公开/公告号CN101812103A
专利类型发明专利
公开/公告日2010-08-25
原文格式PDF
申请/专利权人 中国科学院上海有机化学研究所;
申请/专利号CN200910247567.8
申请日2009-12-30
分类号C07H17/08;C12N9/00;C12N15/52;C12N15/76;C12P19/62;A61K31/7048;A61P31/04;A61P35/00;
代理机构上海新天专利代理有限公司;
代理人邬震中
地址 200032 上海市徐汇区枫林路354号
入库时间 2023-12-18 00:35:33
法律状态公告日
法律状态信息
法律状态
2023-06-02
专利权的转移 IPC(主分类):C07H17/08 专利号:ZL2009102475678 登记生效日:20230523 变更事项:专利权人 变更前权利人:中国科学院上海有机化学研究所 变更后权利人:国科新研国际技术转移有限公司 变更事项:地址 变更前权利人:200032 上海市徐汇区枫林路354号 变更后权利人:202152 上海市崇明区三星镇北星公路1999号3号楼196-8室(上海玉海棠科技园区)
专利申请权、专利权的转移
2012-08-29
授权
授权
2010-10-13
实质审查的生效 IPC(主分类):C07H17/08 申请日:20091230
实质审查的生效
2010-08-25
公开
公开
技术领域
本发明属于生物技术工程领域,具体涉及基因工程改造6-MSA合成酶功能,在重组菌株组合生物合成新化合物的发酵,分离,结构鉴定,活性测定。
背景技术
天然化合物是药物发现和发展的主要源泉,如抗感染的青霉素和红霉素、抗肿瘤的博来霉素和埃托霉素、抗寄生虫的阿维菌素和免疫抑制剂环孢菌素等,在过去几十年里一直是人类治疗疾病的主要药物。所以以天然化合物为基础研制和开发新药一直是化学界和医药界关注的重点领域。其中微生物来源的天然产物占重要的部分,目前已报道的微生物天然产物有数千种之多,其中聚酮类和非核糖体聚肽类占了相当大的比例。
螺环乙酰乙酸内酯抗生素家族是聚酮类天然产物中非常有特色的一类,它们中的绝大多数是由放线菌产生,具有良好的抗菌活性。其中代表性分子Chlorothricin(CHL,图1)是一个新型的大环内酯类抗生素,首次分离自菌株Streptomyces antibioticusTü99,结构特点是由糖苷配基的大环骨架部分含有[6,6]并环和[6,5]螺环(图1):(1)反式的十氢萘环,(2)4-羟乙酰乙酸内酯和一个环己烯环形成的螺环(螺环乙酰乙酸内酯家族天然产物由此得名),侧链糖基和六甲基水杨酸组成(Helv.Chim.Acta 52(1969)127-142)。氯丝菌素具有良好的抗感染活性,其抗菌活性归结于对丙酮酸羧化酶催化的anaplerotic CO2固定的抑制。进一步的研究表明,CHL与Bacillus subtilis细胞膜磷脂存在相互作用,具有抗胆固醇的活性。最新研究还表明其具有潜在的抗肿瘤活性(J.Antibiot.34,1101-1106,J.Antibiot.36,668-670J.Antibiot.44,207-212)。
氯丝菌素独特的化学结构和丰富的生物活性引起了化学、生物和医药界的关注,如何以之作为先导化合物发展更有价值的临床药物成为各方努力的方向。但是,其复杂的化学结构为化学合成和结构衍生带来了巨大的挑战,由于过多的手性中心和集中的官能团,化学合成对于CHL及其类似物的生产前景非常有限,作为有机合成的重要补充,组合生物合成技术的发展为复杂天然产物及其类似物的获得提供了新方法。通过对其生物合成基因的克隆,揭示了其包括大环骨架、侧链糖基和六甲基水杨酸等结构单元在内的生物合成机制(Chemistry&Biology 13,575-585),在此基础上运用组合生物合成的原理,合理修饰其生物合成的代谢途径,探索获得所需要的新型的生物活性提高的结构类似物。
发明内容
本发明的目的是提供一种新型的产生活性提高的氯丝菌素类似物的方法,具体涉及一种对六-甲基水杨酸合成酶的KR结构域的氨基酸残基突变,最终在氯丝菌素产生菌的重组菌株中产生活性提高的螺环乙酰乙酸内酯类抗生素。
具体如下:
首先用PCR的方法在Streptomyces antibioticus DSM40725中得到编码六-甲基水杨酸合成酶KR结构域的1.9KbDNA片段,然后连入pSP72的载体中得到含有KR片段的质粒,下一步从PCR得到的质粒中重切出含有突变位点的KR结构域片段,替代野生型的6-MSAS的KR结构域一起连入表达载体pTGV2中,然后把此pTGV2衍生的质粒导入到异源表达模式菌株Streptomyces albus中检测产物。结果1540位的Y突变成F的重组菌株中产生OSA。上述方法简单易行,只需常规的分子生物学手段,就可以很快得到功能改变的蛋白,并在异源宿主中检测突变的产物。本发明提供的点突变蛋白的方法,完全不依赖于任何商业化的突变试剂盒,而是一种简单,廉价,高效的点突变蛋白的方法。
本发明还提供了一种在Streptomyces antibioticus DSM40725 chlB1基因敲除的突变菌株中产生两个的新的氯丝菌素类似物的策略和应用。具体方法是我们把上述在Streptomyces albus中异源表达产生OSA的表达质粒导入到Streptomyces antibioticusDSM40725chlB1基因敲除的突变菌株的中,发酵检测,分离纯化,结构鉴定,初步的生物活性测定得到了两个活性提高一倍的绿丝菌素的类似物7和8。
附图说明
图1表示Chlorothricin(CHL),deschloro-CHL,以及本发明所产生的新化合物7和8的结构。
图2表示几种含有一型重复PKS合成的芳香单元的天然产物,Cholorothricin,Maduroprptin,polyketomycin,Avilamycin,Calicheamicin。
图3表示真菌和细菌6-MSAS的结构域组成以及其结构域选择性失活产生的产物的推测,(A)6-MSAS的功能结构域的组成。(B)真菌和细菌6-MSAS的合成路线以及DH突变(H947F)的ChlB1产生6-MSA;KR突变(Y1450F)的ChlB1产生OSA;真菌KR突变(G1387A,G1389P,G1392A)的6-MSAS产生TAL。
图4表示HPLC分析DH和KR突变后的ChlB1在Streptomyces albus中异源表达的产物。(I)Streptomyces albus含有pTGV2空质粒;(II)6-MSA标准品;(III)Streptomyces albus含有野生型6-MSAS(ChlB1)表达质粒产生6-MSA,产量约为2mg/ml;(IV)Streptomyces albus含有DH突变(H947A)的ChlB1表达质粒不产生6-MSA;(V)Streptomyces albus含有DH突变(H947F)的ChlB1表达质粒继续产生6-MSA,产量约为0.3mg/ml;(VI)TAL标准品;(VII)OSA标准品;(VIII)Streptomycesalbus含有KR突变(G1389A)的ChlB1表达质粒不产生6-MSA,TAL,OSA;(IV)Streptomyces albus含有KR突变(G1387A,G1389P,G1392A)的ChlB1表达质粒不产生6-MSA,TAL,OSA;(X)Streptomyces albus含有KR突变(Y1540F)的ChlB1表达质粒产生OSA,产量约为0.3mg/ml。
图5表示6-MSA和OSA的酰基单元的后修饰以及转移到DMCHL糖基上形成deschloro-CHL;CHL;7和8的示意图。
图6表示HPLC分析Streptomyces antibioticus DSM40725(ΔchlB1)中新的螺环乙酰乙酸内酯的产生(I)野生菌株Streptomyces antibioticus DSM40725产生deschloro-CHL,CHL;(II)突变菌株Streptomyces antibioticus DSM40725(ΔchlB1)产生DMCHL;(III)KR突变(Y1540F)的ChlB1表达质粒互补Streptomyces antibioticusDSM40725(ΔchlB1)菌株产生7,8和DMCHL。a,deschloro-CHL;b,CHL;C,DM-CHL;d,7;and e,8。
图7表示新化合物7的1H-1H COSY以及7和8的OSA单元的HMBC示意图。
图8表示化合物71H-NMR图谱
图9表示化合物81H-NMR图谱
图10表示新化合物7的H-H COSY
图11表示新化合物7的HSQC
图12表示新化合物7的HMBC
图13表示新化合物7ROESY
图14表示新化合物8的OSA单元的HMBC图谱。表明化合物8的结构是CHL的结构且在6-MSA单元苯环的对位增加了氧原子。
图15表示将MSS4.3导入到Streptomyces antibioticus DSM40725(ΔchlB1)中发酵产物的HPLC分析:(I)MSS4.3质粒互补Streptomyces antibioticus DSM40725(ΔchlB1)菌株产生OSA以及痕量的7和8;(II)OSA标准品。
图16化合物7的LC-MS
图17化合物8的LC-MS
图18表示新化合物的抗Bacillus subtilis活性测定:1,deschloro-CHL;2,CHL;3,7;4,8。(I)各化合物5μg溶于200μl甲醇后加入牛筋杯;(II)各化合物10μg溶于200μl甲醇后加入牛筋杯,37度,12小时。7和8的抗菌活性分别比deschloro-CHL和CHL提高了1倍左右。
符号说明
附图1中,Chlorothricin(CHL):氯丝菌素;deschloro-CHL:脱氯氯丝菌素;7和8:氯丝菌素的类似物。
附图2中,PKS:聚酮。
附图3中,6-MSAS,六-甲基水杨酸;AT:酰基转移酶;KS:酮基合成酶;DH:脱氢酶;KR:酮基还原酶;ACP:酰基载体蛋白。
附图4中,Streptomyces albus:异源表达的模式菌株,属于链霉菌属;ChlB1:氯丝菌素生物合成基因簇中六-甲基水杨酸合成酶;HPLC:高效液相色谱;OSA:苔色酸;TAL:2羟基4-甲基-2-吡喃酮。
附图5中,CHL:氯丝菌素;deschloro-CHL:脱氯氯丝菌素;DM-CHL:脱六-甲基水杨酸氯丝菌素。
附图6中,Streptomyces antibioticus DSM40725氯丝菌素产生菌,属于链霉菌属。7和8:新化合物。
附图7中,1H-1H COSY:氢-氢二维相关谱;HMBC:多键碳氢关系。
附图81H-NMR:核磁共振氢谱。
附图11中,HSQC:异核单量子相干谱
附图13中,NOESY:二维核奥弗豪泽增强谱。
附图15中,MSS4.3:苔色酸合成酶的基因(aviM)克隆到表达载体pWHM3中。
附图17中,LC-MS:高效液相色谱和质谱联用。
附图18中,Bacillus subtilis:枯草芽孢杆菌。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应知道,这些实例仅用于说明本发明而不用于限制本发明的范围。本发明的其它方面由于本文的公开内容,对本领域的技术人员而言是显而易见的,下列实施例中未注明具体条件的实验方法,通常按照公开发表的文献中所述的条件。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用,但不能限制本方面的内容。
实施例1 ChlB1的DH和KR结构域点突变和异源表达
1.DH和KR结构域点突变的ChlB1的获得
1)DH结构域的突变
失活ChlB1的DH结构域,选择突变其947的组氨酸残基,将其突变成甘氨酸或者苯丙氨酸。首先取两对设计好的引物,引物序列如下:
947位H突变成A
上游:5’-C TAC CCC GGC AGC GCC ACC ATC AAC GGC ACG-3’
下游:5’-CGT GCC GTT GAT GGT GGC GCT GCC GGG GTA G-3’
947位H突变成F
上游:5’-C TAC CCC GGC AGC TTC ACC ATC AAC GGC ACG-3’
下游:5’-CGT GCC GTT GAT GGT GAA GCT GCC GGG GTA G-3’
以pAL1084(野生型DH结构域的片段克隆到pSP72中)为模板模板,dNTP,DMSO,无酶水,高保真的primestar酶及其缓冲液组成PCR反应体系按照设计好的程序进行PCR。PCR产物为含有突变的DH结构域片段的质粒,将其进行凝胶电泳分离,切胶回收纯化,加入限制性内切酶Dpn I切碎作为模板的质粒pAL1084。然后吸取5μl酶切体系转化E.coli DH5,挑取单克隆菌落于LB培养液(含有Amp抗生素)中培养过夜,至菌液较浓。提取质粒送测序,检测947位的H是否突变成的相应的氨基酸。然后从测序突变的质粒中用BglII/AvrII切出1.0kb的片段连入pAL1087(chlB1克隆到pTGV2中)的相同的酶切位点中,得到DH的突变的pTGV2衍生质粒pAL1088,pAL1089。
2)KR的突变
失活ChlB1的KR结构域,选择三种策略①1389位的G突变成A,②1387的G突变成A,1389的G突变成P,1392位的G突变成A,③1540位的Y突变成F。
取三对设计好的引物,引物序列如下:
①1389位的G突变成A
上游:5’-C ACC GGC GGA CTG GCC ACC CTC GGC CTG G-3’
下游:5’-C CAG GCC GAG GGT GGC CAG TCC GCC GGT G-3’
②1387的G突变成A,1389的G突变成P,1392位的G突变成A
上游:5’-C ACC GGC GCA CTG CCC ACC CTC GCC CTG G-3’
下游:5’-C CAG GGC GAG GGT GGG CAG TGC GCC GGT G-3’
③1540位的Y突变成F
上游:5’-GGC CAG GCC GCC TTC GGC TCC GCC AAC G-3’
下游:5’-C GTT GGC GGA GCC GAA GGC GGC CTG GCC-3’
以pAL1085(野生型KR结构域的片段克隆到pSP72中)为模板模板,dNTP,DMSO,无酶水,高保真的primestar酶及其缓冲液组成PCR反应体系按照设计好的程序进行PCR。PCR产物为含有突变的KR结构域的质粒,将其进行凝胶电泳分离,切胶回收纯化,加入限制性内切酶Dpn I切碎作为模板质粒pAL1085。然后吸取5ul酶切体系转化E.coli DH5,挑取单克隆菌落入LB培养液(含有Amp抗生素)中培养过夜,至菌液较浓。提取质粒送测序,检测1387G,1389G,1392G是否突变成的相应的氨基酸。然后从测序突变的质粒中用AvrII/HindIII切出1.9kb的片段连入pAL1087的相同的酶切位点中,分别得到KR的突变的pTGV2衍生质粒pAL1090,pAL1091,pAL1092。
2.DH和KR结构域突变的ChlB1的在异源表达
1)菌株ET12567与菌株Streptomyces albus J1074的属间接合转移。
将上述质粒pAL1088,pAL1089,pAL1090,pAL1091,pAL1092转化ET12567,培养含有适当质粒的ET12567至OD600=0.4-0.6,25ml培养液中的细菌离心收集,用等体积的LB洗两次,重悬于1ml LB中,作为大肠杆菌供体细胞。取适量冻存于-80℃的Streptomycesalbus J1074的20%甘油孢子悬液500μL,用500μl的TES缓冲液洗两次,重悬于等体积的TES缓冲液,50℃热激10分钟使孢子萌发。再加等体积的TSB,37℃温育2-5小时,离心重悬于1.5ml LB培养基中作为链霉菌受体细胞。将不同浓度的受体细胞100μL与等体积的供体细胞混合直接涂布在含有10mM氯化镁的MS(2.0g甘露糖醇,2.0g黄豆饼粉,2.0g琼脂,自来水定容至100ml)平板上,30℃培养20小时后,用无菌水轻轻洗涤平板表面以洗去大部分大肠杆菌,在每一平板的表面覆盖1ml含萘啶酮酸(终浓度为50μg/ml)和相应抗生素(Tsr 50μg/ml))的无菌水。30℃培养5天以上挑取接合子。
2)接合子的筛选.
挑单菌落于TSB(Tsr 50μg/ml)培养,用菌液PCR验证导入质粒的正确性。
3)发酵和处理:
将培养至对数生长期的细菌(约48hr)转接0.5%的菌液于R5A(蔗糖100g/l;硫酸镁0.25g/l;六水氯化镁10.12g/l;葡萄糖10g/l;Hy-case amino 0.1g/l;酵母提取物5g/l;Mops 21g/l;2ml R5微量元素;调pH=6.85,高压灭菌)的液体培养基中继续培养120hr,把所有的发酵物(包括菌体和菌液)的PH调至2-3,超声15min(10s/50s);用滤纸滤去大部分菌体,用等体积的乙酸乙酯萃取两次,旋干,重旋于甲醇中。HPLC分析发酵产物。
HPLC分析检测条件:
仪器:Agilent 1100HPLC系统
柱子:Phenomenex C18 column(4.63250nm,part number 00F-3300-E0,S/N115575-1
检测波长:UV=220nm
流动相条件:V=1mL/min;A=H2O(1‰TFA);B=CH3CN(1‰TFA)
Min A%/B%
0, 90%A/10%B
5, 80%A/20%B
25,30%A/70%B
26,5%A/95%B
29,5%A/95%B
30,90%A/10%B
经过LC-MS的分析,分别检测到947位H突变成A的重组菌株中,不产生6-MSA;947位H突变成F的重组菌株中,产生6-MSA;1389位的G突变成A的重组菌株中,不产生OSA,TAL(2羟基4-甲基-2-吡喃酮),6-MSA;1387的G突变成A,1389的G突变成P,1392位的G突变成A的重组菌株中,不产生OSA,TAL,6-MSA;1540位的Y突变成F的重组菌株中,产生OSA。
实施例2,KR突变的ChlB1互补ChlB1突变株产生新的CHL的类似物
1,E.coli ET12567和Streptomyces antibioticus DSM40725(ΔchlB1)和之间的属间接合转移
从经过转化的E.coli ET12567培养平板上挑取单菌落接到试管当中培养过夜,吸取0.5ml的菌液接到25ml LB,置于37℃摇床中培养至OD600为0.3-0.4,或者0.4-0.6。离心收集菌体,用等体积的LB培养基洗涤两次,离心收集菌体并悬浮于1ml LB培养基中。作为DNA供体。
取出-80℃,20%甘油保存的Streptomyces antibioticus DSM40725(ΔchlB1)的孢子悬液(3×109个/mL),8,000rpm离心3分钟去除上清,然后用0.5ml TES buffer(0.05M,pH 8.0)洗涤两次,500μl TES buffer(0.05M,pH 8.0)重悬,50℃热休克10分钟以激发孢子萌发,然后加入500μl TSB,混匀,37℃温育4-5hr,离心收集孢子并悬浮于1.5ml LB broth中。作为受体菌。
取100μl受体菌和100μl供体菌混合,然后涂在两块AS-1或者MS平板上(含有10mM的MgCl2),另外将同样处理过的孢子涂在两块AS-1或者MS平板上(含有10mM的MgCl2),分别做为阳性对照和阴性对照。接合转移的平板在30℃培养16-20小时以后采用无菌水轻轻洗涤平板表面以除去绝大部分大肠杆菌,在每一平板的表面加盖1ml ddH2O(含有Tsr终浓度为50μg/ml,Nalidixic acid终浓度威50μg/ml),30℃培养3-5天后挑取接合子。
将获得的接合子接种到液体培养基TSB(Tsr=50μg/ml)中,30℃振荡约28hr。取出200μl涂布在R2YE(不含蔗糖)(Tsr=50μg/ml),30℃培养5天,收孢子,保存于-80℃。
2,KR突变的ChlB1互补Streptomyces antibioticus DSM40725(ΔchlB1)的重组菌株的发酵
在MS(20ml,Tsr=50μg/ml)平板上涂链霉菌孢子100-200μl(~109/ml),30℃培养10天;将培养好的固体培养基冷冻抽干,捣碎后加50ml无水甲醇,超声破菌10分钟(超声10秒,间隔50秒),搅拌器搅拌1小时;过滤(或离心)收集有机相,培养基用50ml无水甲醇再萃取一次,搅拌1小时,收集并合并两次的有机相;旋蒸除去甲醇,得到深褐色残余物,用1ml无水甲醇溶解,离心后HPLC分析。
分析突变菌株产生了两个全新的峰,LC-MS检测两个化合很可能是CHL的类似物,命名为7和8。
实施例3,突变菌株的新化合物的发酵分离纯化,结构鉴定以及生物活性的测定
小量发酵突变株时,HPLC和LC-MS检测到可能的两个新化合物7和8,为了鉴定这两个化合物的结构,我们对突变株进行了大量发酵,发酵方法见实例2,发酵完培养基冷冻抽干后,捣碎加等体积无水甲醇,超声破菌10分钟(超声10秒,间隔50秒),搅拌器搅拌1小时,过滤(或离心)收集甲醇,;旋蒸除去甲醇,用1/5体积水分散,调pH到2.0-3.0,用等体积乙酸乙酯萃取三次,减压抽干得到深褐色膏状物。膏状物第一步先进行粗分,用100-200目粗硅胶拌样膏状物,油泵抽干,上300-400目硅胶预装的正相柱,梯度洗脱条件为:
洗脱剂 配比
乙酸乙酯∶石油醚
80%/20%
60%/40%
40%/60%
20%/80%
纯乙酸乙酯
100%
二氯甲烷∶甲醇
95%/5%
90%/10%
纯丙酮
100%
发现两个新化合物出现在40%乙酸乙酯/60%石油醚;20%乙酸乙酯/80%石油醚和100%乙酸乙酯的洗脱部分中,把上述三部分的洗脱液减压抽干溶于3ml甲醇,上C18材料的高压反相柱,洗脱顺序为:
水∶甲醇 配比
95%/5%
90%/10%
85%/15%
80%/20%
60%/40%
50%/50%
40%/60%
100%
两个新化合物出现在80%水/20%甲醇,60%水/40%甲醇这两部分,收集这两部分,减压抽干,容易于3ml甲醇,然后HPLC半制备,按上述的HPLC的洗脱条件,收集两个新峰,最后把HPLC制备完的样品溶于3ml甲醇,上样superdex凝胶柱,用100%的甲醇洗脱,最终得到纯的两个新化合物7和8。新化合物7,8和CHL的ESI-MS/MS图谱表明7的加钠分子离子峰为959.17;8的加钠分子离子峰为993.05;CHL的加钠分子离子峰为977.36。7,8和CHL都含有特征碎片峰:535,665,777;7有碎片峰447;8有碎片峰481;CHL有碎片峰465;说明新化合物7和8的结构是在6-甲基水杨酸单元增加了个氧原子。新化合物油泵抽干溶于500μl氘代甲醇或者氘代氯仿中,进行1H-NMR,13C-NMR,HMBC,HSQC,CONSY,NOESY数据采集,分析新化合物7,8和CHL的1H-NMR图谱显示7结构中两个单峰化学位移分别为6.24and 6.29,说明含有两个芳香氢;8结构中一个单峰化学位移为6.53,说明含有一个芳香氢;CHL结构中一个单峰化学位移6.76,一个双峰化学位移7.37(耦合常数为8.9赫兹),说明含有三个芳香氢。三个结果进行对比得出7和8的结构和CHL的差别是6-MSA单元苯环上增加了个氧原子。13C-NMR图谱。图谱表明7和CHL具有相同的碳骨架。7的H-H COSY(图10),HSQC(图11),HMBC(图12),和ROESY(图13),表明7的结构是deschloro-CHL的结构且在6-MSA单元苯环的对位增加了氧原子。8的OSA单元的HMBC(图14)图谱,表明化合物8的结构是CHL的结构且在6-MSA单元苯环的对位增加了氧原子,综上所述,确定了化合物的结构。对7和8化合物进行了抗菌活性的测定,发现新化合物7和8抗革兰氏阳性菌Bacillussubtilis的活性分别比deschloro-CHL和CHL的活性提高一倍左右。说明7和8在6-MSA单元的对位引入的羟基,增加了化合物的水溶性,提高了其生物活性。
以下根据本发明内容提供的基因和蛋白序列:
氨基酸/核苷酸序列表:
SEQUENCE LISTING
<110>上海有机化学研究所
<120>基因工程改造6-甲基水杨酸合成酶并组合生物合成螺环乙酰乙酸内酯类抗生素
<130>说明书,权利要求书
<160>2
<170>PatentIn version 3.3
<210>1
<211>5271
<212>DNA
<213>Streptomyces antibioticus DSM40725
<220>
<221>CDS
<222>(1)..(5271)
<400>1
gtg cag agt cac gac gtt gcc cgt gcg ggc ggc agg gaa gtc gtc gag 48
Val Gln Ser His Asp Val Ala Arg Ala Gly Gly Arg Glu Val Val Glu
1 5 10 15
gag ccg atc gcc gtg ctc ggg atg gcg tgc cgg ttc gca ggt ggt gcc 96
Glu Pro Ile Ala Val Leu Gly Met Ala Cys Arg Phe Ala Gly Gly Ala
20 25 30
gac acc ctg gag gcg ttc tgg gag ttg ctg ctg gag ggc cgg gac ggc 144
Asp Thr Leu Glu Ala Phe Trp Glu Leu Leu Leu Glu Gly Arg Asp Gly
35 40 45
atc ggt gag gtg cct gag aag cgg tgg cgc gcc tac gag gag gcc ggc 192
Ile Gly Glu Val Pro Glu Lys Arg Trp Arg Ala Tyr Glu Glu Ala Gly
50 55 60
ccc gat cat gcg gcg gcg gtg cgg agg gcg acg cgg tgg ggt ggg ttc 240
Pro Asp His Ala Ala Ala Val Arg Arg Ala Thr Arg Trp Gly Gly Phe
65 70 75 80
ctc gat gac atc gag ggg ttc gac gcg gag ttc ttc ggg ttg tcg ccg 288
Leu Asp Asp Ile Glu Gly Phe Asp Ala Glu Phe Phe Gly Leu Ser Pro
85 90 95
cgt gag gcg gag ttg atg gat ccg cag cag cgg ttg ctg ctg gag gtg 336
Arg Glu Ala Glu Leu Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Val
100 105 110
gcg tgg gag gcg ttg gag cac gcg ggt att gcg ccg cgg gag ttg gcg 384
Ala Trp Glu Ala Leu Glu His Ala Gly Ile Ala Pro Arg Glu Leu Ala
115 120 125
ggg acg gac gcg ggt gtg ttc gtg ggg atc ggt tcg gat gat tac ggc 432
Gly Thr Asp Ala Gly Val Phe Val Gly Ile Gly Ser Asp Asp Tyr Gly
130 135 140
cgg cgg ttg ttg gag gat ctg ccg ggg atc gag gcg tgg acg ggg atc 480
Arg Arg Leu Leu Glu Asp Leu Pro Gly Ile Glu Ala Trp Thr Gly Ile
145 150 155 160
ggc agt gcg atg tgt gcg gcg gcg aac cgg atc tcg tat gcg ctg gat 528
Gly Ser Ala Met Cys Ala Ala Ala Asn Arg Ile Ser Tyr Ala Leu Asp
165 170 175
ctg aag ggg ccg agt ctg gcg gtg gac acg gcg tgt tcg gcg tcg ttg 576
Leu Lys Gly Pro Ser Leu Ala Val Asp Thr Ala Cys Ser Ala Ser Leu
180 185 190
gtg gcg gtg cat ctg gcg tgt cag agt ctg cgg gcg ggt gag agt gag 624
Val Ala Val His Leu Ala Cys Gln Ser Leu Arg Ala Gly Glu Ser Glu
195 200 205
gtg tcg ctc gcg gcg ggt gtg aat ctg atg atc tca ccg ggg ttg acg 672
Val Ser Leu Ala Ala Gly Val Asn Leu Met Ile Ser Pro Gly Leu Thr
210 215 220
ctg acg ctg gat gcg gcg ggt gcg acg gcg ccg gac ggg cgg tcg aag 720
Leu Thr Leu Asp Ala Ala Gly Ala Thr Ala Pro Asp Gly Arg Ser Lys
225 230 235 240
tcc ttc gat gcc tcc gcg gac ggt tat ggc cgg ggc gag ggg tgt ggg 768
Ser Phe Asp Ala Ser Ala Asp Gly Tyr Gly Arg Gly Glu Gly Cys Gly
245 250 255
ctg ctc gtg ctg aag cgg ttg tcg gac gcg gtg cgg gac ggg gat ccg 816
Leu Leu Val Leu Lys Arg Leu Ser Asp Ala Val Arg Asp Gly Asp Pro
260 265 270
gtg ctg gcg gtg atc cgg ggc agt tcg gtg aac cag gac ggg aag acg 864
Val Leu Ala Val Ile Arg Gly Ser Ser Val Asn Gln Asp Gly Lys Thr
275 280 285
aac ggg atc atg gcg ccg agt ggt tcg gcg cag gag cat gtg ctg gat 912
Asn Gly Ile Met Ala Pro Ser Gly Ser Ala Gln Glu His Val Leu Asp
290 295 300
ctg gcg tgc cgg cgg gcg ggg gtg gat ccg gcg tcg gtg gat tac gtc 960
Leu Ala Cys Arg Arg Ala Gly Val Asp Pro Ala Ser Val Asp Tyr Val
305 310 315 320
gag gcg cat ggc acg ggg acg cgg ctt gga gac ccg ttg gaa gcg ggt 1008
Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro Leu Glu Ala Gly
325 330 335
gcg ctg agc gcg gtg ttc ggg cgg ggg cgg ccc aag gat gag ccg tgt 1056
Ala Leu Ser Ala Val Phe Gly Arg Gly Arg Pro Lys Asp Glu Pro Cys
340 345 350
ctg atc ggt tcg gtg aag tcg aac atc ggg cat ctg gag gcg gcg gcg 1104
Leu Ile Gly Ser Val Lys Ser Asn Ile Gly His Leu Glu Ala Ala Ala
355 360 365
ggg att gcg agc ctg atc aag gcg acg ctg gcg ttg agc aag gga gag 1152
Gly Ile Ala Ser Leu Ile Lys Ala Thr Leu Ala Leu Ser Lys Gly Glu
370 375 380
atc ccg ccg agt ctg aac ttc tcg cag ggc aat ccg gcg atc gac tgg 1200
Ile Pro Pro Ser Leu Asn Phe Ser Gln Gly Asn Pro Ala Ile Asp Trp
385 390 395 400
gcg gag tcc ggg ctg cgg gtg gtg acc gag cgg acg gcc tgg ccc gag 1248
Ala Glu Ser Gly Leu Arg Val Val Thr Glu Arg Thr Ala Trp Pro Glu
405 410 415
cgg gag gac cga ccg gtc cgt gcg ggc gtt tcc ggc ttc ggc tat ggc 1296
Arg Glu Asp Arg Pro Val Arg Ala Gly Val Ser Gly Phe Gly Tyr Gly
420 425 430
ggc acc atc gcg cat gtg gtc atg gag cag gcg cct gag gtg agt cgg 1344
Gly Thr Ile Ala His Val Val Met Glu Gln Ala Pro Glu Val Ser Arg
435 440 445
ccc gat gac gcg gcg ggt gat gag ggg tct gcc gag gtc gtg acg gag 1392
Pro Asp Asp Ala Ala Gly Asp Glu Gly Ser Ala Glu Val Val Thr Glu
450 455 460
cgg ctg ttc ccg ctc tcg ggt gga acg cag gcc gga ctc cgg gcg tat 1440
Arg Leu Phe Pro Leu Ser Gly Gly Thr Gln Ala Gly Leu Arg Ala Tyr
465 470 475 480
gcg gga cgc ctc gcg gac cgg ctg tcg gac gac gac gcc gag gaa ctg 1488
Ala Gly Arg Leu Ala Asp Arg Leu Ser Asp Asp Asp Ala Glu Glu Leu
485 490 495
ccc ctg gag tcg gtc ggg cac acc ctg gcc ttg cgc agg tcg gcg ctg 1536
Pro Leu Glu Ser Val Gly His Thr Leu Ala Leu Arg Arg Ser Ala Leu
500 505 510
gcg cac cgg gcc gcc gtc gtg gcc tcg gac cgc aag gac ctg gtg gcc 1584
Ala His Arg Ala Ala Val Val Ala Ser Asp Arg Lys Asp Leu Val Ala
515 520 525
aag ctg cgg ttg atc acg ctg ggg gag cag acc cgg gaa gcc gtg atc 1632
Lys Leu Arg Leu Ile Thr Leu Gly Glu Gln Thr Arg Glu Ala Val Ile
530 535 540
ggg tcg gta ccc tcc gat gcc ggt gcg ggg ccg gtg tgg gtg ttc tcc 1680
Gly Ser Val Pro Ser Asp Ala Gly Ala Gly Pro Val Trp Val Phe Ser
545 550 555 560
ggg cat ggt tcg cag tgg tcg ggg atg ggg cgt gaa ctg ctg gcg tcc 1728
Gly His Gly Ser Gln Trp Ser Gly Met Gly Arg Glu Leu Leu Ala Ser
565 570 575
gag ccc gcg ttc gca gcg gtg atc gac gag atc gat ccc gtt ttc cgt 1776
Glu Pro Ala Phe Ala Ala Val Ile Asp Glu Ile Asp Pro Val Phe Arg
580 585 590
gcg gag atc ggg ttc tcg gcc cgg cag gct ctg ctc gac ggt gac ttc 1824
Ala Glu Ile Gly Phe Ser Ala Arg Gln Ala Leu Leu Asp Gly Asp Phe
595 600 605
gac acc gtc gac cgt gtt cag acg atg att ttc gcg gtg cag gtc gcg 1872
Asp Thr Val Asp Arg Val Gln Thr Met Ile Phe Ala Val Gln Val Ala
610 615 620
ctg gcg gcg gtc tgg cac tct tat ggt gcc gcc ccg tcg gcg gtg atc 1920
Leu Ala Ala Val Trp His Ser Tyr Gly Ala Ala Pro Ser Ala Val Ile
625 630 635 640
ggg cac tcc gtg ggg gag atc gcg gcg gct gtg gcg gcg ggt gcg ctg 1968
Gly His Ser Val Gly Glu Ile Ala Ala Ala Val Ala Ala Gly Ala Leu
645 650 655
tcg ctg acg gac gga gcg cgg ctg atc tgc cgc cgc tcc cga ctc ttg 2016
Ser Leu Thr Asp Gly Ala Arg Leu Ile Cys Arg Arg Ser Arg Leu Leu
660 665 670
cgg cgg gtg gcc ggc cag gga gcg atg gct atg gcg agc atc tcc ttc 2064
Arg Arg Val Ala Gly Gln Gly Ala Met Ala Met Ala SerIle Ser Phe
675 680 685
gag gag gcg gcc gag cgg ctg gcg ggc cgt acg gat gtg gtg ccg gcg 2112
Glu Glu Ala Ala Glu Arg Leu Ala Gly Arg Thr Asp Val Val Pro Ala
690 695 700
att gcc gcg tcc ccg ctc tcc gcg gtc gtg gca ggt gac cct gca gcg 2160
Ile Ala Ala Ser Pro Leu Ser Ala Val Val Ala Gly Asp Pro Ala Ala
705 710 715 720
atc aac gcg ctg atc gac gag tgg cag gca cag gac atc cag atg cgc 2208
Ile Asn Ala Leu Ile Asp Glu Trp Gln Ala Gln Asp Ile Gln Met Arg
725 730 735
cgg gtc gcc tcg gac gtg gcc ttc cac agc ccg cac atg gac ccg ctg 2256
Arg Val Ala Ser Asp Val Ala Phe His Ser Pro His Met Asp Pro Leu
740 745 750
ctc acc gaa atc gcg gcc gct gcc gag gac ttg acg ccg cgc cag ccc 2304
Leu Thr Glu Ile Ala Ala Ala Ala Glu Asp Leu Thr Pro Arg Gln Pro
755 760 765
gaa ctc ccg gtg tac tcc acg gcc atg gag gac ccc cgc tcc cag gcg 2352
Glu Leu Pro Val Tyr Ser Thr Ala Met Glu Asp Pro Arg Ser Gln Ala
770 775 780
acc ctc gac ggc tcc tac tgg gcc gcc aac ctg cgt aac ccg gtg cgg 2400
Thr Leu Asp Gly Ser Tyr Trp Ala Ala Asn Leu Arg Asn Pro Val Arg
785 790 795 800
ttg cag ccg gcg gtg acg gcg gcg gtc gag gac ggc cac cgc gcg ttc 2448
Leu Gln Pro Ala Val Thr Ala Ala Val Glu Asp Gly His Arg Ala Phe
805 810 815
atc gaa gtg tcc gcg cat ccc gtg gtc acg cac tcc atc ggc gag acg 2496
Ile Glu Val Ser Ala His Pro Val Val Thr His Ser Ile Gly Glu Thr
820 825 830
ctc tcc gag ctc ggc cag gag gac gcc ttc acc ggc tcc tcc ctg cgc 2544
Leu Ser Glu Leu Gly Gln Glu Asp Ala Phe Thr Gly Ser Ser Leu Arg
835 840 845
cgc aac cag ccc gaa cgc gcc acc ctc ctg tcc gcc gtc ggc gcg gcg 2592
Arg Asn Gln Pro Glu Arg Ala Thr Leu Leu Ser Ala Val Gly Ala Ala
850 855 860
cac tgc cat ggc atc gcg gtg gac tgg gcg cgt ctg cac ccg acc ggt 2640
His Cys His Gly Ile Ala Val Asp Trp Ala Arg Leu His Pro Thr Gly
865 870 875 880
gac ctg gtc gcc ctg ccg ctg gtg gcc tgg cag cgc agc ccg cac tgg 2688
Asp Leu Val Ala Leu Pro Leu Val Ala Trp Gln Arg Ser Pro His Trp
885 890 895
cac gag cgg gcc tcc gcc gcc acc ggc cag ggc ttg cag cac gac ctt 2736
His Glu Arg Ala Ser Ala Ala Thr Gly Gln Gly Leu Gln His Asp Leu
900 905 910
gac tcc cac gcg ctg ctc ggg ccg cgc gtc ccg gtc gcg gga cgg ccg 2784
Asp Ser His Ala Leu Leu Gly Pro Arg Val Pro Val Ala Gly Arg Pro
915 920 925
ctg gaa ctg tgg cgc aca ctg ctc gac gac gag acg cgc ccc tac ccc 2832
Leu Glu Leu Trp Arg Thr Leu Leu Asp Asp Glu Thr Arg Pro Tyr Pro
930 935 940
ggc agc gcc acc atc aac ggc acg gag atc gtg ccc gcc gcc gtc ctg 2880
Gly Ser Ala ThrIle Asn Gly Thr Glu Ile Val Pro Ala Ala Val Leu
945 950 955 960
atc aac acg ttc ctc gac gcg gca cgc gcc gcc gac ggg gcc cgc ccg 2928
Ile Asn Thr Phe Leu Asp Ala Ala Arg Ala Ala Asp Gly Ala Arg Pro
965 970 975
gtc ctg cgg gac atg gcg ctg cgg ctg ccg ctg atc acc acc gag cgg 2976
Val Leu Arg Asp Met Ala Leu Arg Leu Pro Leu Ile Thr Thr Glu Arg
980 985 990
cgc gaa ctc cag gtc gtc agg gac gac aac tcc ttg cgt ctg gcc tcg 3024
Arg Glu Leu Gln Val Val Arg Asp Asp Asn Ser Leu Arg Leu Ala Ser
995 1000 1005
cgt tca ctg gag gac ggt gcc gcg tgg ctg acc cac acc acc gcc 3069
Arg Ser Leu Glu Asp Gly Ala Ala Trp Leu Thr His Thr Thr Ala
1010 1015 1020
acc gcc gca ccg gcg ggc agc ggc gaa gcg ctc cag gac ctg gcc 3114
Thr Ala Ala Pro Ala Gly Ser Gly Glu Ala Leu Gln Asp Leu Ala
1025 1030 1035
gcc ggt gcc gtg ttg cgc ccg gcg gac ccg ggt gat gtg cag cgc 3159
Ala Gly Ala Val Leu Arg Pro Ala Asp Pro Gly Asp Val Gln Arg
1040 1045 1050
cac ctg acc tcg gtg ggc gtg ccg acc atg gga ttt gag tgg acc 3204
His Leu Thr Ser Val Gly Val Pro Thr Met Gly Phe Glu Trp Thr
1055 1060 1065
atc gag gaa ctc gcc cgg agc gag ggc atg ttg gcc gca cgt gtg 3249
Ile Glu Glu Leu Ala Arg Ser Glu Gly Met Leu Ala Ala Arg Val
1070 1075 1080
agt gtc gag cgg ccg cag cgg gcc cag gag acg tgg gcg ccc ttg 3294
Ser Val Glu Arg Pro Gln Arg Ala Gln Glu Thr Trp Ala Pro Leu
1085 1090 1095
ctg gac gcc gcg ctg tcc atc gcg ccg acg gcc atc ccc ggc ccg 3339
Leu Asp Ala Ala Leu Ser Ile Ala Pro Thr Ala Ile Pro Gly Pro
1100 1105 1110
ccg gcc ctg cgc atg gtg gcc tcc ttc gag gag atc gtc acc gaa 3384
Pro Ala Leu Arg Met Val Ala Ser Phe Glu Glu Ile Val Thr Glu
1115 1120 1125
ggc gcc ccg ccg gcc ggt ccg gcg acc atc cag gtc gcg gcc gac 3429
Gly Ala Pro Pro Ala Gly Pro Ala Thr Ile Gln Val Ala Ala Asp
1130 1135 1140
ccg gtc cac gag aac acc gtc gac gtc cgg atc gcc gac acc gac 3474
Pro Val His Glu Asn Thr Val Asp Val Arg Ile Ala Asp Thr Asp
1145 1150 1155
ggg cag gcc gtg gcg tgg gtg cgc ggc ctg cgc tac gac ggc atg 3519
Gly Gln Ala Val Ala Trp Val Arg Gly Leu Arg Tyr Asp Gly Met
1160 1165 1170
gac cag ggc ggc atg acg gcg gcg cac ccc cgc gac ctg gtc ttc 3564
Asp Gln Gly Gly Met Thr Ala Ala His Pro Arg Asp Leu Val Phe
1175 1180 1185
gag atg gcc tgg cgg ccc ttc gag gcc ccc gcg ccg cag gac gtg 3609
Glu Met Ala Trp Arg Pro Phe Glu Ala Pro Ala Pro Gln Asp Val
1190 1195 1200
tcc gcc cgc cgg atc gtc ctg atc gcc gca cac gac gtg aag ccc 3654
Ser Ala Arg Arg Ile Val Leu Ile Ala Ala His Asp Val Lys Pro
1205 1210 1215
ctg cgc acg gcc ctc acc cgt gcc ggc gct cac gtc gac gtc ggg 3699
Leu Arg Thr Ala Leu Thr Arg Ala Gly Ala His Val Asp Val Gly
1220 1225 1230
ctg gac ggc acg cte gac gag aac acc gac gtc gtc gtg gtg ccc 3744
Leu Asp Gly Thr Leu Asp Glu Asn Thr Asp Val Val Val Val Pro
1235 1240 1245
gac ctc acc gcg gac atc ccc gtc ccc gag gcc gca gcc cgt tcc 3789
Asp Leu Thr Ala Asp Ile Pro Val Pro Glu Ala Ala Ala Arg Ser
1250 1255 1260
gca tgg ctg ctg ctg agc acc gcg cag cgc atc gcc gcc ctg gac 3834
Ala Trp Leu Leu Leu Ser Thr Ala Gln Arg Ile Ala Ala Leu Asp
1265 1270 1275
acc ctg cgc ttc ccc cgc ctg tgg tgc ctg acc acc gca gtc cgt 3879
Thr Leu Arg Phe Pro Arg Leu Trp Cys Leu Thr Thr Ala Val Arg
1280 1285 1290
gaa agc cag gcc gaa acc cac ctc gcg cag tcc acc ctg tgg ggc 3924
Glu Ser Gln Ala Glu Thr His Leu Ala Gln Ser Thr Leu Trp Gly
1295 1300 1305
ctg ggc cgg gtg atc gcg ggc gag cac agc gaa ctg tgg ggc ggc 3969
Leu Gly Arg Val Ile Ala Gly Glu His Ser Glu Leu Trp Gly Gly
1310 1315 1320
gtc atc gac ctg gcc ccc ggc acc ccg gac gcc acc acc ctg ctc 4014
Val Ile Asp Leu Ala Pro Gly Thr Pro Asp Ala Thr Thr Leu Leu
1325 1330 1335
agc gtc ctg cac acc ggc ggc ggc gag gac gtc atc gcc ctc cgc 4059
Ser Val Leu His Thr Gly Gly Gly Glu Asp Val Ile Ala Leu Arg
1340 1345 1350
gac ggc acc gcc acc acg gcc cgc ctc acc acg acg caa cgc gag 4104
Asp Gly Thr Ala Thr Thr Ala Arg Leu Thr Thr Thr Gln Arg Glu
1355 1360 1365
ccc act ggc acc ccg ctg gaa tgc cgg gcg gac gga acg tac ctg 4149
Pro Thr Gly Thr Pro Leu Glu Cys Arg Ala Asp Gly Thr Tyr Leu
1370 1375 1380
atc acc ggc gga ctg ggc acc ctc ggc ctg gaa gtc gcc ggc cgg 4194
Ile Thr Gly Gly Leu Gly Thr Leu Gly Leu Glu Val Ala Gly Arg
1385 1390 1395
ctc gcc gaa cgc ggc gcc cgc cgt ctc gtc ctc gcc gga cgc acc 4239
Leu Ala Glu Arg Gly Ala Arg Arg Leu Val Leu Ala Gly Arg Thr
1400 1405 1410
gga ctg cca ccc cgc tcc acc tgg ggc gag acc acc gac acg cac 4284
Gly Leu Pro Pro Arg Ser Thr Trp Gly Glu Thr Thr Asp Thr His
1415 1420 1425
acc agg cag cgc atc gag gcc gtc aag gcc ctc gaa gac cag ggc 4329
Thr Arg Gln Arg Ile Glu Ala Val Lys Ala Leu Glu Asp Gln Gly
1430 1435 1440
gtc acc gtc cgt gtc atc ccc ctc gac atc acc gac acg gcc aag 4374
Val Thr Val Arg Val Ile Pro Leu Asp Ile Thr Asp Thr Ala Lys
1445 1450 1455
gcc gcc gaa cag ctc acc ccc gac gcc ctg ggc ctg cca ccc atc 4419
Ala Ala Glu Gln Leu Thr Pro Asp Ala Leu Gly Leu Pro Pro Ile
1460 1465 1470
cgc ggc atc gtc cac ctc gcc ggc gtc ctc gac aac cgc atg gtg 4464
Arg Gly Ile Val His Leu Ala Gly Val Leu Asp Asn Arg Met Val
1475 1480 1485
acc gcg gtc gac gag aca tcc ctg cgc acc gtg ctg cgg ccc aag 4509
Thr Ala Val Asp Glu Thr Ser Leu Arg Thr Val Leu Arg Pro Lys
1490 1495 1500
gcc gac ggc gcc tgg acc ctg cac acc ctc ttc ccg ccc ggc acc 4554
Ala Asp Gly Ala Trp Thr Leu His Thr Leu Phe Pro Pro Gly Thr
1505 1510 1515
atc gac ttc ctg atc ctg ttc tcc tcc tgc ggc cag ctc ctc ggc 4599
Ile Asp Phe Leu Ile Leu Phe Ser Ser Cys Gly Gln Leu Leu Gly
1520 1525 1530
ctg ccc ggc cag gcc gcc tac ggc tcc gcc aac gcc ttc ctc gac 4644
Leu Pro Gly Gln Ala Ala Tyr Gly Ser Ala Asn Ala Phe Leu Asp
1535 1540 1545
gcc ctc gcc gtc cac cgc aac acc acc acc ccg acc gcc gcc gac 4689
Ala Leu Ala Val His Arg Asn Thr Thr Thr Pro Thr Ala Ala Asp
1550 1555 1560
acc acc agc ttc ggc tgg acc tcc tgg cgc ggc cag ggc atg gcc 4734
Thr Thr Ser Phe Gly Trp Thr Ser Trp Arg Gly Gln Gly Met Ala
1565 1570 1575
gtc aac gac gtc gtc gac gcc gaa ctg cgc gcc cga ggc gtc acc 4779
Val Asn Asp Val Val Asp Ala Glu Leu Arg Ala Arg Gly Val Thr
1580 1585 1590
gac atc acc acc cag gaa gcc ttc gcc gcc tgg gac ttc gcc gca 4824
Asp Ile Thr Thr Gln Glu Ala Phe Ala Ala Trp Asp Phe Ala Ala
1595 1600 1605
caa cac ggc ccc gga aac tac ccc gtc cta cgc cgg ctg ccc cac 4869
Gln His Gly Pro Gly Asn Tyr Pro Val Leu Arg Arg Leu Pro His
1610 1615 1620
gag ccg gac atg gac cag ctc ccc ctc ctc agc gag atc cac cac 4914
Glu Pro Asp Met Asp Gln Leu Pro Leu Leu Ser Glu Ile His His
1625 1630 1635
acc cag ccc acc gcc ccc acc tcc ggc gcc gca acc gac tcc tac 4959
Thr Gln Pro Thr Ala Pro Thr Ser Gly Ala Ala Thr Asp Ser Tyr
1640 1645 1650
gcg ggc ctc gcc ccc gac gaa ctg cgc gcc cgc ctc atc gac gag 5004
Ala Gly Leu Ala Pro Asp Glu Leu Arg Ala Arg Leu Ile Asp Glu
1655 1660 1665
gtc gcc gca cac atc tcg gcc gag atg aaa ctc gcc gcc tcc cag 5049
Val Ala Ala His Ile Ser Ala Glu Met Lys Leu Ala Ala Ser Gln
1670 1675 1680
ctc gac cac cgc aag tcc ctg gtc gag cag ggc ctg gac tcg gtg 5094
Leu Asp His Arg Lys Ser Leu Val Glu Gln Gly Leu Asp Ser Val
1685 1690 1695
atg acg atc gtg atc cgg cgc cgc ctg gag aag tgg ttc ggt cac 5139
Met Thr Ile Val Ile Arg Arg Arg Leu Glu Lys Trp Phe Gly His
1700 1705 1710
aaa ctc ccc gcg acc ctg ctg tgg cac cag ccc acc gtc acc gcc 5184
Lys Leu Pro Ala Thr Leu Leu Trp His Gln Pro Thr Val Thr Ala
1715 1720 1725
atc agc gaa cac ctg gcc gaa ctc ctg gcc ccc acc acg tcc cag 5229
Ile Ser Glu His Leu Ala Glu Leu Leu Ala Pro Thr Thr Ser Gln
1730 1735 1740
ccc gac aac acg gca ccc gcc gaa ccg gcg gca acg gcc tga 5271
Pro Asp Asn Thr Ala Pro Ala Glu Pro Ala Ala Thr Ala
1745 1750 1755
<210>2
<211>1756
<212>PRT
<213>Streptomyces antibioticus DSM40725
<400>2
Val Gln Ser His Asp Val Ala Arg Ala Gly Gly Arg Glu Val Val Glu
1 5 10 15
Glu Pro Ile Ala Val Leu Gly Met Ala Cys Arg Phe Ala Gly Gly Ala
20 25 30
Asp Thr Leu Glu Ala Phe Trp Glu Leu Leu Leu Glu Gly Arg Asp Gly
35 40 45
Ile Gly Glu Val Pro Glu Lys Arg Trp Arg Ala Tyr Glu Glu Ala Gly
50 55 60
Pro Asp His Ala Ala Ala Val Arg Arg Ala Thr Arg Trp Gly Gly Phe
65 70 75 80
Leu Asp Asp Ile Glu Gly Phe Asp Ala Glu Phe Phe Gly Leu Ser Pro
85 90 95
Arg Glu Ala Glu Leu Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Val
100 105 110
Ala Trp Glu Ala Leu Glu His Ala Gly Ile Ala Pro Arg Glu Leu Ala
115 120 125
Gly Thr Asp Ala Gly Val Phe Val Gly Ile Gly Ser Asp Asp Tyr Gly
130 135 140
Arg Arg Leu Leu Glu Asp Leu Pro Gly Ile Glu Ala Trp Thr Gly Ile
145 150 155 160
Gly Ser Ala Met Cys Ala Ala Ala Asn Arg Ile Ser Tyr Ala Leu Asp
165 170 175
Leu Lys Gly Pro Ser Leu Ala Val Asp Thr Ala Cys Ser Ala Ser Leu
180 185 190
Val Ala Val His Leu Ala Cys Gln Ser Leu Arg Ala Gly Glu Ser Glu
195 200 205
Val Ser Leu Ala Ala Gly Val Asn Leu Met Ile Ser Pro Gly Leu Thr
210 215 220
Leu Thr Leu Asp Ala Ala Gly Ala Thr Ala Pro Asp Gly Arg Ser Lys
225 230 235 240
Ser Phe Asp Ala Ser Ala Asp Gly Tyr Gly Arg Gly Glu Gly Cys Gly
245 250 255
Leu Leu Val Leu Lys Arg Leu Ser Asp Ala Val Arg Asp Gly Asp Pro
260 265 270
Val Leu Ala Val Ile Arg Gly Ser Ser Val Asn Gln Asp Gly Lys Thr
275 280 285
Asn Gly Ile Met Ala Pro Ser Gly Ser Ala Gln Glu His Val Leu Asp
290 295 300
Leu Ala Cys Arg Arg Ala Gly Val Asp Pro Ala Ser Val Asp Tyr Val
305 310 315 320
Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro Leu Glu Ala Gly
325 330 335
Ala Leu Ser Ala Val Phe Gly Arg Gly Arg Pro Lys Asp Glu Pro Cys
340 345 350
Leu Ile Gly Ser Val Lys Ser Asn Ile Gly His Leu Glu Ala Ala Ala
355 360 365
Gly Ile Ala Ser Leu Ile Lys Ala Thr Leu Ala Leu Ser Lys Gly Glu
370 375 380
Ile Pro Pro Ser Leu Asn Phe Ser Gln Gly Asn Pro Ala Ile Asp Trp
385 390 395 400
Ala Glu Ser Gly Leu Arg Val Val Thr Glu Arg Thr Ala Trp Pro Glu
405 410 415
Arg Glu Asp Arg Pro Val Arg Ala Gly Val Ser Gly Phe Gly Tyr Gly
420 425 430
Gly Thr Ile Ala His Val Val Met Glu Gln Ala Pro Glu Val Ser Arg
435 440 445
Pro Asp Asp Ala Ala Gly Asp Glu Gly Ser Ala Glu Val Val Thr Glu
450 455 460
Arg Leu Phe Pro Leu Ser Gly Gly Thr Gln Ala Gly Leu Arg Ala Tyr
465 470 475 480
Ala Gly Arg Leu Ala Asp Arg Leu Ser Asp Asp Asp Ala Glu Glu Leu
485 490 495
Pro Leu Glu Ser Val Gly His Thr Leu Ala Leu Arg Arg Ser Ala Leu
500 505 510
Ala His Arg Ala Ala Val Val Ala Ser Asp Arg Lys Asp Leu Val Ala
515 520 525
Lys Leu Arg Leu Ile Thr Leu Gly Glu Gln Thr Arg Glu Ala Val Ile
530 535 540
Gly Ser Val Pro Ser Asp Ala Gly Ala Gly Pro Val Trp Val Phe Ser
545 550 555 560
Gly His Gly Ser Gln Trp Ser Gly Met Gly Arg Glu Leu Leu Ala Ser
565 570 575
Glu Pro Ala Phe Ala Ala Val Ile Asp Glu Ile Asp Pro Val Phe Arg
580 585 590
Ala Glu Ile Gly Phe Ser Ala Arg Gln Ala Leu Leu Asp Gly Asp Phe
595 600 605
Asp Thr Val Asp Arg Val Gln Thr Met Ile Phe Ala Val Gln Val Ala
610 615 620
Leu Ala Ala Val Trp His Ser Tyr Gly Ala Ala Pro Ser Ala Val Ile
625 630 635 640
Gly His Ser Val Gly Glu Ile Ala Ala Ala Val Ala Ala Gly Ala Leu
645 650 655
Ser Leu Thr Asp Gly Ala Arg Leu Ile Cys Arg Arg Ser Arg Leu Leu
660 665 670
Arg Arg Val Ala Gly Gln Gly Ala Met Ala Met Ala Ser Ile Ser Phe
675 680 685
Glu Glu Ala Ala Glu Arg Leu Ala Gly Arg Thr Asp Val Val Pro Ala
690 695 700
Ile Ala Ala Ser Pro Leu Ser Ala Val Val Ala Gly Asp Pro Ala Ala
705 710 715 720
Ile Asn Ala Leu Ile Asp Glu Trp Gln Ala Gln Asp Ile Gln Met Arg
725 730 735
Arg Val Ala Ser Asp Val Ala Phe His Ser Pro His Met Asp Pro Leu
740 745 750
Leu Thr Glu Ile Ala Ala Ala Ala Glu Asp Leu Thr Pro Arg Gln Pro
755 760 765
Glu Leu Pro Val Tyr Ser Thr Ala Met Glu Asp Pro Arg Ser Gln Ala
770 775 780
Thr Leu Asp Gly Ser Tyr Trp Ala Ala Asn Leu Arg Asn Pro Val Arg
785 790 795 800
Leu Gln Pro Ala Val Thr Ala Ala Val Glu Asp Gly His Arg Ala Phe
805 810 815
Ile Glu Val Ser Ala His Pro Val Val Thr His Ser Ile Gly Glu Thr
820 825 830
Leu Ser Glu Leu Gly Gln Glu Asp Ala Phe Thr Gly Ser Ser Leu Arg
835 840 845
Arg Asn Gln Pro Glu Arg Ala Thr Leu Leu Ser Ala Val Gly Ala Ala
850 855 860
His Cys His Gly Ile Ala Val Asp Trp Ala Arg Leu His Pro Thr Gly
865 870 875 880
Asp Leu Val Ala Leu Pro Leu Val Ala Trp Gln Arg Ser Pro His Trp
885 890 895
His Glu Arg Ala Ser Ala Ala Thr Gly Gln Gly Leu Gln His Asp Leu
900 905 910
Asp Ser His Ala Leu Leu Gly Pro Arg Val Pro Val Ala Gly Arg Pro
915 920 925
Leu Glu Leu Trp Arg Thr Leu Leu Asp Asp Glu Thr Arg Pro Tyr Pro
930 935 940
Gly Ser Ala Thr Ile Asn Gly Thr Glu Ile Val Pro Ala Ala Val Leu
945 950 955 960
Ile Asn Thr Phe Leu Asp Ala Ala Arg Ala Ala Asp Gly Ala Arg Pro
965 970 975
Val Leu Arg Asp Met Ala Leu Arg Leu Pro Leu Ile Thr Thr Glu Arg
980 985 990
Arg Glu Leu Gln Val Val Arg Asp Asp Asn Ser Leu Arg Leu Ala Ser
995 1000 1005
Arg Ser Leu Glu Asp Gly Ala Ala Trp Leu Thr His Thr Thr Ala
1010 1015 1020
Thr Ala Ala Pro Ala Gly Ser Gly Glu Ala Leu Gln Asp Leu Ala
1025 1030 1035
Ala Gly Ala Val Leu Arg Pro Ala Asp Pro Gly Asp Val Gln Arg
1040 1045 1050
His Leu Thr Ser Val Gly Val Pro Thr Met Gly Phe Glu Trp Thr
1055 1060 1065
Ile Glu Glu Leu Ala Arg Ser Glu Gly Met Leu Ala Ala Arg Val
1070 1075 1080
Ser Val Glu Arg Pro Gln Arg Ala Gln Glu Thr Trp Ala Pro Leu
1085 1090 1095
Leu Asp Ala Ala Leu Ser Ile Ala Pro Thr Ala Ile Pro Gly Pro
1100 1105 1110
Pro Ala Leu Arg Met Val Ala Ser Phe Glu Glu Ile Val Thr Glu
1115 1120 1125
Gly Ala Pro Pro Ala Gly Pro Ala Thr Ile Gln Val Ala Ala Asp
1130 1135 1140
Pro Val His Glu Asn Thr Val Asp Val Arg Ile Ala Asp Thr Asp
1145 1150 1155
Gly Gln Ala Val Ala Trp Val Arg Gly Leu Arg Tyr Asp Gly Met
1160 1165 1170
Asp Gln Gly Gly Met Thr Ala Ala His Pro Arg Asp Leu Val Phe
1175 1180 1185
Glu Met Ala Trp Arg Pro Phe Glu Ala Pro Ala Pro Gln Asp Val
1190 1195 1200
Ser Ala Arg Arg Ile Val Leu Ile Ala Ala His Asp Val Lys Pro
1205 1210 1215
Leu Arg Thr Ala Leu Thr Arg Ala Gly Ala His Val Asp Val Gly
1220 1225 1230
Leu Asp Gly Thr Leu Asp Glu Asn Thr Asp Val Val Val Val Pro
1235 1240 1245
Asp Leu Thr Ala Asp Ile Pro Val Pro Glu Ala Ala Ala Arg Ser
1250 1255 1260
Ala Trp Leu Leu Leu Ser Thr Ala Gln Arg Ile Ala Ala Leu Asp
1265 1270 1275
Thr Leu Arg Phe Pro Arg Leu Trp Cys Leu Thr Thr Ala Val Arg
1280 1285 1290
Glu Ser Gln Ala Glu Thr His Leu Ala Gln Ser Thr Leu Trp Gly
1295 1300 1305
Leu Gly Arg Val Ile Ala Gly Glu His Ser Glu Leu Trp Gly Gly
1310 1315 1320
Val Ile Asp Leu Ala Pro Gly Thr Pro Asp Ala Thr Thr Leu Leu
1325 1330 1335
Ser Val Leu His Thr Gly Gly Gly Glu Asp Val Ile Ala Leu Arg
1340 1345 1350
Asp Gly Thr Ala Thr Thr Ala Arg Leu Thr Thr Thr Gln Arg Glu
1355 1360 1365
Pro Thr Gly Thr Pro Leu Glu Cys Arg Ala Asp Gly Thr Tyr Leu
1370 1375 1380
Ile Thr Gly Gly Leu Gly Thr Leu Gly Leu Glu Val Ala Gly Arg
1385 1390 1395
Leu Ala Glu Arg Gly Ala Arg Arg Leu Val Leu Ala Gly Arg Thr
1400 1405 1410
Gly Leu Pro Pro Arg Ser Thr Trp Gly Glu Thr Thr Asp Thr His
1415 1420 1425
Thr Arg Gln Arg Ile Glu Ala Val Lys Ala Leu Glu Asp Gln Gly
1430 1435 1440
Val Thr Val Arg Val Ile Pro Leu Asp Ile Thr Asp Thr Ala Lys
1445 1450 1455
Ala Ala Glu Gln Leu Thr Pro Asp Ala Leu Gly Leu Pro Pro Ile
1460 1465 1470
Arg Gly Ile Val His Leu Ala Gly Val Leu Asp Asn Arg Met Val
1475 1480 1485
Thr Ala Val Asp Glu Thr Ser Leu Arg Thr Val Leu Arg Pro Lys
1490 1495 1500
Ala Asp Gly Ala Trp Thr Leu His Thr Leu Phe Pro Pro Gly Thr
1505 1510 1515
Ile Asp Phe Leu Ile Leu Phe Ser Ser Cys Gly Gln Leu Leu Gly
1520 1525 1530
Leu Pro Gly Gln Ala Ala Tyr Gly Ser Ala Asn Ala Phe Leu Asp
1535 1540 1545
Ala Leu Ala Val His Arg Asn Thr Thr Thr Pro Thr Ala Ala Asp
1550 1555 1560
Thr Thr Ser Phe Gly Trp Thr Ser Trp Arg Gly Gln Gly Met Ala
1565 1570 1575
Val Asn Asp Val Val Asp Ala Glu Leu Arg Ala Arg Gly Val Thr
1580 1585 1590
Asp Ile Thr Thr Gln Glu Ala Phe Ala Ala Trp Asp Phe Ala Ala
1595 1600 1605
Gln His Gly Pro Gly Asn Tyr Pro Val Leu Arg Arg Leu Pro His
1610 1615 1620
Glu Pro Asp Met Asp Gln Leu Pro Leu Leu Ser Glu Ile His His
1625 1630 1635
Thr Gln Pro Thr Ala Pro Thr Ser Gly Ala Ala Thr Asp Ser Tyr
1640 1645 1650
Ala Gly Leu Ala Pro Asp Glu Leu Arg Ala Arg Leu Ile Asp Glu
1655 1660 1665
Val Ala Ala His Ile Ser Ala Glu Met Lys Leu Ala Ala Ser Gln
1670 1675 1680
Leu Asp His Arg Lys Ser Leu Val Glu Gln Gly Leu Asp Ser Val
1685 1690 1695
Met Thr Ile Val Ile Arg Arg Arg Leu Glu Lys Trp Phe Gly His
1700 1705 1710
Lys Leu Pro Ala Thr Leu Leu Trp His Gln Pro Thr Val Thr Ala
1715 1720 1725
Ile Ser Glu His Leu Ala Glu Leu Leu Ala Pro Thr Thr Ser Gln
1730 1735 1740
Pro Asp Asn Thr Ala Pro Ala Glu Pro Ala Ala Thr Ala
1745 1750 1755
机译: 大环内酯(3S,9S,15S)-(6E,12E)-3,9,15-三甲基-4,10,16-三氧杂环六癸癸-6,12-二烯-1,5,8,11,14-五酮和获得大环内酯(3S,9S,15S)-(6E,12E)-3,9,15-三甲基-4,10,16-三氧杂六环六癸-6,12-二烯-1,5,8的方法,11,14-戊酮
机译: 大环内酯(3S,9S,15S)-(6E,12E)-3,9,15-三甲基-4,10,16-三氧杂环六癸癸-6,12-二烯-1,5,8,11,14-五酮和获得大环内酯(3S,9S,15S)-(6E,12E)-3,9,15-三甲基-4,10,16-三氧杂六环六癸-6,12-二烯-1,5,8的方法,11,14-戊酮
机译: 用于制备(s)-(-)-2-三氟甲基-4-(3-氰基苯基)-4,6,7,8-四氢-5(1H)-喹诺酮酸4的药物组合物组成方法-氰基苯基)2-三氟甲基-5-氧代-1,4,5,6,7,8--六氢喹啉-3-羧酸,4,4-三氟乙酰乙酰氨基异硼酸,血胺基酮茴香酸2,4,4 2-氰基乙基4 -三氟乙酰乙酸和用于尿失禁的治疗及该化合物的用途