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Small molecular near-infrared fluorescent protein and fusion protein thereof

A fluorescent protein and near-infrared light technology, which is applied in the field of fluorescent markers, can solve the problems of insufficient brightness, large protein molecular weight, and few types of near-infrared fluorescent proteins, and achieves the effect of excellent stability.

Active Publication Date: 2019-12-17
HUAZHONG AGRI UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to overcome the technical deficiencies in the prior art that there are not many types of near-infrared fluorescent proteins, the emission wavelength is relatively single, the brightness is not high enough, and the molecular weight of the protein is large, and a small molecule near-infrared fluorescent protein is provided. protein

Method used

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  • Small molecular near-infrared fluorescent protein and fusion protein thereof
  • Small molecular near-infrared fluorescent protein and fusion protein thereof
  • Small molecular near-infrared fluorescent protein and fusion protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1BD

[0035] Vector construction, expression and property determination of embodiment 1 BDFPs mutant

[0036] Mutation starting template BDFP1.6 (SEQ ID NO.1, ie ApcF2(20-169)-

[0037] F30L / S46T / I51V / N72C / Y82C / Y92M / D101G / E107G / L109M / L113F / G125C / T127A / S130G / N136K / V143A / T151A / V160I / V161A / E163V) PCC720sp. Nuclear subunit protein ApcF2. BDFP1.6, evolved from BDFP1.1 (ApcF2(20-169)-S46T / I51V / N72C / Y82C / Y92M / N136K / V160I / V161A). The fluorescence range of BDFP1.6 is blue-shifted from near-infrared light to far-red light; especially its effective brightness is even better than that of commonly used iRFP670, while its molecular weight is only half of its size.

[0038] Through long-term creative research, it was found that the 113th, 125th and 127th amino acid residues have an important influence on the spectral properties of BDFPs protein. These residues in BDFP1.1 are leucine (L), glycine (G) and threonine (T), respectively, while phenylalanine (F), cysteine ​​(C) in BDFP1.6 and alanine...

Embodiment 2BD

[0077] The stability of embodiment 2BDFPs mutant fluorescent protein

[0078] In this example, the resistance of fluorescent proteins to in vitro conditions, such as acid-base, denaturing conditions, high temperature, and photobleaching, was tested. The results are shown in Figure 6 middle.

[0079] Figure 6 (a) shows the stability of BDFPs, IFP2.0 and iRFP720 in pH2-9 acid-base environment, the fluorescence intensity of BDFPs only changes by 40% in the pH range from 9 to 2, while IFP2.0 at pH3. At 5 o'clock, the fluorescence of iRFP720 has been completely quenched at pH 2. This shows that BDFP1.9 is relatively stable at low pH, and has higher stability than IFP2.0 and iRFP720 under acidic conditions.

[0080] Figure 6 (b) shows the fluorescence intensities of BDFPs, IFP2.0 and iRFP720 in different concentrations of guanidine hydrochloride solutions. It can be seen from the figure that the fluorescence of IFP2.0, iRFP720 and BDFP1.1 has been completely quenched in 4M ...

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Abstract

The invention discloses a small molecular near-infrared fluorescent protein. The near-infrared fluorescent protein comprises an amino acid sequence of a BDFP near-infrared fluorescent protein, and comprises mutations at the 24th, 27th, 30th, 31st and 38th sites of amino acid. The amino acid sequence of the BDFP far-infrared fluorescent protein is as shown in 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 forbeing used as a protein fusion tag sequence compared with the existing near-infrared fluorescent protein.

Description

technical field [0001] The invention belongs to the technical field of fluorescent markers, and more specifically relates to a small-molecule near-infrared fluorescent protein and a fusion protein thereof. Background technique [0002] Far-red light (FR) or near-infrared light (NIR) has low light absorption and light scattering in animal tissues, and has high penetration. It is the spectral region with the greatest ability to penetrate most tissues such as skin. Fluorescent proteins with such luminescent pigment groups are more suitable for deep imaging of living animal tissues, and are more ideal fluorescent markers for in vivo imaging. [0003] At present, there are mainly two types of fluorescent markers, both of which have a molecular weight of about 35kD. One is derived from green fluorescent protein (GFP), which can self-catalyze to form a chromophore, but the spectral range has certain limitations. The maximum fluorescence emission wavelength is generally around 670n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/795C07K19/00C12N15/31C12N15/62G01N1/28G01N21/64A61K49/00
CPCA61K49/0045C07K14/795C07K2319/00G01N1/28G01N21/6486
Inventor 周明夏坤付卫雷佟顺刚
Owner HUAZHONG AGRI UNIV
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