CN104592360B - 碱性抗菌肽及其靶向设计和应用 - Google Patents
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Abstract
本发明属于生物技术领域，公开了碱性抗菌肽及其靶向设计和应用，所述碱性抗菌肽由至少一种碱性氨基酸和一种或两种疏水氨基酸组成，所述碱性抗菌肽的氨基酸的个数为12～24个，其中，碱性氨基酸的比例大于33.3%，并对大肠杆菌、绿脓杆菌、金黄色葡萄球菌、多重耐药性金黄色葡萄球菌、酿酒酵母等具有广谱的抑菌作用；将上述碱性抗菌肽制成靶向抗菌肽，所述靶向抗菌肽在0.1mM杀肺癌细胞的效率高达100.3%；本发明所述抗菌肽或靶向抗菌肽可用于抑菌和抗癌，可广泛用于医疗、农业、食品防腐剂等领域。
Description
技术领域
本发明涉及生物技术领域，更具体地，涉及碱性抗菌肽及其靶向设计和应用。
背景技术
抗生素(antibiotics)是一类天然或人工合成的化合物，能够杀死细菌或者可以抑制细菌的生长。随着科学技术的不断发展，抗生素的定义也不断被扩充，其中抗微生物，包括抗真菌等化合物都被纳入到抗生素的范围。
人类自1940年发现第一种抗生素青霉素(penicillin)并将其应用于临床之后，就开始了抗生素治疗的新时代。许多曾经严重危害人类生命健康的感染性疾病因抗生素的使用而得到了有效的控制，并大幅降低了婴儿出生的死亡率和手术后的感染率，人类的平均寿命也得以延长15～20年。因此，各种各样的抗生素已经成为多数疾病的治疗中必不可少的药物。
然而，随之而来的是抗生素的滥用导致的严重的问题：抗生素的耐药性(Drugresistance)。在临床上，耐药性是指病原体及癌细胞等对化学治疗药物敏感性降低。而抗生素的耐药性主要指当微生物暴露在抗生素环境中时，仍然能够生存并进行繁殖的现象。出现耐药性的原因是，在自然选择的压力下，拥有抗性基因的菌株会成为优势菌株存活下来。这些抗性基因通常存在于质粒中，而对于微生物(尤其是细菌)，抗性基因可以通过转化、转导等现象进行转移并迅速复制，使一个菌落迅速获得抗性。
研究表明，研制一种新型抗生素大约需要十年或更长的时间，而细菌产生耐药性的时间却不足两年，新药的研制速度远远跟不上细菌耐药性产生的速度。而一旦出现拥有多种抗性基因的“超级细菌”，人们将对其无药可用。早在1976年，肺炎链球菌就被发现对青霉素产生了耐药。而在2010年在南亚地区发现的新型超级病菌NDM-1，所有现存的抗生素都对其不起作用。目前为止，NDM-1引起的疾病还找不到有效的治疗方法，而且有不断蔓延的趋势，引起了广泛关注。
因此在医疗卫生上迫切需要高效、低毒、高选择性广谱抑菌药物的研究和开发以保护人类的健康。而研究表明许多生物的基因组中也有编码抗生素的基因。这些基因编码的多为一些短肽，称为抗菌肽。抗菌肽一般携带正电荷，其具有抑菌活性强、不易产生耐药性等特点。抗菌肽一般长为10到40个氨基酸，经常形成过膜通道并经常具有溶血性、毒性，缺乏靶向抗菌抗癌特性。如果能够人工设计抗菌肽，则能够开发新的抗生素的资源，有效地解决当前医学上抗生素耐药性的问题。
发明内容
本发明所要解决的技术问题是克服现有抗菌肽经常具有的溶血性、膜通透性和毒性的缺陷，提供一种碱性抗菌肽。
本发明的第二个目的是提供上述碱性抗菌肽的靶向设计方法。
本发明的第三个目的是提供一种由上述碱性抗菌肽设计得到的靶向抗菌肽，所述靶向抗菌肽可靶向抗菌、抗癌。
本发明的第四个目的是提供上述碱性抗菌肽或靶向抗菌肽的应用。
本发明的目的是通过以下技术方案予以实现的：
一种碱性抗菌肽，所述碱性抗菌肽由至少一种碱性氨基酸和一种或两种疏水氨基酸组成，其中，所述碱性抗菌肽的氨基酸的个数为12～24个，碱性氨基酸的比例大于33.3％。
碱性氨基酸带有正电荷，一般可结合氯离子等负离子。加上一些疏水氨基酸，就可以结合到细胞膜上，破坏细胞膜的完整性，通过改变膜透性产生抑菌杀菌作用。现有技术中也有研究碱性氨基酸和疏水氨基酸所得到的抗菌肽，但所述抗菌肽的设计通常都需要考虑肽的二级结构，二级结构同时还会影响最终合成的碱性抗菌肽的活性，这不仅会增加抗菌肽的合成成本，也会影响抗菌肽的实际应用；另外，现有技术所合成的碱性抗菌肽通常形成过膜通道，多有溶血性，对人体是不安全的。
更优选地，本发明所述碱性抗菌肽氨基酸的个数为20个以上，其碱性氨基酸的比例在45％以上时所得到的抗菌肽效果更好；同时，本发明所述碱性抗菌肽是非高通透型的肽(通常是低通透型)且不裂解人体红细胞，无溶血活性，一般不形成高效过膜通道，因此对人体是安全的。
申请人通过大量的筛选和研究发现，将疏水氨基酸置于抗菌肽的一端时，碱性抗菌肽的抗菌效果很好；具体地，所述疏水氨基酸位于碱性抗菌肽的一端，其疏水氨基酸的个数为3～8个。
优选地，本发明所述碱性氨基酸选自赖氨酸和精氨酸的一种或两种；所述疏水氨基酸选自亮氨酸和苯丙氨酸的一种或两种；更优选地，本发明所述碱性抗菌肽由亮氨酸和赖氨酸组成，或者由亮氨酸和精氨酸组成，或者由苯丙氨酸和赖氨酸组成，或者由亮氨酸、赖氨酸和精氨酸组成，或者由亮氨酸、赖氨酸和苯丙氨酸组成，或者由苯丙氨酸、赖氨酸和精氨酸组成。
本发明所述碱性抗菌肽有上万亿种组成，且抗菌肽的设计不用考虑氨基酸的二级结构和物化性质，只要由上述氨基酸组成和保持高的碱性氨基酸比例，就可以形成活性高的碱性抗菌肽。
优选地，所述抗菌肽的氨基酸序列如SEQ ID NO：1～48所示。
更优选地，所述碱性抗菌肽的序列为Lm(L/K/R)n、Fm(L/K)n、Fm(F/K)n或(L/K)n Lm，其中，12≤n≤24，m≤8。
优选地，所述抗菌肽中氨基酸可以是D型，也可以是L型，还可以同时具有L型和D型。
一种靶向抗菌肽，所述靶向抗菌肽由上述任意一种抗菌肽与抗体互补决定区通过若干个亮氨酸连接获得；具体得，所述靶向抗菌肽结构组成为抗菌肽-Lo-抗体互补决定区；其中o为亮氨酸的个数，o≥4。
优选地，所述靶向抗菌肽的序列如SEQ ID NO：49～62所示。
需要说明的是，仅仅有个别抗菌肽接上某些抗体互补决定区成为靶向抗菌肽后，会使不裂解人体红细胞的抗菌肽变为裂解人体红细胞的靶向抗菌肽。因此，开发靶向抗菌肽药物时要进行适当筛选。
另外，本发明的碱性抗菌肽和靶向抗菌肽均具有抗大肠杆菌(MG1655)，绿脓杆菌(1.2464，北京中国普通微生物菌种保藏管理中心)，金黄色葡萄球菌(ATCC6538)，多重耐药金黄色葡萄球菌Y5(张颖等，一起食物中毒事件的金黄色葡萄球菌分子分型研究，中华预防医学杂志，2008，42(9):672-676；Ran He et al.A combinatorial yeast overlay methodfor the isolation of antibacterial oligopeptides,Proceedings of the NationalAcademy of Sciences,India Section B:Biological Sciences,2014,84(4):1069–1075)，酿酒酵母(INVSc1)等的效果。
因此，本发明所述碱性抗菌肽和靶向抗菌肽可用于抑菌，也可用于制备抑菌药物。上述抗菌肽中，当赖氨酸个数等于或超过肽的50％时，对大肠杆菌较为有效。高于或低于50％，对金黄色葡萄球菌和绿脓杆菌都有效；长度为16至24个氨基酸之间的抗菌肽对上述3个菌都有效。
同时，本发明所述碱性抗菌肽和靶向抗菌肽同样具有抗癌的作用，本发明所述碱性抗菌肽和靶向抗菌肽具有抗肺癌细胞A549的活性和对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用；所述靶向抗菌肽在较低的浓度下具有比对照抗菌肽更强的抗癌活性，特别是编号为P59的抗菌肽，在0.1mM杀肺癌细胞的效率高达100.3％；因此本发明所述靶向抗菌肽可以用于导向抑菌或导向抗癌，也可用于制备抑菌或抗癌药物。
对于靶向抗菌肽(编号为P49，P50，P59，P60)而言，中间的亮氨酸个数为4。这几个肽在低浓度具有比对照抗菌肽P2或P11更强的杀癌活性，其用到的抗体互补决定区为CD47蛋白质的抗体的互补决定区3和2(Yasufumi Kikuchi,Shinsuke Uno,Yasuko Kinoshita,et al.HUMANIZED ANTI-CD47 ANTIBODY.European Patent Application EP1693385)，其序列分别为：ARGGYYTYDDWG和YIYPYNDGTKYNEKFKD。科学家检测过的人类癌细胞都表达CD47，相比于正常细胞通常以较高水平表达(平均大约高3倍多)。将本发明所述抗菌肽与CD47蛋白的抗体互补决定区相连接，可以将抗菌肽带到癌细胞位置。
本发明所述抗菌肽与CD47蛋白的抗体互补决定区必须通过亮氨酸相连接，发明人通过实验发现，由编号为P2的碱性抗菌肽直接连接CD47蛋白的抗体互补决定区形成的靶向抗菌肽(编号为P63)完全丧失抑菌活性；另外，靶向抗菌肽P64和P65是由碱性抗菌肽、苯丙氨酸或异亮氨酸和CD47蛋白的抗体互补决定区形成，而完全丧失对大肠杆菌和绿脓杆菌的抑菌活性。与P10相比，P61和P62中间有亮氨酸连接而保留了较强抑菌活性。
与现有技术相比，本发明具有以下有益效果：
本发明提供了一种碱性抗菌肽，所述碱性抗菌肽由至少一种碱性氨基酸和一种或两种疏水氨基酸组成；所述碱性抗菌肽的氨基酸的个数为12～24个，其中，碱性氨基酸的比例大于33.3％；本发明所述碱性抗菌肽不用考虑氨基酸的二级结构和物化性质，所述抗菌肽可任意组合；是非高通透型的肽(通常低通透型)且不裂解人体红细胞，无溶血活性，一般不形成高效过膜通道，因此对人体是安全的。同时对大肠杆菌、绿脓杆菌、金黄色葡萄球菌、多重耐药性金黄色葡萄球菌具有广谱的抑菌作用；将上述碱性抗菌肽制成靶向抗菌肽，所述靶向抗菌肽在0.1mM杀肺癌细胞的效率高达100.3％；本发明所述抗菌肽或靶向抗菌肽可用于抑菌和抗癌，可广泛用于医疗、农业、食品防腐剂等领域。
附图说明
图1为抗菌肽的溶血活性。
图2为靶向抗菌肽的溶血活性。
图3为80μM不同的抗菌肽水解ONPG的速率。
图4为80μM不同的抗菌肽水解ONPG的速率。
图5为肽对多重耐药金黄色葡萄球菌Y5抑菌实验结果。
图6为肽对绿脓杆菌抑菌实验结果。
图7为肽对酿酒酵母INVSc1抑菌圈实验结果。
图8为抗菌肽对肺癌细胞系A549的杀伤作用。
图9为靶向抗菌肽对肺癌细胞系A549的杀伤作用。
图10为抗菌肽对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用。
图11为靶向抗菌肽对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用。
图12为靶向抗菌肽对SV40病毒永生化的人支气管上皮细胞株16HBE14o–的杀伤作用。
具体实施方式
下面结合说明书附图和具体实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的简单修改或替换，均属于本发明的范围；若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1最小抑菌浓度(MIC)测定
按表1的多肽序列，由上海波泰生物科技有限公司采用fmoc固相合成法合成各种多肽，使用RP-HPLC纯化；所述多肽包括抗菌肽和靶向抗菌肽，其中靶向抗菌肽是由抗菌肽与抗体互补决定区通过若干个亮氨酸连接获得。
所用的细菌为大肠杆菌(MG1655)，绿脓杆菌(1.2464，北京中国普通微生物菌种保藏管理中心)，金黄色葡萄球菌(ATCC6538)，多重耐药金黄色葡萄球菌Y5(张颖等，一起食物中毒事件的金黄色葡萄球菌分子分型研究，中华预防医学杂志，2008，42(9):672-676；RanHe et al.A combinatorial yeast overlay method for the isolation ofantibacterial oligopeptides,Proceedings of the National Academy of Sciences,India Section B:Biological Sciences,2014,84(4):1069–1075)，
最小抑菌浓度(MIC)测定过程如下：
(1)接种细菌单菌落于常规LB液体培养基中，37℃，200转/分钟摇床过夜培养11小时，并测菌落形成单位。
(2)将各肽(抗菌肽和靶向抗菌肽)分别设置四个终浓度：40μM、80μM、160μM和320μM；每个肽每个浓度设置3个平行。
(3)用常规LB液体培养基稀释培养过夜的菌液得到106菌落形成单位的使用菌液；实验组：每孔多肽和菌液各50微升，对照组1：100微升无菌培养基；对照组2：无菌培养基和菌液各50微升；对照组3：50微升的菌液+与样品相对应的相应量的DMSO和培养基；对照组4：50微升的菌液+与样品相对应的相应量的水和培养基。
(4)将加好样品的96孔板在37℃恒温箱培养20小时后，用酶标仪在492纳米及620纳米处测OD值。
(5)数据分析
计算公式：用492纳米处的OD值计算。
抑制率％＝100×[1-(肽与菌液实验组-不加菌液对照组)/(水或者DMSO与菌液对照组-不加菌液对照组)]
当抑菌率达到79.5％时，记录为MIC(最小抑菌浓度)，结果见表1。
表1抗菌肽对多种细菌的最小抑菌浓度
*-表示未测定；编号的括号里标明DMSO，表示为DMSO溶解的肽。未标注为水溶肽；无活性：所有4个浓度抑菌率低于15％；促进生长：所有4个浓度抑菌率低于-20％；括号内表示为最大抑菌率。氨基酸小写为D型。a：ARGGYYTYDDWG(CD47蛋白质的抗体的互补决定区3。b：YIYPYNDGTKYNEKFKD(CD47蛋白质的抗体的互补决定区2。
实施例2溶血性实验
1.血液处理
(1)采集新鲜人血5毫升放入含有0.5毫升3.8％柠檬酸钠管中，吹打抗凝管中的血液，使其与抗凝剂充分混匀。
(2)将上述血液在2000转/分钟条件下离心8分钟，去上清。用10mM，pH＝7.4的PBS漂洗血液，2000转/分钟离心5分钟，弃上清，重复操作，直到红细胞悬液清亮透彻无血清等杂质为止，弃去上清。然后按照5％(v/v)溶于PBS中，得到红细胞悬浮液。
2.抗菌肽处理：将抗菌肽溶于PBS，最后得到终浓度为40μM、80μM、160μM、320μM的抗菌肽溶液。
3.体系混合
吸取50微升处理过的抗菌肽溶液加入到96孔板中，每肽每浓度3个平行，之后用排枪吸取倒在培养皿上的红细胞悬液，每孔50微升加入到96孔板中，全部加完后密封96孔板，放入摇床，37℃，200转/分钟震荡1小时。
将20μM、40μM、80μM和160μM的天蚕素按照上述方法同红细胞悬浮液混合，阳性对照为1％Triton X-100；阴性对照为PBS溶液。
4.数据分析
将震荡培养后的96孔板在2000转/分钟条件下离心8分钟，吸取60微升上清液平行转移到新的96孔板中，酶标仪检测540纳米波长处的OD值。
溶血百分值＝(A540样品-A540阴性对照)/(A540阳性对照-A540阴性对照)×100％。
实验结果如图1和图2。
实施例3大肠杆菌膜通透性实验
β-半乳糖苷酶是一种水解酶，位于细菌的细胞质中，可以将邻硝基苯β-D-半乳吡喃糖苷(ONPG)水解成半乳糖和邻硝基苯酚(呈黄色)。在体系中加入一定量的ONPG，通过测量培养液在420纳米的吸光值的变化，可以判断ONPG的水解度，从而测知β-半乳糖苷酶是否水解ONPG。一般情况下，由于酶位于细胞内部，而ONPG不能进入细胞内。但一旦细胞膜通透性改变时，ONPG进入细胞内并水解，培养液迅速变成黄色，A420值在短时间内快速升高。因此，可以利用此法来检测抗菌肽对细胞膜通透性的影响。膜通透性高说明抗菌肽能形成过膜通道。
本发明所述通透性实验过程如下：
(1)挑取大肠杆菌ML-35单菌落过夜培养，并测量其菌落形成单位；根据测量的菌落形成单位数值，取适量体积的过夜培养物于EP管中，10000转离心1分钟，并用10mM磷酸钠(含0.1M NaCl)缓冲液重悬。重复3次，用足量磷酸钠缓冲液将菌体重悬至菌落形成单位＝2×107。
(2)取10微升10mM肽(包括抗菌肽和靶向抗菌肽)，加入490微升磷酸钠缓冲液，得到200μM、500微升的肽稀释液；取10微升水或DMSO代替肽，加入90微升磷酸钠缓冲液作为对照组。
(3)取200微升肽稀释液，加入50微升ONPG溶液，得到肽-ONPG混合液；在对照组中也加入50微升ONPG；用肽-ONPG混合液加96孔板，每孔50微升，每个样三个重复；取4℃保存的菌液，用排枪吸取50微升加入每孔。按此方法，最终实验组每孔溶液体积100微升，肽终浓度80μM，ONPG终浓度1.5mM，菌浓度1x107菌落形成单位；形成实验组每孔肽终浓度为40μM时方法同上。
室温加完菌液后迅速转移至多功能酶标仪，测量其在37℃下420纳米的吸光值，从第一次测量起，每隔10分钟测量一次。测量13次。记录数据并分析，结果如图3、图4。
实施例4抑菌圈实验
肽对多重耐药金黄色葡萄球菌Y5和绿脓杆菌抑菌圈实验：分别接入金黄色葡萄球菌Y5和绿脓杆菌(1.2464)菌液，使菌液浓度约为1×108菌落形成单位/毫升，倒入平板，冷却。
1.样品处理：
金黄色葡萄球菌平板每个孔加入40微升、5mM肽，阴性对照为40微升DMSO/水，阳性对照为10微升5毫克/毫升Amp+30微升水。
绿脓杆菌平板每个孔加入25微升、5mM肽，阴性对照为25微升水/DMSO，阳性对照为10微升、5毫克/毫升Amp+15微升水。
将处理好的样品在37℃恒温培养16小时后记录是否有抑菌圈及其大小结果(如图5、6和表2)。
表2多肽对金黄色葡萄球菌和绿脓杆菌的抑菌圈大小
注：以上和以下抑菌圈直径都不包括打孔器直径；a：ARGGYYTYDDWG是CD47蛋白质的抗体的互补决定区3；b：YIYPYNDGTKYNEKFKD是CD47蛋白质的抗体的互补决定区2
实施例5酿酒酵母INVSc1抑菌圈实验
配置YPD培养基:葡萄糖2％，蛋白胨2％，，酵母膏1％，固体培养基还要加琼脂2％；2.划线培养酿酒酵母INVSc1单菌落；接种单菌落30℃摇床培养24小时后取出放4℃保存；测菌落形成单位。
融化YPD固体培养基，等培养基不太热的时候加入上述测过菌落形成单位的菌液，按照菌液：YPD培养基＝1：20，使酵母菌液浓度为1×106个/毫升；倒入直径为15厘米的平板约50毫升/板；冷却后用直径为5毫米打孔器打孔，用镊子将孔中的培养基丢弃，每孔加入40微升、10mM肽，阴性对照为40微升的水/DMSO阴性对照，阳性对照为10微升、10毫克/升的苯菌灵+30微升水。用封口膜封好平板之后放入30℃恒温箱培养约18小时后拍照并记录抑菌圈大小。结果如图7和表3。
表3酿酒酵母抑菌圈大小
实施例6肽对细胞活力的影响
肺癌细胞系A549见参考文献(Lieber M,Smith B,Szakal A et al.A continuoustumor-cell line from a human lung carcinoma with properties of type IIalveolar epithelial cells.Int J Cancer,2006,17(1):62-70)。细胞培养方法如美国菌种保藏中心所述。永生化的人支气管上皮细胞株16HBE14o–细胞见参考文献(Cozens A L,Yezzi M J,Kunzelmann K,Ohrui T,Chin L,Eng K,Finkbeiner W E,Widdicombe J H,andGruenert D C."CFTR expression and chloride secretion in polarized immortalhuman bronchial epithelial cells."American Journal of Respiratory Cell andMolecular Biology,1994,10(1):38-47)培养于DMEM/F12培养基中，A549细胞培养于DMEM(高糖)培养基中，并且两种培养基中都加入10％的胎牛血清。
细胞活力采用3–(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazoliumbromide(MTT；MBCHAM)比色法测定，具体步骤参见参考文献(Warshamana-Greene GS,LitzJ,Buchdunger E et al.The insulin-like growth factor-I receptor kinaseinhibitor,NVP-ADW742,sensitizes small cell lung cancer cell lines to theeffects of chemotherapy.Clin Cancer Res,2005,11(4):1563-1571)。主要步骤：16HBE14o–和A549细胞培养于96孔板中至指数生长期。然后对各孔细胞施以指定的不同浓度的肽。处理24小时后，用MTT法测定在492纳米的吸光度值。最后根据公式：细胞活力抑制率(％)＝(对照组492纳米吸光值-实验组492纳米吸光值)/对照组492纳米吸光值×00％)来计算出各种肽对细胞活力的抑制率，结果如图8～12。
SEQUENCE LISTING
<110> 中山大学
<120> 碱性抗菌肽及其靶向设计和应用
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<210> 11
<211> 24
<212> PRT
<213> P11
<400> 11
Leu Leu Leu Leu Leu Lys Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Lys Lys Lys Lys Lys
20
<210> 12
<211> 24
<212> PRT
<213> P12
<400> 12
Leu Leu Leu Leu Leu Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
1 5 10 15
Lys Lys Lys Lys Lys Lys Lys Lys
20
<210> 13
<211> 24
<212> PRT
<213> P13
<400> 13
Leu Leu Leu Leu Leu Lys Lys Leu Leu Lys Leu Leu Lys Leu Leu Leu
1 5 10 15
Lys Leu Leu Lys Leu Leu Lys Lys
20
<210> 14
<211> 24
<212> PRT
<213> P14
<400> 14
Lys Lys Leu Leu Lys Lys Leu Leu Lys Leu Lys Lys Lys Leu Lys Leu
1 5 10 15
Leu Lys Leu Lys Leu Lys Leu Lys
20
<210> 15
<211> 23
<212> PRT
<213> P15
<400> 15
Lys Leu Lys Lys Lys Leu Lys Lys Lys Lys Leu Lys Leu Lys Leu Leu
1 5 10 15
Lys Lys Leu Leu Lys Leu Lys
20
<210> 16
<211> 24
<212> PRT
<213> P16
<400> 16
Lys Lys Lys Leu Lys Lys Lys Leu Lys Leu Lys Lys Leu Lys Lys Leu
1 5 10 15
Leu Lys Leu Lys Leu Lys Lys Lys
20
<210> 17
<211> 24
<212> PRT
<213> P17
<400> 17
Lys Lys Lys Lys Lys Lys Lys Leu Lys Leu Lys Lys Leu Lys Lys Leu
1 5 10 15
Leu Lys Lys Lys Lys Lys Lys Lys
20
<210> 18
<211> 24
<212> PRT
<213> P18
<400> 18
Lys Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys Leu Lys Lys Lys Lys
1 5 10 15
Lys Lys Lys Lys Leu Leu Leu Leu
20
<210> 19
<211> 24
<212> PRT
<213> P19
<400> 19
Lys Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys Leu Lys Lys Lys Lys
1 5 10 15
Lys Lys Leu Leu Leu Leu Leu Leu
20
<210> 20
<211> 20
<212> PRT
<213> P20
<400> 20
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Lys Lys Lys
1 5 10 15
Leu Lys Lys Lys
20
<210> 21
<211> 24
<212> PRT
<213> P21
<400> 21
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Lys Leu Lys Lys Lys
20
<210> 22
<211> 24
<212> PRT
<213> P22
<400> 22
Leu Leu Leu Leu Leu Leu Lys Lys Leu Lys Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys
20
<210> 23
<211> 24
<212> PRT
<213> P23
<400> 23
Leu Leu Leu Leu Leu Leu Lys Lys Leu Lys Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys
20
<210> 24
<211> 24
<212> PRT
<213> P24
<400> 24
Leu Leu Leu Leu Leu Arg Arg Leu Arg Leu Arg Arg Leu Leu Arg Arg
1 5 10 15
Leu Leu Leu Arg Arg Leu Arg Arg
20
<210> 25
<211> 24
<212> PRT
<213> P25
<400> 25
Leu Leu Leu Leu Leu Leu Arg Arg Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
Leu Arg Arg Leu Arg Arg Arg Arg
20
<210> 26
<211> 24
<212> PRT
<213> P26
<400> 26
Leu Leu Leu Leu Leu Leu Leu Arg Arg Leu Arg Arg Arg Arg Arg Arg
1 5 10 15
Leu Arg Arg Arg Arg Leu Arg Arg
20
<210> 27
<211> 24
<212> PRT
<213> P27
<400> 27
Leu Leu Leu Leu Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10 15
Leu Arg Arg Leu Arg Arg Arg Arg
20
<210> 28
<211> 24
<212> PRT
<213> P28
<400> 28
Leu Leu Leu Leu Arg Arg Arg Arg Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
Leu Arg Arg Arg Leu Arg Arg Arg
20
<210> 29
<211> 24
<212> PRT
<213> P29
<400> 29
Leu Leu Leu Arg Arg Arg Arg Arg Arg Leu Arg Arg Leu Arg Arg Arg
1 5 10 15
Leu Arg Arg Arg Arg Arg Arg Arg
20
<210> 30
<211> 12
<212> PRT
<213> P30
<400> 30
Leu Leu Leu Leu Leu Arg Arg Arg Arg Leu Arg Arg
1 5 10
<210> 31
<211> 16
<212> PRT
<213> P31
<400> 31
Leu Leu Leu Leu Leu Arg Arg Leu Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
<210> 32
<211> 24
<212> PRT
<213> P32
<400> 32
Arg Arg Arg Arg Leu Arg Arg Arg Leu Arg Arg Leu Arg Arg Arg Arg
1 5 10 15
Arg Arg Arg Leu Leu Leu Leu Leu
20
<210> 33
<211> 24
<212> PRT
<213> P33
<400> 33
Arg Arg Arg Arg Leu Arg Arg Leu Arg Leu Arg Arg Leu Arg Arg Leu
1 5 10 15
Leu Arg Leu Leu Arg Arg Arg Arg
20
<210> 34
<211> 24
<212> PRT
<213> P34
<400> 34
Leu Leu Leu Leu Leu Arg Arg Arg Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
Leu Arg Arg Arg Arg Arg Arg Arg
20
<210> 35
<211> 16
<212> PRT
<213> P35
<400> 35
Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10 15
<210> 36
<211> 24
<212> PRT
<213> P36
<400> 36
Leu Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Leu Arg Arg Leu Arg
1 5 10 15
Arg Arg Leu Arg Arg Arg Arg Arg
20
<210> 37
<211> 24
<212> PRT
<213> P37
<400> 37
Arg Arg Leu Arg Arg Arg Leu Leu Arg Leu Arg Arg Arg Leu Arg Leu
1 5 10 15
Leu Arg Leu Arg Leu Arg Leu Arg
20
<210> 38
<211> 24
<212> PRT
<213> P38
<400> 38
Phe Phe Phe Phe Phe Lys Lys Phe Lys Phe Lys Lys Phe Phe Lys Lys
1 5 10 15
Phe Phe Phe Lys Lys Phe Lys Lys
20
<210> 39
<211> 24
<212> PRT
<213> P39
<400> 39
Phe Phe Phe Phe Phe Phe Phe Lys Lys Phe Lys Lys Lys Lys Lys Lys
1 5 10 15
Phe Lys Lys Lys Lys Phe Lys Lys
20
<210> 40
<211> 24
<212> PRT
<213> P40
<400> 40
Phe Phe Phe Lys Lys Lys Lys Lys Lys Phe Lys Lys Phe Lys Lys Lys
1 5 10 15
Phe Lys Lys Lys Lys Lys Lys Lys
20
<210> 41
<211> 24
<212> PRT
<213> P41
<400> 41
Lys Lys Lys Lys Phe Lys Lys Phe Lys Phe Lys Lys Phe Lys Lys Phe
1 5 10 15
Phe Lys Phe Phe Lys Lys Lys Lys
20
<210> 42
<211> 23
<212> PRT
<213> P42
<400> 42
Lys Lys Lys Lys Phe Lys Lys Phe Lys Phe Lys Lys Phe Lys Lys Phe
1 5 10 15
Lys Lys Phe Lys Lys Lys Lys
20
<210> 43
<211> 23
<212> PRT
<213> P43
<400> 43
Lys Lys Lys Phe Lys Lys Lys Phe Lys Phe Lys Lys Phe Lys Lys Phe
1 5 10 15
Phe Lys Phe Lys Lys Lys Lys
20
<210> 44
<211> 23
<212> PRT
<213> P44
<400> 44
Lys Lys Lys Lys Phe Lys Lys Lys Phe Phe Lys Lys Phe Lys Lys Phe
1 5 10 15
Lys Phe Lys Phe Lys Lys Lys
20
<210> 45
<211> 24
<212> PRT
<213> P45
<400> 45
Lys Lys Lys Lys Phe Lys Lys Phe Lys Phe Lys Lys Phe Lys Lys Phe
1 5 10 15
Phe Lys Phe Phe Lys Lys Lys Lys
20
<210> 46
<211> 16
<212> PRT
<213> P46
<400> 46
Leu Leu Leu Leu Arg Arg Arg Lys Lys Arg Arg Arg Arg Lys Lys Arg
1 5 10 15
<210> 47
<211> 24
<212> PRT
<213> P47
<400> 47
Phe Phe Phe Phe Phe Lys Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Lys Lys Lys Lys Lys
20
<210> 48
<211> 16
<212> PRT
<213> P48
<400> 48
Phe Phe Phe Phe Arg Arg Arg Lys Lys Arg Arg Arg Arg Lys Lys Arg
1 5 10 15
<210> 49
<211> 40
<212> PRT
<213> P49
<400> 49
Leu Leu Leu Leu Leu Lys Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Lys Lys Lys Lys Lys Leu Leu Leu Leu Ala Arg Gly Gly
20 25 30
Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 50
<211> 45
<212> PRT
<213> P50
<400> 50
Leu Leu Leu Leu Leu Lys Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Lys Lys Lys Lys Lys Leu Leu Leu Leu Tyr Ile Tyr Pro
20 25 30
Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp
35 40 45
<210> 51
<211> 40
<212> PRT
<213> P51
<400> 51
Leu Leu Leu Leu Leu Arg Arg Arg Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
Leu Arg Arg Arg Arg Arg Arg Arg Leu Leu Leu Leu Ala Arg Gly Gly
20 25 30
Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 52
<211> 42
<212> PRT
<213> P52
<400> 52
Leu Leu Leu Leu Leu Arg Arg Arg Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
Leu Arg Arg Arg Arg Arg Arg Arg Leu Leu Leu Leu Leu Leu Ala Arg
20 25 30
Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 53
<211> 44
<212> PRT
<213> P53
<400> 53
Leu Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Leu Arg Arg Leu Arg
1 5 10 15
Arg Arg Leu Arg Arg Arg Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu
20 25 30
Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 54
<211> 50
<212> PRT
<213> P54
<400> 54
Leu Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Leu Arg Arg Leu Arg
1 5 10 15
Arg Arg Leu Arg Arg Arg Leu Arg Arg Arg Arg Arg Leu Leu Leu Leu
20 25 30
Leu Leu Leu Leu Leu Leu Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp
35 40 45
Trp Gly
50
<210> 55
<211> 52
<212> PRT
<213> P55
<400> 55
Leu Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Leu Arg Arg Leu Arg
1 5 10 15
Arg Arg Leu Arg Arg Arg Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu
20 25 30
Leu Leu Leu Leu Leu Leu Leu Leu Ala Arg Gly Gly Tyr Tyr Thr Tyr
35 40 45
Asp Asp Trp Gly
50
<210> 56
<211> 44
<212> PRT
<213> P56
<400> 56
Leu Leu Leu Leu Leu Arg Arg Leu Arg Leu Arg Arg Arg Leu Arg Arg
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu
20 25 30
Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 57
<211> 54
<212> PRT
<213> P57
<400> 57
Leu Leu Leu Leu Leu Arg Arg Arg Arg Arg Arg Leu Arg Arg Leu Arg
1 5 10 15
Arg Arg Leu Arg Arg Arg Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu
20 25 30
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Ala Arg Gly Gly Tyr Tyr
35 40 45
Thr Tyr Asp Asp Trp Gly
50
<210> 58
<211> 52
<212> PRT
<213> P58
<400> 58
Arg Arg Arg Arg Leu Arg Arg Leu Arg Leu Arg Arg Leu Arg Arg Leu
1 5 10 15
Leu Arg Leu Leu Arg Arg Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu
20 25 30
Leu Leu Leu Leu Leu Leu Leu Leu Ala Arg Gly Gly Tyr Tyr Thr Tyr
35 40 45
Asp Asp Trp Gly
50
<210> 59
<211> 40
<212> PRT
<213> P59
<400> 59
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys Leu Leu Leu Leu Ala Arg Gly Gly
20 25 30
Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 60
<211> 45
<212> PRT
<213> P60
<400> 60
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys Leu Leu Leu Leu Tyr Ile Tyr Pro
20 25 30
Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp
35 40 45
<210> 61
<211> 40
<212> PRT
<213> P61
<400> 61
Lys Lys Lys Lys Leu Lys Lys Leu Lys Leu Lys Lys Leu Lys Lys Leu
1 5 10 15
Leu Lys Leu Leu Lys Lys Lys Lys Leu Leu Leu Leu Ala Arg Gly Gly
20 25 30
Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 62
<211> 44
<212> PRT
<213> P62
<400> 62
Lys Lys Lys Lys Leu Lys Lys Leu Lys Leu Lys Lys Leu Lys Lys Leu
1 5 10 15
Leu Lys Leu Leu Lys Lys Lys Lys Leu Leu Leu Leu Tyr Ile Tyr Pro
20 25 30
Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Asp
35 40
<210> 63
<211> 36
<212> PRT
<213> P63
<400> 63
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys Ala Arg Gly Gly Tyr Tyr Thr Tyr
20 25 30
Asp Asp Trp Gly
35
<210> 64
<211> 42
<212> PRT
<213> P64
<400> 64
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys Phe Phe Phe Phe Phe Phe Ala Arg
20 25 30
Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 65
<211> 42
<212> PRT
<213> P65
<400> 65
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Leu Lys Lys
1 5 10 15
Leu Lys Lys Leu Lys Lys Lys Lys Ile Ile Ile Ile Ile Ile Ala Arg
20 25 30
Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly
35 40
<210> 66
<211> 24
<212> PRT
<213> P66
<400> 66
Leu Leu Leu Leu Leu Leu Lys Lys Lys Leu Lys Lys Lys Thr Lys Lys
1 5 10 15
Leu Lys Lys Thr Lys Lys Lys Lys
20
<210> 67
<211> 28
<212> PRT
<213> P67
<400> 67
Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu
1 5 10 15
Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly
20 25
Claims (2)
1.一种靶向抗菌肽，其特征在于，所述靶向抗菌肽结构组成为碱性抗菌肽-Lo-抗体互补决定区；其中o为亮氨酸的个数，o≥4；所述靶向抗菌肽的序列如SEQ ID NO：59所示。
2.权利要求1所述靶向抗菌肽在制备抑菌或/和抗癌药物方面的应用。
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| CN201510009981.0A CN104592360B (zh) | 2015-01-07 | 2015-01-07 | 碱性抗菌肽及其靶向设计和应用 |
| PCT/CN2015/097734 WO2016110177A1 (zh) | 2015-01-07 | 2015-12-17 | 碱性抗菌肽及其靶向设计和应用 |
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| CN104592360B (zh) * | 2015-01-07 | 2018-05-29 | 中山大学 | 碱性抗菌肽及其靶向设计和应用 |
| JP6823790B2 (ja) | 2015-06-30 | 2021-02-03 | チェイン アンチマイクロバイアルズ オイＣｈａｉｎ Ａｎｔｉｍｉｃｒｏｂｉａｌｓ Ｏｙ | 新規な抗菌ペプチド、その変異体及び使用 |
| CN106589135B (zh) * | 2016-11-25 | 2018-08-28 | 东北农业大学 | 一种靶向抗菌肽及其制备方法和应用 |
| CN109705195B (zh) * | 2019-01-31 | 2021-12-14 | 东北农业大学 | 一种大肠杆菌靶向抗菌肽ki-qk及制备方法和应用 |
| CN110615829B (zh) * | 2019-09-29 | 2022-07-19 | 深圳市三浦天然化妆品有限公司 | 一种自组装抗菌肽水凝胶 |
| CN112778401B (zh) * | 2021-01-25 | 2022-10-18 | 中国农业大学 | 一种辛酸酰化修饰抗菌肽及其应用 |
| CN114133429B (zh) * | 2021-11-22 | 2022-06-24 | 哈尔滨吉象隆生物技术有限公司 | 一种杀灭肿瘤细胞的多肽及其应用 |
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| WO2005044857A1 (ja) * | 2003-11-11 | 2005-05-19 | Chugai Seiyaku Kabushiki Kaisha | ヒト化抗cd47抗体 |
| EP2108372A1 (en) * | 2008-04-09 | 2009-10-14 | Forschungszentrum Borstel Leibniz-Zentrum für Medizin und Biowissenschaften | Novel antimicrobial peptides |
| CN102816245A (zh) * | 2012-08-23 | 2012-12-12 | 郭铠 | 一组合成抗菌蛋白及其制备方法和应用 |
| CN103421090B (zh) * | 2013-07-31 | 2016-01-13 | 中国石油大学(华东) | 一种新型抗菌肽 |
| CN104592360B (zh) * | 2015-01-07 | 2018-05-29 | 中山大学 | 碱性抗菌肽及其靶向设计和应用 |
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| WO2016110177A1 (zh) | 2016-07-14 |
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