CN107828769A - 一种耐热裂解酶MMPpgh和编码此酶的多核苷酸 - Google Patents
一种耐热裂解酶MMPpgh和编码此酶的多核苷酸 Download PDFInfo
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- CN107828769A CN107828769A CN201710897681.XA CN201710897681A CN107828769A CN 107828769 A CN107828769 A CN 107828769A CN 201710897681 A CN201710897681 A CN 201710897681A CN 107828769 A CN107828769 A CN 107828769A
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- lyases
- mmppgh
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- enzyme
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Abstract
本发明公开了一种耐热裂解酶MMPpgh和编码此酶的多核苷酸,该酶的氨基酸序列如SEQ ID NO:1所示;该裂解酶能够抑制革兰氏阳性和阴性细菌的生长,其在37~75℃范围内具有催化活性,在56~65℃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MMPpgh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。
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。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MMPpgh,该裂解酶能够抑制革兰氏阳性和阴性细菌的生长,其在37~75℃范围内具有催化活性,其来源于亚栖热菌(Meiothermus)噬菌体MMP17,该耐热裂解酶MMPpgh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MMPpgh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37~75℃范围内均具有催化活性,在56~65℃催化活性最高,可适用于高温环境的杀菌,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MMPpgh基因PCR产物电泳示意图,其中泳道1为Marker,泳道2为阴性对照,泳道3为MMPpgh基因PCR产物,其大小为633bp;
图2是本发明中重组质粒MMPpgh/pET28a双酶切图谱示意图,其中泳道1为Marker,泳道2为未经双酶切的重组质粒MMPpgh/pET28a条带;泳道3为重组质粒MMPpgh/pET28a双酶切后的两个条带;
图3是本发明中耐热裂解酶MMPpgh的蛋白表达检测示意图,泳道1为GenStar M221-01蛋白Marker,泳道2、3为诱导后的表达菌株超声破碎后的上清液;
图4是本发明耐热裂解酶MMPpgh蛋白的纯化结果检测示意图,其中泳道1为GenStarM221-01蛋白Marker,泳道2为总蛋白,泳道3为空载总蛋白,泳道4为挂柱透过液,泳道5为30mM咪唑洗脱液,泳道6为150mM咪唑洗脱液,泳道7为750mM咪唑洗脱液;
图5是本发明中温度对耐热裂解酶MMPpgh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MMPpgh活性的影响结果示意图。
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MMPpgh的克隆和表达
1、裂解酶基因的扩增,(以Meiothermus TG17的噬菌体MMP17基因组DNA为模板)
(1)嗜热噬菌体MMP17裂解酶MMPpgh基因的扩增所用引物序列如下:
正向引物:5'- CCGGAATTCATGCGCATCGTTCATCCC -3'
反向引物:5'- CCCAAGCTTTTGCAATGCGCGATTTGT -3';
(2)扩增体系如下:
表1:扩增反应体系
;
(3)扩增条件如下:
将反应体系混匀,先在95℃预变性3min,然后在95℃变性40 s,58℃退火30 s,72℃延伸40 s,30个循环后,72℃延伸8min。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),MMPpgh基因PCR产物大小为633bp。
2、裂解酶基因MMPpgh PCR产物的胶回收
(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)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:
①菌种活化:无菌接种环蘸取-80℃冻存的菌种保存液,三线法接种于氨苄LB平板,37℃培养12-16小时;
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7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。
(2)耐热裂解酶MMPpgh基因片段和pET-28a质粒的双酶切
①酶切体系如下:
表2:酶切体系
;
②反应条件:37℃烘箱酶切5h左右,回收MMPpgh基因片段和酶切后的pET-28a质粒。(3)重组表达载体的构建、MMPpgh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MMPpgh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MMPpgh蛋白在大肠杆菌中的诱导表达
将构建好的重组载体MMPpgh/pET28a转化至E.coliRosetta 菌株,得到表达菌株pET28a-mmppgh/ Rosetta;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5mL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MMPpgh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);将SDS-PAGE胶取出,加入R-250考马斯亮蓝染色液,沸水浴30min;染色完成后,进行脱色液脱色,脱色2次每次沸水浴30min,观察结果并拍照分析(见图3)。
5、耐热裂解酶MMPpgh重组蛋白的纯化
利用上述方法大量诱导含重组质粒MMPpgh/pET28a的Rossetta菌株,菌液经离心收集大肠杆菌菌体(4℃,4,500rpm,20min)。用30mM咪唑溶液悬浮菌体后进行超声波破碎,4℃,13,000rpm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,分别用10倍柱体积的(150mM、500 mM、750 mM)咪唑洗脱柱子,再用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 mMTris-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)。
6、耐热裂解酶MMPpgh重组蛋白的浓度及活性测定
(1)Bradford法测定裂解酶MMPpgh重组蛋白的浓度
取干净的4mL离心管,按顺序标号,空白、标1、标2、标3、标4、标5、标6。分别加入标准蛋白溶液(1mg/mL的标准蛋白BSA溶液)、待测样品溶液、ddH2O、考马斯亮蓝G-250储存液,混匀。用紫外分光光度计测其波长为600nm处的吸光度值,以浓度为0 mg/mL的BSA溶液作为空白对照。以蛋白浓度为横坐标,OD600值为纵坐标,绘制标准曲线。计算公式:待测样品蛋白浓度(mg/mL)= 测定样品的OD600值/标准蛋白的OD600值×标准蛋白的浓度×稀释倍数得标准曲线方程为:y=0.0253x+0.0053(R2=0.9967),将纯化后得裂解酶MMPpgh测其OD600吸光值,测得OD值为0.122,计算得到蛋白浓度为0.231mg/mL。
(2)Folin-酚法测定裂解酶MMPpgh对酪蛋白水解酶活测定
①标准曲线制备
取干净的50mL离心管,按顺序标号,空白、标1、标2、标3、标4、标5。将100μg/mL酪氨酸(mL)标准液与不同体积的ddH2O配成不同标准浓度,总体积为10mL;分别吸取上表中不同浓度的酪氨酸溶液1mL,各加入0.4mol/L碳酸钠溶液5mL,再加入购买的福林试剂1mL;摇匀后放置于40℃水浴锅中,保温发色20min,测量OD值600并记录;得到标准曲线方程y=0.0107x-0.0336(R2=0.9961)。
计算公式Y=A×N×4/10(公式:Y:样品的酶活力,单位U/mL;A:样品测得的OD值,代入标准曲线后计算得到的酪氨酸微克数;N:酶液的稀释倍数;10:反应时间为10min,以1min计;4:反应试剂的总体积,mL)
酶活单位定义:1mL酶液,在一定温度和pH条件下,1min水解酪素产生1μg酪氨酸为一个酶活单位,以U/mL表示。
②酶活测定
吸取样品稀释液1mL,置于40℃水浴锅中预热 2min,然后加入经同样预热的2%酪蛋白1mL,精确保温10min,时间到后,立即再各加入0.4mol/L的三氯乙酸 2mL,以终止反应,继续置于水浴锅中保温20min,使得残余蛋白质沉淀。注射器吸取含有沉淀的溶液,将溶液使用0.45μm滤膜过滤,然后吸取滤液1mL,再加入0.4mol/L碳酸钠5mL,已稀释的福林试剂1mL摇匀,40℃保温发色20min后进行光密度(OD)测定。
测定结果:OD600值为0.215,代入标准曲线方程y=0.0107x-0.0336(R2=0.9961)计算得到酪氨酸含量23.2μg。将所得酪氨酸含量值代入公式Y=A×N×4/10,计算得到酶活为223U/mL。
实施例2:耐热裂解酶MMPpgh部分酶学性质及酶活性实验
1、耐热裂解酶MMPpgh最适作用温度、pH值及金属离子对耐热裂解酶MMPpgh的影响
为研究裂解酶MMPpgh最适作用温度,pH值及金属离子对裂解酶的影响,采用培养至对数期的TG17菌液;将对数生长期的TG17菌液,4500rpm离心20min后,弃上清。沉淀以10mLPBS(pH7.4)悬浮后,超声破碎,离心得沉淀,将沉淀重新使用5mL PBS悬浮。此时TG17细胞壁均为细胞碎片,以此作为裂解酶MMPpgh的反应底物。测得TG17反应底物的初始OD600值=0.241。
(1)耐热裂解酶MMPpgh的最适催化温度
将纯化后的裂解酶MMPpgh加入制备好的TG17反应底物中,然后置于不同温度反应5h,反应温度分别是37℃、45℃、50℃、56℃、65℃、70℃、75℃。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37-75℃,在65℃催化活性最高,可见耐热裂解酶MMPpgh具有明显的高温催化活性。
(2)耐热裂解酶MMPpgh的最适催化pH
将纯化的裂解酶MMPpgh加入制备好的TG17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7-8。
2、耐热裂解酶MMPpgh对菌株生长的影响
按1%接种量,接种E.coli、沙门氏菌、金黄色葡萄球菌和耐药性肺炎克雷伯菌到LB液体培养基中,37℃ 150rpm培养至对数生长期。吸取20μL纯化后的MMPpgh酶液与80μL处于对数生长期的菌液混合,37℃恒温培养50min后,将反应后的混合液稀释到合适的倍数,涂布平板,过夜培养后,统计实验组与空白对照组平板单菌落数,计算致死率。空白对照组为20μLddH2O代替酶液。
将反应后的混合液稀释后涂布平板,由涂布结果知,每组实验组和空白对照组平板上的单菌落数并不一致,计算后得大肠杆菌致死率为52.2%;沙门氏菌致死率为87.5%;金黄色葡萄球菌致死率为53.9%,耐药性肺炎克雷伯菌致死率为89.4%(表3)。
表3:裂解酶MMPpgh对不同菌株生长的影响结果
;
3、不同金属离子对酶活的影响
将纯化的裂解酶MMPpgh加入制备好的TG17底物中,混合液中加入不同金属离子Mn2+、Ca2+、Mg2+、Zn2+、Cu2+、K+,并保持离子浓度为2mmol/L,最适作用温度和最适pH条件下,作用时间5小时,测定OD600值。其中空白实验组中使用ddH2O代替金属离子。我们把能提高酶活的金属离子称为没得激活剂。反之,则称酶的抑制剂。Mg2+、Zn2+对酶有激活作用,尤其是Mg2+离子。而Ca2+、K+、Mn2+对酶有抑制作用(表4)。
表4:金属离子对MMPpgh酶活的影响
。
序列表
<110> 昆明理工大学
<120> 一种耐热裂解酶MMPpgh和编码此酶的多核苷酸
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 210
<212> PRT
<213> 噬菌体MMP17(Phage MMP17)
<400> 1
Met Arg Ile Val His Pro Phe Pro Gln Pro Ala Arg Ala Arg Val Asp
1 5 10 15
Ala Gly Phe Leu Asp Pro Arg Tyr Pro Gln Trp Arg Arg Ala Ala Gly
20 25 30
Leu Ala Pro Ala Glu His Thr Gly Val Asp Tyr Asn Leu Val Gly Thr
35 40 45
Ser Gly Asp Ala Asp Leu Gly Tyr Pro Val Val Ala Met Ala Asp Gly
50 55 60
Ile Val Arg His Ala Arg Ala His Arg Ile Trp Gly Asn Ile Val Leu
65 70 75 80
Leu Glu His Pro Gln Leu Gly Leu Trp Ser Gln Tyr Ala His Leu Tyr
85 90 95
Gln Leu Ala Val Asp Ala Gly Gln Glu Ile Trp Ala Gly Glu Pro Leu
100 105 110
Gly Ser Ile Gly Arg Gly Asp Pro Arg Ala Pro Phe Leu Ala His Leu
115 120 125
His Phe Glu Ile Arg Thr Arg Pro Leu Pro Ala Asp Asn Trp Pro Gly
130 135 140
Met Asn Lys Thr Ala Ile Lys Glu Gly Tyr Leu Asp Pro Glu Thr Trp
145 150 155 160
Leu Lys Gln His Met Ala Thr Glu Arg Arg Phe Thr Arg Gln Gly Leu
165 170 175
Val Leu Trp Leu Pro Asp Gly Lys His Ser Met Pro Gly Lys Thr Ile
180 185 190
Val Asn Leu Asp Asp Pro Thr Leu Val His Val Arg Thr Asn Arg Ala
195 200 205
Leu Gln
210
<210> 2
<211> 636
<212> DNA
<213> 噬菌体MMP17(Phage MMP17)
<400> 2
atgcgcatcg ttcatccctt cccccaacct gcccgagccc gcgtagacgc gggcttttta 60
gatccccgct atccccagtg gcgacgggca gctgggctgg ccccggctga acacacaggg 120
gtggactaca acctggtagg caccagcggt gatgctgacc tgggttatcc ggtggtagca 180
atggccgatg gcattgttcg gcatgcccgt gcgcaccgca tttggggaaa tatcgttctg 240
ctcgagcatc cccaattggg cctgtggagc cagtacgccc atctgtacca gttggccgta 300
gatgcagggc aggaaatctg ggccggggaa ccgctgggca gcatcggcag gggggaccct 360
cgagctccct tcctggccca cctgcatttc gagatacgca cgcgtccgct ccccgccgac 420
aactggccgg ggatgaacaa aactgcgata aaggagggat atctggatcc ggaaacatgg 480
tggctgaagc agcatatggc gaccgagcgg cggttcaccc ggcaggggct cgtcctgtgg 540
ttgccagatg gaaaacacag tatgcctggc aagacaatcg tcaatctaga cgatccaacg 600
ttagtgcatg tgcgtacaaa tcgcgcattg caatag 636
<210> 3
<211> 27
<212> DNA
<213> 人工序列(Artificial)
<400> 3
ccggaattca tgcgcatcgt tcatccc 27
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial)
<400> 4
cccaagcttt tgcaatgcgc gatttgt 27
Claims (2)
1.一种耐热裂解酶MMPpgh,其特征在于: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MMPpgh的多核苷酸,其特征在于:核苷酸序列如SEQID NO:2所示。
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Effective date of registration: 20220926 Address after: 653100 in the plant area of Taishan paper mill, group 3, liangwangba community, Gaocang street, Hongta District, Yuxi City, Yunnan Province Patentee after: Yuxi niuyila Agricultural Technology Co.,Ltd. Address before: 650093 No. 253, Xuefu Road, Wuhua District, Yunnan, Kunming Patentee before: Kunming University of Science and Technology |