CN117430676B - 一种羊毛硫肽类抗菌肽及其应用 - Google Patents
一种羊毛硫肽类抗菌肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种羊毛硫肽类抗菌肽，其结构为：核心肽‑羊毛硫肽基因簇，所述羊毛硫肽基因簇前和基因簇之间插入核糖体结合位点序列，所述核心肽的核苷酸序列如SEQ ID NO.1或SEQ ID NO.1的保守替代序列所示。本发明使用的枯草芽孢杆菌对培养基的要求比较简单，在生产中绝对占有经济优势，噬菌体、质粒都可作为克隆载体，并且，提供的羊毛硫肽具有显著的抑菌活性，可应用于医药、医疗等抗生素替代领域，与现有抗生素相比，其抑菌活性高，制备成本低，具有较好的应用前景。
Description
技术领域
本发明涉及肽类抗生素的应用领域，特别是涉及一种羊毛硫肽类抗菌肽及其应用。
背景技术
抗菌肽是生物体内经诱导产生的一类具有生物活性的小分子多肽，分子量在 3~5kDa 之间，具有热稳定性及广谱抗菌特性。目前全球为抵御致病菌而导致的抗生素乱用问题日益严重，为此寻找一些新型和环境友好型兼备的抗菌物质代替抗生素已成为研究热点。抗菌肽可以快速有效地杀死具有抗生素抗性的细菌，被认为是传统抗生素的最佳替代物。
羊毛硫肽是一类作用于细胞膜上的新型抗菌肽，主要结构特点为含有非编码的翻译后修饰氨基酸–羊毛硫氨酸(Ala)、甲基羊毛硫氨酸、β-甲基羊毛硫氨酸(Aba)和脱氢丙氨酸等(Dha) ，一般大小在10-50个氨基酸，结构上存在由半胱氨酸与脱氢丙氨酸脱氢氨基丁酸形成的含硫羊毛硫氨酸和甲基羊毛硫氨酸环状结构。大部分羊毛硫肽还包含其他的修饰残基或环状结构，例如美杀菌素(mersacidin)和表皮菌素(epidermin)中的 2-硫乙胺基团，肉桂霉素(cinnamycin)中的赖丙氨酸和羟基天冬氨酸，乳杆菌素(lacticin) S 和乳杆菌素 3147 中的 D-丙氨酸，Pep5 和乳杆菌素在 N 端的氧代乙酰及放线菌合成microbisporicin 抗生素中的羟脯氨酸等，此类抗生素近年来才逐渐被人们发现、了解并开发。
但是羊毛硫肽类抗菌肽通常存在抗菌谱较窄和抗逆性不足的缺点，因此开发研究新型高效的抗逆性较强的羊毛硫肽类抗菌肽具有重要的价值。随着高通量测序技术的快速发展，基于大量的微生物基因组数据显示，枯草芽孢杆菌在合成多种新颖的羊毛硫肽类抗菌肽方面，其潜力远超以往的认知。枯草芽孢杆菌中存在很多编码羊毛硫肽类抗菌肽的生物合成基因簇，这些基因簇在野生型菌株通常是沉默的，通过异源表达的方法实现这些羊毛硫肽类抗菌肽的生产对于研发新型高效的羊毛硫肽类抗菌肽具有重要意义。
发明内容
本发明的目的在于克服现有技术的至少一个不足，提供一种羊毛硫肽类抗菌肽及其应用。
本发明所采取的技术方案是：
第一个方面，本发明提供一种羊毛硫肽类抗菌肽，其结构为：前导肽-核心肽-羊毛硫肽基因簇，所述羊毛硫肽基因簇前和基因簇之间插入核糖体结合位点序列，所述导肽-核心肽的核苷酸序列如SEQ ID NO.1或SEQ ID NO.1的保守替代序列所示。
在一些实例中，所述羊毛硫肽基因簇为含修饰酶的LanM基因和含ABC转运子基因LanT基因。
在一些实例中，所述核糖体结合位点插入于LanM基因之前以及LanM基因和LanT基因之间，所述的核糖体结合位点的核苷酸序列如SEQ ID NO.3所示。
在一些实例中，所述羊毛硫肽基因簇的核苷酸序列如SEQ ID NO.4所示。
第二个方面，本发明提供一种基因，编码第一个方面任一项所述的羊毛硫肽类抗菌肽。
在一些实例中，所述基因的核苷酸序列如SEQ ID NO.5所示
第三个方面，本发明提供一种载体，其含有第二个方面所述的基因。
第四个方面，本发明一种基因工程菌，可表达第一个方面所述的羊毛硫肽类抗菌肽。
在一些实例中，所述基因工程菌选自枯草芽孢杆菌。
第五个方面，本发明第一个方面所述的羊毛硫肽类抗菌肽在制备抗细菌制剂中的应用。
本发明的有益效果是：
本发明提供的具有SEQ ID NO.1所示核苷酸序列为新发现序列，之前未有报道，提供的羊毛硫肽对金黄色葡萄球菌和铜绿假单胞菌株具有显著的抑菌活性，可应用于医药、医疗等抗生素替代领域，与现有抗生素相比，其抑菌活性高，制备成本低，具有较好的应用前景。
本发明使用的枯草芽孢杆菌 (B.Subtilis) 作为外源基因的表达宿主，具有如下优点 ：(1) B.Subtilis 是好氧菌不含热源脂多糖 (lipopolysaccharides，LPSs)，有益菌无致病性 ；B.Subtilis被美国FDA认可为安全性菌株。(2)B.Subtilis细胞膜为单层，而大肠杆菌的为双层，分泌的蛋白在 B.Subtilis 体系下比在大肠杆菌体系下更容易回收、纯化，操作简便，许多酶都是 B.Subtilis 产生的分泌到胞外的蛋白。(3) 长期的研究表明，B.Subtilis 没有明显的密码子偏爱性。(4)B.Subtilis 对培养基的要求比较简单，在生产中绝对占有经济优势，噬菌体、质粒都可作为克隆载体，B.Subtilis 有良好的转录翻译系统，可以避免形成错误折叠的蛋白质、包涵体。
附图说明
图1为实施例1的羊毛硫肽重组表达载体图。
图2为金黄色葡萄球菌ATCC6538抑菌活性，W为野生型，S为羊毛硫肽基因工程菌发酵液上清，M为羊毛硫肽基因工程菌菌体超声上清。
图3为铜绿假单胞菌株CMCC(B)10104抑菌活性，W为野生型，C为空白发酵培养基，M为羊毛硫肽基因工程菌菌体超声上清。
具体实施方式
下文的公开提供了许多不同的实施方式或例子用来实现本发明的不同方案。
实施例
1）羊毛硫肽基因工程菌的建立
将自然土壤环境筛选获得的枯草芽孢杆菌测序，通过ORF finder分析，寻找可能的开放阅读框，并对其进行在线Blast比对，获得羊毛硫肽基因簇，为含修饰酶基因LanM和ABC转运子基因LanT的第二类羊毛硫肽。
前导肽和核心肽基因序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示；羊毛硫肽基因簇LanM和LanT如SEQ ID NO.4所示。
人工合成羊毛硫肽：前导肽-核心肽-RBS-LanM-RBS-LanT 核苷酸序列，如SEQ IDNO.5所示，核糖体结合位点序列（RBS）如SEQ ID NO.3所示。
在序列两端分别插入BglII和BamHI内切酶位点，将合成基因与pHY-P43分别用BglII和BamHI进行双酶切，回收酶切产物并进行T4连接，将连接产物转化DH5α，50μg/mLAmp筛选阳性菌并测序验证，获得表达载体pHY-P43-LanMT，表达载体图谱见图1。
2）羊毛硫肽基因工程菌重组表达转化体的获得
将前述重组表达载体pHY-P43-LanMT转化至枯草芽孢杆菌WB800N中，即可获得重组表达转化体。转化方法电转法：将感受态从-80℃冰箱中取出放在冰上，将500ng表达载体与80uL感受态混合后冰上孵育2min，加入预冷的电转杯（1mm）中，电击一次。电转仪设置：2.0kv，1mm,电击1次。电击完毕取出杯子并立即加入1ml RM（LB+0.5M山梨醇+0.38甘露醇），37℃，200rpm，复苏3h后涂布于含有50μg/mL四环素的LB固体培养基上，倒置培养过夜获取单克隆，扩培后用20%终浓度甘油保存于-80℃，测序分析验证正确后，即为重组表达转化体，命名WB800N-LanMT。
3）羊毛硫肽基因工程菌的发酵培养
将重组表达转化体WB800N-LanMT接种于在LB液体培养基(添加50μg/ml四环素)中培养，37℃和220rpm下过夜培养；将培养物以1:100比例转接入50mL新鲜LB培养基(每升成分为蛋白胨10g±0.1；酵母提取物 5 g±0.1；NaCl 10g±0.1，250mL摇瓶)中，并在37℃下生长24h。当600nm处的光密度(OD600)达到约6时，12000rpm、4℃离心10min，弃上清，得到羊毛硫肽基因工程菌，菌体储存于-20℃。
4）重组表达羊毛硫肽的高密度发酵制备
接种重组突变菌株WB800N-LanMT于3mL液体LB培养基中，37℃、220rpm震荡过夜培养后，按1%左右的比例接种于400mL液体LB培养基中，培养至OD600达到4时作为种子液，接入2L的发酵培养基中进行高密度发酵。
初始温度37℃，搅拌速度300rpm，通气量1.5vvm，pH为6.8，之后不断提高搅拌转速最大至1000rpm。培养4小时左右，培养基中的碳源消耗完全，按照DO进行反馈补料。补料后温度降为25℃，溶氧保持30%以上，补料24小时后放罐。使用离心机8000rpm离心10min弃上清得到的菌体湿细胞，即得到羊毛硫肽。
羊毛硫肽抑菌活性测试
采用双层牛津杯抑菌圈法测定抗菌肽的抑菌活性。向平板倒10 mL LB固体培养基，凝固后，将牛津杯垂直放置于培养基表面。
挑金黄色葡萄球菌ATCC6538和铜绿假单胞菌株CMCC(B)10104单菌落于37℃、180rpm 摇瓶培样5-6h，菌液OD600约0.4，分别吸取菌悬液以1：1000加入冷却至50℃的LB固体培养基中摇匀，倒15mL至放有牛津杯的平板表面。静置凝固，取出牛津杯，用移液枪吸取100uL测试液加入到孔中。37℃培养箱静置培养16-17h观察抑菌情况并测定其抑菌圈直径。
金黄色葡萄球菌ATCC6538抑菌活性见图2，W：野生型（抑菌圈0 cm），S：羊毛硫肽基因工程菌发酵液上清（抑菌圈2.86 cm），M：羊毛硫肽基因工程菌菌体超声上清（抑菌圈2.74cm）。
铜绿假单胞菌株CMCC(B)10104抑菌活性见图3，W：野生型（抑菌圈0 cm），C：空白发酵培养基（抑菌圈0cm），M：羊毛硫肽基因工程菌菌体超声上清（抑菌圈1.5 cm）。
羊毛硫肽基因工程菌对金黄色葡萄球菌ATCC6538和铜绿假单胞菌株CMCC(B)10104均具有抑菌活性，对金黄色葡萄球菌的抑菌活性更强。
以上是对本发明所作的进一步详细说明，不可视为对本发明的具体实施的局限。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的简单推演或替换，都在本发明的保护范围之内。
Claims (2)
1.一种羊毛硫肽类抗菌肽，其特征在于，所述羊毛硫肽类抗菌肽的制备方法为：构建含有如SEQ ID NO.5所示的核苷酸序列的载体，然后将该载体转化至基因工程菌中，发酵培养基因工程菌即得到所述羊毛硫肽类抗菌肽，其中，所述基因工程菌选自枯草芽孢杆菌。
2.权利要求1所述的羊毛硫肽类抗菌肽在制备抗细菌制剂中的应用，其特征在于，所述细菌选自金黄色葡萄球菌或铜绿假单胞菌。
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| CN106366168A (zh) * | 2016-08-26 | 2017-02-01 | 上海交通大学 | 羊毛硫肽类抗菌肽及其脱氢衍生物的制备方法 |
| CN112812157A (zh) * | 2021-02-03 | 2021-05-18 | 河北省科学院生物研究所 | 一种羊毛硫肽AmylocinA3及其制备方法和应用 |
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| CN116732073A (zh) * | 2022-03-01 | 2023-09-12 | 中国科学院深圳理工大学(筹) | 羊毛硫抗生素高通量筛选方法 |
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| CN106366168A (zh) * | 2016-08-26 | 2017-02-01 | 上海交通大学 | 羊毛硫肽类抗菌肽及其脱氢衍生物的制备方法 |
| CN112812157A (zh) * | 2021-02-03 | 2021-05-18 | 河北省科学院生物研究所 | 一种羊毛硫肽AmylocinA3及其制备方法和应用 |
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| CN115975047A (zh) * | 2022-10-24 | 2023-04-18 | 扬州奥锐特药业有限公司 | 一种重组融合蛋白生产多肽的方法及其应用 |
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