專利名稱：一種新型抗菌多肽及其制備方法
技術(shù)領(lǐng)域：
本發(fā)明涉及一種重組的抗葡萄球菌多肽，編碼該多肽的核苷酸和氨基酸序列。
本發(fā)明還涉及重組抗葡萄球菌多肽的制備方法。
背景技術(shù)：
細(xì)菌感染是人類生命和健康的主要威脅，自磺胺和青霉素問世以來，人類陸續(xù)發(fā)明的抗菌素主要以通過抑制細(xì)菌胞壁合成、抑制或干擾細(xì)菌的核酸和蛋白質(zhì)的代謝與合成途徑來達(dá)到抗菌目的。然而這些抗菌途徑容易誘使細(xì)菌發(fā)生突變而產(chǎn)生抗菌素耐藥性。因此人們一直在致力于開發(fā)新型的抗菌素。在細(xì)菌胞膜上直接形成離子通道而致細(xì)菌死亡是比較有前途的抗菌素開發(fā)方向之一。自然界中，為數(shù)不少的細(xì)菌毒素就是以這種機(jī)制來殺死細(xì)菌的，其模式標(biāo)本就是大腸桿菌(Escherichia coli)分泌的一種毒素蛋白——大腸菌素(colicin)，1952年Jacob發(fā)現(xiàn)其能特異地殺死其它株系的大腸桿菌和相關(guān)株系的某些桿菌，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等發(fā)現(xiàn)了可形成離子通道的大腸菌素可以在人工脂質(zhì)雙分子膜上形成電壓依賴性離子通道，從而在根本上揭示了這一類細(xì)菌毒素的抗菌機(jī)制(Schein et al，.Colicin K acts by forming voltagedependent channels in phospholipid bilayer membranes.Nature 276159-163(1973))。1996年Qiu和Finkelstein等揭示了大腸菌素Ia在人工脂質(zhì)雙分子膜上形成的離子通道開放和關(guān)閉時(shí)的跨膜立體結(jié)構(gòu)(Qiu et al.，Major transmembrane movementassociated with colicin，Ia channel gating.J.Gen.Physiology.107313-328(1996))，為在分子水平上設(shè)計(jì)和制備新型的抗菌素奠定了理論基礎(chǔ)。
近二十年，人們逐漸發(fā)現(xiàn)細(xì)菌也具有隨外界環(huán)境變化而分泌的信號(hào)傳導(dǎo)多肽，制備信號(hào)傳導(dǎo)多肽的胞膜受體及胞內(nèi)調(diào)節(jié)蛋白的基因往往偶聯(lián)在一起，協(xié)調(diào)地控制著細(xì)菌的生長和分裂，但該方向的研究一直進(jìn)展緩慢，比如金黃葡萄球菌的信號(hào)傳導(dǎo)多肽AgrD，一直到1995年才最后搞清楚是一個(gè)八肽(Ji et al.，Celldensity control of staphylococcal virulence mediated by an octapeptide pheromone.Proc.Natl.Acad.Sci.USA 9212055-12059(1995))。1999年發(fā)現(xiàn)金黃色葡萄球菌對數(shù)生長期的早期或中晚期加入人工合成的這種八肽可以刺激或抑制金黃色葡萄球菌的生長(Mayville et al.，Structure activity analysis of synthetic autoinducingthiolactone peptides from staphylococcus aureus resoonsible for virulence.PNAS，961218-1223(1999))。
如上所述，大腸菌素是一種理想的離子通道抗菌素，缺點(diǎn)是只能作用于大腸桿菌等格蘭氏陰性桿菌，如果能利用致病菌特有的信號(hào)傳導(dǎo)肽作為誘導(dǎo)物，將大腸菌素或其水性孔道結(jié)構(gòu)或誘導(dǎo)至特定的細(xì)菌胞膜附近形成離子通道來殺傷該致病菌，應(yīng)該是一種理想的抗菌素開發(fā)方向。

發(fā)明內(nèi)容
本發(fā)明的一個(gè)目的在于提供一種新型的重組抗菌多肽，其能避免誘發(fā)細(xì)菌的本發(fā)明的另一目的在于提供編碼該重組抗菌多肽的核苷酸序列。
本發(fā)明的再一目的在于提供一重組質(zhì)粒，其含有編碼本發(fā)明重組抗菌多肽的核苷酸序列。
本發(fā)明的再一目的在于提供一氨基酸序列，其編碼本發(fā)明的重組抗菌多肽。
本發(fā)明的再一目的在于提供含有本發(fā)明重組抗菌多肽的藥物組合物。
本發(fā)明的再一目的在于提供重組抗菌多肽的制備方法。
根據(jù)本發(fā)明的一方面，將編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因與大腸菌素基因連接，從而獲得編碼重組抗葡萄球菌多肽的基因。
在本發(fā)明的優(yōu)選實(shí)施方案中，分別采用編碼金黃色葡萄球菌和表皮葡萄球菌的信號(hào)傳導(dǎo)多肽的基因作為抗菌多肽的信息素基因，其中編碼金黃色葡萄信號(hào)傳導(dǎo)多肽的基因選自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)；編碼表皮葡萄球菌信號(hào)傳導(dǎo)多肽的基因?yàn)榫哂蠸EQ ID NO.9核苷酸序列的基因。大腸菌素可選自能形成離子通道的大腸菌素El、Ia、Ib、A、B和N或其水性孔道結(jié)構(gòu)域，在本發(fā)明的一個(gè)優(yōu)選實(shí)施例中，將上述信號(hào)傳導(dǎo)多肽基因分別與大腸菌素Ia(SEQ ID NO.11)的羧基端或氨基端連接而形成如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。這些核苷酸序列編碼如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，這些氨基酸序列所編碼的多肽即分別對應(yīng)為本發(fā)明的抗菌多肽Ph-SA1，Ph-SA2，Ph-SA3，Ph-SA4，Ph-SE，和Ph-SAR1。
在構(gòu)建的重組抗菌多肽中，可與靶細(xì)菌胞膜受體結(jié)合的信號(hào)傳導(dǎo)多肽作為誘導(dǎo)物誘導(dǎo)大腸菌素通道結(jié)構(gòu)域穿過細(xì)菌外膜與膜間隙到達(dá)靶細(xì)胞膜附近，然后大腸菌素通道結(jié)構(gòu)域中的水性孔道結(jié)構(gòu)域在靶細(xì)菌胞膜上形成離子通道，使靶細(xì)菌胞內(nèi)內(nèi)容物泄漏而致靶細(xì)菌死亡，從而達(dá)到殺菌的目的。
本發(fā)明中編碼金黃色葡萄球菌信號(hào)傳導(dǎo)多肽的核苷酸序列SEQ ID NO.1所編碼氨基酸序列(SEQ ID NO2)與Genebank U85097號(hào)序列所表達(dá)的氨基酸序列相同；編碼金黃色葡萄球菌信號(hào)傳導(dǎo)多肽的核苷酸序列SEQ ID NO.3所編碼氨基酸序列(SEQ ID NO4)與Genebank AF001782號(hào)序列所表達(dá)的氨基酸序列相同；編碼金黃色葡萄球菌信號(hào)傳導(dǎo)多肽的核苷酸序列SEQ ID NO.5所編碼氨基酸序列(SEQ ID NO6)與Genebank AF001783號(hào)序列所表達(dá)的氨基酸序列相同；編碼金黃色葡萄球菌信號(hào)傳導(dǎo)多肽的核苷酸序列SEQ ID NO.7所編碼氨基酸序列(SEQ ID NO8)與Genebank AF288215號(hào)序列所表達(dá)的氨基酸序列相同；編碼表皮葡萄球菌信號(hào)傳導(dǎo)多肽的核苷酸序列SEQ ID NO.9所編碼氨基酸序列(SEQ ID NO10)與Genebank Z49220號(hào)序列所表達(dá)的氨基酸序列相同。
根據(jù)本發(fā)明的另一方面，提供如

圖1～圖6所示的包含本發(fā)明核苷酸的質(zhì)粒載體，該質(zhì)粒載體是將如上所述的編碼葡萄球菌信號(hào)傳導(dǎo)多肽的核苷酸序列經(jīng)雙鏈寡聚核苷酸點(diǎn)突變技術(shù)插入大腸菌素Ia基因的第626位氨基酸或第1位氨基酸上而形成本發(fā)明的重組質(zhì)粒。
本發(fā)明中，構(gòu)建質(zhì)粒載體的原始質(zhì)粒pSELECTTM-1來自于Promega公司，其中裝載了大腸菌素Ia和immunity蛋白基因，針對金黃色葡萄球菌信號(hào)傳導(dǎo)基因設(shè)計(jì)了四對引物，其序列分別如SEQ ID NO.24～SEQ ID NO32所示；針對表皮葡萄球信號(hào)傳導(dǎo)基因設(shè)計(jì)了一對引物，其序列如SEQ ID NO33和SEQ IDNO34所示。利用雙鏈寡聚核苷酸點(diǎn)突變技術(shù)，按照Strategene公司藥箱操作獲得重組質(zhì)粒，再將獲得的重組質(zhì)粒轉(zhuǎn)染入大腸桿菌TG1工程菌而獲得可產(chǎn)生重組抗菌多肽的工程菌細(xì)胞。
根據(jù)本發(fā)明的再一方面，提供含有本發(fā)明重組抗菌多肽的藥物組合物，可以通過將本發(fā)明多肽添加藥學(xué)上可接受的載體或賦形劑或可選的其它成分而制成適于臨床使用的藥物組合物。
根據(jù)本發(fā)明的又一方面，提供本發(fā)明抗菌多肽的制備方法，將編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因可操作地與大腸菌素基因連接獲得編碼重組抗菌多肽的基因，將獲得的基因?qū)氲奖磉_(dá)系統(tǒng)中進(jìn)行表達(dá)，分離表達(dá)的多肽而獲得本發(fā)明的抗菌多肽。
本發(fā)明的抗菌多肽相較于傳統(tǒng)抗菌素的優(yōu)點(diǎn)在于，其不會(huì)誘導(dǎo)細(xì)菌產(chǎn)生傳統(tǒng)的耐藥性。眾所周知，細(xì)菌可以通過突變產(chǎn)生β-內(nèi)酰胺酶、減少攝入、改變藥物作用位點(diǎn)等方式來改變其細(xì)胞壁結(jié)構(gòu)，改變其蛋白質(zhì)和核酸的代謝等對傳統(tǒng)抗菌素產(chǎn)生耐藥性。但是細(xì)菌卻較難以通過突變來改變其細(xì)胞膜的磷脂雙分子層的結(jié)構(gòu)，而這恰好是本發(fā)明的獨(dú)到之處，本發(fā)明的重組多肽通過直接在靶細(xì)菌的胞膜上形成離子通道而達(dá)到殺菌目的。
附圖的簡要說明圖1示出含有金黃色葡萄球菌信號(hào)傳導(dǎo)多肽Agr D I和大腸菌素Ia的質(zhì)粒pCHCSA1的結(jié)構(gòu)；圖2示出含有金黃色葡萄球菌信號(hào)傳導(dǎo)多肽Agr D II和大腸菌素Ia的質(zhì)粒pCHCSA2的結(jié)構(gòu)；圖3示出含有金黃色葡萄球菌信號(hào)傳導(dǎo)多肽Agr D III和大腸菌素Ia的質(zhì)粒pCHCSA3的結(jié)構(gòu)；圖4示出含有金黃色葡萄球菌信號(hào)傳導(dǎo)多肽Agr D IV和大腸菌素Ia的質(zhì)粒pCHCSA4的結(jié)構(gòu)；圖5示出含有表皮葡萄球菌信號(hào)傳導(dǎo)多肽和大腸菌素Ia的質(zhì)粒pCHCSE的結(jié)構(gòu)；圖6示出含有金黃色葡萄球菌信號(hào)傳導(dǎo)多肽AgrD I和大腸菌素Ia的質(zhì)粒結(jié)構(gòu)，其中Agr D I連接在大腸菌素Ia的第1位氨基酸上；圖7示出包括634個(gè)氨基酸殘基的抗菌多肽的結(jié)構(gòu)；圖8為本發(fā)明抗菌多肽(Ph-SA1)對青霉素敏感金葡菌的抑制實(shí)驗(yàn)結(jié)果，表明青霉素敏感金葡菌的生長只能被青霉素和Ph-SA1抑制。圖中Control，對照，PEN，青霉素，COL，野生型大腸菌素，TG1，無質(zhì)粒的TG1工程菌蛋白，F(xiàn)usionProtein，大腸菌素Ia羧基端連接了其它八肽的重組蛋白，PMC即Ph-SA，抗金葡菌多肽。各實(shí)驗(yàn)組的藥物用量均為100ng/ml；圖9為抗金葡菌多肽(Ph-SA1)對耐甲氧西林金黃色葡萄球菌(ATCC BAA-42)殺菌效果的透射電鏡觀察(放大25,000倍)。Control，對照組，正常形態(tài)菌；Wildtype colicin Ia，野生型大腸菌素Ia處理(50μg/ml)，處理兩小時(shí)后，菌形態(tài)仍然正常；AgrD，金葡菌信號(hào)多肽處理(10μg/ml)，處理兩小時(shí)后，細(xì)菌形態(tài)仍然正常，上端一個(gè)菌體正在分裂；Oxacillin，苯唑西林處理(500μg/ml)，大劑量苯唑西林處理2小時(shí)后，菌胞壁呈溶解形態(tài)；Ph-SA1，抗金葡多肽處理(50μg/ml)，抗金葡多肽處理半小時(shí)后，菌破裂，內(nèi)容物泄漏，而胞壁結(jié)構(gòu)完整；結(jié)論單純使用信號(hào)傳導(dǎo)多肽或野生型大腸菌素處理都不能殺死MRSA，抗金葡多肽卻能殺滅MRSA且殺菌機(jī)制與苯唑西林完全不同；圖10為抗金葡多肽對MRSA(BAA42)感染小鼠體內(nèi)保護(hù)試驗(yàn)結(jié)果之生存曲線；圖11為本發(fā)明抗菌多肽(Ph-SA1和Ph-SE)對表皮葡萄球菌的抑制作用，表明表皮葡萄球菌的生長可被抗金葡多肽部分抑制，可被抗表葡多肽有效的抑制。圖中Control，對照，PEN，青霉素，Ph-SA1，抗金葡多肽，Ph-SE，抗表葡多肽。藥物濃度2μg/ml。
圖12為各種抗菌多肽對耐甲氧西林金葡菌的生長抑制作用，表明耐甲氧西林金葡菌的生長幾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；圖中Control，對照，Oxacillin，苯唑西林，Ph-SA1，Ph-SA2，Ph-SA3，Ph-SA4，分別連接了四種AgrD的抗金葡多肽，Ph-SE，抗表葡多肽，Ph-SAR1，AgrD1信息素連接在大腸菌素Ia氨基端的抗金葡多肽，藥物濃度5μg/ml。
發(fā)明的
具體實(shí)施例方式
下面結(jié)合附圖，通過對本發(fā)明較佳實(shí)施例的描述詳細(xì)說明本發(fā)明。
實(shí)施例1表達(dá)抗菌多肽的質(zhì)粒的構(gòu)建和重組抗菌多肽制備原始質(zhì)粒為裝載了大腸菌素Ia和immunity蛋白基因的pSELECTTM-1商用質(zhì)粒(質(zhì)粒大小8.3kb，Promega公司)(UCSF，P.Gosh贈(zèng)與)。經(jīng)雙鏈寡聚核苷酸點(diǎn)突變技術(shù)(QuickChangeTMKit，Strategeue公司)將編碼葡萄球菌信號(hào)多肽基因插入到大腸菌素Ia基因的I626位點(diǎn)上，制備了抗葡萄球菌工程多肽的一系列突變質(zhì)粒，pCHCSA1、pCHCSA2、pCHCSA3、pCHCSA4和pCHCSE以及Agr DI連接在大腸菌素Ia的第1位氨基酸上的質(zhì)粒(如圖1～圖7所示)。突變質(zhì)粒轉(zhuǎn)染入E.coli TG1工程菌(AECOM，K.Jakes贈(zèng)與)里制備多肽。
突變程序按Strategene QuikChange Site-Directed Mutagenesis Kit(Catalog#200518)藥箱手冊進(jìn)行1.準(zhǔn)備點(diǎn)突變反應(yīng)物5μl 10X buffer2μl(10ng)野生型大腸菌素質(zhì)粒1.25μl(125ng)設(shè)計(jì)的5’-3’寡聚核苷酸引物(見所列引物序列)1.25μl(125ng)設(shè)計(jì)的3’-5’寡聚核苷酸引物(見所列引物序列)1μl dNTP雙蒸水50μl1μl pfu(除質(zhì)粒、引物和雙蒸水外，均為藥箱所備試劑)2.進(jìn)行PCR擴(kuò)增，擴(kuò)增條件變性95℃，35秒，退火53℃，70秒，延伸68℃，17分共20個(gè)循環(huán)；3.加入Dpn1內(nèi)切酶1μl消化母體DNA鏈后(37℃，1小時(shí))，取1μl反應(yīng)物與XL1-Blue感受態(tài)細(xì)胞50μl冰孵30分鐘，行熱沖擊42℃，45秒，再置入冰中2分鐘；4.加入NZY培基0.5ml后220rpm，37℃搖菌1小時(shí)后，取50-100μl反應(yīng)物鋪板(LB培基加1％瓊脂加50μg/ml氨芐青霉素，37C過夜)；5.18小時(shí)后挑菌，循Qiagene，Gibco等公司的各種商用提取質(zhì)粒藥箱提取質(zhì)粒均可，測序確定突變成功。
6.將質(zhì)粒50ng與制備的TG1工程菌感受肽細(xì)胞50μl冰孵30分，42℃，90秒熱沖擊，取50-100μl反應(yīng)物加入LB培基0.5ml，220rpm，37℃搖菌1小時(shí)后鋪板(LB培基加1％瓊脂加50μg/ml氨芐青霉素)37℃孵育，18小時(shí)后挑取菌落。
7.大量增菌，8-16升FB培基，250rpm，37℃，6-8小時(shí)；離心沉淀菌體，4℃，6000g，20分鐘，取4℃、50mM硼酸緩沖液(2mM EDTA+2mM DTT)50-80ml懸浮菌體，加0.2M PMSF 250微升后超聲破碎(4℃，400W，2分鐘)，高速離心破碎菌體(4℃，75000g，1.5小時(shí))，取上清加入硫酸鏈霉素500萬單位沉淀DNA，高速離心(4℃，30000g，10分鐘)后取上清裝入分子量15000的透析袋于4℃，50mM硼酸緩沖液4升透析過夜后，高速離心(4℃，30000g，10分鐘)后上清上樣于CM離子交換柱，4℃，0.3M NaCl+50mM硼酸緩沖液洗脫即可得到重組抗菌多肽。對應(yīng)于以上6種質(zhì)粒，獲得了Ph-SA1、Ph-SA2、Ph-SA3、Ph-SA4、Ph-SE和Ph-SAR1六種重組抗菌多肽。
上述制備質(zhì)粒中所設(shè)計(jì)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實(shí)施例2重組抗菌多肽對青霉素敏感金葡菌的抑制作用細(xì)菌為美國標(biāo)準(zhǔn)菌株，ATCC 25923青霉素敏感金黃色葡萄球菌，菌液5微升(108CFU/ml級(jí)菌量)加入1％胰蛋白胨，1％NaCl，0.5％酵母，0.5％葡萄糖，1％HK2PO4的培養(yǎng)液10ml中，共準(zhǔn)備6組，第一組加入0.3M NaCl+50mM硼酸緩沖液(量與實(shí)驗(yàn)組中加入的抗菌多肽液體量相同)作為對照，第二組加入100ng/ml青霉素G鈉，第三組加入100ng/ml野生型大腸菌素Ia(大腸菌素保存液為0.3M NaCl+50mM硼酸緩沖液)，第四組加入不含質(zhì)粒的TG1工程菌蛋白(保存液為0.3M NaCl+50mM硼酸緩沖液)，第五組加入100ng/ml大腸菌素Ia羧基端連接了其它的八肽的融合蛋白(保存液為0.3M NaCl+50mM硼酸緩沖液)，第六組加入100ng/ml抗金葡菌多肽Ph-SA1(保存液為0.3M NaCl+50mM硼酸緩沖液)。
上述各組液體置于100ml三角燒瓶中，200rpm，37℃生長，每小時(shí)采樣100μl加入96孔酶標(biāo)板中經(jīng)分光光度計(jì)(A 595nm)比色測試細(xì)菌生長濁度，畫出細(xì)菌生長曲線來比較抗菌多肽的抑菌效力，結(jié)果如圖8所示，顯示青霉素敏感金葡菌的生長只能被青霉素和Ph-SA1抑制。
實(shí)施例3抗金葡菌多肽對耐甲氧西林金黃色葡萄球菌(ATCC BAA-42)殺菌效果的透射電鏡觀察(1％磷鎢酸染色，放大25,000倍)。
如圖9所示，Control，對照組，細(xì)菌加入前述培養(yǎng)液中，37℃，200rpm生長2小時(shí)后仍呈現(xiàn)正常形態(tài)；Wild type colicin Ia，細(xì)菌生長條件同對照組，加入野生型大腸菌素Ia(50μg/ml)生長兩小時(shí)后，菌形態(tài)仍然正常；AgrD，細(xì)菌生長條件同對照組，加入金葡菌信號(hào)傳導(dǎo)多肽(10μg/ml)生長兩小時(shí)后，菌形態(tài)仍然正常，上端一個(gè)菌體正在分裂；Oxacillin，細(xì)菌生長條件同對照組，加入苯唑西林(500μg/ml)生長2小時(shí)后，菌胞壁呈溶解形態(tài)；Ph-SA1，細(xì)菌生長條件同對照組，加入抗金葡多肽(50μg/ml)生長半小時(shí)后，菌破裂，內(nèi)容物泄漏，而殘余胞壁結(jié)構(gòu)完整。
結(jié)論單純使用信號(hào)傳導(dǎo)多肽或野生型大腸菌素處理都不能殺死MRSA，抗金葡多肽卻能殺滅MRSA且殺菌機(jī)制與苯唑西林完全不同。
實(shí)施例4重組抗菌多肽的體外抗菌活性以下試驗(yàn)由國家藥品監(jiān)督管理局四川抗菌素研究所藥理研究室完成。
一、實(shí)驗(yàn)材料
(1).藥物①ph-SA1規(guī)格1.5mg/ml，批號(hào)PRE021101，由四川大學(xué)華西醫(yī)學(xué)院提供。
②注射用頭孢唑林鈉白色粉末，規(guī)格0.5g/瓶，批號(hào)020418，華北制藥股份有限公司生產(chǎn)。
③注射用青霉素鈉白色粉末，規(guī)格80萬單位/瓶，批號(hào)00206604，華北制藥股份有限公司生產(chǎn)。
④注射用氨芐西林鈉規(guī)格1g/瓶，批號(hào)F0203329，華北制藥股份有限公司生產(chǎn)。
⑤注射用苯唑西林鈉規(guī)格0.5g/瓶，批號(hào)F0203329，華北制藥股份有限公司生產(chǎn)。
⑥萬古霉素規(guī)格2.5mg/ml，批號(hào)020712，由四川抗菌素工業(yè)研究所制劑室提供。
以上樣品用無菌水溶解稀釋，藥物終濃度為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)、細(xì)菌①臨床分離菌株試驗(yàn)所用菌株均為2002年從四川、北京地區(qū)收集的臨床分離致病菌，所有菌種在收集分離的單位(華西醫(yī)科大學(xué)檢驗(yàn)科及重慶醫(yī)科大學(xué)傳染科細(xì)菌室)均經(jīng)鑒定后，再經(jīng)本室用API系統(tǒng)重新鑒定后使用。
金黃色葡萄菌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)、腸球菌20株、鮑曼不動(dòng)桿菌20株，共274株。
②標(biāo)準(zhǔn)質(zhì)控菌株金黃色葡萄球菌ATCC25923、金黃色葡萄球菌29213是四川抗菌素工業(yè)研究所藥理室保存菌株。
(3)、培養(yǎng)基①M(fèi)-H培養(yǎng)基中國藥品生物制品檢定所產(chǎn)品，M-H肉湯培養(yǎng)基稱取25g加100ml蒸餾水，加熱溶解，分裝，高壓滅菌，116℃，20分鐘。M-H固體培養(yǎng)基稱取36g，加1000m蒸餾水，高壓滅菌，116℃，20分鐘，用于革蘭陽性、陰性需氧菌的藥敏試驗(yàn)。
②腸球菌、鏈球菌用血培養(yǎng)基，即在M-H培養(yǎng)基中加5-10％脫纖維兔血配制而成，5％CO中37℃培養(yǎng)24小時(shí)。
二、試驗(yàn)方法1.最低抑菌濃度MIC的測定采用瓊脂二倍稀釋法測定ph-SA的最低抑菌濃度(MIC)。用多點(diǎn)接種儀(Denley A400)將細(xì)菌接種于含不同藥物濃度的瓊脂平皿表面，每點(diǎn)含菌量約為105CFU/ml，37℃孵育18～24小時(shí)觀察結(jié)果，以無細(xì)菌生長平皿培養(yǎng)基中所含藥物的最低濃度為藥物對該菌的最低抑菌濃度(MIC值)。
三、結(jié)果實(shí)驗(yàn)結(jié)果見表1。
表1 ph-SA1的體外抗菌活性比較菌株 藥物MIC50MIC90MIC范圍(株數(shù))(mg/L) (mg/L) (mg/L)金黃色葡萄球菌ph-SA1 16 ＞16 ＜0.0005～64
MRSA菌株 頭孢唑林＞128＞1281～128青霉素 128 ＞1281～128氨芐西林 16 320.06～64苯唑西林＞128＞1280.5～128萬古霉素0.25 10.004～2金黃色葡萄球菌ph-SA1 0.5 16 0.125～＞64MSSA菌株 頭孢唑林 0.1250.25＜0.015～0.5(88) 青霉素 8640.125～＞128氨芐西林 0.25 1＜0.015～0.25苯唑西林 0.1250.125＜0.015～0.25萬古霉素 0.5 10.03～1表皮葡萄球菌 ph-SA1 ＞64 ＞640.25～＞64MRSE菌株 頭孢唑林 32 ＞1280.06～＞128(25) 青霉素 ＞128＞1288～＞128氨芐西林 16 640.06～64苯唑西林 64 ＞1280.5～＞128萬古霉素 0.5 10.25～2表皮葡萄球菌 ph-SA1 0.25 160.125～32MSSE菌株 頭孢唑林 0.03 0.5＜0.015～0.5(21) 青霉素 4320.125～128氨芐西林 0.03 0.25＜0.015～0.25苯唑西林0.03 0.125＜0.015～0.25萬古霉素 0.5 0.50.06～0.5腸球菌ph-SA1 ＞64 ＞648～＞64(20) 頭孢唑林 32 ＞128
32～＞128青霉素 32 6432～＞128氨芐西林 0.5 160.25～16苯唑西林 16 ＞1288～＞128萬古霉素 120.5～50接表1菌株 藥物MIC50MIC90MIC范圍(株數(shù))(mg/L) (mg/L) (mg/L)銅綠假單胞菌 ph-SA1 16 ＞16 4～＞16(20) 頭孢唑林＞128＞128128～＞128青霉素 ＞128＞128＞128氨芐西林＞128＞12816～＞128苯唑西林＞128＞128＞128萬古霉素＞128＞128＞128鮑曼不動(dòng)桿菌 ph-SA1 32 ＞64 0.5～＞64(20) 頭孢唑林＞128＞128＞128青霉素 ＞128＞128＞128氨芐西林＞128＞12816～＞128苯唑西林＞128＞128＞128萬古霉素＞128＞128128～＞128實(shí)施例5重組抗菌多肽的動(dòng)物體內(nèi)保護(hù)實(shí)驗(yàn)以下試驗(yàn)由國家藥品監(jiān)督管理局四川抗菌素研究所藥理研究室完成。
一、實(shí)驗(yàn)材料(1)·藥物使用藥物同前。
以上樣品用無菌水溶解稀釋，藥物注射終濃度為10mg/kg、5mg/gk、2.5mg/kg。
(2)·細(xì)菌①標(biāo)準(zhǔn)質(zhì)控菌株金黃色葡萄球菌ATCC BAA-42(MRSA)、腸球菌ATCC700802(VRE)是四川抗菌素工業(yè)研究所藥理室保存菌株。
二、實(shí)驗(yàn)方法昆明小鼠170只，雌雄各半，體重18-22克，隨機(jī)分組為腹腔分別注射金黃色葡萄球菌ATCC BAA-42(MRSA)和ATCC 700802(VRE)兩個(gè)大組，其中PH-SA1、青霉素、氨芐西林、頭孢唑林和萬古霉素試驗(yàn)組分別為每組小鼠5只，對照組每組小鼠10只。腹腔注射致死劑量的細(xì)菌后，各藥物試驗(yàn)組小鼠分別自靜脈注射10mg、5mg、2.5mg/kg劑量的藥物一次，每24小時(shí)觀察結(jié)果，連續(xù)7-14天，以小鼠死亡為陽性結(jié)果。
三、結(jié)果5mg、3mg和1mg藥物劑量組結(jié)果顯示，Ph-SA1組小鼠存活率在3個(gè)劑量組中與萬古霉素組小鼠存活率始終保持了恒定的差距，因此在體內(nèi)試驗(yàn)中Ph-SA1對抗耐甲氧西林金黃色葡萄球菌感染的治療效果遠(yuǎn)優(yōu)于萬古霉素。這可能是Ph-SA1既殺菌又抑制細(xì)菌毒素分泌的雙重效果所致。因而該藥在臨床應(yīng)用時(shí)可能具有其它抗菌素?zé)o法比擬的治療效果。其它各抗菌素組小鼠死亡率≥80％，已不具備可比性；對照組于48小時(shí)內(nèi)全部死亡。
實(shí)驗(yàn)結(jié)果見表2和圖10。
表2 Ph-SA1體內(nèi)保護(hù)試驗(yàn)結(jié)果(靜脈給藥)


由表1和表2中結(jié)果可以看出1.MRSA體外抗菌活性比較苯唑西林MIC90測定值比PH-SA1測定值大8倍，比萬古霉素測定值大128倍。PH-SA1分子量(70,0000)是萬古霉素分子量(1,400)的49倍，苯唑西林分子量(400)的175倍。按單位體積內(nèi)相同藥物分子數(shù)標(biāo)定，則PH-SA1對MRSA體外抗菌活性相當(dāng)于萬古霉素對MRSA體外抗菌活性的3倍，相當(dāng)于苯唑西林對MRSA體外抗菌活性的1,400倍。
2.Ph-SA1對MSSA的敏感率(以16mg/L為界限)為91％，對MRSA的敏感率為77％，對MSSE的敏感率為91％，對MRSE的敏感率為40％，對腸球菌的敏感率為30％，對銅綠假單胞菌的敏感率為50％，對鮑曼不動(dòng)桿菌的敏感率為40％。
3.SA感染小鼠體內(nèi)保護(hù)預(yù)實(shí)驗(yàn)中，最小劑藥量(2.5mg/kg)條件下PH-SA1組的死亡率最低(40％)，其它各組死亡率分別為青霉素組60％，氨咔西林組100％，萬古霉素60％，對照組100％。該結(jié)果顯示PH-SA1的保護(hù)率最高(60％)；青霉素和萬古霉素的保護(hù)率次之(40％)。
實(shí)施例6重組抗菌多肽對表皮葡萄球菌生長的影響細(xì)菌為購自中國菌種保存中心(國家醫(yī)藥管理局生物制品檢定所，北京天壇)的表皮葡萄球菌標(biāo)準(zhǔn)菌株，編號(hào)26069，菌液5微升(108CFU/ml級(jí)菌量)加入1％胰蛋白胨，1％NaCl，0.5％酵母，0.5％葡萄糖，1％HK2PO4的培養(yǎng)液10ml中，共準(zhǔn)備4組，第一組加入0.3M NaCl+50mM硼酸緩沖液(量與實(shí)驗(yàn)組中加入的抗菌多肽液體量相同)作為對照，第二組加入2μg/ml青霉素G鈉，余各實(shí)驗(yàn)組各加入2μg/ml抗菌多肽Ph-SA1和Ph-SE(保存液為0.3M NaCl+50mM硼酸緩沖液)。
上述各組液體置于100ml三角燒瓶中，200rpm，37℃生長，每小時(shí)采樣100μl加入96孔酶標(biāo)板中經(jīng)分光光度計(jì)(A 595nm)比色測試細(xì)菌生長濁度，畫出細(xì)菌生長曲線來比較抗菌多肽的抑菌效力，結(jié)果見圖11。
由圖中可見，表皮葡萄球菌的生長可被抗金葡多肽部分抑制，可被抗表葡多肽有效的抑制。
實(shí)施例7幾種重組抗菌多肽抑菌效果的比較以下實(shí)驗(yàn)比較了本發(fā)明制備的6種抗菌多肽的抑菌活性，細(xì)菌為美國標(biāo)準(zhǔn)菌株，ATCC BAA-42耐甲氧西林金黃色葡萄球菌，菌液5微升(108CFU/ml級(jí)菌量)加入1％胰蛋白胨，1％NaCl，0.5％酵母，0.5％葡萄糖，1％HK2PO4的培養(yǎng)液10ml中，共準(zhǔn)備8組，第一組加入0.3M NaCl+50mM硼酸緩沖液(量與實(shí)驗(yàn)組中加入的抗菌多肽液體量相同)作為對照，第二組加入5μg/ml青霉素G鈉，余各實(shí)驗(yàn)組各加入5μg/ml抗菌多肽Ph-SA1、Ph-SA2、Ph-SA3、Ph-SA4、Ph-SE和Ph-SAR1(保存液為0.3M NaCl+50mM硼酸緩沖液)。
上述各組液體置于100ml三角燒瓶中，200rpm，37℃生長，每小時(shí)采樣100μl加入96孔酶標(biāo)板中經(jīng)分光光度計(jì)(A 595nm)比色測試細(xì)菌生長濁度，畫出細(xì)菌生長曲線來比較抗菌多肽的抑菌效力，結(jié)果見圖12。
結(jié)果顯示，耐甲氧西林金葡菌的生長幾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。
以上對本發(fā)明的詳細(xì)描述并不限制本發(fā)明，本領(lǐng)域技術(shù)人員可以根據(jù)本發(fā)明作出各種改變和變形，只要不脫離本發(fā)明的精神，均應(yīng)屬于本發(fā)明所附權(quán)利要求的范圍。
SEQUENCE LISTING<110>成都陽輝生物科技有限責(zé)任公司<120>一種新型抗菌多肽及其制備方法<130>1552<160>33<170>PatentIn version 3.1<210>1<211>24<212>DNA<213>人工序列<220>
<221>CDS<222>(1)..(24)<223>編碼金黃色葡萄球菌Agr D I信號(hào)傳導(dǎo)多肽<400>1tat tcc acc tgt gat ttt ata atg24Tyr Ser Thr Cys Asp Phe Ile Met1 5<210>2<211>8<212>PRT<213>人工序列<400>2Tyr Ser Thr Cys Asp Phe Ile Met1 5<210>3<211>27<212>DNA<213>人工序列<220>
<221>CDS<222>(1)..(27)<223>編碼金黃色葡萄球菌Agr D II信號(hào)傳導(dǎo)多肽<400>3gga gtt aac gca tgt tct tcc ctg ttt 27Gly Val Asn Ala Cys Ser Ser Leu Phe1 5<210>4<211>9<212>PRT<213>人工序列<400>4Gly Val Asn Ala Cys Ser Ser Leu Phe1 5<210>5<211>24<212>DNA<213>人工序列<220>
<221>CDS<222>(1)..(24)<223>編碼金黃色葡萄球菌Agr D III信號(hào)傳導(dǎo)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>編碼金黃色葡萄球菌Agr D IV信號(hào)傳導(dǎo)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>編碼表皮葡萄球菌信號(hào)傳導(dǎo)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>大腸菌素基因<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>編碼抗葡萄球菌多肽<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>編碼抗葡萄球菌多肽<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>編碼抗葡萄球菌多肽<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>編碼抗葡萄球菌多肽<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>編碼抗葡萄球菌多肽<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>編碼抗葡萄球菌多肽<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權(quán)利要求
1.編碼重組抗菌多肽的核苷酸，其特征在于它含有編碼可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域的基因，以及編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因。
2.根據(jù)權(quán)利要求1所述的核苷酸，其中所述編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因連接于所述編碼可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域的基因的C末端。
3.根據(jù)權(quán)利要求1所述的核苷酸，其中所述編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因連接于所述編碼可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域的基因的N末端。
4.根據(jù)權(quán)利要求2或3所述的核苷酸，其中所述可形成離子通道的大腸菌素選自大腸菌素E1、Ia、Ib、A、B和N。
5.根據(jù)權(quán)利要求4所述的核苷酸，其中所述可形成離子通道的大腸菌素為大腸菌素Ia。
6.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)利要求5所述的核苷酸，其中含有SEQ ID NO.22的核苷酸序列。
12.一種重組質(zhì)粒，其特征在于其含有編碼可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域的基因，以及編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因。
13.根據(jù)權(quán)利要求12所述的重組質(zhì)粒，其中含有SEQ ID NO.12的核苷酸序列。
14.根據(jù)權(quán)利要求12所述的重組質(zhì)粒，其中含有SEQ ID NO.14的核苷酸序列。
15.根據(jù)權(quán)利要求12所述的重組質(zhì)粒，其中含有SEQ ID NO.16的核苷酸序列。
16.根據(jù)權(quán)利要求12所述的重組質(zhì)粒，其中含有SEQ ID NO.18的核苷酸序列。
17.根據(jù)權(quán)利要求12所述的重組質(zhì)粒，其中含有SEQ ID NO.20的核苷酸序列。
18.根據(jù)權(quán)利要求12所述的重組質(zhì)粒，其中含有SEQ ID NO.22的核苷酸序列。
19.一種重組抗菌多肽，其含有可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域，以及葡萄球菌信號(hào)傳導(dǎo)多肽。
20.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.根據(jù)權(quán)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.含有權(quán)利要求20～25任何一項(xiàng)所述的重組抗菌多肽的藥物組合物。
27.重組抗菌多肽的制備方法，包括以下步驟將編碼葡萄球菌信號(hào)傳導(dǎo)多肽的基因與編碼可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域的基因可操作地連接獲得編碼重組抗菌多肽的基因；將獲得的基因?qū)氲奖磉_(dá)系統(tǒng)中進(jìn)行表達(dá)；分離表達(dá)的多肽而獲得本發(fā)明的抗菌多肽。
全文摘要
本發(fā)明公開了一種新型抗菌多肽及其制備方法，該重組抗菌多肽，其含有可形成離子通道的大腸菌素或其水性孔道結(jié)構(gòu)域，以及葡萄球菌信號(hào)傳導(dǎo)多肽。本發(fā)明還公開了編碼該重組抗菌多肽的核苷酸序列，以及含有所述核苷酸序列的重組質(zhì)粒。本發(fā)明的新型抗菌多肽相較于傳統(tǒng)抗菌素的優(yōu)點(diǎn)在于，其不會(huì)誘導(dǎo)細(xì)菌產(chǎn)生傳統(tǒng)的耐藥性。細(xì)菌較難以通過突變來改變其細(xì)胞膜的磷脂雙分子層的結(jié)構(gòu)，而本發(fā)明的重組多肽通過直接在靶細(xì)菌的胞膜上形成離子通道而達(dá)到殺菌目的。
文檔編號(hào)C12P21/00GK1532284SQ03120400
公開日2004年9月29日 申請日期2003年3月19日 優(yōu)先權(quán)日2002年11月12日
發(fā)明者丘小慶 申請人:成都陽輝生物科技有限責(zé)任公司