CN107857803A - 天然抗菌肽及其应用 - Google Patents
天然抗菌肽及其应用 Download PDFInfo
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- CN107857803A CN107857803A CN201711261443.6A CN201711261443A CN107857803A CN 107857803 A CN107857803 A CN 107857803A CN 201711261443 A CN201711261443 A CN 201711261443A CN 107857803 A CN107857803 A CN 107857803A
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- Prior art keywords
- antibacterial peptide
- antibacterial
- peptide
- gene
- pichia pastoris
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Abstract
本发明公开了一组来源于真菌的天然抗菌肽，其氨基酸序列分别如SEQ ID NO：3‑4所示。通过检索真菌蛋白数据库，筛选分析获得抗菌肽成熟肽序列，通过对比分析，该组天然抗菌肽与目前已知所有抗菌肽的氨基酸序列存在明显差异，属于新型抗菌肽。该组抗菌肽经密码子优化后实现其在毕赤酵母中的重组表达。抗菌实验结果表明，本发明的抗菌肽对革兰氏阳性菌，尤其是金黄色葡萄球菌ATCC43300，猪链球菌CVCC 3928，CVCC 606具有显著抑菌活性，可应用于抗菌药物、食品添加剂、化妆品、饲料添加剂、防腐剂等领域，具有广阔的应用价值和市场前景。
Description
技术领域
本发明涉及生物医药领域，，具体地说，涉及一组新型的天然抗菌肽及其应用。
背景技术
抗生素对感染性疾病的治疗发挥了重要作用，但由于抗生素滥用，耐药菌株不断增加，现已出现多种超级细菌，病原菌耐药性问题已被世界卫生组织(WHO)列为未来人类健康面临的主要威胁和挑战之一。金黄色葡萄球菌是人类重要病原菌，常引起食物中毒和伤口感染，在动物养殖业中主要引起牛、羊的乳房炎。原料奶中金黄色葡萄球菌易产生金黄色葡萄球菌肠毒素等有害物质而污染乳制品(等，EnterotoxigenicStaphylococcus aureus in bulk milk in Norway.Journal of AppliedMicrobiology.2005,99∶158-166.)，影响乳制品的质量安全，同时对消费者带来安全隐患。耐药性金黄色葡萄球菌(MRSA、VRSA等)能在畜禽和人之间实现交叉感染，很多引起人类感染的MRSA菌株最终发现均来源于动物体内。另外，有研究表明，耐药性金黄色葡萄球菌能在畜禽和人之间实现交叉感染，很多引起人类感染的MRSA菌株最终发现均来源于动物体内。因而寻找新型抗菌物质取代或辅助传统抗生素以应对细菌耐药性问题至关重要。
抗菌肽(antimicrobial peptide)是广泛存在于生物体内具有抗菌活性的多肽，形成生物体第一道先天防御系统，对细菌、真菌、病毒和原虫等均具有杀灭作用。在诸多的抗菌肽中，真菌防御素的研究正逐渐受到关注。真菌防御素对革兰氏阳性细菌具有独特高效的杀菌作用，与传统抗生素之间不存在交叉耐受性，且对人的细胞没有毒害作用。真菌防御素易于通过基因表达进行大量生产，解决了抗菌肽生产成本高的问题。然而，现存的抗菌肽中，仅有0.46％来自真菌。因此，寻找真菌中天然抗菌肽并进行制备是本发明研究的重要内容。
发明内容
本发明的目的是提供一组新型的天然抗菌肽及其应用。
为了实现本发明目的，本发明提供了两个天然抗菌肽P5、P6，分别来自于尖端赛多孢子菌(Scedosporium apiospermum)和伊蒙微小菌(Emmonsia parva UAMH 139)。
本发明还提供上述抗菌肽的编码基因，多肽P5、P6编码基因的核苷酸序列分别如SEQ ID NO：5-6所示。
本发明还提供表达盒、表达载体、克隆载体、转基因细胞系或工程菌，其包括包含编码所述抗菌肽基因序列的核酸。
本发明还提供一种重组毕赤酵母，其是用携带有所述抗菌肽编码基因的表达载体(例如pPICZαA)转化毕赤酵母工程菌，筛选阳性克隆得到的。
在本发明的一个具体实施方式中，所述重组毕赤酵母的构建如下：所述抗菌肽的编码基因经密码子优化后，在优化后的基因序列(分别如SEQ ID NO：5-6所示)的5’端添加XhoI酶切位点和Kex2切割位点，在3’端添加TAA和TAG终止子序列以及XbaI酶切位点，得到基因构建物，再将所述基因构建物插入载体pPICZαA的XhoI和XbaI酶切位点之间构建得到重组表达载体，最后用所述重组表达载体转化毕赤酵母菌X-33，筛选阳性克隆。
本发明还提供利用所述重组毕赤酵母发酵生产天然抗菌肽的方法，其是将上述的重组毕赤酵母发酵培养，分泌产生抗菌肽。
本发明还提供所述抗菌肽在制备广谱抗菌药物或组合物中的应用。
本发明还提供由所述抗菌肽制备的广谱抗菌药物或组合物。其中，所述菌为革兰氏阳性菌，包括但不限于金黄色葡萄球菌、猪链球菌。
本发明进一步提供所述抗菌肽在食品添加剂、化妆品、饲料添加剂领域中的应用。
本发明首次提供了一组来源于烧土火丝菌的天然抗菌肽及其在抗菌方面的应用，尤其针对革兰氏阳性菌具有很好的杀菌效果，尤其是金黄色葡萄球菌ATCC43300，猪链球菌CVCC 3928，CVCC 606具有显著抑菌活性，可应用于抗菌药物、食品添加剂、化妆品、饲料添加剂、防腐剂等领域，具有广阔的应用价值和市场前景。
附图说明
图1为本发明实施例5中P5-P6摇瓶表达120h Tricine-SDS-PAGE电泳图，M：超低分子量蛋白Marker；A,B中1-6：分别为5，6号阳性转化子在摇瓶水平发酵0,24,48,72,96和120h上清(20μL)。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning：a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中使用的酶和试剂：限制性内切酶、TaqDNA聚合酶、T4DNA连接酶等分别购自Biolabs、Invitrogen和Promega公司。四种dNTP购自Promega公司。DNA和蛋白质分子量标准为Biolabs产品。其它常规试剂采用进口分装或国产分析纯。
以下实施例中涉及的培养基和缓冲液配方：
LB培养基：胰蛋白胨10g/L，酵母浸提取物5g/L，NaCl 10g/L；固体LB培养基则加入2％的琼脂糖。
低盐LB培养基：胰蛋白胨10g/L，酵母浸提取物5g/L，NaCl 5g/L；固体低盐LB培养基则加入2％的琼脂粉。
MH培养基：酪蛋白水解物17.5g/L，牛肉浸粉5g/L，淀粉1.5g/L。
MHA培养基：固体MH培养基则加入2％的琼脂粉。
YPD培养基：蛋白胨20g/L，酵母浸提取物10g/L，葡萄糖20g/L；固体YPD培养基则加入2％琼脂粉。
YPDS培养基：蛋白胨20g/L，酵母浸提取物10g/L，山梨醇182.2g/L，葡萄糖20g/L，琼脂粉20g/L。
有关LB培养基、低盐LB、MH、YPD、YPDS等培养基的使用参照Invitrogen公司提供的毕赤酵母操作手册。
20mM磷酸盐缓冲液(A液)：0.4654g Na2HPO4，2.9172gNaH2PO4，加去离子水至950mL，置于磁力搅拌器至完全溶解后调pH5.7，定容至1000mL。
1M NaCl 20mM磷酸盐缓冲液(B液)：0.4654g Na2HPO4，2.9172g NaH2PO4，58.44gNaCl，加去离子水至950mL，置于磁力搅拌器至完全溶解后调pH 5.7，定容至1000mL。
以下实施例中涉及的基因扩增及转化子鉴定方法为PCR法及DNA测序法。
以下实施例中涉及的蛋白检测方法为Tricine-SDS-PAGE，参照(H.Tricine-SDS-PAGE.Nat protoc,2006,1(1)：16-22)。
以下实施例中涉及的蛋白质浓度测定方法为考马斯亮蓝法。
以下实施例中涉及的蛋白分子量的确定方法为MALDI-TOF MS法。
以下实施例中涉及的蛋白纯化的方法为基于离子层析法。
以下实施例中涉及的菌种和质粒见表1。这些菌种是公众可以得到的，无需进行保藏。
表1供试菌种和质粒
实施例1天然抗菌肽与Plectasin类似真菌防御素的发现
通过检索，从UniProt数据库(http：//www.uniprot.org/)真菌蛋白组数据中发现来源于尖端赛多孢子菌(Scedosporium apiospermum)和伊蒙微小菌(Emmonsia parvaUAMH 139)的两条多肽序列(A0A084GET2，A0A0H1BJF3)。该组多肽序列及其功能区域分别如SEQ ID NO：1-2和SEQ ID NO：3-4所示。
将该组多肽全长基因序列用NCBI网站的blastX软件(https：//blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastx&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome)进行序列比对分析结果表明，该基因的编码产物可能为真菌防御素家族抗菌肽前体。进一步将该组基因编码的多肽前体序列用NCBI网站的protein blast软件(https：//blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastp&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome)进行序列比对，并与之前发现的其它真菌防御素家族抗菌肽序列进行比较分析，确定该组抗菌肽成熟过程中酶切位点为-R-G-，从而获得该组抗菌肽成熟肽序列SEQ ID NO：3-4，命名为P5-P6。P5、P6与目前已知所有抗菌肽的氨基酸序列存在差异，属于新型抗菌肽。
实施例2抗菌肽P5、P6基因合成
依据毕赤酵母密码子偏爱性设计抗菌肽P5-P6基因序列(SEQ ID NO：5-6)。为保证表达过程中序列的完整性，在突变体基因序列5’端添加XhoI酶切位点和Kex2切割位点，在3’端添加TAA，TAG终止子序列和XbaI酶切位点。以上序列由上海生工生物工程股份有限公司完成。
实施例3毕赤酵母诱导型表达载体pPICP5～pPICP6的构建
用限制性内切酶XhoI和XbaI分别对合成的基因片段和载体pPICZαA进行双酶切，回收pPICZαA载体片段和抗菌肽基因片段，连接，得到载体pPICP5～pPICP6；
载体pPICP5～pPICP6详细构建过程：利用限制性内切酶XhoI和XbaI分别对合成的基因片段和pPICZαA进行双酶切，酶切体系和条件如下：
以上酶切体系加样完毕后于37℃反应3个小时，2％琼脂糖凝胶电泳检测，电泳条件：130V，30min。电泳完毕在紫外灯下利用手术刀分别切割载体片段与基因片段对应电泳条带并利用天根生物技术有限公司胶回收试剂盒进行DNA片段回收，按试剂盒提供说明书的相关细节进行操作。
利用琼脂糖凝胶电泳检测回收片段并使用定量软件(GeneTools)进行初步定量，按照片段/载体(5∶1)的摩尔比例使用T4DNA连接酶进行连接，体系和条件如下：
以上连接体系加样完毕后于16℃过夜连接，转化E.coli DH5α。转化操作细节如下：
1)连接产物加入100μL E.coli DH5α感受态细胞，冰浴30min；
2)42℃热激90s，立即冰浴2～3min；
3)加入900μL 37℃预热的低盐LB培养基，37℃，80-100rpm恢复培养1h；
4)4000rpm离心5min，吸去700μL上清；
5)重悬菌体后取100-200μL涂布含有25μg/mL Zeocin的LB低盐固体培养基；
6)37℃倒置培养12-16h。
挑取阳性转化子，根据基因序列设计引物，进行菌液PCR验证转化子正确性，PCR体系、条件如下：
PCR体系：
PCR条件：
2％琼脂糖凝胶电泳检测阳性转化子菌液PCR产物，电泳条件：130V，30min。15％甘油管保存含有重组表达载体的E.coli并提取质粒，为线性化电转P.pastoris准备，相关实验细节按照质粒提取试剂盒(天根生物科技有限公司)说明书操作。
实施例4含pPICP5～pPICP6重组酵母菌株的构建
(1)重组载体pPICP5～pPICP6的线性化
利用PmeI对诱导型重组表达载体pPICP5～pPICP6进行线性化，线性化体系和反应条件如下：
以上酶切体系加样完毕后于37℃反应3个小时，2％琼脂糖凝胶电泳检测，电泳条件：130V，30min。电泳完毕检测重组表达载体正确线性化后利用DNA回收试剂盒回收线性化的重组表达载体。
(2)线性化载体的毕赤酵母电转化与鉴定
1)挑取YPD平板上的X-33单菌落，接种至10mL YPD液体培养基，30℃，250rpm，过夜培养；
2)取50μL过夜培养液接种至100mL YPD液体培养基，30℃，250rpm，培养至OD600吸光值为1.2；
3)50mL培养物，4℃，4000rpm，5min离心后加入50mL无菌水重悬；
4)4℃，4000rpm，5min离心后加入25mL无菌水重悬；
5)4℃，4000rpm，5min离心后加入2mL 1M山梨醇重悬；
6)4℃，4000rpm，5min离心后加入100μL 1M山梨醇重悬后即为X-33感受态细胞(以上6步操作须在冰上操作，动作轻柔)；
7)预混80μL X-33感受态细胞和5-10μg线性化载体，转移至冰上预冷的0.2cm电转杯中，冰上放置5min后电转仪操作(1200V，25μF，400Ω)；
8)立即加入1mL 1M山梨醇，混匀；
9)30℃温育1-2h；
10)取100μL温育后的X-33细胞涂布含有100μg/mL Zeocin的YPDS平板，30℃倒置培养2-4天。
挑取YPDS平板上的单菌落接种至100μg/mL Zeocin的YPD液体培养基中，30℃，250rpm，过夜培养。取1mL过夜培养物4℃，12000rpm，5min离心后PBS重悬，沸水浴10min，快速放置于-70℃30min，立即再次沸水浴10min，4℃，12000rpm，5min离心后取上清作为模板进行PCR验证阳性转化子，PCR体系与条件如下：
PCR体系：
PCR条件：
2％琼脂糖凝胶电泳检测阳性转化子菌液PCR产物，电泳条件：120V，30min。将大小正确的阳性转化子一一对应转移至含有100μg/mL Zeocin的YPD平板上以备进一步表达。
实施例5天然抗菌肽P5、P6的表达
(1)转化子的表达
挑取阳性转化子，接种至含有100μg/mL Zeocin的YPD液体培养基，30℃，250rpm振荡培养18-20h；0.5-1％接种量转接至50mL YPD液体培养基，30℃，250rpm振荡培养1天后以4层灭菌纱布替换玻璃纸封口膜包裹摇瓶口，30℃，250rpm振荡培养3天至发酵结束。
(2)重组酵母发酵液抗菌活性检测
抑菌圈实验分析：挑取S.aureusATCC 25923单菌落接种于10mLMH培养基中，37℃，250rpm培养至OD600nm≈0.4，1％接种量转移至50mL MH固体培养基中，混匀，迅速倒19cm×19cm的方形培养皿中，待凝固后，在培养基表面小心放置牛津杯，加入50μL发酵液上清。
(3)重组酵母分泌蛋白水平Tricine-SDS-PAGE检测
对所获得的高活性重组酵母菌株进一步采用Tricine-SDS-PAGE分析重组突变体表达水平，电泳方法参照(2006)。结果见图1。
实施例6天然抗菌肽P5、P6的纯化
(1)阳离子交换层析纯化
Bradford法测定蛋白浓度，4℃，12000rpm离心10min后取上清。将HiPrep SP FF阳离子交换柱(长度16mm，内径10mm，GEHealthcare)利用A液平衡5-10个柱体积后上样。进样完毕后，先用含有20mM，pH6.7的磷酸盐洗脱缓冲液(A液)进行洗脱，待穿透峰洗脱完后，用含有1M NaCl的20mM，pH6.7的磷酸盐洗脱缓冲液(B液)进行洗脱，收集洗脱峰。洗脱步骤为：100％A液，洗脱5个柱体积，为穿透峰；20％A，60％B液，洗脱5个柱体积，为洗脱峰。利用UV215nm监测洗脱情况，Tricine-SDS-PAGE和检测目标产物纯化情况。
(2)1kDa透析袋除盐
收集到的洗脱峰，经1kDa截留分子量透析袋4℃透析，每2h换水一次，换水6次。收集透析后的透析液，低温真空冷冻干燥机(-54℃，0.016MPa)冻干，通过Tricine-SDS-PAGE凝胶电泳检测。
实施例7抗革兰氏阳性菌活性检测
天然抗菌肽P5、P6最小抑菌浓度(MIC，minimum inhibitory concentration)的测定参照临床和实验室标准协会(CLSI，Clinical andLaboratory Standards Institute)制定的方法(WIEGAND等，Agar and broth dilution methods to determine the minimalinhibitory concentration(MIC)of antimicrobial substances.Nature protocols,2008,3(2)：163-175)，根据具体情况略有改动，操作细节如下：
受试菌株的单菌落挑至MH液体培养基中，37℃ 250rpm振荡过夜培养活化后，转接至MH液体培养基中培养至对数生长期(OD600nm＝0.4～0.6)，然后制备成105CFU/mL的菌液，加入96孔无菌细胞培养板内，每孔90μL。
用PBS通过2倍倍比稀释法对抗菌肽P5、P6进行稀释，每孔10μL抗菌肽，使其终浓度分别为128、64、32、16、8、4、2、1、0.5、0.25、0.125和0.0625μg/mL，阴性对照组为PBS代替抗菌肽的受试菌液，空白对照组为无菌MH培养基。每个处理三个平行样。
将培养板置于37℃恒温培养箱孵育16～18h，直至阴性对照孔出现肉眼可见的明显浑浊菌液，能够完全抑制细菌生长的最低浓度即为抗菌肽对受试菌株的MIC值。如出现跳孔或平行样间结果不一致情况，则重新测试。
结果如表2所示，抗菌肽P5、P6对各革兰氏阳性菌均体现出不同程度的较好的抑菌效果。
表2天然抗菌肽P5、P6对革兰氏阳性菌的抗菌活性
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
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Claims (10)
1.天然抗菌肽，其特征在于，其特征在于，所述抗菌肽为多肽P5或P6，其氨基酸序列分别如SEQ ID NO：3-4所示。
2.权利要求1所述抗菌肽的编码基因，其特征在于，多肽P5、P6编码基因的核苷酸序列分别如SEQ ID NO：5-6所示。
3.表达盒、表达载体、克隆载体、转基因细胞系或工程菌，其包括包含编码权利要求1所述抗菌肽基因序列的核酸。
4.重组毕赤酵母，其特征在于，其是用携带有权利要求2所述抗菌肽编码基因的表达载体转化毕赤酵母工程菌，筛选阳性克隆得到的。
5.根据权利要求4所述的重组毕赤酵母，其特征在于，所用表达载体为pPICZαA。
6.根据权利要求5所述的重组毕赤酵母，其特征在于，在权利要求2所述编码基因序列的5’端添加XhoI酶切位点和Kex2切割位点，在3’端添加TAA和TAG终止子序列以及XbaI酶切位点，得到基因构建物，再将所述基因构建物插入载体pPICZαA的XhoI和XbaI酶切位点之间构建得到重组表达载体，最后用所述重组表达载体转化毕赤酵母菌X-33，筛选阳性克隆得到的。
7.利用权利要求4-6任一项所述重组毕赤酵母发酵生产天然抗菌肽的方法。
8.权利要求1所述抗菌肽在制备广谱抗菌药物或组合物中的应用。
9.由权利要求1所述抗菌肽制备的广谱抗菌药物或组合物，其中，所述菌为革兰氏阳性菌，包括金黄色葡萄球菌、猪链球菌。
10.权利要求1所述抗菌肽在食品添加剂、化妆品、饲料添加剂领域中的应用。
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| CN109160939A (zh) * | 2018-09-21 | 2019-01-08 | 铜仁职业技术学院 | 一种生物抗菌肽及其应用 |
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| CN109160939B (zh) * | 2018-09-21 | 2021-05-11 | 铜仁职业技术学院 | 一种生物抗菌肽及其应用 |
| CN110438139A (zh) * | 2019-07-04 | 2019-11-12 | 东北农业大学 | 一种利用毕赤酵母菌制备抗菌肽t9w的方法 |
| EP3811964A1 (en) * | 2019-10-24 | 2021-04-28 | Antinbio, Inc. | Antimicrobial peptide variants and uses thereof |
| US11279737B2 (en) | 2019-10-24 | 2022-03-22 | Antinbio, Inc. | Antimicrobial peptide variants and uses thereof |
| CN111320678A (zh) * | 2020-03-09 | 2020-06-23 | 安亭生物有限责任公司 | 抗菌肽突变体及其应用 |
| CN113666996A (zh) * | 2021-08-26 | 2021-11-19 | 华中农业大学 | 一种中国鲎抗菌肽及其制备方法和应用 |
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