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Preparation method of high fluorescence intensity recombinant phycobiliprotein concatermer

A technology of phycobiliprotein and fluorescence intensity, which is applied in biochemical equipment and methods, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of low fluorescence quantum efficiency, low fluorescence intensity, and limited enhancement of fluorescence intensity, and achieve improved Fluorescence intensity, strong fluorescence signal, low cost effect

Active Publication Date: 2017-07-04
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The document "Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli, Journal of Bioscience and Bioengineering, 107(3), 246–249, 2009" reported a method for producing recombinant allophycocyanin by genetic engineering, but the recombinant allophycocyanin Phycocyanin is a single subunit, covalently bound phycocyanin, and the fluorescence quantum efficiency is low, so the fluorescence intensity is low
The Chinese patent "Preparation method of phycocyanin fluorescent protein combined with phycoerythrin, patent authorization number: 200810025626.2" provides a genetic engineering method to produce recombinant allophycocyanin combined with phycoerythrin. Due to the recombinant The protein is also a single subunit, and the enhancement of fluorescence intensity is limited

Method used

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  • Preparation method of high fluorescence intensity recombinant phycobiliprotein concatermer
  • Preparation method of high fluorescence intensity recombinant phycobiliprotein concatermer
  • Preparation method of high fluorescence intensity recombinant phycobiliprotein concatermer

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Experimental program
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Embodiment 1

[0028] 1. Cloning of genes

[0029] The genes of Synechococcus elongatus BP-1 apcA, Synechocytis sp. PCC 6803 Ho1, Prochlorococcus phage P-SSM2 pebS, Synechococcus elongatus BP-1 cpcS and streptavidin were obtained from the National Center for Biotechnology Information (NCBI) database SA sequence (Accession No. X65082). Therefore, specific primers for amplifying apcA, Ho1, and cpcS were designed respectively (Table 1). Genomic DNA of Synechococcus elongatus BP-1 was used as a template for apcA gene, Genomic DNA of Synechocytissp. The SA gene and linker sequence were artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, during the artificial synthesis of the linker sequence, partial sequences of the SA gene and the apcA gene were added to its 5' end and 3' end, respectively.

[0030] 2. Construction of recombinant plasmids

[0031] 2.1 Fusion (monomer) of streptavidin gene and allophycocyanin α subunit gene and...

Embodiment 2

[0053] 1. Cloning of genes

[0054] Genes of Synechococcus elongatus BP-1 apcA, Synechocytis sp. PCC 6803 Ho1, Prochlorococcus phage P-SSM2 pebS, Synechococcus elongatus BP-1 cpcS and streptavidin were obtained from the National Center for Biotechnology Information (NCBI) database SA sequence (Accession No. X65082). Therefore, specific primers for amplifying apcA, Ho1, and cpcS were designed respectively (Table 1). Genomic DNA of Synechococcus elongatus BP-1 was used as template for apcA gene, genomic DNA of Synechocytissp.PCC 6803 was used as template for Ho1, and genomic DNA of Synechococcus elongatus BP-1 was used as template for cpcS gene. The SA gene and linker sequence were artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, during the artificial synthesis of the linker sequence, partial sequences of the SA gene and the apcA gene were added to its 5' end and 3' end, respectively.

[0055] 2. Constructio...

Embodiment 3

[0078] 1. Cloning of genes

[0079] Genes of Synechococcus elongatus BP-1 apcA, Synechocytis sp. PCC 6803 Ho1, Prochlorococcus phage P-SSM2 pebS, Synechococcus elongatus BP-1 cpcS and streptavidin were obtained from the National Center for Biotechnology Information (NCBI) database SA sequence (Accession No. X65082). Therefore, specific primers for amplifying apcA, Ho1, and cpcS were designed respectively (Table 1). Genomic DNA of Synechococcus elongatus BP-1 was used as template for apcA gene, genomic DNA of Synechocytissp.PCC 6803 was used as template for Ho1, and genomic DNA of Synechococcus elongatus BP-1 was used as template for cpcS gene. The SA gene and linker sequence were artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, during the artificial synthesis of the linker sequence, partial sequences of the SA gene and the apcA gene were added to its 5' end and 3' end, respectively.

[0080] 2. Constructio...

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Abstract

The present invention belongs to the field of fluorescent proteins in biotechnology, and particularly relates to a preparation method of a high fluorescence intensity recombinant phycobiliprotein concatermer. According to the preparation method, a streptavidin gene is linked to an allophycocyanin alpha subunit gene through a linker sequence; on the basis, one or a plurality of allophycocyanin alpha subunit genes are connected in series through linker sequences to form a fusion gene; and the fusion gene, a phycobiliprotein lyase gent and a phycoerythrobilin biosynthetic enzyme gene co-express in Escherichia coli to obtain the recombinant allophycocyanin concatermer with characteristics of biotin binding ability and high fluorescence intensity. According to the present invention, tn the immunofluorescence assay, the recombinant phycobiliprotein concatermer can achieve the strong fluorescent signal compared to the recombinant phycobiliprotein monomer; and the prepared recombinant phycobiliprotein concatermer can be adopted as the fluorescent marker for immunofluorescence detection in the field of biology and biomedicine.

Description

technical field [0001] The invention belongs to the field of recombinant expression and preparation of fluorescent proteins in biotechnology, and in particular relates to a method for preparing recombinant phycobiliprotein concatenations with high fluorescence intensity. Background technique [0002] Phycobiliprotein is an important pigment protein of algae, which has the function of light energy capture and transmission. Each molecule of phycobiliprotein contains two structurally similar polypeptide chains α and β, and the α subunit and β subunit contain about 160-180 amino acid residues, respectively, and the ratio of the two is usually 1:1. Cysteine ​​residues in subunits are covalently bonded to phycobilichromes through thioether bonds, and the type of phycobilichromes and their interactions with apoproteins determine the spectroscopic properties of phycobiliproteins. According to the different absorption spectra, phycobiliproteins can be divided into four categories: p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K19/00
CPCC07K14/795C07K2319/60C12N15/62C12N15/70C12N2800/101C07K19/00
Inventor 陈华新姜鹏武静李富超
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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