CN104726439B - 一种广谱的链球菌裂解酶及其应用 - Google Patents
一种广谱的链球菌裂解酶及其应用 Download PDFInfo
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Abstract
本发明公开了一个能杀灭多种链球菌的裂解酶。采用基因拼接的手段，构建了一个全新的裂解酶ClyR，该酶能高效的杀灭多种链球菌，包括肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等以及多种肠球菌和金黄色葡萄球菌。该酶具有很好的稳定性，对EDTA以及高浓度的NaCl不敏感。而且该酶发挥作用的pH范围很宽，在pH 5‑11都保持高活力。重组蛋白ClyR能很好的在大肠杆菌BL21(DE3)中表达。高剂量的ClyR对小鼠没有明显的毒性，而且，一次给药0.8mg/鼠能完全保护致死剂量的B族链球菌感染的小鼠。该裂解酶能单独，或者与不同形式的试剂和溶液配伍，用于多种链球菌的控制以及治疗由这些菌导致的感染，具有广大的应用前景。
Description
技术领域
本发明属于微生物领域，具体涉及一种能杀灭链球菌、肠球菌和金黄色葡萄球菌的裂解酶和编码基因，以及其在抗生素方面的应用。
背景技术
链球菌是一类球形的革兰氏阳性细菌，能引起人和动物的很多疾病。肺炎链球菌是大叶性肺炎等疾病的主要致病菌，更常在婴幼儿引起支气管肺炎等疾病。化脓链球菌广泛分布于自然界，是人类细菌感染中最重要的病原之一，能引起各种化脓性炎症，包括猩红热，丹毒，新生儿败血症，脑膜炎以及链球菌变态反应等疾病。B族链球菌，如无乳链球菌等，是围产期感染的重要病原菌之一，严重威胁新生儿的健康。变形链球菌是龋齿的主要致病菌，严重威胁人类口腔健康。目前龋齿的预防与治疗还缺乏有效的疫苗。此外，链球菌还能引起许多重要的动物疾病，严重威胁畜牧业。猪链球菌病是一种人畜共患的急性、热性传染病，通常由C、D、E及L群链球菌引起。而奶牛养殖业的头号疾病奶牛乳腺炎，是由无乳链球菌、乳房链球菌、停乳链球菌以及金黄色葡萄球菌等四种主要的致病菌引起的，目前尚缺乏有效的疫苗和应对策略。由上可知，链球菌种类繁多，能引起人和动物的多种疾病。
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25 kD~40 kD，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，已有一些能作用于链球菌的天然裂解酶被报道，这些酶可以在体内、体外较好地杀特定种类的链球菌。但是这些裂解酶通常具有较窄的裂解谱，大多比较难于可溶性表达，或者活性不高，作用pH范围较窄，一般在pH 5-8。目前尚没有能有效裂解变形链球菌的裂解酶报道出来。寻找可溶、大量表达以及广谱的裂解酶对于开发新的抗链球菌的药物，体外控制链球菌的感染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能杀灭多种链球菌的裂解酶。该裂解酶能在体外、体内杀灭链球菌，特别是肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等以及多种肠球菌和金黄色葡萄球菌。为了叙述方便，我们命名为ClyR，其编码基因ClyR。
本发明所提供的链球菌裂解酶的编码基因ClyR的核酸序列如序列表中SEQ.ID.NO.1所示。
本发明所提供的链球菌裂解酶ClyR的蛋白序列如序列表中SEQ.ID.NO.2所示。
本发明还提供一种可溶表达ClyR蛋白质及纯化的方法，其步骤为将ClyR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。
本发明通过体外实验证实了采用ClyR体外杀灭多种链球菌以及金黄色葡萄球菌株的效果，特别是肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等以及多种肠球菌和金黄色葡萄球菌，证明了该裂解酶的高活性与广谱性。还初步试验了ClyR对实验动物模型小鼠感染B族链球菌后的保护效果，以及对细胞毒性的测试，初步证实了其用于开发抗链球菌感染药物的潜能。
本发明有以下的有益效果和优点：
本发明ClyR可用于体内、体外杀灭多种链球菌和葡萄球菌，ClyR能采用大肠杆菌可溶性表达；酶活性高，且能在pH5-11的范围内都具有较高活性；能广谱杀灭链球菌，具有应用于体内抗感染药物的潜能等优点。
附图说明
图1为ClyR基因PCR扩增结果。图中M为标准分子量marker，箭头所指处为750 bp。R为扩增ClyR的条带。
图2为ClyR杀灭停乳链球菌标准菌株ATCC 35666的时间变化结果。实心方块所示为停乳链球菌与ClyR混合后LogCFU随时间的变化趋势，空心圆圈所示为停乳链球菌与缓冲液混合后OD600随时间的变化趋势。空心三角所示为停乳链球菌与ClyR混合后OD600随时间的变化趋势。
图3为ClyR酶活受EDTA影响的结果。将停乳链球菌标准菌株ATCC 35666重悬在不同EDTA的PBS缓冲液中，与相同浓度的ClyR混合后检测OD600随时间的变化趋势。纵坐标表示ClyR在不同条件下的相对酶活。
图4为ClyR酶活受pH影响的结果。将停乳链球菌标准菌株ATCC 35666重悬在不同pH的缓冲液中，与相同浓度的ClyR混合后检测OD600随时间的变化趋势。纵坐标表示ClyR在不同条件下的相对酶活。
图5为ClyR酶活受NaCl影响的结果。将停乳链球菌标准菌株ATCC 35666重悬在不同NaCl的缓冲液中，与相同浓度的ClyR混合后检测OD600随时间的变化趋势。纵坐标表示ClyR在不同条件下的相对酶活。
图6为ClyR体外杀灭链球菌以及葡萄球菌等不同菌株广谱性的结果。纵坐标表示将不同菌株与ClyR混合后37℃孵育15 min，OD600降低的数值。
图7为ClyR体内杀灭链球菌的结果。每组20只小鼠，分别注射致死剂量的无乳链球菌后，实验组于3小时后腹腔注射0.8 mg ClyR。对照组于3小时候腹部注射的等体积的PBS溶液。每天观测各组小鼠的存活率。
图8为ClyR对于细胞毒性的测试结果。分别用浓度为0.1, 0.5和1 mg/ml的ClyR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9为ClyR在牛奶中杀灭链球菌的结果。停乳链球菌ATCC 35666与无菌牛奶混合后用不同浓度的ClyR处理1小时后稀释平板计数。
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，我们设计和人工合成一种新的链球菌裂解酶ClyR。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提供。测序均在上海英骏公司完成。
实施例1、能特异杀灭链球菌的裂解酶的构建
（1）在南京金斯瑞生物技术有限公司全序列合成能表达裂解酶ClyR的ClyR基因DNA序列。合成序列装入pUC57质粒中。以ClyR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- TATACCATGGGCATGGCAGCAAATCTGG -3 (SEQ.ID.NO.3)
NcoI
反向引物：5- ATATCTCGAGTTTGAAGGTACCCCATGCGTTG -3(SEQ.ID.NO.4)
XhoI
以2 μl基因为模板，各加入引物1 μg进行PCR扩增，PCR扩增程序如下：1）94 ℃，5min；2）94 ℃，30 sec，62 ℃，45 sec，72 ℃，45 sec，30个循环；3）72 ℃，10 min；将产物进行凝胶电泳回收，电泳图谱如图1，ClyR的基因大小为759 bp，与所设计的裂解酶的大小一致。
（2）将ClyR基因连入表达质粒pBAD24中得到重组载体pB-ClyR，然后将其转化大肠杆菌BL21(DE3)。
（3）ClyR的表达纯化。
将表达菌株BL21(DE3)/pB-ClyR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1 M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、ClyR杀灭停乳链球菌标准菌株ATCC 35666的验证。
将停乳链球菌过夜培养物，离心收集后用PBS洗涤一次然后溶于PBS中。取一定量的ClyR与上述菌液混合，同时用酶标仪监测混合液在600 nm吸收值的变化以及活菌数目的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。最后得到的裂解曲线见附图2。结果显示ClyR能快速的裂解ATCC 35666菌株从而导致在600 nm吸收值快速降低。
实施例3、ClyR酶活受EDTA影响结果的验证。
将停乳链球菌标准菌株ATCC 35666过夜培养物，离心收集后用PBS洗涤一次然后溶于PBS中。然后将菌悬液等分为多份，分别加入不同浓度的EDTA混匀。取一定量的ClyR与所有上述菌液混合，同时用酶标仪监测混合液在600 nm吸收值的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。检测结束后计算每组与空白组相比较OD600降低的数值，将OD600降低最多的值定义为1，其它组OD600的降低值与之作比较，得到相对酶活数值。最后得到的裂解曲线见附图3。结果显示ClyR酶活受EDTA影响较小。
实施例4、ClyR酶活受pH影响结果的验证。
将停乳链球菌标准菌株ATCC 35666过夜培养物，离心收集后用PBS洗涤一次然后溶于不同pH值的缓冲液中。取一定量的ClyR与所有上述菌液混合，同时用酶标仪监测混合液在600 nm吸收值的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。检测结束后计算每组与空白组相比较OD600降低的数值，将OD600降低最多的值定义为1，其它组OD600的降低值与之作比较，得到相对酶活数值。最后得到的裂解曲线见附图4。结果显示ClyR在pH5-11的范围内具有较好的活性。
实施例5、ClyR酶活受NaCl影响结果的验证。
将停乳链球菌标准菌株ATCC 35666过夜培养物，离心收集后用PBS洗涤一次然后溶于PBS中。然后将菌悬液等分为多份，分别加入不同浓度的NaCl混匀。取一定量的ClyR与所有上述菌液混合，同时用酶标仪监测混合液在600 nm吸收值的变化。同时，用缓冲液与停乳链球菌的混合液作为负对照。检测结束后计算每组与空白组相比较OD600降低的数值，将OD600降低最多的值定义为1，其它组OD600的降低值与之作比较，得到相对酶活数值。最后得到的裂解曲线见附图5。结果显示ClyR酶活受NaCl影响较小。
实施例6、ClyR体外杀灭链球菌以及葡萄球菌等不同菌株广谱性的验证。
将多种链球菌、金黄色葡萄球菌以及其它参照菌株过夜培养，离心收集后用PBS洗涤一次然后溶于PBS中。取一定量的ClyR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15 min。用OD600降低的数值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附图6。结果显示ClyR能快速的杀灭多种链球菌（特别是肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等）以及葡萄球菌而对其它的测试菌株没有裂解作用。
实施例7、ClyR体内杀灭链球菌的验证。
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6×108 无乳链球菌菌株。3 h后，将小鼠分为两组，每组20只。实验组小鼠腹腔注射ClyR 800 μg，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7。结果显示ClyR能有效的杀灭体内的链球菌，从而提高小鼠的存活率。
实施例8、ClyR无细胞毒性的验证。
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ClyR (0.1-1 mg/mL)以及离子霉素（15 mg/mL）和丝裂霉素C（15 mg/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450 nm吸光值。所得到的结果见附图8。结果显示高浓度的ClyR无细胞毒性。
实施例9、ClyR用于牛奶中链球菌清除的验证。
将链球菌过夜培养物离心收集后用PBS洗涤一次然后溶于无菌纯牛奶中。取不同浓度的ClyR与上述菌液混合，混匀后的反应液置于37度作用1小时，然后稀释平板计算其中的活菌数。试验得到的鉴定结果见附图9。结果显示该酶能有效的清除牛奶中的链球菌。
Claims (10)
1.一种链球菌裂解酶ClyR，其蛋白序列如SEQ.ID.NO.2所示。
2.编码权利要求1所述的链球菌裂解酶的基因ClyR，其核酸序列如SEQ.ID.NO.1所示。
3.权利要求1所述的链球菌裂解酶ClyR可溶表达及纯化的方法，其特征为：将ClyR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，再用磷酸盐缓冲液透析处理。
4.权利要求1所述的链球菌裂解酶ClyR在动物和人体外杀灭链球菌、肠球菌以及金黄色葡萄球菌株的用途。
5.根据权利要求4所述的用途，其特征在于，所述链球菌为肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌或马链球菌。
6.根据权利要求4所述的用途，其特征在于，权利要求1所述的链球菌裂解酶ClyR在牛奶中杀灭链球菌的用途。
7.权利要求1所述的链球菌裂解酶ClyR在制备抗链球菌感染药物上的用途。
8.根据权利要求7所述的用途，其特征在于，权利要求1所述的链球菌裂解酶ClyR在制备预防或治疗奶牛乳腺炎药物上的用途。
9.根据权利要求7所述的用途，其特征在于，权利要求1所述的链球菌裂解酶ClyR在制备预防或治疗龋齿药物上的用途。
10.一种抗生素，其中含有权利要求1所述的链球菌裂解酶ClyR。
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| EP3312275A1 (en) | 2018-04-25 |
| AU2016248363A1 (en) | 2017-11-02 |
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| US9993532B2 (en) | 2018-06-12 |
| AU2016248363B2 (en) | 2022-01-20 |
| EP3312275A4 (en) | 2018-09-19 |
| EP3312275B1 (en) | 2022-04-27 |
| CA2981996C (en) | 2022-07-26 |
| WO2016165604A1 (zh) | 2016-10-20 |
| CA2981996A1 (en) | 2016-10-20 |
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