CN110592057A - 嵌合裂解酶ILTphg和编码此酶的多核苷酸 - Google Patents
嵌合裂解酶ILTphg和编码此酶的多核苷酸 Download PDFInfo
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- CN110592057A CN110592057A CN201910923094.2A CN201910923094A CN110592057A CN 110592057 A CN110592057 A CN 110592057A CN 201910923094 A CN201910923094 A CN 201910923094A CN 110592057 A CN110592057 A CN 110592057A
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- Prior art keywords
- lyase
- chimeric
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- gly
- iltphg
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Abstract
本发明公开了一种嵌合裂解酶ILTphg和编码此酶的多核苷酸，其氨基酸序列如SEQ ID NO:1所示，其核苷酸序列如SEQ ID NO:5所示；将该嵌合裂解酶应用在抑制或杀灭革兰氏阳性菌和/或革兰氏阳性菌中，实验结果显示其能够抑制革兰氏阳性和阴性细菌的生长，其在30~50℃范围内具有催化活性，其核苷酸序列可用于构建生产此裂解酶的基因工程菌株且适于工业化生产。
Description
技术领域
本发明属于生物技术领域，具体涉及一种嵌合裂解酶ILTphg，以及编码此酶的核酸序列。
背景技术
近年来，随着抗生素大量滥用，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，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。
然而，天然噬菌体裂解酶存在以下问题。首先，革兰氏阴性菌胞壁肽聚糖外的一层外膜有效的阻挡裂解酶进入宿主细胞壁上发挥作用。因此，在体外，大多数噬菌体裂解酶不能裂解阴性细菌，限制了裂解酶用于治疗阴性细菌导致的感染。其次，噬菌体裂解酶的种属特异性，也在一定程度上限制了其在临床上的应用，需要发展抗菌活性高，较为广谱的裂解酶；最后一些裂解酶在大肠杆菌（Escherichia coli）中表达时，表现出表达量低，可溶性差等问题。可见，这些问题亟待解决，以适应未来发展噬菌体裂解酶抗菌药物的需要。
基于结构上的模块化特点，构建嵌合裂解酶是一种有效解决以上噬菌体裂解酶问题的方法。模块化的裂解酶由两种结构域组成，分别是与底物结合的细胞壁结合域（cellwall binding domain，CBD）和水解底物的EAD，或将催化域与其他结构域组合所得到的重组裂解酶。发展嵌合裂解酶可以使全酶具有更好的性质，如具有更强的杀灭阴性细菌的活性，更广的裂解谱，更好的水溶性等。
大部分嵌合裂解酶的表达都采用多个启动子启动多个基因的办法，而这种方法可能会导致某一种裂解酶表达量过低，而不能达到良好的杀菌效果。
发明内容
针对现有技术存在的不足，本发明提供了一种嵌合裂解酶ILTphg，其是由亚栖热菌（Meiothermust）TG07噬菌体MMP7的裂解酶MMPphg与栖热菌（Thermus）TC16的噬菌体TSP裂解酶TSP-2phg以及连接两个裂解酶的一条linker组成，该结构可以使翻译出来的两个蛋白质保持原有的空间结构，不改变其空间构型，从而保持其结合位点不会发生变化，不会对其酶活性产生影响；linker的分子量很小，通过设计引物利用PCR技术将其连接到裂解酶基因上；该嵌合裂解酶ILTphg的完整氨基酸序列如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；其中裂解酶MMPphg的氨基酸序列如SEQ ID NO:2所示，linker的氨基酸序列如SEQ ID NO:3所示，裂解酶TSP-2phg的氨基酸序列如SEQ ID NO:4所示。
本发明另一目的是提供编码上述嵌合裂解酶ILTphg的多核苷酸，核苷酸序列如SEQ ID NO:5所示；或其互补序列，或具有与SEQ ID NO:5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；该序列由SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8组成。
上述嵌合裂解酶ILTphg能够抑制革兰氏阳性和阴性细菌的生长，其在30~50℃范围内具有催化活性，以及良好的水溶性，并且具有更广的裂解谱、更好的杀菌活性。
本发明的有益效果：
本发明提供的嵌合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药物，其也可作为抑菌剂用于环境的杀菌。
目前报道的裂解酶多数具有专一性的特点，只能裂解一类细菌或某种细菌，而本发明提供的嵌合裂解酶不但对革兰氏阳性菌起作用，还可以对革兰氏阳性菌起作用，具有较好的杀菌活性，有着更广的裂解谱，不但对金黄色葡萄球菌的杀菌效果显著，还对大肠杆菌、沙门氏菌等其他致病菌也有着良好的杀菌效果。
附图说明
图1是本发明裂解酶TSP-2phg基因和MMPphg基因PCR产物胶回收电泳示意图，其中M代表Marker，泳道1为TSP-2phg基因PCR产物胶回收，其大小为501bp，泳道2为MMPphg基因PCR产物胶回收，其大小为633bp；
图2是本发明中连接结果PCR检测电泳示意图，其中M代表Marker，泳道1为阴性对照，泳道2为未连接目的片段的pET-28a的T7启动子PCR结果，泳道3为连接后载体pET-28a-ILTphg的T7启动子PCR结果；
图3是本发明中嵌合裂解酶ILTphg的蛋白表达检测示意图，其中M代表蛋白Marker，泳道1为pET28a-ILTphg/BL21诱导前破碎液，泳道2为pET28a-ILTphg/BL21诱导后破碎液，泳道3为pET28a-ILTphg/BL21诱导后破碎液离心后取的上清液；
图4是本发明嵌合裂解酶ILTphg蛋白的纯化结果检测示意图，其中M代表蛋白Marker，泳道1为总蛋白，2为挂柱透过液，3为上柱后500mM咪唑洗脱液；
图5是本发明嵌合裂解酶ILTphg的活性检测示意图，其中1代表用灭活的嵌合裂解酶ILTphg对照，2、3、4分别为嵌合裂解酶ILTphg酶液、TSP-2phg酶液、MMPphg酶液对金黄色葡萄球菌的抑菌圈；
图6是本发明嵌合裂解酶ILTphg作用后的Thermus TC16及Meiothermust TG07菌体形态的变化示意图；其中：A为裂解酶作用前的Thermus TC16菌体形态，B为裂解酶作用后的Thermus TC16菌体形态，C为裂解酶作用前的Meiothermust TG07菌体形态，D为裂解酶作用后的Meiothermust TG07菌体形态；
图7是本发明嵌合裂解酶ILTphg对金黄色葡萄球菌杀伤作用时间结果示意图；
图8是本发明嵌合裂解酶ILTphg对金黄色葡萄球菌杀伤作用浓度结果示意图。
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：嵌合裂解酶ILTphg的构建和表达
1、裂解酶基因的扩增，（分别以Thermus TG07的噬菌体的MMP7基因组DNA及含有TSP-2基因的质粒为模板）
（1）亚栖热菌噬菌体MMP7的裂解酶MMPphg基因的扩增所用引物序列如下：
正向引物：5'- CCATGGCAATGCGCATCGTTCATCCCTT -3'
反向引物：5'-GCATGTTTAGCGCGTAACGTTCCTCCTCCTCCGAGTCCACCTCCACCTAGT-3'
裂解酶TSP-2phg基因的扩增所用引物序列如下：
正向引物：5'- CCACCTCCACCTAGTCCTCCTCCTCCGAGTATGCGTCTACCGACTAAGAC -3'
反向引物：5'- CCGCTCGAGTTTACCTCCTAGCAACTTG -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94℃预变性4min，然后在94℃变性45s，55℃退火45 s，72℃延伸90s，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1.0％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；
（5）在1%琼脂糖凝胶中进行电泳检测，结果见图1，获得501bp TSP-2phg基因PCR产物，633bp 的MMPphg基因PCR产物；
3、裂解酶TSP-2phg、linker及MMPphg基因片段的连接
采用重叠PCR技术，将裂解酶TSP-2phg、linker及MMPphg基因片段连接在一起；
（1）重叠PCR所用引物序列如下：
正向引物：5'- CCATGGCAATGCGCATCGTTCATCCCTT -3'
反向引物：5'- CCGCTCGAGTTTACCTCCTAGCAACTTG -3'；
（2）扩增体系如下：
表2：扩增反应体系
；
（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，回收目的基因片段。
4、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pET-28a，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）裂解酶ILTphg基因片段的双酶切
酶切体系如下：
表3：反应体系
；
②酶切条件：37 ℃，4h，回收ILTphg基因片段。
（2）带有粘性末端线性载体pET-28a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：
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pET-28a酶切鉴定
酶切体系如下：
表4：反应体系
；
②反应条件：37℃，过夜。
（3）重组表达载体的构建、ILTphg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ILTphg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，即可得到构建好的重组表达载体。
②嵌合裂解酶ILTphg蛋白在大肠杆菌中的诱导表达
将构建好的重组载体ILTphg/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；向余下的菌液加入乳糖（终浓度为1mM），放入37℃、28℃，80rpm摇床诱导培养6小时，取样5mL。
5、嵌合裂解酶ILTphg蛋白 SDS-PAGE检测
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），结果显示嵌合裂解酶ILTpgh为可溶性蛋白。
6、嵌合裂解酶ILTphg重组蛋白的纯化
利用上述方法大量诱导含重组质粒ILTphg/pET28a的BL21菌株，菌液经离心收集大肠杆菌菌体(4℃，8000rpm，10 min)；用PBS溶液悬浮菌体后进行超声波破碎，4℃，13000rpm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，纯化的重组蛋白组分进行SDS-PAGE检测（见图4），结果显示嵌合裂解酶ILTpgh的基因能正确表达出蛋白质。
实施例2：嵌合裂解酶ILTphg的酶活性验证
（1）采用双层平板法培养金黄色葡萄球菌，在未长出菌膜之前向平板上滴加100μL嵌合裂解酶ILTphg酶液(蛋白含量为40pmol/mL)，对照为裂解酶MMPphg（蛋白含量为80pmol/mL）、裂解酶TSP-2phg（蛋白含量为80pmol/mL）及的经121℃灭活20min后的嵌合裂解酶ILTphg(蛋白含量为40pmol/mL)点于平板，于37℃恒温培养箱倒置培养过夜后观察，滴加嵌合裂解酶ILTphg能产生透明的抑菌圈（见图5）。
（2）将栖热菌TC16及亚栖热菌TG07培养至对数期，菌液8000rpm离心10min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菌液中添加400μL嵌合裂解酶ILTphg，37℃反应30min，对照采用经121℃，20min灭活后的嵌合裂解酶ILTphg，制片于光学显微镜下进行镜检，嵌合裂解酶ILTphg对栖热菌TC16及亚栖热菌TG07菌体有明显的裂解作用（见图6）。
（3）对比嵌合裂解酶ILTphg、裂解酶MMPphg及裂解酶TSP pgh 对金黄色葡萄球菌等治病菌生长的影响
按1%接种量，将金黄色葡萄球菌（Staphylococcus aureus）、沙门氏菌（Salmonella）、大肠杆菌（Escherichia coli）、肠炎沙门氏菌（Salmonella enteritidis）、肠炎沙门氏菌亚种（Salmonella enteritidis subspecies）、猪霍乱沙门氏菌（Salmonella cholerae）接种到LB液体培养基中，37℃、150rpm培养至对数生长期；吸取900μl酶液与100μl处于对数生长期稀释至10-5、10-4的菌液混合，37℃恒温培养30min后，将反应后的混合液取100μl涂布平板，过夜培养后，统计实验组与对照组平板单菌落数，计算致死率（表格内划交叉斜线的表示没有抑菌效果）。空白对照组为900μL灭活后离心去掉沉淀的酶液代替酶液；所用裂解酶酶液浓度为：嵌合裂解酶ILTphg酶液(蛋白含量为40pmol/mL)，裂解酶MMPphg、TSP-2phg（蛋白含量为40pmol/mL）；
表5：不同裂解酶的杀菌效果（表中×为无效果）
。
（4）嵌合裂解酶ILTphg对金黄色葡萄球菌的作用时间
将900μl嵌合裂解酶ILTphg酶液（浓度为40pmol/mL）与100μL稀释至适合梯度的金黄色葡萄球菌菌液（生长至对数期，菌浓度为103 cfu/mL）于37℃恒温培养箱分别反应0min，5min，10 min，20 min，30 min，40 min，50 min，60 min后，吸取100 μL涂布计数，空白对照组为900μl灭活后离心去掉沉淀的酶液代替酶液（见图7）。
（5）嵌合裂解酶ILTphg对金黄色葡萄球菌的最低抑菌浓度
按1%接种量，接种金黄色葡萄球菌到LB液体培养基中，37℃、150rpm培养至对数生长期；吸取900μl酶液(浓度分别为5pmol/mL、10pmol/mL、15pmol/mL、20pmol/mL、25pmol/mL、35pmol/ mL、40pmol/ mL)与100μl处于对数生长期稀释至10-5的菌液混合，37℃恒温培养30min后，将反应后的混合液取100μl涂布平板，过夜培养后，统计实验组与对照组平板单菌落数，计算杀伤率；空白对照组为900μL灭活后离心去掉沉淀的酶液代替酶液(见图8)。
序列表
<110> 昆明理工大学
<120> 嵌合裂解酶ILTphg和编码此酶的多核苷酸
<160> 12
<170> SIPOSequenceListing 1.0
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<211> 391
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Ala Gly Phe Leu Asp Pro Arg Tyr Pro Gln Trp Arg Arg Ala Ala Gly
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Leu Ala Pro Ala Glu His Thr Gly Val Asp Tyr Asn Leu Val Gly Thr
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Ile Val Arg His Ala Arg Ala His Arg Ile Trp Gly Asn Ile Val Leu
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Leu Glu His Pro Gln Leu Gly Leu Trp Ser Gln Tyr Ala His Leu Tyr
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Gly Ser Ile Gly Arg Gly Asp Pro Arg Ala Pro Phe Leu Ala His Leu
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His Phe Glu Ile Arg Thr Arg Pro Leu Pro Ala Asp Asn Trp Pro Gly
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Val Leu Trp Leu Pro Asp Gly Lys His Ser Met Pro Gly Lys Thr Ile
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Val Asn Leu Asp Asp Pro Thr Leu Val His Val Arg Thr Asn Arg Ala
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Gln Arg Asn His Glu Gly Ile Pro His Pro Gly Tyr Asp Leu Asn Asn
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Ala Asp Trp Gly Gln Val Gly
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Ile Val Arg His Ala Arg Ala His Arg Ile Trp Gly Asn Ile Val Leu
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Gly Ser Ile Gly Arg Gly Asp Pro Arg Ala Pro Phe Leu Ala His Leu
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His Phe Glu Ile Arg Thr Arg Pro Leu Pro Ala Asp Asn Trp Pro Gly
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Met Asn Lys Thr Ala Ile Lys Glu Gly Tyr Leu Asp Pro Glu Thr Trp
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Leu Lys Gln His Met Ala Thr Glu Arg Arg Phe Thr Arg Gln Gly Leu
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<210> 5
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atgcgcatcg ttcatccctt cccccaacct gcccgagccc gcgtagacgc gggcttttta 60
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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
ctgaagcagc atatggcgac cgagcggcgg ttcacccggc aggggctcgt cctgtggttg 540
ccagatggaa aacacagtat gcctggcaag acaatcgtca atctagacga tccaacgtta 600
gtgcatgtgc gtacaaatcg cgcattgcaa cctcctcctc cgagtccacc tccacctagt 660
cctcctcctc cgagtatgcg tctaccgact aagacttccc gctttggtta tgtgcacggc 720
cagagaaacc acgagggcat tccccaccca ggctatgacc tgaataacgg ccctacgcct 780
actagcgacc ttggtcagcc tgtgtatgcc cctgaggatg gcgtggtggt ctatgcccgg 840
actgggtcag gtacctgggg tgggctggtg gtggtcttgg gcaaaagcgg ctttgcccat 900
cggctaggcc atgtgcgcaa cattcgggtc aaagagggac aggaggtgaa ggaaggccag 960
caggtggccg agattgggga gttcgtcaag gggcttcccc acctgcacta cgacatggtg 1020
gagcccaagg ttatccacac catcagtatc ctgatcaagg ccccttatgt tcggtgggac 1080
ttctggcacg taaactttcc caaactgttt gagcacatgt atgtggaccc ggccaggttt 1140
caccctgagc tggccgactg gggccaagtc ggt 1173
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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
ctgaagcagc atatggcgac cgagcggcgg ttcacccggc aggggctcgt cctgtggttg 540
ccagatggaa aacacagtat gcctggcaag acaatcgtca atctagacga tccaacgtta 600
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cctcctcctc cgagtccacc tccacctagt cctcctcctc cgagt 45
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<212> DNA
<213> 噬菌体TSP(Phage TSP)
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ggcattcccc acccaggcta tgacctgaat aacggcccta cgcctactag cgaccttggt 120
cagcctgtgt atgcccctga ggatggcgtg gtggtctatg cccggactgg gtcaggtacc 180
tggggtgggc tggtggtggt cttgggcaaa agcggctttg cccatcggct aggccatgtg 240
cgcaacattc gggtcaaaga gggacaggag gtgaaggaag gccagcaggt ggccgagatt 300
ggggagttcg tcaaggggct tccccacctg cactacgaca tggtggagcc caaggttatc 360
cacaccatca gtatcctgat caaggcccct tatgttcggt gggacttctg gcacgtaaac 420
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<210> 12
<211> 28
<212> DNA
<213> 人工序列(Artificial)
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ccgctcgagt ttacctccta gcaacttg 28
Claims (3)
1.一种嵌合裂解酶ILTphg，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所述嵌合裂解酶ILTphg的多核苷酸，其特征在于：其核苷酸序列如SEQ ID NO:5所示。
3.权利要求1所述的嵌合裂解酶ILTphg在抑制或杀灭革兰氏阳性菌和/或革兰氏阳性菌中的应用。
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Cited By (3)
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|---|---|---|---|---|
| CN111876400A (zh) * | 2020-08-06 | 2020-11-03 | 昆明理工大学 | 一种常温裂解酶Sly和编码此酶的多核苷酸 |
| CN112143747A (zh) * | 2020-09-09 | 2020-12-29 | 昆明理工大学 | 一种噬菌体裂解酶及其基因、基因重组表达载体与应用 |
| CN115820616A (zh) * | 2022-07-22 | 2023-03-21 | 昆明理工大学 | 一种带荧光标记的噬菌体裂解酶及其应用 |
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| CN111876400B (zh) * | 2020-08-06 | 2022-05-24 | 昆明理工大学 | 一种常温裂解酶Sly和编码此酶的多核苷酸 |
| CN112143747A (zh) * | 2020-09-09 | 2020-12-29 | 昆明理工大学 | 一种噬菌体裂解酶及其基因、基因重组表达载体与应用 |
| CN115820616A (zh) * | 2022-07-22 | 2023-03-21 | 昆明理工大学 | 一种带荧光标记的噬菌体裂解酶及其应用 |
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