CN110079531A - 一种抗菌肽及其应用 - Google Patents
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Abstract
本发明公开了一种抗菌肽，该抗菌肽的基因序列如序列表SEQ ID No.1所示；该抗菌肽的氨基酸序列如序列表SEQ ID No.2所示；该抗菌肽的成熟多肽序列如序列表SEQ ID No.3所示。本发明从粘虫体内鉴定了一种抗菌肽，并进行了体外化学合成，发现体外制备的抗菌肽Cecropin对大肠杆菌具有较好的抑菌效果，为开发治疗大肠杆菌的抗生素替代物提供基础，进而减少因大量抗生素的使用对畜产品和环境造成的污染。
Description
技术领域
本发明属于药物应用技术领域，具体涉及一种抗菌肽及其应用。
背景技术
抗菌肽（Antimicrobial peptides, AMPs）是一种小分子物质，广泛存在于生物体内，是机体免疫的一道重要屏障，可以直接或通过调节机体免疫系统间接杀死病原微生物。研究表明，AMPs具有抗病毒、抗细菌、抗真菌、抗原虫、抗肿瘤、参与免疫调节等多种生物活性。抗菌肽具有多种特点，如自身分子小、水溶性强、稳定性好等，同时不具备抗原性，但有些AMPs具有溶血活性等缺点。天蚕素是最早被发现的AMPs。此后，多种AMPs被研究人员鉴定，AMPs的提取、体外表达、抗菌机制等也受到科学家们的关注和研究。
近些年，抗生素被广泛应用于畜牧养殖、临床医疗中，导致耐药细菌、超级细菌不断出现，抗生素对饮用水、土壤、肉制品等造成污染，进而严重威胁人类的健康。AMPs通常通过感染细菌细胞膜的通透性、作用于细菌DNA、干扰细菌蛋白质的合成等抑制细菌，其独特的抗菌机制不易导致细菌产生耐药性，而且其作为一种小分子多肽不会对机体产生药物残留等副作用，对机体安全性较高。许多研究已经表明，AMPs可以替代抗生素发挥抗菌作用。
大肠杆菌（Escherichia coli）作为一种重要的人畜共患细菌病，在临床上通常导致人和动物产生急性腹泻、多器官炎症，甚至引起败血症，严重威胁畜牧业健康发展和公共卫生安全。临床上大量使用青霉素、链霉素等抗生素，导致一些耐药菌株的出现，从而需要不断加大用药剂量和用药疗程，对环境形成较大的污染和危害。因此，AMPs是治疗和预防大肠杆菌较为理想的抗生素替代物。
发明内容
本发明的目的在于：提供一种抗菌肽，且该抗菌肽对大肠杆菌具有明显的抑制效果。
本发明的目的是以下述方式实现的：
一种抗菌肽，该抗菌肽的基因序列如序列表SEQ ID No.1所示；
该抗菌肽的氨基酸序列如序列表SEQ ID No.2所示；
该抗菌肽的成熟多肽序列如序列表SEQ ID No.3所示。
所述抗菌肽用于制备抗菌剂。
所述抗菌肽用于制备大肠杆菌的抑制剂。
所述抗菌肽用于制备动物用饲料添加剂或食品添加剂。
本发明从粘虫体内鉴定了一种抗菌肽，并进行了体外化学合成，发现体外制备的抗菌肽Cecropin对大肠杆菌具有较好的抑菌效果，为开发治疗大肠杆菌的抗生素替代物提供基础，进而减少因大量抗生素的使用对畜产品和环境造成的污染。
相对于现有技术，本发明的积极效果是：
1、本发明提供了一种新的抗菌肽。
2、本发明体外化学合成该抗菌肽，具有制备简单、快速等优点，为以后抗菌肽的体外化学合成提供支撑。
3、本发明公开的抗菌肽可有效地抑制大肠杆菌的增殖，在大肠杆菌病的治疗中有望替代抗生素的使用，减少肉蛋奶中的抗生素残留，降低抗生素对环境的污染。
附图说明
图1为抗菌肽基因序列、氨基酸序列及其成熟肽的裂解位点，下划线部分为信号肽序列，方框部分为翻译后剪切掉的氨基酸残基，剩余的39个氨基酸为成熟肽序列。
图2为抗菌肽去除核定位信号区化学合成后的质谱分析报告。
图3为抗菌肽去除核定位信号区化学合成后高效液相色谱法分析报告。
图4为抗菌肽对大肠杆菌的抑制效果。1：100 µg/mL卡那霉素；2：500 µg/mL抗菌肽；3：100 µg/mL氨苄青霉素；4：0.9% NaCl溶液；5：100 µg/mL抗菌肽；6：1000 µg/mL抗菌肽。
图5为抗菌肽对大肠杆菌最小抑菌浓度检测结果。
具体实施方式
下面结合附图具体介绍本发明涉及的一种新抗菌肽及其制备方法和应用。本领域技术人员应该理解，下面描述的实施例只是为了更好地理解本发明，并不用来限制本发明。
本发明提供的新抗菌肽来自于粘虫，将田间采集的粘虫送测序公司进行测序，通过对粘虫转录组测序获得的序列进行比对，发现该抗菌肽基因序列。如图1所示，新抗菌肽编码基因长度为192 bp，该抗菌肽含有63个氨基酸残基，与同类抗菌肽比较发现，该抗菌肽含有保守的裂解位点，成熟的抗菌肽为39个氨基酸残基。
如图2和3所示，根据该抗菌肽裂解后的有效序列，体外化学合成多肽序列，通过质谱分析和高效液相色谱法分析结果表明，化学合成多肽与含有39个氨基酸残基的成熟多肽序列一致。
如图4所示，通过平板打孔法检测抗菌肽活性，结果表明，化学合成的抗菌肽对大肠杆菌具有明显的抑制效果。
如图5所示，通过刃天青作为指示剂检测最低抑菌浓度，结果表明，化学合成的抗菌肽对大肠杆菌的最低抑菌浓度为15.6 µg/mL。
下述实施例中的主要原材料来源为：抗菌肽的化学合成与检测由吉尔生化（上海）有限公司完成。大肠杆菌（ATCC25922）购自公司。刃天青等其他试剂和材料均可来源于商业途径。其它试剂和材料所述的实验方法，若无特别说明，均为常规实验方法。
本发明公开的一种抗菌肽及其制备方法和应用，具体实施方法如下：
1.1 抗菌肽的发现
本发明公开的抗菌肽为粘虫新型抗菌肽，通过以下方法获得：以粘虫转录组为数据库，根据已鉴定的抗菌肽序列用BLASTn筛选找到本发明公开的抗菌肽ORF的cDNA序列，并推导出氨基酸序列，根据文献报道综合分析，发现抗菌肽含有保守的裂解位点AP，从而获得成熟的抗菌肽序列（39个氨基酸残基），如图1所示。
1.2抗菌肽的合成
将获得的成熟抗菌肽序列送吉尔生化（上海）有限公司合成，通过质谱分析（如图2所示）和高效液相色谱法分析（如图3所示），发现该成熟抗菌肽可以体外化学合成，而且合成纯度可达98%。
1.3 平板打孔法测抗菌肽的抑菌活性
配制常规固体LB培养基，高压灭菌，取出冷却至约50 ℃时，按1：100体积比加入过夜培养的大肠杆菌菌液，如培养基50 mL，应加入菌液500 µL，摇匀后倒高压过的平皿，完全凝固后用打孔器均匀打孔，然后在各个孔中加入不同浓度的抗菌肽溶液、阳性对照抗生素溶液和阴性对照蒸馏水。将平皿放入37 ℃温箱培养12小时，观察抑菌效果，如图4所示，加入100µg/mL卡那霉素的孔1和加入100 µg/mL氨苄青霉素孔3出现抑菌效果表明阳性对照成立，加入0.9% NaCl溶液的孔4没有抑菌效果表明阴性对照成立，加入100 µg/mL抗菌肽的孔5没有观察到抑菌效果，加入500 µg/mL抗菌肽的孔2和加入1000 µg/mL抗菌肽的孔6出现抑菌效果，表明该体外合成的抗菌肽对大肠杆菌具有抑菌作用。
1.4 最低抑菌浓度的测定
用无菌NaCl溶液（浓度为0.9%）配制浓度为1000 µg/mL的抗菌肽溶液，采用二倍稀释法在96孔板中稀释抗菌肽，每孔50 µL。将大肠杆菌培养至对数生长期，通过菌落计数法确定菌液浓度，然后用无菌液体LB培养基将菌液稀释成2 × 105 CFU/mL。取50 µL稀释好的菌液加入含有不同浓度抗菌肽的孔中。每个抗菌肽浓度设置4个复孔，同时设置NaCl溶液+无菌LB和NaCl溶液+菌液两组作为阴性对照，氨苄青霉素+菌液作为阳性对照。在37 ℃温箱培养16小时，然后每孔加入10 µL 6 mmol /L的刃天青溶液，继续放入37 ℃温箱培养3小时，观察孔中的颜色变化，如图5所示，NaCl+LB组所有孔均是蓝色，NaCl+菌液组所有孔均是红色，说明阴性对照成立；Amp+菌液组所有孔均是蓝色，说明阳性对照成立；四个抗菌肽+菌液的重复组都在抗菌肽浓度为1000×2-7 µg/mL的孔中颜色变为红色，表明该抗菌肽对大肠杆菌的最小抑菌浓度为1000×2-6 µg/mL，即15.6µg/mL。
以上所述的仅是本发明的优选实施方式，应当指出，对于本领域的技术人员来说，在不脱离本发明整体构思前提下，还可以作出若干改变和改进，这些也应该视为本发明的保护范围。
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Claims (4)
1.一种抗菌肽，其特征在于：该抗菌肽的基因序列如序列表SEQ ID No.1所示；
该抗菌肽的氨基酸序列如序列表SEQ ID No.2所示；
该抗菌肽的成熟多肽序列如序列表SEQ ID No.3所示。
2.如权利要求1所述的抗菌肽的应用，其特征在于：所述抗菌肽用于制备抗菌剂。
3.如权利要求2所述的抗菌肽的应用，其特征在于：所述抗菌肽用于制备大肠杆菌的抑制剂。
4.如权利要求1所述的抗菌肽的应用，其特征在于：所述抗菌肽用于制备动物用饲料添加剂或食品添加剂。
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