CN103436514A - 一种耐热裂解酶TSPpgh和编码此酶的多核苷酸 - Google Patents
一种耐热裂解酶TSPpgh和编码此酶的多核苷酸 Download PDFInfo
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- CN103436514A CN103436514A CN2013103632456A CN201310363245A CN103436514A CN 103436514 A CN103436514 A CN 103436514A CN 2013103632456 A CN2013103632456 A CN 2013103632456A CN 201310363245 A CN201310363245 A CN 201310363245A CN 103436514 A CN103436514 A CN 103436514A
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- lyase
- tsppgh
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Abstract
本发明公开了一种耐热裂解酶和编码这种酶的多核苷酸,该裂解酶能够抑制革兰氏阳性和阴性细菌的生长,其在40~80℃范围内具有催化活性,在66.5℃催化活性最高,其核苷酸序列可用于构建生产此裂解酶的基因工程菌株。
Description
技术领域
本发明属于生物技术领域,具体涉及一种耐热裂解酶TSPpgh,以及编码此酶的核酸序列。
背景技术
裂解酶为噬菌体感染宿主后表达释放的一类细胞壁水解酶,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。根据作用于细胞壁肽聚糖共价键位点不同,可以将噬菌体裂解酶分为三类,葡萄糖苷酶(glucoseidase)、酰胺酶(amidase)、内肽酶(endopeptidase),它们分别水解氨基糖之间的糖苷键,N-乙酰胞壁酸和L-丙氨酸之间的酰胺键以及肽交联桥。
目前,很多裂解酶的结构得到了深入研究,分歧杆菌噬菌体裂解酶蛋白分为3个区域,典型的C-端区,与噬菌体内肽酶的C-端细胞壁结合区功能相关;中心区域,包含一个与肽聚糖水解酶相关的结构及N-端区域,常为一系列编码肽酶功能的蛋白,它们每个结构域类型均有明显差别,研究已发现6种可能的N-端肽酶结构区,5种酰胺酶/糖苷酶结构区,和4种C-端细胞壁结合结构区,可能出现的组合至少120种,其中绝大多数结构分布包含一个N-端肽酶结构区,一个中心酰胺酶/胞壁质酶或糖苷转移酶区及C-端区,但是有一个例外,Myrna gp243没有C-端结合区,目前还没有来源于栖热菌高温噬菌体裂解酶的报道。
近年来,随着抗生素大量滥用,逐渐出现了很多耐药性菌株带来的各种问题,人们迫切需要研究新型的抗菌试剂,而细菌的天敌噬菌体给人们提供了一个全新的思路,使人们逐渐开始多方面探索噬菌体裂解酶的抗菌作用。其中裂解酶作为新型抗菌制剂,能够对细菌进行防治、治疗并具有独特的优点。首先,噬菌体裂解酶不会对动物产生毒副作用,因为它只能作用于病原菌宿主而对其他细菌不产生作用。其次,噬菌体裂解酶的催化结构作用于病原宿主菌的细胞壁肽聚糖,病菌对它不会产生耐药性,同时能够与抗生素联合用药,共同发挥作用。2001年Nelson等人实验证明通过重组噬菌体裂解酶纯化能够预防和治疗A型链球菌引起的小鼠粘膜感染。裂解酶能高效裂解细菌,可以抑制细菌的生长,使其有望取代传统的抗生素治疗,特别是对多重耐药性致病菌,也可作为抑菌剂使用于环境的杀菌。
发明内容
本发明旨在提供一种耐热裂解酶TSPpgh,该裂解酶能够抑制革兰氏阳性和阴性细菌的生长,其在40~80℃范围内具有催化活性,其来源于栖热菌(Thermus)噬菌体TSP4,该耐热裂解酶TSPpgh的氨基酸序列如SEQ ID NO:1所示,或具有与SEQ ID NO:1所示的氨基酸序列至少90%的相同性的多肽、类似物或衍生物。
本发明的另一个目的是提供编码耐热裂解酶TSPpgh的多核苷酸,其核苷酸序列如SEQ ID NO:2所示,或其互补序列,或具有与SEQ ID NO:2所示的核苷酸序列至少80%相同性的多核苷酸及其互补序列。
本发明的有益效果:
本发明提供的耐热裂解酶能高效裂解细菌,可以抑制细菌的生长,使其有望取代传统的抗生素治疗,特别是对多重耐药性致病菌,也可作为抑菌剂使用于环境的杀菌。
目前报道的裂解酶多为常温型裂解酶,在常温下具有较高活性,本发明所述裂解酶在40~80℃范围内均具有催化活性,在66.5℃催化活性最高,可适用于高温环境的杀菌,目前未见来源于高温噬菌体的裂解酶报道,其核苷酸序列可用于构建生产此裂解酶的基因工程菌株。
附图说明
图1是本发明耐热裂解酶TSPpgh基因PCR产物电泳示意图,其中M代表Marker,泳道1为TSPpgh基因PCR产物,其大小为501bp;
图2是本发明中重组质粒TSPpgh/pET28a双酶切图谱示意图,其中M代表Marker,泳道1为重组质粒TSPpgh/pET28a双酶切后产生的两个条带;
图3是本发明中耐热裂解酶TSPpgh的蛋白表达检测示意图,其中泳道1为蛋白Marker,泳道2为总蛋白,泳道3为空载pET28a/BL21空载沉淀,泳道4为pET28a-TSPpgh/BL21诱导前沉淀,泳道5为pET28a-TSPpgh/BL21诱导后沉淀,泳道6为pET28a/BL21空载上清,泳道7为pET28a-TSPpgh/BL21诱导前上清,泳道8为pET28a-TSPpgh/BL21诱导后上清;
图4是本发明耐热裂解酶TSPpgh蛋白的纯化结果检测示意图,其中1为蛋白Marker,2为上柱后500mM的磷酸缓冲液过柱后第二管,3为上柱后500mM的磷酸缓冲液过柱后最后一管,4为pET28a/BL21空载,5、6为pET28a-TSPpgh/BL21诱导后上清,7为TSPpgh总蛋白;
图5是本发明中温度对耐热裂解酶TSPpgh活性的影响结果示意图;
图6是本发明不同pH值对耐热裂解酶TSPpgh活性的影响结果示意图;
图7是本发明耐热裂解酶TSPpgh对ThermusTC16菌株生长的影响结果示意图;其中1为TC16原始菌液对照管,2为加入耐热裂解酶TSPpgh的菌液2h,3为未加入耐热裂解酶TSPpgh的菌液2h对照管,4为加入耐热裂解酶TSPpgh的菌液4h,5为未加入耐热裂解酶TSPpgh的菌液4h对照管,6为加入耐热裂解酶TSPpgh的菌液6h,7为未加入耐热裂解酶TSPpgh的菌液6h对照管。
图8是本发明耐热裂解酶TSPpgh对Thermus TC16菌株生长的影响结果示意图;
图9是本发明耐热裂解酶TSPpgh的活性检测示意图,其中1、2、3、4为耐热裂解酶TSPpgh酶液对ThermusTC16的抑菌圈,5代表用灭活的耐热裂解酶TSPpgh对照;
图10是本发明耐热裂解酶TSPpgh作用后的ThermusTC16菌体形态的变化示意图;其中:A为裂解酶作用前的菌体形态;B、C、D为裂解酶作用后的菌体形态;
图11是本发明耐热裂解酶TSPpgh对地衣芽孢杆菌(Bacillus licheniformis)Tamy6菌株,及E.coli菌株生长的影响结果示意图,其中1为未加入耐热裂解酶TSPpgh的大肠杆菌菌液,2为加入耐热裂解酶TSPpgh的大肠杆菌菌液,3为未加入耐热裂解酶TSPpgh的Tamy6菌液,4为加入耐热裂解酶TSPpgh的Tamy6菌株菌液。
具体实施方式
下面通过实施例对本发明作进一步详细说明,但本发明的内容并不局限于此,本实施例中方法如无特殊说明的均按常规方法操作,所用试剂如无特殊说明的采用常规试剂或按常规方法配置的试剂。
实施例1:耐热裂解酶TSPpgh的克隆和表达
1、裂解酶基因的扩增,(以ThermusTC16的噬菌体TSP4基因组DNA为模板)
(1)嗜热噬菌体TSP4裂解酶TSPpgh基因的扩增所用引物序列如下:
正向引物:5'- CATGCCATGGCAATGCGTCTACCGACTAAGAC -3'
反向引物:5'- CCGCTCGAGTTTACCTCCTAGCAACTTGG -3'
(2)扩增体系如下:
表1:扩增反应体系组分
(3)扩增条件如下:
将反应体系混匀,先在94℃预变性4min,然后在94℃变性45s,55℃退火45 s,72℃延伸90 s,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(见图1)。
2、PCR产物的胶回收纯化
(1)在电泳仪中灌制1.0%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。3、重组表达载体的构建
为了把目的基因片断连接到表达载体pET28a,就需要使目的片断带有粘性末端的片断,即带有酶切位点。
(1)耐热裂解酶TSPpgh基因片段的双酶切
表2:反应体系组分
②酶切条件:37 ℃,4h,回收TSPpgh基因片段。
(2) 带有粘性末端线性载体pET28a的制备
为了将目的基因片断连接到表达载体pET28a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:
②增菌并收集菌体:取氨苄青霉素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Amp+-LB培养基中;然后放入37℃培养箱中,摇床培养,过夜;去3ml培养的菌液,5000 rpm,室温离心5 min,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6-10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6-10次,室温放置5分钟,室温13,000 rpm离心10分钟,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),13,000 rpm离心1分钟,弃滤液;
⑦加入500μl去蛋白液PE,13,000 rpm 离心60秒,弃滤液;
⑧加入500μl漂洗液WB,13,000 rpm 离心60秒,弃滤液;
⑨重复步骤⑦一次,13,000 rpm 离心60秒,弃滤液,空柱13,000 rpm 离心2分钟,室温放置3-5分钟,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),室温放置1分钟,13,000 rpm 离心1分钟洗脱质粒。
B、质粒pET32a酶切鉴定
表3:反应体系组分
②反应条件:37℃,过夜。
(3)重组表达载体的构建、TSPpgh蛋白的诱导表达和纯化
①将前面实验得到的带有粘性末端的线性载体pET28a和TSPpgh裂解酶基因片段,通过连接转化并用菌落PCR、酶切鉴定(见图2)及测序验证,即可得到重组表达载体。
②耐热裂解酶TSPpgh蛋白在大肠杆菌中的诱导表达
将构建好的重组载体TSPpgh/pET28a转化大肠杆菌BL21,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OD值0.6-0.8;取出4ml菌液用作对照实验;向余下的菌液加入异丙基-β-D-硫代半乳糖苷IPTG(终浓度为1mM),放入37℃、28℃,80rpm摇床诱导培养6小时,取样5ml。
4、耐热裂解酶TSPpgh蛋白 SDS-PAGE检测
将取出的5ml菌液,6000rpm,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)。
5、耐热裂解酶TSPpgh重组蛋白的纯化
利用上述方法大量诱导含重组质粒TSPpgh/pET28a的BL21菌株,菌液经离心收集大肠杆菌菌体(4℃,5,000x g,10 min)。用30mM咪唑溶液悬浮菌体后进行超声波破碎,4℃,20,000x g离心10 min,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,用缓冲液(50 mM Tris-HCl,pH7.9)透析,酶2小时更换一次透析液,共透析10小时,纯化的重组蛋白组分进行SDS-PAGE检测(见图4)。
实施例2:耐热裂解酶TSPpgh部分酶学性质及酶活性实验
1、耐热裂解酶TSPpgh最适作用温度、pH值及金属离子对耐热裂解酶TSPpgh的影响
为研究最适作用温度,pH值及金属离子对TSPpgh的影响,采用培养至对数期的宿主ThermusTC16的菌液,洗涤3次后收集菌体进行超声波破碎,研磨后制备获得细菌TC16的细胞壁,以TC16细胞壁细胞碎片作为TSPpgh的反应底物,研究其最适催化温度、pH已近金属离子对其的影响。
(1)耐热裂解酶TSPpgh的最适催化温度
将纯化的耐热裂解酶TSPpgh加入制备好的TC16细胞壁底物中,分别置于37℃、45℃、49.5℃、57.1℃、66.5℃、69.8℃、75℃、80℃反应5h,根据反应前后OD600的变化量确定其最适催化温度(图5),如图5所示,其催化温度范围为40-80℃,在66.5℃催化活性最高,可见耐热裂解酶TSPpgh具有明显的高温催化活性。
(2)耐热裂解酶TSPpgh的最适催化pH
将纯化的裂解酶TSPpgh加入制备好的TC16底物中,分别在不同pH值的磷酸盐缓冲液中,相同温度(最适作用温度)下,作用时间5小时,根据反应前后OD600的变化量确定其最适pH(图6),如图6所示,其最适催化pH为6-8。
(3)常见金属离子对耐热裂解酶TSPpgh的影响
将纯化的耐热裂解酶TSPpgh加入制备好的TC16底物中,混合液中加入不同金属离子Na+、Zn2+、Mg2+、Mn2+、Ca2+,并保持离子浓度为0.01mol/L,相同温度(最适作用温度),作用时间5小时,根据反应前后OD600的变化量确定金属离子对其的影响,结果所示,Mg2+对TSPpgh活力有促进作用,Fe2+、Mn2+具有明显抑制作用。
表4: 金属离子对TSPpgh的影响结果
2、耐热裂解酶TSPpgh对栖热菌TC16及其他菌株生长的影响
(1)将ThermusTC16培养到OD≈0.3,分别向三支试管里的菌悬液中各加入50μL 耐热裂解酶TSPpgh,放入65℃恒温摇床培养,于2h、4h、6h分别对三支试管取样进行观察,测得OD600值(见图7、8),可见TSPpgh能明显抑制ThermusTC16菌株的生长。
(2)采用双层平板法培养宿主栖热菌TC16,在未长出菌膜之前向平板上滴加8μL耐热裂解酶TSPpgh酶液(蛋白含量为0.256mg/mL),对照为等量的耐热裂解酶TSPpgh经98℃,20min灭活酶后点于平板,于70℃恒温培养箱倒置培养过夜后观察,滴加耐热裂解酶TSPpgh能产生明显的抑菌圈(见图9)。
(3)栖热菌TC16培养至对数期,6000rpm 10min离心菌液得到菌体,以新鲜DSM88培养基洗涤3次,收集菌体,以PBS溶液进行稀释,然后1mL栖热菌TC16菌液中添加400μL 耐热裂解酶TSPpgh,分为不同时间段取样制片于光学显微镜下进行镜检,耐热裂解酶TSPpgh对栖热菌TC16菌体有明显的裂解作用(见图10)。
(4)耐热裂解酶TSPpgh对Tamy6菌株(革兰氏阳性菌,嗜热地衣芽孢杆菌Bacillus licheniformis,菌种保藏号为CGMCC No.2642)和E.coli生长的影响
Tamy6菌株为耐热芽孢杆菌,为革兰氏阳性菌,其生长最适温度为55℃,DSM88和LB液体培养基分别培养Tamy6菌株和E.coli至对数期,加入耐热裂解酶TSPpgh后,分别在55℃和40℃恒温摇床中振荡培养,转速调为120rpm,2小时后,测量OD600吸光值,耐热裂解酶对这两个菌株的生长均有明显的抑制作用(图11)。
序列表
<110> 昆明理工大学
<120> 一种耐热裂解酶TSPpgh和编码此酶的多核苷酸
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 166
<212> PRT
<213> 噬菌体TSP4
<400> 1
Met Arg Leu Pro Thr Lys Thr Ser Arg Phe Gly Tyr Val His Gly Gln Arg Asn His Glu
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Gly Ile Pro His Pro Gly Tyr Asp Leu Asn Asn Gly Pro Thr Pro Thr Ser Asp Leu Gly
21 30 40
Gln Pro Val Tyr Ala Pro Glu Asp Gly Val Val Val Tyr Ala Arg Thr Gly Ser Gly Thr
41 50 60
Trp Gly Gly Leu Val Val Val Leu Gly Lys Ser Gly Phe Ala His Arg Leu Gly His Val
61 70 80
Arg Asn Ile Arg Val Lys Glu Gly Gln Glu Val Lys Glu Gly Gln Gln Val Ala Glu Ile 81 90 100
Gly Glu Phe Val Lys Gly Leu Pro His Leu His Tyr Asp Met Val Glu Pro Lys Val Ile
101 110 120
His Thr Ile Ser Ile Leu Ile Lys Ala Pro Tyr Val Arg Trp Asp Phe Trp His Val Asn
121 130 140
Phe Pro Lys Leu Phe Glu His Met Tyr Val Asp Pro Ala Arg Phe His Pro Glu Leu Ala
141 150 160
Lys Leu Leu Gly Gly Lys
166
<210> 2
<211> 498
<212> DNA
<213> 噬菌体TSP4
<400> 2
atgcgtctac cgactaagac ttcccgcttt ggttatgtgc acggccagag 50
aaaccacgag ggcattcccc acccaggcta tgacctgaat aacggcccta 100
cgcctactag cgaccttggt cagcctgtgt atgcccctga ggatggcgtg 150
gtggtctatg cccggactgg gtcaggtacc tggggtgggc tggtggtggt 200
cttgggcaaa agcggctttg cccatcggct aggccatgtg cgcaacattc 250
gggtcaaaga gggacaggag gtgaaggaag gccagcaggt ggccgagatt 300
ggggagttcg tcaaggggct tccccacctg cactacgaca tggtggagcc 350
caaggttatc cacaccatca gtatcctgat caaggcccct tatgttcggt 400
gggacttctg gcacgtaaac tttcccaaac tgtttgagca catgtatgtg 450
gacccggcca ggtttcaccc tgagctggcc aagttgctag gaggtaaa 498
<210> 3
<211> 32
<212> DNA
<213> 人工序列
<400> 3
catgccatgg caatgcgtct accgactaag ac 32
<210> 4
<211> 29
<212> DNA
<213> 人工序列
<400> 4
ccgctcgagt ttacctccta gcaacttgg 29
Claims (2)
1.一种耐热裂解酶TSPpgh,其特征在于:其氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述耐热裂解酶TSPpgh的多核苷酸,其特征在于:其核苷酸序列如SEQ ID NO:2所示。
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CN110592056A (zh) * | 2019-09-19 | 2019-12-20 | 昆明理工大学 | 噬菌体裂解酶复合粉剂及其制备方法和应用 |
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