Molecular design method of gene-coded red fluorescent protein

A technology of red fluorescent protein and molecular design, applied in the field of fluorescent protein in biotechnology

Inactive Publication Date: 2010-10-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art has not yet disclosed that after carrying out molecular design on these two types of apoprotein gene fragments capable of binding to PCBs, they are fused with genes hol and pcyA for

Method used

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  • Molecular design method of gene-coded red fluorescent protein

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Embodiment 1

[0017] (1) The heme reductase gene pcyA encoded as alr3707 in Anabaena PCC7120 was cloned into the expression vector pET30 of Novagen Company by molecular biology methods; then the gene was encoded as heme all1897 by molecular design method The terminator of the oxidase gene hol is mutated into a non-terminator amino acid, and a DNA fragment encoding 5 amino acids is designed after the heme oxidase gene hol, and is fused with the heme reductase gene pcyA to obtain a new gene hol: pcyA, through which the fusion gene encodes and synthesizes phycocyanin PCB.

[0018] (2) After the gene fragment gaf2 of the gene fragment gaf2 of the phytochrome from 221-397 of the phytochrome of gene coding in Anabaena PCC7120 from 221-397, design the DNA fragment connection of encoding 5 amino acids by molecular design method, connect with step again (1) The obtained gene hol:pcyA is fused, and the fusion functional gene gaf2:hol:pcyA capable of expressing red fluorescent protein with reversible ...

Embodiment 2

[0022] (1) The heme reductase gene pcyA encoded as slr0116 in Synechocystis sp. PCC6803 was cloned into the expression vector pET30 of Novagen Company by molecular biology methods; then the gene was encoded as heme sll1184 by molecular design The terminator of the oxidase gene hol is mutated into a non-terminator amino acid, and a DNA fragment encoding 5 amino acids is designed after the heme oxidase gene hol, and is fused with the heme reductase gene pcyA to obtain a new gene hol: pcyA, through which the fusion gene encodes and synthesizes phycocyanin PCB.

[0023] (2) in Synechocystis PCC6803, the phytochrome encoded as slr1393 is after the gene fragment of the pigment structure domain gene fragment gaf3 of 441-596, and the DNA fragment of encoding 5 amino acids is designed to be connected by molecular design method, and the step ( 1) The resulting gene hol:pcyA is fused to synthesize a fusion functional gene gaf3:hol:pcyA capable of expressing a red fluorescent protein with...

Embodiment 3

[0027] (1) The heme reductase gene pcyA encoded as alr3707 in Anabaena PCC7120 was cloned into the expression vector pET30 of Novagen Company by molecular biology methods; then the gene was encoded as heme all1897 by molecular design method The terminator of the oxidase gene hol is mutated into a non-terminator amino acid, and a DNA fragment encoding 15 amino acids is designed after the heme oxidase gene hol, and is fused with the heme reductase gene pcyA to obtain a new gene hol: pcyA, through which the fusion gene encodes and synthesizes phycocyanin PCB.

[0028] (2) in Anabaena PCC7120, the phytochrome gene coded as all1069 is from the gene fragment of the pigment domain gene fragment gaf2 of 221-397, and the DNA fragment of encoding 15 amino acids is designed to be connected by molecular design method, and the step ( 1) The obtained gene hol:pcyA is fused, and the fusion functional gene gaf2:hol:pcyA capable of expressing red fluorescent protein with reversible photochromi...

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Abstract

The invention relates to a molecular design method of gene-coded red fluorescent protein, comprising the following steps of: obtaining a corresponding fusion gene hol:pcyA by fusing a heme oxidase gene hol and a heme reducase gene pcyA; coding and synthesizing phycocyanobilin PCB by the fusion gene; generating gene-coded reversible photochromic or non-reversible photochromic red fluorescent protein after fusing the fusion gene hol:pcyA and a genetic fragment of an apoenzyme proteinochrome structure domain which combines the phycocyanobilin PCB again. The molecular designing method of gene-coded red fluorescent protein can be widely used in the biological fields of fluorescent probes, trace labeling or positioning exogenetic organisms, phototherapy, and the like.

Description

technical field [0001] The invention belongs to the field of fluorescent proteins in biotechnology, and in particular relates to molecular design methods of gene-coded reversible photochromic red fluorescent protein and non-reversible photochromic red fluorescent protein. technical background [0002] Green Fluorescent Protein (GFP) is a genetically encoded protein that emits green fluorescence under ultraviolet light. By modifying its molecular structure, the intensity and color of fluorescence emitted by the fluorescent protein can be improved. Therefore, GFP is widely used as a reporter gene in biological fields such as protein labeling and visualization of organisms. However, GFP mainly emits green light under ultraviolet light, which will cause mutation or damage to organisms, which is not conducive to applications in many fields. [0003] Phycocyanobilin (PCB) is the main phycobilin in natural cyanobacteria, which is produced by the conversion of heme under the catal...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/63C07K19/00
Inventor 伍贤军周明张娟赵开弘
Owner HUAZHONG AGRI UNIV
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