CN111718923B - 一种用于治疗白菜软腐病的药物的筛选方法 - Google Patents
一种用于治疗白菜软腐病的药物的筛选方法 Download PDFInfo
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Abstract
本发明提供一种用于治疗白菜软腐病的药物的筛选方法,包括如下步骤:质粒构建,蛋白诱导表达,蛋白分离纯化,蛋白的荧光淬灭反应筛选化合物,确定化合物的最佳抑菌浓度。本发明提供的方法通过荧光光谱法筛选与白菜软腐病菌关键蛋白结合的配体分子,从而得到可治疗白菜软腐病的药物。
Description
技术领域
本发明属于植物保护领域,涉及一种植物病的药物的筛选方法,具体涉及一种用于治疗白菜软腐病的药物的筛选方法。
背景技术
大白菜在食用的蔬菜中占较大比例,自古以来就有“蔬菜之王”的美称,是各地市场销量大且价格看好的蔬菜之一。但软腐病一直是制约大白菜大面积发展和收储的关键因素。白菜软腐病,又叫腐烂病、烂疙瘩、烂葫芦、烂葫芦头病、水烂病、脱帮子,是大白菜生产中一种毁灭性病害,如果发生,可成片绝收。主要为害叶片、柔嫩多汁组织及茎或根部。初呈水浸状或水浸半透明,后变褐软化腐烂,病部渗出黏液,散出臭味。该病不仅在白菜生长后期危害,而且在贮藏期间可继续扩展危害,造成烂窖。
最初发病于接触地面的叶柄和根尖部,叶柄发病部位呈水渍状,外叶失去水分而萎蔫,最后整个植株枯死。生长后期发病时,首先出现水渍状小斑点,叶片半透明,呈油纸状,最后整株软化、腐烂,散发出特殊的恶臭。黄条跳甲、甘蔗黑蟋蟀等虫害严重时,病菌从伤口处大量侵入,导致病害加重。有时,在运输途中发生软化腐烂现象。
造成白菜软腐病的病菌为胡萝卜软腐果胶杆菌胡萝卜亚种PC1菌株(Pectobacterium carotovorum subsp.Carotovorum PC1),是植物细菌性软腐病的主要致病菌,常危害胡萝卜、白菜、马铃薯等植物。除此之外,在北京市顺义区的芹菜软腐病、南京市八卦洲的芦蒿软腐病、甘肃省嘉峪关市的洋葱鳞茎软腐病、山西市晋中的西葫芦软腐病等病害样品中都分离到PC1菌株,说明病原菌宿主范围极广,并带来巨大的经济损失。
为了有效的控制白菜软腐病病害的发生,亟待筛选和发现新型治疗白菜软腐病的药物。
发明内容
为了解决上述问题,本发明的目的在于提供一种用于治疗白菜软腐病的药物的筛选方法,该方法通过荧光光谱法筛选与白菜软腐病菌关键蛋白结合的配体分子,从而得到可治疗白菜软腐病的药物。
为了实现上述目的,本发明提供一种用于治疗白菜软腐病的药物的筛选方法,包括如下步骤:
1)质粒构建:将胡萝卜软腐果胶杆菌胡萝卜亚种PC1菌株的N琥珀酰二氨基庚二酸脱琥珀酰酶的如Seq ID No.1所示的核苷酸序列***到带有T7启动子的大肠杆菌原核表达载体pET28a的Nde1和Xho1酶切位点之间,得到pET28a-PccdapE质粒;
2)PccDapE蛋白诱导表达:将pET28a-PccdapE质粒转化至大肠杆菌BL21(DE3)感受态细胞中,LB培养基37℃培养至OD600在0.6~0.8之间;加入IPTG至浓度为0.1mM,12℃,200rpm条件下,诱导培养48h;
3)使用镍离子螯合树脂柱分离纯化PccDapE蛋白;
4)向PccDapE蛋白溶液中添加待选化合物进行荧光淬灭反应;
5)选择可与PccDapE蛋白发生荧光淬灭反应的化合物配制成梯度浓度的化合物溶液,与大白菜软腐病菌菌液混合,28℃,150rpm摇床培养24h,通过分光光度计确定化合物的最佳抑菌浓度。
优选地,所述步骤4)中荧光淬灭反应的荧光激发波长为280nm;激发和发射狭缝均为5nm,在荧光光度计上记录290~500nm波长范围内的发射光谱。
本发明还提供一种采用上述方法筛选得到的TBFC作为抑制白菜软腐病菌生长的药物的用途。
本发明的有益效果在于:
本发明提供一种用于治疗白菜软腐病的药物的筛选方法,该方法以PccDapE蛋白作为目标蛋白,通过筛选可以与PccDapE蛋白结合的配体化合物,从而得到可以抑制白菜软腐病菌生长的药物,从而为开发治疗白菜软腐病的新型药物提供了筛选思路,并筛选出TBFC作为抑制白菜软腐病菌生长的药物。
附图说明
图1为胡萝卜软腐果胶杆菌胡萝卜亚种中赖氨酸合成路线图。
图2为胡萝卜软腐果胶杆菌胡萝卜亚种的肽聚糖成份结构图。
图3A为表达载体pET28a(+)的质粒图谱及***His-Tag示意图。
图3B为表达载体pET28a(+)的表达区域及His-Tag位点示意图。
图4为PccDapE Ni-NTA亲和层析电泳检测结果图。
图5为PccDapE蛋白加入不同浓度TBFC的荧光猝灭效应图。
图6为TBFC对PccDapE猝灭效应的双对数曲线。
图7为TBFC对白菜软腐病原菌抑制率曲线。
图8为TBFC的大白菜离体抑菌实验。
具体实施方式
下面将对本发明的实施例进行详细、完善的描述,以使本发明的优点和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。
赖氨酸(Lysine,Lys)是人类和哺乳动物的必需氨基酸,不能通过机体本身合成,必须从外部摄取。植物和细菌中Lys都是从L-天冬氨酸(L-aspartate)开始的,白菜等植物利用L-天冬氨酸的代谢产物L-四氢二吡啶甲酸(L-tetrahydrodipicolinate)在ct390基因产物氨基转移酶的作用下直接合成L,L-二氨基庚二酸(L,L,-diaminopimelate,DAP),然后再转化为m-DAP(Jander G,Joshi V.Recent Progress in Deciphering theBiosynthesis of Aspartate-Derived Amino Acids in Plants[J].Molecular plant,2010,003(001):54-65.);白菜软腐菌(Pectobacterium carotovora subsp.carotovora,Pcc)等植物病原细菌则琥珀酰酶途径(Succinylase pathway)合成DAP,然后合成m-DAP(Scapin G,Blanchard J S.Enzymology of bacterial lysine biosynthesis.[J].Advances in Enzymology&Related Areas of Molecular Biology,1998,72(1):279.)最后产生Lys,路线图如图1所示。
如图2所示为胡萝卜软腐果胶杆菌等细菌中细胞壁组成的肽聚糖(Peptdoglycan)结构,从图2可以看出,m-DAP为肽聚糖(Peptdoglycan)的结构组成之一,当m-DAP的合成受到抑制,则细菌无法形成肽聚糖结构,从而造成细胞壁就不完整,导致细菌无法存活。
因此采用胡萝卜软腐果胶杆菌m-DAP前体DAP合成的关键酶N琥珀酰二氨基庚二酸脱琥珀酰酶(PccDapE)作为靶点,可以阻断细菌细胞壁的合成,而抑制胡萝卜软腐果胶杆菌的存活,从而实现治疗白菜软腐病的目标。
荧光光谱是由激发和发射两种光谱组成。激发光谱是根据发光材料在不同波长激发光的作用下测量得到的某一波长处的荧光强度与激发波长的关系。发射光谱是在某一特定波长的激发光作用下,所发射的不同波长处荧光强度的分布情况。荧光光谱法具有高灵敏性、高选择性、样品用量少,操作简单等特征,被认为是研究蛋白质分子构象及蛋白与配体相互作用的最有效方法。根据荧光光谱法可最终测得配体与受体之间的结合常数、结合位点数等参数。荧光猝灭法是荧光光谱法的一种,它是指使荧光物质的量子产率或荧光强度下降的作用,在蛋白与配体相互作用的荧光光谱研究中多数使用的是荧光猝灭法。
材料
1.大肠杆菌BL21(DE3)感受态细胞购自天根生化科技(北京)有限公司
2.LB培养基:1%胰蛋白胨,0.5%酵母粉,1%NaCl,pH7.2,121℃高压灭菌20min。
3.纯度96%的TBFC(4-氧代-4,5,6,7-四氢-1-苯并呋喃-3-羧酸,4-Oxo-4,5,6,7-tetrahydrobenzo[b]furan-3-carboxylic acid)购自华腾制药。
4.PDA培养基(固体):取新鲜去皮土豆200g,将其切成小块加1000mL蒸馏水大火煮烂(约20min,能被玻璃棒戳破即可),四层医用纱布过滤加入葡萄糖20g,琼脂20g,待溶液融化冷却后用蒸馏水补至1L,121℃灭菌,计时20min。
5.PDA培养基(液体):在PDA培养基(固体)基础上去除琼脂即可。
6.大白菜购买自北京垡头京客隆超市。
7.胡萝卜软腐果胶杆菌亚种PC1菌株(Pectobacterium carotovorumsubsp.carotovorum PC1)购买自德国微生物菌种保藏中心(DSMZ),菌株编号为DSM30169。
实施例1选择筛选治疗白菜软腐病的药物靶点
分析胡萝卜软腐果胶杆菌胡萝卜亚种PC1菌株(Pectobacterium carotovorumsubsp.Carotovorum PC1)的全基因组序列(CP001657.1),得到编码N琥珀酰二氨基庚二酸脱琥珀酰酶(succinyl-diaminopimelate desuccinylase)PccDapE的核苷酸编码序列PccdapE如Seq ID No.1所示:
atgtcttgcccggtcatagagctcgctcaacagttaattaaacgcccttccctcagcccgaacgacgagggctgccaggcgctcatgattgaacgcctgacggcgatgggctttaccgtcgaagcaatggattttggcgatacccaaaatttctgggcatggcgcggcacaggaaaaaccctggccttcgccggacacactgacgtagtccccagcggggatgagagccagtggcagcacccgccgtttgagccgatcattcgcgacggcatgctgtacggtcgtggcgcggcggacatgaaaggttcactggccgcgatggtgattgccgccgagcgctttgtcgccgcccacccgagtcatcaagggcgtcttgcgtttctgattacctcggacgaagaagccagcgccgttaacggcacggtaaaagtggttgaggcgctgatggcacgcaacgagcgattggactactgcttggtcggcgagccgtccagtacgcacgtcgtgggcgatgtggtaaaaaatggtcgccgtggctctatcacggctaacctgcgcgtacacggcgtacaggggcacgttgcctaccctcatctggcggacaaccctgttcaccgagcagcaccagcgctgaacgagctcatcgccaccgaatgggatcggggcaacgatttcttcccaccaaccaccatgcagattgccaatattcaagcaggcaccggcagcaataacgtcatccccggcgaactgtttgtgcagtttaatttccgtttcagcaccgaattaacggatgtgttaatccagcagcgcgtcgcggaactgctggatcgccaccagctcaattacaccattgactggaagctttccggccaaccattcctgaccgcacgcggcgaactggtcgatgccgtggtaaatgccgtcaagcactacaatgaggttaccccagagctactgacgaatggtggcacttccgatggccgcttcatcgcacgtatgggcgcgcaggtggttgaactgggccccgttaatgccacgatccataaagtggacgaatgcgtcagcgccgcagatctgcaactgctaagccgcatgtaccagcgcattatggagcagctcatcgcatga
然后,根据三联体密码子推导出PccDapE蛋白的氨基酸序列如Seq ID No.2所示:
MSCPVIELAQQLIKRPSLSPNDEGCQALMIERLTAMGFTVEAMDFGDTQNFWAWRGTGKTLAFAGHTDVVPSGDESQWQHPPFEPIIRDGMLYGRGAADMKGSLAAMVIAAERFVAAHPSHQGRLAFLITSDEEASAVNGTVKVVEALMARNERLDYCLVGEPSSTHVVGDVVKNGRRGSITANLRVHGVQGHVAYPHLADNPVHRAAPALNELIATEWDRGNDFFPPTTMQIANIQAGTGSNNVIPGELFVQFNFRFSTELTDVLIQQRVAELLDRHQLNYTIDWKLSGQPFLTARGELVDAVVNAVKHYNEVTPELLTNGGTSDGRFIARMGAQVVELGPVNATIHKVDECVSAADLQLLSRMYQRIMEQLIA。
其中,核苷酸最后三位为终止密码子不对应氨基酸。
使用ProtParam软件对PccDapE蛋白进行生物信息学分析,以获得该蛋白的酸碱性质,结果为:氨基酸数量(Number of amino acids):375,分子量(MoLecular weight):41055.60,理论等电点(Theoretical pI):5.37。PccDapE氨基酸组成如下表1所示:
表1 PccDapE氨基酸组成
对表1中的氨基酸进行类型分析可知:PccDapE蛋白含:30个强碱性StronglyBasic(+)氨基酸Amino Acids(K,R);43个强酸性Strongly Acidic(-)氨基酸Amino Acids(D,E);143个疏水性氨基酸Hydrophobic Amino Acids(A,I,L,F,W,V);87个极性氨基酸Polar Amino Acids(N,C,Q,S,T,Y);72个其他氨基酸(H,M,G,P,F)。
从PccDapE蛋白的氨基酸类型分析上,PccDapE蛋白为一个富含芳香族氨基酸的弱酸性蛋白质。由于含有芳香族氨基酸W(色氨酸)、Y(酪氨酸)和F(苯丙氨酸)残基的蛋白质在280nm或295nm的激发光的激发下会产生荧光,这种荧光称为内源性荧光,其荧光光谱对环境极为敏感,当化合物改变蛋白质的结构,荧光光谱就会发生改变,因此推测PccDapE蛋白的荧光淬灭剂可作为PccDapE蛋白的抑制剂,从而通过蛋白质和化合物相互作用后荧光光谱的改变来筛选白菜软腐病菌(Pcc)的抑菌剂。
实施例2PccDapE蛋白诱导表达
1.pET28a-PccdapE质粒表达载体的构建
将N琥珀酰二氨基庚二酸脱琥珀酰酶(PccDapE)的如Seq ID No.1所示的核苷酸序列PccdapE***到带有T7启动子的大肠杆菌原核表达载体pET28a的Nde1和Xho1酶切位点之间,得到pET28a-PccdapE质粒,该质粒委托北京赛百胜生物科技有限公司构建,质粒图谱如图3A所示,***位点及His-Tag如图3B所示。
pET28a表达载体在酶切位点两侧带有His标签序列,使得表达出的PccDapE蛋白的N-端和C-端均带有His标签以利于PccDapE蛋白的检测和纯化。
2.载体转化
将pET28a-PccdapE质粒转化至大肠杆菌BL21(DE3)感受态细胞中,方法为:取2-5μl重组质粒pET28a-PccDapE加入到50μL大肠杆菌BL21(DE3)感受态细胞10×1010细胞/毫升溶液中,轻轻吹打混匀,置于冰浴中静置30min后取出离心管,置于42℃恒温水浴锅中热激90s后于冰上放置2~3min。在超净台中取450μL无菌的LB液体培养基加入上述离心管中,用移液器慢慢吹打混匀,置于37℃,200rpm摇床上,振荡培养45min。用移液器吸取100μL菌液涂布至含有100μg/mL卡那霉素的抗性固体平板上,放在超净台中等待液体被吸收后,置于37℃恒温培养箱中倒置培养过夜。
3.PccDapE蛋白大量诱导表达
用无菌接种环挑取含有阳性重组质粒pET28a-PccdapE的单菌落,接种于5mL含有100μg/mL卡那霉素的LB液体培养基中,37℃、200rpm培养过夜后转接到500mL含有100μg/mL卡那霉素的LB液体培养基中,37℃、200rpm培养至菌液OD600=0.6~0.8时,预先在12℃、220rpm条件下培养20分钟,培养基温度降至12℃后,再加入IPTG诱导剂至终浓度为0.1mmoL/L,在12℃、200rpm条件下摇床培养48h。6000rpm,4℃离心30min,舍弃培养液,收集菌体,并将菌体置于-80℃冻存备用。
4.PccDapE蛋白亲和层析纯化
菌体重悬:按约1g菌体用8mL Buffer A(20mM Tris.base,pH 8.0,0.3M NaCl,15mM咪唑,1mMβ-ME.)重悬的比例将上述步骤得到的含PccDapE蛋白的菌体进行重悬,用移液器在冰上反复吹吸几次,直至均质,至于冰浴上备用。
超声破碎细胞:菌体重悬后,在冰浴条件下超声裂解细胞,超声条件为工作5秒,间歇7秒,循环99次,功率为360W。超声完成后取20μL破碎液于离心管中,保存在-20℃冰箱中,用于电泳检测。
冷冻离心:破碎后的细胞,在4℃,15000rpm的高速冷冻离心机中离心30min。分别收集上清液和沉淀,取20μL上清液于离心管中,保存在-20℃冰箱中,用于电泳检测。在沉淀中加等量的Buffer A重悬混匀,取20μL于离心管中,保存在-20℃冰箱中,用于电泳检测。最后将余下的上清液转移至另一洁净离心管中,4℃,15000rpm,离心30min,置于冰上备用。
Ni-NTA纯化PccDapE蛋白:将层析柱连接上蠕动泵,用Buffer A平衡柱子,然后将过滤后的蛋白上清液以0.5mL/min的流速泵入到镍柱上,用另一洁净的烧杯收集穿透液。上完样品后,用Buffer B(20mM Tris.base,pH 8.0,50mMNaCl,15mM imidazole,1Mmβ-ME)清洗直至吸光值<0.05,取一干净烧杯收集穿透液,最后用Buffer C(20mM Tris.base,pH8.0,50mM NaCl,150mMimidazole,1mMβ-ME)收集目的蛋白。将收集的各管洗脱液取样并通过SDS-PAGE检测纯化蛋白的纯度及表达量。
5.SDS-PAGE蛋白电泳
将经Ni-NTA亲和层析柱洗脱下来的蛋白进行SDS-PAGE电泳,考马斯亮蓝染检测,结果如图4所示,其中M为Marker(14.2-116.0KDa);1-4为Ni柱纯化前的PccDapE蛋白电泳结果,其中,1为总菌液的蛋白质,2为沉淀中蛋白质,3为上清中可溶性蛋白,4为未结合到Ni柱上的穿透液中的蛋白;5为柱纯化洗脱液buffer A(20mM Tris.base,pH 8.0,0.3M NaCl,15mM咪唑,1mMβ-ME)的电泳结果;6为柱纯化洗脱液buffer B(20mM Tris.base,pH 8.0,50mM NaCl,15mM imidazole,1Mmβ-ME)的电泳结果;7-9为柱纯化洗脱液buffer C(20mMTris.base,Ph 8.0,50mM NaCl,150mM imidazole,1mMβ-ME)的电泳结果,图中星号为PccDapE蛋白。
从图4可以看出,PccDapE蛋白经大肠杆菌BL(DE3)低温诱导后,沉淀中很少PccDapE,如图4中第2泳道所示;绝大多数PccDapE蛋白存在于上清,如图4中第3泳道所示,说明PccDapE具有良好的可溶性,经Ni-NTA柱纯化后,蛋白质纯度已经达到90%,杂蛋白极少,如图4第5-9泳道所示。从中可以知道PccDapE蛋白的可溶性高,易于通过层析柱纯化。
实施例3通过荧光淬灭技术得到与PccDapE相互作用的化合物
PccDapE能够在280nm激发光作用下产生比较强的内源性荧光,主要是由于PccDapE分子内含有三个发色氨基酸残基:酪氨酸残基(Tyr)、色氨酸残基(Trp)和苯丙氨酸残基(Phe)这,其所占比例分别为1.6%、1.3%和4.0%。
首先测PccDapE蛋白溶液的内源荧光强度,当蛋白质浓度为2.09μM时,PccDapE在335nm处有最大荧光强度λmax=3500。然后将蛋白质加入到ThermoVarioskan Flash多功能荧光分光光度仪的96孔板中,再加入摩尔浓度为20×10-5M的如表2所示化合物,混匀,在335nm扫描各孔的荧光强度,结果发现化合物TBFC使蛋白质的荧光几乎淬灭。因此选择TBFC进行了进一步的研究分析。并用静态猝灭公式计算化合物与蛋白质的结合常数。
表2
向PccDapE蛋白溶液中加入不同用量的20mg/mL TBFC溶液,配制总体积为3mL,PccDapE蛋白最终浓度为2.09μM不变,TBFC终浓度分别为0.00M,6.67×10-5M,8.89×10-5M,11.11×10-5M,13.33×10-5M,15.56×10-5M,17.78×10-5M的试样,25℃水浴30min。然后转移至四通荧光石英比色皿中,以280nm为激发波长,激发和发射狭缝均为5nm,在荧光光度计上记录290~500nm波长范围内的发射光谱。结果如图5所示为不同浓度下的TBFC的荧光猝灭效应图,其中,横坐标为扫描光谱的范围(0-500nm),纵坐标为荧光的发射强度。其中的PccDapE蛋白质浓度2.09μM固定不变,a→g为不同TBFC的浓度(0.00,6.67×10-5,8.89×10-5,11.11×10-5,13.33×10-5,15.56×10-5,17.78×10-5M)下的荧光发射。如图5所示,PccDapE在335nm处出现最大荧光峰,也就是说,当没有抑制剂时(曲线a)PccDapE在335nm处的荧光发射强度最高,随着TBFC浓度的增加(曲线b-g)呈现规律性的减弱。由此表明PccDapE与TBFC发生相互作用,使PccDapE的内源性荧光发生猝灭。TBFC抑制剂浓度越高,荧光发射越弱,说明TBFC抑制了蛋白质的荧光强度。
根据结果分析TBFC对PccDapE的猝灭机制为静态猝灭,静态猝灭是猝灭剂与荧光分子之间通过分子间相互作用形成基态复合物,而导致荧光强度的降低。对于静态猝灭过程,可以用静态猝灭公式得到结合常数:
lg(F0-F)/F=lgK+nlg[Q]
其中:F0为未加入TBFC时PccDapE的荧光强度;F为加入TBFC后PccDapE的荧光强度;K为TBFC与PccDapE的结合常数;n为TBFC与PccDapE的结合位点数;[Q]为TBFC的浓度。以lg(F0-F)/F对lg[Q]作图,得到如图6所示的TBFC对PccDapE猝灭效应的双对数曲线,由直线的截距可以得到TBFC与PccDapE的结合常数K=1.97×106L/moL,由此表明,TBFC与PccDapE具有较强的作用,因此TBFC可以通过与细菌中的PccDapE酶结合,抑制PccDapE酶的活性,从而抑制细菌赖氨酸的生物合成,最终抑制细菌的生长。
实施例4TBFC的抑菌实验
将胡萝卜软腐果胶杆菌胡萝卜亚种PC1菌株(Pectobacterium carotovorumsubsp.carotovorum PC1,菌种编号为DSM-30169,购于德国微生物菌种保藏中心(DSMZ);)接种到PDA培养基(液体)中,28℃,150rpm摇床培养24h。然后将培养好的菌液制备成1×106CFU/ml的菌液,按照1%接种量接种到新鲜的PDA培养基(液体)中备用。
将20mg/ml的TBFC母液用无菌水进行稀释,得到浓度分别为20、17.5、15、12.5、10、7.5、5、2.5mg/ml TBFC溶液待用。
取27支试管,分成A、B、C三组,每组9支,分别编号为1,2,3,4,5,6,7,8,9。向A、B、C各组试管中加入4950μl菌悬液。向编号为1的三支试管中加入50μl20mg/ml的TBFC溶液,以此类推,向2,3,4,5,6,7,8号试管中分别加入17.5、15、12.5、10、7.5、5、2.5mg/ml TBFC溶液50μl。向9号试管加入50μl无菌水。最终得到TBFC终浓度分别为200、175、150、125、100、75、50、25、0μg/ml。将试管放入28℃,150rpm摇床培养24h。
进行不同浓度梯度的TBFC对白菜软腐病原菌的抑制试验,测其OD600如图7所示为TBFC抑制白菜软腐病原菌生长曲线,并用GraphPad Prism 5对数据进行拟合可得TBFC对白菜软腐病原菌的最小抑菌浓度为57.14μg/mL,TBFC抑菌作用效果较好。根据公式:抑制率(%)=100×[(阳性对照组OD600-实验组OD600)÷阳性对照组OD600]计算TBFC对白菜软腐病原菌抑制率,得到如图5所示的TBFC对白菜软腐病原菌抑制率曲线,由图7可知,TBFC对白菜软腐病原菌的抑制率为91.01%。根据《抗菌和抑菌效果评价方法(WST 650—2019)》可知,当抑菌率≥50%~90%,具有抑菌作用,当抑菌率≥90%时,则具有较强抑菌作用。因此,TBFC对白菜软腐病原菌具有较强的抑制作用效果。
实施例5 TBFC的大白菜离体抑菌实验
将菌种接至PDA液体培养基中,28℃,150rpm摇床培养过夜。将培养好的菌液用无菌水稀释成1×108CFU/ml的菌悬液,备用。
将80mg/ml的TBFC母液用无菌水进行倍数稀释,得到浓度分别为80、40、20、10、5mg/ml TBFC溶液待用。
取切成适当大小(3.5cm*5cm)的白菜帮五块、经水洗,待水稍干后,以10%漂白粉溶液作表面消毒,分放在两个灭过菌的上下铺有吸水纸的培养皿(直径15cm)中,用酒精擦过的玻璃棒顺着白菜帮打三排不穿透的孔穴(上下两排孔距白菜帮边缘约1cm,孔间距约为1.5cm)。先以灭过菌的移液器吸取无菌水10μl于菜帮的第一排内孔作为空白对照,再用该移液器吸菌液10μl于第二、三排孔内。待孔内液体干燥后,在第一、二排孔内加10μl无菌水,第三排加等量的TBFC溶液,每个TBFC溶度重复一次,其他浓度以相同方法处理。盖好皿盖,置于26-28℃的温箱中,24小时后检查发病情况,结果如图8所示,其中第一排为空白对照(水+水),第二排为阴性对照(ECC+水),第三排为ECC+TBFC,实验重复一次。
从图8可以看出,第一排孔内无大白菜软腐病菌接种,无发病情况;第二排为阴性对照,接种有大白菜软腐病菌,全部发病;第三排为接种有大白菜软腐病菌同时接种TBFC,当TBFC浓度为5mg/ml时,对于大白菜软腐病菌的抑制效果不显著,当TBFC浓度为10mg/ml及以上时可以明显看出对于大白菜软腐病菌生长的抑制显著。说明TBFC工作浓度在10mg/ml及以上时可以有效抑制大白菜软腐病菌的生长,可以作为大白菜软腐病菌的治疗药物。
从上述实施例可以看出,本发明采用一种用于治疗白菜软腐病的药物的筛选方法,该方法通过荧光光谱法筛选与白菜软腐病菌关键蛋白结合的配体分子,从而得到可治疗白菜软腐病的药物,并通过该方法成功筛选出TBFC作为治疗白菜软腐病的药物。
SEQUENCE LISTING
<110> 北京联合大学
<120> 一种用于治疗白菜软腐病的药物的筛选方法
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1128
<212> DNA
<213> Pectobacterium carotovorum subsp. Carotovorum PC1
<400> 1
atgtcttgcc cggtcataga gctcgctcaa cagttaatta aacgcccttc cctcagcccg 60
aacgacgagg gctgccaggc gctcatgatt gaacgcctga cggcgatggg ctttaccgtc 120
gaagcaatgg attttggcga tacccaaaat ttctgggcat ggcgcggcac aggaaaaacc 180
ctggccttcg ccggacacac tgacgtagtc cccagcgggg atgagagcca gtggcagcac 240
ccgccgtttg agccgatcat tcgcgacggc atgctgtacg gtcgtggcgc ggcggacatg 300
aaaggttcac tggccgcgat ggtgattgcc gccgagcgct ttgtcgccgc ccacccgagt 360
catcaagggc gtcttgcgtt tctgattacc tcggacgaag aagccagcgc cgttaacggc 420
acggtaaaag tggttgaggc gctgatggca cgcaacgagc gattggacta ctgcttggtc 480
ggcgagccgt ccagtacgca cgtcgtgggc gatgtggtaa aaaatggtcg ccgtggctct 540
atcacggcta acctgcgcgt acacggcgta caggggcacg ttgcctaccc tcatctggcg 600
gacaaccctg ttcaccgagc agcaccagcg ctgaacgagc tcatcgccac cgaatgggat 660
cggggcaacg atttcttccc accaaccacc atgcagattg ccaatattca agcaggcacc 720
ggcagcaata acgtcatccc cggcgaactg tttgtgcagt ttaatttccg tttcagcacc 780
gaattaacgg atgtgttaat ccagcagcgc gtcgcggaac tgctggatcg ccaccagctc 840
aattacacca ttgactggaa gctttccggc caaccattcc tgaccgcacg cggcgaactg 900
gtcgatgccg tggtaaatgc cgtcaagcac tacaatgagg ttaccccaga gctactgacg 960
aatggtggca cttccgatgg ccgcttcatc gcacgtatgg gcgcgcaggt ggttgaactg 1020
ggccccgtta atgccacgat ccataaagtg gacgaatgcg tcagcgccgc agatctgcaa 1080
ctgctaagcc gcatgtacca gcgcattatg gagcagctca tcgcatga 1128
<210> 2
<211> 375
<212> PRT
<213> Pectobacterium carotovorum subsp. carotovorum PC1
<400> 2
Met Ser Cys Pro Val Ile Glu Leu Ala Gln Gln Leu Ile Lys Arg Pro
1 5 10 15
Ser Leu Ser Pro Asn Asp Glu Gly Cys Gln Ala Leu Met Ile Glu Arg
20 25 30
Leu Thr Ala Met Gly Phe Thr Val Glu Ala Met Asp Phe Gly Asp Thr
35 40 45
Gln Asn Phe Trp Ala Trp Arg Gly Thr Gly Lys Thr Leu Ala Phe Ala
50 55 60
Gly His Thr Asp Val Val Pro Ser Gly Asp Glu Ser Gln Trp Gln His
65 70 75 80
Pro Pro Phe Glu Pro Ile Ile Arg Asp Gly Met Leu Tyr Gly Arg Gly
85 90 95
Ala Ala Asp Met Lys Gly Ser Leu Ala Ala Met Val Ile Ala Ala Glu
100 105 110
Arg Phe Val Ala Ala His Pro Ser His Gln Gly Arg Leu Ala Phe Leu
115 120 125
Ile Thr Ser Asp Glu Glu Ala Ser Ala Val Asn Gly Thr Val Lys Val
130 135 140
Val Glu Ala Leu Met Ala Arg Asn Glu Arg Leu Asp Tyr Cys Leu Val
145 150 155 160
Gly Glu Pro Ser Ser Thr His Val Val Gly Asp Val Val Lys Asn Gly
165 170 175
Arg Arg Gly Ser Ile Thr Ala Asn Leu Arg Val His Gly Val Gln Gly
180 185 190
His Val Ala Tyr Pro His Leu Ala Asp Asn Pro Val His Arg Ala Ala
195 200 205
Pro Ala Leu Asn Glu Leu Ile Ala Thr Glu Trp Asp Arg Gly Asn Asp
210 215 220
Phe Phe Pro Pro Thr Thr Met Gln Ile Ala Asn Ile Gln Ala Gly Thr
225 230 235 240
Gly Ser Asn Asn Val Ile Pro Gly Glu Leu Phe Val Gln Phe Asn Phe
245 250 255
Arg Phe Ser Thr Glu Leu Thr Asp Val Leu Ile Gln Gln Arg Val Ala
260 265 270
Glu Leu Leu Asp Arg His Gln Leu Asn Tyr Thr Ile Asp Trp Lys Leu
275 280 285
Ser Gly Gln Pro Phe Leu Thr Ala Arg Gly Glu Leu Val Asp Ala Val
290 295 300
Val Asn Ala Val Lys His Tyr Asn Glu Val Thr Pro Glu Leu Leu Thr
305 310 315 320
Asn Gly Gly Thr Ser Asp Gly Arg Phe Ile Ala Arg Met Gly Ala Gln
325 330 335
Val Val Glu Leu Gly Pro Val Asn Ala Thr Ile His Lys Val Asp Glu
340 345 350
Cys Val Ser Ala Ala Asp Leu Gln Leu Leu Ser Arg Met Tyr Gln Arg
355 360 365
Ile Met Glu Gln Leu Ile Ala
370 375
Claims (2)
1.一种用于治疗白菜软腐病的药物的筛选方法,其特征 在于,包括如下步骤:
1)质粒构建:将胡萝卜软腐果胶杆菌胡萝卜亚种PC1菌株的N琥珀酰二氨基庚二酸脱琥珀酰酶的如Seq ID No.1所示的核苷酸序列***到带有T7启动子的大肠杆菌原核表达载体pET28a的Nde1和Xho1酶切位点之间,得到pET28a-PccdapE质粒;
2)PccDapE蛋白诱导表达:将pET28a-PccdapE质粒转化至大肠杆菌BL21(DE3)感受态细胞中,LB培养基37℃培养至OD600在0.6~0.8之间;加入IPTG至浓度为0.1mM,12℃,200rpm条件下,诱导培养48h;
3)使用镍离子螯合树脂柱分离纯化PccDapE蛋白;
4)向PccDapE蛋白溶液中添加待选化合物进行荧光淬灭反应;
5)选择可与PccDapE蛋白发生荧光淬灭反应的化合物配制成梯度浓度的化合物溶液,与大白菜软腐病菌菌液混合,28℃,150rpm摇床培养24h,通过分光光度计确定化合物的最佳抑菌浓度。
2.如权利要求1所述的筛选方法,其特征在于,所述步骤4)中荧光淬灭反应的荧光激发波长为280nm;激发和发射狭缝均为5nm,在荧光光度计上记录290~500nm波长范围内的发射光谱。
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