CN115820616A - 一种带荧光标记的噬菌体裂解酶及其应用 - Google Patents
一种带荧光标记的噬菌体裂解酶及其应用 Download PDFInfo
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Abstract
本发明提供了一种带荧光标记的噬菌体裂解酶及其应用,通过构建重组载体并在基因工程菌株大肠杆菌BL21(DE3)中表达所得到的带荧光标记的噬菌体裂解酶PGTphg,可以作为一种荧光染色剂其荧光活性长期存在,不会淬灭,不用避光保存样品,且节约成本,能大大的提高工作效率,缩短研究时间;该带荧光标记的噬菌体裂解酶可以用于金黄色葡萄球菌生物被膜的防治,具有良好的抑制金黄色葡萄球菌生物被膜形成的生物活性,从而将耐药菌的防控从普通的细菌水平(浮游菌状态),提升到微群落水平(生物被膜状态),具有一定的创新性,值得进一步的推广应用。
Description
技术领域
本发明属于病原微生物防治技术领域,具体公开了一种带荧光标记的噬菌体裂解酶及其应用。
背景技术
细菌生物被膜(Bacterial biofilm,BBF)是细菌在生长过程中为适应生存环境而粘附于物体或活性组织表面,并包被于其自身产生的胞外多糖基质、纤维蛋白、脂蛋白等成分中形成的一种与浮游菌生长方式完全不同的微生物群落,是细菌的一种特殊群体生存形式。由于外部多糖基质等成分的保护作用,存在于生物被膜中的菌体(被膜菌)可以逃避机体免疫系统的作用以及吞噬细胞的捕食,并对抗生素和抗菌药表现出极强的抵抗性。研究表明,生物被膜可以使细菌的耐药性提高10-1000倍,从而导致伤口感染迁延不愈,而且当机体防御系统不能控制它们时,细菌还可以从生物被膜中游出,向外播散到达其它部位引起新的感染,反易复发,极难根治。另外,某些致病菌,特别是金黄色葡萄球菌,还会在人体内外或医疗材料表面形成生物被膜,具有很大的危害性,使用抗生素虽然可以杀灭浮游状态的病原菌,但处于生物被膜深处的细菌却难以被清除,这成为解决超级耐药细菌问题的一大难点。
细菌生物被膜耐药性的产生可能是多方面因素共同作用的结果,目前关于生物被膜形成、清除的分子机制以及相关的防控技术研究比较少,这是本技术的重要切入点。由于几乎所有的细菌在一定条件下都可以形成生物被膜,而且生物被膜中细菌除了能够腐蚀管道和金属表面、污染医用材料外,更可导致动植物及人类各种疾病发生,因此,寻求可以替代抗生素的新型生物被膜抑菌剂迫在眉睫。
裂解酶(lysin或endolysin)为噬菌体感染宿主细菌后期表达释放的一类细胞壁水解酶,其通过水解细胞壁肽聚糖而使细菌裂解,并释放出子代噬菌体。目前噬菌体裂解酶的抗菌活性已获得广泛认可,其有望取代传统的抗生素而成为一种新型的杀菌剂。但是噬菌体裂解酶的抗生物被膜活性及其在抑制细菌生物被膜形成中的研究十分匮乏。在实践中,尚未见通过使用噬菌体裂解酶并添加荧光蛋白标签,用于标记裂解酶对金黄色葡萄球菌生物被膜的作用过程,并防治其形成的报道。进一步的,由于生物被膜不同于普通浮游态细菌的特殊三维空间结构、特殊组成成分(多糖、脂类、蛋白等)和屏障效应,目前能用于细菌生物被膜防控的酶制剂开发十分缓慢。
值得注意的是,普通浮游状态细菌和生物被膜菌是完全不同的两种类型,两者在生长方式,存在形式,抗药性等特点截然不同。例如,即使普通非耐药菌,一旦形成生物被膜,也会产生极大极强的耐药性(一般而言,可使耐药性提高10-1000倍)。
发明内容
本发明的第一目的在于提供一种带荧光标记的噬菌体裂解酶在制备用于防治金黄色葡萄球菌生物被膜形成的制剂中的应用,其主要针对解决目前抑制金黄色葡萄球菌生物被膜形成的抗菌剂缺乏的技术问题。
本发明的第二目的在于提供一种带荧光标记的噬菌体裂解酶在制备金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌、沙门氏菌、志贺氏菌和/或枯草芽孢杆菌的抗菌剂中的应用,其能够解决现目前有关于噬菌体裂解酶杀菌剂较少的技术问题。
本发明的第三目的在于提供一种用于防治金黄色葡萄球菌生物被膜形成的制剂,其能够有效杀灭金黄色葡萄球菌的同时,还能对其生物被膜进行标记染色,不仅能大大节约成本,还能够提高工作效率,缩短研究时间。
与现有技术相比,本发明至少具有如下的优点与积极效果:
本发明提供了一种带荧光标记的噬菌体裂解酶及其应用,其应用基因工程技术构建特异性生产带荧光标记的噬菌体裂解酶PGTphg的转基因大肠杆菌,采用单一质粒完成荧光活性融合噬菌体裂解酶的组合表达,避免了多质粒在工程菌中的稳定性及各基因表达的不同步问题,具有操作简单,成本低、可行性高等优点,为裂解酶的工业化生产应用奠定基础;本发明通过构建重组载体并在基因工程菌株大肠杆菌BL21(DE3)中表达所得到的带荧光标记的噬菌体裂解酶PGTphg,可以作为一种荧光染色剂其荧光活性长期存在,不会淬灭,不用避光保存样品,且节约成本,能大大的提高工作效率,缩短研究时间;本发明的带荧光标记的噬菌体裂解酶PGTphg,可以用于金黄色葡萄球菌生物被膜的防治,具有良好的抑制金黄色葡萄球菌生物被膜形成的生物活性,从而将耐药菌的防控从普通的细菌水平(浮游菌状态),提升到微群落水平(生物被膜状态),具有创新性,值得进一步的推广应用。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例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为本发明中噬菌体TSP4裂解酶基因PCR产物胶回收电泳图,其中M代表Marker,泳道2和3为噬菌体TSP4裂解酶基因PCR产物胶回收电泳图,其DNA片段大小为501bp;
图2为本发明中连接实验PCR检测电泳图,其中M代表Marker,泳道2为阴性对照,泳道3为连接后重组载体PET28a-PGTphg的T7启动子引物PCR结果;
图3为本发明中裂解酶PGTphg的蛋白诱导表达检测图,泳道1为Marker,泳道2为PGTphg/BL2(DE3)未诱导破碎液,泳道3为PET28a-PGTphg/BL2(DE3)乳糖诱导后破碎取上清液,泳道4为PET28a-PGTphg/BL2(DE3)乳糖诱导后总破碎液;泳道5为PET28a-PGTphg/BL21(DE3)IPTG诱导后破碎上清液;泳道5为PET28a-PGTphg/BL21(DE3)IPTG诱导后总破碎液,目的诱导产物大小约在48kDa;
图4为本发明中含荧光标记的裂解酶PGTphg的蛋白纯化检测图,其中M代表蛋白Marker,泳道1为上柱后500mM咪唑洗脱液,纯化得到的目的产物大小约在48kDa;
图5为本发明中含荧光标记的裂解酶PGTphg吸附并染色金黄色葡萄球菌生物被膜,我们利用荧光显微镜对其荧光染色活性进行检测,即PGTphg作为“荧光染色剂”的效果观察;
图6为本发明中含荧光标记的裂解酶PGTphg对金黄色葡萄球菌ATCC6538生物被膜形成的抑制作用分析;
图7为本发明中含荧光标记的裂解酶PGTphg对多重耐药性金黄色葡萄球菌(1606BL1486)生物被膜形成的抑制作用分析;
图8为本发明中含荧光蛋白标记的裂解酶PGTphg对多重耐药性金黄色葡萄球菌(1606BL1486)生物被膜作用过程进行荧光观察,其中A,作用0分钟(对照);B,作用30分钟;C,作用60分钟。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考具体实施例来详细说明本发明。
本发明提供了一种带荧光标记的噬菌体裂解酶在制备用于防治金黄色葡萄球菌生物被膜形成的制剂中的应用。该带荧光标记的噬菌体裂解酶主要用于制备防治食源性多重耐药金黄色葡萄球菌生物被膜形成的制剂,其能够针对耐药性较强的多重耐药性金黄色葡萄球菌生物被膜的形成,具有良好的抗菌效果。
上述带荧光标记的噬菌体裂解酶包括来源于栖热菌属长尾科噬菌体TSP4的裂解酶和GFP绿色荧光蛋白标签。其分子质量约为48kDa,具有裂解酶活性的同时还具有荧光染色活性,其裂解酶的核苷酸序列如SEQ ID NO.1所示,其裂解酶的氨基酸序列如SEQ IDNO.4所示。
本发明还提供了一种上述带荧光标记的噬菌体裂解酶在制备金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌、沙门氏菌、志贺氏菌和/或枯草芽孢杆菌的抗菌剂中的应用。
本发明还提供了一种用于防治金黄色葡萄球菌生物被膜形成的制剂,包括上述带荧光标记的噬菌体裂解酶。
上述带荧光标记噬菌体裂解酶在制剂中的浓度不低于50μg/ml。
上述制剂还包括8-12mg/ml NaCl、0.15-0.25mg/ml KCl、1.5-2.0mg/ml Na2HPO4、0.2-0.4mg/ml KH2PO4。
上述制剂的pH值为7.3-7.5。其中调节pH值优选用HCl,以避免在制剂内引入新的其他离子。
上述制剂的制备方法包括如下步骤:将缓冲溶液灭菌后与所述带荧光标记的噬菌体裂解酶混匀,即得到制剂。其中灭菌的方法优选为高压蒸汽灭菌和过滤除菌,高压蒸汽灭菌的参数为15psi、20min,以保证灭菌的速度和灭菌的成效。
实施例
噬菌体裂解酶PGTphg的分子克隆和重组表达载体的构建
噬菌体裂解酶基因的扩增,(以Thermus TC16的噬菌体TSP4基因组DNA为模板)
栖热菌噬菌体TSP4裂解酶基因的扩增所用引物序列如表1所述:
表1序列表
其中,正向引物和反向引物下划线部分分别代表EcoRI和NotI限制性酶切位点。
(2)PCR扩增体系如下:
表2:PCR扩增反应体系组分
组分 | 用量 |
模板 | 5μl |
引物 | 各3μl |
Mix | 25μl |
ddH<sub>2</sub>O | 补充至50μl |
(3)扩增条件如下:
将反应体系混匀,先在94℃预变性10min,然后在94℃变性45s,58℃退火45s,72℃延伸28s,30个循环后,72℃延伸10min。反应完后取产物3μl,在1%琼脂糖凝胶中进行电泳分析。
2、PCR产物的胶回收纯化
(1)在电泳仪中灌制1%琼脂糖凝胶;
(2)将待分离纯化的PCR产物点样电泳,于适当位置停止电泳;
(3)在紫外灯下切下含该目的片断的凝胶,转移到1.5ml的Ep管中;
(4)用百泰克生物公司胶回收试剂盒进行目的片段的回收,回收方法按说明书操作进行。
(5)在1%琼脂糖凝胶中进行电泳检测胶回收结果(见图1),根据图1结果所示,成功回收,其DNA片段大小为501bp。
3、重组表达载体的构建
(1)带有粘性末端线性载体pET28a-EGFP的制备
为了将目的基因片断连接到表达载体pET28a-EGFP上,就需要使目的片断带有粘性末端的片断,即带有酶切位点。同样,为了使目的片断能插入载体中,也需要使载体带有粘性末端,并且使它们的酶切位点相同。
A、质粒抽提:用质粒提取试剂盒(百泰克),操作步骤如下:
①菌种活化:无菌接种环蘸取-80℃冻存的菌种保存液,三线法接种于卡那LB平板,37℃培养12-16小时;
②增菌并收集菌体:取卡那霉素5μl(终浓度100μg/ml)加入5ml LB培养基中;用接种环挑取阳性克隆,接种于KNa+-LB培养基中;然后放入37℃培养箱中,摇床培养,过夜;取3ml培养的菌液,5000rpm,室温离心5min,使菌体沉淀,弃上清液;
③用250μl溶液P1(含RNA酶)重悬菌体沉淀,涡旋振荡至彻底悬浮;
④加250μl的溶液P2,温和地上下翻转8次使菌体充分裂解,直到溶液变得清亮;
⑤加400μl溶液P3,立即温和地上下翻转8次,室温放5min,室温13,000rpm离心10min,小心取上清;
⑥将吸附柱安置于收集管上,将上一步所得上清液加入吸附柱AC中(吸附柱放入收集管中,溶液太多分可分两次加入),13,000rpm离心1min,弃滤液;
⑦加入500μl去蛋白液PE,13,000rpm离心60s,弃滤液;
⑧加入500μl漂洗液WB,13,000rpm离心60s,弃滤液;
⑨重复步骤⑦一次,13,000rpm离心60s,弃滤液,空柱13,000rpm离心2分钟,室温放置3-5min,除去残留乙醇;
⑩取出吸附柱AC,放入一个干净的离心管中,在吸附膜的中间部位加70μl洗脱缓冲液EB(65℃预热),室温放1min,13,000rpm离心1min洗脱质粒。
(2)TSP4裂解酶基因片段和PET28a-EGFP质粒的双酶切体系如下:
表3:酶切反应体系组分
酶切反应条件:37℃,2h,回收TSP4基因片段和酶切后的PET28a-EGFP质粒。
(3)重组表达载体的连接转化、测序验证
将前面实验得到的带有粘性末端的线性载体pET28a-EGFP和TSP4裂解酶基因片段,通过连接转化并用菌落PCR、T7启动子通用引物PCR鉴定(见图2)及测序验证,即可得到重组表达载体PET28a-PGTphg,根据图2结果显示鉴定转化成功。
试验例1
荧光标记的噬菌体裂解酶PGTphg在大肠杆菌中的诱导表达以及亲和层析柱纯化
1、荧光标记的裂解酶PGTphg重组蛋白在大肠杆菌工程菌株中的诱导表达
将构建好的重组载体PET28a-PGTphg转化大肠杆菌BL21(DE3),含重组质粒的菌株经培养过夜,菌液按1%比例接种到Kan+(终浓度50μg/ml)的LB液体培养基,37℃摇床培养至其OD600值0.6-0.8;取出4ml菌液用作对照实验;向余下的菌液加入IPTG(终浓度为0.5mM)和乳糖(终浓度为0.7g/L),放入20℃,150rpm摇床诱导培养12h,取样5ml检测。
2、SDS-PAGE检测诱导后PGTphg过表达情况
将取出的5ml菌液,8000rpm,离心10min,弃上清,加入终浓度为30mM咪唑溶液悬浮菌体,超声波破碎菌体(功率25%,打3s,停4s,共3min),98℃热裂解10min使菌体破释放菌体内蛋白;配制SDS-PAGE胶,浓缩胶5%,分离胶12%;按照顺序上样进行电泳(浓缩胶80V,30min;分离胶120V,120min),电泳结束进行染色,之后将SDS-PAGE胶取出,加入R250考马斯亮蓝染色液,振荡过夜进行脱色并拍照分析(见图3),根据图3结果所示,目的诱导产物大小约在48kDa。
3、带荧光标记的裂解酶PGTphg的亲和柱纯化
利用上述方法大量诱导含重组质粒PET28a-PGTphg的BL21(DE3)菌株,菌液经离心收集大肠杆菌菌体(4℃,8000rpm,10min)。用PBS溶液悬浮菌体后进行超声波破碎,4℃,13000rpm离心10min,上清用镍柱进行手工纯化,先用10倍柱体积ddH2O清洗柱子,再用10倍柱体积30mM咪唑平衡柱子,样品上柱,用10倍柱体积150mM咪唑洗脱柱子,再用10倍柱体积500mM咪唑洗脱柱子,用10倍柱体积ddH2O清洗柱子,最后用20%无水乙醇填充柱子,纯化的得到的裂解酶PGTphg表达产物进行SDS-PAGE检测(见图4),根据图4结果显示,纯化得到的目的产物大小约在48kDa。
试验例2
将过夜培养的铜绿假单胞菌ATCC27853、金黄色葡萄球菌ATCC6538、大肠杆菌K88CGMCC1.2385、沙门氏菌CMCC(B)50094、枯草芽孢杆菌CMCC(B)63501、志贺氏菌CMCC(B)51105稀释至5×105,并加入EDTA使终浓度1mM均匀混合后,于37℃反应30min,离心,相同体积重悬,取200μL菌液与800μL PGTphg酶液(104μg/mL)于37℃反应30min,取100μL涂布于LB平板上,每个样做三个平行。将平板放于37℃过夜培养,计算单菌落个数。
裂解酶PGTphg对致病菌的杀菌作用见表4:
表4裂解酶PGTphg对一系列致病菌杀菌活性验证
菌株名称 | 菌株编号 | 裂解酶PGTphg杀菌活性 |
金黄色葡萄球菌 | ATCC6538 | ++ |
铜绿假单胞菌 | ATCC27853 | + |
大肠杆菌K88 | CGMCC1.2385 | + |
沙门氏菌 | CMCC(B)50094 | + |
志贺氏菌 | CMCC(B)51105 | + |
枯草芽孢杆菌 | CMCC(B)63501 | + |
其中++表示裂解酶处理后菌体数量降低2个数量级,+表示裂解酶处理后菌体数量降低1个数量级。
根据表4结果显示其对于致病菌金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌、沙门氏菌、志贺氏菌和枯草芽孢杆菌都有很好的杀菌作用,这说明我们纯化得到的带荧光标记的裂解酶PGTphg具有良好的杀菌活性。
试验例3
纯化得到的带荧光标记的噬菌体裂解酶PGTphg可吸附到金黄色葡萄球菌生物被膜并展示了良好的荧光活性
分析步骤如下:
(1)将金黄色葡萄球菌ATCC6538用含有5%葡萄糖的TSB培养基配制成1×106CFU/mL的工作菌液备用;
(2)24孔板中加入2mL的工作菌液,于37℃恒温培养箱培养24h,使其生物被膜形成;
(3)吸出培养液,用PBS洗涤2次去除浮游菌,分别加入500μl(裂解酶浓度为100μg/ml)裂解酶PGTphg,加PBS作为对照,作用12h后去上清,用PBS(PH7.4)洗涤2-3次去除浮游细菌;
(4)用PGTphg在37℃下染色30min,PBS洗涤2次,用甲醇固定15min。去除甲醇溶液,用PBS洗涤2-3次,荧光显微镜观察裂解酶PGTphg对金黄色葡萄球菌生物被膜的染色情况。
如图5所示,我们可以观察到裂解酶PGTphg吸附并作用于金黄色葡萄球菌ATCC6538生物被膜,成功发出绿色荧光。
试验例4
带荧光标记的噬菌体裂解酶PGTphg对金黄色葡萄球菌生物被膜形成的抑制作用
利用XTT还原法测定裂解酶PGTphg作用后金黄色葡萄球菌生物被膜菌体活细胞数的变化,研究PGTphg对于金黄色葡萄球菌生物被膜形成过程的抑制作用。XTT作为线粒体脱氢酶的作用底物,可以被活细胞还原成水溶性的橙黄色甲臢产物。当XTT与电子偶合剂(例如PMS)联合应用时,其所产生的水溶性的甲臢产物的吸光度与活细胞的数量成正比。其步骤如下:
(1)96孔板中加入200μl的MH培养基加过夜培养的金黄色葡萄球菌(ATCC6538)以及多重耐药金黄色葡萄球菌(1606BL1486,本实验室保存),使其菌体浓度达106CFU/mL,并加入裂解酶PGTphg(使其终浓度为50μg/mL),加Kna+(终浓度为50μg/mL)作为阳性对照,PBS作为阴性对照,每个实验做3个平行,37℃培养24h,令其菌体数目增多并贴壁形成生物被膜。
(2)用PBS清洗两次浮游菌后,加入200μl MH培养基,并加入20μl XTT试剂在37℃,黑暗条件下静置培养2h后取出。采用酶标检测仪测定490nm处OD值的变化。进行实验组和PBS对照组比较,并用student t test分析其结果是否具有统计学意义(P值<0.05视为显著差异)。
如图6以及图7所示,经带荧光标记的噬菌体裂解酶PGTphg处理后,与对照PBS相比,金黄色葡萄球菌(ATCC6538)以及多重耐药金黄色葡萄球菌(1606BL1486,本实验室保存)的生物被膜形成均受到显著抑制。
用含有5%葡萄糖的TSB培养基制备金黄色葡萄球菌ATCC6538工作菌液(1×106CFU/mL),加入500μl(裂解酶浓度为100μg/ml)裂解酶PGTphg,37℃下分别作用30分钟和60分钟(并设置作用0分钟对照),观察不同作用时间的实验效果。随后,PBS洗涤2次,用甲醇固定15min。去除甲醇溶液,用PBS洗涤2-3次,通过荧光显微镜进行观察,所得结果如图8所示,根据图8所示,带有荧光标记的噬菌体裂解酶PGTphg对金黄色葡萄球菌的生物被膜作用过程,可以直接通过荧光染色进行观察。总之,上述这些结果表明了本发明中带荧光标记的噬菌体裂解酶PGTphg在制备用于防治金黄色葡萄球菌生物被膜形成的制剂中的应用前景。
综上所述:
本发明提供了一种带荧光标记的噬菌体裂解酶及其应用,其应用基因工程技术构建特异性生产带荧光标记的噬菌体裂解酶PGTphg的转基因大肠杆菌,采用单一质粒完成荧光活性融合噬菌体裂解酶的组合表达,避免了多质粒在工程菌中的稳定性及各基因表达的不同步问题,具有操作简单,成本低、可行性高等优点,为裂解酶的工业化生产应用奠定基础;本发明通过构建重组载体并在基因工程菌株大肠杆菌BL21(DE3)中表达所得到的带荧光标记的噬菌体裂解酶PGTphg,可以作为一种荧光染色剂其荧光活性长期存在,不会淬灭,不用避光保存样品,且节约成本,能大大的提高工作效率,缩短研究时间;本发明的带荧光标记的噬菌体裂解酶PGTphg,可以用于金黄色葡萄球菌生物被膜的防治,具有良好的抑制金黄色葡萄球菌生物被膜形成的生物活性,从而将耐药菌的防控从普通的细菌水平(浮游菌状态),提升到微群落水平(生物被膜状态),具有创新性,值得进一步的推广应用。
以上所描述的实施例是本发明一部分实施例,而不是全部的实施例。本发明的实施例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例。基于本发明中的实施例,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例,都属于本发明保护的范围。
Claims (8)
1.一种带荧光标记的噬菌体裂解酶在制备用于防治金黄色葡萄球菌生物被膜形成的制剂中的应用。
2.根据权利要求1所述的应用,其特征在于,所述带荧光标记的噬菌体裂解酶包括来源于栖热菌属长尾科噬菌体TSP4的裂解酶和GFP绿色荧光蛋白标签。
3.一种如权利要求2所述的带荧光标记的噬菌体裂解酶在制备金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌、沙门氏菌、志贺氏菌和/或枯草芽孢杆菌的抗菌剂中的应用。
4.一种用于防治金黄色葡萄球菌生物被膜形成的制剂,其特征在于,包括如权利要求2所述的带荧光标记的噬菌体裂解酶。
5.根据权利要求4所述的制剂,其特征在于,所述带荧光标记的噬菌体裂解酶在所述制剂中的浓度不低于50μg/ml。
6.根据权利要求5所述的制剂,其特征在于,还包括8-12mg/ml NaCl、0.15-0.25mg/mlKCl、1.5-2.0mg/ml Na2HPO4、0.2-0.4mg/ml KH2PO4。
7.根据权利要求5所述的制剂,其特征在于,所述制剂的pH值为7.3-7.5。
8.一种如权利要求4-7任意一项所述的用于防治金黄色葡萄球菌生物被膜形成的制剂的制备方法,其特征在于,包括如下步骤:将缓冲溶液灭菌后与所述带荧光标记的噬菌体裂解酶混匀,即得到所述制剂。
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