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Gene modified novel BDFP fluorescent protein and fused protein thereof

A fluorescent protein and protein technology, applied in the field of fluorescent markers, can solve the problems of single emission wavelength and cannot be used in effective combination, and achieve the effect of improving effective brightness, improving effective brightness and stable performance.

Active Publication Date: 2019-03-26
GUANGZHOU TEBSUN BIO TECH DEV
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  • 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

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  • Gene modified novel BDFP fluorescent protein and fused protein thereof
  • Gene modified novel BDFP fluorescent protein and fused protein thereof
  • Gene modified novel BDFP fluorescent protein and fused protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Homologous sequence comparison analysis

[0074] 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.

[0075] On the basis of the BDFP1.1 sequence (SEQ ID NO.: 2), L113 was subjected to site-directed saturation mutagenesis; the screened mutants were subjected to continuous random mutation through error-prone PCR reactions 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 -1...

Embodiment 2

[0082] Example 2. Detection of the effective brightness of each BDFP fluorescent protein in HEK 293T cells

[0083] The nucleic acid expressing each mutant (FPs: IRES: eGFP) was constructed in the expression vector pcDNA3.1, and then transiently transfected into HEK 293T cells, and the fluorescence brightness was observed using an inverted fluorescence microscope. The results are shown in figure 2 middle.

[0084] figure 2 a shows the fluorescence images observed under the green channel and FR / NIR channel, respectively. Because BDFP1.1, v1, v2, v3, v4, v7, v8, v14 in FR / NIR channel, 5s exposure time did not collect images with effective brightness, so take 30s exposure time. The exposure time of other mutants was 5s. figure 2 Quantification of the effective luminance of mutants v6 (BDFP1.2), v7 and v8 is shown in b.

[0085] From the above results, it can be seen that the mutants v1, v2, v4, v6(BDFP1.2), v7 and V8 can all observe fluorescence under the FR / NIR channel. ...

Embodiment 3

[0086] Example 3. Further Mutant Screening

[0087] In order to further search for mutants with excellent properties, the inventors conducted another round of mutations. With the same screening criteria as in Example 1, 20 mutants v13-v32 were screened out, as shown in Table 3 below.

[0088] At the same time, the spectral properties of the mutants v13-v32 were detected, and the results are shown in Table 4 above.

[0089] From the results in Table 4, it can be seen that the maximum emission wavelength of v13~v32 is similar to that of v6 (BDFP1.2), both are around 670nm, and the molecular brightness is also higher.

[0090]

[0091]

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Abstract

The invention discloses a far infrared fluorescent protein which comprises an amino acid sequence of a BDFP near infrared fluorescent protein and comprises mutants at amino acids of the 30th, 101st, 107th, 109th, 113th, 130th, 143rd, 151st and 163rd loci. The far infrared fluorescent protein does not comprise mutants at amino acids of the 35th, 120th and 122nd loci. The amino acid sequence of theBDFP near infrared fluorescent protein is as shown in SEQ ID NO: 2. The invention also discloses a fused fluorescent protein of the far infrared fluorescent protein. The invention further discloses anucleic acid which encodes the far infrared fluorescent protein or the fused fluorescent protein thereof and a carrier including the nucleic acid.

Description

technical field [0001] The invention belongs to the field of fluorescent markers. Specifically, the present invention relates to a far-red fluorescent protein and its fusion protein, nucleic acid and vector 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 e...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K19/00C12N15/31C12N15/62
CPCC07K14/43595C07K14/795C07K2319/00
Inventor 夏坤宋建勋卢艳华付卫雷陈彦蓉
Owner GUANGZHOU TEBSUN BIO TECH DEV
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