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Method for improving fluorescence signal intensity of fluorescent protein

A fluorescent protein and fluorescent signal technology, applied in the biological field, can solve the problem that the three-dimensional structure is not easy to form correctly, and achieve the effect of improving the intensity of the fluorescent signal

Inactive Publication Date: 2018-08-17
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the fluorescent protein is expressed in exogenous recombinant cells, the three-dimensional structure is not easy to form correctly, especially in prokaryotic cells.

Method used

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  • Method for improving fluorescence signal intensity of fluorescent protein
  • Method for improving fluorescence signal intensity of fluorescent protein
  • Method for improving fluorescence signal intensity of fluorescent protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of fusion fluorescent protein IGFP

[0032] This example exemplifies a fusion fluorescent protein IGFP spliced ​​with the N-terminal signal peptide of the naturally occurring protein Cry1Ia and the green fluorescent protein GFP, and its amino acid sequence is SEQ ID NO:11.

[0033] The characteristics of the IGFP protein are: the N-terminal signal peptide sequence of Cry1Ia is located at the N-terminal of the green fluorescent protein GFP; there are no other redundant amino acids between the N-terminal signal peptide of Cry1Ia and the amino acid sequence of the green fluorescent protein GFP; the C-terminal of IGFP contains 6 A histidine, used to extract IGFP protein if necessary. This histidine tag sequence is not required to construct the fusion fluorescent protein. The coding polynucleotide sequence of the IGFP protein can be obtained from at least three ways: 1) by using the polynucleotide sequence of the N-terminal signal peptide of coding C...

Embodiment 2

[0034] Example 2: Construction of prokaryotic cell expression vector pAcIGFP fused with fluorescent protein IGFP

[0035] This example exemplifies a prokaryotic cell expression vector for the fusion fluorescent protein described herein, which can be used to monitor the expression pattern of the promoter Pac of the polynucleotide chain encoding the Cry1Ac protein (ie, the promoter of the cry1Ac gene).

[0036] The 5' of the polynucleotide chain encoding the IGFP protein is connected to the promoter Pac of the cry1Ac gene, and the 3' is connected to the terminator Tac of the cry1Ac gene to form a complete expression frame, and this expression frame is inserted into a deletion restriction The pHT304 vector (Olivia Arantes and Didier Lereclus, Gene, 1990) of the Dicer SacI recognition site——p304ΔSacI, forming the pAcIGFP expression vector ( figure 1 ). As a control, the coding polynucleotide sequence of green fluorescent protein (with 6 histidines at the C-terminal) was also conn...

Embodiment 3

[0038] Example 3: Application of expression vector pAcIGFP in Bacillus thuringiensis

[0039] The expression vectors pAcIGFP and pAcGFP were respectively transformed into competent cells of Bacillus thuringiensis by electroporation. The screened single clones were shaken and cultured in liquid LB medium (Luria-Bertani medium) containing 25 μg / mL erythromycin (28.5° C., 200 rpm). At 9, 12, 24, 36, 48, 60, and 72 hours after culture, the fluorescence signal intensity of the cells was monitored respectively. The results showed that the fluorescence signal intensity of the Bacillus thuringiensis cells containing the pAcIGFP vector was consistently about four to six times higher than that of the Bacillus thuringiensis cells containing the pAcGFP ( figure 2 A). This result fully demonstrates that, compared with GFP, IGFP is more suitable for use as a fluorescent marker for Bacillus thuringiensis.

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Abstract

The invention discloses a method for improving fluorescence signal intensity of fluorescent protein. The method includes: using a signal peptide sequence of specific protein or a mutation sequence ofthe signal peptide sequence or chimeric sequence of the signal peptide sequence or a polypeptide sequence designed through artificial simulation to guide expression of the fluorescent protein so as toimprove the fluorescence signal intensity of the fluorescent protein in host cells, wherein the signal peptide sequence of the specific protein specifically refers to a signal peptide sequence of part of naturally existing protein or the mutation sequence of the signal peptide sequence or the chimeric sequence of the signal peptide sequence or the polypeptide sequence designed through artificialsimulation. By the method, the fluorescence signal intensity of the fluorescent protein in the host cells is improved, and fusion protein of this kind can be used as novel complete fluorescent proteinand also can serve as more efficient fluorescent indicating molecule to be used for various researches or research and development of fluorescence-labeled cells or organisms for other purposes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for increasing the fluorescent signal intensity of fluorescent proteins. Background technique [0002] Fluorescent proteins (FPs) are an important class of indicator proteins, classified according to different colors, including but not limited to the following categories: green fluorescent proteins (Green fluorescent proteins), red fluorescent proteins (Red fluorescent proteins), yellow fluorescent proteins ( Yellow fluorescent proteins), orange fluorescent proteins (Orange fluorescent proteins), far-red fluorescent proteins (Far-red fluorescent proteins), cyan (cyan) fluorescent proteins (Cyan fluorescent proteins) and UV-excitable green fluorescent proteins (UV-excitable green fluorescent proteins ) (Nathan C Shaner, Paul A Steinbach and Roger Y Tsien, Nature Method, 2005). Of course, the types or mutants of fluorescent proteins are still increasing. Under the right cond...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/63
CPCC07K14/00C07K2319/02C12N15/63
Inventor 高建华王兴春米怡郭小琴王向英
Owner SHANXI AGRI UNIV
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