Far-red fluorescent protein and fusion protein thereof

A technology of red light fluorescence and fluorescent protein, which is applied in the field of fluorescent markers, can solve the problems of ineffective combination use and single emission wavelength, and achieves the effect of stable performance and improved effective brightness.

Active Publication Date: 2019-01-04
HUAZHONG AGRI UNIV +1
View PDF1 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the BDFP proteins obtained by Ding W L et al. well make up for the above-mentioned shortcomings of the existing far-red or near-infrared fluorescent proteins, the fluorescent emission wavelengths of these proteins are relatively single (all around 710nm), so they cannot be used effectively. Combined use

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Far-red fluorescent protein and fusion protein thereof
  • Far-red fluorescent protein and fusion protein thereof
  • Far-red fluorescent protein and fusion protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Homologous sequence comparison analysis

[0059] figure 1 A comparison of several homologous sequences is shown, in which ApcF2 and its derivative sequence BDFP1.1 are all from Chroococcidiopsis thermalis PCC 7203; ApcB is from Spirulina platensis, which is not suitable for the growth environment of far-red light and near-infrared light. From figure 1 It can be seen that L113 is highly conserved among ApcB, ApcF2 and BDFP1.1.

[0060] On the basis of the BDFP1.1 sequence (SEQ ID NO: 2), L120 was subjected to site-directed saturation mutagenesis; the screened mutants were subjected to continuous random mutation through an error-prone PCR reaction to establish a mutant library. The mutants in the mutant library were screened by using the method of in vitro spectral property detection and live cell imaging according to the following screening criteria: Compared with BDFP1. 660nm; simultaneously with known fluorescent protein marker iRFP670 (ε·Φ fl =10.2mM -...

Embodiment 2

[0067] Example 2. Detection of the effective brightness of each fluorescent protein in live HEK 293T cells

[0068] The nucleic acid (FPs:IRES:eGFP) expressing the mutant BDFP1.1, v3 or v12 (BDFP1.3) was constructed in the expression vector pcDNA3.1, then transiently transfected into HEK 293T cells, and observed using an inverted fluorescence microscope Fluorescence brightness. The results are shown in figure 2 middle.

[0069] figure 2 Fluorescence images observed under the green channel and FR or NIR channel, respectively, are shown. It can be seen that BDFP1.1, v3 or v12 (BDFP1.3) all emit green fluorescence, and BDFP1.1 and v12 (BDFP1.3) emit red fluorescence. Compared with them, the red fluorescence of v3 is weaker.

Embodiment 3

[0070] Embodiment 3. Comparison of the spectral properties of mutant v12 (BDFP1.3) and known fluorescent proteins and the effective brightness in HEK 293T cells

[0071] Fluorescent proteins expressed in E. coli were purified by Ni2+ affinity chromatography, and then their spectral properties were examined. The detection solution environment is KPB (20mM, pH 7.0) and 0.5M NaCl; the excitation light wavelength of the fluorescence emission spectrum is 660nm. The comparison reference substance of molecular brightness is iRFP670(ε·dp fl =10.2mM -1 cm -1 ). In addition, the effective fluorescence (far-red light) of each mutant in HEK293T cells was compared. The results are shown in Table 3 below.

[0072] It can be seen from Table 3 that the emission wavelength of BDFP1.1 is 707nm, while the emission wavelength of BDFP1.3 is 675nm. Therefore, BDFP1.3 can be used in combination with BDFP1.1 for dual fluorescent labeling.

[0073] In addition, BDFP1.3 has an effective brightne...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses far-red fluorescent protein. The far-red fluorescent protein comprises an amino acid sequence of BDFP near-infrared fluorescent protein and mutation at 120th amino acid and isfree of mutation at 113th amino acid, and the amino acid sequence of the BDFP near-infrared fluorescent protein is shown as SED ID NO:2. The invention further discloses fusion fluorescent protein containing the far-red fluorescent protein and also discloses a far-red fluorescent protein or fusion fluorescent protein coding nucleic acid and a carrier with the same.

Description

technical field [0001] The invention belongs to the field of fluorescent markers. Specifically, the present invention relates to a far-red fluorescent protein, a nucleic acid and a carrier encoding the far-red fluorescent protein. 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 animal living 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 the formation of chromophores, but the spectral range is limited, and the maximum fluorescence emission wavelength...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/795C07K19/00C12N15/11C12N15/62C12N15/85
CPCC12N15/85C07K14/405C07K14/795C07K2319/00
Inventor 周明夏坤付卫雷吴明佟顺刚
Owner HUAZHONG AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap