlyTyr130135140AlaArgAspLeuIleValSerAspThrGluLeuGlyArgValGlyAla145150155160LeuCysCysTrpGluHisLeuSerProLeuSerLysTyrAlaLeuTyr165170175SerGlnHisGluAlaIleHisIleAlaAlaTrpProSerPheSerVal180185190TyrSerGluGlnAlaHisAlaLeuSerAlaLysValAsnMetAlaAla195200205SerGlnIleTyrSerValGluGlyGlnCysPheThrIleAlaAlaSer210215220SerValValThrGlnGluThrLeuAspMetLeuGluValGlyGluHis225230235240AsnAlaSerLeuLeuThrValGlyGlyGlySerSerMetIlePheAla245250255ProAspGlyArgThrLeuAlaProTyrLeuProHisAspAlaGluGly260265270LeuIleIleAlaAspLeuAsnMetGluGluIleAlaPheAlaLysAla275280285IleAsnAspProAlaGlyHisTyrSerLysProGluAlaThrArgLeu290295300ValLeuAspLeuGlyHisArgGluProMetThrArgValHisSerGln305310315320SerLeuIleLysGluGluAlaCysGluProLeuThrProSerThrIle325330335AlaProValAlaIleSerGlnIleGlnGluSerAspThrLeuLeuVal340345350GlnGluProSer355<210>17<211>23<212>DNA<213>人工序列<400>17cccatatgcagacaagaaaaatc23<210>18<211>23<212>DNA<213>人工序列<400>18ccaagctttcaggacggttcttg23<210>19<211>46<212>DNA<213>人工序列<400>19cgcaaacttaaacccacacatgcggagcgcaccgtgtttggtgaag46<210>20<211>46<212>DNA<213>人工序列<400>20cttcaccaaacacggtgcgctccgcatgtgtgggtttaagtttgcg46<210>21<211>46<212>DNA<213>人工序列<400>21cgcaaacttaaacccacacattatgagcgcaccgtgtttggtgaag46<210>22<211>46<212>DNA<213>人工序列<400>22cttcaccaaacacggtgcgctcataatgtgtgggtttaagtttgcg46<210>23<211>44<212>DNA<213>人工序列<400>23ccgcaaacttaaacccacagtggcggagcgcaccgtgtttggtg44<210>24<211>44<212>DNA<213>人工序列<400>24caccaaacacggtgcgctccgccactgtgggtttaagtttgcgg44<210>25<211>41<212>DNA<213>人工序列<400>25cgcctggccgtctttttcggtgtacagtgaacaggcccatg41<210>26<211>41<212>DNA<213>人工序列<400>26catgggcctgttcactgtacaccgaaaaagacggccaggcg41<210>27<211>45<212>DNA<213>人工序列<400>27caggcccgcgatgagggctgcgcactgatcgtgtttggtgaaacc45<210>28<211>46<212>DNA<213>人工序列<400>28ggtttcaccaaacacgatcagttgcgcagccctcatcgcgggcctg46<210>29<211>45<212>DNA<213>人工序列<400>29aactcgctctcgctggacagttgggaatttcaacgcattgcccag45<210>30<211>45<212>DNA<213>人工序列<400>30ctgggcaatgcgttgaaattcccaactgtccagcgagagcgagtt45<210>31<211>45<212>DNA<213>人工序列<400>31ggccaatgcctgattgacgacatcggcgagatgctgtggtcgcgc45<210>32<211>45<212>DNA<213>人工序列<400>32gcgcgaccacagcatctcgccgatgtcgtcaatcaggcattggcc45<210>33<211>45<212>DNA<213>人工序列<400>33gagcccatgacacgggttcacatccaaagcctgatcaaggaagag45<210>34<211>45<212>DNA<213>人工序列<400>34ctcttccttgatcaggctttggatgtgaacccgtgtcatgggctc45当前第1页1 2 3 
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