一种新型抗菌多肽及其制备方法

专利名称：一种新型抗菌多肽及其制备方法
技术领域：
本发明涉及一种重组的抗葡萄球菌多肽，编码该多肽的核苷酸和氨基酸序列。
本发明还涉及重组抗葡萄球菌多肽的制备方法。
背景技术：
细菌感染是人类生命和健康的主要威胁，自磺胺和青霉素问世以来，人类陆续发明的抗菌素主要以通过抑制细菌胞壁合成、抑制或干扰细菌的核酸和蛋白质的代谢与合成途径来达到抗菌目的。然而这些抗菌途径容易诱使细菌发生突变而产生抗菌素耐药性。因此人们一直在致力于开发新型的抗菌素。在细菌胞膜上直接形成离子通道而致细菌死亡是比较有前途的抗菌素开发方向之一。自然界中，为数不少的细菌毒素就是以这种机制来杀死细菌的，其模式标本就是大肠杆菌(Escherichia coli)分泌的一种毒素蛋白——大肠菌素(colicin)，1952年Jacob发现其能特异地杀死其它株系的大肠杆菌和相关株系的某些杆菌，如Shigella Sonnei(Jacob et al，)Sur labiosynthese d’une colicine et son mode d’action，Annals of thePasteur Institute.83295-315(1952))。1978年Finkelstein等发现了可形成离子通道的大肠菌素可以在人工脂质双分子膜上形成电压依赖性离子通道，从而在根本上揭示了这一类细菌毒素的抗菌机制(Schein et al，.Colicin K acts by forming voltagedependent channels in phospholipid bilayer membranes.Nature 276159-163(1973))。1996年Qiu和Finkelstein等揭示了大肠菌素Ia在人工脂质双分子膜上形成的离子通道开放和关闭时的跨膜立体结构(Qiu et al.，Major transmembrane movementassociated with colicin，Ia channel gating.J.Gen.Physiology.107313-328(1996))，为在分子水平上设计和制备新型的抗菌素奠定了理论基础。
近二十年，人们逐渐发现细菌也具有随外界环境变化而分泌的信号传导多肽，制备信号传导多肽的胞膜受体及胞内调节蛋白的基因往往偶联在一起，协调地控制着细菌的生长和分裂，但该方向的研究一直进展缓慢，比如金黄葡萄球菌的信号传导多肽AgrD，一直到1995年才最后搞清楚是一个八肽(Ji et al.，Celldensity control of staphylococcal virulence mediated by an octapeptide pheromone.Proc.Natl.Acad.Sci.USA 9212055-12059(1995))。1999年发现金黄色葡萄球菌对数生长期的早期或中晚期加入人工合成的这种八肽可以刺激或抑制金黄色葡萄球菌的生长(Mayville et al.，Structure activity analysis of synthetic autoinducingthiolactone peptides from staphylococcus aureus resoonsible for virulence.PNAS，961218-1223(1999))。
如上所述，大肠菌素是一种理想的离子通道抗菌素，缺点是只能作用于大肠杆菌等格兰氏阴性杆菌，如果能利用致病菌特有的信号传导肽作为诱导物，将大肠菌素或其水性孔道结构或诱导至特定的细菌胞膜附近形成离子通道来杀伤该致病菌，应该是一种理想的抗菌素开发方向。

发明内容
本发明的一个目的在于提供一种新型的重组抗菌多肽，其能避免诱发细菌的本发明的另一目的在于提供编码该重组抗菌多肽的核苷酸序列。
本发明的再一目的在于提供一重组质粒，其含有编码本发明重组抗菌多肽的核苷酸序列。
本发明的再一目的在于提供一氨基酸序列，其编码本发明的重组抗菌多肽。
本发明的再一目的在于提供含有本发明重组抗菌多肽的药物组合物。
本发明的再一目的在于提供重组抗菌多肽的制备方法。
根据本发明的一方面，将编码葡萄球菌信号传导多肽的基因与大肠菌素基因连接，从而获得编码重组抗葡萄球菌多肽的基因。
在本发明的优选实施方案中，分别采用编码金黄色葡萄球菌和表皮葡萄球菌的信号传导多肽的基因作为抗菌多肽的信息素基因，其中编码金黄色葡萄信号传导多肽的基因选自Agr D I(SEQ ID NO.1)、Agr D II(SEQ ID NO.3)和Agr DIII(SEQ ID NO.5)、Agr D IV(SEQ ID NO.7)；编码表皮葡萄球菌信号传导多肽的基因为具有SEQ ID NO.9核苷酸序列的基因。大肠菌素可选自能形成离子通道的大肠菌素El、Ia、Ib、A、B和N或其水性孔道结构域，在本发明的一个优选实施例中，将上述信号传导多肽基因分别与大肠菌素Ia(SEQ ID NO.11)的羧基端或氨基端连接而形成如SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.16、SEQ ID NO.18、SEQ IDNO.20和SEQ ID NO.22的核苷酸序列。这些核苷酸序列编码如SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19、SEQID NO.21和SEQ ID NO.23的氨基酸序列，这些氨基酸序列所编码的多肽即分别对应为本发明的抗菌多肽Ph-SA1，Ph-SA2，Ph-SA3，Ph-SA4，Ph-SE，和Ph-SAR1。
在构建的重组抗菌多肽中，可与靶细菌胞膜受体结合的信号传导多肽作为诱导物诱导大肠菌素通道结构域穿过细菌外膜与膜间隙到达靶细胞膜附近，然后大肠菌素通道结构域中的水性孔道结构域在靶细菌胞膜上形成离子通道，使靶细菌胞内内容物泄漏而致靶细菌死亡，从而达到杀菌的目的。
本发明中编码金黄色葡萄球菌信号传导多肽的核苷酸序列SEQ ID NO.1所编码氨基酸序列(SEQ ID NO2)与Genebank U85097号序列所表达的氨基酸序列相同；编码金黄色葡萄球菌信号传导多肽的核苷酸序列SEQ ID NO.3所编码氨基酸序列(SEQ ID NO4)与Genebank AF001782号序列所表达的氨基酸序列相同；编码金黄色葡萄球菌信号传导多肽的核苷酸序列SEQ ID NO.5所编码氨基酸序列(SEQ ID NO6)与Genebank AF001783号序列所表达的氨基酸序列相同；编码金黄色葡萄球菌信号传导多肽的核苷酸序列SEQ ID NO.7所编码氨基酸序列(SEQ ID NO8)与Genebank AF288215号序列所表达的氨基酸序列相同；编码表皮葡萄球菌信号传导多肽的核苷酸序列SEQ ID NO.9所编码氨基酸序列(SEQ ID NO10)与Genebank Z49220号序列所表达的氨基酸序列相同。
根据本发明的另一方面，提供如

图1～图6所示的包含本发明核苷酸的质粒载体，该质粒载体是将如上所述的编码葡萄球菌信号传导多肽的核苷酸序列经双链寡聚核苷酸点突变技术插入大肠菌素Ia基因的第626位氨基酸或第1位氨基酸上而形成本发明的重组质粒。
本发明中，构建质粒载体的原始质粒pSELECTTM-1来自于Promega公司，其中装载了大肠菌素Ia和immunity蛋白基因，针对金黄色葡萄球菌信号传导基因设计了四对引物，其序列分别如SEQ ID NO.24～SEQ ID NO32所示；针对表皮葡萄球信号传导基因设计了一对引物，其序列如SEQ ID NO33和SEQ IDNO34所示。利用双链寡聚核苷酸点突变技术，按照Strategene公司药箱操作获得重组质粒，再将获得的重组质粒转染入大肠杆菌TG1工程菌而获得可产生重组抗菌多肽的工程菌细胞。
根据本发明的再一方面，提供含有本发明重组抗菌多肽的药物组合物，可以通过将本发明多肽添加药学上可接受的载体或赋形剂或可选的其它成分而制成适于临床使用的药物组合物。
根据本发明的又一方面，提供本发明抗菌多肽的制备方法，将编码葡萄球菌信号传导多肽的基因可操作地与大肠菌素基因连接获得编码重组抗菌多肽的基因，将获得的基因导入到表达系统中进行表达，分离表达的多肽而获得本发明的抗菌多肽。
本发明的抗菌多肽相较于传统抗菌素的优点在于，其不会诱导细菌产生传统的耐药性。众所周知，细菌可以通过突变产生β-内酰胺酶、减少摄入、改变药物作用位点等方式来改变其细胞壁结构，改变其蛋白质和核酸的代谢等对传统抗菌素产生耐药性。但是细菌却较难以通过突变来改变其细胞膜的磷脂双分子层的结构，而这恰好是本发明的独到之处，本发明的重组多肽通过直接在靶细菌的胞膜上形成离子通道而达到杀菌目的。
附图的简要说明图1示出含有金黄色葡萄球菌信号传导多肽Agr D I和大肠菌素Ia的质粒pCHCSA1的结构；图2示出含有金黄色葡萄球菌信号传导多肽Agr D II和大肠菌素Ia的质粒pCHCSA2的结构；图3示出含有金黄色葡萄球菌信号传导多肽Agr D III和大肠菌素Ia的质粒pCHCSA3的结构；图4示出含有金黄色葡萄球菌信号传导多肽Agr D IV和大肠菌素Ia的质粒pCHCSA4的结构；图5示出含有表皮葡萄球菌信号传导多肽和大肠菌素Ia的质粒pCHCSE的结构；图6示出含有金黄色葡萄球菌信号传导多肽AgrD I和大肠菌素Ia的质粒结构，其中Agr D I连接在大肠菌素Ia的第1位氨基酸上；图7示出包括634个氨基酸残基的抗菌多肽的结构；图8为本发明抗菌多肽(Ph-SA1)对青霉素敏感金葡菌的抑制实验结果，表明青霉素敏感金葡菌的生长只能被青霉素和Ph-SA1抑制。图中Control，对照，PEN，青霉素，COL，野生型大肠菌素，TG1，无质粒的TG1工程菌蛋白，FusionProtein，大肠菌素Ia羧基端连接了其它八肽的重组蛋白，PMC即Ph-SA，抗金葡菌多肽。各实验组的药物用量均为100ng/ml；图9为抗金葡菌多肽(Ph-SA1)对耐甲氧西林金黄色葡萄球菌(ATCC BAA-42)杀菌效果的透射电镜观察(放大25,000倍)。Control，对照组，正常形态菌；Wildtype colicin Ia，野生型大肠菌素Ia处理(50μg/ml)，处理两小时后，菌形态仍然正常；AgrD，金葡菌信号多肽处理(10μg/ml)，处理两小时后，细菌形态仍然正常，上端一个菌体正在分裂；Oxacillin，苯唑西林处理(500μg/ml)，大剂量苯唑西林处理2小时后，菌胞壁呈溶解形态；Ph-SA1，抗金葡多肽处理(50μg/ml)，抗金葡多肽处理半小时后，菌破裂，内容物泄漏，而胞壁结构完整；结论单纯使用信号传导多肽或野生型大肠菌素处理都不能杀死MRSA，抗金葡多肽却能杀灭MRSA且杀菌机制与苯唑西林完全不同；图10为抗金葡多肽对MRSA(BAA42)感染小鼠体内保护试验结果之生存曲线；图11为本发明抗菌多肽(Ph-SA1和Ph-SE)对表皮葡萄球菌的抑制作用，表明表皮葡萄球菌的生长可被抗金葡多肽部分抑制，可被抗表葡多肽有效的抑制。图中Control，对照，PEN，青霉素，Ph-SA1，抗金葡多肽，Ph-SE，抗表葡多肽。药物浓度2μg/ml。
图12为各种抗菌多肽对耐甲氧西林金葡菌的生长抑制作用，表明耐甲氧西林金葡菌的生长几乎不能被苯唑西林抑制，Ph-SAR1的抑菌效果最差，抑制效果最好的是Ph-SA1和Ph-SA4，其次是Ph-SE，Ph-SA3和Ph-SA2再次之；图中Control，对照，Oxacillin，苯唑西林，Ph-SA1，Ph-SA2，Ph-SA3，Ph-SA4，分别连接了四种AgrD的抗金葡多肽，Ph-SE，抗表葡多肽，Ph-SAR1，AgrD1信息素连接在大肠菌素Ia氨基端的抗金葡多肽，药物浓度5μg/ml。
发明的
具体实施例方式
下面结合附图，通过对本发明较佳实施例的描述详细说明本发明。
实施例1表达抗菌多肽的质粒的构建和重组抗菌多肽制备原始质粒为装载了大肠菌素Ia和immunity蛋白基因的pSELECTTM-1商用质粒(质粒大小8.3kb，Promega公司)(UCSF，P.Gosh赠与)。经双链寡聚核苷酸点突变技术(QuickChangeTMKit，Strategeue公司)将编码葡萄球菌信号多肽基因插入到大肠菌素Ia基因的I626位点上，制备了抗葡萄球菌工程多肽的一系列突变质粒，pCHCSA1、pCHCSA2、pCHCSA3、pCHCSA4和pCHCSE以及Agr DI连接在大肠菌素Ia的第1位氨基酸上的质粒(如图1～图7所示)。突变质粒转染入E.coli TG1工程菌(AECOM，K.Jakes赠与)里制备多肽。
突变程序按Strategene QuikChange Site-Directed Mutagenesis Kit(Catalog#200518)药箱手册进行1.准备点突变反应物5μl 10X buffer2μl(10ng)野生型大肠菌素质粒1.25μl(125ng)设计的5’-3’寡聚核苷酸引物(见所列引物序列)1.25μl(125ng)设计的3’-5’寡聚核苷酸引物(见所列引物序列)1μl dNTP双蒸水50μl1μl pfu(除质粒、引物和双蒸水外，均为药箱所备试剂)2.进行PCR扩增，扩增条件变性95℃，35秒，退火53℃，70秒，延伸68℃，17分共20个循环；3.加入Dpn1内切酶1μl消化母体DNA链后(37℃，1小时)，取1μl反应物与XL1-Blue感受态细胞50μl冰孵30分钟，行热冲击42℃，45秒，再置入冰中2分钟；4.加入NZY培基0.5ml后220rpm，37℃摇菌1小时后，取50-100μl反应物铺板(LB培基加1％琼脂加50μg/ml氨苄青霉素，37C过夜)；5.18小时后挑菌，循Qiagene，Gibco等公司的各种商用提取质粒药箱提取质粒均可，测序确定突变成功。
6.将质粒50ng与制备的TG1工程菌感受肽细胞50μl冰孵30分，42℃，90秒热冲击，取50-100μl反应物加入LB培基0.5ml，220rpm，37℃摇菌1小时后铺板(LB培基加1％琼脂加50μg/ml氨苄青霉素)37℃孵育，18小时后挑取菌落。
7.大量增菌，8-16升FB培基，250rpm，37℃，6-8小时；离心沉淀菌体，4℃，6000g，20分钟，取4℃、50mM硼酸缓冲液(2mM EDTA+2mM DTT)50-80ml悬浮菌体，加0.2M PMSF 250微升后超声破碎(4℃，400W，2分钟)，高速离心破碎菌体(4℃，75000g，1.5小时)，取上清加入硫酸链霉素500万单位沉淀DNA，高速离心(4℃，30000g，10分钟)后取上清装入分子量15000的透析袋于4℃，50mM硼酸缓冲液4升透析过夜后，高速离心(4℃，30000g，10分钟)后上清上样于CM离子交换柱，4℃，0.3M NaCl+50mM硼酸缓冲液洗脱即可得到重组抗菌多肽。对应于以上6种质粒，获得了Ph-SA1、Ph-SA2、Ph-SA3、Ph-SA4、Ph-SE和Ph-SAR1六种重组抗菌多肽。
上述制备质粒中所设计的寡聚核苷酸序列pCHCSA15’-3’(SEQ ID NO24)gcg aat aag ttc tgg ggt att TAT TCC ACC TGT GAT TTT ATA ATG taa ata aaa tataag aca ggc3’-5’(SEQ ID NO25)gcc tgt ctt ata ttt tat tta CAT TAT AAA ATC ACA GGT GGA ATA aat acc cca gaa cttatt cgcpCHCSA25’-3’(SEQ ID NO26)gcg aat aag ttc tgg ggt att GGA GTT AAC GCA TGT TCT TCC CTG TTT taa ataaaa tat aag aca ggc3’-5’(SEQ ID NO27)gcc tgt ctt ata ttt tat tta AAA CAG GGA AGA ACA TGC GTT AAC TCC aat acc ccagaa ctt att cgcpCHCSA35’-3’(SEQ ID NO28)gcg aat aag ttc tgg ggt att TAT ATA AAC TGT GAT TTT CTTCTG taa ata aaa tataag aca ggc3’-5’(SEQ ID NO29)gcc tgt ctt ata ttt tat tta CAG AAG AAA ATC ACA GTT TAT ATA aat acc cca gaa cttatt cgcpCHCSA45’-3’(SEQ ID NO30)gcg aat aag ttc tgg ggt att TAT TCC ACC TGT TAC TTT ATA ATG taa ata aaa tat
aag aca ggc3’-5’(SEQ ID NO31)gcc tgt ctt ata ttt tat tta CAT TAT AAA GTA ACA GGT GGA ATA aat acc cca gaa cttatt cgcpCHCSE5’-3’(SEQ ID NO32)gcg aat aag ttc tgg ggt att GAT TCC GTT TGT GCA TCC TAT TTT taa ata aaa tataag aca ggc3’-5’(SEQ ID NO33)gcc tgt ctt ata ttt tat tta AAA ATA GGA TGC ACA AAC GGA ATC aat acc cca gaactt att cgc实施例2重组抗菌多肽对青霉素敏感金葡菌的抑制作用细菌为美国标准菌株，ATCC 25923青霉素敏感金黄色葡萄球菌，菌液5微升(108CFU/ml级菌量)加入1％胰蛋白胨，1％NaCl，0.5％酵母，0.5％葡萄糖，1％HK2PO4的培养液10ml中，共准备6组，第一组加入0.3M NaCl+50mM硼酸缓冲液(量与实验组中加入的抗菌多肽液体量相同)作为对照，第二组加入100ng/ml青霉素G钠，第三组加入100ng/ml野生型大肠菌素Ia(大肠菌素保存液为0.3M NaCl+50mM硼酸缓冲液)，第四组加入不含质粒的TG1工程菌蛋白(保存液为0.3M NaCl+50mM硼酸缓冲液)，第五组加入100ng/ml大肠菌素Ia羧基端连接了其它的八肽的融合蛋白(保存液为0.3M NaCl+50mM硼酸缓冲液)，第六组加入100ng/ml抗金葡菌多肽Ph-SA1(保存液为0.3M NaCl+50mM硼酸缓冲液)。
上述各组液体置于100ml三角烧瓶中，200rpm，37℃生长，每小时采样100μl加入96孔酶标板中经分光光度计(A 595nm)比色测试细菌生长浊度，画出细菌生长曲线来比较抗菌多肽的抑菌效力，结果如图8所示，显示青霉素敏感金葡菌的生长只能被青霉素和Ph-SA1抑制。
实施例3抗金葡菌多肽对耐甲氧西林金黄色葡萄球菌(ATCC BAA-42)杀菌效果的透射电镜观察(1％磷钨酸染色，放大25,000倍)。
如图9所示，Control，对照组，细菌加入前述培养液中，37℃，200rpm生长2小时后仍呈现正常形态；Wild type colicin Ia，细菌生长条件同对照组，加入野生型大肠菌素Ia(50μg/ml)生长两小时后，菌形态仍然正常；AgrD，细菌生长条件同对照组，加入金葡菌信号传导多肽(10μg/ml)生长两小时后，菌形态仍然正常，上端一个菌体正在分裂；Oxacillin，细菌生长条件同对照组，加入苯唑西林(500μg/ml)生长2小时后，菌胞壁呈溶解形态；Ph-SA1，细菌生长条件同对照组，加入抗金葡多肽(50μg/ml)生长半小时后，菌破裂，内容物泄漏，而残余胞壁结构完整。
结论单纯使用信号传导多肽或野生型大肠菌素处理都不能杀死MRSA，抗金葡多肽却能杀灭MRSA且杀菌机制与苯唑西林完全不同。
实施例4重组抗菌多肽的体外抗菌活性以下试验由国家药品监督管理局四川抗菌素研究所药理研究室完成。
一、实验材料
(1).药物①ph-SA1规格1.5mg/ml，批号PRE021101，由四川大学华西医学院提供。
②注射用头孢唑林钠白色粉末，规格0.5g/瓶，批号020418，华北制药股份有限公司生产。
③注射用青霉素钠白色粉末，规格80万单位/瓶，批号00206604，华北制药股份有限公司生产。
④注射用氨苄西林钠规格1g/瓶，批号F0203329，华北制药股份有限公司生产。
⑤注射用苯唑西林钠规格0.5g/瓶，批号F0203329，华北制药股份有限公司生产。
⑥万古霉素规格2.5mg/ml，批号020712，由四川抗菌素工业研究所制剂室提供。
以上样品用无菌水溶解稀释，药物终浓度为128mg/L、64mg/L、32mg/L、16mg/L、L8mg/、L4mg/L、2mg/L、1mg/L、0.5mg/L、0.25mg/L、0.125mg/L、0.06mg/L、0.03mg/L、0.015mg/L。
(2)、细菌①临床分离菌株试验所用菌株均为2002年从四川、北京地区收集的临床分离致病菌，所有菌种在收集分离的单位(华西医科大学检验科及重庆医科大学传染科细菌室)均经鉴定后，再经本室用API系统重新鉴定后使用。
金黄色葡萄菌168株(MRSA菌株80株，MSSA菌株88株)、表皮葡萄球菌46株(MRSA菌株25株，MSSE菌株21株)、肠球菌20株、鲍曼不动杆菌20株，共274株。
②标准质控菌株金黄色葡萄球菌ATCC25923、金黄色葡萄球菌29213是四川抗菌素工业研究所药理室保存菌株。
(3)、培养基①M-H培养基中国药品生物制品检定所产品，M-H肉汤培养基称取25g加100ml蒸馏水，加热溶解，分装，高压灭菌，116℃，20分钟。M-H固体培养基称取36g，加1000m蒸馏水，高压灭菌，116℃，20分钟，用于革兰阳性、阴性需氧菌的药敏试验。
②肠球菌、链球菌用血培养基，即在M-H培养基中加5-10％脱纤维兔血配制而成，5％CO中37℃培养24小时。
二、试验方法1.最低抑菌浓度MIC的测定采用琼脂二倍稀释法测定ph-SA的最低抑菌浓度(MIC)。用多点接种仪(Denley A400)将细菌接种于含不同药物浓度的琼脂平皿表面，每点含菌量约为105CFU/ml，37℃孵育18～24小时观察结果，以无细菌生长平皿培养基中所含药物的最低浓度为药物对该菌的最低抑菌浓度(MIC值)。
三、结果实验结果见表1。
表1 ph-SA1的体外抗菌活性比较菌株 药物MIC50MIC90MIC范围(株数)(mg/L) (mg/L) (mg/L)金黄色葡萄球菌ph-SA1 16 ＞16 ＜0.0005～64
MRSA菌株 头孢唑林＞128＞1281～128青霉素 128 ＞1281～128氨苄西林 16 320.06～64苯唑西林＞128＞1280.5～128万古霉素0.25 10.004～2金黄色葡萄球菌ph-SA1 0.5 16 0.125～＞64MSSA菌株 头孢唑林 0.1250.25＜0.015～0.5(88) 青霉素 8640.125～＞128氨苄西林 0.25 1＜0.015～0.25苯唑西林 0.1250.125＜0.015～0.25万古霉素 0.5 10.03～1表皮葡萄球菌 ph-SA1 ＞64 ＞640.25～＞64MRSE菌株 头孢唑林 32 ＞1280.06～＞128(25) 青霉素 ＞128＞1288～＞128氨苄西林 16 640.06～64苯唑西林 64 ＞1280.5～＞128万古霉素 0.5 10.25～2表皮葡萄球菌 ph-SA1 0.25 160.125～32MSSE菌株 头孢唑林 0.03 0.5＜0.015～0.5(21) 青霉素 4320.125～128氨苄西林 0.03 0.25＜0.015～0.25苯唑西林0.03 0.125＜0.015～0.25万古霉素 0.5 0.50.06～0.5肠球菌ph-SA1 ＞64 ＞648～＞64(20) 头孢唑林 32 ＞128
32～＞128青霉素 32 6432～＞128氨苄西林 0.5 160.25～16苯唑西林 16 ＞1288～＞128万古霉素 120.5～50接表1菌株 药物MIC50MIC90MIC范围(株数)(mg/L) (mg/L) (mg/L)铜绿假单胞菌 ph-SA1 16 ＞16 4～＞16(20) 头孢唑林＞128＞128128～＞128青霉素 ＞128＞128＞128氨苄西林＞128＞12816～＞128苯唑西林＞128＞128＞128万古霉素＞128＞128＞128鲍曼不动杆菌 ph-SA1 32 ＞64 0.5～＞64(20) 头孢唑林＞128＞128＞128青霉素 ＞128＞128＞128氨苄西林＞128＞12816～＞128苯唑西林＞128＞128＞128万古霉素＞128＞128128～＞128实施例5重组抗菌多肽的动物体内保护实验以下试验由国家药品监督管理局四川抗菌素研究所药理研究室完成。
一、实验材料(1)·药物使用药物同前。
以上样品用无菌水溶解稀释，药物注射终浓度为10mg/kg、5mg/gk、2.5mg/kg。
(2)·细菌①标准质控菌株金黄色葡萄球菌ATCC BAA-42(MRSA)、肠球菌ATCC700802(VRE)是四川抗菌素工业研究所药理室保存菌株。
二、实验方法昆明小鼠170只，雌雄各半，体重18-22克，随机分组为腹腔分别注射金黄色葡萄球菌ATCC BAA-42(MRSA)和ATCC 700802(VRE)两个大组，其中PH-SA1、青霉素、氨苄西林、头孢唑林和万古霉素试验组分别为每组小鼠5只，对照组每组小鼠10只。腹腔注射致死剂量的细菌后，各药物试验组小鼠分别自静脉注射10mg、5mg、2.5mg/kg剂量的药物一次，每24小时观察结果，连续7-14天，以小鼠死亡为阳性结果。
三、结果5mg、3mg和1mg药物剂量组结果显示，Ph-SA1组小鼠存活率在3个剂量组中与万古霉素组小鼠存活率始终保持了恒定的差距，因此在体内试验中Ph-SA1对抗耐甲氧西林金黄色葡萄球菌感染的治疗效果远优于万古霉素。这可能是Ph-SA1既杀菌又抑制细菌毒素分泌的双重效果所致。因而该药在临床应用时可能具有其它抗菌素无法比拟的治疗效果。其它各抗菌素组小鼠死亡率≥80％，已不具备可比性；对照组于48小时内全部死亡。
实验结果见表2和图10。
表2 Ph-SA1体内保护试验结果(静脉给药)


由表1和表2中结果可以看出1.MRSA体外抗菌活性比较苯唑西林MIC90测定值比PH-SA1测定值大8倍，比万古霉素测定值大128倍。PH-SA1分子量(70,0000)是万古霉素分子量(1,400)的49倍，苯唑西林分子量(400)的175倍。按单位体积内相同药物分子数标定，则PH-SA1对MRSA体外抗菌活性相当于万古霉素对MRSA体外抗菌活性的3倍，相当于苯唑西林对MRSA体外抗菌活性的1,400倍。
2.Ph-SA1对MSSA的敏感率(以16mg/L为界限)为91％，对MRSA的敏感率为77％，对MSSE的敏感率为91％，对MRSE的敏感率为40％，对肠球菌的敏感率为30％，对铜绿假单胞菌的敏感率为50％，对鲍曼不动杆菌的敏感率为40％。
3.SA感染小鼠体内保护预实验中，最小剂药量(2.5mg/kg)条件下PH-SA1组的死亡率最低(40％)，其它各组死亡率分别为青霉素组60％，氨咔西林组100％，万古霉素60％，对照组100％。该结果显示PH-SA1的保护率最高(60％)；青霉素和万古霉素的保护率次之(40％)。
实施例6重组抗菌多肽对表皮葡萄球菌生长的影响细菌为购自中国菌种保存中心(国家医药管理局生物制品检定所，北京天坛)的表皮葡萄球菌标准菌株，编号26069，菌液5微升(108CFU/ml级菌量)加入1％胰蛋白胨，1％NaCl，0.5％酵母，0.5％葡萄糖，1％HK2PO4的培养液10ml中，共准备4组，第一组加入0.3M NaCl+50mM硼酸缓冲液(量与实验组中加入的抗菌多肽液体量相同)作为对照，第二组加入2μg/ml青霉素G钠，余各实验组各加入2μg/ml抗菌多肽Ph-SA1和Ph-SE(保存液为0.3M NaCl+50mM硼酸缓冲液)。
上述各组液体置于100ml三角烧瓶中，200rpm，37℃生长，每小时采样100μl加入96孔酶标板中经分光光度计(A 595nm)比色测试细菌生长浊度，画出细菌生长曲线来比较抗菌多肽的抑菌效力，结果见图11。
由图中可见，表皮葡萄球菌的生长可被抗金葡多肽部分抑制，可被抗表葡多肽有效的抑制。
实施例7几种重组抗菌多肽抑菌效果的比较以下实验比较了本发明制备的6种抗菌多肽的抑菌活性，细菌为美国标准菌株，ATCC BAA-42耐甲氧西林金黄色葡萄球菌，菌液5微升(108CFU/ml级菌量)加入1％胰蛋白胨，1％NaCl，0.5％酵母，0.5％葡萄糖，1％HK2PO4的培养液10ml中，共准备8组，第一组加入0.3M NaCl+50mM硼酸缓冲液(量与实验组中加入的抗菌多肽液体量相同)作为对照，第二组加入5μg/ml青霉素G钠，余各实验组各加入5μg/ml抗菌多肽Ph-SA1、Ph-SA2、Ph-SA3、Ph-SA4、Ph-SE和Ph-SAR1(保存液为0.3M NaCl+50mM硼酸缓冲液)。
上述各组液体置于100ml三角烧瓶中，200rpm，37℃生长，每小时采样100μl加入96孔酶标板中经分光光度计(A 595nm)比色测试细菌生长浊度，画出细菌生长曲线来比较抗菌多肽的抑菌效力，结果见图12。
结果显示，耐甲氧西林金葡菌的生长几乎不能被苯唑西林抑制，Ph-SAR1的抑菌效果最差，抑制效果最好的是Ph-SA1和Ph-SA4，其次是Ph-SE，Ph-SA3和Ph-SA2再次之。
以上对本发明的详细描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变和变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
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<221>CDS<222>(1)..(24)<223>编码金黄色葡萄球菌Agr D III信号传导多肽
<400>5tat ata aac tgt gat ttt ctt ctg 24Tyr Ile Asn Cys Asp Phe Leu Leu1 5<210>6<211>8<212>PRT<213>人工序列<400>6Tyr Ile Asn Cys Asp Phe Leu Leu1 5<210>7<211>24<212>DNA<213>人工序列<220>
<221>CDS<222>(1)..(24)<223>编码金黄色葡萄球菌Agr D IV信号传导多肽<400>7tat tcc acc tgt tac ttt ata atg 24Tyr Ser Thr Cys Tyr Phe Ile Met1 5<210>8<211>8<212>PRT<213>人工序列<400>8Tyr Ser Thr Cys Tyr Phe Ile Met1 5<210>9<211>24<212>DNA<213>人工序列<220>
<221>CDS<222>(1)..(24)<223>编码表皮葡萄球菌信号传导多肽<400>9gat tcc gtt tgt gca tcc tat ttt24Asp Ser Val Cys Ala Ser Tyr Phe1 5<210>10<211>8<212>PRT<213>人工序列<400>10Asp Ser Val Cys Ala Ser Tyr Phe1 5<210>11<211>1878
<212>DNA<213>Escherichia coli<220>
<221>misc_feature<222>(1)..(1878)<223>大肠菌素基因<400>11atgtctgacc ctgtacgtat tacaaatccc ggtgcagaat cgctggggta tgattcagat60ggccatgaaa ttatggccgt tgatatttat gtaaaccctc cacgtgtcga tgtctttcat 120ggtaccccgc ctgcatggag ttccttcggg aacaaaacca tctggggcgg aaacgagtgg 180gttgatgatt ccccaacccg aagtgatatc gaaaaaaggg acaaggaaat cacagcgtac 240aaaaacacgc tcagcgcgca gcagaaagag aatgagaata agcgtactga agccggaaaa 300cgcctctctg cggcgattgc tgcaagggaa aaagatgaaa acacactgaa aacactccgt 360gccggaaacg cagatgccgc tgatattaca cgacaggagt tcagactcct gcaggcagag 420ctgagagaat acggattccg tactgaaatc gccggatatg acgccctccg gctgcataca 480gagagccgga tgctgtttgc tgatgctgat tctcttcgta tatctccccg ggaggccagg 540tcgttaatcg aacaggctga aaaacggcag aaggatgcgc agaacgcaga caagaaggcc 600gctgatatgc ttgctgaata cgagcgcaga aaaggtattc tggacacccg gttgtcagag 660ctggaaaaaa atggcggggc agcccttgcc gttcttgatg cacaacaggc ccgtctgctc 720gggcagcaga cacggaatga cagggccatt tcagaggccc ggaataaact cagttcagtg 780acggaatcgc ttaacacggc ccgtaatgca ttaaccagag ctgaacaaca gctgacgcaa 840cagaaaaaca cgcctgacgg caaaacgata gtttcccctg aaaaattccc ggggcgttca 900tcaacaaatc attctattgt tgtgagcggt gatccgagat ttgccggtac gataaaaatc 960acaaccagcg cagtcatcga taaccgtgca aacctgaatt atcttctgag ccattccggt 1020ctggactata aacgcaatat tctgaatgac cggaatccgg tggtgacaga ggatgtggaa 1080ggtgacaaga aaatttataa tgctgaagtt gctgaatggg ataagttacg gcaaagattg 1140cttgatgcca gaaataaaat cacctctgct gaatctgcgg taaattcggc gagaaataac 1200ctcagtgcca gaacaaatga gcaaaagcat gcaaatgacg ctcttaatgc cctgttgaag 1260gaaaaagaga atatacgtaa ccagctttcc ggcatcaatc agaagatagc ggaagagaaa 1320agaaaacagg atgaactgaa ggcaacgaaa gacgcaatta atttcacaac agagttcctg 1380aaatcagttt cagaaaaata tggtgcaaaa gctgagcagt tagccagaga gatggccggg 1440caggctaaag ggaagaaaat acgtaatgtt gaagaggcat taaaaacgta tgaaaagtac 1500cgggctgaca ttaacaaaaa aattaatgca aaagatcgtg cagcgattgc cgcagccctt 1560gagtctgtga agctgtctga tatatcgtct aatctgaaca gattcagtcg gggactggga 1620tatgcaggaa aatttacaag tcttgctgac tggatcactg agtttggtaa ggctgtccgg 1680acagagaact ggcgtcctct ttttgttaaa acagaaacca tcatagcagg caatgccgca 1740acggctcttg tggcactggt cttcagtatt cttaccggaa gcgctttagg cattatcggg 1800tatggtttac tgatggctgt caccggtgcg ctgattgatg aatcgcttgt ggaaaaagcg 1860aataagttct ggggtatt1878<210>12<211>1905
<212>DNA<213>人工序列<220>
<221>CDS<222>(4)..(1902)<223>编码抗葡萄球菌多肽<400>12atg tct gac cct gta cgt att aca aat ccc ggt gca gaa tcg ctg ggg 48Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1 5 10 15tat gat tca gat ggc cat gaa att atg gcc gtt gat att tat gta aac 96Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn20 25 30cct cca cgt gtc gat gtc ttt cat ggt acc ccg cct gca tgg agt tcc 144Pro Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser35 40 45ttc ggg aac aaa acc atc tgg ggc gga aac gag tgg gtt gat gat tcc 192Phe Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser50 55 60cca acc cga agt gat atc gaa aaa agg gac aag gaa atc aca gcg tac 240Pro Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr65 70 75aaa aac acg ctc agc gcg cag cag aaa gag aat gag aat aag cgt act 288Lys Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr80 85 90 95gaa gcc gga aaa cgc ctc tct gcg gcg att gct gca agg gaa aaa gat 336Glu Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp100 105 110gaa aac aca ctg aaa aca ctc cgt gcc gga aac gca gat gcc gct gat 384Glu Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp115 120 125att aca cga cag gag ttc aga ctc ctg cag gca gag ctg aga gaa tac 432Ile Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr130 135 140gga ttc cgt act gaa atc gcc gga tat gac gcc ctc cgg ctg cat aca 480Gly Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr145 150 155gag agc cgg atg ctg ttt gct gat gct gat tct ctt cgt ata tct ccc 528Glu Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro160 165 170 175cgg gag gcc agg tcg tta atc gaa cag gct gaa aaa cgg cag aag gat 576Arg Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp180 185 190gcg cag aac gca gac aag aag gcc gct gat atg ctt gct gaa tac gag 624Ala Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu195 200 205cgc aga aaa ggt att ctg gac acc cgg ttg tca gag ctg gaa aaa aat 672Arg Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn210 215 220ggc ggg gca gcc ctt gcc gtt ctt gat gca caa cag gcc cgt ctg ctc 720Gly Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu225 230 235ggg cag cag aca cgg aat gac agg gcc att tca gag gcc cgg aat aaa 768Gly Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys240 245 250 255ctc agt tca gtg acg gaa tcg ctt aac acg gcc cgt aat gca tta acc 816Leu Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr260 265 270
aga gct gaa caa cag ctg acg caa cag aaa aac acg cct gac ggc aaa 864Arg Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys275 280 285acg ata gtt tcc cct gaa aaa ttc ccg ggg cgt tca tca aca aat cat 912Thr Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His290 295 300tct att gtt gtg agc ggt gat ccg aga ttt gcc ggt acg ata aaa atc 960Ser Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile305 310 315aca acc agc gca gtc atc gat aac cgt gca aac ctg aat tat ctt ctg 1008Thr Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu320 325 330335agc cat tcc ggt ctg gac tat aaa cgc aat att ctg aat gac cgg aat 1056Ser His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn340 345 350ccg gtg gtg aca gag gat gtg gaa ggt gac aag aaa att tat aat gct 1104Pro Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala355 360 365gaa gtt gct gaa tgg gat aag tta cgg caa aga ttg ctt gat gcc aga 1152Glu Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg370 375 380aat aaa atc acc tct gct gaa tct gcg gta aat tcg gcg aga aat aac 1200Asn Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn385 390 395ctc agt gcc aga aca aat gag caa aag cat gca aat gac gct ctt aat 1248Leu Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn400 405 410 415gcc ctg ttg aag gaa aaa gag aat ata cgt aac cag ctt tcc ggc atc 1296Ala Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile420 425 430aat cag aag ata gcg gaa gag aaa aga aaa cag gat gaa ctg aag gca 1344Asn Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala435 440 445acg aaa gac gca att aat ttc aca aca gag ttc ctg aaa tca gtt tca 1392Thr Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser450 455 460gaa aaa tat ggt gca aaa gct gag cag tta gcc aga gag atg gcc ggg 1440Glu Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly465 470 475cag gct aaa ggg aag aaa ata cgt aat gtt gaa gag gca tta aaa acg 1488Gln Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr480 485 490 495tat gaa aag tac cgg gct gac att aac aaa aaa att aat gca aaa gat 1536Tyr Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp500 505 510cgt gca gcg att gcc gca gcc ctt gag tct gtg aag ctg tct gat ata 1584Arg Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile515 520 525tcg tct aat ctg aac aga ttc agt cgg gga ctg gga tat gca gga aaa 1632Ser Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys530 535 540ttt aca agt ctt gct gac tgg atc act gag ttt ggt aag gct gtc cgg 1680Phe Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg545 550 555aca gag aac tgg cgt cct ctt ttt gtt aaa aca gaa acc atc ata gca 1728Thr Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala560 565 570 575
ggc aat gcc gca acg gct ctt gtg gca ctg gtc ttc agt att ctt acc 1776Gly Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr580 585 590gga agc gct tta ggc att atc ggg tat ggt tta ctg atg gct gtc acc 1824Gly Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr595 600 605ggt gcg ctg att gat gaa tcg ctt gtg gaa aaa gcg aat aag ttc tgg 1872Gly Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp610 615 620ggt att tat tcc acc tgt gat ttt ata atg taa 1905Gly Ile Tyr Ser Thr Cys Asp Phe Ile Met625 630<210>13<211>633<212>PRT<213>人工序列<400>13Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly Tyr1 5 10 15Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn Pro20 25 30Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser Phe35 40 45Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser Pro50 55 60Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr Lys65 70 75 80Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr Glu85 90 95Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp Glu100 105 110Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp Ile115 120 125Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr Gly130 135 140Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr Glu145 150 155 160Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro Arg165 170 175Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp Ala180 185 190Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu Arg195 200 205Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn Gly
210 215 220Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu Gly225 230 235 240Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys Leu245 250 255Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr Arg260 265 270Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys Thr275280 285Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His Ser290 295 300Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile Thr305 310 315 320Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu Ser325 330 335His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn Pro340 345 350Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala Glu355 360 365Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg Asn370 375 380Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn Leu385 390 395 400Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn Ala405 410 415Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile Asn420 425 430Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala Thr435 440 445Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser Glu450 455 460Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly Gln465 470 475 480Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr Tyr485 490 495Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp Arg500505 510Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile Ser515 520 525
Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys Phe530 535 540Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg Thr545 550 555 560Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala Gly565 570575Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr Gly580 585 590Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr Gly595 600 605Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp Gly610 615 620Ile Tyr Ser Thr Cys Asp Phe Ile Met625 630<210>14<211>1908<212>DNA<213>人工序列<220>
<221>CDS<222>(4)..(1905)<223>编码抗葡萄球菌多肽<400>14atg tct gac cct gta cgt att aca aat ccc ggt gca gaa tcg ctg ggg 48Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1 5 10 15tat gat tca gat ggc cat gaa att atg gcc gtt gat att tat gta aac 96Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn20 25 30cct cca cgt gtc gat gtc ttt cat ggt acc ccg cct gca tgg agt tcc144Pro Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser35 40 45ttc ggg aac aaa acc atc tgg ggc gga aac gag tgg gtt gat gat tcc192Phe Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser50 55 60cca acc cga agt gat atc gaa aaa agg gac aag gaa atc aca gcg tac240Pro Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr65 70 75aaa aac acg ctc agc gcg cag cag aaa gag aat gag aat aag cgt act288Lys Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr80 85 90 95gaa gcc gga aaa cgc ctc tct gcg gcg att gct gca agg gaa aaa gat336Glu Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp100 105 110gaa aac aca ctg aaa aca ctc cgt gcc gga aac gca gat gcc gct gat384Glu Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp115 120 125att aca cga cag gag ttc aga ctc ctg cag gca gag ctg aga gaa tac432Ile Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr130 135 140
gga ttc cgt act gaa atc gcc gga tat gac gcc ctc cgg ctg cat aca 480Gly Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr145 150 155gag agc cgg atg ctg ttt gct gat gct gat tct ctt cgt ata tct ccc 528Glu Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro160 165 170 175cgg gag gcc agg tcg tta atc gaa cag gct gaa aaa cgg cag aag gat 576Arg Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp180 185 190gcg cag aac gca gac aag aag gcc gct gat atg ctt gct gaa tac gag 624Ala Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu195 200 205cgc aga aaa ggt att ctg gac acc cgg ttg tca gag ctg gaa aaa aat 672Arg Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn210 215 220ggc ggg gca gcc ctt gcc gtt ctt gat gca caa cag gcc cgt ctg ctc 720Gly Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu225 230 235ggg cag cag aca cgg aat gac agg gcc att tca gag gcc cgg aat aaa 768Gly Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys240 245 250 255ctc agt tca gtg acg gaa tcg ctt aac acg gcc cgt aat gca tta acc 816Leu Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr260 265 270aga gct gaa caa cag ctg acg caa cag aaa aac acg cct gac ggc aaa 864Arg Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys275 280 285acg ata gtt tcc cct gaa aaa ttc ccg ggg cgt tca tca aca aat cat 912Thr Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His290 295 300tct att gtt gtg agc ggt gat ccg aga ttt gcc ggt acg ata aaa atc 960Ser Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile305 310 315aca acc agc gca gtc atc gat aac cgt gca aac ctg aat tat ctt ctg1008Thr Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu320 325 330 335agc cat tcc ggt ctg gac tat aaa cgc aat att ctg aat gac cgg aat1056Ser His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn340 345 350ccg gtg gtg aca gag gat gtg gaa ggt gac aag aaa att tat aat gct1104Pro Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala355 360 365gaa gtt gct gaa tgg gat aag tta cgg caa aga ttg ctt gat gcc aga1152Glu Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg370 375 380aat aaa atc acc tct gct gaa tct gcg gta aat tcg gcg aga aat aac1200Asn Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn385 390 395ctc agt gcc aga aca aat gag caa aag cat gca aat gac gct ctt aat1248Leu Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn400 405 410 415gcc ctg ttg aag gaa aaa gag aat ata cgt aac cag ctt tcc ggc atc1296Ala Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile420 425 430aat cag aag ata gcg gaa gag aaa aga aaa cag gat gaa ctg aag gca1344Asn Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala435 440 445
acg aaa gac gca att aat ttc aca aca gag ttc ctg aaa tca gtt tca 1392Thr Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser450 455 460gaa aaa tat ggt gca aaa gct gag cag tta gcc aga gag atg gcc ggg 1440Glu Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly465 470 475cag gct aaa ggg aag aaa ata cgt aat gtt gaa gag gca tta aaa acg 1488Gln Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr480 485 490 495tat gaa aag tac cgg gct gac att aac aaa aaa att aat gca aaa gat 1536Tyr Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp500 505 510cgt gca gcg att gcc gca gcc ctt gag tct gtg aag ctg tct gat ata 1584Arg Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile515 520 525tcg tct aat ctg aac aga ttc agt cgg gga ctg gga tat gca gga aaa 1632Ser Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys530 535 540ttt aca agt ctt gct gac tgg atc act gag ttt ggt aag gct gtc cgg 1680Phe Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg545 550 555aca gag aac tgg cgt cct ctt ttt gtt aaa aca gaa acc atc ata gca 1728Thr Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala560 565 570 575ggc aat gcc gca acg gct ctt gtg gca ctg gtc ttc agt att ctt acc 1776Gly Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr580 585 590gga agc gct tta ggc att atc ggg tat ggt tta ctg atg gct gtc acc 1824Gly Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr595 600 605ggt gcg ctg att gat gaa tcg ctt gtg gaa aaa gcg aat aag ttc tgg 1872Gly Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp610 615 620ggt att gga gtt aac gca tgt tct tcc ctg ttt taa 1908Gly Ile Gly Val Asn Ala Cys Ser Ser Leu Phe625 630<210>15<211>634<212>PRT<213>人工序列<400>15Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly Tyr1 5 10 15Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn Pro20 25 30Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser Phe35 40 45Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser Pro50 55 60Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr Lys65 70 75 80Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr Glu
85 90 95Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp Glu100 105 110Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp Ile115 120 125Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr Gly130 135 140Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr Glu145 150 155 160Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro Arg165 170 175Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp Ala180 185 190Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu Arg195 200 205Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn Gly210 215 220Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu Gly225 230235 240Glu Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys Leu245 250 255Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr Arg260 265 270Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys Thr275 280 285Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His Ser290 295 300Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile Thr305 310 315 320Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu Ser325 330 335His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn Pro340 345 350Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala Glu355 360 365Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg Asn370 375 380Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn Leu385 390 395 400
Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn Ala405 410 415Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile Asn420 425 430Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala Thr435 440 445Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser Glu450 455 460Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly Gln465 470 475 480Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr Tyr485 490 495Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp Arg500 505 510Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile Ser515 520 525Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys Phe530 535 540Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg Thr545 550 555 560Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala Gly565 570 575Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr Gly580 585 590Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr Gly595 600 605Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp Gly610 615 620Ile Gly Val Asn Ala Cys Ser Ser Leu Phe625 630<210>16<211>1905<212>DNA<213>人工序列<220>
<221>CDS<222>(4)..(1902)<223>编码抗葡萄球菌多肽<400>16atg tct gac cct gta cgt att aca aat ccc ggt gca gaa tcg ctg ggg 48Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1 5 10 15
tat gat tca gat ggc cat gaa att atg gcc gtt gat att tat gta aac 96Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn20 25 30cct cca cgt gtc gat gtc ttt cat ggt acc ccg cct gca tgg agt tcc144Pro Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser35 40 45ttc ggg aac aaa acc atc tgg ggc gga aac gag tgg gtt gat gat tcc192Phe Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser50 55 60cca acc cga agt gat atc gaa aaa agg gac aag gaa atc aca gcg tac240Pro Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr65 70 75aaa aac acg ctc agc gcg cag cag aaa gag aat gag aat aag cgt act288Lys Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr80 85 90 95gaa gcc gga aaa cgc ctc tct gcg gcg att gct gca agg gaa aaa gat336Glu Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp100 105 110gaa aac aca ctg aaa aca ctc cgt gcc gga aac gca gat gcc gct gat384Glu Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp115 120 125att aca cga cag gag ttc aga ctc ctg cag gca gag ctg aga gaa tac432Ile Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr130 135 140gga ttc cgt act gaa atc gcc gga tat gac gcc ctc cgg ctg cat aca480Gly Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr145 150 155gag agc cgg atg ctg ttt gct gat gct gat tct ctt cgt ata tct ccc528Glu Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro160 165 170 175cgg gag gcc agg tcg tta atc gaa cag gct gaa aaa cgg cag aag gat576Arg Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp180 185 190gcg cag aac gca gac aag aag gcc gct gat atg ctt gct gaa tac gag624Ala Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu195 200 205cgc aga aaa ggt att ctg gac acc cgg ttg tca gag ctg gaa aaa aat672Arg Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn210 215 220ggc ggg gca gcc ctt gcc gtt ctt gat gca caa cag gcc cgt ctg ctc720Gly Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu225 230 235ggg cag cag aca cgg aat gac agg gcc att tca gag gcc cgg aat aaa768Gly Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys240 245 250 255ctc agt tca gtg acg gaa tcg ctt aac acg gcc cgt aat gca tta acc816Leu Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr260 265 270aga gct gaa caa cag ctg acg caa cag aaa aac acg cct gac ggc aaa864Arg Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys275 280 285acg ata gtt tcc cct gaa aaa ttc ccg ggg cgt tca tca aca aat cat912Thr Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His290 295 300tct att gtt gtg agc ggt gat ccg aga ttt gcc ggt acg ata aaa atc960Ser Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile305 310 315
aca acc agc gca gtc atc gat aac cgt gca aac ctg aat tat ctt ctg1008Thr Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu320 325 330 335agc cat tcc ggt ctg gac tat aaa cgc aat att ctg aat gac cgg aat1056Ser His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn340 345 350ccg gtg gtg aca gag gat gtg gaa ggt gac aag aaa att tat aat gct1104Pro Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala355 360 365gaa gtt gct gaa tgg gat aag tta cgg caa aga ttg ctt gat gcc aga1152Glu Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg370 375 380aat aaa atc acc tct gct gaa tct gcg gta aat tcg gcg aga aat aac1200Asn Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn385 390 395ctc agt gcc aga aca aat gag caa aag cat gca aat gac gct ctt aat1248Leu Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn400 405 410 415gcc ctg ttg aag gaa aaa gag aat ata cgt aac cag ctt tcc ggc atc1296Ala Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile420 425 430aat cag aag ata gcg gaa gag aaa aga aaa cag gat gaa ctg aag gca1344Asn Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala435 440 445acg aaa gac gca att aat ttc aca aca gag ttc ctg aaa tca gtt tca1392Thr Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser450 455 460gaa aaa tat ggt gca aaa gct gag cag tta gcc aga gag atg gcc ggg1440Glu Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly465 470 475cag gct aaa ggg aag aaa ata cgt aat gtt gaa gag gca tta aaa acg1488Gln Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr480 485 490 495tat gaa aag tac cgg gct gac att aac aaa aaa att aat gca aaa gat1536Tyr Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp500 505 510cgt gca gcg att gcc gca gcc ctt gag tct gtg aag ctg tct gat ata1584Arg Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile515 520 525tcg tct aat ctg aac aga ttc agt cgg gga ctg gga tat gca gga aaa1632Ser Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys530 535 540ttt aca agt ctt gct gac tgg atc act gag ttt ggt aag gct gtc cgg1680Phe Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg545 550 555aca gag aac tgg cgt cct ctt ttt gtt aaa aca gaa acc atc ata gca1728Thr Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala560 565 570 575ggc aat gcc gca acg gct ctt gtg gca ctg gtc ttc agt att ctt acc1776Gly Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr580 585 590gga agc gct tta ggc att atc ggg tat ggt tta ctg atg gct gtc acc1824Gly Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr595 600 605ggt gcg ctg att gat gaa tcg ctt gtg gaa aaa gcg aat aag ttc tgg1872Gly Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp610 615 620ggt att tat ata aac tgt gat ttt ctt ctg taa1905
Gly Ile Tyr Ile Asn Cys Asp Phe Leu Leu625 630<210>17<211>633<212>PRT<213>人工序列<400>17Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly Tyr1 5 10 15Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn Pro20 25 30Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser Phe35 40 45Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser Pro50 55 60Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr Lys65 70 75 80Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr Glu85 90 95Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp Glu100 105 110Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp Ile115 120 125Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr Gly130 135 140Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr Glu145 150 155 160Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro Arg165 170 175Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp Ala180 185 190Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu Arg195 200 205Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn Gly210 215 220Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu Gly225 230 235 240Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys Leu245 250 255Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr Arg260 265 270
Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys Thr275 280 285Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His Ser290 295 300Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile Thr305 310 315 320Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu Ser325 330 335His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn Pro340 345 350Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala Glu355 360 365Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg Asn370 375 380Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn Leu385 390 395 400Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn Ala405 410 415Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile Asn420 425 430Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala Thr435 440 445Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser Glu450 455 460Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly Gln465 470 475 480Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr Tyr485 490 495Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp Arg500 505 510Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile Ser515 520525Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys Phe530 535 540Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg Thr545 550 555 560Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala Gly565 570 575
Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr Gly580 585 590Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr Gly595 600 605Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp Gly610 615 620Ile Tyr Ile Asn Cys Asp Phe Leu Leu625 630<210>18<211>1905<212>DNA<213>人工序列<220>
<221>CDS<222>(4)..(1902)<223>编码抗葡萄球菌多肽<400>18atg tct gac cct gta cgt att aca aat ccc ggt gca gaa tcg ctg ggg 48Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1 5 10 15tat gat tca gat ggc cat gaa att atg gcc gtt gat att tat gta aac 96Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn20 25 30cct cca cgt gtc gat gtc ttt cat ggt acc ccg cct gca tgg agt tcc144Pro Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser35 40 45ttc ggg aac aaa acc atc tgg ggc gga aac gag tgg gtt gat gat tcc192Phe Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser50 55 60cca acc cga agt gat atc gaa aaa agg gac aag gaa atc aca gcg tac240Pro Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr65 70 75aaa aac acg ctc agc gcg cag cag aaa gag aat gag aat aag cgt act288Lys Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr80 85 90 95gaa gcc gga aaa cgc ctc tct gcg gcg att gct gca agg gaa aaa gat336Glu Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp100 105 110gaa aac aca ctg aaa aca ctc cgt gcc gga aac gca gat gcc gct gat384Glu Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp115 120 125att aca cga cag gag ttc aga ctc ctg cag gca gag ctg aga gaa tac432Ile Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr130 135 140gga ttc cgt act gaa atc gcc gga tat gac gcc ctc cgg ctg cat aca480Gly Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr145 150 155gag agc cgg atg ctg ttt gct gat gct gat tct ctt cgt ata tct ccc528Glu Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro160 165 170175cgg gag gcc agg tcg tta atc gaa cag gct gaa aaa cgg cag aag gat576Arg Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp180 185 190
gcg cag aac gca gac aag aag gcc gct gat atg ctt gct gaa tac gag 624Ala Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu195 200 205cgc aga aaa ggt att ctg gac acc cgg ttg tca gag ctg gaa aaa aat 672Arg Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn210 215 220ggc ggg gca gcc ctt gcc gtt ctt gat gca caa cag gcc cgt ctg ctc 720Gly Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu225 230 235ggg cag cag aca cgg aat gac agg gcc att tca gag gcc cgg aat aaa 768Gly Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys240 245 250 255ctc agt tca gtg acg gaa tcg crt aac acg gcc cgt aat gca tta acc 816Leu Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr260 265 270aga gct gaa caa cag ctg acg caa cag aaa aac acg cct gac ggc aaa 864Arg Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys275 280 285acg ata gtt tcc cct gaa aaa ttc ccg ggg cgt tca tca aca aat cat 912Thr Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His290 295 300tct att gtt gtg agc ggt gat ccg aga ttt gcc ggt acg ata aaa atc 960Ser Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile305 310 315aca acc agc gca gtc atc gat aac cgt gca aac ctg aat tat ctt ctg 1008Thr Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu320 325 330 335agc cat tcc ggt ctg gac tat aaa cgc aat att ctg aat gac cgg aat 1056Ser His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn340 345 350ccg gtg gtg aca gag gat gtg gaa ggt gac aag aaa att tat aat gct 1104Pro Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala355 360 365gaa gtt gct gaa tgg gat aag tta cgg caa aga ttg ctt gat gcc aga 1152Glu Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg370 375 380aat aaa atc acc tct gct gaa tct gcg gta aat tcg gcg aga aat aac 1200Asn Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn385 390 395ctc agt gcc aga aca aat gag caa aag cat gca aat gac gct ctt aat 1248Leu Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn400 405 410 415gcc ctg ttg aag gaa aaa gag aat ata cgt aac cag ctt tcc ggc atc 1296Ala Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile420 425 430aat cag aag ata gcg gaa gag aaa aga aaa cag gat gaa ctg aag gca 1344Asn Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala435 440 445acg aaa gac gca att aat ttc aca aca gag ttc ctg aaa tca gtt tca 1392Thr Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser450 455 460gaa aaa tat ggt gca aaa gct gag cag tta gcc aga gag atg gcc ggg 1440Glu Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly465 470 475cag gct aaa ggg aag aaa ata cgt aat gtt gaa gag gca tta aaa acg 1488Gln Ala Lys Gly Lys Lys Ile Arg asn Val Glu Glu ala Leu Lys Thr480 485 490 495tat gaa aag tac cgg gct gac att aac aaa aaa att aat gca aaa gat 1536
Tyr Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp500 505 510cgt gca gcg att gcc gca gcc ctt gag tct gtg aag ctg tct gat ata1584Arg Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile515 520 525tcg tct aat ctg aac aga ttc agt cgg gga ctg gga tat gca gga aaa1632Ser Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys530 535 540ttt aca agt ctt gct gac tgg atc act gag ttt ggt aag gct gtc cgg1680Phe Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg545 550 555aca gag aac tgg cgt cct ctt ttt gtt aaa aca gaa acc atc ata gca1728Thr Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala560 565 570 575ggc aat gcc gca acg gct ctt gtg gca ctg gtc ttc agt att ctt acc1776Gly Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr580 585 590gga agc gct tta ggc att atc ggg tat ggt tta ctg atg gct gtc acc1824Gly Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr595 600 605ggt gcg ctg att gat gaa tcg ctt gtg gaa aaa gcg aat aag ttc tgg1872Gly Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp610 615 620ggt att tat tcc acc tgt tac ttt ata atg taa1905Gly Ile Tyr Ser Thr Cys Tyr Phe Ile Met625 630<210>19<211>633<212>PRT<213>人工序列<400>19Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly Tyr1 5 10 15Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn Pro20 25 30Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser Phe35 40 45Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser Pro50 55 60Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr Lys65 70 75 80Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr Glu85 90 95Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp Glu100 105 110Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp Ile115 120 125Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr Gly130 135 140
Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr Glu145 150 155 160Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro Arg165 170 175Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp Ala180 185 190Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu Arg195 200 205Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn Gly210 215 220Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu Gly225 230 235 240Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys Leu245 250 255Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr Arg260 265 270Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys Thr275 280 285Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His Ser290 295 300Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile Thr305 310 315 320Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu Ser325 330 335His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn Pro340 345 350Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala Glu355 360 365Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg Asn370 375 380Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn Leu385 390 395 400Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn Ala405 410 415Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile Asn420 425 430Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala Thr435 440 445
Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser Glu450 455 460Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly Gln465 470 475 480Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr Tyr485 490 495Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp Arg500 505 510Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile Ser515 520 525Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys Phe530 535 540Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg Thr545 550 555 560Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala Gly565 570 575Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr Gly580 585 590Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr Gly595 600 605Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp Gly610 615 620Ile Tyr Ser Thr Cys Tyr Phe Ile Met625 630<210>20<211>1905<212>DNA<213>人工序列<220>
<221>CDS<222>(4)..(1902)<223>编码抗葡萄球菌多肽<400>20atg tct gac cct gta cgt att aca aat ccc ggt gca gaa tcg ctg ggg 48Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1 5 10 15tat gat tca gat ggc cat gaa att atg gcc gtt gat att tat gta aac 96Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn20 25 30cct cca cgt gtc gat gtc ttt cat ggt acc ccg cct gca tgg agt tcc 144Pro Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser35 40 45ttc ggg aac aaa acc atc tgg ggc gga aac gag tgg gtt gat gat tcc 192Phe Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser50 55 60
cca acc cga agt gat atc gaa aaa agg gac aag gaa atc aca gcg tac 240Pro Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr65 70 75aaa aac acg ctc agc gcg cag cag aaa gag aat gag aat aag cgt act 288Lys Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr80 85 90 95gaa gcc gga aaa cgc ctc tct gcg gcg att gct gca agg gaa aaa gat 336Glu Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp100 105 110gaa aac aca ctg aaa aca ctc cgt gcc gga aac gca gat gcc gct gat 384Glu Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp115 120 125att aca cga cag gag ttc aga ctc ctg cag gca gag ctg aga gaa tac 432Ile Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr130 135 140gga ttc cgt act gaa atc gcc gga tat gac gcc ctc cgg ctg cat aca 480Gly Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr145 150 155gag agc cgg atg ctg ttt gct gat gct gat tct ctt cgt ata tct ccc 528Glu Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro160 165 170 175cgg gag gcc agg tcg tta atc gaa cag gct gaa aaa cgg cag aag gat 576Arg Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp180 185 190gcg cag aac gca gac aag aag gcc gct gat atg ctt gct gaa tac gag 624Ala Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu195 200 205cgc aga aaa ggt att ctg gac acc cgg ttg tca gag ctg gaa aaa aat 672Arg Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn210 215 220ggc ggg gca gcc ctt gcc gtt ctt gat gca caa cag gcc cgt ctg ctc 720Gly Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu225 230 235ggg cag cag aca cgg aat gac agg gcc att tca gag gcc cgg aat aaa 768Gly Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys240 245 250 255ctc agt tca gtg acg gaa tcg ctt aac acg gcc cgt aat gca tta acc 816Leu Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr260 265 270aga gct gaa caa cag ctg acg caa cag aaa aac acg cct gac ggc aaa 864Arg Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys275 280 285acg ata gtt tcc cct gaa aaa ttc ccg ggg cgt tca tca aca aat cat 912Thr Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His290 295 300tct att gtt gtg agc ggt gat ccg aga ttt gcc ggt acg ata aaa atc 960Ser Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile305 310 315aca acc agc gca gtc atc gat aac cgt gca aac ctg aat tat ctt ctg1008Thr Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu320 325 330 335agc cat tcc ggt ctg gac tat aaa cgc aat att ctg aat gac cgg aat1056Ser His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn340 345 350ccg gtg gtg aca gag gat gtg gaa ggt gac aag aaa att tat aat gct1104Pro Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala355 360 365gaa gtt gct gaa tgg gat aag tta cgg caa aga ttg ctt gat gcc aga1152
Glu Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg370 375 380aat aaa atc acc tct gct gaa tct gcg gta aat tcg gcg aga aat aac 1200Asn Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn385 390 395ctc agt gcc aga aca aat gag caa aag cat gca aat gac gct ctt aat 1248Leu Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn400 405 410 415gcc ctg ttg aag gaa aaa gag aat ata cgt aac cag ctt tcc ggc atc 1296Ala Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile420 425 430aat cag aag ata gcg gaa gag aaa aga aaa cag gat gaa ctg aag gca 1344Asn Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala435 440 445acg aaa gac gca att aat ttc aca aca gag ttc ctg aaa tca gtt tca 1392Thr Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser450 455 460gaa aaa tat ggt gca aaa gct gag cag tta gcc aga gag atg gcc ggg 1440Glu Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly465 470 475cag gct aaa ggg aag aaa ata cgt aat gtt gaa gag gca tta aaa acg 1488Gln Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr480 485 490 495tat gaa aag tac cgg gct gac att aac aaa aaa att aat gca aaa gat 1536Tyr Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp500 505 510cgt gca gcg att gcc gca gcc ctt gag tct gtg aag ctg tct gat ata 1584Arg Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile515 520 525tcg tct aat ctg aac aga ttc agt cgg gga ctg gga tat gca gga aaa1632Ser Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys530 535 540ttt aca agt ctt gct gac tgg atc act gag ttt ggt aag gct gtc cgg 1680Phe Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg545 550 555aca gag aac tgg cgt cct ctt ttt gtt aaa aca gaa acc atc ata gca 1728Thr Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala560 565 570 575ggc aat gcc gca acg gct ctt gtg gca ctg gtc ttc agt att ctt acc 1776Gly Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr580 585 590gga agc gct tta ggc att atc ggg tat ggt tta ctg atg gct gtc acc 1824Gly Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr595 600 605ggt gcg ctg att gat gaa tcg ctt gtg gaa aaa gcg aat aag ttc tgg 1872Gly Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp610 615 620ggt att gat tcc gtt tgt gca tcc tat ttt taa 1905Gly Ile Asp Ser Val Cys Ala Ser Tyr Phe625 630<210>21<211>633<212>PRT<213>人工序列<400>21Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly Tyr1 5 10 15
Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn Pro20 25 30Pro Arg Val Asp Val Phe His Gly Thr Pro Pro Ala Trp Ser Ser Phe35 40 45Gly Asn Lys Thr Ile Trp Gly Gly Asn Glu Trp Val Asp Asp Ser Pro50 55 60Thr Arg Ser Asp Ile Glu Lys Arg Asp Lys Glu Ile Thr Ala Tyr Lys65 70 75 80Asn Thr Leu Ser Ala Gln Gln Lys Glu Asn Glu Asn Lys Arg Thr Glu85 90 95Ala Gly Lys Arg Leu Ser Ala Ala Ile Ala Ala Arg Glu Lys Asp Glu100 105 110Asn Thr Leu Lys Thr Leu Arg Ala Gly Asn Ala Asp Ala Ala Asp Ile115 120 125Thr Arg Gln Glu Phe Arg Leu Leu Gln Ala Glu Leu Arg Glu Tyr Gly130 135 140Phe Arg Thr Glu Ile Ala Gly Tyr Asp Ala Leu Arg Leu His Thr Glu145 150 155 160Ser Arg Met Leu Phe Ala Asp Ala Asp Ser Leu Arg Ile Ser Pro Arg165 170 175Glu Ala Arg Ser Leu Ile Glu Gln Ala Glu Lys Arg Gln Lys Asp Ala180 185 190Gln Asn Ala Asp Lys Lys Ala Ala Asp Met Leu Ala Glu Tyr Glu Arg195 200 205Arg Lys Gly Ile Leu Asp Thr Arg Leu Ser Glu Leu Glu Lys Asn Gly210 215 220Gly Ala Ala Leu Ala Val Leu Asp Ala Gln Gln Ala Arg Leu Leu Gly225 230 235 240Gln Gln Thr Arg Asn Asp Arg Ala Ile Ser Glu Ala Arg Asn Lys Leu245 250 255Ser Ser Val Thr Glu Ser Leu Asn Thr Ala Arg Asn Ala Leu Thr Arg260 265 270Ala Glu Gln Gln Leu Thr Gln Gln Lys Asn Thr Pro Asp Gly Lys Thr275 280 285Ile Val Ser Pro Glu Lys Phe Pro Gly Arg Ser Ser Thr Asn His Ser290 295 300Ile Val Val Ser Gly Asp Pro Arg Phe Ala Gly Thr Ile Lys Ile Thr305 310 315 320
Thr Ser Ala Val Ile Asp Asn Arg Ala Asn Leu Asn Tyr Leu Leu Ser325 330 335His Ser Gly Leu Asp Tyr Lys Arg Asn Ile Leu Asn Asp Arg Asn Pro340 345 350Val Val Thr Glu Asp Val Glu Gly Asp Lys Lys Ile Tyr Asn Ala Glu355 360 365Val Ala Glu Trp Asp Lys Leu Arg Gln Arg Leu Leu Asp Ala Arg Asn370 375 380Lys Ile Thr Ser Ala Glu Ser Ala Val Asn Ser Ala Arg Asn Asn Leu385 390 395 400Ser Ala Arg Thr Asn Glu Gln Lys His Ala Asn Asp Ala Leu Asn Ala405 410 415Leu Leu Lys Glu Lys Glu Asn Ile Arg Asn Gln Leu Ser Gly Ile Asn420 425 430Gln Lys Ile Ala Glu Glu Lys Arg Lys Gln Asp Glu Leu Lys Ala Thr435 440 445Lys Asp Ala Ile Asn Phe Thr Thr Glu Phe Leu Lys Ser Val Ser Glu450 455 460Lys Tyr Gly Ala Lys Ala Glu Gln Leu Ala Arg Glu Met Ala Gly Gln465 470 475 480Ala Lys Gly Lys Lys Ile Arg Asn Val Glu Glu Ala Leu Lys Thr Tyr485 490 495Glu Lys Tyr Arg Ala Asp Ile Asn Lys Lys Ile Asn Ala Lys Asp Arg500 505 510Ala Ala Ile Ala Ala Ala Leu Glu Ser Val Lys Leu Ser Asp Ile Ser515 520 525Ser Asn Leu Asn Arg Phe Ser Arg Gly Leu Gly Tyr Ala Gly Lys Phe530 535 540Thr Ser Leu Ala Asp Trp Ile Thr Glu Phe Gly Lys Ala Val Arg Thr545 550 555 560Glu Asn Trp Arg Pro Leu Phe Val Lys Thr Glu Thr Ile Ile Ala Gly565 570 575Asn Ala Ala Thr Ala Leu Val Ala Leu Val Phe Ser Ile Leu Thr Gly580 585 590Ser Ala Leu Gly Ile Ile Gly Tyr Gly Leu Leu Met Ala Val Thr Gly595 600 605Ala Leu Ile Asp Glu Ser Leu Val Glu Lys Ala Asn Lys Phe Trp Gly610 615 620
Ile Asp Ser Val Cys Ala Ser Tyr Phe625 630<210>22<211>1905<212>DNA<213>人工序列<220>
<221>CDS<222>(4)..(1902)<223>编码抗葡萄球菌多肽<400>22atg tat tcc acc tgt gat ttt ata atg tct gac cct gta cgt att aca 48Tyr Ser Thr Cys Asp Phe Ile Met Ser Asp Pro Val Arg Ile Thr1 5 10 15aat ccc ggt gca gaa tcg ctg ggg tat gat tca gat ggc cat gaa att 96Asn Pro Gly Ala Glu Ser Leu Gly Tyr Asp Ser Asp Gly His Glu Ile20 25 30atg gcc gtt gat att tat gta aac cct cca cgt gtc gat gtc ttt cat144Met Ala Val Asp Ile Tyr Val Asn Pro Pro Arg Val Asp Val Phe His35 40 45ggt acc ccg cct gca tgg agt tcc ttc ggg aac aaa acc atc tgg ggc192Gly Thr Pro Pro Ala Trp Ser Ser Phe Gly Asn Lys Thr Ile Trp Gly50 55 60gga aac gag tgg gtt gat gat tcc cca acc cga agt gat atc gaa aaa240Gly Asn Glu Trp Val Asp Asp Ser Pro Thr Arg Ser Asp Ile Glu Lys65 70 75agg gac aag gaa atc aca gcg tac aaa aac acg ctc agc gcg cag cag288Arg Asp Lys Glu Ile Thr Ala Tyr Lys Asn Thr Leu Ser Ala Gln Gln80 85 90 95aaa gag aat gag aat aag cgt act gaa gcc gga aaa cgc ctc tct gcg336Lys Glu Asn Glu Asn Lys Arg Thr Glu Ala Gly Lys Arg Leu Ser Ala100 105 110gcg att gct gca agg gaa aaa gat gaa aac aca ctg aaa aca ctc cgt384Ala Ile Ala Ala Arg Glu Lys Asp Glu Asn Thr Leu Lys Thr Leu Arg115 120 125gcc gga aac gca gat gcc gct gat att aca cga cag gag ttc aga ctc432Ala Gly Asn Ala Asp Ala Ala Asp Ile Thr Arg Gln Glu Phe Arg Leu130 135 140ctg cag gca gag ctg aga gaa tac gga ttc cgt act gaa atc gcc gga480Leu Gln Ala Glu Leu Arg Glu Tyr Gly Phe Arg Thr Glu Ile Ala Gly145 150 155tat gac gcc ctc cgg ctg cat aca gag agc cgg atg ctg ttt gct gat528Tyr Asp Ala Leu Arg Leu His Thr Glu Ser Arg Met Leu Phe Ala Asp160 165 170 175gct gat tct ctt cgt ata tct ccc cgg gag gcc agg tcg tta atc gaa576Ala Asp Ser Leu Arg Ile Ser Pro Arg Glu Ala Arg Ser Leu Ile Glu180 185 190cag gct gaa aaa cgg cag aag gat gcg cag aac gca gac aag aag gcc624Gln Ala Glu Lys Arg Gln Lys Asp Ala Gln Asn Ala Asp Lys Lys Ala195 200 205gct gat atg ctt gct gaa tac gag cgc aga aaa ggt att ctg gac acc672Ala Asp Met Leu Ala Glu Tyr Glu Arg Arg Lys Gly Ile Leu Asp Thr210 215 220cgg ttg tca gag ctg gaa aaa aat ggc ggg gca gcc ctt gcc gtt ctt720Arg Leu Ser Glu Leu Glu Lys Asn Gly Gly Ala Ala Leu Ala Val Leu225 230 235gat gca caa cag gcc cgt ctg ctc ggg cag cag aca cgg aat gac agg768
Asp Ala Gln Gln Ala arg Leu Leu Gly Gln Gln Thr Arg Asn Asp Arg240 245 250 255gcc att tca gag gcc cgg aat aaa ctc agt tca gtg acg gaa tcg ctt 816Ala Ile Ser Glu Ala arg Asn Lys Leu Ser Ser Val Thr Glu Ser Leu260 265 270aac acg gcc cgt aat gca tta acc aga gct gaa caa cag ctg acg caa 864Asn Thr Ala Arg Asn ala Leu Thr Arg Ala Glu Gln Gln Leu Thr Gln275 280 285cag aaa aac acg cct gac ggc aaa acg ata gtt tcc cct gaa aaa ttc 912Gln Lys Asn Thr Pro Asp Gly Lys Thr Ile Val Ser Pro Glu Lys Phe290 295 300ccg ggg cgt tca tca aca aat cat tct att gtt gtg agc ggt gat ccg 960Pro Gly Arg Ser Ser Thr Asn His Ser Ile Val Val Ser Gly Asp Pro305 310 315aga ttt gcc ggt acg ata aaa atc aca acc agc gca gtc atc gat aac 1008Arg Phe Ala Gly Thr Ile Lys Ile Thr Thr Ser Ala Val Ile Asp Asn320 325 330 335cgt gca aac ctg aat tat ctt ctg agc cat tcc ggt ctg gac tat aaa 1056Arg Ala Asn Leu Asn Tyr Leu Leu Ser His Ser Gly Leu Asp Tyr Lys340 345 350cgc aat att ctg aat gac cgg aat ccg gtg gtg aca gag gat gtg gaa 1104Arg Asn Ile Leu Asn Asp Arg Asn Pro Val Val Thr Glu Asp Val Glu355 360 365ggt gac aag aaa att tat aat gct gaa gtt gct gaa tgg gat aag tta 1152Gly Asp Lys Lys Ile Tyr Asn Ala Glu Val Ala Glu Trp Asp Lys Leu370 375 380cgg caa aga ttg ctt gat gcc aga aat aaa atc acc tct gct gaa tct 1200Arg Gln Arg Leu Leu Asp Ala Arg Asn Lys Ile Thr Ser Ala Glu Ser385 390 395gcg gta aat tcg gcg aga aat aac ctc agt gcc aga aca aat gag caa 1248Ala Val Asn Ser Ala Arg Asn Asn Leu Ser Ala Arg Thr Asn Glu Gln400 405 410 415aag cat gca aat gac gct ctt aat gcc ctg ttg aag gaa aaa gag aat 1296Lys His Ala Asn Asp Ala Leu Asn Ala Leu Leu Lys Glu Lys Glu Asn420 425 430ata cgt aac cag ctt tcc ggc atc aat cag aag ata gcg gaa gag aaa 1344Ile Arg Asn Gln Leu Ser Gly Ile Asn Gln Lys Ile Ala Glu Glu Lys435 440 445aga aaa cag gat gaa ctg aag gca acg aaa gac gca att aat ttc aca 1392Arg Lys Gln Asp Glu Leu Lys Ala Thr Lys Asp Ala Ile Asn Phe Thr450 455 460aca gag ttc ctg aaa tca gtt tca gaa aaa tat ggt gca aaa gct gag 1440Thr Glu Phe Leu Lys Ser Val Ser Glu Lys Tyr Gly Ala Lys Ala Glu465 470 475cag tta gcc aga gag atg gcc ggg cag gct aaa ggg aag aaa ata cgt 1488Gln Leu Ala Arg Glu Met Ala Gly Gln Ala Lys Gly Lys Lys Ile Arg480 485 490 495aat gtt gaa gag gca tta aaa acg tat gaa aag tac cgg gct gac att 1536Asn Val Glu Glu Ala Leu Lys Thr Tyr Glu Lys Tyr Arg Ala Asp Ile500 505 510aac aaa aaa att aat gca aaa gat cgt gca gcg att gcc gca gcc ctt 1584Asn Lys Lys Ile Asn Ala Lys Asp Arg Ala Ala Ile Ala Ala Ala Leu515 520525gag tct gtg aag ctg tct gat ata tcg tct aat ctg aac aga ttc agt 1632Glu Ser Val Lys Leu Ser Asp Ile Ser Ser Asn Leu Asn Arg Phe Ser530 535 540cgg gga ctg gga tat gca gga aaa ttt aca agt ctt gct gac tgg atc 1680Arg Gly Leu Gly Tyr Ala Gly Lys Phe Thr Ser Leu Ala Asp Trp Ile
545 550 555act gag ttt ggt aag gct gtc cgg aca gag aac tgg cgt cct ctt ttt 1728Thr Glu Phe Gly Lys Ala Val Arg Thr Glu Asn Trp Arg Pro Leu Phe560 565 570 575gtt aaa aca gaa acc atc ata gca ggc aat gcc gca acg gct ctt gtg 1776Val Lys Thr Glu Thr Ile Ile Ala Gly Asn Ala Ala Thr Ala Leu Val580 585 590gca ctg gtc ttc agt att ctt acc gga agc gct tta ggc att atc ggg 1824Ala Leu Val Phe Ser Ile Leu Thr Gly Ser Ala Leu Gly Ile Ile Gly595 600 605tat ggt tta ctg atg gct gtc acc ggt gcg ctg att gat gaa tcg ctt 1872Tyr Gly Leu Leu Met Ala Val Thr Gly Ala Leu Ile Asp Glu Ser Leu610 615 620gtg gaa aaa gcg aat aag ttc tgg ggt att taa 1905Val Glu Lys Ala Asn Lys Phe Trp Gly Ile625 630<210>23<211>633<212>PRT<213>人工序列<400>23Tyr Ser Thr Cys Asp Phe Ile Met Ser Asp Pro Val Arg Ile Thr Asn1 5 10 15Pro Gly Ala Glu Ser Leu Gly Tyr Asp Ser Asp Gly His Glu Ile Met20 25 30Ala Val Asp Ile Tyr Val Asn Pro Pro Arg Val Asp Val Phe His Gly35 40 45Thr Pro Pro Ala Trp Ser Ser Phe Gly Asn Lys Thr Ile Trp Gly Gly50 55 60Asn Glu Trp Val Asp Asp Ser Pro Thr Arg Ser Asp Ile Glu Lys Arg65 70 75 80Asp Lys Glu Ile Thr Ala Tyr Lys Asn Thr Leu Ser Ala Gln Gln Lys85 90 95Glu Asn Glu Asn Lys Arg Thr Glu Ala Gly Lys Arg Leu Ser Ala Ala100 105 110Ile Ala Ala Arg Glu Lys Asp Glu Asn Thr Leu Lys Thr Leu Arg Ala115 120 125Gly Asn Ala Asp Ala Ala Asp Ile Thr Arg Gln Glu Phe Arg Leu Leu130 135 140Gln Ala Glu Leu Arg Glu Tyr Gly Phe Arg Thr Glu Ile Ala Gly Tyr145 150 155 160Asp Ala Leu Arg Leu His Thr Glu Ser Arg Met Leu Phe Ala Asp Ala165 170 175Asp Ser Leu Arg Ile Ser Pro Arg Glu Ala Arg Ser Leu Ile Glu Gln180 185 190
Ala Glu Lys Arg Gln Lys Asp Ala Gln Asn Ala Asp Lys Lys Ala Ala195 200 205Asp Met Leu Ala Glu Tyr Glu Arg Arg Lys Gly Ile Leu Asp Thr Arg210 215 220Leu Ser Glu Leu Glu Lys Asn Gly Gly Ala Ala Leu Ala Val Leu Asp225 230 235 240Ala Gln Gln Ala Arg Leu Leu Gly Gln Gln Thr Arg Asn Asp Arg Ala245 250 255Ile Ser Glu Ala Arg Asn Lys Leu Ser Ser Val Thr Glu Ser Leu Asn260 265 270Thr Ala Arg Asn Ala Leu Thr Arg Ala Glu Gln Gln Leu Thr Gln Gln275 280 285Lys Asn Thr Pro Asp Gly Lys Thr Ile Val Ser Pro Glu Lys Phe Pro290 295 300Gly Arg Ser Ser Thr Asn His Ser Ile Val Val Ser Gly Asp Pro Arg305 310 315 320Phe Ala Gly Thr Ile Lys Ile Thr Thr Ser Ala Val Ile Asp Asn Arg325 330 335Ala Asn Leu Asn Tyr Leu Leu Ser His Ser Gly Leu Asp Tyr Lys Arg340 345 350Asn Ile Leu Asn Asp Arg Asn Pro Val Val Thr Glu Asp Val Glu Gly355 360 365Asp Lys Lys Ile Tyr Asn Ala Glu Val Ala Glu Trp Asp Lys Leu Arg370 375 380Gln Arg Leu Leu Asp Ala Arg Asn Lys Ile Thr Ser Ala Glu Ser Ala385 390 395 400Val Asn Ser Ala Arg Asn Asn Leu Ser Ala Arg Thr Asn Glu Gln Lys405 410 415His Ala Asn Asp Ala Leu Asn Ala Leu Leu Lys Glu Lys Glu Asn Ile420 425 430Arg Asn Gln Leu Ser Gly Ile Asn Gln Lys Ile Ala Glu Glu Lys Arg435 440 445Lys Gln Asp Glu Leu Lys Ala Thr Lys Asp Ala Ile Asn Phe Thr Thr450 455 460Glu Phe Leu Lys Ser Val Ser Glu Lys Tyr Gly Ala Lys Ala Glu Gln465 470 475 480Leu Ala Arg Glu Met Ala Gly Gln Ala Lys Gly Lys Lys Ile Arg Asn485 490 495
Val Glu Glu Ala Leu Lys Thr Tyr Glu Lys Tyr Arg Ala Asp Ile Asn500 505 510Lys Lys Ile Asn Ala Lys Asp Arg Ala Ala Ile Ala Ala Ala Leu Glu515 520 525Ser Val Lys Leu Ser Asp Ile Ser Ser Asn Leu Asn Arg Phe Ser Arg530 535 540Gly Leu Gly Tyr Ala Gly Lys Phe Thr Ser Leu Ala Asp Trp Ile Thr545 550 555 560Glu Phe Gly Lys Ala Val Arg Thr Glu Asn Trp Arg Pro Leu Phe Val565 570 575Lys Thr Glu Thr Ile Ile Ala Gly Asn Ala Ala Thr Ala Leu Val Ala580 585 590Leu Val Phe Ser Ile Leu Thr Gly Ser Ala Leu Gly Ile Ile Gly Tyr595 600 605Gly Leu Leu Met Ala Val Thr Gly Ala Leu Ile Asp Glu Ser Leu Val610 615 620Glu Lys Ala Asn Lys Phe Trp Gly Ile625 630<210>24<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>24gcgaataagt tctggggtat ttattccacc tgtgatttta taatgtaaat aaaatataag60acaggc 66<210>25<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>25gcctgtctta tattttattt acattataaa atcacaggtg gaataaatac cccagaactt60attcgc 66<210>26<211>69<212>DNA<213>人工序列<220>
<221>misc_feature
<222>(1)..(69)<223>引物<400>26gcgaataagt tctggggtat tggagttaac gcatgttctt ccctgtttta aataaaatat60aagacaggc69<210>27<211>69<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(69)<223>引物<400>27gcctgtctta tattttattt aaaacaggga agaacatgcg ttaactccaa taccccagaa60cttattcgc69<210>28<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>28gcgaataagt tctggggtat ttatataaac tgtgattttc ttctgtaaat aaaatataag60acaggc 66<210>29<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>29gcctgtctta tattttattt acagaagaaa atcacagttt atataaatac cccagaactt60attcgc 66<210>30<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>30gcgaataagt tctggggtat ttattccacc tgttacttta taatgtaaat aaaatataag60acaggc 66
<210>31<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>31gcctgtctta tattttattt acattataaa gtaacaggtg gaataaatac cccagaactt60attcgc 66<210>32<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>32gcgaataagt tctggggtat tgattccgtt tgtgcatcct atttttaaat aaaatataag60acaggc 66<210>33<211>66<212>DNA<213>人工序列<220>
<221>misc_feature<222>(1)..(66)<223>引物<400>33gcctgtctta tattttattt aaaaatagga tgcacaaacg gaatcaatac cccagaactt60attcgc 6权利要求
1.编码重组抗菌多肽的核苷酸，其特征在于它含有编码可形成离子通道的大肠菌素或其水性孔道结构域的基因，以及编码葡萄球菌信号传导多肽的基因。
2.根据权利要求1所述的核苷酸，其中所述编码葡萄球菌信号传导多肽的基因连接于所述编码可形成离子通道的大肠菌素或其水性孔道结构域的基因的C末端。
3.根据权利要求1所述的核苷酸，其中所述编码葡萄球菌信号传导多肽的基因连接于所述编码可形成离子通道的大肠菌素或其水性孔道结构域的基因的N末端。
4.根据权利要求2或3所述的核苷酸，其中所述可形成离子通道的大肠菌素选自大肠菌素E1、Ia、Ib、A、B和N。
5.根据权利要求4所述的核苷酸，其中所述可形成离子通道的大肠菌素为大肠菌素Ia。
6.根据权利要求5所述的核苷酸，其中含有SEQ ID NO.12的核苷酸序列。
7.根据权利要求5所述的核苷酸，其中含有SEQ ID NO.14的核苷酸序列。
8.根据权利要求5所述的核苷酸，其中含有SEQ ID NO.16的核苷酸序列。
9.根据权利要求5所述的核苷酸，其中含有SEQ ID NO.18的核苷酸序列。
10.根据权利要求5所述的核苷酸，其中含有SEQ ID NO.20的核苷酸序列。
11.根据权利要求5所述的核苷酸，其中含有SEQ ID NO.22的核苷酸序列。
12.一种重组质粒，其特征在于其含有编码可形成离子通道的大肠菌素或其水性孔道结构域的基因，以及编码葡萄球菌信号传导多肽的基因。
13.根据权利要求12所述的重组质粒，其中含有SEQ ID NO.12的核苷酸序列。
14.根据权利要求12所述的重组质粒，其中含有SEQ ID NO.14的核苷酸序列。
15.根据权利要求12所述的重组质粒，其中含有SEQ ID NO.16的核苷酸序列。
16.根据权利要求12所述的重组质粒，其中含有SEQ ID NO.18的核苷酸序列。
17.根据权利要求12所述的重组质粒，其中含有SEQ ID NO.20的核苷酸序列。
18.根据权利要求12所述的重组质粒，其中含有SEQ ID NO.22的核苷酸序列。
19.一种重组抗菌多肽，其含有可形成离子通道的大肠菌素或其水性孔道结构域，以及葡萄球菌信号传导多肽。
20.根据权利要求19所述的抗菌多肽，其中包含SEQ ID NO.13的氨基酸序列。
21.根据权利要求19所述的抗菌多肽，其中包含SEQ ID NO.15的氨基酸序列。
22.根据权利要求19所述的抗菌多肽，其中包含SEQ ID NO.17的氨基酸序列。
23.根据权利要求19所述的抗菌多肽，其中包含SEQ ID NO.19的氨基酸序列。
24.根据权利要求19所述的抗菌多肽，其中包含SEQ ID NO.21的氨基酸序列。
25.根据权利要求19所述的抗菌多肽，其中包含SEQ ID NO.23的氨基酸序列。
26.含有权利要求20～25任何一项所述的重组抗菌多肽的药物组合物。
27.重组抗菌多肽的制备方法，包括以下步骤将编码葡萄球菌信号传导多肽的基因与编码可形成离子通道的大肠菌素或其水性孔道结构域的基因可操作地连接获得编码重组抗菌多肽的基因；将获得的基因导入到表达系统中进行表达；分离表达的多肽而获得本发明的抗菌多肽。
全文摘要
本发明公开了一种新型抗菌多肽及其制备方法，该重组抗菌多肽，其含有可形成离子通道的大肠菌素或其水性孔道结构域，以及葡萄球菌信号传导多肽。本发明还公开了编码该重组抗菌多肽的核苷酸序列，以及含有所述核苷酸序列的重组质粒。本发明的新型抗菌多肽相较于传统抗菌素的优点在于，其不会诱导细菌产生传统的耐药性。细菌较难以通过突变来改变其细胞膜的磷脂双分子层的结构，而本发明的重组多肽通过直接在靶细菌的胞膜上形成离子通道而达到杀菌目的。
文档编号C12P21/00GK1532284SQ03120400
公开日2004年9月29日 申请日期2003年3月19日 优先权日2002年11月12日
发明者丘小庆 申请人:成都阳辉生物科技有限责任公司