CN116656646A - 一种芽孢杆菌群体感应淬灭酶AiiA酶及其基因序列与应用 - Google Patents
一种芽孢杆菌群体感应淬灭酶AiiA酶及其基因序列与应用 Download PDFInfo
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Abstract
本发明提供一种芽孢杆菌群体感应淬灭酶AiiA酶,其氨基酸序列如SEQ ID NO.1所示,其编码基因的核苷酸序列如SEQ ID NO.2所示。该芽孢杆菌群体感应淬灭酶AiiA酶的酶活性高,能够高效降解高丝氨酸内酯AHL,从而显著解决在疾病防治中使用抗生素易导致细菌产生耐药性的问题。
Description
技术领域
本发明涉及一种芽孢杆菌群体感应淬灭酶AiiA酶及其基因序列与应用,属于芽孢杆菌群体感应淬灭酶AiiA酶制备技术领域。
背景技术
群体感应(Quorum sensing,QS),又可以称之为“自诱导(Autoinduction)”,是自然界中,微生物为了竞争资源进化出一种依赖种群密度的精细交流机制。最初,Alexander在1965年通过研究肺炎链球菌DNA的吸收机制发现第一个群感因子,这是一种由肺炎链球菌合成并分泌的多肽分子,可以引起某些基因的表达来促进外源DNA进入其细胞。直至1994年,Fuqua首次使用了“群体感应”这个术语,他发现并报道了海洋细菌费氏弧菌(Vibrofischeri)的发光现象,利用了LuxR和LuxI蛋白进行细胞间通讯。之后的其他研究中,QS现象被发现广泛存在于各种微生物群体中。其中微生物通过分泌和响应特定的物理或化学信号分子,引发细胞运动、化学生物合成和生物膜形成等行为。QS中细菌产生和释放的化学信号分子,称为自诱导剂(Autoinducer,AI),当细菌密度升高时信号分子的浓度也会随着升高。由于信号分子可以被细胞感知,这使得整个群体能够在信号分子达到某个临界浓度(对应于特定细胞密度)后发起协同行动。
细菌的众多生物学功能受到群体感应系统的调节与控制。研究表明,一些物质能够通过干扰群感系统来关闭病原菌的毒力表达,而不是单纯的限制细胞的生长,从而不会使致病菌显示出克服药物毒性、复杂的超感染和抗生素耐药性的性状。在革兰氏阴性菌中,高丝氨酸内酯AHL是调控群体感应系统的关键信号分子。群体感应淬灭酶AiiA酶是一种能够降解AHL的群体感应淬灭酶。研究群体感应的淬灭,开发高效的群体感应淬灭酶AiiA酶制剂是一种潜在预防细菌耐药性的有效方案。但是,现有的AiiA酶的酶活较低,难以满足实际应用的需要。
发明内容
本发明提供了一种芽孢杆菌群体感应淬灭酶AiiA酶及其基因序列与应用,可以有效解决上述问题
本发明是这样实现的:
一种芽孢杆菌群体感应淬灭酶AiiA酶,其氨基酸序列如SEQ ID NO.1所示。
作为进一步改进的,芽孢杆菌群体感应淬灭酶AiiA酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
一种上述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因,其核苷酸序列如SEQ IDNO.2所示。
一种重组表达载体,包含上述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因。
作为进一步改进的,所述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因插入到质粒上的限制性酶切位点之间,所述限制性内切酶包括NdeI限制性内切酶和/或XhoI限制性内切酶。
一种重组表达工程菌,包含上述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因。
作为进一步改进的,将上述的重组表达载体转化感受态细胞而得到。
一种芽孢杆菌群体感应淬灭酶AiiA酶的制备方法,培养上述的重组表达工程菌,诱导重组蛋白酶的表达,回收并纯化所表达的芽孢杆菌群体感应淬灭AiiA酶。
一种芽孢杆菌群体感应淬灭酶AiiA酶在杀菌中的应用。
一种芽孢杆菌群体感应淬灭酶AiiA酶在水产养殖中的应用。
本发明的有益效果是:
本发明的芽孢杆菌群体感应淬灭酶AiiA酶的酶活性高,能够高效降解高丝氨酸内酯AHL(调控群体感应系统的关键信号分子),从而显著解决在疾病防治中使用抗生素易导致细菌产生耐药性的问题,能够有效用于防治水产养殖出现的一些细菌感染疾病问题。
附图说明
为了更清楚地说明本发明实施方式的技术方案,下面将对实施方式中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明实施例5提供的AiiA酶基因扩增产物的电泳结果图,其中,2泳道为本发明实施例1所述AiiA酶,6泳道为野生型,其他泳道为其他突变型:(a)为蛋白上清、(b)为纯化蛋白。
图2是AHL的分子结构。
图3是本发明实施例3提供的工程菌菌落PCR验证结果。
图4是本发明实施例6提供的酶活检测结果。其中,14为本发明实施例的AiiA酶,wt为野生型,其他为其他突变型。
图5是本发明实施例7提供的AiiA酶对溶藻弧菌抑菌效果的验证。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚,下面将结合本发明实施方式中的附图,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式是本发明一部分实施方式,而不是全部的实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。因此,以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。
实施例1芽孢杆菌AiiA酶基因的筛选
基因筛选过程如下:
筛选质粒的构建
(1)生物砖lux pR-RBS1.0-lysis-TT的构建
质粒pSB1A2作XbaI/SpeI双酶切,RBS1.0-lysis-TT(由金斯瑞合成)作XbaI/SpeI双酶切,胶回收后通过T4DNA连接酶在4℃过夜连接,获得重组质粒pSB1A2-RBS1.0-lysis-TT;提取质粒pSB1A2-RBS1.0-lysis-TT,做XbaI/PstI的双酶切产生后端插入段RBS1.0-lysis-TT和约2000bp的质粒骨架pSB1A2,胶回收其中后端插入段条带RBS1.0-lysis-TT;质粒pSB1A2作XbaI/SpeI双酶切,lux pR(由金斯瑞合成)作XbaI/SpeI双酶切,胶回收后通过T4DNA连接酶在4℃过夜连接,获得重组质粒pSB1A2-lux pR;提取质粒pSB1A2-lux pR(质粒由美国麻省理工学院Genetic Parts注册中心提供),做SpeI/PstI的双酶切生成约2100bp的后端载体pSB1A2-lux pR;连接上述两个片段,转化到DH5ɑ中,涂布在含有氨苄青霉素的平板上,做2个平行板,第二天挑单菌落,培养12-16h,提取质粒做EcoRI和PstI的双酶切和EcoRI的单酶切,跑胶验证,单酶切的条带大小为约2450bp,双酶切的条带分别为约400bp的目的段lux pR-RBS1.0-RFP-TT和2100bp左右的质粒骨架pSB1A2。将该工程菌保存于-20℃甘油管,该菌带有lux pR-RBS1.0-lysis-TT的质粒。
(2)生物砖lux pR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT的构建
提取质粒pSB1A2-lux pR-RBS1.0-lysis-TT(本质粒由上述方法1构建获得),做XbaI/PstI双酶切产生后端插入段lux pR-RBS1.0-lysis-TT和约2000bp的质粒骨架pSB1A2,胶回收其中后端插入段条带lux pR-RBS1.0-lysis-TT;质粒pSB1A2作XbaI/SpeI双酶切,J23108-RBS1.0-aiiA-TT(由金斯瑞合成)作XbaI/SpeI双酶切,胶回收后通过T4DNA连接酶在4℃过夜连接,获得重组质粒pSB1A2-J23108-RBS1.0-aiiA-TT;提取质粒pSB1A2-J23108-RBS1.0-aiiA-TT,做SpeI/PstI的双酶切生成约2100bp的后端载体J23108-RBS1.0-aiiA-TT;连接上述两个片段,转化到DH5ɑ中,涂布在含有氨苄青霉素的平板上,做2个平行板,第二天挑单菌落,培养12-16h,提取质粒做EcoRI和PstI的双酶切和EcoRI的单酶切,跑胶验证,单酶切的条带大小为约3400bp,双酶切的条带为约1300bp的目的段lux pR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT和2100bp左右的质粒骨架pSB1A2。将该工程菌保存于-20℃甘油管,该菌带有lux pR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT的质粒。
(3)生物砖
J23101-RBS1.0-luxR-TT-luxpR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT的构建
质粒pSB1A2作XbaI/SpeI双酶切,通过PCR在RBS1.0-lysis-TT的N端添加J23108胶回收产物作XbaI/SpeI双酶切,胶回收后通过T4DNA连接酶在4℃过夜连接,获得重组质粒pSB1A2-J23101-RBS-luxR-TT;提取质粒pSB1A2-J23101-RBS-luxR-TT;做EcoRI/SpeI的双酶切产生前端插入段J23101-RBS1.0-luxR-TT和约2000bp的质粒骨架pSB1A2,胶回收其中后端插入段条带J23101-RBS1.0-luxR-TT;提取pSB1A2-lux pR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT(本质粒由上述方法2构建所得),做EcoRI/XbaI的双酶切生成前端载体luxpR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT和pSB1A2;连接上述两个片段,转化到DH5ɑ中,涂布在含有氨苄青霉素的平板上,做2个平行板,第二天挑单菌落,培养12-16h,提取质粒做EcoRI和PstI的双酶切和EcoRI的单酶切,跑胶验证。单酶切的条带大小为约4500bp,双酶切的条带分别为约2400bp的目的段J23101-RBS1.0-luxR-TT-luxpR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT和2100bp左右的质粒骨架pSB1A2。说明本筛选质粒构建完成,条带大小正确。将该工程菌保存于-20℃甘油管,该菌带有J23101-RBS1.0-luxR-TT-lux pR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT的质粒。
其中,筛选质粒的基因序列为SEQ ID NO:1,luxpR启动子J23101的基因序列为SEQID NO:2,luxpR启动子J23108的基因序列为SEQ ID NO:3,核糖体结合位点B0034的基因序列为SEQ ID NO:4,终止子TT的基因序列为SEQ ID NO:5,luxpR蛋白的基因序列为SEQ IDNO:6,lysis致死蛋白的基因序列为SEQ ID NO:7,野生型AiiA酶的基因序列为SEQ ID NO:8。
高通量蛋白质定向进化
1)易错PCR条件确定
利用软件Snapgene设计易错PCR引物AiiA-F,AiiA-R,上游引物包含SpeI酶切位点而下游引物含有PstI酶切位点,设计原则同一般PCR:AiiA-F:5’-GCGACTAGTGAAAGAGGAGAAATACTAGATGAC-3’(SEQ ID NO:16)
AiiA-R:5’-GCGCTGCAGTATATAAACGCAGAAAGG-3’(SEQ ID NO:17)
PCR反应体系如表1所示,本例中的dNTP是按照dATP、dGTP 2.5mM,dCTP、dTTP10mM的比例配置的。PCR反应条件为:94℃预变性10min;94℃变性30s,58℃退火30s,72℃延伸1min,30~35个循环;72℃延伸10min。
表1易错PCR反应体系
2)利用易错PCR扩增目的基因
按实施例1)所述,确定好易错PCR的反应条件后,对AiiA基因进行扩增。通过OMEGA试剂商提供的胶回收试剂盒对其进行胶回收。
3)线性化载体的制备
通过双酶切获得线性化载体,按实施例1获得的载体J23101RBS1.0-luxR-TT-luxpR-RBS1.0-lysis-TT-J23108-RBS1.0-aiiA-TT,对其做SpeⅠ和PstⅠ双酶切,通过琼脂糖凝胶电泳回收线性化的载体。
同样地,对于易错PCR扩增获得的目的基因做SpeⅠ和PstⅠ双酶切,并通过琼脂糖凝胶电泳回收酶切产物。
双酶切反应均在37℃条件下反应4h。双酶切体系如表2所示:
表2酶切反应体系
4)重组质粒的构建
分别测定线性化载体与酶切产物的浓度,通过T4连接酶进行连接。
酶连接的反应条件:16℃下反应6h或者在4℃的冰箱中存放12h,在0.2mL PCR管中配置的样品、药品加入体积可以按照表3进行添加。
V1,V2由(公式1)、(公式2)计算所得
V1+V2=8μL (公式2)
其中V1、V2分别为插入基因和载体的体积,M1、M2分别为基因的摩尔质量,c1、c2为以ng/μL为单位的基因片段浓度。
表3酶连接反应体系
5)突变文库的构建
将本实施例得到的AiiA重组突变质粒立即转化入大肠杆菌E.coli DH5α感受态细胞中,涂于含有AHL100 nM和100μg/ml的氨苄青霉素的LB平板上,37℃培养12~16h,根据平板上存活的单菌个数来获得AiiA突变菌株。
在本发明构建的筛选质粒中,当AHL降解酶AiiA表达量低或者活性低时,外加的AHL足够与LuxR蛋白结合,使得细菌裂解蛋白基因lysis表达,导致DNA促旋酶中毒从而使宿主细菌大量死亡。反之,当AHL降解酶AiiA存在时,外加的AHL被充分降解,则裂解蛋白lysis的表达保持在低水平,因而宿主细胞可以生长。当群体感应淬灭酶AiiA的活性更强时,能降解的AHL更多,宿主细胞就能在更高的AHL浓度环境下存活。所以,可以通过在筛选平板上菌株的存活来获得高效群体感应淬灭酶的突变株。
其中,AiiA突变菌株中的一突变AiiA酶基因序列如下所示:
ATGACAGTAAAGAAGCTTTATTTCGTCCCAGCAGGTCGTAGTATGTTGGATCATTCGTCTGTTAATAGTACATTAACACCAGGAGAATTATTAGACTTACCGGTTTGGTGTTATCTTTTGGAGACTGAAGAAGGACCTATTTTAGTAGATACAGGTATGCCAGAAAGTGCAGTTAATAATGAAGGTCTTTTTAACGGTACATTTGTCGAAGGGCAGGTTTTACCGAAAATGACTGAAGAAGATAGAATCGTGAATATTTTAAAACGGGTTGGTTATGAGCCGGAAGACCTTCTTTATATTATTAGTTCTCACTTGCATTTTGATCATGCAGGAGGAAATGGCGCTTTTATAAATACACCAATCATTGTACAGCGTGCTGAATATGAGGCGGCGCAGCATAGCGAAGAATATTTGAAAGAATGTATATTGCCGAATTTAAACTACAAAATCATTGAAGGTGATTATGAAGTCGTACCAGGAGTTCAATTATTGCATACACCAGGCCATACTCCAGGGCATCAATCGCTATTAATTGAGACAGAAAAATCCGGTCCTGTATTATTAACGATTGATGCATCGTATACGAAAGAGAATTTTGAAAATGAAGTGCCATTTGCGGGATTTGATTCAGAATTAGCTTTATCTTCAATTAAACGTTTAAAAGAAGTGGTGATGAAAGAGAAGCCGATTGTTTTCTTTGGACATGATATAGAGCAGGAAAGGGGATGTAAAGTGTTCCCTGAATATATATAATAA(SEQ ID NO:9)。
该基因也可以由金斯瑞公司合成,在野生型AiiA酶基因的基础上再进行点突变(40号碱基的T突变为A)。
实施例2芽孢杆菌AiiA酶工程菌的构建
对突变AiiA酶基因(SEQ ID NO:9)进行PCR扩增,分别用限制性内切酶NdeI和XhoI消化PCR产物,然后用Takara连接试剂盒(T4连接酶)在pET28a载体上的多个克隆位点之间连接。表达克隆分别由T7启动子驱动,质粒上的His标记编码序列将组氨酸编码到靶蛋白的N端。阳性克隆在大肠杆菌DH5α中扩增,转移到大肠杆菌BL21中进行蛋白表达。
(1)无菌条件下,将5-10μL酶连产物或质粒加入50μL感受态细胞中,轻轻用移液枪吹打来混匀质粒与感受态细胞。
(2)将上述加入质粒的感受态细胞插入在冰中30min,期间准备42℃水浴对感受态进行热击45-60s,随后继续将离心管插在冰中2min。
(3)无菌条件下,在感受态细胞中加入450μL灭菌LB培养基,放置在37℃,200r/min摇床中震荡培养1h。
(4)将上述菌液涂布于含有氨苄青霉素(和AHL)的平板上,用涂棒将菌液缓慢推干后,用封口膜对培养皿的边缘进行封口,最后将涂好的平板放在37℃恒温箱中培养16-24h。
实施例3工程菌鉴定、保存与活化
挑取过夜培养的平板上长出的单菌落于20mL培养基中,加入氨苄青霉素至终浓度50μg/mL,然后放置在37℃摇床恒温培养12h左右后提质粒做单双酶切,跑胶鉴定,验证结果如图3所示,条带大小正确。
无菌条件下,在甘油管中加入200μL 40%的甘油和已经培养了12-16h的需要保藏的菌种培养液800μL,用移液枪轻轻吹打混匀后,在贴在甘油甘油管外的标签纸上作上菌种名称、抗性和日期,置于-20℃保存。
在灭过菌的LB培养基中加入氨苄青霉素溶液到100ng/mL,然后从甘油管中用移液枪吸取10μL保藏的菌种接入,在摇床中设置参数为37℃,200r/min中震荡培养大约12个小时,可以通过培养基的浑浊程度来判断是否需要多培养几个小时。
实施例4芽孢杆菌AiiA酶的表达与纯化
(1)将质粒的载体更换成为可以进行IPTG诱导的pET-28a(+)商业载体后导入E.coli BL21(DE3),经单克隆纯培养后进行菌种-80℃保藏。
(2)20mL LB培养基中按照1%接种量活化菌种,加入40mg/mL卡那霉素20μL,打开摇床设置参数为37℃、200rpm,培养12h。
(3)200mL的LB培养基中按照接种量10%加入活化培养基,加入40mg/mL卡那霉素200μL,打开摇床设置参数为37℃、200rpm培养至OD600=0.5,加入1M的IPTG 450μL,打开摇床设置参数为30℃,200rpm诱导培养24-36h。
(4)使用冷冻离心机在4℃,8000rpm,5min条件下收集诱导培养后的菌体,丢弃上清液。在使用PBS缓冲液将底部湿菌体洗涤两次后,再用上样缓冲液将菌体悬浮起来。随后将其置于冰浴中,超声破碎仪破碎细胞(超声破碎80次,200W,超声3s,间隔6s,探头置于液面下方1cm)。
(5)将已经破碎过的菌体悬浮液放在冷冻离心机下条件为4℃,30min,8000rpm下,通过离心力使细胞碎片沉淀在底部,澄清的上清液就是实验所需要的粗酶液。
由于pET-28a(+)载体上带有His-tag标签,利用上海七海复泰生物科技有限公司的His·Bind Resin纯化柱进行蛋白纯化。首先,用10mL的上样缓冲液平衡纯化柱,然后把样品上样在平衡好的纯化柱上。样品注入完成后,我们使用10mL上样缓冲液进行柱子的清洗,之后使用10-20mL洗杂缓冲液将杂质蛋白质从纯化柱上洗脱下去。使用5mL洗脱缓冲液将目的蛋白质洗脱下来,收集到的洗脱液就是我们的纯酶。最后,用超滤管对纯化的蛋白进行浓缩和脱盐。浓缩和脱盐方法:(1)用0.45μm滤膜来除去纯化样品和上样缓冲液中的颗粒杂质;(2)然后把过滤后的纯化样品加入超滤管的内管中,离心(4℃,5000g,20min),至内管样品体积小于1.5mL,弃外管滤液;(3)重复第二步1次,直至去除所有的咪唑和盐离子,将除盐后的酶液4℃保存;(4)清洗超滤管。
表4纯化蛋白浓度
实施例5进行变性聚丙烯酰胺凝胶电泳SDS-PAGE验证
目标蛋白(AiiA)通过纯化手段的纯化情况可以用SDS-PAGE进行判断。使用Willget10%预制胶,100mA,80V电压下30min后更换为150V高压跑胶60min。跑胶结束后把蛋白胶放于染色皿中加入考马斯亮蓝染色液,摇床45r/min,1h。洗净后进行脱色步骤,使用考马斯亮蓝脱色液,摇床45r/min,1h。结束后将脱色过后的蛋白质凝胶成像分析仪进行分析。结果如图1所示。
根据图1第6泳道结果可判断,所述AiiA酶约28.00kDa
经测序,其氨基酸的序列如下:
MTVKKLYFVPAGRSMLDHSSVNSTLTPGELLDLPVWCYLLETEEGPILV
DTGMPESAVNNEGLFNGTFVEGQVLPKMTEEDRIVNILKRVGYEPEDLLYIIS
SHLHFDHAGGNGAFINTPIIVQRAEYEAAQHSEEYLKECILPNLNYKIIEGDY
EVVPGVQLLHTPGHTPGHQSLLIETEKSGPVLLTIDASYTKENFENEVPFAGF
DSELALSSIKRLKEVVMKEKPIVFFGHDIEQERGCKVFPEYI(SEQ ID NO:10)。
实施例6芽孢杆菌AiiA酶酶活检测
在底物信号分子(3-oxo-C6-HSL)的存在下,测定光密度随反应时间的变化,从而反映出AiiA酶活性。1个酶活力单位(U)(μmol/min):1min降解1μmol3-oxo-C6-HSL所需的酶量。酶的蛋白含量使用考马斯亮蓝法进行测定,595nm。
(1)反应体系含2X染液(2mM HEPES pH 7.5、200mM NaSO4、0.4mM ZnSO4,100μM苯酚红),50μL纯化蛋白;
(2)10μL信号分子(3-oxo-C6-HSL)溶液,反应的总体积是200μL,OD557测定间隔为10s,共60次;
(3)空白对照组缺少纯化过后的酶,其他反应条件不变。
酶活测定结果如图4所示,本发明中的芽孢杆菌AiiA酶(横坐标14)相比于野生型(横坐标wt)相对比活力有所提升。
实施例7芽孢杆菌AiiA酶在水产养殖中的应用
革兰氏阴性细菌群体感应基因回路最少含有一对费氏弧菌(V.fischeri)QS蛋白的同系物(LuxI和LuxR)。LuxI类蛋白是自诱导物合成酶,利用来源于酰基载体蛋白的酰基和S-腺苷甲硫氨酸的高丝氨酸内酯,合成自诱导物酰基高丝氨酸内酯化合物(AHL)作为信号分子,如图2。在较高的细胞密度时,积累的AHL分子与LuxR类蛋白结合形成复合物,此复合物随后激活靶基因转录。利用这种群体感应机制,革兰氏阴性菌可以有效地将基因表达与细胞种群密度的波动结合起来。已知具有LuxI/luxR蛋白或其同系物的QS系统有:铜绿假单胞菌(Pseudomonas aeruginosa)LasI/LasR-RhlI/RhlR毒力系统、根癌农杆菌(Agrobacterium tumefaciens)TraI/TraR毒力系统、胡萝卜软腐欧文(氏)菌(Erwiniacarotovora)ExpI/ExpR-CarI/CarR毒力/抗生素系统等。
溶藻弧菌ATCC33787是一种革兰氏阴性菌,具有群体感应作用机制,会造成对虾等水产养殖物患急性肝胰腺坏死综合症(AHPND),AHPND多发于虾苗投放后30-35天内并可以观察到虾苗突然大量死亡,又被称为早期死亡综合症(EMS)。急性肝胰腺坏死综合症在斑节对虾和南美白对虾中更具有易感性。
AHPND具有传播速度快、传播范围广、死亡率高等特点,因此,该病一旦产生造成的损失极其惨重。该病自2009年在中国首次爆发并被报道以来,就对中国养殖业造成了不可挽回的重大经济损失,随后陆续在越南和马来西亚、泰国、墨西哥、菲律宾、南美洲以及美国等地大规模爆发从而产生对虾大幅减产等重大影响。
AiiA基因表达产生淬灭酶AiiA,会降解环境中的AHL,从而阻遏AHL与luxR蛋白结合。
AiiA酶对溶藻弧菌ATCC33787抑菌作用验证:
(1)在LB培养基中培养溶藻弧菌ATCC33787。
(2)吸取每菌液1mL。用1mL 150mM EDTA孵育30min。
(3)6500rpm离心10min以除去EDTA,弃上清液。然后加入1mL相应的培养基,重悬沉淀。
(4)重复步骤(3)两次。
(5)稀释溶液,稀释浓度为10-3,10-4,10-5,10-6。
(6)每种稀释浓度的溶液按以下三种方式处理,然后用于琼脂斑点实验:
a.使用无菌水处理;
b.使用10%NaOH处理20分钟;
c.使用AiiA酶液处理。
(7)在37℃过夜培养。
(8)观察琼脂斑点实验的结果。
将溶藻弧菌从1/10-3稀释至1/10-6。阴性对照组使用EDTA和无菌水处理,阳性对照组使用碱液处理。将处理过后的样品分别用于斑点试验,然后观察平板上菌落生长情况。如图5所示,结果显示:当菌液经AiiA酶液处理后,溶藻弧菌在平板上基本不生长。表明AiiA酶对溶藻弧菌有杀灭作用,且与用碱裂解处理的杀灭结果一致。
综上所述,本研究描述了一种芽孢杆菌群体感应淬灭酶AiiA酶,并对其进行了异源表达,考察其QQ降解AHL的能力。通过降解AHLs介导病原菌的QS,该酶对革兰氏阴性菌溶藻弧菌ATCC33787具有显著的抑制作用,从而可抑制在水产养殖中鱼、鳖、蛙等水产养殖动物由溶藻弧菌ATCC33787引起的细菌性疾病。结果表明,该AiiA酶可作为防治水产养殖中革兰氏阴性菌溶藻弧菌ATCC33787引起的细菌性疾病的有效途径。
本发明所涉及的核苷酸和氨基酸序列如下:
SEQ ID NO:1:
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGAAAGAGGA
GAAATACTAGATGAAAAACATAAATGCCGACGACACATACAGAATAATTAA
TAAAATTAAAGCTTGTAGAAGCAATAATGATATTAATCAATGCTTATCTGAT
ATGACTAAAATGGTACATTGTGAATATTATTTACTCGCGATCATTTATCCTCA
TTCTATGGTTAAATCTGATATTTCAATCCTAGATAATTACCCTAAAAAATGG
AGGCAATATTATGATGACGCTAATTTAATAAAATATGATCCTATAGTAGATTA
TTCTAACTCCAATCATTCACCAATTAATTGGAATATATTTGAAAACAATGCT
GTAAATAAAAAATCTCCAAATGTAATTAAAGAAGCGAAAACATCAGGTCT
TATCACTGGGTTTAGTTTCCCTATTCATACGGCTAACAATGGCTTCGGAATG
CTTAGTTTTGCACATTCAGAAAAAGACAACTATATAGATAGTTTATTTTTAC
ATGCGTGTATGAACATACCATTAATTGTTCCTTCTCTAGTTGATAATTATCGA
AAAATAAATATAGCAAATAATAAATCAAACAACGATTTAACCAAAAGAGA
AAAAGAATGTTTAGCGTGGGCATGCGAAGGAAAAAGCTCTTGGGATATTT
CAAAAATATTAGGTTGCAGTGAGCGTACTGTCACTTTCCATTTAACCAATG
CGCAAATGAAACTCAATACAACAAACCGCTGCCAAAGTATTTCTAAAGCA
ATTTTAACAGGAGCAATTGATTGCCCATACTTTAAAAATTAATAACACTGAT
AGTGCTAGTGTAGATCACTACTAGAGCAGGCATCAAATAAAACGAAAGGC
TCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC
TCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTA
TATACTAGAGACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGT
TATAGTCGAATAAATACTAGAGAAAGAGGAGAAATACTAGAGATGAAAAA
AATAACAGGGATTATTTTATTGCTTCTTGCAGCCATTATTCTTGCTGCATGT
CAGGCAAACTATATCCGTGATGTTCAGGGCGGGACAGTATCACCGTCGTC
AACTGCTGAACTGACCGGAGTGGAAACGCAGTAATACTAGAGCCAGGCA
TCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCT
GTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCG
GGTGGGCCTTTCTGCGTTTATATACTAGAGTTTACAGCTAGCTCAGTCCTA
GGTATTATGCTAGCTACTAGTGAAAGAGGAGAAATACTAGATGACAGTAA
AGAAGCTTTATTTCGTCCCAGCAGGTCGTTGTATGTTGGATCATTCGTCTG
TTAATAGTACATTAACACCAGGAGAATTATTAGACTTACCGGTTTGGTGTTA
TCTTTTGGAGACTGAAGAAGGACCTATTTTAGTAGATACAGGTATGCCAGA
AAGTGCAGTTAATAATGAAGGTCTTTTTAACGGTACATTTGTCGAAGGGCA
GGTTTTACCGAAAATGACTGAAGAAGATAGAATCGTGAATATTTTAAAAC
GGGTTGGTTATGAGCCGGAAGACCTTCTTTATATTATTAGTTCTCACTTGCA
TTTTGATCATGCAGGAGGAAATGGCGCTTTTATAAATACACCAATCATTGT
ACAGCGTGCTGAATATGAGGCGGCGCAGCATAGCGAAGAATATTTGAAAG
AATGTATATTGCCGAATTTAAACTACAAAATCATTGAAGGTGATTATGAAG
TCGTACCAGGAGTTCAATTATTGCATACACCAGGCCATACTCCAGGGCATC
AATCGCTATTAATTGAGACAGAAAAATCCGGTCCTGTATTATTAACGATTGA
TGCATCGTATACGAAAGAGAATTTTGAAAATGAAGTGCCATTTGCGGGATT
TGATTCAGAATTAGCTTTATCTTCAATTAAACGTTTAAAAGAAGTGGTGAT
GAAAGAGAAGCCGATTGTTTTCTTTGGACATGATATAGAGCAGGAAAGGG
GATGTAAAGTGTTCCCTGAATATATATAATAATACTAGAGCCAGGCATCAAA
TAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGT
TTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGG
GCCTTTCTGCGTTTATATA
SEQ ID NO:2:
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC
SEQ ID NO:3:
TCTAGAGAAAGACGAGATATACTAGATG
SEQ ID NO:4:
AAAGAGGAGAAA
SEQ ID NO:5:
GCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCG
TTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTC
ACCTTCGGGTGGGCCTTTCTGCGTTTATA
SEQ ID NO:6:
ATGAAAAACATAAATGCCGACGACACATACAGAATAATTAATAAAATTAAA
GCTTGTAGAAGCAATAATGATATTAATCAATGCTTATCTGATATGACTAAAA
TGGTACATTGTGAATATTATTTACTCGCGATCATTTATCCTCATTCTATGGTT
AAATCTGATATTTCAATCCTAGATAATTACCCTAAAAAATGGAGGCAATATT
ATGATGACGCTAATTTAATAAAATATGATCCTATAGTAGATTATTCTAACTCC
AATCATTCACCAATTAATTGGAATATATTTGAAAACAATGCTGTAAATAAAA
AATCTCCAAATGTAATTAAAGAAGCGAAAACATCAGGTCTTATCACTGGG
TTTAGTTTCCCTATTCATACGGCTAACAATGGCTTCGGAATGCTTAGTTTTG
CACATTCAGAAAAAGACAACTATATAGATAGTTTATTTTTACATGCGTGTAT
GAACATACCATTAATTGTTCCTTCTCTAGTTGATAATTATCGAAAAATAAAT
ATAGCAAATAATAAATCAAACAACGATTTAACCAAAAGAGAAAAAGAATG
TTTAGCGTGGGCATGCGAAGGAAAAAGCTCTTGGGATATTTCAAAAATATT
AGGTTGCAGTGAGCGTACTGTCACTTTCCATTTAACCAATGCGCAAATGA
AACTCAATACAACAAACCGCTGCCAAAGTATTTCTAAAGCAATTTTAACA
GGAGCAATTGATTGCCCATACTTTAAAAAT
SEQ ID NO:7:
ATGAAAAAAATAACAGGGATTATTTTATTGCTTCTTGCAGCCATTATTCTTG
CTGCATGTCAGGCAAACTATATCCGTGATGTTCAGGGCGGGACAGTATCAC
CGTCGTCAACTGCTGAACTGACCGGAGTGGAAACGCAGTAA
SEQ ID NO:8:
ATGACAGTAAAGAAGCTTTATTTCGTCCCAGCAGGTCGTTGTATGTTGGAT
CATTCGTCTGTTAATAGTACATTAACACCAGGAGAATTATTAGACTTACCG
GTTTGGTGTTATCTTTTGGAGACTGAAGAAGGACCTATTTTAGTAGATACA
GGTATGCCAGAAAGTGCAGTTAATAATGAAGGTCTTTTTAACGGTACATTT
GTCGAAGGGCAGGTTTTACCGAAAATGACTGAAGAAGATAGAATCGTGAA
TATTTTAAAACGGGTTGGTTATGAGCCGGAAGACCTTCTTTATATTATTAGT
TCTCACTTGCATTTTGATCATGCAGGAGGAAATGGCGCTTTTATAAATACA
CCAATCATTGTACAGCGTGCTGAATATGAGGCGGCGCAGCATAGCGAAGA
ATATTTGAAAGAATGTATATTGCCGAATTTAAACTACAAAATCATTGAAGG
TGATTATGAAGTCGTACCAGGAGTTCAATTATTGCATACACCAGGCCATAC
TCCAGGGCATCAATCGCTATTAATTGAGACAGAAAAATCCGGTCCTGTATT
ATTAACGATTGATGCATCGTATACGAAAGAGAATTTTGAAAATGAAGTGCC
ATTTGCGGGATTTGATTCAGAATTAGCTTTATCTTCAATTAAACGTTTAAAA
GAAGTGGTGATGAAAGAGAAGCCGATTGTTTTCTTTGGACATGATATAGA
GCAGGAAAGGGGATGTAAAGTGTTCCCTGAATATATA
以上所述仅为本发明的优选实施方式而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种芽孢杆菌群体感应淬灭酶AiiA酶,其特征在于,其氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的芽孢杆菌群体感应淬灭酶AiiA酶,其特征在于,其编码基因的核苷酸序列如SEQ ID NO.2所示。
3.一种权利要求1或2所述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因,其特征在于,其核苷酸序列如SEQ ID NO.2所示。
4.一种重组表达载体,其特征在于,包含权利要求3所述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因。
5.根据权利要求4所述的重组表达载体,其特征在于,所述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因插入到质粒上的限制性酶切位点之间,所述限制性内切酶包括NdeI限制性内切酶和/或XhoI限制性内切酶。
6.一种重组表达工程菌,其特征在于,包含权利要求3所述的芽孢杆菌群体感应淬灭酶AiiA酶的编码基因。
7.根据权利要求6所述重组表达工程菌,其特征在于,将权利要求4或5所述的重组表达载体转化感受态细胞而得到。
8.一种芽孢杆菌群体感应淬灭酶AiiA酶的制备方法,其特征在于,培养权利要求6或7所述的重组表达工程菌,诱导重组蛋白酶的表达,回收并纯化所表达的芽孢杆菌群体感应淬灭AiiA酶。
9.一种芽孢杆菌群体感应淬灭酶AiiA酶在杀菌中的应用。
10.一种芽孢杆菌群体感应淬灭酶AiiA酶在水产养殖中的应用。
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