CN116768989A - 一种维生素b12核糖开关突变体及高通量检测维生素b12的方法 - Google Patents
一种维生素b12核糖开关突变体及高通量检测维生素b12的方法 Download PDFInfo
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Abstract
本发明公开了一种维生素B12核糖开关突变体及高通量检测维生素B12的方法，属于生物技术领域。本发明基于一种广泛响应维生素B12类化学物的核糖开关突变体构建了高通量检测维生素B12的方法，将维生素B12的浓度转换为可视化、易检测的荧光强度。利用本发明，不仅可以对氰钴胺、羟钴胺、甲钴胺、腺苷钴胺的浓度进行快速地单一检测，还可以对不同类型的维生素B12进行快速地同时检测，具有非常高的应用潜力。
Description
技术领域
本发明涉及一种维生素B12核糖开关突变体及高通量检测维生素B12的方法，属于生物技术领域。
背景技术
核糖开关是一种位于mRNA特定区域的分子，其通过感知代谢物浓度的变化调控目标基因的表达。核糖开关由适体(Aptamer domain)域和表达平台(expression platform)两部分组成，适体域能折叠成多维结构，感知目标代谢物浓度的变化，并特异性的结合目标代谢物，表达平台位于适体域的下游，通常和适体域具有一定的重叠。当目标代谢物与适体域相结合后，表达平台的RNA空间结构发生改变，调控基因表达。核糖体开关具有响应时间短、代谢负担小、特异性高等特点，已经被用于细菌代谢工程。
维生素B12(Vitamin B12)，又名钴胺素，是一类对动物具有生物活性的类咕啉同功维生素的总称。其中，氰钴胺(C63H88CoN14O14P)是我们通过食物与营养补充的维生素B12的主要形式。羟钴胺(C62H89CoN13O15P)用于治疗维生素缺乏症，也用于治疗氰化物中毒。甲钴胺(C63H91CoN13O14P)为内源性维生素B12，其对神经元的传导作用良好，可通过甲基转换反应促进核酸-蛋白-脂肪代谢，其作为甲硫氨酸合成酶的辅酶，可使高半胱氨酸转化为甲硫氨酸，参与脱氧核苷合成胸腺嘧啶过程，可促进核酸、蛋白合成，促进轴索内输送和轴索再生及髓鞘的形成，修复被损害的神经组织。腺苷钴胺(C72H100CoN18O17P)是一种维生素B12辅酶，主要用于巨幼红细胞性贫血、营养不良性贫血、妊娠期贫血，亦用于多发性神经炎、神经根炎、营养性神经疾患等神经性疾患以及放射线和药物引起的白细胞减少症。
微生物合成维生素B12可产生不同类型的维生素B12，而维生素B12目前大多依赖HPLC等检测方法，其成本高、耗时长、环境不友好，难以广泛应用于维生素B12的高通量检测。尽管目前有可识别氰钴胺等维生素B12的核糖开关，但这些核糖开关的特异性强，仅能检测一种核糖开关。因此，快速检测胞内合成的多种类型维生素的含量成为亟待解决的问题。
发明内容
为解决上述问题，本发明提供了一种维生素B12核糖开关突变体及基于其的一种对多种类型维生素B12进行快速检测的方法，尤其是该核糖开关突变体与荧光蛋白序列结合，可根据氰钴胺、羟钴胺、甲钴胺和腺苷钴胺的含量不同产生不同强度的荧光响应，解决了现有维生素B12检测困难的问题，有利于实现维生素B12的高通量检测。
本发明的第一个目的是提供一种维生素B12核糖开关突变体，其核苷酸序列如SEQID NO.2所示。具体如下：
GTCAAATAGGTGCCGGTCCGTGAACAACAGCCGGCTTAAAAGGGACACCGGTAAAAGCCGGTGAGGTCCCGCCACTGTACTTGGCGCCAAGAGCCAGGATACCTGCCTGTTTGATCAGCACGAATTCTGCGAGGACAGATGATGTGT。
本发明的第二个目的是提供上述维生素B12核糖开关突变体在基因表达调控中的应用。
本发明的第三个目的是提供上述维生素B12核糖开关突变体在维生素B12合成菌株的高通量筛选中的应用。
进一步地，所述维生素B12核糖开关突变体下游连接识别标记编码基因。
进一步地，用于不同维生素B12的单一检测或混合检测。
进一步地，所述维生素B12包括氰钴胺、羟钴胺、甲钴胺、腺苷钴胺中的一种或几种。
本发明的第四个目的是提供一种检测维生素B12的方法，包括以下步骤：
S1、将编码识别标记的基因连接在核苷酸序列如SEQ ID NO.2所示的核糖开关下游，获得重组质粒；
S2、将含有所述重组质粒的重组菌与待检测体系共孵育或将所述重组质粒导入待检测菌株中，通过识别标记实现维生素B12的检测。
进一步地，所述识别标记包括但不限于荧光蛋白、抗性基因等，本发明的一个实施例中选用绿色荧光蛋白进行测试。
进一步地，所述核糖开关与编码识别标记的基因由诱导型启动子调控表达。
进一步地，检测体系中含有诱导剂。
进一步地，所述重组菌的宿主或待检测菌株包括但不限于大肠杆菌、枯草芽孢杆菌等。
进一步地，所述维生素B12包括氰钴胺、羟钴胺、甲钴胺、腺苷钴胺中的一种或几种。
本发明的有益效果：
本发明构建了响应维生素B12浓度的人工核糖开关突变体并将该核糖开关的下游连接荧光蛋白基因，将维生素B12的浓度转换为可视化、易检测的荧光强度。利用本发明，不仅可以对氰钴胺、羟钴胺、甲钴胺、腺苷钴胺的浓度进行快速地单一检测，还可以对不同类型的维生素B12进行快速地同时检测，具有非常高的应用潜力。
附图说明
图1为质粒pACYC-Ribo B12 M1-eGFP的图谱。
图2为维生素B12核糖开关突变体作用原理示意图。
图3为野生型维生素B12核糖开关与核糖开关突变体在分别添加50nM氰钴胺、羟钴胺、甲钴胺、腺苷钴胺下的eGFP发出的荧光强度。
图4为在大肠杆菌中，分别添加0-200nM氰钴胺、羟钴胺、甲钴胺、腺苷钴胺下的eGFP发出的荧光强度。
图5为在大肠杆菌中，混合添加50nM氰钴胺、羟钴胺、甲钴胺、腺苷钴胺下的eGFP发出的荧光强度。
图6为在枯草芽孢杆菌中，混合添加50nM氰钴胺、羟钴胺、甲钴胺、腺苷钴胺下的eGFP发出的荧光强度。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
下述实施例中涉及的序列如下：
编码枯草芽孢杆菌维生素B12核糖开关的核苷酸序列如SEQ ID NO.1所示。
编码维生素B12核糖开关突变体M1的核苷酸序列如SEQ ID NO.2所示。
编码eGFP的核苷酸序列如SEQ ID NO.3所示。
引物序列如SEQ ID NO.4-9所示。
实施例1：表征维生素B12浓度的人工核糖开关突变体载体构建与筛选
维生素B12核糖开关筛选自枯草芽孢杆菌，其为转录“ON”型核糖开关。当维生素B12存在时，其与核糖开关结合后，核糖开关下游基因可正常转录与翻译；当维生素B12不存在时，核糖开关自身构象改变，形成转录终止子，RNA聚合酶无法通过，使得下游基因无法转录从而抑制该基因的表达。为增强核糖开关对维生素B12的亲和力并获取可识别多种类型维生素B12，以枯草芽孢杆菌的维生素B12核糖开关为模板，并通过索莱宝生物科技有限公司的随机突变试剂盒对该核糖开关进行基于易错PCR的随机突变，以筛选出亲和力更强、特异性较广的人工核糖开关突变体。
模板核糖开关序列如SEQ ID NO.1所示。易错PCR的扩增体系为：2x Mut RandomSystem 25μL、Mut Enhancer 5μL、1μL上下游引物以及1μL模板。易错PCR的扩增条件为：95℃2min，94℃30S，56℃1min，72℃15S，72℃7min；20个循环。突变体的扩增引物如SEQ IDNO.4和SEQ ID NO.5所示。eGFP片段及载体片段通过普通PCR扩增制备。PCR扩增体系为：12.5μLprimerstar max、9.5μL ddH2O、1μL上下游引物以及1μL模板。扩增采用3步循环：98℃10S,56℃15S,72℃5S；30个循环。具体地以SEQ ID NO.6和SEQ ID NO.7为引物，以合成的eGFP基因为模板，在高保真复制酶的作用下扩增CH-eGFP序列；以SEQ ID NO.8和SEQ IDNO.9为引物，以pACYCduet-1质粒为模板扩增CH-pACYC序列。扩增结束后，PCR产物在1％琼脂糖胶中进行DNA凝胶电泳，待电泳结束后，用紫外凝胶成像仪观察电泳结果，用手术刀小心切取大小正确的条带胶块，通过诺唯赞胶回收试剂盒对扩增产物进行回收。依次扩增并回收核糖开关突变体片段、CH-eGFP和CH-pACYC的DNA片段，这三个片段通过一步克隆构建重组产物。重组产物通过化学转化进入大肠杆菌DH5α感受态中，转化菌体在LB(加有34mg/mL的氯霉素)固体平板上过夜生长。收集平板上生长的单菌落并置于LB(加有34mg/mL的氯霉素)液体培养基中，在37℃、200rpm下培养6h。之后，通过诺唯赞的质粒提取试剂盒提取突变文库质粒，并通过化学转化将突变文库质粒转入大肠杆菌BL21(DE3)中。
将转化子接种于10mL LB(加有34mg/mL的氯霉素)液体培养基中，37℃下培养至0D600＝0.6。然后，加入0.5mM的IPTG诱导维生素B12核糖开关及eGFP基因表达。同时，分别加入底物50nM的氰钴胺、羟钴胺、甲钴胺和腺苷钴胺。在37℃下以160r.min-1培养摇培3h后，收集菌体并重悬于5mL 1×PBS缓冲液中。通过流式细胞仪对菌体的荧光值进行分选(激发波长为488nm、发射波长507nm)。最后，分选出的维生素B12突变体M1(序列如SEQ ID NO.2所示)在50nM的氰钴胺、羟钴胺、甲钴胺和腺苷钴胺诱导条件下，所激发的荧光强度均显著高于枯草芽孢杆菌的维生素B12核糖开关(WT)，具体见图3，其中，组1-4分别为氰钴胺、羟钴胺、甲钴胺和腺苷钴胺。
实施例2：单一类型维生素B12的检测
将含有pACYC-Ribo B12 M1-eGFP的大肠杆菌BL21(DE3)菌株在5mL LB液体培养基(加有34mg/mL的氯霉素)中，37℃过夜活化。取1％的过夜活化的菌株接种于10mL LB(加有34mg/mL的氯霉素)液体培养基中，37℃下培养至0D600＝0.6。然后，加入0.5mM的IPTG诱导维生素B12核糖开关及eGFP基因表达。同时，分别加入底物0nM、50nM、100nM、150nM、200nM的氰钴胺、羟钴胺、甲钴胺和腺苷钴胺。在37℃下以160r.min-1培养摇培3h后，收集菌体并重悬于5mL 1×PBS缓冲液中。吸取200μL缓冲液置于黑色酶标板中，在酶标仪下(激发波长为488nm、发射波长507nm)检测样品的荧光强度。
0nM的底物添加样品作为空白对照，并作为维生素B12核糖开关在OFF状态下所激活的GFP蛋白荧光强度，而维生素B12核糖开关在不同样品诱导下的ON/OFF以GFP荧光强度倍数(不同浓度的维生素B12诱导下的荧光强度除以空白对照的荧光值)来表征。
经过检测(图4)，维生素B12在0-150nM的氰钴胺的诱导下有着较好的响应，150nM的氰钴胺的诱导下ON/OFF为120；维生素B12在0-150nM的羟钴胺的诱导下有着较好的响应，150nM的氰钴胺的诱导下ON/OFF为310；维生素B12在0-150nM的甲钴胺的诱导下有着较好的响应，150nM的甲钴胺的诱导下ON/OFF为150；维生素B12在0-200nM的腺苷钴胺的诱导下有着较好的响应，200nM的腺苷钴胺的诱导下ON/OFF为490。
实施例3：不同类型维生素B12的混合检测
将含有质粒已成功转化pACYC-Ribo B12 M1-eGFP的大肠杆菌BL21(DE3)菌株在5mL LB(加有34mg/mL的氯霉素)液体培养基中，37℃过夜活化。取1％的过夜活化的菌株接种于10mL LB(加有34mg/mL的氯霉素)液体培养基中，37℃下培养至0D600＝0.6。然后，加入0.5mM的IPTG诱导维生素B12核糖开关及eGFP基因表达，之后进行维生素B12的混合检测。空白组仍为0nM的底物添加样品；对照组为分别添加50nM的氰钴胺、羟钴胺、甲钴胺、腺苷钴胺的单一检测样品；实验组为组合添加50nM的氰钴胺、羟钴胺、甲钴胺、腺苷钴胺的混合检测样品。空白组、对照组、实验组样品添加完毕后，菌株在37℃下以160r.min-1培养摇培3h后，收集菌体并重悬于5mL 1×PBS缓冲液中。吸取200μL缓冲液放入黑色酶标板中，在酶标仪(激发波长为488nm、发射波长507nm)下检测样品的荧光强度。经过检测(图5，其中，组1为对照组-氰钴胺；组2为对照组-羟钴胺；组3为对照组-甲钴胺；组4为对照组-腺苷钴胺；组5为实验组)，实验组的ON/OFF显著高于对照组，表明该维生素B12核糖开关可用于不同类型维生素B12的混合检测。
实施例4：维生素核糖开关在枯草芽孢杆菌中的应用
为验证维生素B12核糖开关对维生素B12的含量的通用性快速检测，将pACYC-RiboB12 M1-eGFP通过化学转化导入枯草芽孢杆菌感受态细胞中。转化产物涂布于LB(加有34mg/mL的氯霉素)固体平板上过夜生长。挑取单菌落并将其接种于LB(加有34mg/mL的氯霉素)液体培养基中以160r.min-1摇培12h。经过平板筛选与菌液PCR验证后，成功导入质粒的转化子进行后续实验。
将已成功转化的枯草芽孢杆菌在5mL LB(加有34mg/mL的氯霉素)液体培养基中，37℃过夜活化。取1％的过夜活化的菌株接种于10mL LB(加有34mg/mL的氯霉素)液体培养基中，37℃下培养至0D600＝0.6。然后，加入0.5mM的IPTG诱导维生素B12核糖开关及eGFP基因表达，之后进行维生素B12的混合检测。空白组为0nM的底物添加；对照组分别为单一样品且浓度为50nM的氰钴胺、羟钴胺、甲钴胺和腺苷钴胺；实验组为浓度为50nM的氰钴胺、羟钴胺、甲钴胺和腺苷钴胺的混合样品。在样品添加完毕后，菌株在37℃下以160r.min-1摇培8h后，收集菌体并重悬于5mL 1×PBS缓冲液中。吸取200μL缓冲液放入黑色酶标板中，在酶标仪下(激发波长为488nm、发射波长507nm)检测样品的荧光强度。如图6所示(组1为对照组-氰钴胺；组2为对照组-羟钴胺；组3为对照组-甲钴胺；组4为对照组-腺苷钴胺；组5为实验组)，实验组维生素B12核糖开关的ON/OFF显著高于对照组，表明维生素B12核糖开关在枯草芽孢杆菌中可对不同类型的维生素B12进行混合检测。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种维生素B12核糖开关突变体，其特征在于：所述维生素B12核糖开关突变体的核苷酸序列如SEQ ID NO.2所示。
2.权利要求1所述维生素B12核糖开关突变体在基因表达调控中的应用。
3.权利要求1所述维生素B12核糖开关突变体在维生素B12合成菌株的高通量筛选中的应用。
4.根据权利要求3所述的应用，其特征在于：所述维生素B12核糖开关突变体下游连接识别标记编码基因。
5.根据权利要求3所述的应用，其特征在于：用于不同维生素B12的单一检测或混合检测。
6.一种检测维生素B12的方法，其特征在于，包括以下步骤：
S1、将编码识别标记的基因连接在核苷酸序列如SEQ ID NO.2所示的核糖开关下游，获得重组质粒；
S2、将含有所述重组质粒的重组菌与待检测体系共孵育或将所述重组质粒导入待检测菌株中，通过识别标记实现维生素B12的检测。
7.根据权利要求6所述的方法，其特征在于：所述识别标记包括荧光蛋白、抗性基因中的至少一种。
8.根据权利要求6所述的方法，其特征在于：所述核糖开关与编码识别标记的基因由诱导型启动子调控表达。
9.根据权利要求6所述的方法，其特征在于：所述重组菌的宿主或待检测菌株包括大肠杆菌或枯草芽孢杆菌。
10.根据权利要求6所述的方法，其特征在于：所述维生素B12包括氰钴胺、羟钴胺、甲钴胺、腺苷钴胺中的一种或几种。
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| CN104212826A (zh) * | 2014-08-11 | 2014-12-17 | 浙江工商大学 | 一种维生素b12核糖体开关载体的构建方法 |
| CN112694991A (zh) * | 2020-12-29 | 2021-04-23 | 河北华北制药华恒药业有限公司 | 一种产维生素b12的菌株及其应用 |
| CN113138236A (zh) * | 2021-03-31 | 2021-07-20 | 山东英盛生物技术有限公司 | 一种定量检测血清中维生素b12的方法及预处理试剂盒 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104212826A (zh) * | 2014-08-11 | 2014-12-17 | 浙江工商大学 | 一种维生素b12核糖体开关载体的构建方法 |
| CN112694991A (zh) * | 2020-12-29 | 2021-04-23 | 河北华北制药华恒药业有限公司 | 一种产维生素b12的菌株及其应用 |
| CN113138236A (zh) * | 2021-03-31 | 2021-07-20 | 山东英盛生物技术有限公司 | 一种定量检测血清中维生素b12的方法及预处理试剂盒 |
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