CN104878023A - 一个新型细菌抗逆功能基因及其表达产物与应用 - Google Patents
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Abstract
一个新型细菌抗逆功能基因及其表达产物与应用，所述基因的核苷酸序列如SED ID NO：1所示。本发明还包括新型细菌抗逆功能基因的表达产物分支硫醇抗氧化物酶MPx与应用。本发明之新型细菌抗逆基因可克隆到表达载体pET28a上，然后转到BL21(DE3)菌株中表达，并用温和破碎法纯化表达产物，即可得到分支硫醇抗氧化物酶MPx，该表达产物可显著提高细菌的抗逆性。实验证明，在100mM过氧化氢、11mM异丙苯-OOH和120mMLA-OOH处理下，Δmpx突变体的存活率下降约20％～35％，野生型中Pmpx的启动子活性在35mM H2O2，5.5mM异丙苯-OOH和60mMLA-OOH处理下分别提高了38.7％，44.5％和32.3％。
Description
技术领域
本发明涉及一个新型细菌抗逆功能基因及其表达产物应用。
背景技术
长期暴露于不同类型或不同来源环境刺激物诱发的活性氧自由基(ROS)胁迫下，促使细胞乃至整个机体进化出一套强有力对抗氧化应激损伤的抗氧防御机制，来防止ROS及其代谢产物对机体的有害影响，这些抗氧防御机制除了直接对内源性ROS的产生进行调控外，还可通过细胞内的抗氧化物酶系统和小分子量抗氧化剂(Low Molecular Mass Antioxidant，LMMA)来清除过多的ROS。其中，小分子量抗氧化剂LMMA作为生命系统中一种重要的抗氧化剂，能保护细胞远离ROS直接或间接诱发产生的氧化危害，维持着细胞内氧化还原平衡和蛋白硫醇/二硫化物的正常比率。LMMA拥有很多抗氧化酶所没有的优势，例如LMMA是小分物质，很容易渗透通过细胞内的膜结构，从而与其作用的靶点更加接近。此外，细胞自身还能调节LMMA的浓度，同时对于多种ROS都具有很强的清除能力。而且LMMA的作用效果具有协同性，LMMA之间的内部关联对于抗氧胁迫极为重要。这种小分子量抗氧化剂的内在来源是生物合成和生物反应过程中产生的，除了乙酰辅酶A(CoA)和半胱氨酸(Cys)外，现在发现的生物体内LWMA主要有谷胱甘肽(glutathione，GSH)、卵类硫醇(Ovothiol，OSH)、麦角硫醇(Ergothioneine，ESH)、芽孢硫醇(Bacillithiol，BSH)、分枝硫醇(Mycothiol，MSH)等小分子量硫醇。其中MSH在发挥抗氧胁 迫功能的时候会对一些蛋白进行硫醇化修饰，蛋白S-硫醇化修饰是机体应对氧化应激的一种重要的硫醇保护和氧化还原开关机制，是机体在氧化应激应答下一种翻译后的可逆修饰。S-硫醇化修饰是硫醇与蛋白巯基之间形成的混合二硫键，可避免蛋白活性位点Cys过度氧化成不可逆的磺酸，一旦胁迫结束，混合二硫键又可被相关的酶还原而恢复到正常巯基状态。
目前，研究发现MSH合成缺陷的谷氨酸棒杆菌突变株对诱发ROS产生的抗生素、氧化剂、重金属及烷化剂等极为敏感，这说明MSH涉及细胞的保护作用，即S-分枝硫醇化修饰可能存在。谷氨酸棒杆菌除了作为系统生物学研究中的典型模式生物外，更是一种在发酵工业中得到广泛应用的重要的工业微生物，主要用于氨基酸、维生素、核苷酸等的生产。谷氨酸棒杆菌自身不具备合成GSH的代谢途径，而是以MSH为胞内主要硫醇，研究表明MSH的生理功能相似于GSH，也能维持胞内的氧化还原平衡，涉及活性氧和活性氮、抗生素、氧化剂、烷化剂、重金属、酸、有机溶剂、芳香化合物和异源物质的清除或解毒；并且MSH过量合成可明显提高谷氨酸棒杆菌在多种环境应激下的鲁棒性和存活率，显然对抗逆境的鲁棒性决定了谷氨酸棒杆菌生产性能的优劣，MSH的抗环境胁迫性能就成为塑造谷氨酸棒杆菌生物鲁棒性的重要手段。因此，如何提高该菌在逆境下的鲁棒性也成为众多研究的中心问题。谷棒中分支硫醇过氧化物酶MPx与含硫的谷胱甘肽过氧化物酶具有很高的序列相似性，通过使用不同的质子给予体分析它潜在的过氧化物酶活性来研究分支硫醇过氧化物酶的功能，分支硫醇 过氧化物酶Mpx通过Trx/TrxR系统以及Mrx1/Mtr/MSH再生系统降低过氧化氢和烷基过氧化氢的含量，然而，仅MSH不能有效的降低分支硫醇过氧化物酶，分支硫醇过氧化物酶体系的催化机制涉及Cys36与Cys79之间的内部二硫键的形成，这个二硫键是与硫氧还蛋白相互作用的枢纽，对于分枝硫醇氧化还原酶系统来说，催化循环涉及经单硫醇反应机理Cys36次磺酸硫醇化修饰与去硫醇化修饰。Mpx的表达显著增强细菌对各种过氧化物的耐受性，降低蛋白质的羰基化和细胞内活性氧类物质的积累，Mpx的表达直接受胁迫应答的ECF转录起始因子SigH的激活。由此可见，谷氨酸棒状杆菌中分支硫醇抗氧化物酶(Mpx)代表着一种既利用Mrx又利用Trx还原系统的新的谷胱甘肽过氧化物酶类型，谷氨酸棒状杆菌中分支硫醇抗氧化物酶(Mpx)的合成对于其抗氧胁迫具有重要意义。
发明内容
本发明所要解决的技术问题是，提供一个新型细菌抗逆功能基因及其表达产物与应用，该细菌抗逆功能基因的表达产物分支硫醇抗氧化物酶MPx，可显著增强细菌的抗逆性。
本发明解决其技术问题采用的技术方案是：
本发明之一个新型细菌抗逆功能基因，其核苷酸序列如SED ID NO：1所示。
本发明之一种分支硫醇抗氧化物酶MPx，由所述新型细菌抗逆功能基因编码的蛋白，氨基酸序列如SED ID NO：2所示。
本发明之一种分支硫醇抗氧化物酶MPx的制备方法，包括以下步 骤：
(1)获得目的基因：以含MPx基因的谷氨酸棒状杆菌的基因组DNA为模板，按基因序列设计一对引物，
Mpx-F：CGCGGATCCATGACTTCTATTCATGACATC(下划线为BamHI的酶切位点)，如SED ID NO：3所示；
Mpx-R：ACGCGTCGACTTAGATAGGGAGATTCTCCTC(下划线为SalI的酶切位点)，如SED ID NO：4所示，在上游和下游引物分别引入不同的酶切位点，经PCR循环获得所需基因片段，如SED ID NO：1所示；
(2)构建重组表达载体：提取表达质粒后，用限制性内切酶BamHI和SalI进行双酶切，酶切产物经琼脂糖电泳后，切胶回收；在T4 DNA连接酶作用下与载体pET28a连接；
(3)获得含重组表达质粒的表达菌种：将步骤(2)中所述连接产物转化大肠杆菌E.coli JM109，根据重组载体的抗Km特性，菌落pcr验证，碱裂解法抽提质粒，双酶切初步鉴定；测序验证目的基因的插入方向及阅读框架均正确，然后以重组质粒DNA转化表达宿主菌BL21(DE3)的感受态菌株中；
(4)诱导靶蛋白的表达及蛋白的纯化：将含有重组质粒的E.coli BL21(DE3)菌株接种到LB液体培养基中，37℃、160～220rpm摇床培养到OD600为0.4～0.6，接着加入终浓度为0.5～0.7mM IPTG，18～26℃，160～220rpm摇床诱导表达8～10小时；4℃离心收集菌体，用50mM，pH 6～8的PBS缓冲液洗2～3次后，重新悬浮PBS缓冲液中，冰水中超声破碎细胞；最后将菌液，4℃离心20～30min 取上清，上清过NTA-Ni亲和柱纯化蛋白，取纯化后蛋白经过处理后，采用凝胶浓度为12％的SDS-PAGE聚丙烯酰胺凝胶检测表达结果，即成。
本发明之一种新型细菌抗逆功能基因在增强细菌的抗逆性中的应用。
本发明之一种分支硫醇抗氧化物酶MPx在抗氧胁迫中的应用。
本发明之新型细菌抗逆基因可克隆到表达载体pET28a上，然后转到BL21(DE3)菌株中表达，温和破碎菌体并纯化表达产物，即可得到分支硫醇抗氧化物酶MPx，该表达产物可显著提高细菌的抗逆性。实验证明，在100mM过氧化氢、11mM异丙苯-OOH和120mMLA-OOH处理下，Δmpx突变体的存活率下降约20％～35％，野生型中Pmpx的启动子活性在35mMH2O2，5.5mM异丙苯-OOH和60mMLA-OOH处理下分别提高了38.7％，44.5％和32.3％。
附图说明
图1是本发明实施例提供的分支硫醇抗氧化物酶MPx表达载体的构建过程示意图。
图2是本发明实施例提供的SDS-PAGE的结果示意图。
图3是野生型在不同浓度过氧化物处理下的存活率。
图4是在一定浓度过氧化物处理下，野生型，突变体，互补型菌株和过表达菌株的存活率。
图5是一定浓度过氧化物处理下，野生型，突变体，互补型菌株和过表达菌株细胞内的ROS水平。
图6是在氧化胁迫下Δmpx和野生型细胞中蛋白羰基化水平。
图7是在不同浓度过氧化物诱导下，启动子活性变化。
具体实施方式
下面结合实施例对本发明进一步加以说明。
实施例1：分支硫醇抗氧化物酶MPx的制备
(1)获得目的基因：以含MPx基因的谷氨酸棒状杆菌的基因组DNA为模板，按基因序列设计一对引物，
Mpx-F：CGCGGATCCATGACTTCTATTCATGACATC(下划线为BamHI的酶切位点)；Mpx-R：ACGCGTCGACTTAGATAGGGAGATTCTCCTC(下划线为SalI的酶切位点)，在上游和下游引物分别引入不同的酶切位点，PCR反应体系详见表，PCR反应条件为：95℃预变性4min，94℃变性50S，55℃退火1min，72℃延伸1min/kb，30个循环后72℃延伸10min，经PCR循环获得所需基因片段，如SED ID NO：1所示。
表-PCR反应体系：
| Content | Volume |
| 2×Reaction Mix | 8μl |
| Primer 1(10μM) | 1μl |
| Primer 2(10μM) | 1μl |
| DNA | 2μl |
| Golden DNA Pol | 0.4μl |
| ddH2O | Up to 20μl |
(2)构建重组表达载体：提取表达质粒后，用限制性内切酶BamHI和SalI进行双酶切，酶切产物经琼脂糖电泳后，切胶回收；在T4 DNA连接酶作用下与载体pET28a连接；
(3)获得含重组表达质粒的表达菌种：将步骤(2)中所述连接产物转化大肠杆菌E.coli JM109，根据重组载体的抗Km特性，菌落PCR验证，碱裂解法抽提质粒，双酶切初步鉴定，测序验证目的基因的插入方向及阅读框架均正确，然后以重组质粒DNA转化表达宿主菌BL21(DE3)的感受态菌株中；
(4)诱导靶蛋白的表达及蛋白的纯化：将含有重组质粒的E.coli BL21(DE3)菌株接种到LB液体培养基中，37℃、220rpm摇床培养到OD600为0.4，加入终浓度为0.5mM IPT6，24℃，160rpm摇床诱导表达10小时；4℃，10,000g离心收集菌体，用50mM PBS缓冲液(pH 7.4)洗2次后，重新悬浮PBS缓冲液中，冰水中超声破碎细胞；12,000g，4℃离心30min取上清，上清过NTA-Ni亲和柱纯化蛋白。
(5)检测纯化的的分支硫醇抗氧化物酶MPx活性：由于NADPH的氧化，340nm处的吸光度会降低，所以通过监测340nm处吸光度的降低来测定过氧化物酶的活性。分支硫醇过氧化物酶的催化性能的测定使用还原硫氧还蛋白Trx-生成系统(4μM硫氧还蛋白还原酶(TRXR)和40μM硫氧还蛋白)，分枝硫醇氧化还原酶Mrx1系统(4μM的氧化型分枝硫醇还原酶Mtr，240μM分支硫醇MSH和40μM分枝硫醇氧化还原酶Mrx1)或者分支硫醇MSH系统(4μM氧化型分枝硫醇还原酶和500μM分支硫醇MSH.)作为电子给予体。反应总体积为500ul，含有50mM Tris-HCl缓冲液(pH为7.5)，1mM EDTA，250μM NADPH，0.5μM MPx(野生型(WT)或它的突变体)，和电子给予 体(Trx系统，Mrx1系统或者MSH系统)。预孵育五分钟，加入200uM或者500uM的过氧化氢底物反应开始。在其他底物是饱和浓度时改变底物的浓度来获得不同的底物催化参数。(0-150μM Trx或者Mrx1，0-3.0mM MSH，0-1000μM过氧化氢底物)。NADPH的氧化作为A340被监测，再减去在Mpx不存在的情况下自发的还原速率就得到酶活性。使用NADPH摩尔吸光系数(ε340＝6220M-1·cm-1)将每秒每微摩尔的酶所氧化的NADPH的微摩尔数量计算出来。每一个底物浓度下重复三次平行试验。使用GraphPad棱镜5程序用非线性回归计算Mpx的在Trx硫氧还蛋白，Mrx1，MSH，或过氧化氢底物分别作为电子给予体时的kcat和Km值。过氧化物酶活性还通过使用亚铁二甲酚橙检测(FOX)测定。催化的线性速率范围内，在不同时间点测定过氧化物的消耗。反应混合液含有的成分如上面所述。该反应通过加入过氧化物来引发，在不同的时间间隔间隔，通过加入16.7μl的HCl(1M)到83.3μl反应混合物中停止反应。然后，将所得混合物(100μl)与900μl FOX试剂混合(FOX试剂由88mg对羟基甲苯、7.6mg二甲酚橙、9.8mg(NH4)2Fe(SO4)2·6H20、90μl甲醇和10μl 250mM的硫酸)后，在37℃温育30分钟，560nm处测吸光度，重复3次。
实施例2：谷氨酸棒状杆菌中MPx作为抗氧化剂的作用
(1)Mpx在缓解氧胁迫方面生理学功能是通过测量不同氧化剂处理下Δmpx突变体的细胞存活率来估算的。使用的氧化剂的浓度是野生型的死亡率为30％～50％情况下的浓度，见图3。相比于野生型，在 100mM过氧化氢、11mM异丙苯-OOH和120mMLA-OOH处理下，Δmpx突变体的存活率下降约20％～35％，见图4。然而，在互补菌株中，敏感的表型是完全反转的。此外，MPX过表达明显增加野生型菌株对过氧化氢，异丙苯-OOH和LA-OOH挑战(Figure1A)的抗氧化的能力。
(2)在氧化应急条件下Mpx还原Ros的作用：在不同菌株中，Ros的水平被用一种被动扩散进入细胞的膜渗透性染料DCFH-DA检测。用H2O2，Cumene-OOH和LA-OOH处理30分钟后，Δmpx突变体显示ROS水平明显高于野生型(P≤0.05)见图5。此外，通过导入一个过表达的mpx质粒，Δmpx突变体中的Ros水平完全恢复和野生型相当，见图1。由图5可知，mpx可以通过降低细胞内各种氧化剂引起的ROS的积累进行抗氧化功能。
(3)测试mpx是否通过减少对蛋白的氧化性损伤来保护细胞：对在氧化胁迫下生长的Δmpx和野生型细胞中分离的总蛋白进行蛋白羰基化的测定，正如预期的，在过氧化氢，异丙苯-OOH和LA-OOH处理30分钟之后，见图6。由图6可知，野生型中蛋白质羰基化水平显著低于Δmpx突变体。
(4)证明mpx促进谷氨酸棒在氧化应激条件下的存活率：通过染色体Pmpx：lacZ融合报告基因分析在各种氧化剂的应答下mpx的表达情况，相比于未处理的对照，野生型中Pmpx的启动子活性在35mM H2O2，5.5mM异丙苯-OOH和60mMLA-OOH处理下分别提高了38.7％，44.5％和32.3％，见图7。由图7可知，Pmpx：lacZ融合基因的表达在响应于测试的氧化剂时表现出剂量依赖性增加。
综上，通过各种氧化剂对MPX的诱导表明，一个转录调控因子可能参与了其表达的调节。
Claims (5)
1.一个新型细菌抗逆功能基因，其特征在于，所述基因的核苷酸序列如SED ID NO：1所示。
2.一种分支硫醇抗氧化物酶MPx，其特征在于，由所述权利要求1所述的新型细菌抗逆功能基因编码的蛋白，氨基酸序列如SED ID NO：2所示。
3.一种如权利要求2所述的分支硫醇抗氧化物酶MPx的制备方法，其特征在于，包括以下步骤：
(1)获得目的基因：以含MPx基因的谷氨酸棒状杆菌的基因组DNA为模板，按基因序列设计一对引物，其中，Mpx-F序列如SED ID NO：3所示；Mpx-R序列如SED ID NO：4所示，在上游和下游引物分别引入BamHI和SalI的酶切位点，经PCR循环获得所需基因片段，如SED IDNO：1所示；
(2)构建重组表达载体：提取表达质粒后，用限制性内切酶BamHI和SalI进行双酶切，酶切产物经琼脂糖电泳后，切胶回收；在T4 DNA连接酶作用下与载体pET28a连接；
(3)获得含重组表达质粒的表达菌种：将步骤(2)中所述连接产物转化大肠杆菌E.coli JM109，根据重组载体的抗Km特性，菌落pcr验证，碱裂解法抽提质粒，双酶切初步鉴定；测序验证目的基因的插入方向及阅读框架均正确，然后以重组质粒DNA转化表达宿主菌BL21(DE3)的感受态菌株中；
(4)诱导靶蛋白的表达及蛋白的纯化：将含有重组质粒的E.coliBL21(DE3)菌株接种到LB液体培养基中，37℃、160～220rpm摇床培养到OD600为0.4～0.6，接着加入终浓度为0.5～0.7mM IPTG，18～26℃，160～220rpm摇床诱导表达8～10小时；4℃离心收集菌体，用50mM，pH 6～8的PBS缓冲液洗2～3次后，重新悬浮PBS缓冲液中，冰水中超声破碎细胞；最后将菌液，4℃离心20～30min取上清，上清过NTA-Ni亲和柱纯化蛋白，取纯化后蛋白经过处理后，采用凝胶浓度为12％的SDS-PAGE聚丙烯酰胺凝胶检测表达结果，即成。
4.根据权利要求1所述的新型细菌抗逆功能基因在增强细菌的抗逆性中的应用。
5.根据权利要求2所述的分支硫醇抗氧化物酶MPx在抗氧胁迫中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510194757.3A CN104878023A (zh) | 2015-04-22 | 2015-04-22 | 一个新型细菌抗逆功能基因及其表达产物与应用 |
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| CN110055302A (zh) * | 2019-03-27 | 2019-07-26 | 昆明理工大学 | 检测抗菌粉体材料诱发活性氧在细胞内富集水平的方法 |
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| WO2008142034A2 (en) * | 2007-05-22 | 2008-11-27 | Basf Plant Science Gmbh | Plants with increased tolerance and/or resistance to environmental stress and increased biomass production |
| CN101952305A (zh) * | 2007-12-19 | 2011-01-19 | 巴斯夫植物科学有限公司 | 具有增加的产量和/或增加的环境胁迫耐受性(iv-bm)的植物 |
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| WO2008142034A2 (en) * | 2007-05-22 | 2008-11-27 | Basf Plant Science Gmbh | Plants with increased tolerance and/or resistance to environmental stress and increased biomass production |
| WO2008142034A3 (en) * | 2007-05-22 | 2009-03-05 | Basf Plant Science Gmbh | Plants with increased tolerance and/or resistance to environmental stress and increased biomass production |
| CN101952305A (zh) * | 2007-12-19 | 2011-01-19 | 巴斯夫植物科学有限公司 | 具有增加的产量和/或增加的环境胁迫耐受性(iv-bm)的植物 |
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| CN110055302A (zh) * | 2019-03-27 | 2019-07-26 | 昆明理工大学 | 检测抗菌粉体材料诱发活性氧在细胞内富集水平的方法 |
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