WO2020125545A1 - 大斯托克斯橙色荧光蛋白LSSmKO1及其应用 - Google Patents

大斯托克斯橙色荧光蛋白LSSmKO1及其应用 Download PDF

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WO2020125545A1
WO2020125545A1 PCT/CN2019/124979 CN2019124979W WO2020125545A1 WO 2020125545 A1 WO2020125545 A1 WO 2020125545A1 CN 2019124979 W CN2019124979 W CN 2019124979W WO 2020125545 A1 WO2020125545 A1 WO 2020125545A1
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fluorescent protein
large stokes
orange fluorescent
mkoκ
orange
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储军
郭育奇
张楚秋
刘丰
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Shenzhen Institute of Advanced Technology of CAS
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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  • the invention relates to the technical field of bio-optics and molecular imaging, in particular, to a large Stokes orange fluorescent protein LSSmKO1 and the application of the large Stokes orange fluorescent protein LSSmKO1.
  • the development of the large Stokes fluorescent protein can effectively solve this problem.
  • This kind of protein can realize the same excitation light to excite three or more fluorescent proteins at the same time, so as to obtain the fluorescence of different wavelength regions, and achieve the purpose of imaging three or more fluorescent proteins at the same time.
  • the excitation peak of the large Stokes orange fluorescent protein (LSSOFP) is very close to the green fluorescent protein (GFP) and the large Stokes red fluorescent protein (LSSRFP), and a beam of light with a specific wavelength can be found to excite three With proteins, the emission peaks of the three are far apart, the emission spectra overlap less, and the fluorescent signals can be received separately, thereby realizing the simultaneous three-color imaging of living cells.
  • a large Stokes orange fluorescent protein is provided, the large Stokes orange fluorescent protein is a mutant orange fluorescent protein mKO ⁇ , compared with the amino acid sequence of mKO ⁇ , The large Stokes orange fluorescent protein has a mutation site: V160D.
  • the application of Big Stokes orange fluorescent protein in single-photon, multi-photon and super-resolution microscopic imaging is provided.
  • the application of Big Stokes orange fluorescent protein in a bioluminescence resonance energy transfer system is provided.
  • the application of large Stokes orange fluorescent protein in imaging based on random single molecule localization super-resolution technology is provided.
  • the large Stokes fluorescent protein LSSmKO1 provided by the present invention has the following advantages:
  • Figure 1 is a comparison diagram of the amino acid sequence of the large Stokes orange fluorescent protein LSSmKO1 and the amino acid sequence of the orange fluorescent protein mKO ⁇ ;
  • Figure 2 is the DNA sequence of the large Stokes orange fluorescent protein LSSmKO1 gene
  • Figure 3 is the excitation and emission spectra of the large Stokes orange fluorescent protein LSSmKO1;
  • Figure 4 is a high performance liquid chromatography peak diagram of the large Stokes orange fluorescent protein LSSmKO1.
  • inventive concept will be described in more detail with reference to the drawings.
  • inventive concept can be implemented in various different forms, and should not be interpreted as being limited to the technical solutions shown here only. Rather, these embodiments are provided as examples so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. Therefore, experimental steps, conditions, instruments, etc., which are unnecessary for those of ordinary skill in the art to fully understand the various aspects and features of the inventive concept, may not be described. If no specific conditions are indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be used. The reagents or instruments used do not indicate the manufacturer, are all conventional products that can be obtained through commercial purchase.
  • the Stokes shift refers to the difference between the maximum emission wavelength and the maximum excitation wavelength of the fluorescent substance. If the difference is greater than 100 nm, the fluorescent protein is considered to have a large Stokes shift characteristic. In the multicolor simultaneous imaging experiment, the Stokes fluorescent protein can ensure the simultaneous fluorescence imaging of two or more fluorescent proteins with different emission spectra, and effectively reduce the spectral crosstalk between different fluorescent proteins.
  • mKeima is a red fluorescent protein with emission and absorption peaks at 440nm and 620nm, respectively, and cyan fluorescent protein (CFP) and mKeima are excited using 458nm light.
  • CFP cyan fluorescent protein
  • mKeima has two excitation peaks. In addition to being excited at 440nm, it can also be excited at 584nm, which causes some difficulties for multicolor imaging.
  • Piatkevich KD et al. obtained the fluorescent protein mutants LSSmKatel and LSSmKate2 in 2010. These two mutants have the same excitation wavelength, but different emission wavelengths, so they can be better used to carry out Two-photon in vivo observation of physiological and biochemical reactions such as metastasis of tumor cells (Piatkevich KD, Hulit J, Subach OM, Wu B, Abdulla A, Segall JE, Verkhusha VV.
  • mBeRFP Compared with LSS-mKate2 and mKeima, mBeRFP has the advantages of high light intensity, good light stability and fast maturity (Yang J, Wang L, Yang F, Luo H, Xu L, Lu J, Zeng S, Zhang Z.mBeRFP, an improved large shifts red fluorescent protein.PLoS One, 2013, 8(6): e64849.). Guan et al. obtained a monomeric fluorescent protein hmKeima8.5 with a large Stokes shift in 2015.
  • This protein and the blue-green fluorescent protein mTFP1 form an efficient fluorescent protein pair for multiphoton and multicolor imaging
  • the wavelengths of the emission peaks and peaks of the two fluorescent proteins differ by 120nm, therefore, crosstalk can be effectively avoided, and the brightness of hmKeima8.5 is also significantly superior to its previous generation mKeima (Guan Y, Meurer M, Raghavan S , Rebane A, Lindquist JR, Santos S, Kats I, Davidson MW, Mazitschek R, Hughes TE, Drobizhev M, Knop M, Shah JJ. , 2015, 26(11): 2054-66.).
  • the existing Big Stokes orange fluorescent protein is only one kind of LSSmOrange, which was obtained by Shcherbakova DM and Hink MA in 2012.
  • the excitation and emission peaks of this protein are 437nm and 572nm, respectively.
  • the excitation peak of this protein is very close to the green fluorescent protein (GFP) and the large Stokes red fluorescent protein (LSSRFP). Therefore, a beam of light with a specific wavelength can be found to excite three proteins at the same time, which can be used for three-color simultaneous live cells Imaging experiment.
  • GFP green fluorescent protein
  • LSSRFP large Stokes red fluorescent protein
  • the emission peak of LSSmOrange is relatively close to the emission peak of LSSRFP, and there is a certain spectrum overlap, which causes a large crosstalk.
  • the present invention provides a large Stokes orange fluorescent protein, which is named LSSmKO1.
  • the fluorescent protein was selected by site-directed mutagenesis of orange fluorescent protein mKO ⁇ .
  • FIG. 1 shows a comparison diagram of the amino acid sequence of the large Stokes orange fluorescent protein LSSmKO1 according to the inventive concept and the amino acid sequence of the orange fluorescent protein mKO ⁇ , in which the mutated amino acid positions are marked in gray.
  • the large Stokes shifted fluorescent protein LSSmKO1 has the following mutation site: V160D.
  • This mutation site reflects the large Stokes shift characteristic of LSSmKO1, specifically, the 160th valine of the mKO ⁇ amino acid sequence is replaced by aspartic acid.
  • the amino acid sequence of LSSmKO1 has the following mutation sites compared to the amino acid sequence of orange fluorescent protein mKO ⁇ : M11T, E40A, T42K, R44K, A56S, L59I, A63S , H68N, V70A, E76A, L96M, S105K, Y118I, I134V, N137K, E167V, C174V, F178I, G196S, V200E, K202I, V211Y, H217S, S218Y, 219-228: SNLGMDELYK.
  • These mutation sites reflect the characteristics of LSSmKO1's maturity rate, brightness and monosomy.
  • the mutation site described above may specifically be: the methionine at position 11 of the amino acid sequence of mKO ⁇ is replaced by threonine, the glutamic acid at position 40 is replaced by glycine, and the threonine at position 42 is replaced by Lysine is substituted, arginine at position 44 is replaced by lysine, alanine at position 56 is replaced by serine, leucine at position 59 is replaced by isoleucine, position 63 Of alanine is replaced by serine, histidine at position 68 is replaced by asparagine, valine at position 70 is replaced by alanine, and glutamic acid at position 76 is replaced by alanine , The leucine at position 96 was replaced by methionine, the serine at position 105 was replaced by lysine, the tyrosine at position 118 was replaced by isoleucine, and the isoleucine at position 134 was replaced by va Is replaced by lysine, aspara
  • Phenylalanine at position 196 was replaced by isoleucine
  • glycine at position 196 was replaced by serine
  • valine at position 200 was replaced by glutamic acid
  • lysine at position 202 was replaced by isoleucine
  • valine at position 211 was replaced by tyrosine
  • histidine at position 217 was replaced by serine
  • serine at position 218 was replaced by tyrosine
  • serine at positions 219-228 was added, Asparagine, leucine, glycine, methionine, aspartic acid, glutamic acid, leucine, tyrosine, lysine.
  • the present invention also provides a gene encoding the above-mentioned large Stokes fluorescent protein LSSmKO1.
  • a gene encoding the above-mentioned large Stokes fluorescent protein LSSmKO1.
  • the DNA base sequence of the gene is shown in FIG. 2, and the base sequence can be obtained by gene synthesis.
  • the present invention also provides the application of the above-mentioned large Stokes fluorescent protein LSSmKO1 in single-photon and multi-photon micro-color imaging.
  • the fluorescent protein can be used in single-photon and multi-photon microimaging together with green fluorescent protein while being excited by monochromatic light for multicolor imaging and applied to light sheet-type nonlinear optical microscopy.
  • the present invention also provides the application of the above-mentioned large Stokes fluorescent protein LSSmKO1 in a new bioluminescence resonance energy transfer system BRET.
  • the acceptor in this system may be luciferase NanoLuc
  • the donor may be the above-mentioned fluorescent protein LSSmKO1.
  • the emission spectrum of luciferase NanoLuc generally overlaps with the absorption spectrum of LSSmKO1. Therefore, if the distance between the two proteins is close enough, the energy generated by the oxidation reaction intermediate of luciferase NanoLuc will be transferred by bioluminescence resonance energy.
  • the principle is transferred to LSSmKO1, which excites the latter, so that the system emits the emission spectrum of LSSmKO1.
  • the present invention also provides the application of the above-mentioned large Stokes fluorescent protein LSSmKO1 in the imaging of super-resolution technology based on random single molecule localization.
  • the sample is illuminated by the excitation light, and the points on the image will not be bright when two close points are bright at the same time, and super resolution can be achieved by positioning.
  • a high-resolution image can be obtained by positioning hundreds of thousands of single molecules through thousands of pictures. .
  • the mKO ⁇ gene was amplified by polymerase chain reaction, and then the mutants were expressed and screened on a constitutive expression vector.
  • the expression strain used was Stellar (purchased from Agilent Technologies). To ensure the integrity of the library, each mutation There are 10 clones in the body, and finally, the fluorescent properties of the mutants are detected by the naked eye and the blue LED excitation light through the orange acrylic filter, so as to screen out the monoclonal protein expressing the fluorescent protein of the large Stokes shift, which is the above-mentioned Das Tox shifts the fluorescent protein LSSmKO1.
  • the excitation spectrum of LSSmKO1 is represented by a dotted line, and the emission spectrum thereof is represented by a solid line.
  • the excitation and emission peaks of LSSmKO1 are around 445nm and 568nm, respectively.
  • LSSmKO1 matures significantly faster, under the same incubation time and temperature, LSSmKO1 will be brighter than LSSmOrange first.
  • LSSmKO1 was extracted and purified, and detected by high performance liquid chromatography and size exclusion column, it can be confirmed that the peak time of LSSmKO1 is very close to Fusionred, which is obviously later than the diploid LSSmKate2. Fusionred is currently recognized as a fluorescent protein with good monomericity, and its peak time is very close to indicate that LSSmKO1 is also a fluorescent protein with good monomericity. Since LSSmKO1 is a monomeric fluorescent protein, it will not cause mutual aggregation of fluorescent protein molecules under in vitro conditions.
  • the present invention provides the application of the large Stokes orange fluorescent protein LSSmKO1 and the gene encoding the above-mentioned large Stokes fluorescent protein LSSmKO1 and the large Stokes orange fluorescent protein LSSmKO1.
  • the large Stokes fluorescent protein LSSmKO1 provided by the present invention has the following advantages:

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Abstract

提供了大斯托克斯橙色荧光蛋白LSSmKO1及其应用。所述大斯托克斯橙色荧光蛋白是一种突变的橙色荧光蛋白mKOκ，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有突变位点：V160D。

Description

大斯托克斯橙色荧光蛋白LSSmKO1及其应用 技术领域
本发明涉及生物光学与分子影像学技术领域，具体地，涉及一种大斯托克斯橙色荧光蛋白LSSmKO1以及该大斯托克斯橙色荧光蛋白LSSmKO1的应用。
背景技术
在使用荧光蛋白进行活细胞或活体成像时，往往难以实现多色同时成像，若能突破这一限制，则可实现对多个生物快速反应事件的检测，将极大推动神经生物学的发展。
在单光子成像中，可采用由不同激发波长的光激发多种荧光蛋白来实现多色同时成像。然而，在多光子成像中，添加一个二级钛蓝宝石雷射或光学参数振荡器的费用较高，并且难以将它们调控到一起，因此，荧光蛋白的三色或多色同时成像较难实现。目前同时成像多个生物分子事件的显微镜方法主要依赖对激光的动态调制，如果想做多色的荧光同时成像就要求多套重复的光学器件以及更加精确的同步控制，然而这方面的很多技术难关还没有被攻破，所以要实现荧光蛋白的三色或多色同时成像非常困难。因此，在基本不改变原有显微系统的情况下开发出一种可用于活体多通道同时成像的荧光蛋白来实现该功能显得十分必要。
然而，开发出大斯托克斯荧光蛋白可有效的解决这一问题。该类蛋白可实现同一激发光同时激发三种或多种荧光蛋白，从而得到不同波长区域的荧光，达到三种或多种荧光蛋白同时成像的目的。然而，大斯托克斯橙色荧光蛋白(LSSOFP)的激发峰与绿色荧光蛋白(GFP)和大斯托克斯红色荧光蛋白(LSSRFP)很接近，可找到一束特定波长的光同时激发三种蛋白，使三者的发射峰相隔较远，发射光谱重叠较少，荧光信号可分别被接收，从而实现活细胞的三色同时成像。
可是，现在已有的LSSOFP种类非常少，并且现有的LSSOFP的发射光谱与LSSRFP的发射光谱重叠较大，成像串扰严重，所以开发出一种新的发射光谱蓝移的LSSOFP很有必要。
发明内容
根据本发明构思的一些实施例，提供了一种大斯托克斯橙色荧光蛋白，所述大斯托克斯橙色荧光蛋白是一种突变的橙色荧光蛋白mKOκ，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有突变位点：V160D。
根据本发明构思的一些实施例，提供了大斯托克斯橙色荧光蛋白在单光子、多光子和超分辨显微成像中的应用。
根据本发明构思的一些实施例，提供了大斯托克斯橙色荧光蛋白在生物发光共振能量转移系统中的应用。
根据本发明构思的一些实施例，提供了大斯托克斯橙色荧光蛋白在基于随机单分子定位的超分辨技术成像中的应用。
本发明提供的大斯托克斯荧光蛋白LSSmKO1具有以下优点：
(1)具有极好的单体性；
(2)相对于其它具有大斯托克斯位移的橙色荧光蛋白来说具有亮度高、成熟时间短的优点；
(3)具有大斯托克斯位移，可用于单光子、多光子和超分辨显微多色同时成像，因此可以与绿色荧光蛋白mTFP2一起同时被单色光以双光子的模式激发，提高了双光子成像的分辨率，应用于光片式非线性光学显微成像；
(4)可以应用于新的生物发光共振能量转移系统BRET，其作为供体与受体荧光素酶NanoLuc结合，可在生物体内形成一种新的高灵敏的生物发光共振能量转移系统BRET，提高了活体成像的深度和灵敏度。
附图说明
通过下面结合附图对本发明的实施例的描述，本发明的上述和其它目的和特点将会变得更加清楚，在附图中：
图1是大斯托克斯橙色荧光蛋白LSSmKO1的氨基酸序列与橙色荧光蛋白mKOκ的氨基酸序列对比图；
图2是大斯托克斯橙色荧光蛋白LSSmKO1基因的DNA序列；
图3是大斯托克斯橙色荧光蛋白LSSmKO1的激发光谱图和发射光谱图；
图4是大斯托克斯橙色荧光蛋白LSSmKO1的高效液相色谱峰图。
具体实施方式
在下文中，将参照附图更详细地描述本发明构思。然而，本发明构思可以以各种不同的形式来实施，并且不应该被解释为局限于仅在这里示出的技术方案。相反地，提供这些实施例作为示例，使得本公开将是彻底的和完整的，并将把本发明构思充分地传达给本领域技术人员。因此，可以不描述对于本领域普通技术人员完全理解本发明构思的各个方面和特征而言是不必要的实验步骤、条件和仪器等。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则这里用的所有术语(包括技术术语和科学术语)具有与本发明所属领域的普通技术人员通常理解的含义相同的含义。还将理解的是，术语(诸如在通用字典中定义的术语)应该被解释为具有与它们在相关领域的上下文和本说明书中的意思一致的意思，并且不应以理想化和/或过于形式化的含义来解释它们，除非这里明确地如此定义。
斯托克斯位移指的是荧光物质的最大发射波长和最大激发波长之差。该差值若大于100nm，则认为该荧光蛋白具有大斯托克斯位移的特性。大斯托克斯荧光蛋白在多色同时成像实验中，可保证两个或多个不同发射光谱的荧光蛋白同时荧光成像，并且有效地减少不同荧光蛋白之间的光谱串扰。
最早报道的大斯托克斯位移的荧光蛋白为mKeima，它是一种红色荧光蛋白，其发射峰与吸收峰分别位于440nm和620nm，采用458nm的光激发青色荧光蛋白(CFP)和mKeima，可达到同时激发两个荧光分子的目的，从而实现单光源的多色成像(Kogure T，Karasawa S，Araki T，Saito K，Kinjo M，Miyawaki A.A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.Nat Biotechnol，2006，24：577-581)。但mKeima的缺点在于，它有两个激发峰，除了能在440nm处被激发，还能在584nm处被激发，这对多色成像造成了一些困难。为了克服mKeima的这一缺点，Piatkevich K.D.等人于2010年得到了荧光蛋白突变体LSSmKatel和LSSmKate2，这两个突变体具有同样的激发波长，但发射波长却不同，因此可较好的利用它们进行双光子活体观察肿瘤细胞的转移等生理生化反应(Piatkevich KD，Hulit J，Subach OM，Wu B，Abdulla A，Segall JE，Verkhusha VV. Monomeric red fluorescent proteins with a large Stokes shift.Proc Natl Acad Sci USA，2010，107：5369-5374)。Jie Yang等人于2013年得到了荧光蛋白突变体mBeRFP，它是一种红色荧光蛋白，其发射峰与吸收峰分别位于446nm和611nm处，可以采用458nm的光激发CFP和mBeRFP。mBeRFP与LSS-mKate2和mKeima相比具有光强度高、光稳定性好、成熟快等优点(Yang J，Wang L，Yang F，Luo H，Xu L，Lu J，Zeng S，Zhang Z.mBeRFP，an improved large stokes shift red fluorescent protein.PLoS One，2013，8(6)：e64849.)。Guan Y等人于2015年得到了一种单体的具有大斯托克斯位移的荧光蛋白hmKeima8.5，该蛋白与蓝绿色荧光蛋白mTFP1组成高效的荧光蛋白对用于多光子和多色成像，所述两种荧光蛋白的发射峰峰值所在的波长相差120nm，因此，可以有效的避免串扰，且hmKeima8.5在亮度上也明显优于它的前一代mKeima(Guan Y，Meurer M，Raghavan S，Rebane A，Lindquist JR，Santos S，Kats I，Davidson MW，Mazitschek R，Hughes TE，Drobizhev M，Knop M，Shah JV.Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.Mol Biol Cell，2015，26(11)：2054-66.)。
现有的大斯托克斯橙色荧光蛋白只有LSSmOrange一种，该蛋白是2012年由Shcherbakova DM和Hink MA研究得到。该蛋白的激发峰和发射峰分别为437nm和572nm。该蛋白的激发峰与绿色荧光蛋白(GFP)和大斯托克斯红色荧光蛋白(LSSRFP)很接近，因此，可找到一束特定波长的光同时激发三种蛋白，可用于活细胞三色同时成像的实验。但是LSSmOrange的发射峰与LSSRFP的发射峰波长相对较近，有一定光谱重叠，造成串扰较大，所以开发出一种发射光谱蓝移的大斯托克斯橙色荧光蛋白很有必要(Shcherbakova DM，Hink MA，Joosen L，Gadella TW，Verkhusha VV.An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging.J Am Chem Soc，2012，134(18)：7913-23.)。
为了解决上述存在的技术问题，本发明提供了一种大斯托克斯橙色荧光蛋白，命名该荧光蛋白为LSSmKO1。该荧光蛋白是通过由橙色荧光蛋白mKOκ定点诱变筛选得到的。图1示出了根据发明构思的大斯托克斯橙色荧光蛋白LSSmKO1的氨基酸序列与橙色荧光蛋白mKOκ的氨基酸序列的对比图，其中，突变的氨基酸位点用灰色标出。
如图1中所示，与mKOκ的氨基酸序列相比，大斯托克斯位移荧光蛋白LSSmKO1具有以下突变位点：V160D。该突变位点体现了LSSmKO1的大斯托 克斯位移特性，具体为mKOκ氨基酸序列的第160位的缬氨酸被天冬氨酸所取代。
在一个示例性实施例中，如图1中所示，LSSmKO1的氨基酸序列与橙色荧光蛋白mKOκ的氨基酸序列相比，还具有以下突变位点：M11T，E40A，T42K，R44K，A56S，L59I，A63S，H68N，V70A，E76A，L96M，S105K，Y118I，I134V，N137K，E167V，C174V，F178I，G196S，V200E，K202I，V211Y，H217S，S218Y，219-228：SNLGMDELYK。这些突变位点体现了LSSmKO1的成熟率、亮度和单体性等特征。
另外，上面所描述的突变位点具体可以为：mKOκ的氨基酸序列的第11位的蛋氨酸被苏氨酸所取代，第40位的谷氨酸被甘氨酸所取代，第42位的苏氨酸被赖氨酸所取代，第44位的精氨酸被赖氨酸所取代，第56位的丙氨酸被丝氨酸所取代，第59位的亮氨酸被异亮氨酸所取代，第63位的丙氨酸被丝氨酸所取代，第68位的组氨酸被天冬酰胺所取代，第70位的缬氨酸被丙氨酸所取代，第76位的谷氨酸被丙氨酸所取代，第96位的亮氨酸被蛋氨酸所取代，第105位的丝氨酸被赖氨酸所取代，第118位的酪氨酸被异亮氨酸所取代，第134位的异亮氨酸被缬氨酸所取代，第137位的天冬酰胺被赖氨酸所取代，第167位的谷氨酸被缬氨酸所取代，第174位的半胱氨酸被缬氨酸所取代，第178位的苯丙氨酸被异亮氨酸所取代，第196位的甘氨酸被丝氨酸所取代，第200位的缬氨酸被谷氨酸所取代，第202位的赖氨酸被异亮氨酸所取代，第211位的缬氨酸被酪氨酸所取代，第217位的组氨酸被丝氨酸所取代，第218位的丝氨酸被酪氨酸所取代，第219-228位加入了丝氨酸、天冬酰胺、亮氨酸、甘氨酸、蛋氨酸、天冬氨酸、谷氨酸、亮氨酸、酪氨酸、赖氨酸。
另一方面，本发明还提供了编码上述大斯托克斯荧光蛋白LSSmKO1的基因。根据密码子的简并性，在上述大斯托克斯荧光蛋白LSSmKO1的氨基酸序列确定的情况下，所述基因的DNA碱基序列有很多种情况，其均属于本发明的保护范围。这里，所述基因的DNA碱基序列如图2所示，该碱基序列可以通过基因合成的方式获得。
另一方面，本发明还提供了上述大斯托克斯荧光蛋白LSSmKO1在单光子和多光子显微多色成像中的应用。例如，该荧光蛋白可以与绿色荧光蛋白一起在单光子和多光子显微成像中有效的同时被单色光激发进行多色成像并应用于光片式非线性光学显微成像。
另一方面，本发明还提供了上述大斯托克斯荧光蛋白LSSmKO1在新的生物发光共振能量转移系统BRET中的应用。例如，该系统中受体可以为荧光素酶NanoLuc，供体可以为上述的荧光蛋白LSSmKO1。荧光素酶NanoLuc的发射光谱大体与LSSmKO1的吸收光谱重叠，因此，如果这两种蛋白间的距离足够近，则荧光素酶NanoLuc的氧化反应中间体所产生的能量将通过生物发光共振能量转移的原理转移给LSSmKO1，从而激发后者，使得该系统发射出LSSmKO1的发射光谱。
另一方面，本发明还提供了上述大斯托克斯荧光蛋白LSSmKO1在基于随机单分子定位的超分辨技术成像中的应用。例如，用该荧光蛋白标记细胞中的结构和细胞器后，利用激发光照射样品，得到图像上的点不会有两个靠得很近的点同时亮，就可以通过定位的方式实现超分辨。虽然一次定位只能得到少数几个分子，但是通过数千张图片对数十万个单分子的定位，就可以获得一张高分辨率的图像。。
在下文中，将对本发明的特征、性能及其实现方法进行详细描述。
1、橙色荧光蛋白mKOκ的定点诱变
将mKOκ基因通过聚合酶链式反应扩增，然后在组成型表达载体上对突变体进行表达和筛选，所用表达菌株为Stellar(购买自安捷伦科技公司)，为确保文库的完整性，每个突变体设置10个克隆，最后通过肉眼分辨以及蓝光LED激发光透过橙色丙烯酸滤波器检测突变体的荧光性质，从而筛选出表达大斯托克斯位移的荧光蛋白的单克隆，即为上述大斯托克斯位移荧光蛋白LSSmKO1。
2、LSSmKO1的激发光谱和发射光谱检测实验
首先采用B-PER II(购买自皮尔斯公司)对表达LSSmKO1的菌株进行裂解，然后采用HisPur Cobalt Resin(购买自皮尔斯公司)纯化蛋白，紧接着通过Econo-Pac 10DG重力流色谱柱(购买自美国Bio-Rad公司)去盐。完成以上蛋白纯化步骤后，使用Infinite M1000多功能酶标仪检测LSSmKO1的激发光谱和发射光谱，其结果示于图3中。
3、LSSmKO1的单体性检测实验
首先采用B-PER II(购买自皮尔斯公司)对表达LSSmKO1的菌株进行裂解，然后采用HisPur Cobalt Resin(购买自皮尔斯公司)纯化蛋白，紧接着通过Econo-Pac 10DG重力流色谱柱(购买自美国Bio-Rad公司)去盐。完成以上蛋 白纯化步骤后，统一将荧光蛋白的浓度浓缩成10mg/ml，放入到进样瓶中，用岛津高效液相色谱仪和分子排阻柱子检测荧光蛋白的单体性，其结果示于图4中。
4、LSSmKO1的光谱性质和单体性
参照图3，LSSmKO1的激发光谱图由虚线表示，其发射光谱图由实线表示。LSSmKO1的激发峰和发射峰分别在大约445nm和568nm处。此外，与现在常用的LSSmOrange相比，LSSmKO1的成熟速度明显变快，在相同的培养时间和温度下，LSSmKO1会比LSSmOrange先变亮。
参照图4，将LSSmKO1蛋白提取纯化后，通过高效液相色谱和分子排阻柱检测后，可以确认，LSSmKO1出峰的时间与Fusionred非常接近，明显晚于二倍体的LSSmKate2。Fusionred是目前公认单体性很好的荧光蛋白，与它出峰的时间很接近说明LSSmKO1也是单体性很好的荧光蛋白。由于LSSmKO1是一种单体的荧光蛋白，所以在体外条件下不会引起荧光蛋白分子的相互聚集。
因此，本发明提供了大斯托克斯橙色荧光蛋白LSSmKO1以及编码上述大斯托克斯荧光蛋白LSSmKO1的基因以及大斯托克斯橙色荧光蛋白LSSmKO1的应用。
本发明提供的大斯托克斯荧光蛋白LSSmKO1具有以下优点：
(1)具有极好的单体性；
(2)相对于其它具有大斯托克斯位移的橙色荧光蛋白来说具有亮度高、成熟时间短的优点；
(3)具有大斯托克斯位移，可用于单光子、多光子和超分显微多色同时成像，因此可以与绿色荧光蛋白mTFP2一起同时被单色光以双光子的模式激发，提高了双光子成像的分辨率，应用于光片式非线性光学显微成像；
(4)可以应用于新的生物发光共振能量转移系统BRET，作为供体与受体荧光素酶NanoLuc结合，可在生物体内形成一种新的高灵敏的生物发光共振能量转移系统BRET，提高了活体成像的深度和灵敏度。
Figure PCTCN2019124979-appb-000001
Figure PCTCN2019124979-appb-000002

Claims (18)

  1. 一种大斯托克斯橙色荧光蛋白，其中：所述大斯托克斯橙色荧光蛋白是一种突变的橙色荧光蛋白mKOκ，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有突变位点：V160D。
  2. 根据权利要求1所述的大斯托克斯橙色荧光蛋白，其中，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有以下突变位点：M11T、E40A，T42K，R44K，A56S，L59I，A63S，H68N，V70A，E76A，L96M，S105K，Y118I，I134V，N137K，E167V，C174V，F178I，G196S，V200E，K202I，V211Y，H217S，S218Y，219-228：SNLGMDELYK。
  3. 根据权利要求2所述的大斯托克斯橙色荧光蛋白，其中，所述大斯托克斯橙色荧光蛋白的基因的DNA碱基序列为：
    Figure PCTCN2019124979-appb-100001
  4. 一种大斯托克斯橙色荧光蛋白在单光子、多光子和超分辨显微成像中的应用，其中：所述大斯托克斯橙色荧光蛋白是一种突变的橙色荧光蛋白mKOκ，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有突变位点：V160D。
  5. 根据权利要求4所述的应用，其中，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有以下突变位点：M11T、E40A，T42K，R44K，A56S，L59I，A63S，H68N，V70A，E76A，L96M，S105K，Y118I，I134V，N137K，E167V，C174V，F178I，G196S，V200E，K202I，V211Y，H217S，S218Y，219-228： SNLGMDELYK。
  6. 根据权利要求5所述的应用，其中，所述大斯托克斯橙色荧光蛋白的基因的DNA碱基序列为：
    Figure PCTCN2019124979-appb-100002
  7. 一种大斯托克斯橙色荧光蛋白在生物发光共振能量转移系统中的应用，其中：所述大斯托克斯橙色荧光蛋白是一种突变的橙色荧光蛋白mKOκ，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有突变位点：V160D。
  8. 根据权利要求7所述的应用，其中，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有以下突变位点：M11T、E40A，T42K，R44K，A56S，L59I，A63S，H68N，V70A，E76A，L96M，S105K，Y118I，I134V，N137K，E167V，C174V，F178I，G196S，V200E，K202I，V211Y，H217S，S218Y，219-228：SNLGMDELYK。
  9. 根据权利要求8所述的应用，其中，所述大斯托克斯橙色荧光蛋白的基因的DNA碱基序列为：
    Figure PCTCN2019124979-appb-100003
    Figure PCTCN2019124979-appb-100004
  10. 根据权利要求7所述的应用，其中，在生物发光共振能量转移系统中，受体为荧光素酶NanoLuc，供体为所述大斯托克斯橙色荧光蛋白。
  11. 根据权利要求8所述的应用，其中，在生物发光共振能量转移系统中，受体为荧光素酶NanoLuc，供体为所述大斯托克斯橙色荧光蛋白。
  12. 根据权利要求9所述的应用，其中，在生物发光共振能量转移系统中，受体为荧光素酶NanoLuc，供体为所述大斯托克斯橙色荧光蛋白。
  13. 一种大斯托克斯橙色荧光蛋白在基于随机单分子定位的超分辨技术成像中的应用，其中：所述大斯托克斯橙色荧光蛋白是一种突变的橙色荧光蛋白mKOκ，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有突变位点：V160D。
  14. 根据权利要求13所述的应用，其中，与mKOκ的氨基酸序列相比，所述大斯托克斯橙色荧光蛋白具有以下突变位点：M11T、E40A，T42K，R44K，A56S，L59I，A63S，H68N，V70A，E76A，L96M，S105K，Y118I，I134V，N137K，E167V，C174V，F178I，G196S，V200E，K202I，V211Y，H217S，S218Y，219-228：SNLGMDELYK。
  15. 根据权利要求14所述的应用，其中，所述大斯托克斯橙色荧光蛋白的基因的DNA碱基序列为：
    Figure PCTCN2019124979-appb-100005
    Figure PCTCN2019124979-appb-100006
  16. 根据权利要求13所述的应用中，其中，用所述大斯托克斯橙色荧光蛋白标记细胞中的结构和细胞器后，利用激发光照射样品。
  17. 根据权利要求14所述的应用中，其中，用所述大斯托克斯橙色荧光蛋白标记细胞中的结构和细胞器后，利用激发光照射样品。
  18. 根据权利要求15所述的应用中，其中，用所述大斯托克斯橙色荧光蛋白标记细胞中的结构和细胞器后，利用激发光照射样品。
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