CN116496355B - 一种耐盐防弧菌抗菌肽fh-18及其制备方法和应用 - Google Patents
一种耐盐防弧菌抗菌肽fh-18及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种耐盐防弧菌抗菌肽FH‑18及其制备方法和应用，属于生物技术领域。所述抗菌肽FH‑18的氨基酸序列如序列表SEQ ID No.1所示。本发明以高毒性抗菌肽CHY1为模板，通过删除氨基酸缩短肽链长度，氨基酸位置互换形成双亲结构界面，同时也破坏α‑螺旋的双亲性结构，抗菌肽FH‑18显著降低了红细胞溶血性且抗弧菌活性明显提高，治疗指数是CHY1的722倍。通过此方法明显改善了模板肽的细胞毒性，可为替抗产品的选择提供新来源，助力渔业无抗饲料背景下的健康发展。
Description
技术领域
本发明属于生物技术领域，具体涉及一种耐盐防弧菌抗菌肽FH-18及其制备方法和应用。
背景技术
饲料禁抗、养殖限抗和产品无抗政策严格执行，南美白对虾的传统养殖模式遭受巨大挑战，导致弧菌发病骤升为危害对虾安全出池的首要因素，因此，亟需寻找新型绿色、安全、高效的抗生素替代品。
抗菌肽与抗生素具有不同的抑菌机制，前者以“温和式”干扰多种生物性功能的发挥，而非特异性作用靶位点使其丧失功能，而后者倾向作用于菌体单一靶点。抗菌肽在动物体内无残留，对环境无污染，符合对虾安全生产的需要，适合在饲养过程中使用，具有作为新一代绿色饲料添加剂的潜质。目前，对抗菌肽的开发与应用已经在渔业生产和医药卫生领域广泛开展。
chrysophsin-1(CHY1)，一种分离于鱼鳃的嗜酸性颗粒细胞样细胞的抗菌肽，对鱼类和甲壳类动物的革兰氏阴性和革兰氏阳性病原体表现出强大的杀菌活性。具有以下特征：25个氨基酸长度、净正电荷数+9、疏水性氨氨基占48%、α-螺旋结构、耐高盐、溶血性高。CHY1直接用于生产还有改造空间，如缩短氨基酸长度、降低溶血性等。因此以天然抗菌肽为模板进行分子改造成为研发高效抗菌制剂的热点。
发明内容
本发明的目的在于提供一种耐盐防弧菌抗菌肽FH-18及其制备方法和应用；与天然抗菌肽CHY1相比，所述抗菌肽FH-18治疗指数更高，长度缩短，CHY1原有优势基本未变。
本发明的目的通过如下技术实现：一种抗菌肽FH-18，所述抗菌肽FH-18的氨基酸序列FFKHIIILWKHLIHRRRH-NH2，此序列末端羧基酰胺化连接NH2。
如上所述抗菌肽FH-18，制备方法如下：
（1）以CHY1为母板，首先删除序列残基Ala和Gly，缩短成18个氨基酸长度肽；其次在18个氨基酸肽链中，Trp3与Lys9，Leu4与His8，Lys6与Ile10位置互换；最后Ｃ末端酰胺化而生成抗菌肽FH-18，氨基酸序列FFKHIIILWKHLIHRRRH-NH2；
（2）采用固相化学合成法通过多肽合成仪得到肽树脂，将得到的肽树脂经过TFA切割后，得到抗菌肽FH-18；
（3）经过反相高效液相色谱纯化后，即完成抗菌肽FH-18的制备。
如上所述的一种抗菌肽FH-18在制备治疗弧菌属感染性疾病药物中的应用。
通过本方法制备的抗菌肽FH-18的实验技术简单，对得到的抗菌肽FH-18进行抗菌和溶血活性检测，发现抗菌肽FH-18溶血活性降低，长度缩短7个氨基酸，保留耐高盐、杀弧菌能力强的优势。综上所述，FH-18是一种具耐高盐防弧菌高应用价值的抗菌肽，将可促进无抗养殖背景下对虾安全出池。
附图说明
图1为CHY1和FH-18的序列特征。
图2为CHY1和FH-18的螺旋轮图。
图3为CHY1和FH-18的抑菌活性。
图4为CHY1和FH-18的溶血活性、治疗指数及盐稳定性。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1：一种抗菌肽FH-18，所述抗菌肽FH-18的氨基酸序列FFKHIIILWKHLIHRRRH-NH2，此序列末端羧基酰胺化连接NH2。
该抗菌肽FH-18的设计：
依据CHY1序列，首先因α-螺旋结构中3.6个氨基酸旋转1圈(25/3.6≈6.94)，则删除7个残基确保母板C端的RRRH结构位置不变而有利于发挥杀菌作用和节约氨基酸用量或合成成本；而删除Ala残基对抗菌肽疏水性影响不明显，删除Gly残基有利于保持结构稳定，同时两者删除更可保持净电荷数不变。氨基酸序列特征见附图1所示。
其次，在缩短至18个氨基酸序列中，Trp3与Lys9，Leu4与His8，Lys6与Ile10位置互换，以形成双亲面，同时保持破坏肽的α-螺旋的双亲性结构特征，可降低螺旋度而降低模板肽高溶血活性和维持强杀菌活性。如附图1和附图2所示。
实施例2：如实施例1所述抗菌肽FH-18的制备方法，采用固相化学合成法合成抗菌肽FH-18。
1、抗菌肽FH-18的制备从C端到N端逐一进行，通过多肽合成仪来完成。首先将Fmoc-X(X是每个抗菌肽的C端第一个氨基酸)接入到Wang树脂，然后脱去Fmoc基团后得到X-Wang树脂；再将Fmoc-Y-Trt-OH(9-芴甲氧羧基-三甲基-Y，Y为每个抗菌肽C端第二个氨基酸)；按照这个程序依次从C端合成到N端，直至合成完毕，得到脱去Fmoc基团的侧链保护的树脂；
2、在上述得到的肽树脂中，加入切割试剂，20℃避光下反应2h，过滤；沉淀TFA(三氟乙酸)洗涤，将洗液与上述滤液混合，旋转蒸发仪浓缩，再加入10倍左右体积的预冷无水乙醚，-20℃沉淀3h，析出白色粉末物，以2500g离心10min，收集沉淀，再用无水乙醚洗涤沉淀，真空干燥，得到多肽，其中切割试剂由TFA、水和TIS（三异丙基氯硅烷）按照质量比95:2.5:2.5混合而成；
3、使用0.2mol/L硫酸钠（磷酸调节至pH7.5）进行柱平衡30min，用90%乙腈水溶液溶解多肽，过滤，C18反相常压柱，采用梯度洗脱（洗脱剂为甲醇和硫酸钠水溶液按照体积比为30:70~70:30混合），流速为1mL/min，检测波为220nm，收集主峰，冻干；再利用反相C18柱进一步纯化，洗脱液Ａ为0.1%TFA/乙腈溶液；洗脱液Ｂ为0.1%TFA/水溶液，洗脱浓度为50％~90%，洗脱时间为30min，流速为1mL/min，再同上收集主峰，冻干，即得纯化抗菌肽FH-18；
4、抗菌肽FH-18的鉴定：将上述得到的抗菌肽FH-18经过电喷雾质谱法分析，理论分子量与实测分子量基本一致，抗菌肽FH-18的纯度大于95%。
实施例3：如实施例1所述抗菌肽FH-18在制备治疗弧菌属感染性疾病药物中的应用。
该抗菌肽FH-18在应用中生物活性的测定。
1、抗菌活性的测定：将抗菌肽FH-18配置成浓度为2.56mM储存液以备使用。利用微量肉汤稀释法测定FH-18和CHY1的最小抑菌浓度。以0.01%乙酸（含0.2%BSA）作为稀释液，使用二倍稀释法依次配置系列梯度的FH-18和CHY1溶液。取上述溶液100μL置于96孔细胞培养板中，然后分别添加等体积的待测菌液（~105个/mL）于各孔中。分别设置阳性对照（含有菌液而不含有FH-18和CHY1）和阴性对照（既不含菌液也不含FH-18和CHY1）。37℃恒温培养20h，以肉眼未见孔底部有混浊现象的即为最小抑菌浓度。
结果如附图3和附图4所示，FH-18对于几种弧菌表现出不同程度的抑菌活性，与CHY1相比，FH-18对弧菌抑菌活性有提高。从最小抑菌浓度的几何平均数分析而言，FH-18的抑菌效果增加，说明分子设计保留了原肽的高抗菌活性。
2、溶血活性的测定：采集人的新鲜血液1mL，肝素抗凝后溶解到2mL的PBS溶液中，1000g离心5min，收集红细胞；用PBS洗涤3遍，再用10mL的PBS重悬；取50µL红细胞悬液与50µL用PBS溶解的不同浓度的FH-18和CHY1溶液混合均匀，在37℃培养箱内恒温孵育1h；取出后，4℃、1000g离心5min；取出上清液用酶标仪在570nm处测光吸收值；每组取平均值，并比较分析。其中50µL红细胞加50µL的PBS作为阴性对照；50µL红细胞加50µL0.1%的Tritonx-100作为阳性对照。最小溶血浓度是FH-18和CHY1引起5%溶血率时的浓度。
如附图4所示，FH-18在64μM时表现出溶血活性，而对照CHY1在0.25μM浓度时就表现出高的溶血活性，说明FH-18的设计明显降低细胞毒性。另外FH-18保留了CHY1的耐盐性。
综上结果显示，与CHY1相比，FH-18的抗菌活性有所增强，溶血性更低。综合分析抗菌肽的抑菌和溶血活性，可以通过治疗指数（溶血浓度与抑菌浓度的比值）来更全面的评价各个抗菌肽的生物学活性，如附图4所示，FH-18具有更高的治疗指数26.7，是CHY1的722倍。因而设计得到的FH-18抗菌肽具有较高的替代抗生素的发展潜力。
Claims (3)
1.一种抗菌肽FH-18，其特征在于：所述抗菌肽FH-18的氨基酸序列FFKHIIILWKHLIHRRRH-NH2，此序列末端羧基酰胺化连接NH2。
2.如权利要求1所述的一种抗菌肽FH-18的制备方法，其特征在于：制备方法如下：
（1）以CHY1为母板，首先删除序列残基Ala和Gly，缩短成18个氨基酸长度肽；其次在18个氨基酸肽链中，Trp3与Lys9，Leu4与His8，Lys6与Ile10位置互换；最后Ｃ末端酰胺化而生成抗菌肽FH-18，氨基酸序列FFKHIIILWKHLIHRRRH-NH2；
（2）采用固相化学合成法通过多肽合成仪得到肽树脂，将得到的肽树脂经过TFA切割后，得到抗菌肽FH-18；
（3）经过反相高效液相色谱纯化后，即完成抗菌肽FH-18的制备。
3.如权利要求1所述的一种抗菌肽FH-18在制备治疗弧菌属感染性疾病药物中的应用。
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