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Improved red fluorescin and application thereof

A technology of red fluorescent protein and amino acid, which is applied in application, fluorescence/phosphorescence, plant gene improvement, etc., and can solve problems such as application limitation, cell or tissue toxicity, and short wavelength

Active Publication Date: 2018-10-16
北京义翘神州科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of wild-type eqFP578 is still not very ideal, including: 1): It is not suitable for thick specimens and live animal imaging experiments in terms of maturation speed, fluorescence intensity, wavelength, etc.; 2): And it is easy to form tetramers at high concentrations , has certain toxicity to cells or tissues, and its application is limited
3): The wavelength is still short

Method used

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  • Improved red fluorescin and application thereof
  • Improved red fluorescin and application thereof
  • Improved red fluorescin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of the red fluorescent protein eqFP578 mutant library

[0039] Through the analysis of the protein structure of the pacifier sea anemone (Entacmaea quadricolor) red fluorescent protein eqFP578 (see SEQ ID NO.1 for the amino acid sequence) by Discovery studio4.0 software, find out 32 sites that may affect the structure of eqFP578, these 32 sites Yes: L4, K6, N21, G30, P52, L58, M63, H72, L79, E86, I93, L102, T103, F110, N112, I115, V124, N129, M133, A142, N143, G152, S158, G166, H171, F174, K181, H193, R198, M216, K220, H230, these 32 points are staggered and divided into 8 groups (Table 1), and saturation mutations are performed on the amino acids at each site in each group, thus obtaining 8 groups of saturation Mutated gene bank; In addition, the eqFP578 gene was randomly mutated by the error-prone PCR method to obtain a group of mutant gene banks; the mOrange2 gene was randomly mutated by the error-prone PCR method to obtain a group of mutant ge...

Embodiment 2

[0046] Example 2 Screening of red fluorescent protein eqFP578 mutant clones

[0047] Spread the eqFP578-DS library evenly on the ZYM-AT self-inducing medium plate (1% tryptone, 0.5% yeast extract, 25mM Na 2 HPO 4 , 25mM KH 2 PO 4 , 50mM NH 4 Cl, 5mM Na 2 SO 4 , 0.5% glycerol, 0.05% glucose, 0.2% a-lactose, 2mM MgSO 4 , 0.2x trace elements, 1% agar, 50 μg / mL Amp and 10 μg / mL Tet), cultured overnight at 30 ° C, the single clones in the eqFP578-DS library expressed mutant fluorescent proteins, showing different brightness and color. Observe the plate and circle the brighter red colonies with a marker. The next step is to prepare ZYM-AT self-inducing liquid medium, and add 200 μl ZYM-AT liquid medium to each well of a 96-well deep-well plate. Then these bright red colonies were picked out and inoculated into 96-well plates, a total of 1820 clones were picked, and all deep-well plates were placed in a shaker at 30°C and 200rpm for 16 hours for fluorescent protein expression...

Embodiment 3

[0052] Construction and expression of embodiment 3RFPSpark mutant fluorescent protein and FP650 fluorescent protein eukaryotic expression vector

[0053]Using primers RFP-inf-F: 5'GGTACCGCTAGCGGATCCATGGTGAGCAAGGGCGAGGAG 3' (SEQ ID NO.8) and RFP-inf-R: 5'GGCCGCTCTAGACTCGAGTCACTTGTACAGCTCGTCCAT 3' (SEQ ID NO.9) to amplify the RFPSpark gene with the mutant type screened as a template, The infusion enzyme was connected to the eukaryotic expression vector pCMV3, and the correct plasmid pCMV3-RFPSpark was identified. The pCMV3-RFPSpark plasmid with transfection grade purity was purified and transfected into 293H cells.

[0054] The specific steps are as follows: 293H cells are cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide. Before transfection, the cells were divided into 1*10 5 The density of cells / well was inoculated in 24-well plates and used for transfection after 24 hours of culture, and the cell density was 70%-90%. 2μg DNA and a cer...

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Abstract

The invention provides improved red fluorescin and an application thereof and belongs to the field of protein engineering. The improved red fluorescin RFPSpark provided by the invention is obtained through reforming an amino acid sequence eqFP578 of red fluorescin derived from Entacmaea quadricolor and has an amino acid sequence shown in SEQ ID No. 7. Compared with wild type protein, the fluorescin has obviously different excitation and emission spectra and has the characteristics of higher fluorescence intensity, higher mature speed, better stability and longer wavelength; and compared with another modified protein FP650 of eqFP578, the fluorescent brightness of the red fluorescin provided by the invention is 3 times higher than that of the FP650 under equivalent test conditions. The invention further provides the application of the improved red fluorescin. The improved red fluorescin is applicable to analysis of cell marking, animal deep tissue and living animal imaging, multicolor application and the like and has a broad application prospect.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to an improved red fluorescent protein and its application. Background technique [0002] Fluorescent proteins serve as molecular tags and have broad applications in analytical biotechnology and intracellular molecular tracking. Especially in the aspect of cellular molecular imaging, the fluorescent protein is fused to a target protein in the cell to label and analyze the location, distribution and movement of the target protein in the cell and the interaction with other intracellular molecules. The selection of fluorescent labels mainly considers the following aspects: 1) The fluorescent protein has high expression brightness and is non-toxic; 2) The fluorescent protein has good photostability; 3) The fluorescent protein cannot be sensitive to the expression cell or tissue environmental factors; 4) Fluorescent proteins that can be detected in multiple channels and do n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12G01N21/64
CPCC07K14/43595G01N21/6428
Inventor 谢良志孙春昀孙玲玲孟红芳
Owner 北京义翘神州科技股份有限公司
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