Small-molecule near-infrared light fluorescent protein and fusion protein thereof
阅读说明：本技术 一种小分子近红外光荧光蛋白及其融合蛋白 (Small-molecule near-infrared light fluorescent protein and fusion protein thereof ) 是由 周明 夏坤 付卫雷 佟顺刚 于 2019-07-01 设计创作，主要内容包括：本发明公开了一种小分子的近红外光荧光蛋白,所述近红外荧光蛋白包括BDFP近红外光荧光蛋白的氨基酸序列,并且包括在第24位,27位,30位,31位,38位的氨基酸处的突变,所述BDFP远红光荧光蛋白的氨基酸序列如SED ID NO:1～14任一所示。本发明提供的近红外荧光蛋白有效亮度高,分子量小并且是单体结构的近红外荧光蛋白,相比现有近红外荧光蛋白更适合作为蛋白融合标签序列。(The invention discloses a small-molecule near-infrared fluorescent protein, which comprises an amino acid sequence of BDFP near-infrared fluorescent protein and mutations at amino acids at positions 24, 27, 30, 31 and 38, wherein the amino acid sequence of the BDFP far-infrared fluorescent protein is shown as any one of SED ID NO 1-14. The near-infrared fluorescent protein provided by the invention has high effective brightness and small molecular weight, is a near-infrared fluorescent protein with a monomer structure, and is more suitable to be used as a protein fusion tag sequence compared with the existing near-infrared fluorescent protein.)
1. A near-infrared fluorescent protein, which comprises an amino acid sequence of a BDFP near-infrared fluorescent protein and mutations at amino acids at positions 24, 27, 30, 31 and 38, wherein the BDFP far-infrared fluorescent protein is a BDFP protein series taking ApcF2(Chroococci thermophilis sp.PCC7203) as a template.
2. The near-infrared fluorescent protein of claim 1, wherein the sequence of the BDFP far-red fluorescent protein is shown in any one of SEQ ID NO 1-14.
3. The near-infrared fluorescent protein of claim 1 or 2, wherein the amino acid at position 24 is mutated to arginine; the amino acid at position 27 is mutated to glutamine; the amino acid at position 30 is mutated into glutamine; the amino acid at the 31 st position is mutated into glutamine; valine at position 38 was mutated to arginine.
4. The NIR fluorescent protein of claim 3, wherein valine at position 24 is mutated to arginine; leucine 27 is mutated to glutamine; leucine at position 30 is mutated to glutamine; leucine at position 31 is mutated to glutamine; valine at position 38 was mutated to arginine.
5. The near-infrared fluorescent protein of claim 4, wherein the amino acid sequence of the near-infrared fluorescent protein is shown in SEQ ID NO. 15.
6. A fusion fluorescent protein, which comprises the near-infrared fluorescent protein of any one of claims 1 to 5.
7. A nucleic acid encoding the near-infrared fluorescent protein of any one of claims 1 to 5 or the fusion fluorescent protein of claim 6.
8. A vector comprising the nucleic acid of claim 7.
9. The near-infrared fluorescent protein of any one of claims 1 to 5 or the fusion fluorescent protein of claim 6 for use in cell fluorescence localization.
10. Use of the near-infrared fluorescent protein according to any one of claims 1 to 5 or the fusion fluorescent protein according to claim 6 for deep imaging of living tissue of an animal.
Technical Field
The invention belongs to the technical field of fluorescent markers, and particularly relates to a small-molecule near-infrared fluorescent protein and a fusion protein thereof.
Background
Far-red (FR) or near-infrared (NIR) light has low light absorption and light scattering in animal tissue, high penetration, and is the spectral region with the greatest ability to penetrate most tissues, such as skin. The fluorescent protein with the luminescent pigment group is more suitable for deep imaging of living animal tissues and is a more ideal fluorescent marker for living body imaging.
At present, the fluorescent markers are mainly of two types, and the molecular weight is about 35 kD. One is derived from Green Fluorescent Protein (GFP) which can form chromophore by autocatalysis, but the spectrum range has a certain limitation, and the maximum fluorescence emission wavelength is generally about 670nm, such as a marker TagRFP 675. Another receptor protein, bacterial photopigment protein (BphP), which is present in thousands of bacteria. The BphP mainly uses Biliverdin (BV) with a linear tetrapyrrole structure as a chromophore; meanwhile, biliverdin BV widely exists in thousands of eukaryotes, which means that the BphP fluorescent marker can be applied to living animal cells and tissues without any proper or exogenous auxiliary factors. Representative of the BphP-type labels are the iFP series and the iRFP series, and have fluorescence emission wavelengths in the range of 670nm-720nm, such as the IFP2.0 maximum fluorescence emission wavelength of 714 nm.
Phycobiliproteins (phycobi1iprotein) have fluorescence emission in the far-red range, a mechanism similar to bacterial photopigment protein (BphP), and are derived mainly from non-covalently bound Phycocyanobilin (PCB). Typical phycobiliprotein fluorescent markers, such as ApcA, smuRFP, ApcF2, have a maximum fluorescence emission wavelength of 698 nm.
Ding W L et al obtained several new fluorescent phycobiliproteins after genetic modification based on the sequence of core subunit ApcF2 of phycobilisome and named BDFP, these BDFP proteins can be covalently bound with biliverdin BV, the performance is more stable than ApcF2, in addition, the molecules of these BDFP proteins are the smallest, about 15kD, the maximum fluorescence emission wavelength is about 710 nm.
Although the BDFP protein obtained by Ding W L et al well compensates the above-mentioned disadvantages of the existing far-red or near-infrared fluorescent proteins, the fluorescence emission wavelengths of these proteins are relatively single (both around 710 nm), and thus they cannot be effectively used in combination. Therefore, the fluorescent protein with higher brightness, more various spectral properties and excellent property is obtained through genetic engineering modification, and the method has very important significance.
Disclosure of Invention
The invention aims to solve the technical problems of few kinds of near-infrared fluorescent proteins, single emission wavelength, low brightness and large protein molecular weight in the prior art, and provides a small-molecular near-infrared fluorescent protein.
Another technical problem to be solved by the invention is to provide a fusion fluorescent protein.
The invention also aims to solve the technical problem of providing nucleic acid for coding the near-infrared fluorescent protein or the fusion fluorescent protein.
The invention also solves the technical problem of providing a vector comprising the nucleic acid.
The invention also aims to solve the technical problem of providing the application of the infrared light fluorescent protein or the fusion fluorescent protein in the aspect of cell fluorescence localization.
The invention also aims to solve the technical problem of providing the application of the infrared light fluorescent protein or the fusion fluorescent protein in deep imaging of animal living tissues.
The purpose of the invention is realized by the following technical scheme:
Provided is a near-infrared fluorescent protein comprising the amino acid sequence of BDFP near-infrared fluorescent protein and mutations at the amino acids at positions 24, 27, 30, 31 and 38, wherein the BDFP far-infrared fluorescent protein is a BDFP protein series with ApcF2 (Chococcidiopsis thermolis sp.PCC7203) as a template.
More specifically, the sequence of the BDFP far-red light fluorescent protein is shown in any one of SEQ ID NO 1-14.
Further, the amino acid at position 24 is mutated to arginine; the amino acid at position 27 is mutated to glutamine; the amino acid at position 30 is mutated into glutamine; the amino acid at the 31 st position is mutated into glutamine; valine at position 38 was mutated to arginine.
Preferably, the valine at position 24 is mutated to arginine; leucine 27 is mutated to glutamine; leucine at position 30 is mutated to glutamine; leucine at position 31 is mutated to glutamine; valine at position 38 was mutated to arginine.
More preferably, the amino acid sequence of the near-infrared light fluorescent protein is shown as SEQ ID NO. 15.
Provides a fusion fluorescent protein, wherein the fusion fluorescent protein comprises the near-infrared fluorescent protein.
Providing a nucleic acid encoding the near-infrared fluorescent protein or the fusion fluorescent protein.
There is provided a vector comprising the nucleic acid described above.
Provides the application of the near-infrared fluorescent protein or the fusion fluorescent protein in the aspect of cell fluorescence localization.
Provides the application of the near infrared fluorescent protein or the fusion fluorescent protein in deep imaging of animal living tissues.
The invention has the beneficial effects that:
The invention provides various micromolecular near infrared light fluorescent proteins, the emission wavelength is about 694nm to 705nm, and the characteristic of high brightness of the existing BDFP1.6 is kept, wherein the BDFP1.9 is a monomer structure, has the minimum molecular weight (17kD) in the near infrared light fluorescent proteins, has a slight blue shift of a spectrum, and has the effective brightness similar to IFP 2.0. Most BDFPs proteins are dimeric structures. However, in the aspect of application, the fluorescent protein with a monomer structure does not affect the stoichiometry of the target protein, so that the fluorescent protein is more suitable to be used as a protein fusion tag sequence.
The BDFP1.9 near-infrared light fluorescent protein with the monomer structure provided by the invention has excellent stability in low-pH and high-concentration guanidine hydrochloride solution or high-temperature environment, and can resist photobleaching.
the near-infrared Fluorescent Proteins (FPs) are powerful tools for realizing deep imaging, the invention provides more choices for the fluorescent proteins for deep imaging, the near-infrared fluorescent proteins provided by the invention can be used together with other fluorescent proteins, and the small-molecule near-infrared fluorescent proteins are more suitable to be used as protein fusion tag sequences.
Drawings
FIG. 1 comparison of spectra and intensity of BDFPs fluorescent proteins: (a) absorbance and fluorescence spectra of BDFPs, and purifying a protein sample by Ni2+ affinity chromatography; (c) the effective brightness of BDFPs in HEK293t cells was compared to that of iRFP720 and IFP2.0 under the same conditions. The mean near-infrared fluorescence intensity was normalized to mean eGFP fluorescence intensity. Error bar, SEM (n ═ 3, number of images). (d) Comparison of the effective luminance of hek293t with the molecular luminance of near infrared FPs under the same conditions. The effective luminance and molecular luminance of BDFP1.7 were set to 100%.
FIG. 2 analysis of the mimic structure and aggregation of BDFP 1.8. (a) Simulated structure of BDFP1.8, red for amino acids red-shifted from the spectrum, blue and pink for amino acids associated with effective brightness; (b) and (c) local structures at M81K and a127V for BDFP1.8, respectively; (d) the dimeric fluorescent protein ApcE generates an amino acid residue site for polymerization; (e) amino acid sequence homology comparison of ApcE with BDFP 1.8; (f) amino acid sequence homology comparison of BDFP1.6 with BDFP 1.8.
FIG. 3 in vitro polymerization of BDFPs fluorescent protein with BV. Fluorescence enhancement of BDFPs BV in solution. 18 μ M BDFPs were incubated with 0.1(a), (b)1, (c)10 μ M BV in KPB buffer (containing 150mM/L sodium chloride, pH 7.2) using F ═ a1-A2exp-ktThe equation fits the increase in fluorescence index. (d) Effective Brightness and mean k values (t) of BDFPs in HEK293t cells50％Ln2/k), the effective luminance of BDFP1.7 is set to 1.
FIG. 4 shows the molecular size and intracellular fluorescence intensity of BDFPs fluorescent protein. (a) The result of exclusion chromatography of the BDFPs fluorescent protein; (b) results of exclusion chromatography using protein markers; (c) SDS-PAGE results of BDFPs fluorescent protein; (d) fluorescence microscopic imaging of the plasmid of the eGFP, mCherry and BDFPs fused fluorescent protein after expression in HeLa cells; (e) fluorescence intensity ratio of several fusion fluorescent proteins in the smooth endoplasmic reticulum of HeLa cells.
FIG. 5 retention time of fluorescence of BDFPs with IFP2.0, iRFP720 in photobleaching treatment in HEK293T cells: BDFPs, IFP2.0 and iRFP720 were expressed in HEK293T cells and detected 24 hours after transfection by photobleaching treatment and the retention time of fluorescence was detected under continuous illumination with a 640nm diode laser (maximum output power 77% at 100 mW).
FIG. 6 in vitro stability comparisons of BDFPs with IFP2.0, iRFP 720. (a) Stability of BDFPs with IFP2.0 and iRFP720 in acid-base environment of pH 2-9; (b) stability of BDFPs with IFP2.0, iRFP720 with guanidine hydrochloride (GdnHCl) in different concentrations of solution (pH 7.2); (c) BDFPs and IFP2.0, iRFP720 at 80 ℃ high temperature stability; (d) retention time of fluorescence in the photobleaching treatment of BDFPs and IFP2.0, iRFP 720: photobleaching of FPs in KPB buffer (pH 7.2) under illumination of 100WHBO103W/2 lamp, BDFPs and IFP2.0 Using near-infrared Filter set (. lamda.) (ex650/45nm and λem710/50nm), iRFP720 employs a near infrared filter set (λ)ex650/45nm and λem720/40nm), the light was focused through a C-Apochromat immersion lens (100 x, numerical aperture 1.2) onto a Zeiss Axioscope a1 microscope equipped with a cool-snap HQ2CCD camera, and the fluorescence intensity curve fit was attenuated with a single index.
Detailed Description
The technical solutions of the present invention are further described below with reference to specific examples and drawings, but the present invention is not limited to these specific embodiments. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.