CN104805066B - 一种葡萄球菌裂解酶及其应用 - Google Patents

一种葡萄球菌裂解酶及其应用 Download PDF

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CN104805066B
CN104805066B CN201510252947.6A CN201510252947A CN104805066B CN 104805066 B CN104805066 B CN 104805066B CN 201510252947 A CN201510252947 A CN 201510252947A CN 104805066 B CN104805066 B CN 104805066B
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staphylococcus
lyases
clyo
staphylococcus aureus
methicillin
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CN104805066A (zh
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危宏平
杨航
余军平
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Targeted Antibiotics Biopharmaceutical Technology Co ltd
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Wuhan Faye Gile C Biological Science And Technology Co Ltd
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Abstract

本发明公开了一种能杀灭葡萄球菌的裂解酶及其应用，属于生物制剂领域。本发明公开了该酶的氨基酸序列和编码基因序列。该酶发挥作用的pH范围宽，在pH4‑11都保持有裂解葡萄球菌的活力，采用编码基因构建的重组蛋白酶能在大肠杆菌BL21(DE3)中可溶表达。该酶能用于在体外高效杀灭各种葡萄球菌，包括临床分离的甲氧西林敏感的金黄色葡萄球菌和甲氧西林耐药的金黄色葡萄球菌。该酶能用作治疗体内葡萄球菌感染的抗生素。所公开的酶也能快速裂解葡萄球菌的细胞壁并释放胞内的物质，如三磷酸腺苷和DNA等，利用所释放的物质来对葡萄球菌进行检测。

Description

一种葡萄球菌裂解酶及其应用
技术领域
本发明属于生物制剂领域，具体涉及一种能杀灭葡萄球菌，特别是金黄色葡萄球菌裂解酶及其在葡萄球菌杀灭、检测等方面的应用。
背景技术
根据生化反应和产生色素不同，葡萄球菌可分为金黄色葡萄球菌(staphylococcus aureus)、表皮葡萄球菌(staphylococcus epidermidis)和腐生葡萄球菌(staphylococcus saprophytics)等三种。其中金黄色葡萄球菌多为致病菌，表皮葡萄球菌偶尔致病，腐生葡萄球菌一般不致病。金黄色葡萄球菌(staphylococcus aureus)是一种常见的革兰氏阳性球菌，能引起人和动物的许多严重的感染，同时也是医院感染常见的病原体之一。葡萄球菌易于对抗生素产生抗性，包括常见的各种抗生素，以及新型的抗微生物制剂。耐甲氧西林金黄色葡萄球菌(MRSA)的出现和广泛传播给临床治疗带来了前所未有的挑战。为了应对葡萄球菌的耐药性问题，常需要提取葡萄球菌基因组DNA，用于PCR检测临床菌株是否是MRSA等等。然而，由于葡萄球菌细胞壁坚厚，常规的蛋清溶菌酶对其没有明显的溶壁作用，而具有良好溶壁效果的溶葡萄球菌素价格昂贵，难以普遍应用。
噬菌体裂解酶是双链DNA噬菌体感染宿主细菌后晚期表达的一种细胞壁水解酶。裂解酶大小通常为25kD～40kD，在结构上由两个独立的功能域构成，N-端的催化功能域，和一个决定细胞结合位点的C-端细胞壁结合功能域(CBD)，二者之间由一个小片段链接。序列分析表明，同一类裂解酶的催化域高度保守，而细胞结合域可变，这为构建新的嵌合裂解酶提供了可能。裂解酶具有很高的特异性，只能特异性的识别和裂解特定种类的细菌。此外，裂解酶作用位点很保守，加上噬菌体与细菌共同进化的特异性，宿主菌很难对其产生抗性。裂解酶的这些特点，为其用于临床上耐药细菌的控制和治疗提供了理论的可行性。到目前为止，已有一些能作用于金黄色葡萄球菌的天然裂解酶以及嵌合裂解酶被报道，这些酶可以在体内、体外较好地杀灭金黄色葡萄球菌。但是这些裂解酶大多比较难于可溶性表达，或者活性不高，或者不能适应高蛋白环境(如牛奶等)，作用pH范围较窄，一般在pH 5-8。寻找能可溶、大量表达以及高活性的裂解酶对于开发新的抗葡萄球菌的药物，体外控制葡萄球菌的感染，裂解葡萄球菌的细胞壁用于检测等等都具有非常重要的意义。
发明内容
本发明所要解决的技术问题是提供一种可溶表达、活性高的葡萄球菌裂解酶及其应用。该种裂解酶能在体外、体内杀灭葡萄球菌，特别是金黄色葡萄球菌。为了叙述方便，我们命名为ClyO，其编码基因为ClyO。
本发明提供的葡萄球菌裂解酶的编码基因ClyO的核酸序列如序列表中SEQ.ID.NO.1所示。
本发明提供的葡萄球菌裂解酶ClyO的蛋白序列如序列表中SEQ.ID.NO.2所示。
本发明还公开了一种可溶表达ClyO蛋白质及纯化的方法，其步骤为：将ClyO基因克隆后连入表达载体pBAD24中，然后将表达质粒转化大肠杆菌BL21(DE3)中进行表达，所表达的蛋白质先经过离子交换纯化，再用磷酸盐(PBS)缓冲液透析处理。
本发明证实了该裂解酶ClyO的高活性与对葡萄球菌的广谱性。本发明公开了ClyO体外杀灭表皮葡萄球菌、腐生葡萄球菌、马胃葡萄球菌、头葡萄球菌、白色葡萄球菌、木糖葡萄球菌、松鼠葡萄球菌、溶血性葡萄球菌、产色葡萄球菌、以及金黄色葡萄球菌的用途。所述金黄色葡萄球菌包括临床分离的甲氧西林敏感的金黄色葡萄球菌和甲氧西林耐药的金黄色葡萄球菌。本发明还初步试验了ClyO对实验动物模型小鼠感染金黄色葡萄菌后的保护效果，以及对细胞毒性的测试，初步证实了其用于制备治疗葡萄球菌感染药物的潜能。
本发明还进一步公开了裂解酶ClyO在葡萄球菌检测和鉴定中的用途。用裂解酶ClyO与待测细菌作用后，释放菌内的三磷酸腺苷(ATP)或DNA等胞内物质，通过检测释放的ATP或DNA来用于葡萄球菌的检测和鉴定。
本发明还进一步公开了利用ClyO快速裂解葡萄球菌细胞壁并释放胞内ATP的特点，采用荧光素酶检测释放的ATP来进行葡萄球菌的快速鉴定的方法。其步骤为：将待测细菌与ClyO混合后，加入荧光素酶及其底物于37摄氏度孵育，同时用酶标仪检测混合液发光强度的变化，同时用没有加入ClyO的菌混合液作为阴性对照。
本发明还进一步公开了利用ClyO快速裂解葡萄球菌细胞壁并释放胞内DNA的特点，以裂解液中释放的DNA为模板，采用PCR方法对葡萄球菌进行各种检测和鉴定的方法。作为例子，提供了用于MRSA鉴定和分型的检测结果。其步骤为：将待测细菌与ClyO混合后于37摄氏度孵育10-15min，对混合液10000g离心1min。取上清做模板，加入MRSA鉴定的两对引物(分别针对femB和mecA)进行PCR来对菌株是否为MRSA进行鉴定；对鉴定为MRSA的菌株，则进一步加入SCCmec分型所需的5对引物进行PCR，对MRSA菌株进行分型。
本发明的裂解酶具有以下的突出效果和优点：
ClyO能在体内、体外杀灭各种葡萄球菌，包括多种临床分离的金黄色葡萄球菌和耐药金黄色葡萄球菌(MRSA)菌株；ClyO细胞毒性低，具有应用于体内作为抗感染药物的潜能；ClyO能采用大肠杆菌可溶性表达，适合于今后的发酵生产；ClyO的酶活性高，且在pH4-11的范围内都具有较高活性。
下面结合具体的实施例对本发明做进一步的详细说明。
附图说明
图1为ClyO基因的PCR扩增结果。图中M泳道为标准分子量marker，个条带的大小标记在其左侧。O泳道为所扩增ClyO的条带。
图2.ClyO杀灭金黄色葡萄球菌标准菌株CCTCC AB91118的结果。虚线所示为金黄色葡萄球菌与ClyO混合后OD600随时间的变化趋势，实线所示为金黄色葡萄球菌与缓冲液混合后OD600随时间的变化趋势。空心圆圈所示为金黄色葡萄球菌与缓冲液混合后对数值(LogCFU)随时间的变化趋势。
图3.ClyO体外杀灭多种葡萄球菌以及对不同种类菌株特异性的结果。纵坐标表示将不同菌株与ClyO混合后37摄氏度孵育30min，OD600降低的速率。
图4.ClyO在牛奶中杀灭耐甲氧西林金黄色葡萄球菌(MRSA)菌株的结果。纵坐标表示将不同菌株与ClyO混合后37摄氏度孵育60min后细菌数目降低的对数值。
图5为ClyO清除小鼠烫伤皮肤组织上感染的耐甲氧西林金黄色葡萄球菌(MRSA)的结果。每组20只小鼠，在烫伤的皮肤组织上分别接种高浓度的MRSA，24小时后分别用PBS以及100mg ClyO处理。处理24小时后切下皮肤组织，稀释平板计算单位皮肤组织中的细菌载量。对照组为接种细菌后不做任何处理。
图6为ClyO体内杀灭金黄色葡萄球菌的结果。每组20只小鼠，分别注射致死剂量的金黄色葡萄球菌后，实验组于3小时后腹腔注射1mg ClyO。对照组于3小时后腹腔注射等体积的PBS溶液。每天观测各组小鼠的存活率。
图7为ClyO对于细胞毒性的测试结果。分别用浓度为0.1，0.5和1mg/ml的ClyO作用于Caco-2细胞没有观察到明显的细胞毒性。其中离子霉素和丝裂霉素C为对细胞有毒性的阳性对照。
图8为ClyO用于基于ATP释放的葡萄球菌快速检测结果。图中黑色直线代表空白对照，上升的曲线代表不同的金黄色葡萄球菌菌株。
图9为ClyO用于MRSA鉴定的PCR检测结果。三角所示位置为femB基因，星号所示位置为mecA基因。同时具有femB基因和mecA基因的为耐甲氧西林金黄色葡萄球菌MRSA。只具有femB基因的为甲氧西林敏感的金黄色葡萄球菌MSSA。只具有mecA基因的为金黄色葡萄球菌的其它菌株。
图10为ClyO用于耐甲氧西林金黄色葡萄球菌(MRSA)分型的PCR检测结果。SCCmecI型菌株PCR所得条带应为415bp。SCCmec II型菌株PCR所得条带应为937bp。SCCmec III型菌株PCR所得条带应为518bp。SCCmec IV型菌株PCR所得条带应为415bp和937bp的两条带。SCCmec V型菌株PCR所得条带应为518bp和359bp的两条带。
图11为ClyO用于模拟皮肤消毒后降低皮肤表面葡萄球菌的数目。当接种量<100CFU时，用ClyO处理后能完全消除。
具体实施方式
通过对葡萄球菌噬菌体裂解酶的催化域和细胞结构域氨基酸序列的分析，本发明设计和人工合成了能表达一种葡萄球菌裂解酶ClyO的基因序列，并通过体外重组到大肠杆菌中表达，获得了该酶。下列实施例中所用的方法如无特别说明均是常规的实验方法。实验中所用到的引物均由上海英骏公司合成。测序均在上海英骏公司完成。所采用的金黄色葡萄球菌标准菌株CCTCC AB91118购自武大菌株保藏中心，表皮葡萄球菌ATCC 12228、参考菌株购于广东微生物保藏中心、其它均为临床分离菌株，来源于武汉的几个医院，并经过美国Biolog自动微生物鉴定仪鉴定。临床菌株的耐药性经过甲氧西林纸片稀释法进行培养验证。临床分离的金葡菌均在baird-parker琼脂基础(购自广东环凯微生物科技有限公司)上划线分离，然后挑单菌落于TSB培养基中过夜培养，供测试。
实施例1、能杀灭葡萄球菌的裂解酶的构建。
(1)在上海生工生物工程有限公司全序列合成能表达裂解酶ClyO的ClyO基因DNA序列。合成序列装入pUC57质粒中。以ClyO基因为模板，在目的基因的两端分别加入NcoI和XhoI的酶切位点，引物设计如下：
正向引物：5-TTAACCATGGGCATGGCACTGCCTAAAACG-3(SEQ.ID.NO.3)
NcoI
反向引物：5-ATATCTCGAGTTTAAATGTACCCCAAGC-3(SEQ.ID.NO.4)
XhoI
以2μl基因为模板，各加入引物1μg进行PCR扩增，PCR扩增程序如下：1)94℃，5min；2)94℃，30sec，62℃，45sec，72℃，45sec，30个循环；3)72℃，10min；将产物进行凝胶电泳回收，电泳图谱如图1，ClyO的基因大小为777bp。与所设计的裂解酶基因片段的大小一致。
(2)将ClyO基因连入表达质粒pBAD24中得到重组载体pB-ClyO，然后将其转化大肠杆菌BL21(DE3)。
(3)ClyO的表达纯化。
将表达菌株BL21(DE3)/pB-ClyO用0.2％的L-阿拉伯糖低温诱导表达。收集菌体后超声破碎，取上清用33％硫酸铵沉淀，将沉淀溶于PBS后于PBS中透析过夜。透析液即有明显的杀菌活性。
透析后的粗提液，或者是超声后的上清，过HiTrap Q Sepharose FF column(GEHealthcare)，收集柱流出物。将柱流出物过HiTrap SP Sepharose FF column，然后用1M的NaCl梯度洗脱，分段收集洗脱峰。将有活性的各管混合后于PBS中透析过夜，即为纯化后的酶液。
实施例2、ClyO杀灭金黄色葡萄球菌标准菌株CCTCC AB91118的验证
将金黄色葡萄球菌过夜培养物，离心收集后用磷酸缓冲液(PBS)洗涤一次，再重悬于PBS溶液中。取一定量的ClyO与上述菌液混合，同时用酶标仪监测混合液在600nm吸收值的变化。用缓冲液与金黄色葡萄球菌的混合液作为负对照。最后得到的裂解曲线见图2。同时，在处理不同时间后稀释平板计算活菌数目。结果显示ClyO能快速的裂解CCTCC AB91118菌株，导致菌液的浊度降低，表现为菌液在600nm波长的吸收值快速降低。
实施例3、ClyO体外多种葡萄球菌以及对不同种类菌株特异性的验证。
为了验证ClyO的杀菌谱，我们选择了几种发明者实验室保存的非葡萄球菌菌株和临床分离的金黄色葡萄球菌、表皮葡萄球菌、腐生葡萄球菌、马胃葡萄球菌、松鼠葡萄球菌、溶血性葡萄球菌、产色葡萄球菌以及其它菌株一起测试。首先将测试的菌株分别过夜培养，离心收集后用PBS洗涤一次，然后重悬于PBS中。取一定量的ClyO与上述菌液分别混合，同时用酶标仪监测混合液在600nm吸收值的变化30min。用OD600降低的速率表示不同菌株的裂解效果。同时，用缓冲液与菌液的混合液作为负对照。试验得到的杀灭效果见图3。结果显示ClyO能快速的杀灭临床分离的多种葡萄球菌而对其它种类的测试菌株没有裂解作用。
实施例4、ClyO在牛奶中杀灭多种葡萄球菌菌株的效果。
将临床分离得到的多型MRSA菌过夜培养，离心收集后用PBS洗涤一次后，重悬于无菌巴氏消毒奶中。取一定量的ClyO与上述菌液分别混合后在37度放置60min，然后稀释平板计算其中的活菌数目。试验得到的杀灭效果见图4。结果显示ClyO能在牛奶中快速的杀灭多种葡萄球菌菌株。
实施例5、ClyO清除小鼠烫伤皮肤组织上感染的耐甲氧西林金黄色葡萄球菌(MRSA)的效果。
实验中所用小鼠为6周大的BALB/c雌性鼠，重约20到22克。在实验小鼠(60只)背部用80度的开水处理10s造成皮肤烫伤。之后在烫伤这种上接种1×107的MRSA菌株。24小时后，将小鼠分为三组，每组20只。实验组小鼠分别用无菌PBS，或者100mg ClyO处理，对照组则不做任何处理。24小时后计算单位烫伤皮肤组织中的细菌载量，所得到的结果见图5。结果显示ClyO能有效降低烫伤皮肤组织中MRSA菌的载量。
实施例6、ClyO体内杀灭金黄色葡萄球菌的验证。
实验中所用小鼠为6周大的BALB/c雌性鼠，重约20到22克。在实验小鼠(40只)腹腔注射6×107金黄色葡萄球菌临床分离菌株WHS11081。3小时后，将小鼠分为两组，每组20只。实验组小鼠腹腔注射ClyO 1000μg，对照组则注射PBS缓冲液。每天观测小鼠的存活率，所得到的结果见附图6。结果显示ClyO能有效的杀灭体内的金黄色葡萄球菌，从而提高小鼠的存活率。
实施例7、ClyO细胞毒性的测试。
将Caco-2细胞以每孔5×103的浓度接种到96孔板中，培养24小时后，向孔板中加入一定浓度的ClyO(0.1-1mg/mL)以及离子霉素(15mg/mL)和丝裂霉素C(15mg/mL)。继续培养24小时。培养结束后，向孔板中加入染色剂WST-8，静置后在酶标仪上读取450nm吸光值。所得到的结果见图7。结果显示即使高浓度的ClyO也无细胞毒性，而作为阳性对照的离子霉素和丝裂霉素均显示了较高的细胞毒性。
实施例8、ClyO用于基于ATP释放的葡萄球菌快速检测
将金黄色葡萄球菌和大肠杆菌的过夜培养物离心收集，用PBS洗涤沉淀一次后重悬于PBS中。取一定量的ClyO与上述菌液混合，同时加入0.25μM的荧光素酶和5μM的荧光素底物(其中含有12.5μM辅酶A)。混匀后的反应液置于37度，用酶标仪监测发光强度的变化。试验得到的鉴定结果见图8。结果显示所测试的金黄色葡萄球菌菌液都有不同程度的发光，原因显然是由于ClyO能裂解金葡菌的细胞壁导致了胞内ATP的释放，荧光素酶能利用所释放的ATP催化底物发光，而作为空白对照的大肠杆菌溶液，由于ClyO不能裂解其细胞壁，没有ATP释放，从而表现为没有发光产生。
实施例9、ClyO用于金黄色葡萄球菌MRSA菌株的PCR鉴定
将临床分离的金黄色葡萄球菌过夜培养物离心收集后，用PBS洗涤沉淀一次后重悬于PBS中。取一定量的ClyO与上述菌液混合，37摄氏度裂解10-15min。将裂解后的溶液10000g离心1min，取上清1μl做模板，各加入引物1μg进行PCR扩增。扩增femB基因所用引物为FemB-F(5’-TTACAGAGTTAACTGTTACC-3’)(SEQ.ID.NO.5)和FemB-R(5’-ATACAAATCCAGCACGCTCT-3’)(SEQ.ID.NO.6)；扩增mecA基因所用引物为MecA-F(5’-GTAGAAATGACTGAACGTCCGATAA-3’)(SEQ.ID.NO.7)和MecA-R(5’-CCAATTCCACATTGTTTCGGTCTAA-3’)(SEQ.ID.NO.8)。PCR扩增程序如下：1)94℃，5min；2)94℃，30sec，50℃，45sec，72℃，60sec，30个循环；3)72℃，10min；将产物进行凝胶电泳分析，电泳图谱如图9。femB基因大小为651bp，mecA基因为310bp。耐甲氧西林金葡菌(MRSA)应同时具有两个基因，甲氧西林敏感的金葡菌(MSSA)应只有femB基因。只具有mecA基因的为金葡菌的其它菌株。结果显示用ClyO作用金黄色葡萄球菌后的裂解液上清做模板，可以方便的用于PCR鉴定MRSA菌株。
实施例10、ClyO用于MRSA分型的PCR鉴定的验证
将临床分离的MRSA菌过夜培养物离心收集后，用PBS洗涤沉淀一次后重悬于PBS中。取一定量的ClyO与上述菌液混合，37摄氏度裂解10-15min。将裂解后的溶液10000g离心1min，取上清1μl做模板，各加入分型所需的引物1μg进行PCR扩增，PCR扩增程序如下：1)94℃，5min；2)94℃，30sec，50℃，30sec，72℃，30sec，30个循环；3)72℃，10min；将产物进行凝胶电泳分析，电泳图谱如图10。结果显示用ClyO作用后的裂解液上清做模板，可以方便的用于MRSA的PCR分型鉴定。各种型别的MRSA菌株均能得到很好的鉴定。其中分型所需的4对引物及各型的基因特征如下表所示。
实施例11、ClyO用于模拟皮肤消毒后降低皮肤表面葡萄球菌的数目
将不同浓度的表皮葡萄球菌涂于小鼠裸露的皮肤表面，自然干燥后进行测试。测试时，实验组先用含有10μg/ml ClyO的PBS溶液擦拭，等10s-2min后，再用75％酒精直接擦拭皮肤表面，反复数次后将棉球放入葡萄球菌液体培养基中，过夜培养，然后稀释平板计数。对照组只采用75％酒精擦拭皮肤表面，反复数次后将棉球放入葡萄球菌液体培养基中，过夜培养，然后稀释平板计数。结果统计图见图11。结果显示采用ClyO能有效清除皮肤表面的表皮葡萄球菌。

Claims (9)

1.一种能裂解葡萄球菌的裂解酶，其氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述裂解酶的基因，其核酸序列如SEQ ID NO.1所示。
3.权利要求1所述的裂解酶的可溶表达与纯化的方法，其特征在于，将权利要求2所述基因克隆后连入表达载体pBAD24中，然后将表达质粒转化大肠杆菌BL21(DE3)中进行表达，所表达的蛋白质先经过离子交换柱纯化，再用磷酸盐缓冲液透析处理。
4.权利要求1所述的裂解酶在制备杀灭表皮葡萄球菌、腐生葡萄球菌、马胃葡萄球菌、头葡萄球菌、白色葡萄球菌、松鼠葡萄球菌、溶血性葡萄球菌、产色葡萄球菌、以及金黄色葡萄球菌的药剂中的用途。
5.根据权利要求4所述的用途，其特征在于，所述金黄色葡萄球菌包括甲氧西林敏感的金黄色葡萄球菌和甲氧西林耐药的金黄色葡萄球菌。
6.权利要求1所述的裂解酶在作为治疗葡萄球菌感染药物中活性成分的用途。
7.权利要求1所述的裂解酶在制备葡萄球菌检测和鉴定的药剂中的用途，其特征在于，用权利要求1所述裂解酶与待测细菌作用后，释放葡萄球菌内的ATP或DNA，通过检测释放的ATP或DNA来用于葡萄球菌的检测和鉴定。
8.根据权利要求7所述的用途，其特征在于，将待测细菌与权利要求1所述裂解酶混合后，加入荧光素酶及其底物于37摄氏度孵育，同时用酶标仪检测混合液发光强度的变化，同时用没有加入权利要求1所述裂解酶的待测菌混合液作为阴性对照。
9.根据权利要求7所述的用途，其特征在于，将待测细菌与权利要求1所述裂解酶混合后于37摄氏度孵育10-15min，对混合液10000g离心1min，取上清做模板，加入耐甲氧西林金黄色葡萄球菌鉴定的两对引物进行PCR来对菌株是否为耐甲氧西林金黄色葡萄球菌进行鉴定；对鉴定为耐甲氧西林金黄色葡萄球菌的菌株，则进一步加入SCCmec分型所需的5对引物进行PCR，对耐甲氧西林金黄色葡萄球菌菌株进行分型。
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