CN104403003A - Gene encoding nicotinamide adenine dinucleotide fluorescent probe and preparation method and application thereof - Google Patents
Gene encoding nicotinamide adenine dinucleotide fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN104403003A CN104403003A CN201410499305.1A CN201410499305A CN104403003A CN 104403003 A CN104403003 A CN 104403003A CN 201410499305 A CN201410499305 A CN 201410499305A CN 104403003 A CN104403003 A CN 104403003A
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Abstract
本发明涉及烟酰胺腺嘌呤二核苷酸基因编码荧光探针及其制备方法和应用。一方面,本发明涉及烟酰胺腺嘌呤二核苷酸的检测探针,具体涉及烟酰胺腺嘌呤二核苷酸的重组荧光融合蛋白检测探针。在一个具体方面,本发明涉及还原型烟酰胺腺嘌呤二核苷酸(NADH)的重组荧光融合蛋白检测探针;在另一个具体方面,本发明涉及氧化型烟酰胺腺嘌呤二核苷酸(NAD+)的重组荧光融合蛋白检测探针;在又一方面,本发明涉及还原型和氧化型烟酰胺腺嘌呤二核苷酸比率的重组荧光融合蛋白检测探针。本发明也涉及上述检测探针的制备方法及其分别在检测NADH、NAD+和NADH/NAD+比率中的应用。
The invention relates to a fluorescent probe encoded by nicotinamide adenine dinucleotide gene and its preparation method and application. On the one hand, the present invention relates to a detection probe for nicotinamide adenine dinucleotide, in particular to a detection probe for a recombinant fluorescent fusion protein of nicotinamide adenine dinucleotide. In one specific aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe of reduced nicotinamide adenine dinucleotide (NADH); in another specific aspect, the present invention relates to oxidized nicotinamide adenine dinucleotide (NADH) NAD+) recombinant fluorescent fusion protein detection probe; In yet another aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe for the ratio of reduced and oxidized nicotinamide adenine dinucleotide. The present invention also relates to the preparation method of the above detection probe and its application in detecting NADH, NAD+ and NADH/NAD+ ratio respectively.
Description
本申请是申请号201110288807.6,申请日为2011年9月26日,名为“烟酰胺腺嘌呤二核苷酸基因编码荧光探针及其制备方法和应用”的发明专利申请的分案申请。母案的全部内容在此通过引用全文纳入本文用于所有目的。This application is the application number 201110288807.6, the application date is September 26, 2011, and it is a divisional application of the invention patent application named "nicotinamide adenine dinucleotide gene-encoded fluorescent probe and its preparation method and application". The entire content of the parent case is hereby incorporated by reference in its entirety for all purposes.
技术领域technical field
本发明涉及烟酰胺腺嘌呤二核苷酸的检测探针,具体涉及烟酰胺腺嘌呤二核苷酸的重组荧光融合蛋白检测探针。在一个具体方面,本发明涉及还原型烟酰胺腺嘌呤二核苷酸(NADH)的重组荧光融合蛋白检测探针;在另一个具体方面,本发明涉及氧化型烟酰胺腺嘌呤二核苷酸(NAD+)的重组荧光融合蛋白检测探针;在又一方面,本发明涉及还原型和氧化型烟酰胺腺嘌呤二核苷酸比率的重组荧光融合蛋白检测探针。本发明也涉及上述检测探针的制备方法及其分别在检测NADH、NAD+和NADH/NAD+比率中的应用。The invention relates to a detection probe for nicotinamide adenine dinucleotide, in particular to a detection probe for a recombinant fluorescent fusion protein of nicotinamide adenine dinucleotide. In one specific aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe of reduced nicotinamide adenine dinucleotide (NADH); in another specific aspect, the present invention relates to oxidized nicotinamide adenine dinucleotide (NADH) NAD+) recombinant fluorescent fusion protein detection probe; In yet another aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe for the ratio of reduced and oxidized nicotinamide adenine dinucleotide. The present invention also relates to the preparation method of the above detection probe and its application in detecting NADH, NAD+ and NADH/NAD+ ratio respectively.
背景技术Background technique
NAD+及NADH作为辅酶,是呼吸链的重要组成部分,它参与了呼吸链中的电子传递过程(Rich,P.R.等,Biochem Soc Trans.2003,V.31(6),pp.1095-1105)。在呼吸链的氧化还原反应中,NAD+作为质子的载体,当其接受了其他分子传递过来的一个电子后,由最初的氧化态转变为还原态,该反应的最终产物为NADH,而NADH可以作为还原剂向其他分子提供电子(Belenky,P.等,Trends inBiochemical Sciences.2007,V.32(1),pp.12-19)。近来的研究表明,NAD(H)不仅参与能量代谢、物质合成以及抗氧化作用,还涉及到体内的钙稳态、基因表达、免疫作用以及细胞衰老与死亡等等,而NAD(H)在其中均有着至关重要的作用,因此NAD(H)本身及其代谢所涉及的众多酶类也成为了药物设计的靶标(Sauve,A.A.等,J Pharmacol Exp Ther.2008,V.324(3),pp.883-893)。As coenzymes, NAD + and NADH are an important part of the respiratory chain, and they participate in the electron transfer process in the respiratory chain (Rich, PR, etc., Biochem Soc Trans.2003, V.31(6), pp.1095-1105) . In the redox reaction of the respiratory chain, NAD + is the carrier of the proton. When it accepts an electron from other molecules, it changes from the initial oxidation state to the reduction state. The final product of this reaction is NADH, and NADH can Donates electrons to other molecules as a reducing agent (Belenky, P. et al., Trends in Biochemical Sciences. 2007, V.32(1), pp.12-19). Recent studies have shown that NAD(H) is not only involved in energy metabolism, material synthesis and anti-oxidation, but also involved in calcium homeostasis, gene expression, immune function, cell aging and death, etc., and NAD(H) is involved in Both play a vital role, so NAD(H) itself and the many enzymes involved in its metabolism have also become targets for drug design (Sauve, AA, etc., J Pharmacol Exp Ther.2008, V.324(3), pp. 883-893).
但是,大多数活细胞内的NAD(H)的总量大约为10-6M~10-3M,而且NAD+/NADH的比例也随着细胞内状态不同而不尽相同(Lin,S.J.等,CurrentOpinion in Cell Biology.2003,V.15(2),pp.241-246),因此这给NAD(H)的测定带来了极大的不便。较早的检测方法主要是利用NADH在340nm紫外光区有特征吸收,由此建立了紫外分光光度法,该方法存在两个主要的缺陷:1、有效灵敏度受仪器精密度所限,约为10-7M;2、在复杂体系中,不能有效地区分NADH与NADPH。随后,根据NAD+作为辅酶,在电子传递过程中接受电子转变为NADH的特性,发展出了一系列酶学检测方法。其他如HPLC分析、电化学法、毛细管电泳、荧光成像等方法也常见诸于各种文献报道中。然而,大部分的方法或者对单个细胞中靶标分子的灵敏度不足,或者不能进行亚细胞器定位。特别需要指出的是,这些现有方法均存在一个主要的缺陷,即需要对样品进行裂解、分离、纯化等操作,而NADH本身又极易氧化,在一系列繁琐的操作中极易将误差引入,导致最终显示的结果与实际存在出入。另外,这些现有方法不能应用于活体动物或细胞,不能进行实时地检测,这限制了这些方法在临床疾病诊断及药物前体研究等邻域的应用。目前在活体或细胞上只能采用NADH自发荧光来检测(Zhang,Q.H.等,Science.2002,V.295(5561),pp.1895-1897),而这种传统方法存在以下严重缺陷:首先,已知细胞对NAD+/NADH和NADP+/NADPH的调控是相对独立的,正常状态下NAD+/NADH的比例大约在700:1,而NADP+/NADPH的比例在1:200;其次,它们的氧化还原势存在着巨大的区别,这反映出NADH与NADPH分别在能量代谢与合成代谢中扮演截然不同的角色;第三,NADH与NADPH自发荧光完全不可区分,利用自发荧光进行成像测量获得的结果是NADH与NADPH之和,鉴于NADH含量很低且大部分以蛋白质结合形式存在,所以NADH自发荧光数据实质反映的是蛋白质结合的NADPH的浓度(Zhang,Q.H.等,Science.2002,V.295(5561),pp.1895-1897);第四,由于NADH激发波长位于紫外区(340nm)且自发荧光较弱,需要复杂昂贵的仪器如用于临床监测的仪器CritiView,再加上紫外光对组织的穿透力很弱并会造成细胞损伤,这些光学特性严重制约了自发荧光监测的应用。However, the total amount of NAD(H) in most living cells is about 10 -6 M ~ 10 -3 M, and the ratio of NAD + /NADH varies with different intracellular states (Lin, SJ et al. , Current Opinion in Cell Biology.2003, V.15(2), pp.241-246), so this brings great inconvenience to the determination of NAD(H). The earlier detection method is mainly based on the characteristic absorption of NADH in the 340nm ultraviolet region, thus establishing the ultraviolet spectrophotometric method. There are two main defects in this method: 1. The effective sensitivity is limited by the precision of the instrument, which is about 10 -7 M; 2. In a complex system, NADH and NADPH cannot be effectively distinguished. Subsequently, according to the characteristics of NAD + as a coenzyme, which accepts electrons and transforms into NADH in the process of electron transfer, a series of enzymatic detection methods were developed. Other methods such as HPLC analysis, electrochemical method, capillary electrophoresis, and fluorescence imaging are also commonly found in various literature reports. However, most methods either have insufficient sensitivity for target molecules in single cells or cannot perform subcellular organelle localization. In particular, it should be pointed out that there is a major defect in these existing methods, that is, the sample needs to be cracked, separated, purified, etc., and NADH itself is extremely easy to oxidize, and errors are easily introduced in a series of tedious operations. , resulting in discrepancies between the final displayed results and the actual existence. In addition, these existing methods cannot be applied to living animals or cells, and cannot be detected in real time, which limits the application of these methods in the fields of clinical disease diagnosis and drug precursor research. At present, only NADH autofluorescence can be used to detect in vivo or cells (Zhang, QH et al., Science.2002, V.295(5561), pp.1895-1897), and this traditional method has the following serious defects: It is known that cells regulate NAD + /NADH and NADP + /NADPH relatively independently. Under normal conditions, the ratio of NAD + /NADH is about 700:1, while the ratio of NADP+/NADPH is 1:200; secondly, their There is a huge difference in redox potential, which reflects that NADH and NADPH play distinct roles in energy metabolism and anabolism, respectively; third, NADH and NADPH autofluorescence are completely indistinguishable, and the results obtained by imaging measurements using autofluorescence is the sum of NADH and NADPH. Since the content of NADH is very low and most of it exists in protein-bound form, the NADH autofluorescence data essentially reflects the concentration of protein-bound NADPH (Zhang, QH, etc., Science.2002, V.295( 5561), pp.1895-1897); Fourth, because the NADH excitation wavelength is in the ultraviolet region (340nm) and the autofluorescence is weak, complex and expensive instruments such as the CritiView for clinical monitoring are required, and the ultraviolet light is used for tissue These optical properties severely limit the application of autofluorescence monitoring.
因此,本领域亟需发展一种特异性NADH检测技术,特别是一种适合生理水平和亚细胞水平的特异性NADH检测技术。Therefore, there is an urgent need in this field to develop a specific NADH detection technology, especially a specific NADH detection technology suitable for physiological and subcellular levels.
而相对于传统的小分子染料检测技术以及迅速发展的量子点检测技术,荧光蛋白检测技术在大多数的活体细胞成像方面具有独一无二的压倒性优势,它能够通过遗传导入至细胞、组织乃至整个器官中,因此荧光蛋白能够作为一个全细胞标记物或基因启动激活的指示器。Compared with the traditional small molecule dye detection technology and the rapidly developing quantum dot detection technology, the fluorescent protein detection technology has a unique overwhelming advantage in most live cell imaging. It can be genetically introduced into cells, tissues and even whole organs. Therefore, fluorescent proteins can be used as whole-cell markers or indicators of gene activation.
绿色荧光蛋白最初是从维多利亚多管发光水母(Aequorea victoria)中提取,野生型的AvGFP由238个氨基酸构成,分子量约为26kD。当前的研究确认,天然GFP蛋白中第65~67位的三个氨基酸Ser-Tyr-Gly能够自发形成一个荧光生色基团:对-羟基苯亚甲基咪唑啉酮(p-hydroxybenzylideneimidazolinone),是其主要的发光位置。野生型AvGFP的光谱特征十分复杂,其荧光激发的主峰在395nm处,而在475nm处另有一个附峰,后者振幅强度约为前者的1/3。在标准的溶液条件下,395nm处的激发可产生508nm处的发射,而475nm处的激发产生的最大发射波长位于503nm(Heim,R.等,Proc Natl Acad Sci U S A.1994,V.91(26),pp.12501-12504)。Green fluorescent protein was originally extracted from Aequorea victoria. The wild-type AvGFP consists of 238 amino acids and has a molecular weight of about 26kD. Current research has confirmed that the three amino acids Ser-Tyr-Gly at positions 65-67 in the natural GFP protein can spontaneously form a fluorescent chromophore: p-hydroxybenzylideneimidazolinone, which is Its main luminous position. The spectral characteristics of wild-type AvGFP are very complex, the main peak of its fluorescence excitation is at 395nm, and there is another auxiliary peak at 475nm, the amplitude of the latter is about 1/3 of the former. Under standard solution conditions, excitation at 395nm produces emission at 508nm, while excitation at 475nm produces a maximum emission wavelength at 503nm (Heim, R. et al., Proc Natl Acad Sci U S A.1994, V.91 (26), pp. 12501-12504).
随着对GFP蛋白突变的研究日渐深入,利用分子生物学技术,目前已经发展出多种表现突出的GFP衍生物,通过在野生型GFP基础上进行不同的单点突变或者组合,可获得诸如增强型GFP(S65T,F64L)、YFP(T203Y)、CFP(Y66W)等。而借助对GFP蛋白序列的重新排列,将原第145-238位氨基酸部分作为新蛋白的N端,原第1-144位氨基酸作为新蛋白的C端,两片段间通过一小段具有柔性的短肽链连接,形成一个对空间变化敏感的环状排列荧光蛋白(circularpermutation fluorescent protein),在此基础上对原蛋白T203Y进行的点突变就形成了环状排列的黄色荧光蛋白cpYFP(Nagai,T.等,Proc Natl Acad Sci U S A.2001,V.98(6),pp.3197-3202)。With the deepening of research on GFP protein mutations, using molecular biology techniques, a variety of outstanding GFP derivatives have been developed. By performing different single-point mutations or combinations on the basis of wild-type GFP, such as enhanced Type GFP (S65T, F64L), YFP (T203Y), CFP (Y66W), etc. With the help of the rearrangement of the GFP protein sequence, the original 145-238 amino acid part is used as the N-terminal of the new protein, and the original 1-144 amino acid is used as the C-terminal of the new protein. Peptide chains are connected to form a circular permutation fluorescent protein (circular permutation fluorescent protein) that is sensitive to spatial changes. On this basis, the point mutation of the original protein T203Y forms a circular permutation of yellow fluorescent protein cpYFP (Nagai, T. etc., Proc Natl Acad Sci U S A.2001, V.98(6), pp.3197-3202).
由于对荧光蛋白研究日益深入,相关的一些基于荧光的分析检测技术也获得了进一步的发展。例如当前常用的荧光共振能量转移(FRET)技术,该技术主要原理是当两个荧光发色基团在足够靠近时,当供体分子吸收一定频率的光子后被激发到更高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移(即发生能量共振转移)。FRET是一种非辐射能量跃迁,通过分子间的电偶极相互作用,将供体激发态能量转移到受体激发态的过程,使供体荧光强度降低,而受体可以发射更强于本身的特征荧光(敏化荧光),也可以不发荧光(荧光猝灭)。当前对绿色荧光蛋白的进一步研究发现,衍生自绿色荧光蛋白突变体的青色荧光蛋白(CFP)和黄色荧光蛋白(YFP)是一对表现出色的供体/受体对。CFP的发射光谱与YFP的吸收光谱有相当的重叠,当它们足够接近时,用CFP的吸收波长激发,CFP的发色基团将会把能量高效率地共振转移至YFP的发色基团上,所以CFP的发射荧光将减弱或消失,主要发射将是YFP的荧光。两个发色基团之间的能量转换效率与它们之间的空间距离的6次方成反比,对空间位置的改变非常灵敏。因此现有研究报道利用基因工程重组手段将期望研究的蛋白基因两端分别与CFP与YFP融合表达出一个全新的融合蛋白,该蛋白与其专一性的靶标分子的结合所产生的空间变化即通过荧光的变化所直观地显现。Due to the increasingly in-depth research on fluorescent proteins, some related fluorescence-based analysis and detection technologies have also been further developed. For example, the currently commonly used fluorescence resonance energy transfer (FRET) technology, the main principle of this technology is that when two fluorescent chromophores are close enough, when the donor molecule absorbs photons of a certain frequency, it is excited to a higher electronic energy state. , before the electron returns to the ground state, the energy is transferred to the adjacent acceptor molecule through the dipole interaction (that is, energy resonance transfer occurs). FRET is a non-radiative energy transition. Through the electric dipole interaction between molecules, the energy of the excited state of the donor is transferred to the excited state of the acceptor, so that the fluorescence intensity of the donor is reduced, and the acceptor can emit stronger than itself The characteristic fluorescence (sensitized fluorescence) can also not fluoresce (fluorescence quenching). Further research on the current GFP found that cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), derived from GFP mutants, are a well-performing donor/acceptor pair. The emission spectrum of CFP overlaps considerably with the absorption spectrum of YFP. When they are close enough, the chromophore of CFP will resonantly transfer energy to the chromophore of YFP when excited by the absorption wavelength of CFP. , so the emission fluorescence of CFP will weaken or disappear, and the main emission will be the fluorescence of YFP. The energy conversion efficiency between two chromophores is inversely proportional to the sixth power of the spatial distance between them, and is very sensitive to changes in spatial position. Therefore, existing research reports use genetic engineering recombination methods to fuse the two ends of the desired protein gene with CFP and YFP to express a brand new fusion protein. Fluorescence changes are visualized visually.
因此,本文所用的荧光蛋白序列可以来自于维多利亚多管发光水母(Aequoreavictoria)的荧光蛋白及其衍生物,包括但不局限于这些突变体:黄色荧光蛋白(YFP)、绿色荧光蛋白(GFP)、青色荧光蛋白(CFP)等的序列,其中优选黄色荧光蛋白YFP的序列,更优选环状排列的黄色荧光蛋白cpYFP的序列。Therefore, the fluorescent protein sequence used herein can be derived from the fluorescent protein of Aequoreavictoria and its derivatives, including but not limited to these mutants: yellow fluorescent protein (YFP), green fluorescent protein (GFP), The sequence of cyan fluorescent protein (CFP), etc., wherein the sequence of yellow fluorescent protein YFP is preferred, and the sequence of yellow fluorescent protein cpYFP arranged in a circle is more preferred.
本技术中所涉及的另一蛋白,YdiH蛋白(又称为Rex蛋白)是一种本领域已知的细菌转录抑制蛋白,分子量为23kDa,它能调控发酵和厌氧呼吸。常见的YdiH蛋白来自嗜热水生菌(Thermus aquaticus)(SEQ ID NO:1NCBI GenBank:AF061257.1)、天蓝色链霉菌(Streptomyces coelicolor)(SEQ ID NO:2NCBIGenBank:AL939116.1)或枯草芽孢杆菌(Bacillus subtilis)(SEQ ID NO:3NCBIGenBank:AL009126.3),YdiH蛋白首次获得鉴定是Brekasis和Paget等人于2003年在天蓝色链霉菌(Streptomyces coelicolor)中发现的,这是一种广泛存在于革兰氏阳性菌中的一种对氧化还原敏感的调控蛋白。对于天蓝色链霉菌(S.coelicolor)YdiH(Rex)蛋白的研究显示,这是一种典型的含有Rossmann结构域的NAD(H)结合蛋白。其中关键的Rossmann结构域是一种主要存在于核苷酸结合蛋白中的蛋白质超二级结构,是典型的辅因子NAD(H)结合结构域,以各类辅因子NAD结合蛋白为代表。该结构主要由6个β折叠通过2对α螺旋以有序的β-α-β-α-β形式组成。因为每个Rossmann结构域只能结合一个核苷酸分子,因此类似NAD这类的二核苷酸结合蛋白的结构域中存在两个成对的Rossmann部分。当前研究显示天蓝色链霉菌YdiH(Rex)蛋白能够直接感应胞质NADH/NAD+比率的变化,而在有氧环境下,当细胞内NADH/NAD+比率处于低水平时,YdiH(Rex)蛋白可抑制其靶基因(cydABC、nuoA-D和rexhemACD)的转录,而NADH/NAD+比率的升高能够使Rex从其操纵子位点解离,在这一动态过程中YdiH(Rex)蛋白的空间构象会随着环境的变化而发生改变(Brekasis,D.等,EMBO J.2003,V.22(18),pp.4856-4865)。因此Rex蛋白是一个很好的细胞内NADH检测探针的候选者。同时,最近Wang等人对枯草芽孢杆菌YdiH(Rex)蛋白进行结晶并对其作用机理和功能进行了部分研究,结果显示源自枯草芽孢杆菌的YdiH(Rex)蛋白是一个同源二聚体蛋白,由两个功能结构域构成,其中N端结构域(1-85位残基)是一个DNA结合域,而C端结构域(86-215位残基)是一个典型的Rossmann折叠,它能够结合NADH(Wang,E.等,Mol Microbiol.2008,V.69(2),pp.466-478)。Another protein involved in this technology, YdiH protein (also known as Rex protein) is a bacterial transcriptional repressor protein known in the art, with a molecular weight of 23kDa, which can regulate fermentation and anaerobic respiration. Common YdiH proteins are from Thermus aquaticus (SEQ ID NO: 1 NCBI GenBank: AF061257.1), Streptomyces coelicolor (SEQ ID NO: 2 NCBI GenBank: AL939116.1) or Bacillus subtilis (Bacillus subtilis) (SEQ ID NO: 3NCBIGenBank: AL009126.3), the YdiH protein was first identified in Streptomyces coelicolor by Brekasis and Paget et al. in 2003. It is a widely present in A redox-sensitive regulatory protein in Gram-positive bacteria. The study on YdiH(Rex) protein of Streptomyces coelicolor (S.coelicolor) showed that it is a typical NAD(H) binding protein containing Rossmann domain. Among them, the key Rossmann domain is a protein super secondary structure mainly present in nucleotide binding proteins, which is a typical cofactor NAD(H) binding domain, represented by various cofactor NAD binding proteins. The structure mainly consists of six β-sheets in an ordered β-α-β-α-β form through 2 pairs of α-helices. Because each Rossmann domain can only bind one nucleotide molecule, there are two paired Rossmann moieties in the domain of dinucleotide binding proteins like NAD. The current study shows that the YdiH(Rex) protein of Streptomyces coelicolor can directly sense the change of cytoplasmic NADH/NAD + ratio, and under aerobic environment, when the intracellular NADH/NAD + ratio is at a low level, the YdiH(Rex) protein It can inhibit the transcription of its target genes (cydABC, nuoA-D and rexhemACD), and the increase of NADH/NAD + ratio can make Rex dissociate from its operator site. During this dynamic process, the YdiH(Rex) protein The spatial conformation will change with the environment (Brekasis, D. et al., EMBO J.2003, V.22(18), pp.4856-4865). So Rex protein is a good candidate for intracellular NADH detection probe. At the same time, Wang et al. recently crystallized the Bacillus subtilis YdiH (Rex) protein and conducted some studies on its mechanism of action and function. The results showed that the YdiH (Rex) protein derived from Bacillus subtilis is a homodimeric protein , consists of two functional domains, in which the N-terminal domain (residues 1-85) is a DNA binding domain, while the C-terminal domain (residues 86-215) is a typical Rossmann fold, which can Binds NADH (Wang, E. et al., Mol Microbiol. 2008, V.69(2), pp.466-478).
虽然YdiH(Rex)蛋白本身对环境内的氧化还原状态敏感,但是其自身发生的变化并不能直观的显示出并被外界所捕获,而借助于荧光蛋白这一工具,我们可以很好地通过将两者进行融合表达,获得一个全新的基因编码的荧光探针,利用YdiH(Rex)蛋白感受环境内氧化还原状态的变化并将这一变化传递至荧光蛋白,通过荧光蛋白产生荧光与否以及荧光的强弱,对环境中氧化还原状态改变进行实时且直观地描述。Although the YdiH (Rex) protein itself is sensitive to the redox state in the environment, its own changes cannot be displayed intuitively and captured by the outside world. With the help of the fluorescent protein tool, we can use the The two are fused and expressed to obtain a brand-new gene-encoded fluorescent probe, which uses the YdiH (Rex) protein to sense changes in the redox state in the environment and transmits this change to the fluorescent protein, which produces fluorescence and fluorescence The intensity of the redox state in the environment is described in real time and intuitively.
综上所述,我们认为,利用包含YdiH蛋白的重组荧光融合蛋白能够满足在生理水平和亚细胞水平上检测NADH的迫切需要。In summary, we believe that the use of recombinant fluorescent fusion proteins containing YdiH proteins can meet the urgent needs of detecting NADH at the physiological and subcellular levels.
不应认为对本文所述参考文献的引用或讨论意味着承认这些参考文献是本发明的现有技术。Citation or discussion of references described herein should not be construed as an admission that such references are prior art to the present invention.
发明内容Contents of the invention
下文以及本申请权利要求中提及具体序列信息时,各“SEQ ID No”后所列编号均为母案:中国专利申请201110288807.6中的序列编号。由于内容删减,相关序列在本申请所提交的序列表中的编号有所调整,具体对应关系如下表。When specific sequence information is mentioned below and in the claims of this application, the numbers listed after each "SEQ ID No" are the parent case: the sequence number in Chinese patent application 201110288807.6. Due to content deletion, the numbers of related sequences in the sequence listing submitted by this application have been adjusted, and the specific corresponding relationship is shown in the following table.
一方面,本发明提供一种遗传编码的NADH荧光探针,其内含有对环境内NADH敏感的多肽,和通过光谱性质的改变对环境内NADH进行表现的部分。在一个实施方式中,所述通过光谱性质的改变对环境内NADH进行表现的部分是荧光蛋白序列或其衍生物。在另一个实施方式中,所述对NADH敏感的多肽是具有如下特征的多肽,或其功能片段或NADH结合结构域:In one aspect, the present invention provides a genetically encoded NADH fluorescent probe, which contains a polypeptide sensitive to NADH in the environment and a part that expresses NADH in the environment through changes in spectral properties. In one embodiment, the part that expresses NADH in the environment through changes in spectral properties is a fluorescent protein sequence or a derivative thereof. In another embodiment, the polypeptide sensitive to NADH is a polypeptide having the following characteristics, or a functional fragment thereof or an NADH binding domain:
(1)具有NADH结合特性的Rossman结构域;和/或(1) a Rossman domain with NADH binding properties; and/or
(2)来源于对NADH敏感的转录调控因子Rex家族蛋白。(2) Derived from NADH-sensitive transcriptional regulator Rex family proteins.
在一个优选实施方式中,所述对NADH敏感的多肽可具有以下特征:In a preferred embodiment, the polypeptide sensitive to NADH may have the following characteristics:
(1)含有来自于细菌的转录调控因子Rex蛋白基因ydiH的多肽,该多肽的编码序列可以是SEQ ID No:1、2或3;(1) A polypeptide containing the transcription regulator Rex protein gene ydiH from bacteria, the coding sequence of the polypeptide can be SEQ ID No: 1, 2 or 3;
(2)在至少85个氨基酸残基内任何与(1)所述序列具有95%相同性的同源或非同源序列;(2) any homologous or non-homologous sequence having 95% identity with the sequence described in (1) within at least 85 amino acid residues;
(3)在至少85个氨基酸残基内任何与(1)所述序列具有90%相同性的同源或非同源序列;(3) any homologous or non-homologous sequence having 90% identity with the sequence described in (1) within at least 85 amino acid residues;
(4)在至少85个氨基酸残基内任何与(1)所述序列具有70%相同性的同源或非同源序列;(4) any homologous or non-homologous sequence having 70% identity with the sequence described in (1) within at least 85 amino acid residues;
(5)在至少85个氨基酸残基内任何与(1)所述序列具有50%相同性的同源或非同源序列;(5) any homologous or non-homologous sequence having 50% identity with the sequence described in (1) within at least 85 amino acid residues;
(6)在至少85个氨基酸残基内任何与(1)所述序列具有40%相似性的同源或非同源序列;或(6) any homologous or non-homologous sequence having 40% similarity to the sequence described in (1) within at least 85 amino acid residues; or
(7)在至少85个氨基酸残基内任何与(1)所述序列具有35%相似性的同源或非同源序列。(7) Any homologous or non-homologous sequence having 35% similarity to the sequence described in (1) within at least 85 amino acid residues.
在另一实施方式中,本发明荧光探针可以包含具有NADH结合特性的Rossman结构域B和荧光蛋白序列A、A1和/或A2,其组合形式可以是:In another embodiment, the fluorescent probe of the present invention may comprise a Rossman domain B with NADH binding properties and fluorescent protein sequences A, A1 and/or A2, and the combination thereof may be:
(1)B-A-B;(1) B-A-B;
(2)B-A-B-B;(2) B-A-B-B;
(3)A1-B-A2,其中A1和A2可以相同或不同;A1可以是来自于维多利亚多管发光水母的荧光蛋白或其衍生物的氨基酸序列,A2可以是来自于维多利亚多管发光水母的另一荧光蛋白或其衍生物的氨基酸序列;(3) A1-B-A2, wherein A1 and A2 can be the same or different; A1 can be the amino acid sequence of a fluorescent protein or a derivative thereof from Aequorea victoria, and A2 can be the amino acid sequence of a fluorescent protein from Aequorea victoria The amino acid sequence of another fluorescent protein or derivative thereof;
(4)B的第一部分-A-B的第二部分;其中A插入在B的柔性区域内,因而将B分割成B的第一部分和B的第二部分,B的第一部分和B的第二部分构成完整的B结构域;或(4) First part of B-A-Second part of B; where A is inserted within the flexible region of B, thus dividing B into first part of B and second part of B, first part of B and second part of B constitute the complete B domain; or
(5)B的第一部分-A-B的第二部分-B;其中A插入在B的柔性区域内,因而将B分割成B的第一部分和B的第二部分,B的第一部分和B的第二部分构成完整的B结构域。(5) First part of B-A-Second part of B-B; where A is inserted in the flexible region of B, thus dividing B into first part of B and second part of B, first part of B and second part of B The two parts constitute the complete B domain.
在又一实施方式中,本发明荧光探针也可以具有以下结构:In yet another embodiment, the fluorescent probe of the present invention may also have the following structure:
A1-B1-接头1-FM-接头2-B2,A 1 -B 1 -connector 1 -FM-connector 2 -B 2 ,
其中,A1是YdiH蛋白的第一结构域,优选包含枯草芽孢杆菌属YdiH蛋白的氨基酸序列的氨基酸1-84(SEQ ID NO:14)或嗜热水生菌属YdiH蛋白的氨基酸序列的氨基酸1-79(SEQ ID NO:15)或其变异体;B1是YdiH蛋白的第二结构域,优选包含枯草芽孢杆菌属YdiH蛋白的氨基酸序列的氨基酸85-194(SEQ ID NO:16)或嗜热水生菌属YdiH蛋白的氨基酸序列的氨基酸80-189(SEQ ID NO:17)或其变异体;B2是YdiH蛋白的第三结构域,优选包含枯草芽孢杆菌属YdiH蛋白的氨基酸序列的氨基酸120-215(SEQ ID NO:18)或嗜热水生菌属YdiH蛋白的氨基酸序列的氨基酸114-211(SEQ ID NO:19)或其变异体;Wherein, A1 is the first structural domain of YdiH protein, preferably comprises the amino acid 1-84 (SEQ ID NO: 14) of the aminoacid sequence of the amino acid sequence of Bacillus subtilis YdiH protein or the amino acid of the aminoacid sequence of the thermophilic bacteria YdiH protein 1-79 (SEQ ID NO: 15) or variant thereof; B 1 is the second domain of the YdiH protein, preferably comprising amino acid 85-194 (SEQ ID NO: 16) of the amino acid sequence of the Bacillus subtilis YdiH protein or Amino acids 80-189 (SEQ ID NO: 17) or variants thereof of the amino acid sequence of the Thermophylla YdiH protein; B2 is the third domain of the YdiH protein, preferably comprising the amino acid sequence of the Bacillus subtilis YdiH protein Amino acids 120-215 (SEQ ID NO: 18) or amino acids 114-211 (SEQ ID NO: 19) of the amino acid sequence of the Thermophilic genus YdiH protein or variants thereof;
FM是荧光团,可以是YFP、GFP、CFP等以及以这些蛋白为基础的变异体,优选YFP,更优选cpYFP;FM is a fluorophore, which can be YFP, GFP, CFP, etc. and variants based on these proteins, preferably YFP, more preferably cpYFP;
接头1可以存在或不存在;存在时,接头1可以是任何氨基酸序列,优选长度不超过4个氨基酸,例如包含氨基酸T、S、A、G或由这四个氨基酸中任意1至4个构成的任意组合,例如氨基酸序列SAG或TS等,但不限于此种组合;Linker 1 may or may not be present; when present, Linker 1 may be any amino acid sequence, preferably not more than 4 amino acids in length, for example comprising amino acids T, S, A, G or consisting of any 1 to 4 of these four amino acids Any combination of , such as amino acid sequence SAG or TS, etc., but not limited to such a combination;
接头2可以存在或不存在;存在时,接头2可以是任何氨基酸序列,优选长度不超过3个氨基酸,例如包含氨基酸G、T、G或由这四个氨基酸中任意1至4个构成的任意组合,例如包含氨基酸序列GTG,但不限于此种组合。Linker 2 may or may not be present; when present, linker 2 may be any amino acid sequence, preferably no more than 3 amino acids in length, for example comprising amino acids G, T, G or any sequence consisting of any 1 to 4 of these four amino acids. Combinations, for example, include the amino acid sequence GTG, but are not limited to such combinations.
在一个实施方式中,本发明还提供一种荧光探针,其包含荧光团以及YdiH蛋白或YdiH蛋白的任何片段、衍生物或类似物。在另一实施方式中,本发明还提供一种荧光探针,其包含荧光团以及YdiH蛋白的变异体。本发明还提供一种荧光探针,其包含荧光团以及YdiH蛋白的可溶性片段。In one embodiment, the present invention also provides a fluorescent probe comprising a fluorophore and YdiH protein or any fragment, derivative or analog of YdiH protein. In another embodiment, the present invention also provides a fluorescent probe comprising a fluorophore and a variant of YdiH protein. The present invention also provides a fluorescent probe comprising a fluorophore and a soluble fragment of YdiH protein.
在另一实施方式中,本发明还提供了一种遗传编码的NAD+荧光探针,其内含有对环境内NAD+敏感的多肽,和通过光谱性质的改变对环境内NAD+进行表现的部分。在一个具体实施方式中,所述NAD+荧光探针包含SEQ ID NO:129。In another embodiment, the present invention also provides a genetically encoded NAD+ fluorescent probe, which contains a polypeptide sensitive to NAD+ in the environment and a part that expresses NAD+ in the environment through changes in spectral properties. In a specific embodiment, the NAD+ fluorescent probe comprises SEQ ID NO: 129.
在又一实施方式中,本发明还提供了一种遗传编码的NADH/NAD+比率荧光探针,其内含有对环境内NADH/NAD+比率敏感的多肽,和通过光谱性质的改变对环境内NADH/NAD+比率进行表现的部分。在一个具体实施方式中,所述NADH/NAD+比率荧光探针包含SEQ ID NO:148。In yet another embodiment, the present invention also provides a genetically encoded NADH/NAD+ ratio fluorescent probe, which contains a polypeptide sensitive to the NADH/NAD+ ratio in the environment, and changes the NADH/NAD+ ratio in the environment by changing the spectral properties. The NAD+ ratio performs the performance part. In a specific embodiment, the NADH/NAD+ ratio fluorescent probe comprises SEQ ID NO: 148.
另一方面,本发明提供一种融合蛋白,其包含本发明荧光探针。在一个实施方式中,所述融合蛋白包含本发明荧光探针和各种特异性亚细胞定位信号,所述定位信号可将目标蛋白定位于指定的亚细胞器内。In another aspect, the present invention provides a fusion protein comprising the fluorescent probe of the present invention. In one embodiment, the fusion protein comprises the fluorescent probe of the present invention and various specific subcellular localization signals, and the localization signals can localize the target protein in specified subcellular organelles.
另一方面,本发明提供一种核酸序列,其包含编码本发明荧光探针或融合蛋白的核苷酸序列。在一个具体实施方式中,本发明提供一种核酸序列,其包含编码荧光蛋白的核苷酸序列和编码对NADH敏感的蛋白质的核苷酸序列。In another aspect, the present invention provides a nucleic acid sequence comprising a nucleotide sequence encoding the fluorescent probe or fusion protein of the present invention. In a specific embodiment, the present invention provides a nucleic acid sequence comprising a nucleotide sequence encoding a fluorescent protein and a nucleotide sequence encoding a protein sensitive to NADH.
在一个优选实施方式中,所述编码对NADH敏感的蛋白质的核苷酸序列是编码具有如下特征的多肽或其功能片段或NADH结合结构域的核苷酸序列:In a preferred embodiment, the nucleotide sequence encoding a protein sensitive to NADH is a nucleotide sequence encoding a polypeptide having the following characteristics or a functional fragment thereof or an NADH binding domain:
(1)具有NADH结合特性的Rossman结构域;和/或(1) a Rossman domain with NADH binding properties; and/or
(2)来源于对NADH敏感的转录调控因子Rex家族蛋白。(2) Derived from NADH-sensitive transcriptional regulator Rex family proteins.
在另一个优选实施方式中,本发明核酸序列可以包含具有NADH结合特性的Rossman结构域的编码序列b和荧光蛋白的编码序列a、a1和/或a2,其组合形式可以是:In another preferred embodiment, the nucleic acid sequence of the present invention may comprise the coding sequence b of the Rossman domain with NADH binding properties and the coding sequence a, a1 and/or a2 of the fluorescent protein, and the combination thereof may be:
(1)b-a-b;(1) b-a-b;
(2)b-a-b-b;(2) b-a-b-b;
(3)a1-b-a2,其中a1和a2可以相同或不同;a1可以是来自于维多利亚多管发光水母的荧光蛋白或其衍生物的编码序列,a2可以是来自于维多利亚多管发光水母的另一荧光蛋白或其衍生物的编码序列;(3) a1-b-a2, wherein a1 and a2 can be the same or different; a1 can be the coding sequence of a fluorescent protein or a derivative thereof from Aequorea victoria, and a2 can be from Aequorea victoria the coding sequence of another fluorescent protein or derivative thereof;
(4)b的第一部分-a-b的第二部分;其中a插入在b的柔性区域内,因而将b分割成b的第1部分和b的第二部分,b的第一部分和b的第二部分构成完整的b结构域;(4) The first part of b-a-the second part of b; where a is inserted in the flexible region of b, thus dividing b into the first part of b and the second part of b, the first part of b and the second part of b Partially constitutes the complete b domain;
(5)b的第一部分-a-b的第二部分-b;其中a插入在b的柔性区域内,因而将b分割成b的第1部分和b的第二部分,b的第一部分和b的第二部分构成完整的b结构域。(5) The first part of b-a-the second part of b-b; where a is inserted in the flexible region of b, thus dividing b into the first part of b and the second part of b, the first part of b and the The second part constitutes the complete b-domain.
本发明还涉及上述核酸序列的互补序列和变异体,其可包含编码本发明荧光探针或融合蛋白的片段、类似物、衍生物、可溶性片段和变异体的核酸序列或其互补序列。The present invention also relates to complementary sequences and variants of the aforementioned nucleic acid sequences, which may include nucleic acid sequences or complementary sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the fluorescent probe or fusion protein of the present invention.
在又一方面,本发明还提供一种表达载体,其包含与表达控制序列操作性连接的本发明核酸序列。所述表达控制序列可以是复制起点、启动子、增强子、操纵子、终止子、核糖体结合位点等。In yet another aspect, the present invention also provides an expression vector comprising the nucleic acid sequence of the present invention operably linked to an expression control sequence. The expression control sequence may be an origin of replication, a promoter, an enhancer, an operator, a terminator, a ribosome binding site, and the like.
在又一方面,本发明还提供一种宿主细胞,其包含本发明表达载体。In yet another aspect, the present invention also provides a host cell comprising the expression vector of the present invention.
在又一方面,本发明还提供一种制备本发明荧光探针或融合蛋白的方法,包括以下步骤:In yet another aspect, the present invention also provides a method for preparing the fluorescent probe or fusion protein of the present invention, comprising the following steps:
a.将本发明表达载体转移到宿主细胞中,a. transferring the expression vector of the present invention into a host cell,
b.在适合所述宿主细胞表达的条件下培养所述宿主细胞,和b. culturing said host cell under conditions suitable for expression by said host cell, and
c.由所述宿主细胞分离所述荧光探针或融合蛋白。c. Isolating said fluorescent probe or fusion protein from said host cell.
本发明还提供本发明荧光探针或融合蛋白在检测NADH中的应用。在一个实施方式中,本发明提供本发明荧光探针或融合蛋白在体外或体内检测NADH中的应用。在一个实施方式中,本发明提供本发明荧光探针或融合蛋白在亚细胞水平检测NADH中的应用。在一个实施方式中,本发明提供本发明荧光探针或融合蛋白在原位检测NADH中的应用。在另一个实施方式中,本发明提供本发明荧光探针或融合蛋白在筛选药物中的应用,所述药物可用于调节对象的NADH水平。在另一个实施方式中,本发明提供本发明荧光探针或融合蛋白在诊断疾病中的应用,所述疾病与NADH水平有关。The present invention also provides the application of the fluorescent probe or fusion protein of the present invention in detecting NADH. In one embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in detecting NADH in vitro or in vivo. In one embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in the detection of NADH at the subcellular level. In one embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in in situ detection of NADH. In another embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in screening drugs that can be used to regulate the NADH level of a subject. In another embodiment, the present invention provides the use of the fluorescent probe or fusion protein of the present invention in the diagnosis of diseases that are related to NADH levels.
本发明还提供了一种检测NADH的试剂盒,其中包含本发明荧光探针或融合蛋白。所述检测可以在体内、体外、亚细胞或原位水平进行。本发明还提供一种筛选药物的试剂盒,所述药物可用于调节对象的NADH水平,所述试剂盒包含有效量的本发明荧光探针或融合蛋白。本发明还提供了一种用于检测与NADH水平有关的疾病的试剂盒,所述试剂盒包含有效量的本发明融合蛋白。在使用时,本领域技术人员能够根据所述融合蛋白的活性方便地确定所述的有效量。The present invention also provides a kit for detecting NADH, which contains the fluorescent probe or fusion protein of the present invention. The detection can be performed at the in vivo, in vitro, subcellular or in situ level. The present invention also provides a kit for screening drugs that can be used to regulate the NADH level of a subject, and the kit includes an effective amount of the fluorescent probe or fusion protein of the present invention. The present invention also provides a kit for detecting diseases related to NADH level, said kit comprising an effective amount of the fusion protein of the present invention. In use, those skilled in the art can conveniently determine the effective amount according to the activity of the fusion protein.
本发明中的蛋白质和核酸序列优选以分离形式提供,更优选地被纯化至均质。The proteins and nucleic acid sequences of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
附图说明Description of drawings
下面结合附图和实施例对本发明作进一步说明。The present invention will be further described below in conjunction with drawings and embodiments.
图1SDS-PAGE鉴定从大肠杆菌(E.coli)中分离纯化的F-rex2。Figure 1 SDS-PAGE identification of F-rex2 isolated and purified from Escherichia coli (E.coli).
图2为烟酰胺腺嘌呤二核苷酸荧光探针F-rex2的基本光谱特性。Figure 2 shows the basic spectral characteristics of the fluorescent probe F-rex2 of nicotinamide adenine dinucleotide.
图3-1为还原性与氧化性烟酰胺腺嘌呤二核苷酸比率探针对NADH、NAD+结合的响应特性。Figure 3-1 shows the response characteristics of the reducing and oxidizing nicotinamide adenine dinucleotide ratio probes to the binding of NADH and NAD + .
图3-2为还原性与氧化性烟酰胺腺嘌呤二核苷酸比率探针对不同NADH与NAD+比率的测定结果。Figure 3-2 is the measurement results of different ratios of NADH and NAD + by reducing and oxidizing nicotinamide adenine dinucleotide ratio probes.
图3-3为NADH/NAD+比率探针在体外模拟生理条件下各吡啶核苷酸类似物对此探针的影响检测。Figure 3-3 is the detection of the influence of various pyridine nucleotide analogues on the NADH/NAD+ ratio probe under simulated physiological conditions in vitro.
具体实施方式Detailed ways
I.定义:I. Definition:
在给出数值或范围时,本文所用术语“约”指该数值或范围在给定数值或范围的20%以内、10%以内和5%以内。As used herein, the term "about" when a value or range is given means that the value or range is within 20%, within 10%, and within 5% of the given value or range.
本文所用术语“包含”、“包括”和其等同形式包括“含有”以及“由……组成”的含义,例如“包含”X的组合物可仅由X组成或可含有其它物质,例如X+Y。As used herein, the terms "comprising", "comprising" and their equivalents include the meanings of "containing" and "consisting of", for example a composition "comprising" X may consist solely of X or may contain other substances, such as X+ Y.
在本发明中,术语“YdiH蛋白”指YdiH蛋白(又称为Rex蛋白)是一种本领域已知的细菌转录抑制蛋白,分子量为23kDa,它能调控发酵和厌氧呼吸。这是一种广泛存在于革兰氏阳性菌中的一种对氧化还原敏感的调控蛋白,并且是一种典型的含有Rossmann结构域的NAD(H)结合蛋白。其中关键的Rossmann结构域是一种主要存在于核苷酸结合蛋白中的蛋白质超二级结构,是典型的结合辅因子NAD(H)的活性区域,以各类辅因子NAD结合蛋白为代表。该结构主要由6个β折叠通过2对α螺旋以有序的β-α-β-α-β形式组成。因为每个Rossmann结构域只能结合一个核苷酸分子,因此类似NAD这类的二核苷酸结合蛋白的结构域中存在两个成对的Rossmann部分。YdiH(Rex)蛋白能够直接感应胞质NADH/NAD+比率的变化,而在有氧环境下,当细胞内NADH/NAD+比率处于低水平时,YdiH(Rex)蛋白可抑制其靶基因(cydABC、nuoA-D和rexhemACD)的转录,而NADH/NAD+比率的升高能够使Rex从其操纵子位点解离,在这一动态过程中YdiH(Rex)蛋白的空间构象会随着环境的变化而发生改变。本发明中所涉及的“YdiH蛋白”可以包含核苷酸序列SEQ ID NO:1或SEQID NO:2或SEQ ID NO:3编码的氨基酸序列。本发明中所涉及的“柔性区域”是指蛋白质高级结构中存在的一些特定的如Loop结构域等结构,这些结构域相比于其他的蛋白质高级结构具有更高的移动性和柔性,并且能够导致结构域发生动态变化,而蛋白质在这些区域也存在着极大地发生空间构象改变的趋势。本发明所涉及的柔性区域主要指T-rex(即嗜热水生菌(Thermus aquaticus)来源的蛋白质Rex)中的V113-G119区域,以及D188-G192这段区域。In the present invention, the term "YdiH protein" means that YdiH protein (also known as Rex protein) is a bacterial transcriptional repressor protein known in the art, with a molecular weight of 23 kDa, which can regulate fermentation and anaerobic respiration. This is a redox-sensitive regulatory protein widely present in Gram-positive bacteria, and is a typical NAD(H)-binding protein containing a Rossmann domain. Among them, the key Rossmann domain is a protein super secondary structure mainly present in nucleotide-binding proteins, and is a typical active region that binds the cofactor NAD(H), represented by various cofactor NAD-binding proteins. The structure mainly consists of six β-sheets in an ordered β-α-β-α-β form through 2 pairs of α-helices. Because each Rossmann domain can only bind one nucleotide molecule, there are two paired Rossmann moieties in the domain of dinucleotide binding proteins like NAD. YdiH(Rex) protein can directly sense the change of cytoplasmic NADH/NAD + ratio, and under aerobic environment, when the intracellular NADH/NAD + ratio is at low level, YdiH(Rex) protein can repress its target gene (cydABC , nuoA-D and rexhemACD), and the increase of NADH/NAD + ratio can make Rex dissociate from its operator site. In this dynamic process, the spatial conformation of YdiH (Rex) protein will change with the environment change to change. The "YdiH protein" involved in the present invention may comprise the amino acid sequence encoded by the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3. The "flexible region" involved in the present invention refers to some specific structures such as Loop domains in the high-level structure of proteins. Compared with other high-level protein structures, these domains have higher mobility and flexibility, and can This leads to dynamic changes in the structural domains, and the protein also has a tendency to greatly change the spatial conformation in these regions. The flexible region involved in the present invention mainly refers to the V113-G119 region and the D188-G192 region of T-rex (ie, the protein Rex derived from Thermus aquaticus).
本文所用术语“荧光探针”是指与荧光蛋白融合的对环境内NADH敏感的多肽,所述对环境内NADH敏感的多肽具体可以是YdiH蛋白,其利用YdiH中专一性的NADH结合结构Rossman结构域与NADH结合后产生的构象变化引起的荧光蛋白的构象变化,进而导致产生或消失的荧光或产生的荧光发生改变,并借助不同NADH浓度下测定的荧光蛋白的荧光绘制标准曲线,进而检测并分析NADH的存在和/或水平。The term "fluorescent probe" as used herein refers to a polypeptide sensitive to NADH in the environment fused with a fluorescent protein. The polypeptide sensitive to NADH in the environment can specifically be a YdiH protein, which utilizes the specific NADH binding structure Rossman in YdiH The conformational change of the fluorescent protein caused by the conformational change after the binding of the structural domain to NADH leads to the generation or disappearance of the fluorescence or the change of the generated fluorescence, and a standard curve is drawn with the fluorescence of the fluorescent protein measured at different NADH concentrations to detect And analyze the presence and/or level of NADH.
本文所用术语“融合蛋白”与“荧光融合蛋白”和“重组荧光融合蛋白”同义,指包含第一种多肽或蛋白质或者其片段、类似物或衍生物的氨基酸序列,以及异源多肽或蛋白质(即,不同于第一种多肽或蛋白质或者其片段、类似物或衍生物的第二种多肽或蛋白质或者其片段、类似物或衍生物)的氨基酸序列的多肽或蛋白质。在一个实施方式中,融合蛋白包含与异源蛋白质、多肽或肽融合的荧光蛋白。按照这个实施方式,异源蛋白质、多肽或肽可能是或不是不同类型荧光蛋白。在一个实施方式中,与融合于异源蛋白质、多肽或肽之前的原始多肽或蛋白质的活性相比,融合蛋白保持或提高了活性。在一个具体实施方式中,融合蛋白包含与异源蛋白质、多肽或肽融合的荧光探针,所述异源蛋白质、多肽或肽可以是特异性亚细胞定位信号。The term "fusion protein" as used herein is synonymous with "fluorescent fusion protein" and "recombinant fluorescent fusion protein", and refers to an amino acid sequence comprising a first polypeptide or protein or a fragment, analog or derivative thereof, and a heterologous polypeptide or protein (ie, a polypeptide or protein having an amino acid sequence different from that of a first polypeptide or protein or a fragment, analog or derivative thereof). In one embodiment, the fusion protein comprises a fluorescent protein fused to a heterologous protein, polypeptide or peptide. According to this embodiment, the heterologous protein, polypeptide or peptide may or may not be a different type of fluorescent protein. In one embodiment, the fusion protein maintains or increases the activity compared to the activity of the original polypeptide or protein prior to fusion to the heterologous protein, polypeptide or peptide. In a specific embodiment, the fusion protein comprises a fluorescent probe fused to a heterologous protein, polypeptide or peptide, which may be a specific subcellular localization signal.
本文所用术语“荧光团”与“荧光蛋白”同义,指自身发出荧光或在照射下发出荧光的蛋白质。荧光蛋白常常用作检测手段,例如生物技术领域常用的绿色荧光蛋白GFP及由该蛋白突变衍生出的BFP、CFP、YFP、cpYFP等。The term "fluorophore" as used herein is synonymous with "fluorescent protein" and refers to a protein that fluoresces by itself or under illumination. Fluorescent proteins are often used as detection methods, such as the green fluorescent protein GFP commonly used in the field of biotechnology and BFP, CFP, YFP, cpYFP, etc. derived from mutations of this protein.
本文所用术语“GFP”指绿色荧光蛋白,最初是从维多利亚多管发光水母(Aequorea victoria)中提取,野生型的AvGFP由238个氨基酸构成,分子量约为26kD,其氨基酸序列为SEQ ID No:20。当前的研究确认,天然GFP蛋白中第65~67位的三个氨基酸Ser-Tyr-Gly能够自发形成一个荧光生色基团:对-羟基苯亚甲基咪唑啉酮(p-hydroxybenzylideneimidazolinone),是其主要的发光位置。野生型AvGFP的光谱特征十分复杂,其荧光激发的主峰在395nm处,而在475nm处另有一个附峰,后者振幅强度约为前者的1/3。在标准的溶液条件下,395nm处的激发可产生508nm处的发射,而475nm处的激发产生的最大发射波长位于503nm。The term "GFP" used herein refers to green fluorescent protein, which was originally extracted from Aequorea victoria. The wild-type AvGFP consists of 238 amino acids and has a molecular weight of about 26kD. Its amino acid sequence is SEQ ID No: 20 . Current research has confirmed that the three amino acids Ser-Tyr-Gly at positions 65-67 in the natural GFP protein can spontaneously form a fluorescent chromophore: p-hydroxybenzylideneimidazolinone, which is Its main luminous position. The spectral characteristics of wild-type AvGFP are very complex, the main peak of its fluorescence excitation is at 395nm, and there is another auxiliary peak at 475nm, the amplitude of the latter is about 1/3 of the former. Under standard solution conditions, excitation at 395nm produces emission at 508nm, while excitation at 475nm produces a maximum emission at 503nm.
本文所用术语“YFP”指黄色荧光蛋白,该蛋白衍生自绿色荧光蛋白GFP,其氨基酸序列与GFP同源性高达90%以上,YFP相比于GFP关键改变在于第203位氨基酸由苏氨酸突变为酪氨酸(T203Y)。相比于原始的AvGFP,YFP的主激发峰的波长红移至514nm而发射波长则改变为527nm。在此基础上对YFP第65位氨基酸进行定点突变(S65T)可获得荧光增强型黄色荧光蛋白EYFP,典型的EYFP氨基酸序列为SEQ ID No:21。而对EYFP蛋白序列的重新排列,将原第145-238位氨基酸部分作为新蛋白的N端,原第1-144位氨基酸作为新蛋白的C端,两片段间通过一小段具有柔性的短肽链VDGGSGGTG连接,形成一个对空间变化敏感的环状排列黄色荧光蛋白cpYFP(circular permutation yellow fluorescent protein),典型的cpYFP氨基酸序列为SEQ ID No:22。The term "YFP" used herein refers to yellow fluorescent protein, which is derived from the green fluorescent protein GFP, and its amino acid sequence is more than 90% homologous to GFP. Compared with GFP, the key change of YFP is that the 203rd amino acid is mutated by threonine It is tyrosine (T203Y). Compared with the original AvGFP, the wavelength of the main excitation peak of YFP was red-shifted to 514nm and the emission wavelength was changed to 527nm. On this basis, site-directed mutation (S65T) of the 65th amino acid of YFP can obtain the fluorescence-enhanced yellow fluorescent protein EYFP, and the typical amino acid sequence of EYFP is SEQ ID No: 21. For the rearrangement of the EYFP protein sequence, the original 145-238 amino acid part is used as the N-terminal of the new protein, and the original 1-144 amino acid is used as the C-terminal of the new protein, and a small flexible short peptide is passed between the two fragments The chain VDGGSGGTG is connected to form a circular permutation yellow fluorescent protein cpYFP (circular permutation yellow fluorescent protein) that is sensitive to spatial changes. The typical amino acid sequence of cpYFP is SEQ ID No: 22.
在本发明中,与荧光团融合的YdiH蛋白可以是分离自枯草芽孢杆菌或嗜热水生菌或天蓝色链霉菌的天然YdiH蛋白的全长或其片段,优选是天然枯草芽胞杆菌属YdiH蛋白的氨基酸1-215或嗜热水生菌属YdiH蛋白的氨基酸1-211或天蓝色链霉菌属YdiH蛋白的氨基酸1-259,更优选枯草芽胞杆菌属YdiH蛋白的氨基酸1-215或嗜热水生菌属YdiH蛋白的氨基酸1-211。In the present invention, the YdiH protein fused with a fluorophore can be the full length or fragment thereof of a natural YdiH protein isolated from Bacillus subtilis or thermophilic bacteria or Streptomyces coelicolor, preferably a natural Bacillus subtilis YdiH protein Amino acid 1-215 of or the amino acid 1-211 of the thermophilic genus YdiH protein or the amino acid 1-259 of the Streptomyces coelicolor genus YdiH protein, more preferably the amino acid 1-215 of the subtilis genus YdiH protein or the thermophilic Amino acids 1-211 of the Phytophthora YdiH protein.
“接头”指在本发明多肽、蛋白质或核酸中连接两个部分的氨基酸或核酸序列。在本发明多肽或蛋白质中进行连接时,接头的长度不大于6个氨基酸,优选不大于4个氨基酸,更优选是3个氨基酸。在本发明核酸中进行连接时,接头的长度不大于18个核苷酸,优选不大于12个核苷酸,更优选是9个核苷酸。"Linker" refers to an amino acid or nucleic acid sequence that joins two parts in a polypeptide, protein or nucleic acid of the invention. When connecting in the polypeptide or protein of the present invention, the length of the linker is not more than 6 amino acids, preferably not more than 4 amino acids, more preferably 3 amino acids. When linking is performed in the nucleic acid of the present invention, the length of the linker is not more than 18 nucleotides, preferably not more than 12 nucleotides, more preferably 9 nucleotides.
提到某多肽或蛋白时,本发明所用术语“变异体”包括具有所述多肽或蛋白相同功能、但序列不同的变异体。这些变异体包括(但并不限于):在所述多肽或蛋白的序列中缺失、插入和/或取代一个或多个(通常为1-30个,较佳地1-20个,更佳地1-10个,最佳地1-5个)氨基酸,以及在其C末端和/或N末端添加一个或数个(通常为20个以内,较佳地为10个以内,更佳地为5个以内)氨基酸获得的序列。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变多肽或蛋白的功能。在本领域中,性能相似的氨基酸往往指具有相似侧链的氨基酸家族,在本领域已有明确定义。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。又比如,在C末端和/或N末端添加一个或数个氨基酸通常也不会改变多肽或蛋白的功能。本领域技术人员公知,在基因克隆操作中,常常需要设计合适的酶切位点,这势必在所表达的多肽或蛋白末端引入了一个或多个不相干的残基,而这并不影响目的多肽或蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化,常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内,例如,包括但不限于,适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签,或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。多肽或蛋白的变异体可包括:同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严谨条件下能与所述多肽或蛋白的DNA杂交的DNA所编码的多肽或蛋白、以及利用抗所述多肽或蛋白的抗血清获得的多肽或蛋白。这些变异体还可包含与所述多肽或蛋白的序列相同性为至少约70%、至少约75%、至少约80%、至少约85%、至少约90%、至少约95%、至少约98%、至少约99%或100%的多肽或蛋白。When referring to a certain polypeptide or protein, the term "variant" used in the present invention includes variants that have the same function of the polypeptide or protein but have different sequences. These variants include (but are not limited to): one or more deletions, insertions and/or substitutions (usually 1-30, preferably 1-20, more preferably 1-10, preferably 1-5) amino acids, and one or several (usually within 20, preferably within 10, more preferably 5) added at its C-terminal and/or N-terminal within 1) amino acid sequence obtained. For example, in this field, substitutions with amino acids with similar or similar properties generally do not change the function of the polypeptide or protein. In this field, amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in this field. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), amino acids with acidic side chains (e.g. aspartic acid, glutamic acid), Amino acids with side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g. threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). For another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the polypeptide or protein. Those skilled in the art know that in gene cloning operations, it is often necessary to design suitable restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, which does not affect the purpose Activity of polypeptide or protein. Another example is to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminal, C-terminal or the recombinant protein. Within other suitable regions within the protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tag, or proteolytic enzyme site for factor Xa or thrombin or enterokinase. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, those capable of hybridizing to the DNA of the polypeptide or protein under high or low stringency conditions Polypeptide or protein encoded by DNA, and polypeptide or protein obtained by using antiserum against said polypeptide or protein. These variants may also comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the polypeptide or protein. %, at least about 99%, or 100% of the polypeptide or protein.
在两种或多种多肽或核酸分子序列中,术语“相同性”或“相同性百分数”指在比较窗口或指定区域上,采用本领域已知方法如序列比较算法,通过手工比对和目测检查来比较和比对最大对应性时,两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如,60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同)。例如,适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法,分别可参见Altschul等(1977)Nucleic Acids Res.25:3389和Altschul等(1990)J.Mol.Biol.215:403。In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "identity percentage" refers to the comparison window or specified region, using methods known in the art such as sequence comparison algorithms, by manual alignment and visual inspection Two or more sequences or subsequences that are identical or in which a specified percentage of amino acid residues or nucleotides are identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the same). For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403.
本文所用术语“可溶性片段”通常指具有所述蛋白全长序列的至少约10个连续氨基酸,通常至少约30个连续氨基酸,较佳地至少约50个连续氨基酸,更佳地至少约80个连续氨基酸,最佳地至少约100个连续氨基酸的片段。The term "soluble fragment" as used herein generally refers to at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, of the full-length sequence of the protein. Amino acids, optimally stretches of at least about 100 contiguous amino acids.
本文所用术语“功能片段”、“衍生物”和“类似物”是指基本上保持与本发明天然YdiH相同的生物学功能或活性的蛋白。本发明的YdiH的功能片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的蛋白,或(iii)成熟蛋白与另一个化合物(比如延长蛋白半衰期的化合物,例如聚乙二醇)融合所形成的蛋白,或(iv)附加的氨基酸序列融合到此蛋白序列而形成的蛋白(如前导序列或分泌序列或用来纯化此蛋白的序列或蛋白原序列,或与抗原IgG片段的形成的融合蛋白)。根据本文的教导,这些功能片段、衍生物和类似物属于本领域熟练技术人员公知的范围。The terms "functional fragment", "derivative" and "analogue" used herein refer to a protein that substantially maintains the same biological function or activity as the native YdiH of the present invention. The functional fragments, derivatives or analogs of YdiH of the present invention may be (i) proteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acids The residue may or may not be encoded by the genetic code, or (ii) the protein has a substitution group in one or more amino acid residues, or (iii) the mature protein is combined with another compound (such as a compound that extends the half-life of the protein , such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the protein sequence (such as a leader or secretory sequence or a sequence or proprotein sequence used to purify the protein, Or the fusion protein formed with the antigen IgG fragment). Based on the teaching herein, these functional fragments, derivatives and analogs are within the scope of those skilled in the art.
所述类似物与天然YdiH蛋白的差别可以是氨基酸序列上的差异,也可以是不影响序列的修饰形式上的差异,或者兼而有之。这些蛋白包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到,如通过辐射或暴露于诱变剂而产生随机诱变,还可通过定点诱变法或其他已知分子生物学的技术得到。The difference between the analogue and the natural YdiH protein may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology.
所述类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物,以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解,本发明的YdiH蛋白并不限于上述列举的代表性蛋白、片段、衍生物和类似物。修饰(通常不改变一级结构)形式包括:体内或体外的蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化,如那些在蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的蛋白。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。The analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the YdiH protein of the present invention is not limited to the representative proteins, fragments, derivatives and analogs listed above. Modified (usually without altering primary structure) forms include: chemically derivatized forms of proteins such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications during protein synthesis and processing or during further processing steps. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.
本发明所用术语“核酸”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。The term "nucleic acid" as used in the present invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.
提到核酸时,本文所用术语“变异体”可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括简并变异体、取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个核酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码的蛋白的功能。本发明核酸可包含与所述核酸序列的序列相同性为至少约70%、至少约75%、至少约80%、至少约85%、至少约90%、至少约95%、至少约98%、至少约99%或100%的核苷酸序列。As used herein, the term "variant" when referring to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a nucleic acid, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of its encoded protein. Nucleic acids of the invention may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, At least about 99% or 100% nucleotide sequence.
如此处所用,术语“在严谨条件下杂交”是用来描述典型的相互间至少60%同源的核苷酸序列仍可相互杂交的杂交和清洗条件。优选的,严谨条件为这样的条件,在此条件下相互间有至少65%、更优的至少70%、且甚至更优选的至少80%或更高同源性的序列一般仍可相互杂交。此严谨条件为本领域普通技术人员所公知。严谨条件的一个优选,非限制性实例为:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,0℃;或(2)杂交时加有变性剂,50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的相同性至少在90%以上,更好是95%以上时才发生杂交。As used herein, the term "hybridizes under stringent conditions" is used to describe hybridization and wash conditions typically under which nucleotide sequences that are at least 60% homologous to each other can still hybridize to each other. Preferably, stringent conditions are those under which sequences having at least 65%, more preferably at least 70%, and even more preferably at least 80% or more homology to each other generally still hybridize to each other. Such stringent conditions are well known to those of ordinary skill in the art. A preferred, non-limiting example of stringent conditions is: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 0° C.; or (2) hybridization with With denaturant, 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, better Hybridization occurs when it is more than 95%.
本发明还涉及与上述的序列杂交的核酸片段。如本文所用,“核酸片段”的长度至少含15个核苷酸,较好是至少30个核苷酸,更好是至少50个核苷酸,最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)。The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR).
本发明荧光探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据本发明所公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。The full-length sequence or fragments of the fluorescent probe or fusion protein of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离和纯化得到有关多肽或蛋白。Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant polypeptide or protein is obtained by isolating and purifying from the proliferated host cell by conventional methods.
此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
目前,已经可以完全通过化学合成来得到编码本发明蛋白(或其片段、衍生物、类似物或变异体)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如载体)和细胞中。可通过突变PCR或化学合成等方法将突变引入本发明蛋白序列中。At present, the DNA sequence encoding the protein of the present invention (or its fragments, derivatives, analogs or variants) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.
本文所用的术语“表达载体”和“重组载体”可互换使用,指本领域熟知的原核或真核载体,例如细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体,这些载体能够在宿主体内复制和稳定,这些重组载体的一个重要特征是通常含有表达控制序列。本文所用术语“表达控制序列”指调控目的基因的转录、翻译和表达的可以与目的基因操作性连接的元件,可以是复制起点、启动子、标记基因或翻译控制元件,包括增强子、操纵子、终止子、核糖体结合位点等,表达控制序列的选择取决于所用的宿主细胞。在本发明中适用的重组载体包括但不限于细菌质粒。在重组表达载体中,“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本发明融合蛋白编码序列和合适的转录/翻译控制信号的表达载体的方法。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。这些启动子的代表性例子有:大肠杆菌的lac或trp启动子;λ噬菌体PL启动子;真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTR和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。As used herein, the terms "expression vector" and "recombinant vector" are used interchangeably and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, Retroviral or other vectors, which are capable of replicating and stabilizing in the host, an important feature of these recombinant vectors is that they usually contain expression control sequences. The term "expression control sequence" as used herein refers to elements that regulate the transcription, translation and expression of the gene of interest that can be operably linked to the gene of interest, and can be an origin of replication, a promoter, a marker gene or a translation control element, including enhancers, operators , terminator, ribosome binding site, etc., the choice of expression control sequence depends on the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In a recombinant expression vector, "operably linked" means that a nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the fusion protein coding sequences of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
本领域普通技术人员将理解重组表达载体的设计可取决于如欲转化的宿主细胞的选择、所需的蛋白质表达水平等因素。此外,重组表达载体优选地包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如用于真核细胞的二氢叶酸还原酶、新霉素抗性,或用于大肠杆菌的四环素或氨苄青霉素抗性。Those of ordinary skill in the art will appreciate that the design of the recombinant expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, and the like. In addition, the recombinant expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cells, neomycin resistance, Or tetracycline or ampicillin resistance for E. coli.
在一种实施方式中,将本发明荧光探针或融合蛋白的编码序列经BamHI和HindIII双酶切后与BamHI和HindIII双酶切的pRSETb载体连接,得到大肠杆菌重组表达载体。可以将本发明的表达载体转移到宿主细胞中,以产生包括融合蛋白的蛋白或肽。此种转移过程可用转化或转染等本领域技术人员熟知的常规技术进行。In one embodiment, the coding sequence of the fluorescent probe or fusion protein of the present invention is double-digested with BamHI and HindIII and then ligated with the pRSET b vector cut with BamHI and HindIII to obtain a recombinant expression vector for Escherichia coli. Expression vectors of the present invention can be transferred into host cells to produce proteins or peptides, including fusion proteins. This transfer process can be carried out by conventional techniques such as transformation or transfection, which are well known to those skilled in the art.
本文在所用术语“宿主细胞”又称为受体细胞,是指能够接收和容纳重组DNA分子的细胞,是重组基因扩增的场所,理想的受体细胞应该满足易于获取和增殖两个条件。本发明的“宿主细胞”可包括原核细胞和真核细胞,具体包括细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。The term "host cell" used herein is also called recipient cell, which refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for recombinant gene amplification. Ideal recipient cells should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells and mammalian cells.
本发明的表达载体可用于在原核或真核细胞中表达本发明荧光探针或融合蛋白。从而,本发明涉及已导入本发明表达载体的宿主细胞、优选大肠杆菌。宿主细胞可为任何原核或真核细胞,代表性例子有:大肠杆菌,链霉菌属,鼠伤寒沙门氏菌的细菌细胞,真菌细胞如酵母,植物细胞,果蝇S2或Sf9的昆虫细胞,CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等,其中包括但不限于上述的那些宿主细胞。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞,此类细胞已为本领域熟知并常用,例如各种大肠杆菌细胞和酵母细胞。在本发明的一个实施方式中,选用大肠杆菌BL21构建表达本发明融合蛋白的宿主细胞。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。The expression vector of the present invention can be used to express the fluorescent probe or fusion protein of the present invention in prokaryotic or eukaryotic cells. Thus, the present invention relates to a host cell, preferably Escherichia coli, into which the expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell, representative examples are: bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS , 293 cells, or animal cells of Bowes melanoma cells, etc., including but not limited to those host cells mentioned above. The host cells are preferably various cells that are conducive to gene product expression or fermentative production, and such cells are well known and commonly used in the art, such as various Escherichia coli cells and yeast cells. In one embodiment of the present invention, Escherichia coli BL21 is selected to construct a host cell expressing the fusion protein of the present invention. Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.
本文所用术语“转化”和“转染”、“接合”和“转导”意指本领域内公知的各种将外源核酸(例如,线性DNA或RNA(例如,线性化载体或无载体的单独的基因构建体))或载体形式的核酸(例如,质粒、粘粒、噬菌体、噬粒、噬菌粒、转座子或其它DNA)导入宿主细胞的技术,包括磷酸钙或氯化钙共沉淀、DEAE-甘露聚糖-介导的转染、脂转染、天然感受态、化学介导的转移或电穿孔。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主细胞是真核细胞时,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" mean various transformations of exogenous nucleic acid (e.g., linear DNA or RNA (e.g., linearized vector or vector-free vector) known in the art. Techniques for introducing a single gene construct)) or a nucleic acid in the form of a vector (for example, a plasmid, cosmid, phage, phagemid, phagemid, transposon, or other DNA) into a host cell, including calcium phosphate or calcium chloride co- Precipitation, DEAE-mannan-mediated transfection, lipofection, native competence, chemical-mediated transfer, or electroporation. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired. When the host cells are eukaryotic cells, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
可以用适合所述宿主细胞表达的常规方法培养获得的转化细胞,表达本发明融合蛋白。根据所用的宿主细胞,培养中所用的培养基可以是各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。The obtained transformed cells can be cultured to express the fusion protein of the present invention by conventional methods suitable for the expression of said host cells. The medium used in the culture may be various conventional ones depending on the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
在上面的方法中的重组蛋白可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离或纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant protein in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated or purified by various separation methods by taking advantage of its physical, chemical and other properties, if necessary. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
在一个实施方式中,通过包含本发明融合蛋白编码序列的大肠杆菌发酵生产本发明荧光探针或融合蛋白,并通过硫酸铵沉降,离子交换层析和凝胶层析纯化得到了纯形式的本发明荧光探针或融合蛋白。In one embodiment, the fluorescent probe or fusion protein of the present invention is produced by fermentation of Escherichia coli comprising the coding sequence of the fusion protein of the present invention, and purified by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography to obtain the present invention in pure form. Invent fluorescent probes or fusion proteins.
本发明荧光探针或融合蛋白的用途包括但不限于:检测NADH、在生理状态下检测NADH、在亚细胞水平检测NADH、原位检测NADH、筛选药物、诊断与与NADH水平有关的疾病等。The application of the fluorescent probe or fusion protein of the present invention includes but not limited to: detection of NADH, detection of NADH in physiological state, detection of NADH at subcellular level, detection of NADH in situ, screening of drugs, diagnosis of diseases related to NADH level, etc.
在本文中,浓度、含量、百分数和其它数值均可用范围的形式表示。也应理解,使用这种范围形式只是为了方便和简洁,应该被弹性地借读为包括范围上下限所明确提及的数值,还应包括该范围内包括的所有单个数值或子范围,就好像明确提及各个数值和子范围那样。Concentrations, amounts, percentages, and other numerical values may be expressed herein in the form of ranges. It should also be understood that the use of this range format is for convenience and brevity only and should be read flexibly to include the values expressly recited by the upper and lower limits of the range, and also to include all individual values or subranges subsumed within that range, as if expressly Individual values and subranges are referred to as such.
实施例Example
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等,《分子克隆:实验室指南》(美国纽约州:冷泉港实验室出版社(Cold SpringHarbor Laboratory Press),1989);或按照制造厂商所建议的条件进行。在本文中,除非另外说明,百分比和份数均按重量计算。The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook etc., "Molecular Cloning: Laboratory Guide" (New York State, USA: Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), 1989); Or follow the conditions recommended by the manufacturer. Herein, percentages and parts are by weight unless otherwise indicated.
I.实验材料和试剂I. Experimental Materials and Reagents
试剂:除特别标注,其他均来自上海国药集团化学试剂有限公司(中国上海)。Reagents: Unless otherwise noted, all others were from Shanghai Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
PCR扩增所使用的Taq酶、缓冲液、dNTP;分子生物实验中所使用的蛋白酶、缓冲液、T4DNA连接酶、T4DNA连接酶缓冲液、T4多聚核苷酸激酶(PNK)、T4PNK缓冲液,均来自立陶宛维尔纽斯的富酶泰斯公司(Fermentas)。Taq enzyme, buffer, dNTP used in PCR amplification; protease, buffer, T4DNA ligase, T4DNA ligase buffer, T4 polynucleotide kinase (PNK), T4PNK buffer used in molecular biology experiments , both from Fermentas, Vilnius, Lithuania.
实施例1pRSETb-ydiH(189)-YFP-ydiH(190)的构建和表达Construction and expression of embodiment 1 pRSET b -ydiH(189)-YFP-ydiH(190)
1.扩增cpYFP的核酸序列:1. Amplify the nucleic acid sequence of cpYFP:
以pMD19-cpYFP为模板,利用引物cpYFP F和cpYFP R扩增黄色荧光蛋白(cpYFP)的编码序列,引物序列(引物由上海生工生物工程有限公司(中国上海)合成)如下:Using pMD19-cpYFP as a template, using primers cpYFP F and cpYFP R to amplify the coding sequence of yellow fluorescent protein (cpYFP), the primer sequence (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. (Shanghai, China)) is as follows:
P1:PstI GAATCTGCAGGCTACAACAGCCACAACGTCTATATC(SEQ IDNO:29)P1: PstI GAAT CTGCAG GCTACAACAGCCACAACGTCTATATC (SEQ ID NO: 29)
P2:KpnI CCAAGCTTCGGGGTACCGTTGTACTCCAGCTTGTG(SEQ IDNO:30)P2: KpnICCAAGCTTCGGGGTACCGTTGTACTCCAGCTTGTG (SEQ ID NO: 30)
PCR反应体系为The PCR reaction system is
PCR反应条件为:The PCR reaction conditions are:
将PCR扩增产物在1%的琼脂糖凝胶中电泳20分钟,得到约750bp的cpYFP片段。利用上海生工DNA片段回收纯化试剂盒(上海生物工程有限公司,中国上海)按照厂商说明书从凝胶中回收和纯化cpYFP片段。The PCR amplification product was electrophoresed in 1% agarose gel for 20 minutes to obtain a cpYFP fragment of about 750 bp. The cpYFP fragment was recovered and purified from the gel using the Shanghai Sangon DNA Fragment Recovery and Purification Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions.
2.扩增嗜热水生菌(Thermus aquaticus)属YdiH蛋白目的基因序列:2. Amplify the target gene sequence of YdiH protein from Thermus aquaticus:
嗜热水生菌属YdiH蛋白基因T-ydiH委托上海捷瑞生物工程有限公司(中国上海)合成(按照NCBI Genbank数据中记录的基因全序列进行合成,NCBI GenbankAF061257.1)。The YdiH protein gene T-ydiH of the genus Thermophysis was entrusted to Shanghai Jierui Bioengineering Co., Ltd. (Shanghai, China) to synthesize (synthesized according to the full sequence of the gene recorded in the NCBI Genbank data, NCBI GenbankAF061257.1).
以上述基因为模板,利用引物ydiH 1F和ydiH 2R扩增嗜热水生菌的YdiH蛋白基因(T-ydiH)全长,其中引物ydiH 1F和ydiH 2R扩增获得N端含有BamHI酶切位点C端含有HindIII酶切位点的T-YdiH蛋白基因(T-ydiH)全长片段T-yidH,引物ydiH 1F和ydiH 2R序列如下:Using the above gene as a template, use primers ydiH 1F and ydiH 2R to amplify the full length of the YdiH protein gene (T-ydiH) of thermophilic bacteria, wherein the primers ydiH 1F and ydiH 2R amplify the N-terminal containing the BamHI restriction site The C-terminus contains the full-length fragment T-yidH of the T-YdiH protein gene (T-ydiH) with a HindIII restriction site, and the sequences of primers ydiH 1F and ydiH 2R are as follows:
P3:BamHI CCGGATCCGATGAATAAGGATCAATCAAAAATTC(SEQ IDNO:31)P3: BamHI CC GGATCC GATGAATAAGGATCAATCAAAAATTC (SEQ ID NO: 31)
P4:HindIII CCCAAGCTTCTATTCGATTTCCTCTAAAAC(SEQ ID NO:32)P4: HindIII CCC AAGCTT CTATTCGATTTCCTCTAAAAC (SEQ ID NO: 32)
PCR反应体系为The PCR reaction system is
PCR反应条件为:The PCR reaction conditions are:
将PCR扩增产物在1%的琼脂糖凝胶中电泳30分钟,得到大小约为700bp的ydiH1片段,利用上海生工DNA片段回收纯化试剂盒(上海生物工程有限公司,中国上海)按照厂商说明书回收和纯化T-ydiH片段。The PCR amplified product was electrophoresed in 1% agarose gel for 30 minutes to obtain a ydiH1 fragment with a size of about 700 bp, which was purified using the Shanghai Sangon DNA Fragment Recovery Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions Recovery and purification of T-ydiH fragments.
3.目的基因与载体的连接3. Ligation of target gene and vector
将回收纯化的PCR片段T-ydiH以及载体质粒pRSETb分别进行双酶切,体系如下:The recovered and purified PCR fragment T-ydiH and the vector plasmid pRSET b were subjected to double enzyme digestion respectively, and the system was as follows:
反应条件:37℃,5小时。Reaction conditions: 37°C, 5 hours.
反应结束后,50μl反应体系中加入10μl 6×上样缓冲液终止反应。然后通过琼脂糖凝胶电泳分离目的片段,利用上海生工DNA片段回收纯化试剂盒(上海生物工程有限公司,中国上海)按照厂商说明书回收并纯化片段。After the reaction, 10 μl of 6× loading buffer was added to the 50 μl reaction system to terminate the reaction. Then, the target fragment was separated by agarose gel electrophoresis, and the fragment was recovered and purified using the Shanghai Sangon DNA Fragment Recovery and Purification Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions.
将回收到的T-ydiH双酶切产物及载体质粒pRSETb双酶切产物连接,体系如下Ligate the recovered T-ydiH double digestion product with the carrier plasmid pRSET b double digestion product, the system is as follows
反应条件:16℃,过夜。从而形成连接产物pRSETb-ydiH。Reaction conditions: overnight at 16°C. Thus forming the ligation product pRSET b -ydiH.
以上述经过验证的pRSETb-ydiH为模板,利用引物T-ydiH(L190)F和T-ydiH(F189)R扩增pRSETb-ydiH序列全长,其中引物T-ydiH(L190)F和T-ydiH(F189)R扩增获得N端含有PstI酶切位点C端含有KpnI酶切位点的pRSETb-ydiH序列全长片段yidH-pRSETb,引物T-ydiH(L190)F和T-ydiH(F189)R序列如下:Using the above verified pRSET b -ydiH as a template, use primers T-ydiH(L190)F and T-ydiH(F189)R to amplify the full length of the pRSET b -ydiH sequence, where primers T-ydiH(L190)F and T -ydiH(F189)R amplified to obtain pRSET b containing a PstI restriction site at the N - terminus and a KpnI restriction site at the C-terminus. The sequence of ydiH(F189)R is as follows:
P5:KpnI ATAGGTACCGGCCTGGCCGGCCTGACCCGGCTG(SEQ IDNO:33)P5: KpnI ATA GGTACC GGCCTGGCCGGCCTGACCCGGCTG (SEQ ID NO: 33)
P6:PstI ATACTGCAGAGAAGTCCACGTTCTCCACGGCCACCTC(SEQ ID NO:34)P6: PstI ATA CTGCAG AGAAGTCCACGTTCTCCACGGCCACCTC (SEQ ID NO: 34)
最后,利用以下条件对上述yidH-pRSETb片段和经过验证的cpYFP片段双酶切:Finally, the above yidH-pRSET b fragment and the verified cpYFP fragment were double digested using the following conditions:
反应条件:37℃,5小时。Reaction conditions: 37°C, 5 hours.
反应结束后,50μl反应体系中加入10μl 6×上样缓冲液终止反应。然后通过琼脂糖凝胶电泳分离目的片段,利用上海生工DNA片段回收纯化试剂盒(上海生物工程有限公司,中国上海)按照厂商说明书回收并纯化片段。After the reaction, 10 μl of 6× loading buffer was added to the 50 μl reaction system to terminate the reaction. Then, the target fragment was separated by agarose gel electrophoresis, and the fragment was recovered and purified using the Shanghai Sangon DNA Fragment Recovery and Purification Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions.
如上所述,将回收到的yidH-pRSETb和cpYFP的双酶切产物连接,从而形成最终连接产物pRSETb-ydiH(189)-YFP-ydiH(190)。As described above, the recovered yidH-pRSET b and cpYFP double digestion products were ligated to form the final ligation product pRSET b -ydiH(189)-YFP-ydiH(190).
取菌落PCR鉴定为阳性的克隆,采用通用引物测序,由北京六合华大基因科技股份有限公司上海分公司进行测序。测定的序列用Vector NTI 8.0进行数据比对分析。The clones identified as positive by colony PCR were sequenced with universal primers and sequenced by Beijing Liuhe Huada Gene Technology Co., Ltd. Shanghai Branch. The determined sequences were compared and analyzed using Vector NTI 8.0.
4.转化4. Conversion
将重组质粒pRSETb-ydiH(189)-YFP-ydiH(190)转化入感受态的大肠杆菌(E.coli)BL21(DE3)pLysS(购自天根生化,中国北京)中,获得重组菌BL-Frex,具体方法如下:Transform the recombinant plasmid pRSET b -ydiH(189)-YFP-ydiH(190) into competent Escherichia coli (E.coli) BL21(DE3)pLysS (purchased from Tiangen Biochemical, Beijing, China) to obtain the recombinant strain BL -Frex, the specific method is as follows:
(i)在洁净条件下,取1μl质粒或10μl连接产物加入100μl感受态中,冰浴45分钟;(i) Under clean conditions, take 1 μl of plasmid or 10 μl of ligation product and add it to 100 μl of competent cells, and place in ice bath for 45 minutes;
(ii)冰浴后,迅速于42℃水浴中热激90~120秒;(ii) After ice bathing, quickly heat shock in a water bath at 42°C for 90-120 seconds;
(iii)再冰浴5分钟;(iii) ice bath for 5 minutes;
(iv)加入800ul LB液体培养基,37℃、220rpm摇床复苏1小时;(iv) Add 800ul LB liquid medium, recover on a shaker at 37°C and 220rpm for 1 hour;
(v)4000rpm、常温离心5分钟后,弃去上清;(v) After centrifuging at 4000rpm and normal temperature for 5 minutes, discard the supernatant;
(vi)用少量的新鲜LB重悬沉淀,随后将全部菌液均匀涂布于所需的LB平板上,37℃倒置培养过夜。(vi) Resuspend the pellet with a small amount of fresh LB, then evenly spread all the bacterial solution on the desired LB plate, and culture it upside down at 37°C overnight.
采用常规的菌落PCR方法筛选阳性克隆,并转入5ml含有相应抗性的LB液体培养基中,37℃、220rpm过夜培养。重组菌BL-Perex在LB培养基中37℃培养至菌体浓度OD为0.8,加入0.1mM IPTG,18℃诱导表达20小时,用Ni2+亲和层析柱(通用电气,瑞典乌普萨拉)从菌体裂解液中分离纯化F-rex2蛋白,经SDS-PAGE鉴定,只在约50kD处有一条蛋白条带,为F-rex2蛋白(图1),图1中1为Ni2+亲和层析柱分离纯化的F-rex2蛋白,2为标记物。Positive clones were screened by conventional colony PCR method, and transferred to 5 ml of LB liquid medium containing the corresponding resistance, and cultured overnight at 37°C and 220rpm. Recombinant strain BL-Perex was cultured in LB medium at 37°C until the bacterial cell concentration OD was 0.8, added 0.1mM IPTG, induced expression at 18°C for 20 hours, and used Ni 2+ affinity chromatography column (General Electric, Uppsa, Sweden) Pull) Separation and purification of F-rex2 protein from thalline lysate, through SDS-PAGE identification, only a protein band is arranged at about 50kD place, is F-rex2 protein (Fig. 1), 1 is Ni 2+ in Fig. 1 The purified F-rex2 protein was separated by an affinity chromatography column, and 2 was a marker.
实施例2.ydiH(189)-YFP-ydiH(190)衍生系列探针Example 2. ydiH(189)-YFP-ydiH(190) derived series probes
探针构建原理Probe construction principle
利用构建pRSETb-ydiH(189)-YFP-ydiH(190)等探针的中间过渡质粒为模板,根据定点突变的原理,进行衍生系列探针的构建。Using the intermediate transition plasmids for constructing probes such as pRSET b -ydiH(189)-YFP-ydiH(190) as templates, a series of derivative probes were constructed according to the principle of site-directed mutagenesis.
突变文库的建立Construction of mutant library
1.引物设计(上海生工)1. Primer design (Shanghai Sangong)
2.PCR扩增2. PCR amplification
利用定点突变PCR进行截短突变及定点突变。Truncation and site-directed mutagenesis were performed using site-directed mutagenesis PCR.
突变PCR扩增体系(引物、酶、dNTP等来自富酶泰斯公司):Mutant PCR amplification system (primers, enzymes, dNTPs, etc. are from Fuzyces Co.):
3.DNA片段分离、纯化3. Separation and purification of DNA fragments
DpnI消化DpnI digestion
首先利用DpnI酶(来自富酶泰斯公司)在37℃处理上述PCR扩增片段3小时,以便去除潜在的模板质粒污染。然后,使反应体系在80℃变性失活20分钟。经变性失活的反应混合物可以直接用于后续的分子生物学实验。Firstly, the above PCR amplified fragment was treated at 37°C for 3 hours with DpnI enzyme (from Fuzyces Co., Ltd.) in order to remove potential template plasmid contamination. Then, the reaction system was denatured and inactivated at 80° C. for 20 minutes. The denatured and inactivated reaction mixture can be directly used in subsequent molecular biology experiments.
DNA片段磷酸化Phosphorylation of DNA fragments
在ATP存在下,利用T4多聚核苷酸激酶(T4polynucleotide kinase,T4PNK)(来自富酶泰斯公司)在37℃处理1小时,以使DNA核糖环的5’-OH磷酸化,以便于片段环化自连。然后,使反应体系在75℃变性失活10分钟。变性失活的反应混合物可以直接用于后续的分子生物学实验。In the presence of ATP, use T4 polynucleotide kinase (T4polynucleotide kinase, T4PNK) (from Fuzitaisi company) to treat for 1 hour at 37°C to phosphorylate the 5'-OH of the DNA ribose ring to facilitate fragmentation Circular self-connection. Then, the reaction system was denatured and inactivated at 75° C. for 10 minutes. The denatured and inactivated reaction mixture can be directly used in subsequent molecular biology experiments.
连接connect
用T4DNA连接酶(来自富酶泰斯公司)将经过磷酸化处理的DNA片段(突变的DNA片段pRSETb-ydiH-YFP或pRSETb-YFP-ydiH)进行环化自连(16℃,过夜)。Circular self-ligation of phosphorylated DNA fragments (mutated DNA fragments pRSET b -ydiH-YFP or pRSET b -YFP-ydiH) with T4 DNA ligase (from Fuzydase) (16°C, overnight) .
突变体质粒鉴定Mutant plasmid identification
取菌落PCR筛选为阳性的克隆,采用通用引物测序,由北京六合华大基因科技股份有限公司上海分公司完成。测定的序列用Vector NTI 8.0进行数据比对分析。Colony PCR screened positive clones were taken and sequenced with universal primers, which was completed by Shanghai Branch of Beijing Liuhe Huada Gene Technology Co., Ltd. The determined sequences were compared and analyzed using Vector NTI 8.0.
构建探针系列Build a Probe Series
根据上述方法可进一步获得下述探针系列,并予以分别编号。According to the above method, the following probe series can be further obtained and numbered respectively.
实施例3还原型和氧化型烟酰胺腺嘌呤二核苷酸比率荧光探针测定NADH/NAD+比率的变化Example 3 Fluorescent Probes for the Ratio of Reduced and Oxidized Nicotinamide Adenine Dinucleotide Determination of Changes in NADH/NAD+ Ratio
还原型和氧化型烟酰胺腺嘌呤二核苷酸比率荧光探针的结构是,在Trex的F189和L190这两处氨基酸中间插入cpYFP,此探针的序列是SEQ ID NO:148,制备方法同实施例2。该探针仅对NADH和NAD+有响应,对NADH类似物没有任何响应,当采用485nm激发可发现NAD和NADH结合均能导致探针528nm发射荧光均有增强,但是采用420nm激发,仅NADH的结合能够致使探针发生响应。由于探针420nm激发与485nm激发,均能于528nm处产生发射荧光,因此利用不同波长的荧光激发,测定其这两者在528nm出发射荧光的强度比值(420nm/485nm)可发现仅NADH的结合导致探针荧光比值的响应为上升,而NAD+的结合导致其响应为下降(图3-1)。当NADH和NAD保持总浓度不变,调整二者的相互比例时,该趋势变化更为明显,且不随总浓度的变化而变化(图3-2)。The structure of the reduced and oxidized nicotinamide adenine dinucleotide ratio fluorescent probe is that cpYFP is inserted between the amino acids F189 and L190 of Trex. The sequence of this probe is SEQ ID NO: 148, and the preparation method is the same as Example 2. The probe only responds to NADH and NAD + , and has no response to NADH analogues. When using 485nm excitation, it can be found that the combination of NAD and NADH can lead to the enhancement of the 528nm emission fluorescence of the probe, but using 420nm excitation, only NADH Binding can render the probe responsive. Since the probes are excited at 420nm and 485nm, both can emit fluorescence at 528nm, so using different wavelengths of fluorescence excitation, measuring the intensity ratio (420nm/485nm) of the two emission fluorescence at 528nm can find that only the binding of NADH The response of probe fluorescence ratio is increased, while the binding of NAD + causes its response to decrease (Fig. 3-1). When the total concentration of NADH and NAD is kept constant and the mutual ratio of the two is adjusted, the trend change is more obvious and does not change with the change of the total concentration (Figure 3-2).
当含有20uM的NADP,NADPH,ADP和ATP时,[NADH]/[NAD]按照一定比例去滴定,我们发现变化的响应倍数和变化趋势均没有受其影响,说明NADP,NADPH,ADP和ATP四种类似物对探针没有影响(如图3-3)。When containing 20uM NADP, NADPH, ADP and ATP, [NADH]/[NAD] was titrated according to a certain ratio, and we found that the response times and changing trends were not affected by it, indicating that NADP, NADPH, ADP and ATP four The analogs have no effect on the probe (as shown in Figure 3-3).
因此该探针既可以做还原型和氧化型烟酰胺腺嘌呤二核苷酸比率探针,还可以单独做还原型烟酰胺腺嘌呤二核苷酸探针。Therefore, the probe can be used as a ratio probe of reduced form and oxidized form of nicotinamide adenine dinucleotide, and can also be used as a reduced form of nicotinamide adenine dinucleotide probe alone.
其它实施方式other implementations
本说明书描述了许多实施方式。然而应理解,本领域技术人员通过阅读本说明书获知不背离本发明的构思和范围的各种改进。因此,这些其他实施方式也应包括在所附权利要求书的范围内。This specification describes a number of implementations. However, it should be understood that various modifications without departing from the spirit and scope of the present invention will come to those skilled in the art from reading this specification. Accordingly, such other implementations should also be included within the scope of the appended claims.
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